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This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg)Nanyang Technological University, Singapore.
Effects of thrombin and its related peptidefragments on neutrophils
Lim, Chun Hwee
2018
Lim, C. H. (2018). Effects of thrombin and its related peptide fragments on neutrophils.Doctoral thesis, Nanyang Technological University, Singapore.
https://hdl.handle.net/10356/87781
https://doi.org/10.32657/10220/46840
Downloaded on 23 Nov 2020 18:49:21 SGT
EFFECTS OF THROMBIN AND ITS RELATED PEPTIDE
FRAGMENTS ON NEUTROPHILS
LIM CHUN HWEE
Interdisciplinary Graduate School
NTU Institute for Health Technologies
2018
0
1
EFFECTS OF THROMBIN AND ITS RELATED
PEPTIDE FRAGMENTS ON NEUTROPHILS
LIM CHUN HWEE
Interdisciplinary Graduate School
NTU Institute for Health Technologies
A thesis submitted to the Nanyang Technological University in partial
fulfillment of the requirement for the degree of
Doctor of Philosophy
2018
2
3
Statement of Originality
I hereby certify that the work embodied in this thesis is the result of
original research and has not been submitted for a higher degree to any
other University or Institution.
15 / 08 / 2018
Date Lim Chun Hwee
4
Supervisor Declaration Statement
I have reviewed the content and presentation style of this thesis and
declare it is free of plagiarism and of sufficient grammatical clarity to be
examined. To the best of my knowledge, the research and writing are
those of the candidate except as acknowledged in the Author Attribution
Statement. I confirm that the investigations were conducted in accord
with the ethics policies and integrity standards of Nanyang Technological
University and that the research data are presented honestly and without
prejudice.
15 / 08 / 2018
Date Artur Schmidtchen
5
Authorship Attribution Statements
1) Chapter 2, excluding unpublished data where indicated, is published
as ‘Lim, C. H., Puthia, M., Butrym, M., Tay, H. M., Lee, M. Z. Y., Hou,
H. W. and Schmidtchen, A. Thrombin-derived host defence peptide
modulates neutrophil rolling and migration in vitro and functional
response in vivo. Sci Rep 7, 11201, doi:10.1038/s41598-017-11464-x
(2017).’
The contributions of the co-authors are as follows:
I co-designed the project with Professor Schmidtchen.
I co-designed the in vitro neutrophil rolling experiments with HM Tay
and HW Hou. Cell treatments were prepared by me before they
assayed the cells for their rolling propensities. We interpreted the
results together.
I co-designed the in vivo experiments with M Butrym and M Puthia. M
Butrym and M Puthia performed the in vivo experiments and the
respective downstream processing of tissues and analyses. We
interpreted the results together.
MZY Lee was an intern who assisted in the chemotactic assays and
respective flow cytometry analyses under my directions.
I performed the rest of the experiments independently.
I prepared the manuscript drafts. Professor Schmidtchen revised the
drafts.
I am the corresponding author.
6
2) Chapter 3 is published as ‘Lim. C. H., Adav, S. S., Sze, S. K., Choong,
Y. K., Saravanan, R. and Schmidtchen, A. Thrombin and plasmin alter
the proteome of neutrophil extracellular traps. F Immunol 9, 1554,
doi:10.3389/fimmu.2018.01554 (2018).’
The contributions of the co-authors are as follows:
I co-designed the project with Professor Schmidtchen.
SS Adav and I co-designed the workflow from cell preparation to mass
spectrometry.
I prepared the biological materials used for mass spectrometry
SS Adav, Professor Schmidtchen and I interpreted the mass
spectrometry data.
SK Sze provided critical inputs on the use of mass spectrometry data.
YK Choong and I designed and planned the gel mobility shift
experiments.
R Saravanan contributed to the discussion on thrombin and plasmin
interactions with DNA.
SS Adav wrote the methods for mass spectrometry.
I did all the other validation and in silico work.
I prepared the manuscript drafts. Professor Schmidtchen revised the
drafts.
I am the corresponding author.
15 / 08 / 2018
Date Lim Chun Hwee
7
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Acknowledgements
I would like to thank the Interdisciplinary Graduate School for granting me
this opportunity to explore my academic ambitions. Also, I would like to
thank Lee Kong Chian School of Medicine and the supporting staff for
hosting me and always ensuring smooth operation of the laboratories.
I would like to first thank Professor Artur Schmidtchen who readily agreed
to meet and provide me with this opportunity to be part of his fascinating
ideas and plans. I would also like to thank my former undergraduate
project’s supervisor Dr Ng Lai Guan who is always supportive and agreed
to co-supervise me on this journey. To Professor Sten Ohlson, whom had
left NTU since, thank you for your good humour, encouragements and
advice throughout my stay here. Special thanks to Professor David
Laurence Becker who has always been prompt in providing his support.
To my laboratory mates cum friends at the Lee Kong Chian School of
Medicine, your positive energy and good cheer have dotted my journey
with memories which will accompany me for the rest of my life.
Big thanks to my family for their sacrifice, love and advice which created
my current self for whom I will always be grateful for.
Last, but definitely not least, to my best friend and confidante who is now
my wife, Janet – you have been the unwavering support and my best critic.
We have been through tough journeys together, and this would not have
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been smooth sailing without your sacrifice and encouragements. Thank
you, and I look forward to embarking on many great journeys with you.
11
Table of Contents
A List of publications ...................................................................... 15
B Summary ...................................................................................... 17
C List of abbreviations .................................................................... 21
1 Introduction .................................................................................. 31
1.1 The human skin .......................................................................... 31
1.2 Skin wounds and its socio-economic impacts ............................. 33
1.3 Wound healing ............................................................................ 38
1.4 Neutrophils in inflammation and host defence ............................ 40
1.4.1 Neutrophil development .................................................... 40
1.4.2 Neutrophil recruitment....................................................... 43
1.4.3 Neutrophil antimicrobial responses ................................... 47
1.4.4 Neutrophil extracellular traps ............................................ 52
1.4.5 Neutrophils in healing ....................................................... 56
1.5 Aims ............................................................................................ 59
1.6 Scope ......................................................................................... 61
2 Thrombin-derived host defence peptides modulate neutrophil
functions in vitro and in vivo ............................................................. 65
2.1 Abstract ...................................................................................... 65
2.2 Introduction ................................................................................. 66
2.3 Materials and methods ............................................................... 71
2.3.1 Materials ........................................................................... 71
2.3.2 Ethics statement ............................................................... 72
2.3.3 Isolation of polymorphonucleated cells (PMNs) ................ 72
2.3.4 Peptide uptake by PMNs .................................................. 73
2.3.5 LDH cytotoxicity assay ...................................................... 74
12
2.3.6 Assessment of PMN surface markers ............................... 74
2.3.7 Luminol-based ROS measurement ................................... 75
2.3.8 Flow cytometry-based ROS assessment .......................... 76
2.3.9 Enzyme-linked immune-sorbent assay (ELISA) for soluble
CD62L .......................................................................................... 77
2.3.10 PMN rolling characterisation .......................................... 77
2.3.11 Transwell migration assay ............................................. 78
2.3.12 ROS assessment in vivo ............................................... 79
2.3.13 Neutrophil infiltration ...................................................... 79
2.3.14 Statistical analyses ........................................................ 80
2.4 Results ........................................................................................ 81
2.4.1 GKY25 is selectively taken up by PMNs ........................... 81
2.4.2 GKY25 inhibits LPS-induced PMN activation .................... 83
2.4.3 GKY25 induces specific CD62L shedding without activating
PMNs .......................................................................................... 86
2.4.4 GKY25-induced shedding of CD62L affects PMN rolling in
vitro .......................................................................................... 88
2.4.5 GKY25 reduces PMN chemotactic responses against IL8 in
a CXCR1/2-independent manner ................................................... 90
2.4.6 GKY25 ameliorates LPS-induced local ROS production in
vivo .......................................................................................... 92
2.4.7 FYT21 modulates PMNs more effectively than HVF18 ..... 94
2.5 Discussion .................................................................................. 98
2.6 Acknowledgements................................................................... 105
2.7 Author contributions .................................................................. 106
2.8 Disclosure ................................................................................. 107
2.9 Supplementary information ....................................................... 108
13
3 Thrombin and plasmin alter the proteome of neutrophil
extracellular traps............................................................................. 115
3.1 Abstract .................................................................................... 115
3.2 Introduction ............................................................................... 116
3.3 Materials and Methods ............................................................. 119
3.3.1 Materials ......................................................................... 119
3.3.2 Ethics statement ............................................................. 120
3.3.3 PMNs isolation ................................................................ 120
3.3.4 NET induction ................................................................. 121
3.3.5 LC-MS/MS and data analysis ......................................... 121
3.3.6 SDS-PAGE and protein detection ................................... 125
3.3.7 DNA gel mobility shift assay ........................................... 126
3.3.8 DNA protection ............................................................... 126
3.3.9 Immunofluorescence....................................................... 127
3.3.10 Peptigrams .................................................................. 128
3.3.11 Statistical analysis ....................................................... 128
3.4 Results ...................................................................................... 129
3.4.1 Identification of NET-associated proteins ........................ 129
3.4.2 Thrombin and plasmin can bind to NETs ........................ 131
3.4.3 Effects of thrombin and plasmin on NET-associated histones
........................................................................................ 133
3.4.4 Effects of thrombin and plasmin on NET-associated
neutrophil elastase ....................................................................... 136
3.5 Discussion ................................................................................ 138
3.6 Acknowledgements................................................................... 145
3.7 Author contributions .................................................................. 145
3.8 Conflict of interest ..................................................................... 145
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3.9 Supplementary Information ....................................................... 146
4 Significance, future work and conclusions ............................. 153
4.1 TCPs: balance between therapy, immunomodulation and host
evasion ............................................................................................... 153
4.2 NETs: trapping biomarkers? ..................................................... 157
4.3 Concluding remarks .................................................................. 162
5 References.................................................................................. 165
15
A. List of publications
i. Lim, C. H., Puthia, M., Butrym, M., Tay, H. M., Lee, M. Z. Y., Hou, H.
W. and Schmidtchen, A. Thrombin-derived host defence peptide
modulates neutrophil rolling and migration in vitro and functional
response in vivo. Sci Rep 7, 11201, doi:10.1038/s41598-017-11464-x
(2017).
ii. Lim. C. H., Adav, S. S., Sze, S. K., Choong, Y. K., Saravanan, R. and
Schmidtchen, A. Thrombin and plasmin alter the proteome of
neutrophil extracellular traps. F Immunol 9, 1554,
doi:10.3389/fimmu.2018.01554 (2018).
16
17
B. Summary
Wounds are highly proteolytic environments containing proteases from
host cells as well as colonising bacteria. In this milieu, novel host defence
peptides derived from host proteins such as cytokines and coagulation
pathway proteins are being generated. For example, proteolysis of the
serum protein thrombin forms C-terminal peptides that have antibacterial
and anti-endotoxic properties. In addition, these peptides were shown to
have immunomodulatory effects on monocytes, but whether they have
any effects on the other major innate immune cells, neutrophils, was not
known. Hence, we aimed to address the immunomodulatory potential of
these C-terminal truncations on neutrophil functions. We show that the
prototypic thrombin C-terminal peptide GKY25 can attenuate neutrophil
responses to lipopolysaccharides (LPS) and the cytokine interleukin-8 in
vitro. Independently, GKY25 induced the shedding of neutrophil adhesion
molecule CD62L, thus reducing neutrophil surface tethering capacity in
vitro. Furthermore, subcutaneous or intraperitoneal GKY25 reduced
neutrophil response to subcutaneous LPS administration in vivo. This
demonstrates the therapeutic potential of GKY25 in modulating host
response in endotoxin-mediated conditions.
Of interest was to investigate whether endogenous C-terminal peptides
can also modulate neutrophils’ activities. Using two C-terminal truncated
peptides – HVF18 and FYT21 – generated by the proteolysis of thrombin
by neutrophil and Pseudomonas aeruginosa elastases respectively, we
show that FYT21 was more effective at inhibiting LPS-induced neutrophil
18
response at physiologically relevant concentrations. Furthermore, FYT21,
even more effectively than GKY25, reduced neutrophil surface CD62L.
These indicate a possible host evasion mechanism where FYT21 reduces
inflammatory responses to facilitate P. aeruginosa colonisation.
Neutrophils’ presence in wounds extend beyond their inflammatory
functions; they produce neutrophil extracellular traps (NETs), an
extracellular network of DNA decorated with neutrophil-derived proteins.
NET-associated proteins have been argued to trap and kill bacteria, as
well as being implicated in autoimmune complications. However, in vitro
assessments of its proteome (or NETome) had excluded external factors’
influence and non-neutrophil proteins from analyses. Using a
reductionistic approach, we applied physiological concentrations of
thrombin and plasmin, two of the more abundant serum proteins
representing the different stages of wounding, to NETs. Using a proteomic
approach, we identified 164 NET-associated proteins, where majority of
the proteins were shared between treatments but were different in
abundances. We show that most classical NET-proteins were reduced in
the presence of thrombin and plasmin, possibly through direct proteolytic
effects (i.e. on histones) or other regulatory mechanism (i.e. for neutrophil
elastase). This indicates that the NETome, under various inflammatory
conditions, can be dynamically different. We propose that by elucidating
these differences in a physiological context, it could potentially lead to
discovery of novel diagnostic or therapeutic markers relevant to specific
diseases where NETs are implicated.
19
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C. List of abbreviations
#
5-LOX: 5-lipoxygenase
α-ELANE: anti-neutrophil elastase antibody
α-H2B: anti-histone H2B antibody
α-H3: anti-histone H3 antibody
α-H4: anti-histone H4 antibody
α-PLG: anti-plasminogen antibody
α-VFR17: anti-thrombin C-terminal peptide VFR17 (VFRLKKWIQKVIDQFGE)
A
ADAM: A disintegrin and metalloproteinase
AF: Alexa-fluor
AF488-α-goat: AF488-conjugated donkey polyclonal anti-goat IgG antibody
AF568-α-rabbit: AF568-conjugated goat polyclonal anti-rabbit IgG antibody
AMP: Antimicrobial peptide
APC: Allophycocyanin
ATPase: Adenosine triphosphotase
AUC: Area under the curve
B
BCA: Bicinchoninic acid
BSA: Bovine serum albumin
BV: Brilliant violet
C
C/EBP: CCAAT enhancer-binding protein
Ca2+: Calcium ions
CaCl2: Calcium chloride
CD-: Cluster of differentiation
CDK: Cyclin-D kinase
CCL: CC-motif ligand chemokine
CF: Cystic fibrosis
CGD: Chronic granulomatous disease
CPPD: Calcium pyrophosphate dihydrate
22
CR1: Complement receptor 1; CD35
CXCL: CXC-motif ligand chemokine
CXCR: CXC-motif ligand chemokine receptor
Cy: Cyanine
D
DAMPs: Damage-associated molecular patterns
DAPI: 4’6-diamidino-2-phenylindole
DMSO: Dimethyl sulfoxide
DNA: Deoxyribonucleic acid
DNase I: DNA endonuclease I
E
ECM: Extracellular matrix
EDTA: Ethylenediaminetetraacetic acid
ELANE: Neutrophil elastase
ELISA: Enzyme-linked immunosorbent assay
emPAI: exponentially modified protein abundance index
F
FBS: Foetal bovine serum
FcγR: Fc-gamma receptor
FDR: False discovery rate
FITC: Fluorescein isothiocyanate
fMLP: N-formyl-methionyl-leucyl-phenylalanine
FPR1: Formyl peptide receptor-1
FYT21: Thrombin C-terminal peptide FYTHVFRLKKWIQKVIDQFGE
G
G-CSF: Granulocyte-colony stimulating factor
G-CSFR: Granolocyte-colony stimulating factor receptor
GKY25: Thrombin C-terminal peptide GKYGFYTHVFRLKKWIQKVIDQFGE
GPCR: G-protein coupled receptor
GTPase: Guanosine triphosphotase
23
H
H+: Hydrogen ion
H2O: Water
H2O2: Hydrogen peroxide
H2DCFDA: 2’,7’-dichlorodihydrofluorescein diacetate
H2B: Histone 2B
H3: Histone 3
H3.3: Histone 3.3
H4: Histone 4
hCAP-18: human cathelicidin protein-18
HCD: Higher energy collision dissociation
HDP: Host defence peptide
HIV-1: Human immunodeficiency virus-1
HMGB-1: High-mobility group box-1
HOCl: Hypochlorous oxide
HRP: Horseradish peroxidase
HRP-α-goat: HRP-conjugated donkey polyclonal anti-goat IgG antibody
HRP-α-rabbit: HRP-conjugated goat polyclonal anti-rabbit IgG antibody
HSCs: Haematopoietic stem cells
HVF18: Thrombin C-terminal peptide HVFRLKKWIQKVIDQFGE
I
i.p.: Intraperitoneal
IC: Immune complexes
ICAM-1: intercellular adhesion molecule 1
IgG: Immunoglobulin-G
IgM: Immunoglobulin-M
IL1β: Interleukin-1β
IL8: Interleukin-8
IL10: Interleukin-10
IRAK-4: Interleukin-1 receptor associated kinase-4
IVE25: Thrombin N-terminal peptide IVEGSDAEIGMSPWQVMLFRKSPQE
24
L
LC-MS/MS: liquid chromatography mass spectrometry
LDH: Lactate dehydrogenase
LL37: human cathelicidin; LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES
LPS: Lipopolysaccharides
LTA: Lipoteichoic acid
LTB4: Leukotriene B4
LXA4: Lipoxin A4
M
MDR: Multi-drug resistant
MFI: Median fluorescence intensity
MMP: Metalloproteinase
MNase: Micrococcal nuclease
MPO: Myeloperoxidase
N
NADPH: Nicotinamide adenine dinucleotide phosphate
NETs: Neutrophil extracellular traps
NETome: Proteome of NETs
NETosis: Process of NET formation
NIMP-R14: Anti-neutrophil antibody
NOX: NADPH oxidase
O
O2●-: Superoxides
OCT: Optimum Cutting Temperature
OmpA: Outer membrane protein A (from Escherichia coli)
P
PAD4: Protein arginine deiminase 4
PAMPs: Pathogen-associated molecular patterns
PB: Pacific blue
PBS: Phosphate buffered saline
PDMS: polydimethylsiloxane
25
PE: Phycoerythrin
PerCP: Peridinin chlorophyll protein complex
PFA: Paraformaldehyde
PMA: Phorbol 12-myristate 13-acetate
PMNs: Polymorphonucleated cells
ppm: Parts per million
PR3: Proteinase 3
PRR: Pattern-recognition receptor
PSGL1: P-selectin glycoprotein ligand 1
PVDF: polyvinylidene fluoride
R
ROS: Reactive oxygen species
RPMI: Rosewell Park Memorial Institute; cell culture medium
S
s.c.: Subcutaneous
sCD62L: Soluble CD62L
SDS-PAGE: Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
Serpin: Serine protease inhibitor
SNAP-23: Synaptosome-associated protein-23
SNARE: Soluble N-ethylmaleimide-sensitive factor attachment protein
receptor
SOD: Superoxide dismutase
T
TAE: Tris-acetate ethylenediaminetetraacetic acid
TAMRA: Tetramethylrhodamine
TAN: Tumour-associated neutrophils
TBS: Tris buffered saline
TBS-T: TBS with 0.1% Tween-20 (v/v)
TCP: Thrombin C-terminal peptide
TFPI-2: Tissue factor pathway inhibitor-2
TGFβ: Transforming growth factor-β
TLR: Toll-like receptor
26
TNFα: Tumour necrosis factor-α
T-GKY25: TAMRA-labelled GKY25
T-IVE25: TAMRA-labelled IVE25
U
US: United States
V
VAMP: Vesicle-associated membrane protein
VEGF: Vaso-endothelial growth factor
W
WHO: World Health Organization
27
28
29
– CHAPTER 1 –
Introduction
30
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1 Introduction
1.1 The human skin
The human skin is arguably the largest organ of the human body and
provides the first layer of defence against external assaults by microbes.
The skin has 3 layers – the epidermis, dermis and subcutaneous layers
(Fig. 1.1). The avascularised epidermis is the outermost layer and is
made of up ~80% keratinocytes and other cells such as melanocytes
(pigment cells), Merkel cells (superficial mechanosensory cells) and
Langerhans cells (dendritic cells) are also present. The proliferating
centre for keratinocytes is at the stratum basale, or the basal layer of the
epidermis, where epidermal stem cells are situated. In the stratum
spinosum, where keratinocytes differentiate and mature, they produce
and modify keratin fibres intracellularly which eventually translocate to the
plasma membrane. Overtime, these keratinocytes start to undergo
intracellular degradation to remove organelles and their nuclei.
Throughout this process, they are constantly compressed to form simple
squamous epithelium. Finally, as these cells die, they become
corneocytes, or the basic units of the stratified and cornified layer
otherwise known as the stratum corneum. It takes a keratinocyte ~14 days
to mature and become a corneocyte, and another ~14 days to traverse
the stratum corneum to be finally exposed to the environment1.
32
Figure 1.1: The human skin. Figure is adapted with permission under the Creative
Commons Attribution 4.0 International License from OpenStax, Anatomy &
Physiology. OpenStax CNX. Feb 26, 2016. Copyright © 1999-2018, Rice University.
Available from: http://cnx.org/contents/14fb4ad7-39a1-4eee-ab6e-
Underlying the epidermis is the highly vascularised dermis separated by
the basement membrane, or basal lamina, made up of mainly laminin and
Type IV collagen. The dermis is composed of an extracellular matrix
consisting proteoglycans, hyaluronan and dense connective tissues
produced by dermal fibroblasts to provide the skin with mechanical and
tensile support. The papillary layer of the dermis lies in proximity with the
basal lamina connecting the epidermis. Here, loose connective tissues
consisting Type I and III collagen and elastic fibres predominate. Denser
Type I collagen fibrils and elastic fibres form the deeper reticular layer2.
Appendages such as the sebaceous and sweat glands as well as hair
33
follicles transverse the dermis and epidermis. Cellular components and
structures from other systems are also present, such as the circulatory
system (microvasculature), nervous system (e.g. sensory corpuscles)
and immune system (e.g. dendritic cells, macrophages, mast cells, T
cells). Collectively, the epidermis and dermis act as a physical barrier
against environmental infectious agents, regulate body temperature and
osmolarity, retain water and provide first line immuno-surveillance1.
The hypodermis, or subcutaneous layer, forms the innermost layer of the
skin lying just above skeletal muscles. Majority of the cells are adipocytes.
Larger blood vessels penetrate this layer before invagination into the
dermis to form microvasculature. Also, the larger lymphatic vessels are
present in the adipose tissues and run parallel at the hypodermal-dermal
junction before branching into smaller vessels in the dermis2. As adipose
tissues, the hypodermis is an energy source and provides thermal
insulation and impact absorption from external forces to protect major
internal organs, muscles and bones1.
1.2 Skin wounds and its socio-economic impacts
Being the largest exposed organ of the human body, the skin is constantly
subjected to environmental assaults, at times resulting in injuries and
wounds. These injuries can be classified as closed and open wounds,
where the former refers to injuries related to impacts with blunt surfaces
leading to capillary damages and ‘bleeding’ without the breaking of skin
34
(i.e. contusions or bruises). Conversely, injuries which result in the
breaking of the skin surface are open wounds3.
Table 1.1: Types of open skin wounds.
WOUND TYPES DESCRIPTIONS CLASSIFICATIONS
Abrasions3
Loss of the epithelium due to friction between the skin and contact surface.
Usually superficial
Puncture4
High impact tangential penetration of the skin by a sharp object.
Partial to full thickness; could penetrate deeper tissues
Incisions3
An intentional cut to tear open the skin by using sharp instruments, such as during surgery. Not usually a concern following surgical re-approximation of the wound edges.
Usually full thickness; could penetrate deeper tissues
Lacerations3
Tear or opening of the skin by mechanical force. Could be caused by sharp objects or high shear.
Partial to full thickness; could penetrate deeper tissues
Ulcerations5
Sloughing of necrotic tissues leading to discontinuity of the skin barrier.
Partial to full thickness; could extend into deeper tissues
Burns6
Caused by contacts with high temperatures, corrosive chemicals, radiation and electricity.
Range from 1st to 3rd degree burns corresponding to superficial, partial and full thickness burns, respectively7.
There are different types of open wounds (Table 1.1). Superficial wounds
usually involve the loss of the stratum corneum resulting in low grade
inflammation and minimal bleeding. These wounds are commonly caused
by abrasions or mild burns to the skin. Deeper injuries exposing the
subcutaneous layer are considered partial thickness wounds, while
penetration into the subcutaneous layer to expose the skeletal muscles
35
are full thickness wounds. Such injuries are often accompanied by
considerable amount of bleeding and inflammation.
The socio-economic impacts of wounds, particularly chronic wounds,
cannot be underestimated. Chronic wounds, by definition, are wounds
that do not heal after 3 months from the onset. In the United States (US)
alone, it is estimated that 6.5 million people suffered from some forms of
chronic wounds, with an annual expenditure approximated at US$25
billion to treat and manage these wounds. Globally, it is estimated that 1-
2% of the population in developed countries will experience chronic
wounds at least once throughout their lifetimes8.
Chronic wounds often refer, but not restricted, to ulcerative skin wounds.
Although they differ in aetiology, chronic wounds are commonly
associated as co-morbidity to old-age and chronic diseases such as
diabetes and immobility. Pressure ulcers, as an example, affect people
with poor mobility, including the elderly and bed-ridden patients. They
occur predominantly in regions of bony prominence (e.g. elbows, sacrum)
when constant pressure, friction and shear force are applied8,9. In the US,
2.5 million pressure ulcers are treated annually in acute care facilities
alone. In addition, prevalence of nosocomial pressure ulcers in critical
care settings stands at approximately 22% in the US and 20% in Europe,
whereas those in acute and long-term care facilities come up to 15% and
29%, respectively, in the US. Treatment for pressure ulcers can be hefty
too – individual full-thickness ulcer can cost up to US$70,000 to treat.
36
Collectively, the US estimated an annual expenditure of US$11 billion to
treat pressure ulcers. Furthermore, litigations using acquired pressure
ulcers as evidence against poor patient care in long-term facilities have
proven favourable for 87% of these cases8, thus spelling wider social
issues pertaining to patient care and management.
Other, more prevalent chronic wounds or ulcers are located to the lower
extremities. They can be caused by vascular insufficiencies resulting in
venous or arterial leg ulcers, with the former implicated in 60-80% of all
chronic leg ulcers10,11. Venous leg ulcers affect roughly 600,000
individuals in the US annually and cost approximately US$9,600 to treat
per case, surmounting to a total cost of US$2.5-3.5 billion. In addition to
costs, venous ulcers led to approximately 2 million lost workdays per year
and US$2 billion lost wages. More remarkably, up to one-third of these
affected patients experienced recurrence, a figure which applies to
Europeans as well8.
Another common lower extremity chronic wounds are diabetic foot ulcers.
Diabetes (both type 1 and 2) affect 7.8%, >20% and 5-7% of the
populations in the US, Middle East and Europe, respectively8. Like
venous and arterial ulcers, diabetic patients often have some forms of
angiopathies, often due to accompanying cardiovascular diseases. This
is further complicated by neuropathies impairing these patients’ motor,
autonomic and sensory abilities12. Common areas of occurrence are the
bottom of the feet where primary wounds can go undetected due to the
37
patients’ dulled senses. Estimation has put the occurrence rate at up to
25% of all diabetic patients8, with a further 15-20% of these patients
requiring lower extremity amputations12. In the US, foot ulcers can cost
up to approximately US$20,000 per episode, with each amputation
procedure costing up to US$38,000.
These problems are further compounded by an ageing global population
and higher incidences of obesity. A report in 2016 finds that 8.5% of the
global population, or 617 million people, were aged 65 years and above.
This number is projected to almost triple to 1.6 billion people by 2050,
although the rate of ageing is not uniform between countries (e.g. Africa
would still have a relatively young population of <7% aged 65 years and
above by 2050). Due to this shift in global population and the positive
correlation between old age, chronic diseases (e.g. diabetes,
cardiovascular diseases) and immobility, the socio-economic burden is
only expected to increase13.
Separately, increased prevalence of obesity predicts higher prevalence of
other co-morbidities, again including chronic and metabolic diseases. The
World Health Organization (WHO) estimates in 2016 that 1.9 billion adults
over the age of 18 are overweight, with more than 650 million obese (13%
of total adult population)14. Should the current trend continue, it is
estimated that 20% of total adult population will be obese by 203015.
There are now growing evidence that obesity can impede wound healing,
38
hence potentially complicating the primary causes of chronic wound
manifestations as described earlier8.
Wounds resulting from external insults must also be taken into
consideration. These include surgical, traumatic and burn wounds which,
when left untreated or without proper management, can result in infection.
Surgical site infections for example, constitute up to 20% of all nosocomial
infections and can prolong hospital stays by an average of 9.7 days16.
Estimation in the US shows that 1.2 million require varying degrees of
treatments for burn wounds, with 100,000 requiring hospitalisation and
5,000 dying from burns wounds annually. Relatedly, 75% of all burn-
related deaths are as a result of sepsis or other infectious complications
in patients with severe burns covering more than 40% of the total body
surface area6.
Taken together, wounds not only have severe health impacts, it has
tremendous socio-economic impacts as well. Understanding how wounds
develop, where they affect and how they resist treatments can have huge
contributions to devising new diagnostic, prognostic and/or therapeutic
strategies.
1.3 Wound healing
Wound healing can be defined in 4 stages – haemostasis, inflammation,
proliferation and remodelling – which involve interactions at both
molecular and cellular levels17. A 3-stage wound healing model combining
haemostasis and inflammation is also commonly used18. The first stage
39
of wound healing is haemostasis, where the coagulation cascade is
activated following platelet aggregation at the site of vasculature damage
and their release of clotting factors17. The main activity involves the
activation of thrombin which converts fibrinogen into insoluble fibrin fibres
to trap erythrocytes and platelets, thus forming blood clots19. In concert,
the release of damage- and/or pathogen-associated molecular patterns
(DAMPs / PAMPs) and cytokines and chemokines by resident cells
initiated the inflammatory phase20. Neutrophils, the most abundant innate
immune cells in circulation, respond immediately to these danger signals.
Their primary functions are to kill and contain infectious micro-organisms
and further augment inflammation21. Monocytes recruitment to the injury
and their transition into inflammatory macrophages follow which enhance
and maintain effective inflammation by cytokines production, extracellular
matrix degradation, antimicrobial activities and phagocytic capacities18,22.
The whole inflammatory process usually lasts up to 2 days depending on
the severity and management of the wound18.
As inflammation starts to reduce, the proliferative phase takes over with
the aim to close the wound. Macrophages play a key role in coordinating
this transition by their ability to sense the microenvironment and respond
accordingly. Upon sufficient clearance of any infecting micro-organisms,
reduction in inflammatory cytokines, and neutrophil apoptosis, localised
macrophages ‘code-switch’ to produce anti-inflammatory and tissue
repairing mediators such as interleukin (IL)-10, transforming growth factor
(TGF)-β and vaso-endothelial grow factors (VEGFs)22,23. Under the
40
influence of these growth mediators, fibroblasts proliferate, and some
differentiate into myofibroblasts. They produce and reconstitute the
extracellular matrix predominantly with Type III collagen. Angiogenesis
occurs as well to re-supply the injury site with nutrients and oxygen. These
form visible red granulation tissues which act as scaffolds for
keratinocytes at the wound edge during re-epithelialisation. These
keratinocytes migrate toward the centre of the wound, further pushed
forward by the hyper-proliferating cells behind and eventually, close the
wound and restore the epithelium18.
As the wound closes, tissue remodelling proceeds underneath,
particularly in the dermis. By this time, excess cells such as fibroblasts
and macrophages undergo either apoptosis or become resident tissue
cells. Metalloproteinases (MMPs) produced by resident macrophages
and fibroblasts degrade Type III collagen to allow replacement with Type
I collagen, particularly in the reticular layer of the dermis18,24. Remodelling
can take up to 1 year before the injured site resembles the previous
uninjured state. However, the full tensile strength is never restored4.
1.4 Neutrophils in inflammation and host defence
1.4.1 Neutrophil development
Neutrophils are the most abundant innate cells in circulation, representing
40-70% of all circulating leukocytes in humans. They have a half-life of
about 8 hours under homeostasis. In circulation, neutrophils function to
sense danger, and if any, mount effective inflammatory responses. During
41
such inflammatory processes, neutrophils may have extended half-lives
and they usually undergo necrotic or apoptotic cell death at the site of
inflammation. However, without stimulation, neutrophils are programmed
to home back to the bone marrow to be cleared and recycled25.
Production of neutrophils begin in the bone marrow where haematopoietic
stem cells (HSCs) differentiate into lympho-myeloid precursors mediated
by the increased transcription factor PU.126. These cells further
differentiate into granulocyte-monocyte precursors and with the increase
in the transcription factors CCAAT enhancer-binding proteins (C/EBP),
particularly C/EBP-α, further differentiate along the granulocytic pathway.
Importantly, C/EBP-α drives the transcription of granulocyte-colony
stimulating factor receptor (G-CSFR) which receives signal from the
ligand G-CSF essential for neutrophil development27,28.
Granulopoiesis derives from the fact that neutrophils, or granulocytes
which also include eosinophils and basophils, form intracellular granules
as they differentiate and mature. Neutrophils form 3 major granules –
primary (azurophilic), secondary (specific) and tertiary (gelatinase)
granules which are classified based on the time of their appearance
during granulopoiesis and their contents. For example, primary granules,
or azurophilic granules, are formed the earliest during granulopoiesis, are
rich in myeloperoxidase (MPO) and can be stained with azure A. Apart
from MPO, they also express the surface marker CD63. Secondary and
tertiary granules packed later are peroxidase-negative and further
42
classified based on the higher levels of either lactoferrin or gelatinase,
respectively. Additionally, secondary granules express CD66b on its
surface but not tertiary granules. Finally, a fourth class of granules termed
secretory vesicles are formed following mature neutrophil endocytosis.
These vesicles are identified mainly by their surface markers such as
alkaline phosphatase and CD35 and contain plasma proteins including
albumin29,30. Counter-intuitively, the exocytosis of these granules goes in
the reverse order (secretory primary) during neutrophil recruitment and
stimulation, which is described in section 1.4.2.
The release of neutrophils from the bone marrow proceeds following their
maturation. G-CSF is essential for granulopoiesis and can reduce
CXCL12 expression in the bone marrow to indirectly drive neutrophil
mobilisation31,32. CXCL12 is a ligand of CXCR4 which facilitates the
retention and homing of neutrophils in the bone marrow33-35. Indeed, the
oscillation of CXCR2 and CXCR4 expression has been demonstrated to
coordinate neutrophil release and retention in the bone marrow
respectively. In CXCR2 and CXCR4 double knockout models, neutrophils
were found to be constitutively released, showing that CXCR2 is not
required for neutrophil release. However, the concurrent lack of CXCR4
is potentially important in retaining neutrophils within the bone marrow35.
Indeed, studies have also shown that at later stages of granulopoiesis,
CXCR4 downregulates from neutrophil surface while CXCR2 upregulates
to release the mature neutrophils from the bone marrow36,37.
43
1.4.2 Neutrophil recruitment
Mature neutrophils in circulation constantly survey for danger signals from
within the vasculature. This sensing activity occurs mostly in the
microvasculature (e.g. post-capillary venules) embedded in tissues where
neutrophils engage in weak tethering and rolling along the vaso-
endothelial walls on which danger signals can attach. The tethering and
rolling activities are mediated by the weak adhesion molecules such as
CD62L (or L-selectin) and P-selectin glycoprotein ligand (PSGL)-1
expressed on neutrophils with E-selectin on endothelial cells38. This weak
interaction can be easily disrupted by homeostatic circulatory flow rate38,
hence discouraging prolonged attachment of neutrophils to the
endothelial walls in the absence of inflammatory signals.
Injuries or inflammation kickstart the neutrophil recruitment cascade
which begins with neutrophil arrest from circulatory flow conditions.
Danger signals such as IL8 can be attached or expressed on endothelial
luminal surface to allow detection by rolling neutrophils. Upon detection
of such signals, neutrophils begin to exocytose primarily secretory
vesicles which store the strong adhesion molecule CD11b/CD18 and
express it onto the surface39. The expressed CD11b/CD18 then interacts
with the endothelial intercellular adhesion molecule (ICAM)-1, which is
also upregulated following inflammatory stimulation on endothelial cells40-
43, to allow strong neutrophil-endothelial attachment44-46. The neutrophil
attachment, with the concomitant shedding of CD62L from neutrophil
surface47,48, slows the immune cells into a crawl as neutrophils probe for
44
exit sites to extravasate from the endothelium. This is facilitated by an
increase in endothelial permeability which could be attributed to oxidative
stress49 (i.e. from neutrophils), inflammatory cytokines50 (e.g. tumour
necrosis factor [TNF]-α), DAMPs41,51 (e.g. mitochondrial DNA, high-
mobility group box [HMGB]-1) and PAMPs52,53 (e.g. LPS). However,
independent factors may have minimal effects on vascular permeability in
vivo54; for example, TNF-α has no effect on increasing vascular
permeability in perfused rat lungs ex vivo55. Nevertheless, in a complex
inflammatory environment, they can contribute collectively to allow
effective neutrophil egress from circulation.
Next, in order for neutrophils to transmigrate across the interstitial
following their exit from circulation, they need to detach from the extra-
luminal side of endothelial cells. For this to happen, a stronger, more
sustainable chemoattractant gradient must be present. In the initial phase
of injury, DAMPs and PAMPs stimulate the production of CXC
chemokines (e.g. IL8) and lipid mediator leukotriene B4 (LTB4) by resident
tissue cells. The early-infiltrating neutrophils then further amplify these
chemo-attractants and sustain the recruitment process as they migrate
toward the inflammatory or injured sites. In fact, these chemokines can
act on neutrophils in a paracrine and autocrine manner to stimulate their
chemotaxis and chemokine generation56-58. In addition, neutrophils
produce a myriad of pro-inflammatory cytokines such as IL1β and TNF-α
to stimulate other tissue resident cells, such as inflammatory T cells,
macrophages and skin cells which assist to sustain the inflammation20.
45
The chemo-attractant axis which facilitates neutrophil recruitment is
however still incompletely understood. Using the immune complexes (IC)-
induced arthritic K/BxN mouse model knocked out of LTB4 receptors,
Chou et al (2010) described a lipid-cytokine-chemokine axis (i.e. LTB4
IL1β CCL3 and CXCL2) which initiated and sustained the arthritis59.
However, in an IC-induced peritonitis model, Ramos CD et al (2006)
highlighted the initial involvement of the chemokine CXCL2, then the
cytokine TNF-α and finally LTB460 – a complete opposite from the K/BxN
model. Another sterile inflammation model using high-power laser to
induce focal tissue damage in the mouse ear shows that LTB4 is not
necessary for proximal neutrophil recruitment but is essential to recruit
neutrophils distal from the injury site61. Wounds, in this regard, possibly
resemble the laser-induced tissue damage. Regardless however, there
appears to be spatial, temporal and sequential differences in the
involvement of chemo-attractants between inflammatory conditions56.
As neutrophils migrate across the interstitial, they degranulate in a
sequential and logical manner to release factors needed for navigating
through the dense connective tissues first and exerting antimicrobial
pressure later. Contents of these granules potentiate this sequential
process; secretory vesicles are abundant with membrane adhesion
proteins such as CD11b/CD18 which is required for neutrophil
attachment; gelatinase-rich tertiary and secondary granules facilitate the
degradation of dense matrix proteins and finally, primary granules, which
contain highly toxic compounds such as serine proteases and
46
antimicrobial proteins, are released later at the injury site or fused with
phagosomes intracellularly62. Early studies have described the
sensitivities of respective granules to intracellular calcium (Ca2+) leading
to the hierarchical exocytosis of secretory vesicles, tertiary, secondary
and finally primary granules63,64. This is later attributed to the formation of
soluble N-ethylmaleimide-sensitive factor attachment protein receptor
(SNARE) complex involving vesicle-associated membrane protein
(VAMP)-2 and the 23 kDa synaptosome-associated protein (SNAP-23),
both of which found to predominate in the secretory vesicles, tertiary
granules and secondary granules. Abundance of VAMP-2 on these
granules decreases in this order, possibly contributed by its increasing
transcription over the duration of neutrophil maturation37. These granule
membrane SNARE proteins primarily target the plasma membrane
SNARE protein, syntaxin-4, leading to their rapid and extensive
exocytosis. Conversely, primary granules, devoid of VAMP-2 and SNAP-
23 but richer in VAMP-7, have lower exocytic propensities62,65,66.
However, whether SNAP-23 or VAMP-7 plays any role in the reduced
exocytosis is not fully understood. Regardless, this process allows the
regulated release of the relevant proteins during neutrophil migration.
Furthermore, the ‘non-release’ of primary granules, but preferential fusion
with phagosomes, further localises and concentrates their contents where
they are most relevant (described in section 1.4.3).
47
1.4.3 Neutrophil antimicrobial responses
Neutrophils at the injury site engage a range of antimicrobial responses
to eradicate any invading microbes, primarily by phagocytosis-mediated
intracellular killing by reactive oxygen species (ROS) production and
fusion with primary granules. The first step to phagocytosis is antigen
recognition by the repertoire of receptors expressed on the neutrophil
surface. Microbes can be directly recognised through pattern-recognition
receptors (PRRs) such as Toll-like receptors (TLRs) and C-type lectin.
Furthermore, microbes can be opsonised by immunoglobulin (Ig)-G and
complement factors C1q, C3b and C4b which are recognised by Fc-
gamma receptors (FcγRs) and complement receptor 1 (CR1; or CD35),
respectively67-69. Opsonisation by IgG and IgM also potentiates the
invading microbes to complement-mediated killing70. Phagocytosis then
proceeds when intracellular cytoskeleton re-assembles to form
phagosomal cup as the plasma membrane extends and engulfs the
microbial particle71.
Fully ingested microbial particle forms the early phagosome which is
inherently not antimicrobial72; a study shows that phagocytosed Neiserria
gonorrhoeae delayed and reduced the fusion with primary granules,
leading to increased viability of the bacteria. Restoration of this fusion
increases the killing capacity of neutrophils mediated by serine proteases
supplied by these granules73. Also, a study shows that a Mycobacterium
tuberculosis by-product can inhibit phagosomal acidification and fusion
with lysosomes in macrophages, hence first describing the evasive
48
mechanism of the bacterium from phagocytic killing74. Unlike
macrophages however, neutrophils’ mature phagosomes have near
neutral pH despite insertion of ATPase to pump H+ into the phagosome
lumen and fusion with the acidic primary granules. In fact, early
phagosomes undergo transient alkalinisation. The near-neutral pH could
be largely attributed to the consumption of free H+ during neutrophil
respiratory burst (i.e. ROS), leading to reduced insertion of ATPase and
increased leakiness of the phagosomal membrane72,75,76.
The formation of mature phagosomes in neutrophils can be mediated by
the GTPase Rab5a, where its interaction with syntaxin-4 and
microtubules possibly enables early phagosome bidirectional migration
along the microtubules. Exactly how Rab5a functions is still incompletely
understood72,77. The subsequent phagosome-granule fusion is facilitated
through the interactions between syntaxin-4 on early phagosomes77 and
possibly VAMP-7 on primary granules62.
There are two main modes of phagosome-mediated killing – respiratory
bursts and antimicrobial proteins – both of which require the fusion with
primary granules. Superoxides (O2●-) are the first ROS generated by the
NADPH oxidase (NOX) complex in the phagosomal lumen. The NOX
complex consists of cytosolic p47phox, p67phox and p40phox recruited to the
other NOX components gp91phox (or NOX2), gp22phox and GTPase Rac1
found on the phagosomal membranes. Then, O2●- is catalysed by
superoxide dismutase (SOD) to form hydrogen peroxide (H2O2), which is
49
finally chlorinated by MPO (provided by primary granules) to form
hypochlorous oxide (HOCl), the active component in bleach which exerts
the antimicrobial effect75. Importance of the NOX-dependent antimicrobial
activity is exemplified in patients with chronic granulomatous disease
(CGD) where they have dysfunction in either of the NOX subunits (mainly
gp91phox, p47phox, p67phox and p22phox). These patients suffer from chronic
infections by bacteria and fungi, particularly Staphylococcus aureus and
Aspergillus fumigatus as they are most susceptible to the NOX-dependent
killing78.
In fact, the second mode of phagosomal killing by antimicrobial proteins
is synergised by the precedence of active NOX. The highly oxidative
environment resulting from NOX activity raises the pH of mature
phagosomes to near neutral pH suitable for antimicrobial proteins
functions. This is evident where CGD neutrophils have acidic
phagosomes which impair their antimicrobial activities, probably due
dysfunctional respiratory burst leading to H+ accumulation78. Here,
primary granules supply antimicrobial proteins/peptides, also known as
host defence peptides (HDPs; e.g. bactericidal/permeability increasing
protein, LL37, defensins and azurocidin) and proteases (e.g. neutrophil
elastase, cathepsin G and proteinase 3)62. HDPs, when released or in
phagosomes, exert direct antimicrobial pressure to control bacteria
dissemination. Even during homeostasis, constitutive HDPs released
assist to regulate skin commensals (activities of HDPs are described in
more detail in Chapter 2.2). Besides, neutrophil-derived proteases can
50
exert direct antimicrobial pressure too. For example, neutrophil elastase
(ELANE) was shown to proteolyse the outer membrane protein A (OmpA)
of Escherichia coli which compromises the bacterial membrane integrity
leading to their lysis79. ELANE alone or with Cathepsin G and proteinase
3 (PR3) were also shown to directly kill Klebsiella pneumoniae or
Streptococcus pneumoniae, respectively, although their mode of
antimicrobial effects was not elucidated80,81. Conversely, Cathepsin G
consists of antimicrobial domains which are independent from its protease
activities82. In fact, a mimetic peptide modelled after one of the domains
was shown to have broad-spectrum antimicrobial effects83,84. Despite
these evidence, direct antimicrobial functions of neutrophil-derived
proteases remain incompletely understood. Their indirect antimicrobial
effects through proteolysis however appear to be more relevant from a
physiological perspective. For example, PR3 is known to cleave human
cathelicidin protein-18 (hCAP-18) into LL37 in the extracellular space85.
Similarly, ELANE can process plasma factors extracellularly including
complement C3, thrombin and tissue factor pathway inhibitor-2 (TFPI-2)
to generate antimicrobial peptides86-89. Likewise, cathepsin G had been
shown to process thrombin to generate the related antimicrobial
peptides87. These proteases can also target virulence factors of microbes
and attenuate their infectious propensities90. Collectively, the act of
engulfing bacteria and the formation of functional phagosomes are
fundamentals to neutrophils’ intracellular antibacterial activities.
51
ROS generation and primary granular contents also participate in
neutrophils’ extracellular antimicrobial activities. NOX components
previously sequestered in secondary and tertiary granules can
translocate to plasma membrane surface during neutrophil degranulation
as previously described, which can also recruit the remaining cytosolic
NOX members91. Primary granule exocytosis, in contrast to its fusion with
phagosome mediated by the GTPase Rab5a, is instead mediated by the
GTPase Rac292,93. It is also differentiated by its dependence on the
intracellular Ca2+ flux as opposed to Ca2+-independent late-phase fusion
with the phagosome93,94. Release of the granular contents is logical, as
the pH in acute wounds ranges from 6.5-8.5, thus creating an
environment suitable for neutrophil proteases’ activity. It must however be
highlighted that an alkaline environment can also promote bacterial
growth and increased protease activity leading to collateral tissue damage
as commonly seen in chronic wounds95. Likewise, HDPs released by
neutrophils including LL37 and defensins can also participate in
antimicrobial responses, but the significance they play can be influenced
by environmental factors (described in more detail in Chapter 2.2).
Neutrophils also produce ectosomes, or micro-vesicles, derived from the
plasma membrane first observed by Stein et al (1991) when neutrophils
were subjected to sub-lytic complement attack96. Subsequent studies
reveal that these ectosomes can be formed spontaneously under culture
conditions or following stimulation by opsonised bacteria, ionophores,
phorbol 12-myristate 13-acetate (PMA) and N-formyl-methionyl-leucyl-
52
phenylalanine (fMLP)97-99. Ectosomes formed under different situations
are also highly heterogenous97,100. In the context of antibacterial
response, Timar et al (2013) show that only opsonised bacteria or
zymosan can induce the release of bacteriostatic ectosomes. However,
these ectosomes are only effective against Staphylococcus aureus and
Escherichia coli and not Proteus mirabilis98. Such antimicrobial activity
might be attributed to the antimicrobial proteins packed within the
ectosomes, including MPO, neutrophil elastase, lactoferrin and
defensins98-100. These proteins possibly undertake direct antimicrobial
activities, but how MPO, in the absence of ectosomal NOX activity97, is
involved remains to be studied. Furthermore, how primary granule
proteins are packed into ectosomes is also unclear.
1.4.4 Neutrophil extracellular traps
Perhaps the most intriguing and widely-studied exudate of neutrophil
currently is neutrophil extracellular traps (NETs). First described by
Brinkman et al (2004), NETs are decondensed and unfragmented DNA
structure decorated with a plethora of neutrophil-derived proteins101. Its
formation, or NETosis, is inducible by PMA, Ca2+ ionophores, a variety of
microbes and activated platelets101-110. In addition, host factors can also
induce NETosis, including the human cathelicidin LL37, monosodium
urate (MSU) crystals, amyloid fibrils and immune complexes111-115. NETs,
or cell-free DNA, have also been observed in vivo, particularly from
patients with burn and diabetic wounds116,117, and biological samples from
53
patients with cystic fibrosis, sepsis, primary amyloidosis, autoimmune
diseases and infections, among others113,118-122.
NETosis can be a ‘suicidal’ or ‘vital’ process123. As the term suggests,
suicidal NETosis describes the formation of NETs following neutrophil cell
death, usually during the later stages of neutrophil activation. This form of
NETosis is predominant and NOX-dependent, where ROS production
plays a major role. How exactly ROS facilitates this process is unclear;
one possibility is through its disruption of nuclear membrane, resulting in
the expansion of the multi-lobed nucleus and allowing cytosol-nuclear
content mixing. The latter activity is particularly important, as cytosolic /
granular proteins such as neutrophil elastase has been shown to
translocate to the nucleus to facilitate NET formation124. Moreover,
neutrophils isolated from patients with Papillon-Lefévre syndrome, which
do not produce serine proteases including neutrophil elastase, had
defective NETosis125. This might be further supported by the fact that
neutrophils deficient in the elastase inhibitor, serine protease inhibitor B1
(SerpinB1), are hypersusceptible to NETosis126. NETosis was also shown
to require the citrullination of histones by the protein arginine deiminase 4
(PAD4) activated by Ca2+ in the nucleus to facilitate DNA decondensation
and the subsequent NET formation127,128, but whether it plays a
downstream role in response to ROS generation remains debatable129.
The alternative ‘vital’ NETosis undertakes a ROS-independent process,
where vesicular trafficking of DNA to the extracellular space is more
54
prominent104,123. Here, neutrophils release DNA without plasma
membrane rupture, leaving anuclear neutrophils to continue its
chemotactic and phagocytic functions130. This process is more rapid but
not all neutrophils undergo vital NETosis, suggesting that there might be
subpopulations favouring or primed towards this route. Vital NETosis can
be triggered by S. aureus both in vitro and in vivo104,130, Candida albicans
in vitro131, and also Ca2+ ionophores in vitro132. On the latter, because
PAD4 activation is Ca2+-dependent, stimulation with ionophores might
bypass NOX activation133, although this remains to be investigated. Also,
whether the signalling and downstream molecular players (e.g. elastase
and PAD4), or other possible mechanisms, facilitate this process is yet to
be elucidated.
While PAD4 activity seems to be crucial for NET formation, there are
evidence to suggest that NETosis can occur independent of PAD4
activity. For example, citrullination of histones was not observed in PMA-
induced NETosis129, suggesting that other forms of chromatin
decondensation are responsible (e.g. ELANE translocation into the
nucleus as described). Recent studies have also shown that PAD4 is not
essential for MSU or Candida albicans-induced NETosis112,134. In fact,
MSU-induced NETosis is also ROS-independent112. More interestingly,
calcium pyrophosphate dihydrate (CPPD) crystals implicated in
pseudogout is PAD4-dependent but ROS-independent135. Taken
together, NETosis appears to be a dynamic process dependent on the
55
stimuli which could influence the underlying molecular mechanism not yet
fully understood or anointed.
Brinkmann et al (2004) first described the antibacterial properties of NETs
against S. aureus and Shigella flexneri and attributed this effect to the
NET-bound histones101. Since, numerous studies have sought to
elucidate the antimicrobial activities of NETs against other microbes such
as bacteria (e.g. Pseudomonas aeruginosa, Neiserria gonorrhoeae and
Streptococcus pneumoniae)136-138, fungi (e.g. C. albicans and C.
glabrata)108,139, viruses (e.g. human immunodeficiency virus-1; HIV-1)109
and parasites (e.g. Leishmania amazonesis)110. Studies have shown that
the antimicrobial functions are attributed to NET-bound histones,
antimicrobial proteins and even the DNA itself101,105,138,139, suggesting that
NET-mediated antimicrobial activities might be accumulative and
multifaceted. Evasion mechanisms employed by a host of bacteria might
provide clues on the importance of these components, where majority
involves various nuclease activities on the DNA lattice of NETs137,140,141,
among others138,142. Interestingly, whether NETs are truly microbicidal is
still being debated, as Menegazzi et al (2012) demonstrate that S. aureus
and C. albicans were viable upon their release from NETs following
DNase I digestion. Therefore, they propose that NETs merely trap
microbes and prevent their growth143. A recent study using washed and
purified NETs incubated with P. aeruginosa and S. aureus supports the
‘trapping’ effect instead of the bactericidal properties of NETs144. Taken
56
together, there remains a gap to investigate and elucidate the exact NET-
mediated antimicrobial mechanism.
1.4.5 Neutrophils in healing
Neutrophils, as first responders to injuries and inflammation, participate
in a range of activities to mount an effective immune response and thus,
host defence. However, neutrophil pro-inflammatory responses often
cause collateral damage to the host in addition to the eradication of
invading micro-organisms due to their non-specific nature (e.g. ROS,
serine proteases, MMPs, NETs-mediated cytotoxicity)145. In fact, when
neutrophils were depleted in a mouse wound model, the wound healed
quicker with less fibrosis, albeit in relatively sterile conditions146,147. During
the later stages of inflammation, neutrophils can undergo apoptosis and
necrosis (including NETosis) which are phagocytosed by macrophages
along with other debris to stimulate the latter’s transition from the pro-
inflammatory M1 phenotype to the anti-inflammatory M2 phenotype148,149.
This is however subjected to the absence of pathogens or their derivatives
in the ingested materials by macrophages106,149-151. M2 macrophages
produce anti-inflammatory cytokines such as IL10 and TGFβ that affect
infiltrating neutrophils as well152. IL10 has been shown to act on
neutrophils in a paracrine / autocrine loop and further induce neutrophil
apoptosis153. TGFβ, likewise, can reduce the pro-inflammatory status of
neutrophils as shown in tumour-associated neutrophils (TAN) when
exposed to tumour-derived TGFβ154. In addition, infiltrating neutrophils in
the damaged tissues of myocardial infarction in mice at 7 days post-injury
57
were described to express anti-inflammatory factors and TGFβ reduced
the pro-inflammatory cytokine profile of isolated neutrophils155.
Neutrophils can also produce pro-resolving lipid mediators when
stimulated toward the anti-inflammatory phenotype. For example, instead
of LTB4, neutrophils can produce lipoxin A4 (LXA4). Although the
generation of both mediators diverges from the 5-lipoxygenase (5-LOX)
pathway156, LXA4 is readily inactivated by the highly-oxidative
environment during inflammation157, hence perhaps being more relevant
during the resolution phase of wounding. Other mediators include
resolvins and protectins. They can further inhibit neutrophil recruitment by
reducing their vaso-endothelial adhesion properties and sensitivity to
chemokines. These lipid mediators can also drive neutrophil apoptosis
directly or indirectly by inhibiting MPO-induced neutrophil survival, as is
the case for LXA4158. A recent report shows that delivery of exogenous
LXA4 accelerated the healing of skin ulcers, thus highlighting its
importance and therapeutic potential159. Hence, although neutrophils are
commonly associated to the inflammatory phase of wounding, there are
evidence to suggest that they play pro-resolving roles as well.
The involvement of neutrophils hence cannot be underestimated. This is
prominent in aged mice where neutrophils had reduced chemotactic
properties leading to delayed wound healing160. Similarly, neutrophil
depletion in older mice in situ impeded wound healing161. In the context
of dysfunctional neutrophils, CGD patients suffer from recurrent infections
58
and may have impaired wound healing162,163. The hyper-inflammatory
state contributed by neutrophils also attenuates wound healing, where
increased neutrophil infiltration, MPO activity and extracellular matrix
(ECM) degradative enzymes are implicated in various chronic wounds /
ulcers164-166. NETs found in burn wounds are pro-coagulative and might
contribute to thrombosis often associated with these patients167-169.
Dysregulated neutrophil activities are also commonly associated to poor
healing in diabetic wounds117,170-172. All of these indicate that dysregulated
neutrophil functions can impede proper wound healing.
Contrasting evidence on the activity of diabetic neutrophils exist however;
infected wounds of diabetic mice had reduced local MPO activity despite
normal neutrophil infiltration against infected wounds of wild-type mice170.
Yet, there are substantial evidence to show that diabetic human
neutrophils undergo NETosis more spontaneously117,171,172. Supporting
evidence include increased superoxide production from neutrophils
isolated from patients with poorly-controlled diabetes173 and increased
NETosis of diabetic neutrophils stimulated by the Ca2+ ionophore
ionomycin in a PAD4-dependent manner172. In contrast, there are also
studies to show reduced inducible-NETosis with the ionophore A23187
and LPS by diabetic neutrophils117,171. The study by Fadini et al (2016)
using A23187 measured NETs indirectly by NET-associated elastase and
MPO activities which had discrepancies, especially in spontaneous
NETosis of neutrophils isolated from diabetic patients117. Furthermore,
NETosis stimulated by different agonists may present a different
59
proteomic profile112, hence it may not be accurate to use enzymatic
activities to quantify NETosis. With regard to LPS, it remains controversial
on its NET-stimulatory properties174,175. Finally, NETs were less inducible
in diabetic neutrophils with the bacteria Burkholderia pseudomallei176.
These suggest that there are fundamental differences between the NET-
inducing agonists, in vitro versus in vivo NETosis, microbial burden on
neutrophil activities in diabetes and even the quantification methods.
Nevertheless, the presence of NETs in diabetic mice wounds can impair
wound healing and their disruption by nuclease or PAD4 inhibition can
improve wound healing117,172.
Taken together, neutrophils not only participate in initiating and sustaining
inflammation, they play crucial pro-resolving roles following successful
inflammation and eradication of micro-organisms. Dysregulated
neutrophil functions in various conditions can impair wound healing,
whether it is due to poor inflammation, insufficient clearance of colonising
microbes or hyper and prolonged inflammation.
1.5 Aims
In relation to wounds and inflammations, neutrophils are critical players.
Therapeutics targeting neutrophils directly are far and few between, with
examples of inhibiting neutrophil trafficking or granulopoiesis by blocking
CXCR2 or G-CSF receptor in treating tumours, respectively177. Likewise
for wounds, targeting chemokines have been shown to be effective in
preclinical wound models, but little have yet made it through to clinical
60
investigations178. On the spectrum of treating infection-related conditions,
roscovitine, a cyclin D kinase (CDK)-7 and -9 inhibitor, is being trialled to
treat P. aeruginosa-infected cystic fibrosis (CF) patients by promoting
neutrophil apoptosis and alveolar macrophage phagolysosomal
antibacterial function179. Degrading neutrophil-derived DNA or NETs
using nebulised DNase is already being used to treat CF patients which
improves lung functions180. NETs are also implicated in delayed diabetic
wound healing in humans and preclinical evidence have shown promise
in using nucleases to treat these wounds117,172.
Still, one of the primary causes of sustained neutrophil-mediated
inflammation is infection, and the various host evasion strategies
developed by pathogens remains widely investigated. Therefore,
targeting only the immune system or pathogens might be inadequate or
inefficient in complicated cases. The novel thrombin-derived HDP has
been shown to be antibacterial, anti-endotoxic and can modulate
monocyte / macrophages activities87,181,182. Hence, we hypothesised that
the HDP can also modulate neutrophil function(s) and aimed to elucidate
any potential immunomodulatory effect(s). Its ability to kill bacteria and
modulate immunity, potentially neutrophils as well, positions the HDP as
a promising therapeutic agent for inflammatory conditions.
NETs being prevalent in wounds can delay wound healing and are
implicated in other non-infectious inflammatory conditions such as
systemic lupus erythematosus and rheumatoid arthritis117,167,172,183. Yet,
61
there continues to be debates on its mechanism of induction, antimicrobial
properties and impacts on other regulatory pathways. We speculated that
NETs induced under different inflammatory environments can be exposed
and affected by the different sets of proteins involved. Therefore, using a
reductionistic approach, we examined the proteome of NETs under the
exogenous influence of two serum abundant proteins. It was thus aimed
that this proof-of-concept study can shed light on the dynamicity of NETs,
or its associated proteins more specifically, and its potential implications.
1.6 Scope
With targeting innate immunity as the objective, neutrophils were selected
as they make up the largest circulating leukocyte population in humans.
These neutrophils were isolated from human peripheral blood and
subjected to in vitro functional assays such as flow cytometry, ROS
generation, chemotaxis assay and microfluidics flow assay. Fluorescence
microscopy was employed for the visualisation of compound uptake or
co-localisation with neutrophil structures, and of neutrophil infiltration into
the mouse skin in vivo. In vivo imaging for ROS production using the IVIS
system was utilised to examine inflammatory response in mice.
Proteomics study on NETs was conducted using the high-resolution liquid
chromatography mass spectrometry (LC-MS/MS) followed by in vitro
validation by western blotting. The tryptic peptide profiles from LC-MS/MS
were further examined using in silico tools.
62
63
– CHAPTER 2 –
THROMBIN-DERIVED HOST DEFENCE
PEPTIDES MODULATE NEUTROPHIL
FUNCTIONS IN VITRO AND IN VIVO
Reprinted (adapted) with permission from:
Lim, C. H. et al. Thrombin-derived host defence peptide modulates
neutrophil rolling and migration in vitro and function response in vivo. Sci
Rep 7, 11201, doi:10.1038/s41598-017-11464-x (2017). Copyright ©
2017, Springer Nature.
64
65
2 Thrombin-derived host defence peptides modulate
neutrophil functions in vitro and in vivo
2.1 Abstract
Host defence peptides derived from the C-terminus of thrombin are
proteolytically generated by enzymes released during inflammation and
wounding. In this work, we studied the effects of the prototypic thrombin
C-terminal peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE), on
neutrophil functions. In vitro, GKY25 was shown to reduce LPS-induced
neutrophil activation. In addition, the peptide induced CD62L shedding on
neutrophils without inducing their activation. Correspondingly, GKY25-
treated neutrophils showed reduced attachment and rolling behaviour on
surfaces coated with the CD62L ligand E-selectin. The GKY25-treated
neutrophils also displayed a reduced chemotactic response against the
chemokine IL-8. Furthermore, in vivo, mice treated with GKY25 exhibited
a reduced local ROS response against LPS. Finally, the two wound-
associated thrombin C-terminal truncations HVF18 and FYT21 had
different immunomodulatory potentials, with FYT21 exhibiting more
effective LPS inhibition and CD62L shedding effects at physiological
concentrations. Taken together, our results show that GKY25 can
modulate neutrophil functions in vitro and in vivo.
66
2.2 Introduction
Antimicrobial peptides (AMPs), also known as host defence peptides
(HDPs), play important roles in innate defence in the skin and during
wounding184. They are mostly cationic and amphipathic molecules with
direct and rapid antimicrobial actions against a broad-spectrum of
microbes including both Gram-negative and Gram-positive bacteria,
viruses, as well as fungi184-186. There are 3 classes of HDPs based on
their secondary confirmations, namely the α-helical (e.g. cathelicidins), β-
sheets (e.g. defensins) or free-form (e.g. indolicidin) in aqueous solution.
Due to their cationicity, these HDPs readily and selectively bind to the
anionic bacterial surfaces covered with peptidoglycan, LPS (in Gram-
negative bacteria) and lipoteichoic acid (LTA; in Gram-positive bacteria).
This is followed by the adoption of the HDPs’ respective secondary
conformations which confer their amphipathicity. As the most prominent
example, α-helical HDPs have a cationic face which binds to the bacterial
surface, while the hydrophobic face facilitates the penetration of the
bacteria membrane by interacting with the lipid membrane’s hydrophobic
side chains184,187,188.
HDPs’ antimicrobial effectiveness and strengths are in their unity where,
upon crossing the local threshold concentration, they can disrupt bacterial
membranes. HDPs engage either of the 3 modes-of-action – the “barrel-
stave”, “toroidal pore” or “carpet” model184,188. Ehrenstein and Lecar (1977)
first described the “barrel-stave” model adopted mostly by the α-helical
67
class of HDPs. In this model, the high amphipathic fidelity of the α-helical
HDPs mediates the transmembrane hydrophobic interactions between
the hydrophobic face of the HDP with the lipid side chains, thus forming
the “staves”. Subsequent congregation of these staves creates pores (i.e.
the barrels), hence increasing the leakiness of the bacterial membrane
and as a result, kill them188,189. In the “toroidal pore” model which can be
adopted by various HDPs, their cumulative interactions with the
exteriorised phospho-heads of the bacterial membrane cause inward
bending of the lipid side chains, thus forming pores on the membrane184.
Finally, the “carpet” model describes a detergent-like mechanism, i.e.
disintegrating the membrane by creating micelles from the membranal
phospholipid bilayer. This model can also be adopted by the different
classes of HDPs188.
In the skin, HDPs, including LL37 and β-defensins, are constitutively
expressed by a variety of cells such the epithelial cells, keratinocytes and
sebocytes190. Importantly, these HDPs keep pathogens at bay, as studies
have demonstrated that cathelicidin-deficient mice were more susceptible
to skin infections by pathogenic bacteria and viruses191,192. They also act
as important regulators of the skin microbial flora; for example, an
impairment of S. aureus-induced LL37 production is implicated in atopic
dermatitis which leads to the bacteria’s increased colonisation on the skin
and increasing the host’s susceptibility to skin infections193,194. Conversely,
the skin microflora can ‘feedback’ and regulate these HDPs expression;
studies have shown that bacterial flagellin and lipopeptides can regulate
68
human keratinocytes’ psoriasin and human β-defensins expressions195,196.
LTA from commensal skin bacteria can also induce murine epithelial mast
cell production of cathelicidins which protects the mice against vaccinia
virus infections197.
During inflammation, infiltrating neutrophils further contribute cathelicidins
and α-defensins to significantly increase HDPs in the extracellular space.
These HDPs can also be taken by macrophages and exert their
antimicrobial effects in the phagosomes198. In the interstitium however,
HDPs antimicrobial activities can be diminished due to the presence of
divalent cations and glycans199, thereby probably restricting their
antimicrobial activities to intracellular killing in phagosomes. Subsequent
investigations on HDPs show that they can otherwise modulate neutrophil
activities. For example, LL37 can chemotactically attract neutrophils
possibly through formyl peptide receptor-1 (FPR1) or CXCR2200,201,
although debates on exactly which receptor LL37 acts on exist. Likewise,
β-defensins can chemoattract monocytes through CCR2, a receptor also
expressed by neutrophils suggesting possible chemotactic function for
neutrophils as well202. In addition, LL37 can delay neutrophil apoptosis,
thereby extending their pro-inflammatory activities203. On this note, LL37
has been shown to also increase neutrophils’ phagocytosis, ROS
production and α-defensins release204,205. Furthermore, LL37 can
facilitate NET formation111, and was shown to bind and protect NETs
against nuclease degradation206. HDPs hence, as an immuno-modulator,
have the potential to affect neutrophil activities on several levels.
69
As such, HDPs have gained significant interests as therapeutics due to
their dual roles in killing micro-organisms and modulating host
responses184,187,207,208. This has led to the discovery of several novel
HDPs sequestered within host factors such as Cathepsin G82-84 and the
chemokines IL8209 and CXCL14210, among others211. Knowledge of HDPs’
physicochemical properties have also led to the design of novel HDPs,
such as the mimetic AMP DRGN-1 based on the Komodo dragon’s
histone H1 and the nanoengineered antimicrobial peptide polymer
effective against colistin and multi-drug resistant Acinetobacter
baumannii212,213. Similarly, our colleagues in Lund University, Sweden,
described a new set of HDPs derived from the C-terminal end of thrombin.
These antibacterial thrombin C-terminal peptides (TCPs) which are
cationic, α-helical and amphipathic87,88,214. In addition, these TCPs were
demonstrated to be anti-endotoxic and can modulate monocyte /
macrophage activities181,182,215. Furthermore, the prototypic TCP, GKY25
(GKYGFYTHVFRLKKWIQKVIDQFGE) improved mice survivability
against LPS and P. aeruginosa87,181.
HDPs, particularly the α-helical LL37, have wide-ranging
immunomodulatory effects on neutrophils as described above. Since
these TCPs can modulate monocytes and macrophages and adopt the α-
helical structure, we hypothesised that they can also modulate neutrophils,
the more abundant leukocytes present in the blood and the early
responders to an injury or inflammation. Using various functional assay
tools, we show that the prototypic peptide GKY25 reduces neutrophil
70
rolling and chemotactic responses in vitro through the shedding of CD62L.
We also demonstrate the efficacy of GKY25 in reducing neutrophil-
derived ROS response against LPS in vivo. Taken together, our results
show for the first time that GKY25 can modulate neutrophil responses in
vitro and in vivo216. In addition, we provide preliminary evidence that 2
separate TCPs identified from wound fluids, HVF18 and FYT21 – which
can be generated following thrombin proteolysis by elastases from
neutrophils and P. aeruginosa respectively88 – have different
immunomodulatory potentials on neutrophils. This provides clues on a
possible strategy that the bacteria P. aeruginosa might undertake to
evade the host immune responses.
71
2.3 Materials and methods
2.3.1 Materials
Polymorphoprep was purchased from Axis-Shield, Scotland. Unlabelled
and tetramethylrhodamine (TAMRA)-labelled GKY25
(GKYGFYTHVFRLKKWIQKVIDQFGE) and IVE25
(IVEGSDAEIGMSPWQVMLFRKSPQE), and unlabelled HVF18
(HVFRLKKWIQKVIDQFGE) and FYT21 (FYTHVFRLKKWIQKVIDQFGE)
were ordered from Biopeptide, USA. The fluorescently-labelled antibodies
Pacific Blue (PB)- or Brilliant Violet 510 (BV510)-anti-CD45 (clone HI30),
fluorescein isothiocyanate (FITC)-anti-CD66b (clone G10F5),
allophycocyanin (APC)-anti-CD11b (clone ICRF44), phycoerythrin (PE) /
cyanine (Cy)-7-anti-CD62L (clone DREG-56), PE-anti-PSGL1 (clone
KPL-1), PE/Cy5-anti-CXCR1 (clone 8F1) and peridinin chlorophyll protein
complex (PerCP)/Cy5.5-anti-CXCR2 (clone 5E8) were from BioLegend,
USA. The rat monoclonal anti-neutrophil antibody (NIMP-R14) was from
Abcam, USA. Alexa-fluor (AF)-568 anti-rat IgG was from Life
Technologies, USA. Rosewell Park Memorial Institute (RPMI)-1640
medium without phenol red, the live-dead stain 4’6-diamidino-2-
phenylindole (DAPI) and intracellular ROS fluorescent probe 2’,7’-
dichlorodihydrofluorescein diacetate (H2DCFDA) were bought from
Thermo-Fisher Scientific, USA. GM6001 was purchased from Millipore,
USA. Lactate dehydrogenase (LDH) cytotoxicity kit was purchased from
Pierce, USA. Luminol, horseradish peroxidase (HRP), calcium chloride
(CaCl2), LPS from Escherichia coli O111:B4 and PMA were bought from
72
Sigma-Aldrich, USA. ELISA for soluble CD62L (sCD62L) was from
RayBiotech, USA. E-selectin used for rolling assay was from Peprotech,
USA. The 96-well transwell plates with 3.0 µm inserts were from Corning,
USA. The human chemokine IL8 was from Miltenyi Biotech, Germany.
The ROS probe L-012 was obtained from Wako Chemicals, Germany.
The Optimum Cutting Temperature (OCT) compound was bought from
VWR, USA.
2.3.2 Ethics statement
Human whole blood samples were collected with written informed consent
obtained from each human subject who participated. All methods
involving the use of human blood samples, including the blood collection
process, were performed in accordance with the guidelines and
regulations as reviewed and approved by the Nanyang Technological
University Institutional Review Board, Singapore (IRB-2014-10-041). The
animal models were approved by the Laboratory Animal Ethics
Committee of Malmö/Lund (permit numbers M89-14 and M88-14) and
experiments were conducted in accordance with the guidelines of the
Swedish Animal Welfare Act SFS 1988:534.
2.3.3 Isolation of polymorphonucleated cells (PMNs)
Neutrophils, or PMNs, were isolated from whole blood anti-coagulated
with sodium citrate. Briefly, approximately 2 parts of blood were layered
on 1 part of Polymorphprep before centrifugation at 500x g, 23oC for 30
min (without brakes). The layer containing PMNs was harvested before
73
erythrocyte lysis with Erythrocyte Lysis Buffer (eBioscience, USA). PMNs
were then re-suspended in RPMI supplemented with 10% foetal bovine
serum (FBS). Flow cytometry was then performed on either LSR-II or
LSR-Fortessa-X20 (Becton-Dickinson, USA) and the analyses were
performed using Flowjo (Treestar, USA). PMNs were defined as
CD45+CD66b+ cells and percentage of CD66b+ cells over live CD45+ was
determined to be >95%, unless otherwise stated, before the start of
experiments.
2.3.4 Peptide uptake by PMNs
Uptake of IVE25 or GKY25 was assessed with flow cytometry and
confocal microscopy. PMNs (5 X 105 cells/ml) were incubated with 5 µM
of TAMRA-labelled GKY25 (T-GKY25) or (T-IVE25) for the indicated
periods of time at 37oC. PMNs were then stained with FITC-anti-CD66b
and 10 µm DAPI in FACS buffer containing phosphate-buffered saline
(PBS) supplemented with 5% FBS before flow cytometry using either the
LSR-II or LSR-Fortessa-X20. Median fluorescence intensities (MFIs) of
TAMRA were determined from live, CD66b+ cells for analyses.
For the visualisation of peptide uptake, 200,000 total cells were first
washed with Tris buffered saline (TBS), fixed with 4% paraformaldehyde
(PFA) and washed again with TBS-T (0.1% Triton-X in TBS). Then, PMNs
were re-suspended in TBS-T and centrifuged onto 0.1 µg/ml poly-L-lysine
coated coverslips in a 24-well plate. Finally, the coverslips were mounted
onto the DAPI-containing mounting medium Fluoroshield (Thermo-
74
Scientific, USA) and imaged with the LSM 800 with Airyscan confocal
microscope (Zeiss, Germany).
2.3.5 LDH cytotoxicity assay
Cytotoxic effects of TCPs were assessed with the LDH release assay.
PMNs were first treated with the indicated concentrations of IVE25,
GKY25, HVF18 or FYT21 for 1 hr at 37oC on a 96-well flat-bottom plate.
The supernatants were then used for LDH assay according to
manufacturer’s instructions. Absorbance readings at 490 and 680 nm
were acquired using the Cytation 3 Cell-Imaging Multi-Mode reader
(Research Instruments, USA). LDH activity was determined by
subtracting absorbance reading at 680 nm from the reading at 490 nm
(A490 – A680). Per cent cytotoxicity was calculated as below:
%Cytotoxicity =Compound treated LDH activity−Spontaneous LDH activity
Maximum LDH activity−Spontaneous LDH activity X 100
2.3.6 Assessment of PMN surface markers
PMN surface markers were assessed by flow cytometry. Isolated PMNs
(5 X 105 cells/ml) were treated with either the TCPs IVE25, GKY25,
HVF18 or FYT21 at indicated concentrations, 10 ng/ml LPS or 12.5 ng/ml
IL8 as indicated for 1 hr at 37oC with periodic light shaking. PMNs (1 X
106 cells/ml) pre-incubated in 1:25 ratio by volume of H2O, DMSO or 100
μM GM6001 for 30 min at 37oC were also similarly treated, as indicated,
at a final cell density of 5 X 105 cells/ml. To assess PMN activation, cells
were stained with combinations of 1:200 dilution of FITC-anti-CD66b,
75
APC-anti-CD11b, PE/Cy7-anti-CD62L and PE-anti-PSGL1 with 10 µM of
DAPI diluted in FACS buffer. For analysis of expression of CXCR1 and
CXCR2, treated PMNs were stained with 1:200 dilution of FITC-anti-
CD66b and 1:100 dilution of PE/Cy5-anti-CXCR1 or PerCP/Cy5.5-anti-
CXCR2 with 10 µM DAPI diluted in FACS buffer. For whole blood PMN
assessment, blood was treated with 20 μM IVE25 or GKY25 in the
presence or absence of 10 ng/ml LPS for 2 hrs at 37oC with periodic light
shaking. Erythrocytes were then lysed with Erythrocyte Lysis Buffer
before staining with the respective fluorescently-labelled antibodies and
DAPI diluted in FACS buffer. Finally, flow cytometry was performed using
the LSR-II or LSR-Fortessa-X20. The MFIs of the respective markers
were determined from live, CD66b+ cells for statistical analyses.
2.3.7 Luminol-based ROS measurement
PMNs’ ROS generation was assessed based on luminol
chemiluminescence. ROS assay medium containing 100 µM luminol and
2.4 U/ml HRP in RPMI with 10% FBS was first prepared and used as the
dilution medium for all treatment conditions. Final working concentration
was 50 µM luminol and 1.2 U/ml HRP after the addition of treatment-
containing ROS assay medium to the cell suspension. The assay was
performed in a white, flat bottom 96-well plate.
First, PMNs (1 X 106 cells/ml) were pre-warmed to 37oC for 30 min. Next,
the PMN suspension was added to the ROS assay medium containing
either 50 nM PMA (positive control), 20 ng/ml LPS, 10 µM IVE25 or 10
76
µM in 1:1 ratio by volume. For the pre-treatment of PMNs, PMNs (5 X 105
cells/ml) were first treated with 10 ng/ml LPS, 5 µM IVE25 or 5 µM GKY25
for 30 min at 37oC, washed, re-suspended to 1 X 106 cells/ml then added
to ROS assay medium containing 20 ng/ml LPS in 1:1 ratio by volume.
Chemiluminescence was read immediately with the Cytation 3 Cell-
Imaging Multi-Mode reader every 3 min for 3 hrs in 37oC. Area under the
curve (AUC) was then determined from the chemiluminescence plot over
time for statistical analyses.
2.3.8 Flow cytometry-based ROS assessment
PMNs’ ROS generation was also assessed using flow cytometry. PMNs
(1 X 106 cells/ml) were pre-incubated with 10 μM H2DCFDA for 30 min at
37oC before co-treatment with IVE25, GKY25, HVF18 or FYT21 at
indicated concentrations and 10 ng/ml LPS for 1 hr at 37oC. PMNs treated
with 25 nM PMA was used as positive control. Cells were then stained
with APC-anti-CD11b (Suppl. Fig. 2B), PE/Cy7-anti-CD62L (Suppl. Fig.
2C) and DAPI. Flow cytometry was then performed on the LSR-Fortessa-
X20. As H2DCFDA is excited and emits fluorescence at the same
wavelengths as FITC, a separate sample without H2DCFDA was stained
with FITC-anti-CD66b to determine the PMN population in the forward
scatter against side scatter plot. MFIs for H2DCFDA were determined and
used for statistical analyses.
77
2.3.9 Enzyme-linked immune-sorbent assay (ELISA) for soluble
CD62L
To assess whether CD62L is shed, supernatants were collected from
PMNs (5 X 105 cells/ml) treated with 5 μM of GKY25 for 1 hr. The
supernatants were then used for ELISA to measure sCD62L levels
following manufacturer’s instructions, while the cells were subjected to
flow cytometry as described to assess cell surface CD62L levels.
Absorbance readings for ELISA were acquired using the Cytation 3 Cell-
Imaging Multi-Mode reader.
2.3.10 PMN rolling characterisation
PMN rolling was studied using a microfluidic polydimethylsiloxane (PDMS)
straight-channel microdevice (1 cm length by 400 µm width by 60 µm
height) previously reported by Hou et al (2016). Briefly, the PDMS
microdevice was pre-coated with 50 μg/ml E-selectin for 1 hr at 4°C before
blocking with 0.5% bovine serum albumin (BSA) in PBS for 30 min at room
temperature. For the assay, PMNs (1 X 106 cells/ml) were first treated
with 5 μM of IVE25 or GKY25 for 1 hr at 37oC. Then, 20 μM of CaCl2 was
added to facilitate the cells’ binding and rolling on the E-selectin
functionalised channel. Phase contrast images were captured at 0.5 s
intervals for 30 s with 20x magnification using the MetaMorph software
(Molecular Devices).
The rolling velocity of individual cell was determined with MATLAB
(Mathworks®). Briefly, image contrast was adjusted, and contrast
78
thresholding was applied for cell segmentation and background
elimination. Cells which appeared on less than 10 frames were discarded.
Then, blob analysis was applied to calculate approximate cell size and
centroid position from segmented regions using 8-connected pixels
criterion to eliminate non-cell components. The centroid positions of the
segmented cells were compared with consecutive frames using K-nearest
neighbourhood to determine the rolling distance, which was used to
calculate rolling velocity by dividing the distance by sampling time (0.5 s)
217.
2.3.11 Transwell migration assay
PMN chemotactic function was assessed using 96-well transwell plates
with 3.0 µm pore inserts. PMNs (5 X 105 cells/ml) were treated with 12.5
ng/ml IL8 or 5 µM of either IVE25 or GKY25 for 1 hr at 37oC. The cells
were then washed and reconstituted to 1 X 106 cells/ml. Then, 75 µl of the
cell suspension was added to the respective upper chambers, with the
lower chambers containing 235 µl of 12.5 ng/ml IL8. Spontaneous
migration control was included with the bottom chamber containing only
culture medium. PMNs were then allowed to migrate to the lower chamber
for 2 hrs at 37oC. Next, 200 µl of cells from the lower chamber was stained
with 2 µg/ml Hoechst 33342 in a 96-well plate and the cells were counted
with the INCell Analyzer 2200 (GE Healthcare, USA). In addition, 200 μl
of washed cells from each condition was stained and counted to represent
complete migration. Per cent of migrated cells against total cells was
determined. The assay was performed 11 times with PMNs isolated from
79
6 individuals. Each assay was normalised against its positive control
(untreated PMNs migration against IL8) and the results were pooled for
statistical analysis.
%𝑀𝑖𝑔𝑟𝑎𝑡𝑖𝑜𝑛 = 𝑀𝑖𝑔𝑟𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠
𝑇𝑜𝑡𝑎𝑙 𝑐𝑒𝑙𝑙 𝑐𝑜𝑢𝑛𝑡 𝑋 100%
2.3.12 ROS assessment in vivo
Male BALB/c mice between 8-10 weeks old were used. The back of the
mice was shaved for imaging purpose. Mice were pre-treated with 100 μg
GKY25 (1 mg/ml in deionised water) intraperitoneally (i.p.). Fifteen
minutes after GKY25 treatment, 100 μg LPS (1 mg/ml in deionised water)
was injected subcutaneously (s.c.) in the scruff of the neck. Fifty minutes
after LPS injection, the ROS probe L-012 was injected i.p. (100 μl, 5
mg/ml). The presence of ROS in the mice was imaged 10 min after L-
012 injection using an IVIS SPECTRUM/200 Imaging System (Caliper
Life Sciences, USA). Data acquisition and analyses were performed
using Living Image version 4.4 (Caliper Life Sciences). Results are
expressed as total flux (photons/second).
2.3.13 Neutrophil infiltration
Male BALB/c mice between 8-10 weeks old were used. In the first setup,
mice were pre-treated with 100 μg GKY25 (1 mg/ml in deionised water)
i.p. as above. Fifteen minutes after GKY25 treatment, 100 μg LPS (1
mg/ml in deionised water) was injected s.c. in the scruff of the neck. In the
second experimental setup, mice were injected subcutaneously with 5 μg
80
LPS in the absence or presence of 100 μg GKY25. In both cases, the
mice were sacrificed after 1 hr, after which skin tissues from the LPS-
injection site were harvested and embedded in OCT compound. Then, 10
μm cryo-sections were obtained and fixed in in cold acetone-methanol
(1:1 v/v). After fixing, sections were blocked with 1% BSA and stained with
the anti-neutrophil antibody NIMP-R14 followed by secondary staining
with AF568 anti-rat IgG. Nuclei were then stained with 50 μM DAPI.
Finally, images were taken using the Olympus Optical AX60 fluorescence
microscope (Olympus, USA). Four random microscopic views from each
stained section were scored independently by 2 observers on a scale of
0-10 for neutrophil infiltration, where 0 is no neutrophil infiltration and 10
is maximum neutrophil infiltration.
2.3.14 Statistical analyses
Statistical analyses were performed on Prism version 6.0 (GraphPad,
USA). The respective statistical tests performed were as indicated.
Results were presented in Mean (SD). Statistical significance was
represented in the form of p values as such: * p < 0.05, ** p < 0.01, *** p
< 0.001 or **** p < 0.0001.
81
2.4 Results
2.4.1 GKY25 is selectively taken up by PMNs
As a first step to elucidate possible immunomodulatory effects of GKY25
on PMNs, we studied the peptide-PMN interaction using flow cytometry
and confocal microscopy. Flow cytometry analysis demonstrated a time-
dependent uptake of TAMRA-labelled GKY25 (T-GKY25) by PMNs (Fig.
2.1A). Using T-IVE25 as a negative control, which was observed to have
slightly increasing MFIs from PMNs over time, MFIs of T-GKY25 from
PMNs were significantly higher, demonstrating a more selective uptake of
GKY25. The uptake of T-GKY25 was further confirmed by confocal
microscopy, where only T-GKY25 and not T-IVE25 was detected in the
cytoplasm of the cells (Fig. 2.1B).
82
Figure 2.1: Uptake of GKY25 by PMNs and analysis of LDH release. Uptake of
GKY25 was assessed by (A) flow cytometry and (B) confocal microscopy. (A) PMNs
(5 X 105 cells/ml) were incubated with 5 µM T-IVE25 or T-GKY25 for 0, 30, 60, 120
min at 37oC or on ice for 120 min before flow cytometry (n = 3). (B) PMNs (5 X 105
cells/ml, 200,000 cells in total) were incubated with 5 µM T-IVE25 or T-GKY25 for 120
min before confocal imaging. (C) PMNs incubated with 1, 5, 10, 15, 20, 25 or 30 µM
IVE25 or GKY25 for 1 hr were assessed for cytotoxicity using the LDH release assay.
(---) Baseline. One-way ANOVA. Figures are representative of 2 (1A-B) or 3 (1C)
independent experiments.
GKY25 / DAPI IVE25 / DAPI
(A)T-IVE25 (cold)
T-GKY25 (cold)
0 30 60 90 1200
100
200
300
400
500
Time (min)
MF
I (T
AM
RA
)
T-IVE25
T-GKY25
(B)
(C)
1 5 10 15 20 25 30 1 5 10 15 20 25 30
-20
0
20
40
60
80
%C
yto
toxic
ity
ns
****
********
****
IVE25 (uM) GKY25 (uM)
ns ns
83
HDPs have been reported to be cytotoxic to mammalian cells218, therefore
we set out to examine the potential cell permeabilising effects of GKY25.
The results show that 1 hr treatment with GKY25 was non-cytotoxic at
doses up to 10 µM and displayed dose-dependent cytotoxicity from 15-30
μM (Fig. 2.1C). GKY25 at 5 µM was also non-cytotoxic for up to 4 hrs
(Supp. Fig. S2.1A). In addition, treatment with 5 µM of GKY25 for 1 hr
did not significantly reduce PMNs ability to generate ROS when compared
to LPS-treated PMNs against PMA, a strong chemical stimulator for ROS
generation (Supp. Fig. 2.1B), thus indicating that PMNs remained
functionally active at this level of GKY25 in vitro. Taken together, the
results indicate that GKY25 is taken up by PMNs, is non-cytotoxic at
doses at 10 µM or below, and that it does not affect the innate functional
responses of PMNs.
2.4.2 GKY25 inhibits LPS-induced PMN activation
Previous studies have shown that TCPs, including GKY25, can bind to
both LPS and monocytes/macrophages and inhibit endotoxic effects in
cell models in vitro as well as in mouse models in vivo87,181,182. Here, we
explored the potential anti-endotoxic effects of GKY25 on neutrophils.
First, the expression levels of activation markers’ on PMNs in response to
LPS with or without added GKY25 or the control peptide IVE25 were
assessed. Three surface markers were selected to assess neutrophil
activation – CD66b, which is upregulated as a degranulation marker219,
CD11b which mediates neutrophil adhesion during activation, and CD62L
which is shed upon neutrophil activation47,48. As expected, LPS induced
84
upregulation of CD66b and CD11b with a concomitant downregulation of
CD62L (Fig. 2.2A). In the presence of GKY25 however, the LPS-
mediated change in the expression levels of these activation markers was
significantly inhibited, with levels close to those found in untreated cells
(baseline). The control peptide IVE25 did not show these inhibitory effects
(Fig. 2.2A). Next, we examined the changes in PMN markers in whole
blood in response to LPS in the presence or absence of GKY25. Doses
of GKY25 up to 20 µM were used as its cytotoxicity and haemolytic
activities were reduced in whole blood87. Similarly, while LPS alone
induced significant changes to the activation markers, GKY25 but not
IVE25 abolished the effect dose-dependently (Fig. 2.2B and Suppl. Fig.
SFig. 2.2).
As LPS has been reported to induce a low, but still detectable, ROS
response in human PMNs in vitro220-223, we next decided to evaluate the
effects of GKY25 on LPS-induced ROS. LPS treatment of PMNs yielded
an increase in ROS levels, albeit to a lesser extent as compared to PMA-
treated cells (Fig. 2.2C and Suppl. Fig. S2.3A). Consistent with our flow
cytometry data, the LPS-induced increase of ROS was abolished in the
presence of GKY25, in contrast to the effects of the control peptide IVE25
(Fig. 2.2C and Suppl. Fig. S2.3A). A dose-dependent reduction of ROS
was also observed with increasing GKY25 concentrations (Fig. 2.2D). In
addition, we assessed ROS generation by PMNs pre-treated with either
IVE25 or GKY25. Likewise, we observed a reduction of ROS levels in
GKY25 pre-treated PMNs when compared to the cells pre-treated with
85
IVE25 (Fig. 2.2E). Taken together, these results demonstrate that GKY25
inhibits LPS-induced PMN activation.
Figure 2.2: Effects of GKY25 on LPS responses of PMNs. PMN activation markers
were assessed by flow cytometry on (A) isolated PMNS (5 X 105 cells/ml) co-treated
with either 5 µM IVE25 or 5 µM GKY25 and 10 ng/ml LPS for 1 hr at 37oC (n = 3) and
(B) PMNs in whole blood co-treated with either 20 µM IVE25 or 20 µM GKY25 and 10
ng/ml LPS for 2 hrs at 37oC (n = 3). PMNs stimulatory functions were assessed using
the luminol-based ROS measurement – pre-warmed isolated PMNs (5 X 105 cells/ml)
were co-treated with (C) either 5 µM IVE25 or 5 μM GKY25 and 10 ng/ml LPS (n = 5)
and (D) 0.5, 1.0, 2.5, 5.0 or 10 µM GKY25 and 10 ng/ml LPS (n = 5). For positive
control, PMNs in (C) were also treated with 25 nM PMA. (E) PMNs (5 X 105 cells/ml)
pre-treated with 5 µM IVE25 (IVE25 > LPS) or 5 μM GKY25 (GKY25 > LPS) were also
subjected ROS induction with 10 ng/ml LPS (n = 5). ROS measurements were done
every 3 min for 3 hrs at 37oC and AUCs were determined for statistical analyses. (---)
Baseline. One-way ANOVA. Figures are representative of 3 independent
experiments.
-0.
51.
02.
55.
010
.0
0
5
10
15
RO
S(n
orm
alised
AU
C)
GKY25 (uM)
ns
**** ***
***** ****
+10 ng/ml LPS
(A)
(C) (D)
(B)
(E)
-
IVE25
GKY25
0
50
100
150
200
250
Med
ian
FI (C
D66b
)
+ 10 ng/ml LPS
5 uM
ns
****
-
IVE25
GKY25
0
20
40
60
80
100
Med
ian
FI (C
D11b
)
ns
**
+ 10 ng/ml LPS
5 uM
-
IVE25
GKY25
0
50
100
150
200
250
Med
ian
FI (C
D62L
)
ns
****
+ 10 ng/ml LPS
5 uM
-
IVE25
GKY25
0
20
40
60
80
100
Med
ian
FI (C
D11b
)
****
*
+ 10 ng/ml LPS
20 uM
-
IVE25
GKY25
0
200
400
600
800
1000
Med
ian
FI (C
D62L
)
ns
****
+ 10 ng/ml LPS
20 uM
-
IVE25
GKY25
0
50
100
150
200
250
Med
ian
FI (C
D66b
)
*
****
+ 10 ng/ml LPS
20 uM
-
IVE25
GKY25
0
1
2
3
4
550
60
70
RO
S(n
orm
alised
AU
C)
ns
****
+ 10 ng/ml LPS
5 uM
- > L
PS
IVE25
> L
PS
GKY25
> L
PS
0
10
20
30
40
RO
S(n
orm
alised
AU
C)
ns
*
86
2.4.3 GKY25 induces specific CD62L shedding without activating
PMNs
Interestingly, we observed considerable CD62L down-regulation from
PMNs co-treated with LPS and GKY25 (Fig. 2.2A-B) despite no
upregulation in CD66b and CD11b, suggesting that GKY25 could induce
the down-regulation of CD62L without activating PMNs. To this end, we
investigated whether GKY25 has any direct, LPS-independent effects on
PMN functions. First, we assessed PMN activation markers for early
indication of any modulatory effects. Here, neither GKY25 nor IVE25 had
any significant effects on the surface expression of CD66b (Fig. 2.3A-B)
and CD11b (Fig. 2.3C-D); in contrast to the control peptide however,
GKY25 induced significant down-regulation of CD62L (Fig. 2.3E-F).
Surprisingly, while this event indicated PMN activation, no direct ROS
response was detected against either GKY25 or IVE25 (Fig. 2.3G). Next,
we compared the CD62L profiles of PMNs isolated from the blood of 3
individuals. Here, the PMNs had reduced CD62L surface expression
when treated with GKY25 (Fig. 2.4A) and these were matched with
increased sCD62L levels in the respective supernatants, showing that the
surface marker was shed (Fig. 2.4B). Similarly, the other neutrophil weak
adhesion surface molecule, PSGL1, was also significantly reduced
(Suppl. Fig. S2.4A).
87
Figure 2.3: GKY25 induces CD62L shedding and modulates PMNs’ ROS
response against LPS. PMNs treated with 10 ng/ml LPS, 5 µM IVE25 or 5 µM GKY25
for 1 hr at 37oC were assessed by flow cytometry for surface expression of (A-B)
CD66b, (C-D) CD11b and (E-F) CD62L (n = 3). Figures (B), (D) and (F) are
representative histograms for the respective surface markers. (G) Pre-warmed PMNs
(5 X 105 cells/ml) were subjected to ROS assessment with 10 ng/ml LPS, 5 µM IVE25
or 5 µM GKY25 (n = 5). (---) Untreated baseline. One-way ANOVA. Figures are
representative of 3 independent experiments.
As the ectodomain shedding of CD62L can be mediated by leukocyte-
associated metalloproteinases such as a disintegrin and
metalloproteinase (ADAM)-8 and ADAM-17224-226, we next investigated
CtrlLPS IVE25 GKY25
Ctrl Ctrl
Co
un
t
CD66b
Co
un
t
CD11b
Co
un
t
CD62L
+ LPS + IVE25 + GKY25(A) (B)
(C) (D)
(E) (F)
(G)
Ctrl
10 n
g/ml L
PS
IVE25
GKY25
0
200
400
600
800
Med
ian
FI (C
D66b
)
5 uM
****
nsns
Ctrl
10 n
g/ml L
PS
IVE25
GKY25
0
50
100
150
Med
ian
FI (C
D11b
) ****
ns ns
5 uM
Ctrl
10 n
g/ml L
PS
IVE25
GKY25
0
100
200
300
400
Med
ian
FI (C
D62L
)
****
ns
****
5 uM
-
10 n
g/ml L
PS
IVE25
GKY25
0
1
2
3
4
550
60
70
RO
S(n
orm
alised
AU
C) ***
*
ns ns
5 uM
88
whether the GKY25-mediated CD62L shedding was metalloproteinase-
dependent. PMNs were pre-treated with a broad-spectrum
metalloproteinase inhibitor, GM6001, before being subjected to GKY25
treatment. GKY25 mediated the down-regulation of CD62L also in
GM6001 pre-treated PMNs (Fig. 2.4C). This suggests that GKY25 may
trigger an alternative pathway yielding CD62L shedding.
2.4.4 GKY25-induced shedding of CD62L affects PMN rolling in vitro
CD62L is an important adhesion molecule which facilitates PMNs’
tethering and sensing along vascular walls48,224. Since GKY25 induced
PMN shedding of CD62L (Fig. 2.4A-B), we aimed to investigate whether
this can impact PMN rolling in vitro. Rolling behaviour of PMNs treated
with IVE25 or GKY25 was characterised using a microfluidics chamber
coated with E-selectin, an adhesion molecule upregulated during
endothelial inflammation and known to bind to CD62L225,226. Under
physiological flow conditions (~2 dyne/cm2), GKY25-treated PMNs
demonstrated a lower capture rate on the E-selectin-coated surface
versus untreated or IVE25-treated PMNs (Fig. 2.5A-B). Furthermore,
GKY25-treated PMNs had significantly higher mean rolling velocities as
compared to untreated or IVE25-treated PMNs (Fig. 2.5C and Suppl. Fig.
S2.5A-B). These results indicate that the recruitment of GKY25-treated
PMNs during vascular inflammation may be impaired with weaker cell-
surface attachment and higher rolling velocities possibly through the
reduction of CD62L and PSGL-1 interactions with E-selectin.
89
Figure 2.4: GKY25-induced CD62L shedding in presence of GM6001. PMNs (5 X
105 cells/ml) isolated from 3 individuals were treated with 5 μM GKY25 for 1 hr at 37oC.
The cells were subjected to (A) flow cytometry to assess cell surface CD62L (mean ±
SD of triplicate from each individual), whereas (B) the supernatants were used for
ELISA to assess sCD62L (mean of technical duplicate from each individual). Multiple
T-tests. (C) PMNs (1 X 106 cells/ml) were also pre-incubated with 1:25 ratio by volume
of H2O, DMSO or 100 μM GM6001 for 30 min at 37oC before incubation with 5 µM
GKY25 at the cell density of 5 X 105 cells/ml for another 1 hr at 37oC. Flow cytometry
was performed to assess CD62L levels (n = 3). (---) Untreated baseline. Two-way
ANOVA. Figure 4C is representative of 2 independent experiments.
(A) (B) (C)
Untr
eate
d
5 uM
GKY25
0
100
200
300
400
500
Med
ian
FI (C
D62L
)
Donor 1
Donor 2
Donor 3**
****
****
Untr
eate
d
5 uM
GKY25
-2
-1
0
1
2
3
4
[sC
D62L
] n
g/m
l Donor 1Donor 2Donor 3
*
ns***
H2O
DM
SO
GM
6001
0
200
400
600
800
Med
ian
FI (C
D62L
) Ctrl
GKY25****
**
***
****
90
Figure 2.5: GKY25 attenuates neutrophil attachment and rolling in vitro. PMNs
(1 X 106 cells/ml) were treated with 5 µM IVE25 or GKY25 before being subjected to
microfluidics flow assessment. (A) Representative images of attached cells/field.
Channel width = 400 µm. (B) Number of attached cells/field was counted from the
images and (C) rolling velocities were determined from individual cells over the
duration of time-lapsed video. One-way ANOVA. Figures are representative of 3
independent experiments.
2.4.5 GKY25 reduces PMN chemotactic responses against IL8 in a
CXCR1/2-independent manner
Migration across endothelial walls is an important process during
neutrophil recruitment. To further investigate the potential effects of
GKY25 on neutrophil migration, we assessed peptide-treated PMNs’
chemotactic response against the pro-inflammatory chemokine IL8. As
expected, PMNs pre-treated with IL8 significantly impaired chemotaxis
against IL8 as compared to untreated or IVE25 pre-treated PMNs (Fig.
(A)
Ctrl
IVE25
GKY25
0
20
40
60
Cap
ture
d C
ells
/ F
ield
5 uM
***
ns(B) (C)
Ctrl
IVE25
GKY25
0
5
10
15
20
Ro
llin
g V
elo
cit
y (
um
/s) **ns
5 uM
Control IVE25 GKY25
91
2.6A). Similarly, GKY25 pre-treated PMNs showed a significantly reduced
chemotactic response (Fig. 2.6A). As IL8 acts through the receptors
CXCR1 and CXCR2 and HDPs are reported to act through G-protein
coupled receptors (GPCRs; mostly chemokine receptors)200,201,227, we
also assessed receptor expressions on GKY25-treated PMNs.
Interestingly, while IL8 induced surface downregulation of CXCR1 and
CXCR2, GKY25 did not (Fig. 2.6B-C). This suggests that GKY25 may
affect other pathways affecting PMN chemotaxis, possibly downstream of
chemokine receptor activation.
92
Figure 2.6: GKY25 reduces PMN chemotaxis. PMNs (5 X 105 cells/ml) were pre-
treated with 12.5 ng/ml IL8, 5 µM IVE25 or 5 µM GKY25 for 1 hr at 37oC, washed,
then subjected to (A) transwell migration against 12.5 ng/ml IL8 for 2 hrs at 37oC
(pooled normalised data from n = 11 independent experiments; One-way ANOVA with
Dunnett’s test for multiple comparison) or flow cytometry assessment for (B) CXCR1
and (C) CXCR2; (---) untreated baseline, One-way ANOVA. Figures are
representative of 2 (2.6B) or 3 (2.6C) independent experiments.
2.4.6 GKY25 ameliorates LPS-induced local ROS production in vivo
As neutrophils are the first cells to respond to danger signals, we were
interested to translate our in vitro findings on neutrophil responses to a
relevant in vivo situation. Here, we sought to assess local ROS production
induced by LPS as a measure of neutrophil response. LPS was injected
subcutaneously into the dorsal region of untreated or GKY25-treated mice
before ROS measurement. As observed, LPS treatment resulted in a
significant increase in ROS production, localised to the site of injection
(Fig. 2.7A upper panel and 2.7B). In contrast, significantly reduced ROS
production was observed in GKY25-treated mice (Fig. 2.7A lower panel
and 2.7B), showing that GKY25 administered intraperitoneally was able
to inhibit LPS-induced neutrophil responses also in vivo. In addition, to
explore the effects of GKY25 more in detail, the peptide was administered
either as a pre-treatment given intraperitoneally before LPS
(A) (B) (C)
Ctrl
12.5
ng/m
l IL8
IVE25
GKY25
0.0
0.5
1.0
1.5
No
rmalised
Mig
rati
on
***
5 uM
ns*
Ctrl
12.5
ng/m
l IL8
IVE25
GKY25
0
10
20
30
40
50
Med
ian
FI (C
XC
R2)
****
ns ns
5 uM
Ctrl
12.5
ng/m
l IL8
IVE25
GKY25
0
100
200
300
400
500
MF
I (C
XC
R1)
****
nsns
5 uM
93
administration subcutaneously (as above) or co-administered with LPS
subcutaneously. Skin tissues were then harvested and immuno-stained
with anti-neutrophil antibody to assess neutrophil localisation in either
condition. We observed marked neutrophil infiltration in the extravascular
space in response to LPS which was only reduced in the setup when
GKY25 was given subcutaneously (Suppl. Fig. S2.6A-B) and not as pre-
treatment intraperitoneally (Suppl. Fig. S2.6C-D). Taken together,
GKY25 can reduce neutrophil activation by either direct neutrophil
modulation or LPS scavenging, leading to inhibition of LPS-induced
neutrophil responses.
Figure 2.7: GKY25 ameliorates LPS-induced local ROS production in vivo. (A)
Representative in vivo imaging of ROS with L-012 in mice. Using an IVIS SPECTRUM
200 Imaging System, ROS production was imaged and measured in the scruff of the
neck. Mice were pretreated with GKY25 followed by subcutaneous LPS
administration. Luminescent signal was detected 10 min after L-012 injection. (B) The
statistical significance was calculated between sham- and GKY25-treated mice (n = 6
mice). Unpaired T-test.
LP
S o
nly
LP
S +
GK
Y25
(A) (B)
LPS o
nly
LPS +
GKY25
0
2105
4105
6105
To
tal F
lux
(ph
oto
ns/s
eco
nd
)*
Radiance
(p/sec/cm2/sr)
Colour scale
Min = 2.59 x 104
Max = 4.86 x 105
94
2.4.7 FYT21 modulates PMNs more effectively than HVF18
In previous studies, 2 TCPs – HVF18 and FYT21 – were detected in
wound fluids and were shown to be generated specifically following
thrombin proteolysis by neutrophil and P. aeruginosa elastases,
respectively87,88. Both TCPs exhibit antibacterial properties at similar
concentrations against common Gram-positive and Gram-negative
bacteria; however, they have been shown to have differential LPS-
inhibitory effects and uptake by monocytes88. Therefore again, we aimed
to elucidate whether HVF18 and FYT21, as bona fide TCPs, can
modulate neutrophil responses, either directly or against LPS. GKY25
was included for comparison for its immunomodulatory capacities216.
First, we examined the cytotoxic effects of both TCPs on purified PMNs
by measuring extracellular LDH activity. Here, we show that HVF18
began to elicit a cytotoxic effect at 40 μM but only significantly at 80 μM
(Fig. 2.8A; unpublished). On the other hand, FYT21 exerted significant
cytotoxicity already at 20 μM (Fig. 2.8B; unpublished), showing that it is
more cytotoxic than HVF18. This follows a previous report on the more
effective erythrocyte lysis by FYT21 at lower concentrations as compared
to HVF1888.
95
Figure 2.8: Cytotoxicity of HVF18 and FYT21 on PMNs. PMNs (5 X 105 cells/ml)
were treated with (A) 0-140 µM of HVF18 or (B) 0-60 µM of FYT21 for 1 hr at 37oC
before LDH activity assessment as a measure for cytotoxicity (n = 3). One-way
ANOVA. Figures are representative of 3 independent experiments. Unpublished data.
Next, we examined the PMN activation against LPS, with or without either
of the TCPs, by flow cytometry (Fig. 2.9; unpublished). Here, we
assessed the surface markers, CD11b and CD62L, and the ROS-
sensitive intracellular dye H2DCFDA. Expectedly, GKY25 dose-
dependently inhibited LPS-induced changes to the PMN surface markers
(Fig. 2.9A-B). Likewise, LPS-induced PMN ROS response was also
reduced to baseline dose-dependently by GKY25 (Fig. 2.9C). In addition,
GKY25 can independently downregulate PMN surface CD62L at 5.0 μM
without upregulating CD11b, and this corresponds to our previous finding
(Fig. 2.9A-B vs. Fig. 2.3B-C). While PMN CD62L surface level was
significantly higher following co-treatment of LPS and 1.0-5.0 μM of
GKY25 as compared to treatment with LPS alone (Fig. 2.9B), it never
reached the baseline level unlike its effects in LPS-induced CD11b
changes (Fig. 2.9A). This is congruent with our results in Figure 2.2A-B
where PMNs co-treated with LPS and 5.0 μM of GKY25 had baseline
0 5 10 20 30 40 50 60
-10
0
10
20
30
40
50
**
***
****
****
**
****
****
FYT21 (uM)
%C
yto
toxic
ity
nsns
0 20 40 60 80 100
120
140
0
20
40
60
**
****
****
****
HVF18 (uM)
%C
yto
toxic
ity
nsns
ns
(A) (B)
96
levels of CD66b and CD11b, and not CD62L (although still significantly
higher than PMNs treated with LPS alone).
Figure 2.9: LPS-inhibitory effects of HVF18 and FYT21 on PMNs. H2CDFDA-
loaded PMNs (5 X 105 cells/ml) were treated with 0, 1.0, 2.0 or 5.0 μM of (A-C) GKY25,
(D-F) HVF18 or (G-I) FYT21, with or without 10 ng/ml LPS, for 1 hr at 37oC. The
following were assessed by flow cytometry – (A, D, G) CD11b, (B, E, H) CD62L and
(C, F, I) the intracellular ROS-sensitive dye H2DCFDA. n = 3. (---) Untreated baseline
control. (---) 10 ng/ml LPS only positive control. One-way ANOVA. Figures are
representative of 3 independent experiments. Unpublished data.
We then assessed HVF18 and FYT21’s effects on PMNs. In the presence
of different concentrations of HVF18, PMNs co-treated with LPS
continued to express altered surface markers’ levels induced by LPS (Fig.
2.9D-E). In contrast, while 0.5 to 2.0 μM of FYT21 did not inhibit LPS-
induced CD11b and CD62L changes in levels, 5.0 μM of FYT21
significantly reduced LPS-induced CD11b upregulation to near baseline
no LPS
10 n
g/ml L
PS
0
30
60
90
120
MF
I (C
D11b
)
Ctrl
0.5
1.0
2.0
FYT21
5.0
ns
ns
**
no LPS
10 n
g/ml L
PS
0
30
60
90
120
MF
I (C
D11b
)
Ctrl
0.5
1.0
2.0
GKY25
5.0
ns
********
****
*
no LPS
10 n
g/ml L
PS
0
30
60
90
120
MF
I (C
D11b
)
Ctrl
0.5
1.0
2.0
HVF18
5.0
ns
ns
no LPS
10 n
g/ml L
PS
0
50
100
150
MF
I (C
D62L
)
Ctrl
0.5
1.0
2.0
HVF18
5.0
ns
ns
FYT21
no LPS
10 n
g/ml L
PS
0
50
100
150
MF
I (C
D62L
)
Ctrl
0.5
1.0
2.0
5.0
ns *****
****
****
ns
GKY25
no LPS
10 n
g/ml L
PS
0
50
100
150
MF
I (C
D62L
)
Ctrl
0.5
1.0
2.0
5.0
ns
**
**
****
****
ns
no LPS
10 n
g/ml L
PS
0
30
60
90
120
MF
I (H
2D
CF
DA
)
Ctrl
0.5
1.0
2.0
HVF18
5.0
ns
p =0.0627
no LPS
10 n
g/ml L
PS
0
30
60
90
120
MF
I (H
2D
CF
DA
)
Ctrl
0.5
1.0
2.0
FYT21
5.0
****
no LPS
10 n
g/ml L
PS
0
30
60
90
120
MF
I (H
2D
CF
DA
)
Ctrl
0.5
1.0
2.0
GKY25
5.0
****(A)
(D)
(G)
(B)
(E)
(H)
(C)
(F)
(I)
97
level and significantly increased LPS-induced CD62L downregulation
albeit nowhere near baseline level (Fig. 2.9G-H). When PMN ROS
response was assessed, HVF18 did not significantly inhibit LPS-induced
PMN ROS response at all concentrations, although 5.0 μM of HVF18
exhibited signs of inhibition (p value = 0.0627; Fig. 2.9F). Conversely,
FYT21 significantly inhibited LPS-induced ROS response even at 0.5 μM
(Fig. 2.9I), which is at physiologically-relevant concentrations assuming
plasma concentration of prothrombin is approximately 1.4 μM228. When
statistical analyses were performed to compare the 2 TCPs, PMN ROS
response against LPS was significantly reduced at all concentrations of
FYT21 versus HVF18 (p values = 0.0036, 0.0036, 0.0237 and 0.0222 for
0.5, 1.0, 2.0 and 5.0 μM of TCPs, respectively). Therefore, from at least
these results alone, FYT21 seemed to inhibit LPS-induced PMN
activation more effectively than HVF18. Interestingly however, FYT21 did
not restore CD11b and CD62L to near baseline levels despite inhibition
of ROS generation at physiologically-relevant concentrations (0.5 - 2.0
μM), suggesting a complex immunomodulatory effect on neutrophils.
98
2.5 Discussion
In this study, we demonstrate for the first time the immunomodulatory
effects of GKY25 on neutrophils. First, we show that GKY25 can inhibit
LPS-induced neutrophil activation. In addition, GKY25 can modulate
neutrophils independently by inducing the shedding of CD62L, thus
affecting neutrophil attachment and rolling in vitro. Second, GKY25
impaired neutrophil chemotactic response against IL8 independent from
CXCR1 and CXCR2 regulation. Finally, mice treated with GKY25 showed
significant reduction in local LPS-induced ROS production, indicating that
the peptide can also modulate neutrophil immune responses in vivo.
Additionally, we provide preliminary evidence that the P. aeruginosa
elastase-cleaved TCP can modulate neutrophil functions in vitro.
Neutrophil tethering and sensing along endothelial walls are important
processes mediated by weak adhesion molecules such as CD62L and
PSGL1 during homeostasis and inflammation. Notably, GKY25 induced
both adhesion molecules’ shedding on neutrophil surface without the
concomitant upregulation of CD66b and CD11b that would otherwise
indicate neutrophil activation47,229 (Fig. 2.3A-F, Fig. 2.4A-B and Suppl.
Fig. S2.4). Consequently, GKY25-treated PMNs presented reduced initial
attachment and increased rolling velocities on E-selectin-coated surfaces
(Fig. 2.5), presumably due to reduced surface engagement of CD62L and
PSGL1 to E-selectin. Hypothetically, the shed CD62L and PSGL1 may
have played synergistic roles in binding to E-selectin, thus reducing the
99
total available ligand for PMN attachment. Furthermore, GKY25-treated
PMNs displayed a reduced chemotactic response against IL8 in vitro (Fig.
2.6A). This potential modulation on neutrophil recruitment could play
important roles in host defence; early studies by Jutila et al. (1989)
demonstrate that the use of monoclonal antibodies against CD62L
reduced neutrophil recruitment to inflamed peritoneum in mice230. Mice
deficient in CD62L were later shown to have reduced immune infiltration
into inflamed peritoneum and induced hypersensitivity in mouse ears231.
On the contrary, when CD62L was protected from endoproteolytic
cleavage, sustained and increased neutrophil recruitment was observed
in inflamed peritoneum232. Notably, CD62L which primarily acts as a weak
adhesion molecule, has been implicated to hinder CD62L-deficient
neutrophils’ locomotion in vivo233. In addition, a study has shown that
human neutrophils with reduced CD62L expression exhibited reduced
chemotactic response to fMLP234. Indeed, ligation of CD62L on leukocyte
surfaces (including neutrophils) has been reported to induce signal
transduction and activate integrin function as well as enhancing
lymphocytes’ chemotaxis235-237, hence suggesting that the lack of CD62L
on neutrophil surface could also affect their chemotaxis and consequent
recruitment. Similarly, PSGL1 has been shown to mediate neutrophil
tethering both in vitro and in vivo238,239, further suggesting that GKY25-
induced shedding of weak adhesion molecules may synergistically
modulate neutrophil responses during host defence and inflammation.
100
The exact mechanism by which GKY25 induces shedding of these weak
adhesion molecules and whether it directly modulates neutrophil
response to IL8, remains however to be characterised. CD62L is
anchored to calmodulin, which upon cellular activation, exposes CD62L’s
ectodomain region, leading to cleavage by several metalloproteinases,
most notably by the enzyme ADAM17224,225,240,241. Interestingly, GKY25
can induce CD62L shedding despite the presence of the broad-spectrum
metalloproteinase inhibitor GM6001 (Fig. 2.4C). In this context, it should
be noted that studies have shown that CD62L can indeed be shed via
other mechanisms which are protease-independent, such as mechanical
stress under flow conditions242, after cross-linking with anti-CD62L
antibodies or CD62L-specific compounds243,244, during apoptosis225 and
in response to microparticle generation245,246. Thus, it is possible that
GKY25 has other molecular targets related to these pathways.
Integral to our study is the demonstration on GKY25’s immunomodulatory
effects in vivo (Fig. 2.7 and Suppl. Fig. S2.6). Since the results indicated
that GKY25 modulates neutrophil functions in vitro (Fig. 2.2-2.6), we
aimed to study the peptide’s in vivo effects. For this purpose, GKY25 was
injected intraperitoneally followed by LPS injection subcutaneously before
ROS was measured to track neutrophil activation. In this context, it should
be mentioned that LPS has been reported to exert its major effects in
priming of neutrophils, facilitating activation by substances such as N-
formyl-methionyl-leucyl-phenylalanine (fMLP)220-222,247. Hence, it may be
argued that LPS, at physiologically relevant concentrations, is not a strong
101
activator of the respiratory burst. However, LPS has also been reported
per se to induce the release of ROS in human PMNs in vitro220-222 as well
as ex vivo in whole blood248,249, albeit at lower levels relative to those
observed after treatment with strong activators, such as PMA221,247 (see
also Fig. 2.2C and Suppl. Fig. S2.3A). Furthermore, LPS alone can
activate interleukin-1 receptor-associated kinase-4 (IRAK-4)
phosphorylation of p47phox, a subunit of the NADPH oxidase complex
responsible for ROS production in neutrophils223. Indeed, IRAK-4-
deficient neutrophils from patients cannot produce ROS in response to
LPS221. Thus, consistent with previously published results on LPS-
induced ROS generation250,251, we observed an increase in ROS after
subcutaneous injection of LPS in the experimental model used. The ROS
response was reduced significantly in GKY25-treated mice (Fig. 2.7),
suggesting that neutrophils were either not infiltrating, or not being
activated at the site of LPS injection. Analyses of skin sections from an
identical experiment showed that neutrophil levels were unaffected
(Suppl. Fig. S2.6C-D), indicating that the translation of the peptide effects
on chemotaxis in vitro, as observed in Figures 2.5 and 2.6, to the in vivo
situation, needs further studies. Nevertheless, the in vivo data are
compatible with the reduction of LPS-induced activation as observed in
vitro (Fig. 2.2E).
Neutrophil infiltration was however reduced in the subcutaneous model
when LPS and GKY25 were co-administered (Suppl. Fig. S2.6A-B),
which is compatible with direct LPS blocking, thus leading to less LPS-
102
induced neutrophil recruitment. This finding is consistent with previous
reports where LPS and GKY25 were sequentially injected into the
peritoneal space, leading to a reduction of inflammation and increase in
overall survival in mouse models of endotoxin-shock181. Notably, GKY25
significantly reduced cytokine levels, including TNF, in such animal
models181. Of relevance from an in vivo perspective is that TNF has been
reported to sensitise PMNs to LPS, yielding ROS responses to LPS levels
of 1 ng/ml, 10-100 times lower than those needed for stimulation with LPS
alone252. It is therefore possible that an overall reduction of cytokines by
treatment with GKY25 may contribute to the observed inhibitory effects of
the peptide on LPS-induced PMN activation in vivo. Taken together, the
demonstration that PMNs pre-treated with GKY25 showed reduced ROS
response against LPS in vitro (Fig. 2.2E) and in vivo (Fig. 2.7 and Suppl.
Fig. S2.6) indicate that GKY25 affects neutrophil responses at multiple
levels. Of note is that both setups could potentially be translated to clinical
therapies. For example, in certain surgical situations where endotoxin is
at risk of being spread, pre-surgical administration of GKY25 could
hypothetically show beneficial effects.
Additionally, we have preliminarily assessed the immunomodulatory
properties of HVF18 and FYT21, both detected in wound fluids of P.
aeruginosa infected wounds, particularly FYT21. When comparing these
2 TCPs, we found that FYT21 is more cytotoxic, can attenuate LPS-
induced neutrophil stimulation more effectively and can induce CD62L
downregulation independently (Fig. 2.8 and 2.9). However, both TCPs
103
have similar antibacterial capacities, suggesting that this difference might
be restricted to modulation on PMNs and monocytes88, potentially other
mammalian cells as well. Both TCPs also have the same net positive
charge of +2.1 at pH 7.0 (http://pepcalc.com/) and are projected to adopt
a similarly amphipathic α-helical structure (Supp. Fig. S2.7). These might
explain, to some extent, the similarity in antibacterial effectiveness.
Another prominent difference is the peptides’ lengths which might have
rendered FYT21 to more effectively penetrate across the mammalian
cells’ phospholipid membrane than the shorter HVF18. An early report
shows that liposome disruption was dependent on the lengths of
amphipathic helical peptides253, although whether that is the case in our
study remains to be studied. Alternatively, the histidine N-terminal
capping of HVF18 might have affected its helical fidelity as the histidine,
depending on its protonation, has been shown to contribute to helical
stability254,255. Indeed, protonated HVF18 at acidic pH can disrupt
bacterial membrane and kill bacteria more effectively, although these
effects can be attenuated with physiological salt concentrations unlike
GKY25. The longer peptide length and addition of hydrophobic residues
(Y3, F5, Y6) could have rendered GKY25 more resistant to pH and
physiological salt influences. Also, HVF18 has lower CD14-binding affinity
as compared to GKY25 at neutral pH256. Assuming FYT21 behaves like
GKY25 where it is less susceptible to pH and salt influences, it might
explain to some extent the increased LPS-inhibitory and
immunomodulatory effects as compared to HVF18 in our experimental
104
conditions (RPMI + 10% FBS). Clearly though, further investigations into
the structure-activity relationships of both TCPs on mammalian and
bacterial membranes, perhaps also on the membranal proteins and
receptors, are needed to elucidate and differentiate their
immunomodulatory potentials under physiological conditions.
Critical to this finding is its physiological relevance in the case of P.
aeruginosa-infected wounds. FYT21 is the predominant TCP in plasma
treated with P. aeruginosa supernatants and in bacteria-infected wounds’
fluids, both cases most likely contributed by the bacteria’s elastase88.
Intriguingly, FYT21’s antibacterial effects are diminished in the presence
of 10% plasma and in 25% whole blood, suggesting that physiological
conditions would not favour their antibacterial functions which is
congruent with similar studies on other HDPs199. On the contrary, its LPS-
inhibitory effects and neutrophil-modulatory properties at physiologically
relevant concentrations were unaffected in the presence of 10% heat-
inactivated serum (Fig. 2.9). This corresponds with the previous report
investigating monocyte activities against LPS in 10% plasma or 25%
whole blood88. These suggest that FYT21’s immunomodulatory
properties might be more prominent than its antibacterial effects under
physiological conditions. Hence, although speculative, it is likely that
elastase-producing P. aeruginosa can evade or even downplay immune
responses through the generation of the anti-inflammatory FYT21. This
adds to the fact that P. aeruginosa elastase, as a virulence factor, has
been shown to proteolyse surfactant protein A and cytokines to reduce
105
host defence response257,258. Being able to understand exactly how
FYT21 modulates immunity, and to what extent the elastase plays a role
in attenuating inflammation will generate new knowledge on host-
pathogen interactions. This can potentially be useful for future therapeutic
or prognostic developments.
Taken together, this study demonstrates a novel mechanism by which
GKY25 affects neutrophil functions by attenuating their rolling and
migration through the shedding of weak adhesion molecules such as
CD62L in vitro. GKY25 may also modulate neutrophils in vivo, hence
down-regulate immune responses. Future studies are clearly needed to
understand the exact mode of action of GKY25. Additionally, FYT21, a
TCP generated following thrombin proteolysis by P. aeruginosa elastase,
can also affect neutrophils in vitro, but its significance in a wound
environment requires further investigations. Nevertheless, this study
shows that GKY25 can be an interesting candidate for the development
of novel therapeutics against inflammatory and infective conditions.
Targeting TCPs and/or their generation pathways may also be a
therapeutic or prognostic strategy in wound care and management in the
future.
2.6 Acknowledgements
This research was supported by the Lee Kong Chian School of Medicine,
Nanyang Technological University Singapore Start-Up Grant, Singapore
Ministry of Education under its Singapore Ministry of Education Academic
106
Research Fund Tier 1 (2015-T1-001-082), the Swedish Research Council
(project 2012-1883), the Knut and Alice Wallenberg Foundation, and the
Swedish Foundation for Strategic Research. C.H.L. is supported by the
Interdisciplinary Graduate School, Nanyang Technological University,
Singapore. H.W.H. would like to acknowledge support from the NTU-NHG
Metabolic Diseases Collaboration Grant (MDCG/15004) and Lee Kong
Chian School of Medicine Postdoctoral Fellowship. We would like to
acknowledge Dr Ng Lai Guan and Dr Goh Chi Ching at the Singapore
Immunology Network, Agency for Science, Technology and Research,
Singapore, for their valuable inputs on experiments and discussions
concerning the in vivo models. We would also like to extend our thanks to
Assistant Professor Navin Kumar Verma and Dr Ong Seow Theng at Lee
Kong Chian School of Medicine, Nanyang Technological University,
Singapore, for their assistance on utilising the High Content Analysis
platform.
2.7 Author contributions
C.H.L., M.P., M.B., H.M.T., M.Z.Y.L., H.W.H. and A.S. participated in the
planning, design and interpretation of experiments. C.H.L. and M.P. designed
the in vivo experiments, while M.P. and M.B. conducted them and performed the
analyses. H.M.T. and H.W.H. proposed the microfluidics models and performed
the respective experiments and analyses. M.Z.Y.L. assisted in performing the
chemotactic assays and did the related flow cytometry analyses. C.H.L. planned
and performed the rest of in vitro experiments. C.H.L. and A.S. wrote and revised
the manuscript.
107
2.8 Disclosure
A.S. is a founder of in2cure AB, a company developing peptides for
human therapy. The peptide GKY25 is patent protected.
108
2.9 Supplementary information
Supplementary Figure S2.1: Effects of GKY25 on PMN toxicity and functional
ROS response. (A) PMNs (5 X 105 cells/ml) were treated with either IVE25 or GKY25
for up to 4 hrs at 37oC before LDH activity measurement (n = 3). (B) PMNs (1 X 106
cells/ml) were pre-treated with either LPS, IVE25 or GKY25 for 1hr at 37oC before
being subjected to 25 nM PMA stimulation for ROS response (n = 5). One-way
ANOVA. Figures are representative of 2 independent experiments.
Supplementary Figure 2.2: Dose-dependent assessment of GKY25 inhibition of LPS-induced surface markers’ changes on neutrophils in whole blood. Whole blood was treated with 5, 10 or 20 µM of (A) GKY25 or (B) IVE25 only or with 10 ng/ml for 1 hr at 37oC. Erythrocytes were lysed before flow cytometry analyses. (left) CD66b, (centre) CD11b and (right) CD62L. (---) Untreated baseline. One-way ANOVA. Figures are representative of 3 independent experiments.
-
IVE25
GKY25
-0.06
-0.04
-0.02
0.00
%C
yto
tox
icit
yns ns
5 uM
(A) (B)
Contr
ol
10 n
g/ml L
PS
IVE25
GKY25
0
20
40
60
80
100
RO
S
(no
rmalised
AU
C)
5 uM
** * **
ns
no LPS
10 n
g/ml L
PS
0.0
0.5
1.0
1.5
2.0
2.5
GKY25
Med
ian
Flu
ore
scen
ce In
ten
sit
y
(CD
66b
)
Ctrl
5 uM
10 uM
20 uMns
****
****
****
no LPS
10 n
g/ml L
PS
0.0
0.5
1.0
1.5
2.0
2.5
Med
ian
Flu
ore
scen
ce In
ten
sit
y
(CD
11b
)
ns
*
****
****
no LPS
10 n
g/ml L
PS
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Med
ian
Flu
ore
scen
ce In
ten
sit
y
(CD
62L
)
ns
****
*
no LPS
10 n
g/ml L
PS
0.0
0.5
1.0
1.5
2.0
2.5
IVE25
Med
ian
Flu
ore
scen
ce In
ten
sit
y
(CD
66b
)
Ctrl
5 uM
10 uM
20 uM
ns
ns
no LPS
10 n
g/ml L
PS
0.0
0.5
1.0
1.5
2.0
2.5
Med
ian
Flu
ore
scen
ce In
ten
sit
y
(CD
11b
)
ns
ns
no LPS
10 n
g/ml L
PS
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Med
ian
Flu
ore
scen
ce In
ten
sit
y
(CD
62L
)
**** **
ns
(A)
(B)
109
Supplementary Figure S2.3: Assessment of GKY25 inhibition of LPS-induced
ROS generation by flow cytometry. PMNs (1 X 106 cells/ml) pre-incubated with 10
μM H2DCFDA for 30 min at 37oC were treated with 25 nM PMA for 15 min as positive
control or co-treated with 5 μM IVE25 or 5 μM GKY25 with 10 ng/ml LPS for 1 hr at
37oC before flow cytometry assessment for (A) H2DCFDA, (B) CD11b or (C) CD62L.
(---) Untreated baseline. One-way ANOVA. Figures are representative of 2
independent experiments.
Supplementary Figure S2.4: Modulation of PSGL1 by GKY25. PMNs (5 X 105
cells/ml) isolated from 3 individuals were treated with 5 μM GKY25 for 1 hr at 37oC
before flow cytometry. Multiple T-tests.
(A) (B) (C)
5 uM
+ 10 ng/ml LPS
Ctrl
25 n
M P
MA
10 n
g/ml L
PS
IVE25
GKY25
0
50
100
150
200
250
MF
I (D
CF
DA
)
**
****
***ns
****
5 uM
+ 10 ng/ml LPS
Ctrl
25 n
M P
MA
10 n
g/ml L
PS
IVE25
GKY25
0
50
100
150
MF
I (C
D11b
)
****
ns
ns
ns
5 uM
+ 10 ng/ml LPS
Ctrl
25 n
M P
MA
10 n
g/ml L
PS
IVE25
GKY25
0
50
100
150
MF
I (C
D62L
)
****
ns
**** ****
****
Untr
eate
d
5 uM
GKY25
0
200
400
600
800
Med
ian
FI (C
D162)
Donor 1
Donor 2
Donor 3
**
****
**
110
Supplementary Figure S2.5: Rolling frequencies of PMNs on E-selectin coated
PDMS microdevice. PMNs (1 X 106 cells/ml) were treated with 5 µM IVE25 or GKY25
before being subjected to microfluidics flow assessment. Rolling frequencies of
GKY25-treated PMNs were plotted with (A) untreated PMNs and (B) IVE25-treated
PMNs. Figures were representative of 3 independent experiments.
0.5
1.5
2.5
3.5
4.5
5.5
6.5
7.5
8.5
9.5
10
.511
.512
.513
.514
.515
.516
.517
.5
0
2
4
6
8
10
Rolling Velocities (um/s)
Rela
tive F
req
uen
cy
(%)
IVE25
GKY25
0.5
1.5
2.5
3.5
4.5
5.5
6.5
7.5
8.5
9.5
10
.511
.512
.513
.514
.515
.516
.517
.5
0
2
4
6
8
10
Rolling Velocities (um/s)
Rela
tive F
req
uen
cy
(%)
Ctrl
GKY25
(A) (B)
111
Supplementary Figure S2.6: Neutrophil infiltration in vivo. (A-B) Mice were
administered with LPS in the absence or presence of GKY25 before being sacrificed
after 1 hr (n = 4). (C-D) Mice were also pre-treated with GKY25 intraperitoneally before
LPS administration subcutaneously (n = 3). (A, C) Skin sections were stained with
anti-neutrophil antibody (NIMP-R14, red) and DAPI (blue) and (B, D) used for scoring
for neutrophil infiltration. Unpaired T-test. Scale bar = 50 µM.
LP
S o
nly
LP
S +
GK
Y25
LPS only
LPS + G
KY25
0
2
4
6
8
10
Neu
tro
ph
il In
vasio
n S
co
re *
(A) (B)
DAPI Neutrophils
LP
S o
nly
GK
Y25 >
LP
S
DAPI Neutrophils
(C) (D)
LPS only
GKY25
> L
PS
0
2
4
6
8
10N
eu
tro
ph
il In
vasio
n S
co
re
ns
112
Supplementary Figure S2.7: Projected helical wheels of HVF18 and FYT21.
Projections were performed using the freely-available online programme, available at:
http://lbqp.unb.br/NetWheels/
HVF18 FYT21
113
– CHAPTER 3 –
THROMBIN AND PLASMIN ALTER THE
PROTEOME OF NEUTROPHIL
EXTRACELLULAR TRAPS
Reprinted (adapted) with permission from:
Lim, C. H. et al. Thrombin and plasmin alter the proteome of neutrophil
extracellular traps. F Immunol 9, 1554, doi:10.3389/fimmu.2018.01554
(2018). Copyright © 2018, Lim, Adav, Sze, Choong, Saravanan and
Schmidtchen, Frontiers Media S.A.
114
115
3 Thrombin and plasmin alter the proteome of
neutrophil extracellular traps
3.1 Abstract
Neutrophil extracellular traps (NETs) consist of a decondensed DNA
scaffold decorated with neutrophil-derived proteins. The proteome of
NETs, or “NETome”, has been largely elucidated in vitro. However,
components such as plasma and extracellular matrix proteins may affect
the NETome under physiological conditions. Here, using a reductionistic
approach, we explored the effects of two proteases active during injury
and wounding, human thrombin and plasmin, on the NETome. Using
high-resolution mass spectrometry, we identified a total of 164 proteins,
including those previously not described in NETs. The serine proteases,
particularly thrombin, were also found to interact with DNA and bound to
NETs in vitro. Among the most abundant proteins were those identified
previously, including histones, neutrophil elastase and antimicrobial
proteins. We observed reduced histone (H2B, H3 and H4) and neutrophil
elastase levels upon the addition of the two proteases. Analyses of NET-
derived tryptic peptides identified subtle changes upon protease
treatments. Our results provide evidence that exogenous proteases,
present during wounding and inflammation, influence the NETome. Taken
together, regulation of NETs and their proteins under different
physiological conditions may affect their roles in infection, inflammation,
and the host response.
116
3.2 Introduction
In 2004, Brinkmann et al observed a novel extracellular structure formed
by neutrophils which they coined neutrophil extracellular traps (NETs).
They are made up of decondensed DNA as the structural backbone for
various neutrophil-derived proteins such as histones and neutrophil
elastase (ELANE)101. NETs are exuded in response to stimuli such as
microorganisms, chemical derivatives including PMA and calcium
ionophores, LPS and even activated endothelial cells and platelets.
Studies have shown that NETs exert antimicrobial effects on various
micro-organisms including bacteria, fungi and parasites259. In vivo, they
have been identified in several inflammatory conditions and are also
implicated in the pathogenesis of autoimmune diseases183.
Two separate proteomics studies identified a total of 24139 and 29260 NET-
associated proteins, respectively, predominated by histones, ELANE and
several antimicrobial proteins or peptides. In the experimental set-ups of
these studies however, only neutrophil-derived proteins were
reported139,260. Separately, non-proteomics-based studies have
described the binding of the complement component C1q to NETs261. We
have also preliminarily assessed the interaction of the prototypic TCP
GKY25 with NETs / DNA and have shown that GKY25 can protect DNA
against nuclease degradation (Supp. Fig. S3.1; unpublished). Therefore,
the full repertoire of NETs’ proteins has not yet been elucidated,
especially under physiological and inflammatory conditions.
117
NETs have been identified during wounding, where other proteins are
also variably regulated117,172,262,263. In vitro, NET-associated proteins,
particularly enzymes, have been described to be biologically
active260,264,265. On this note, proteins such as MPO, ELANE and MMP9,
all of which found to be present on NETs, were assessed to be active in
wound fluids harvested from infected, non-healing venous leg ulcers166.
When the proteomes of fluids isolated from healing and non-healing
wounds were compared, neutrophil-derived serine proteases were
observed to be exclusively present in non-healing wound fluids263,
indicating sustained and dysregulated inflammation in these wounds.
Apart from neutrophil-derived proteins, major serum proteins such as
thrombin can also be modulated in wounds; in a porcine wounding model,
thrombin levels were high within the first 3 days of wounding and lowered
by day 7262. This regulation is important as it facilitates the transition to
the later fibrinolytic phase of wound healing mediated by plasmin266. From
this perspective, NETs and its associated proteins which were identified
during wounding might be affected by the highly-proteolytic environment.
Hence, in a reductionistic approach, we aimed to assess the effects of 2
major serum serine proteases, thrombin and plasmin – each functioning
on the opposite spectrum of wounding – on the proteome of NETs. Using
high-resolution liquid chromatography-mass spectrometry (LC-MS/MS),
molecular biology and bio-informatics tools, we therefore explored
changes of NET-associated proteins after addition of thrombin or plasmin.
We observed differences in the abundances of multiple NET-proteins,
118
demonstrating that the NETome is dynamically regulated and shows
qualitative and quantitative changes depending on the environment267.
119
3.3 Materials and Methods
3.3.1 Materials
Sodium citrate-containing tubes used for blood collection were from
Becton-Dickinson, USA. Polymorphprep was purchased from Axis-Shield,
Scotland. The Erythrocyte Lysis Buffer was from eBioscience, USA. The
fluorescently-labelled antibodies used for flow cytometry – BV510-anti-
CD45 (clone HI30) and FITC-anti-CD66b (clone G10F5) – were from
BioLegend, USA. RPMI-1640 cell culture medium without phenol red, the
live-dead stain DAPI, Sytox Green, DNA endonuclease I (DNase I),
micrococcal nuclease (MNase), Proteinase K, Pierce Protease Inhibitor
Mini Tablets, Pierce Silver Stain Kit, Pierce BCA (bicinchoninic acid)
Protein Assay Kit, Supersignal West Dura Extended Duration Substrate,
Supersignal West Femto Maximum Sensitivity Substrate and SYBR Safe
were from Thermo-Fisher Scientific, USA. TAMRA-labelled and
unlabelled GKY25 were from Biopeptide, USA. PMA, poly-L-lysine and
Fluoroshield with DAPI mounting medium were bought from Sigma-
Aldrich, USA. Purified thrombin and plasmin were from Innovative
Research, USA. Sequencing grade modified trypsin was from Promega,
USA. Recombinant histones H2B, H3.3 and H4 were from New England
Biolabs, USA. Purified ELANE was from Merck, Germany. The 4-20%
polyacrylamide Mini-PROTEAN Tris-Glycine precast gels and Immun-
Blot polyvinylidene fluoride (PVDF) membrane were from Bio-Rad, USA.
The E.Z.N.A. DNA/RNA Isolation Kit was from Omega Bio-Tek, USA. The
rabbit polyclonal antibody recognising the thrombin C-terminal peptide (α-
120
VFR17; VFRLKKWIQKVIDQFGE) was from Innovagen AB, Sweden.
Antibodies from Abcam, USA include – rabbit polyclonal anti-H2B (α-H2B),
anti-H3 (α-H3), anti-H4 (α-H4) and anti-ELANE (α-ELANE) antibodies;
goat polyclonal anti-plasminogen antibody (α-PLG); HRP-conjugated
donkey polyclonal anti-goat IgG antibody (HRP-α-goat); AF568 goat
polyclonal anti-rabbit IgG antibody (AF568-α-rabbit) and AF488 donkey
polyclonal anti-goat IgG antibody (AF488-α-goat). The HRP-conjugated
goat polyclonal anti-rabbit IgG antibody (HRP-α-rabbit) was from Agilent,
USA. For blocking of western blots or immunofluorescence staining,
either 3% milk (w/v), 3% or 5% BSA (w/v) was dissolved in TBS-T (TBS
with 0.1% v/v Tween-20) and used, as indicated.
3.3.2 Ethics statement
Human whole blood samples were obtained from participating subjects
who gave written informed consent. The process of blood sample
collection and methods used thereafter were performed in accordance
with the guidelines and regulation recommended and approved by the
Nanyang Technological University Singapore’s Institutional Review Board
(IRB-2014-10-041).
3.3.3 PMNs isolation
Whole blood obtained in sodium citrate-containing tubes by venepuncture
were used for PMNs isolation with Polymorphprep according to
manufacturer’s instructions. Briefly, approximately 1 part of blood was
layered onto 1 part of Polymorphoprep before centrifugation at 500x g for
121
30 min at 25oC without brakes. The separated layer containing PMNs was
then harvested, treated with Erythrocyte Lysis Buffer, washed and
resuspended in RPMI-1640. The purity of isolated PMNs was determined
by flow cytometry (LSR-Fortessa-X20; Becton-Dickinson, USA) and the
acquired data was analysed with Flowjo (Treestar, USA). PMN isolates
at >97% were used, as defined by the percentage of CD66b+ cells over
live CD45+ cells.
3.3.4 NET induction
Isolated PMNs (1 X 106 cells/ml; 2 million cells) were seeded in 6-well
plates and treated with 25 nM PMA for a total of 4 hours at 37oC to induce
NET formation. At the 2-hour time-point, 1 µM thrombin or plasmin was
added, whereas the control wells were left as they were. The wells were
not washed during this period. Next, the wells were washed gently 3 times
with pre-warmed RPMI before further treatments with 20 U/ml DNase I in
RPMI (+DNase) or RPMI alone (-DNase) for 1 hour at 37oC. The
supernatants in the respective wells were then carefully aspirated, added
with protease inhibitor and processed for mass spectrometry or SDS-
PAGE (Fig. 3.1). A total of 3 biological replicates from 3 human subjects
were prepared. The amounts of proteins in each sample were estimated
using the Pierce BCA Protein Assay Kit.
3.3.5 LC-MS/MS and data analysis
To prepare the samples for LC-MS/MS, 40 μg of the NET proteins were
subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis
122
(SDS-PAGE; 5% polyacrylamide stacking gel with 12% polyacrylamide
separating gel). Electrophoresis was first run at 80V for 20 min, then at
100V for another 20 min. Next, each sample lane was separately sliced
and cut into pieces (approximately 1 mm2), washed with buffer (75%
acetonitrile containing 25 mM ammonium bicarbonate) and de-stained
completely. Then, these gel pieces were reduced with 10 mM dithiothreitol
and alkylated with 55 mM iodoacetamide in the dark. They were later
dehydrated with 100% acetonitrile and subjected to sequencing grade
modified trypsin digestion at 37°C, overnight. The peptides were
extracted using 50% acetonitrile / 5% acetic acid. Finally, the extracted
peptides were desalted and vacuum-centrifuged to dryness.
Figure 3.1: General experimental workflow. (A) PMNs were isolated from 3
independent human whole blood samples by density centrifugation and (B) treated
with 25 nM PMA for 2 hours to induce NETs. PMNs were left untreated or
supplemented with 1 μM thrombin or plasmin and incubated for another 2 hours. (C)
After gently washing the wells, they were further treated with 20 U/ml DNase I or with
RPMI only for 1 hour at 37oC to digest the DNA scaffold. The proteins extracted were
then processed for (D) LC-MS/MS or (E) in some cases, subjected to validation by
SDS-PAGE / western blotting.
123
For LC-MS/MS, the peptides were reconstituted in 0.1% formic acid (FA).
Three biological replicates and their technical duplicates were separated
and analysed in a LC-MS/MS system comprising of a Dionex Ultimate
3000 RSLC nano-HPLC system, coupled to an online Q-Exactive mass
spectrometer (Thermo-Fisher Scientific, USA). Five micro-litres of each
sample were injected into an acclaim peptide trap column via the auto-
sampler of the Dionex RSLC nano-HPLC system. The flow rate was
maintained at 300 nl/ml. The mobile phase A (0.1% FA in 5% acetonitrile)
and mobile phase B (0.1% FA in acetonitrile) were used to establish a 60-
minute gradient. Peptides were analysed on a Dionex EASY-spray
column (PepMap® C18, 3um, 100 A) using an EASY nanospray source.
The electrospray potential was set at 1.5 kV. Full MS scan in the range of
350-1600 m/z was acquired at a resolution of 70,000 at m/z 200, with a
maximum ion accumulation time of 100 ms. Dynamic exclusion was set
to 30 seconds. Resolution for MS/MS spectra was set to 35,000 at m/z
200. The AGC setting was 1E6 for the full MS scan and 2E5 for the MS2
scan. The 10 most intense ions above 1000 count threshold were chosen
for higher energy collision dissociation (HCD) fragmentation. The
maximum ion accumulation time was 120 ms. An isolation width of 2 Da
was used for the MS2 scan. Single and unassigned charged ions were
excluded from MS/MS. For HCD, normalized collision energy was set to
28. The underfill ratio was defined as 0.1%.
For data analysis, the raw data files were converted into the Mascot
generic file format using Proteome Discoverer version 1.4 (Thermo
124
Electron, Germany) with the MS2 spectrum processor for the de-isotoping
of the MS/MS spectra. The concatenated target-decoy UniProt human
database (total sequences 92867, total residues 36953354, downloaded
on 25 July 2016) was used for data searches. Data search was performed
using in-house Mascot server (version 2.4.1, Matrix Science, USA). Static
peptide modification was carbamidomethylation of cysteine residues
(+ 57.021 Da), and the dynamic peptide modifications were oxidation of
methionine residues (+ 15.995 Da) and deamidation of asparagine and
glutamine residues (+ 0.984 Da). Two missed cleavage sites of trypsin
and mass tolerances of 10 parts per million (ppm) for peptide precursors
were allowed. A mass tolerance of 0.8 Da was set for fragmented ions in
Mascot searches. Quantification was performed using exponentially
modified protein abundance index (emPAI) scores reported by Mascot
search engine which is based on equations as stated below268.
𝑃𝐴𝐼 =𝑁𝑜𝑏𝑠𝑑
𝑁𝑜𝑏𝑠𝑏𝑙
where Nobsd and Nobsbl are the number of observed peptides per protein
and the number of observable peptides per protein, respectively. The
emPAI is defined as follows.
𝑒𝑚𝑃𝐴𝐼 = 10𝑃𝐴𝐼 − 1
Subsequently, the identified proteins dataset was exported into Microsoft
Excel for further analysis. The false discovery rate (FDR) was calculated
using in-house build program as 2 × Md / (Md + Mt), where Md represents
125
the number of decoy matches, and Mt is the number of target matches.
Proteins with FDR ≤ 1 were considered for further analysis. To identify
NET-associated proteins, we adopted the workflow to eliminate proteins
that were constitutively released from our culture conditions without
DNase I (Suppl. Fig. S3.2). Finally, the hierarchical clustering of true
NET-associated protein abundances was generated using GenePattern
2.0269.
3.3.6 SDS-PAGE and protein detection
Equal amounts of NET-proteins, where indicated, were loaded onto 4 -
20% polyacrylamide gel for SDS-PAGE. Then, the gel was used for either
silver staining with the Pierce Silver Stain Kit (according to
manufacturer’s instructions) or transferred onto PVDF membranes for
western blotting. For the detection of thrombin and plasmin, the blots
were first blocked with 5% or 3% milk/TBS-T respectively. Then, the blots
were incubated with α-VFR17 (1:5000 in 5% milk/TBS-T) and α-PLG
(1:1000 in 3% milk/TBS-T) respectively. Finally, HRP-α-rabbit antibody
(1:10000 in 5% milk/TBS-T) and HRP-α-goat antibody (1:10000 in 3%
milk/TBS-T) were used to detect the respective primary antibodies. For
the detection of histones, the blots were first blocked with 5% BSA/TBS-
T. Then, they were incubated with α-H2B (1:1000), α-H3 (1:1000) and α-
H4 (1:5000) diluted in 5% BSA/TBS-T. The primary antibodies were than
detected with HRP-α-rabbit (1:10000) diluted in 1% BSA/TBS-T. For the
detection of neutrophil elastase, the blot was first blocked with 3%
milk/TBS-T and probed with α-ELANE (1:1000) diluted in 3% milk/TBS-
126
T. Finally, α-ELANE was detected with HRP-α-rabbit antibody (1:10000)
diluted in 3% milk/TBS-T. All the blots were developed using the
enhanced chemiluminescence method with either the Supersignal West
Dura or Femto Substrate and visualised with the ChemiDoc Imaging
System (Bio-Rad, USA).
3.3.7 DNA gel mobility shift assay
DNA was purified from isolated PMNs using the E.Z.N.A. DNA/RNA
Isolation Kit according to manufacturer’s instructions. Then, 20 µg/ml of
the DNA was incubated alone or with 1.0, 2.0, 4.0, 8.0 or 12.0 μM of
thrombin or plasmin in RPMI for 1 hour at 37oC. This was followed by gel
electrophoresis on a 0.5% agarose (w/v) gel in Tris acetate-EDTA (TAE)
buffer with SYBR Safe which was then visualised with the ChemiDoc
Imaging System (Bio-Rad, USA).
3.3.8 DNA protection
DNA protection was assessed by gel electrophoresis and DNA
quantification by Sytox Green staining. First, 2 μg/ml DNA was incubated
with 0, 2.5, 5.0, 10 or 20 μM GKY25 for 30 min at 37oC. Then, either buffer
or 0.2 U/ml MNase was added for another 15 min incubation at 37oC. To
heat-inactivate the MNase, samples were incubated at 65oC for 30 min
before transferring to ice. Finally, buffer or 0.25 mg/ml Proteinase K was
added for a 90 min incubation at 37oC to digest bound or unbound GKY25
to expose the DNA. For gel electrophoresis, presumed 100 ng of DNA
was loaded onto 0.8% agarose (w/v) gel in TAE buffer with SYBR Safe
127
which was then visualized with the ChemiDoc Imaging System (Bio-Rad,
USA). To quantify the free DNA, 1 μg/ml DNA was incubated with 50 nM
Sytox Green for 15 min in the dark before the measurement of
fluorescence intensities at 480ex / 530em with the Cytation 3 Cell-Imaging
Multi-Mode reader (Research Instruments, USA).
3.3.9 Immunofluorescence
PMNs (5 X 105 cells/ml; 100,000 cells) were first seeded onto 0.1 mg/ml
poly-L-lysine-coated coverslips in 24-well plates before NET induction
with PMA and treatment with 1 µM of thrombin or plasmin as described
above (section 3.3.4). For the assessment of GKY25 binding to NETs,
seeded PMNs were treated with 25 nM PMA for 3 hrs at 37oC, washed
once gently with pre-warmed RPMI, and incubated with 5 μM TAMRA-
labelled GKY25 for another 1 hr at 37oC. Then, the wells were gently
washed 2 times with pre-warmed RPMI, fixed with 4% PFA and washed
another 2 times with TBS-T. The samples were blocked with 3%
BSA/TBS-T before incubation with the respective primary and
fluorescently-labelled secondary antibodies diluted in 3% BSA/TBS-T.
The dilutions used were – α-H2B (1:350), α-VFR17 (1:1000), α-
plasminogen (1:500), AF568-goat polyclonal α-rabbit IgG (1:1000) and
AF488-donkey polyclonal α-goat IgG (1:1000). Finally, the coverslips
were mounted using the Fluoroshield with DAPI mounting medium.
128
3.3.10 Peptigrams
Peptigrams were generated (available at http://bioware.ucd.ie/peptigram/)
and used to compare the tryptic peptides detected with modifications.
Each tryptic peptide detected within the sample with peptide scores of
more than 20 were summed (if more than 1 of the same peptide was
detected) to give an overall view of the peptide’s abundance. The data
were then processed for peptides corresponding to their respective
proteins as described270.
3.3.11 Statistical analysis
Statistical analyses were performed on the proteins’ emPAI scores using
the Prism software version 7.0 (GraphPad, USA). One-way ANOVA with
Dunnett’s correction for multiple comparison was applied unless
otherwise indicated. Data was presented as Mean (SD). Statistical
significance based on p values were –# p < 0.1 (not significant), * p < 0.05,
** p < 0.01, *** p < 0.001 and **** p < 0.0001.
129
3.4 Results
3.4.1 Identification of NET-associated proteins
Previous reports of the proteome of NETs described the identification and
quantification of NET-associated proteins from purified neutrophils only
and without additional, exogenous components139,260. Here, we aimed to
investigate whether the coagulation proteases – thrombin and plasmin –
have any effects on the NETome. The experiments were performed in the
absence of serum to exclude potential influences from other serum
proteases. PMA was used as the NET-inducing agent to provide a basal,
standardised, NETome for comparison. Thrombin and plasmin were
added at 1 µM which closely corresponded to their plasma concentrations
of 1.4 µM228 and 1.6 µM271, respectively. Following NET induction
procedures as described in Figure 3.1, we first analysed whether DNase
I was able to release NET-associated proteins by SDS-PAGE followed by
silver staining (Fig. 3.2A-B). We observed significantly more proteins in
DNase I treated samples (Fig. 3.2A) as compared to the non-DNase I
treated material (Fig. 3.2B). This indicated that most of these proteins
were released upon DNase I treatment and thus, NET-bound.
Next, the samples were sent for high-resolution LC-MS/MS to identify the
proteins present. To correctly identify the NET-associated proteins, we
adopted a data processing workflow to ensure that proteins non-
specifically released into the medium during DNase I treatment were
screened out (i.e. from cell membranes or those attached to the wells)
130
(Suppl. Fig. S3.2). Protein correlations of the samples between donors
were also performed (>0.79, Suppl. Fig. S3.3) and demonstrated good
data reproducibility for the experiments.
Figure 3.2: Silver stains and initial comparison of NET-proteins. (A-B) Proteins
extracted from NETs were quantified and loaded (20 μg) onto 4-20% Tris-Glycine
gradient gels. Silver staining was then performed – (A) DNase I treated samples and
(B) non-DNase I treated samples. (C) Proteins identified as NET-bound (as illustrated
in Suppl. Fig. 1) from untreated, 1 μM thrombin- or 1 μM plasmin-treated samples
were compared using Venn diagram.
Consequently, 164 NET-associated proteins were identified (Fig. 3.2C),
a number higher than previously reported139,260. The higher ranked
proteins identified with high protein scores were congruent with those
previously reported as observed in Table 3.1. In addition, we identified
proteins not described to be present on NETs previously, such as the
serine protease inhibitor (Serpin)-B1, lipocalin-2 and complement C3
(Table 3.1). Peptides corresponding to the antimicrobial proteins
cathelicidin and S100A12 were also detected; however, they were
excluded during data analysis due to the FDR ≤ 1 applied.
131
Table 3.1: Comparison of NET-associated proteins identified. Studies from Urban
et al (2009) and O’Donoghue et al (2013) were compared against the present study.
Y – identified in the studies; Y# - identified in plasmin-treated samples only; red box – not identified in the
study; blue box – newly identified or previously identified in only 1 of the 2 studies.
3.4.2 Thrombin and plasmin can bind to NETs
The two S1 peptidases thrombin and plasmin have been reported to
interact with DNA aptamers272 and short oligos273. In the initial
132
experiments, we explored whether these proteins can bind to the DNA of
NETs. Notably, based on the mass spectrometry data obtained, we
detected the presence of thrombin and plasmin in the NET samples
treated with the respective enzyme (Fig. 3.3A-B). Moreover, the presence
of the two proteases was further validated by western blotting, where
antibodies specific for thrombin (~37 kDa) and plasmin (~40 kDa),
detected these proteins in the respective NET samples under reducing
conditions (Fig. 3.3C). To investigate whether the two proteases interact
with DNA, thrombin and plasmin were incubated with purified PMN DNA
and analysed by a gel mobility shift assay. We observed reduced band
mobility after addition of 1.0 µM thrombin and complete DNA retardation
at or above 4.0 μM thrombin. For plasmin however, reduced band mobility
was observed at 8.0 μM plasmin and above, indicating that plasmin
showed a weaker interaction with DNA in comparison with thrombin (Fig.
3.3D). Immunofluorescence microscopy analysis showed that thrombin,
but not plasmin (both at 1 μM), co-localised with NETs (Fig. 3E). In
contrast to thrombin, which was found to associate with NET-like
structures, plasmin was bound to the cell membrane and/or cell debris
(Fig. 3E lower panel), compatible with previous studies showing that
plasmin binds to PMN surfaces274. Notably, when plasmin levels from
plasmin-treated NETs were compared with controls, there was still an
observable increase in plasmin abundance following DNase I treatment,
indicating that plasmin was NET-bound and released upon DNase I
133
treatment (Suppl. Fig. S3.4B). Taken together, the data show that
particularly thrombin can bind to NETs of PMNs.
Figure 3.3: Analysis of NET/DNA-binding of thrombin and plasmin. The raw
emPAI scores from +DNase samples of (A) thrombin and (B) plasmin were compared
across the treatment conditions (n = 3). (C) Western blot analyses on +DNase
samples for α-VRF17 (~37 kDa; upper panel) and α-PLG (~40 kDa; lower panel).
Amounts of proteins loaded were as indicated in the figure. (D) DNA (20 μg/ml) was
either incubated alone or with 1.0, 2.0, 4.0, 8.0 or 12.0 μM of thrombin or plasmin in
RPMI for 1 hour at 37oC. Then, 200 ng of DNA was loaded onto 0.5% agarose gel for
gel electrophoresis. (E) PMNs were treated with PMA only (ctrl) for 4 hours at 37oC or
added with 1 μM thrombin or plasmin after 2 hours with PMA. First row: α-H2B (red);
second and third row: α-VFR17 (red); fourth and fifth row: α-PLG (green). Scale bar:
10 μm.
3.4.3 Effects of thrombin and plasmin on NET-associated histones
In addition to the identification of new NET-associated proteins, we
compared the protein abundances across the 3 treatment conditions by
hierarchical clustering. The proteins were classified into 8 clusters –
proteins enriched in untreated samples (cluster C3 and C5), thrombin-
treated samples (cluster C1, C2 and C4) and plasmin-treated samples
134
(cluster C6) (Fig. 3.4A and Suppl. Fig. S3.5). The clusters C3 and C5
consist of many classically NET-associated proteins such as histones,
ELANE and some antimicrobial proteins (Suppl. Fig. S3.5A). As histones
are significant components of NETs139, three histones – H2B, H3 and H4
(Fig. 3.4B-D) – were selected for analysis by western blotting. The results
show that these NET-associated histones were reduced in the presence
of thrombin and plasmin which corresponded to the obtained mass
spectrometry data (Fig. 3.4E vs. Fig. 3.4B-D).
Figure 3.4: Reduced major histones on NETs by thrombin and plasmin. (A)
Hierarchical clustering of identified NET-associated proteins following data processing
in Suppl. Fig. 1. Enriched proteins are displayed in red, lowered proteins are
displayed in blue and the intermediate values are displayed in the shades of red and
blue. (B-D) The histones’ emPAI scores from thrombin- and plasmin-treated samples
were normalized against the respective donor’s untreated control. Donor-matched
samples are presented (n = 3). (B) emPAI scores for histone H2B subtypes E and F
for each treatment were summed then normalized against the untreated control. (C)
emPAI scores for histone H3 and variant H3.3 for each treatment were summed then
normalized against the untreated control. (D) Normalized histone H4 emPAI scores.
(E) Western blot analyses on +DNase samples using α-H2B (~12 kDa; upper panel),
α-H3 (~11 kDa; middle panel) and α-H4 (~9 kDa; lower panel) antibodies. Amounts of
proteins loaded for each sample were as indicated. (F) Recombinant histones H2B,
H3.3 and H4 (2 μM) were treated with 1 μM thrombin, 1 μM plasmin or 0.25 mg/ml
proteinase K for 2 hours at 37oC before SDS-PAGE with 150 ng of the respective
135
histones and western blotting. Antibodies used were α-H2B (~15 kDa; upper panel),
α-H3 (~15 kDa; middle panel) and α-H4 (~13 kDa; lower panel).
As thrombin and plasmin are proteases, we explored whether their
proteolytic activities resulted in the observed reduction of histone levels.
To address this, the recombinantly produced histones H2B, H3.3 and H4
were incubated with thrombin or plasmin. We observed that the three
types of histones were indeed degraded in the presence of the two
proteases (Fig. 3.4F), which agreed with the immunoblot results (Fig.
3.4E). This suggests that the thrombin- and plasmin-mediated reduction
of the studied NET-associated histones is due to proteolysis.
We next investigated whether treatment with thrombin and plasmin may
alter the patterns of histone peptide fragments detected by mass
spectrometry. To this end, the unique tryptic peptides identified by mass
spectrometry were aligned to the respective full-length histones and
presented in peptigrams270 (Fig. 3.5). Corresponding to the results
presented in Fig. 3.4, we observed that the peptide intensities of the NET-
associated histones were reduced after treatment with thrombin or
plasmin. However, there were no major differences regarding the origins
of the peptide sequences detected for histones H2B and H3 (Fig 3.5A-B).
For histone H4 on the other hand, the peptide from residues 61-78
(VFLENVIRDAVTYTEHAK; 61V-78K) was not detected in all thrombin-
and plasmin-treated samples (Fig. 3.5C), suggesting that this region was
affected by thrombin and plasmin. Taken together, in addition to providing
136
useful information on the peptide sequences detected and their
abundances, these results also reflect the specificity, sensitivity and
reproducibility of our experimental workflow.
Figure 3.5: Peptigrams for histones H2B, H3.3 and H4. Peptigrams were generated
using the sum of the respective peptide scores corresponding to (A) histone H2B
subtype F (Q5QNW6), (B) histone H3 (Q5TEC6) and (C) histone H4 (P62805).
3.4.4 Effects of thrombin and plasmin on NET-associated neutrophil
elastase
Mass spectrometry data showed that ELANE was significantly reduced in
all three NET samples after treatment, particularly with thrombin (Fig.
3.6A). Correspondingly, western blot analysis using polyclonal antibodies
against ELANE showed that it was reduced on NETs following treatments
with the proteases (Fig. 3.6B). In agreement with the observed ELANE
reduced levels, 4 tryptic peptides of ELANE were not detected on the
thrombin- or plasmin-treated NETs from at least 2 donors (Fig. 3.6C and
Suppl. Table S3.1). Two of these 4 peptides were 51G78R and
137
82V91R which lie within the first 100 residues used to generate the
polyclonal antibody against ELANE. These results may explain the
observation that ELANE staining was significantly reduced by the two
proteases as detected by western blotting (Fig. 6B).
Figure 3.6: NET-associated neutrophil elastase is changed by thrombin and
plasmin. (A) The ELANE’s emPAI scores from thrombin- and plasmin-treated
samples were normalized against the respective donor’s untreated control. Donor-
matched samples are presented (n = 3). (B) Western blot analysis for ELANE (α-
ELANE, ~27 kDa) with 60 μg of total protein. (C) Peptigram for ELANE was generated
using the sum of its respective peptide scores.
138
3.5 Discussion
Here, we report for the first time that exogenous proteases such as
thrombin and plasmin can alter the proteome of NETs. In addition, we
were able to identify a higher number of NET-associated proteins than
previously reported. We also provide evidence on the dynamic regulation
of NET-proteins in the presence of thrombin and plasmin, thus providing
a conceptual base for future studies on the complex interactions between
neutrophils, NETs and the microenvironment.
Studies on the proteome of NETs have used nucleases at varying
concentrations (5-20 U/ml) and incubation durations (10-40
min)112,139,260,275. Here, we used 20 U/ml DNase I for 1 hour for more
extensive digestion of NETs. To ensure that we accurately identify NET-
bound proteins with high confidence, emPAI values of proteins detected
in the non-DNase I treated samples were subtracted from DNase I treated
samples (Suppl. Fig. S3.2). As a result, we identified 164 NET-
associated proteins, several previously not described139,260. However, we
did not identify calprotectin S100A12 and the cathelicidin LL37 which
were previously reported to bind to NETs120,139,206,260, although the
corresponding peptides were detected in the LC-MS/MS spectra but were
later excluded for analysis as they fell within the FDR ≤ 1. Nonetheless,
we were able to identify all other proteins, even those identified by either
Urban et al (2009) or O’Donoghue et al (2013) only, such as histone H1
and catalase, respectively (Table 3.1). Moreover, we were able to identify
139
additional proteins, such as the protease inhibitors SerpinA1 and
SerpinB1 which are expressed by neutrophils276,277 and can inhibit
thrombin, ELANE, cathepsin G and other serine proteases278,279. Indeed,
SerpinB1 has been implicated in NET formation and was also recently
identified on NETs112,126. Similarly, lipocalin-2, eosinophil cationic protein
and complement C3 were identified in our NET preparations and have
been reported to be expressed by neutrophils too280-282. The possibility
that the prolonged DNase I incubation time led to release of additional
proteins cannot be excluded; however, the incorporation of non-DNase I
treated controls should mitigate this possibility. Nonetheless, this shows
that by employing sensitive methods and stringent protein exclusion
workflow, we were able to identify previously undisclosed NET-proteins
with good reproducibility.
While thrombin and plasmin were reported to interact with short single-
stranded DNA272,273, their interaction with cell-free DNA remains unclear.
Here, we show that in particular thrombin binds to NETs and/or DNA (Fig.
3.3). Thrombin possesses two distinctive electropositive surface regions
– the fibrinogen-recognition exosite I and heparin-binding exosite II283 –
which have been shown to interact with DNA aptamers272,284. Hence, it is
likely that thrombin binds full-length DNA through these two exosites,
which is of relevance as DNA of NETs was shown to promote thrombin
generation in platelet poor plasma168. We also show that plasmin can bind
to DNA, albeit at higher concentrations than thrombin (Fig. 3.3D),
suggesting that the serine proteases’ DNA-binding affinities are different.
140
Considering the early stages of wounding, the presence of cell-free DNA
or NETs may therefore facilitate thrombin generation and their
subsequent binding to NETs, which is logical from a physiological
perspective as it compartmentalises and thus controls thrombin activity,
possibly to avoid uncontrolled fibrin generation at inflammatory sites.
Conversely, the weak interaction between DNA or NETs and plasmin (or
plasminogen) would ensure this enzyme is more “freely” distributed,
enabling proper fibrinolysis during wounding and inflammation. In this
respect, it is interesting to note that thrombin and plasmin, like ELANE,
belongs to the vast family of S1 peptidases, sharing an overall similar
structure and folding285. Although our results provide evidence that
particularly thrombin binds to NETs, studies on its DNA interaction
domains, binding sequences and affinities are mandated in order to
characterise this interaction in detail. These studies are however outside
the scope of the present study.
In addition, we show that thrombin and plasmin changed the overall
NETome (Fig. 3.2C). The respectively treated samples shared large
overall similarities in terms of the proteins identified (145 of 164 proteins;
88.4%). Of interest is the changes in abundance of these NET-associated
proteins (Fig. 3.4A). We noticed that majority of these classical NET-
associated proteins were reduced when thrombin or plasmin was present
(cluster C3 and C5; Suppl. Fig. S3.5A). For example, various NET-
associated histones seemed to be reduced by both thrombin and plasmin,
compatible with the results showing that both thrombin and plasmin
141
degrade histones (Fig. 3.4B-E). On a separate note, it was also
interesting that the H4 peptide (V61K78) was missing in the thrombin-
and plasmin-treated NETs, which may indicate that the proteases
targeted this region (Fig. 3.5C). It is also interesting to note that plasmin
appears more proteolytically active against histones than thrombin (Fig.
3.4B-F). One possibility is the stronger interaction of thrombin with
DNA/NETs, hence limiting its capacity to proteolyse histones. However,
this is unlikely as its activity against recombinant histones was
considerably weaker versus plasmin (Fig. 3.4F). The other possibility
could be the difference in enzymatic activities between the proteases
against similar targets which is not examined here or previously described
to the best of our knowledge. Drawing from the previous speculation on
their interactions with DNA/NETs and the possible implications however,
their enzymatic specificities and activities against NET-bound proteins
could play complementary roles as well. Considering that LL37 plays
protective role on NETs against nuclease digestion206 and that structural
integrity of NETs is important for its antimicrobial effects140, thrombin’s
interaction with DNA/NETs may confer additional protection. It is also
possible that TCPs can be generated due to the proteolytic pressure
contributed by neutrophils’ proteases, which can then bind to NETs.
Conversely, plasmin’s stronger proteolytic activities may increase NETs
susceptibility to nuclease digestion allowing for proper clearance during
the healing process. Indeed, dysregulated clearance of NETs have been
implicated in autoimmune diseases120,261,286,287, although whether active
142
plasmin (or lack thereof) plays any role is unclear. It is hence important
for future studies to address the interactions between temporal proteases
and NETs, particularly their implications in wounds and other diseases.
Formation of NETs can be triggered by various stimuli, including chemical
inducers such as PMA and calcium ionophores, but also bacteria, fungi
and activated platelets101,104,108,139,275,288-290. Consequently, different
stimuli may adopt different NETosis pathways (as described in Chapter
1.4.4). Therefore, in order to focus on the concept that proteases may
influence the proteome of pre-formed NETs, we selected PMA as the
standard NET-inducing agent. Whether thrombin or plasmin induces
NETosis was not addressed in this study. In this context, it is worth
mentioning that it has been observed that thrombin can induce neutrophil
chemotaxis and aggregation at sub-micromolar concentrations, although
the chemotactic effect was not observed at 1 μM of thrombin291. Moreover,
plasmin has been shown to cause neutrophil aggregation and adhesion
to endothelial surface in vitro at sub-micromolar concentrations292,293,
albeit conflicting evidence exist concerning its adhesion-inducing
effects294. In addition, Ryan et al (1992) did not observe neutrophil lysis
after treatment with plasmin292. Although outside the scope of the present
study, further studies are warranted in order to study the proteases’
possible effects per se on NET formation.
The reduced histone levels on NETs may affect the overall function of
NETs. Histones are antimicrobial295 and contribute to NET-mediated
143
antimicrobial activity101, hence their reduction on NETs may decrease
NET-mediated antimicrobial effects. Simultaneously, levels of other NET-
associated antimicrobial proteins such as defensins and azurocidin were
also modulated (Suppl. Fig. S3.5A), potentially affecting the antimicrobial
effects of NETs. NET-bound or released histones are cytotoxic. Apart
from cellular effects, DNA and/or histones have also been reported to
promote thrombin generation, and cause thrombosis in vivo, in a platelet-
independent or -dependent manner168,296,297. Furthermore, histones can
stabilise clots by increasing fibrin’s resistance to fibrinolysis298. Therefore,
these evidences imply that exogenous proteases, such as thrombin or
plasmin, can modulate NET effects in various physiological contexts.
Interestingly, NET-associated ELANE was also reduced in the presence
of thrombin and plasmin, and several tryptic peptides, present in the
untreated samples, were not detected after subjection to the two
proteases (Fig. 3.6 and Suppl. Table S3.1). Notably, two tryptic peptides
– G51R78 and Q194R220 – occupying 2 of the 3 active sites of
ELANE were missing from at least 2 donors’ samples (Fig. 3.6C and
Suppl. Table S3.1). Intriguingly, It has been reported that murine ELANE
can auto-proteolyse a conserved region near its S1 pocket299, which
corresponds to the peptide Q194R220 occupying the S1 pocket of
human ELANE300. Hence, although speculative, it is possible that
thrombin and plasmin displaces ELANE from NETs thus allowing ELANE
to undergo auto-proteolysis, alternatively it is directly proteolysed by
thrombin and plasmin, as observed for the histones. However, the
144
involvement of other regulatory mechanisms following thrombin and
plasmin addition resulting in the observed reduction of NET-associated
proteins (i.e. ELANE and histones) cannot be ruled out and require further
investigations.
There is a growing interest in studying the NETome under different
inflammatory conditions and their implications. A recent study on NETs
induced by MSU shows that the process of NETosis is ROS-independent
and that NETs are coated with actin, which protects them from nuclease
degradation112. A separate study shows that saliva contained NETs and
could induce NETosis in a ROS-independent manner. Moreover, these
NETs were more nuclease-resistant and could trap bacteria more
effectively than those induced by PMA, although it was not shown whether
other salivary components conferred these effects as such301. Conversely,
NETosis induced by different strains of P. aeruginosa was shown to be
ROS-dependent and lead to generation of NET-proteins similar to those
reported previously (bacterial proteins were however excluded from
analysis)275. Taken together, these reports, as well as our findings,
illustrate that the NETs are dynamic scaffolds and further studies are
merited in order to elucidate the temporal and qualitative changes during
inflammatory-infective states. Knowledge of the dynamic changes of NET
components induced by different stimuli of relevance for different
pathological states could possibly be exploited in the future search for
new disease biomarkers.
145
3.6 Acknowledgements
This study is supported by the Lee Kong Chian School of Medicine,
Nanyang Technological University Singapore Start-Up Grant and the
Singapore Ministry of Education under its Singapore Ministry of Education
Academic Research Fund Tier 1 (2015-T1-001-082). C.H.L. is supported
by the Interdisciplinary Graduate School, Nanyang Technological
University, Singapore. R.S. is supported by the Lee Kong Chian School
of Medicine Post-Doctoral Fellowship 2014 (L0491020).
3.7 Author contributions
C.H.L., S.A. and A.S. participated in the planning, design and
interpretation of experiments, results and validation strategies. C.H.L.
prepared the NETs samples and S.A. further processed them for LC-
MS/MS analysis. S.K.S. provided critical inputs for the LC-MS/MS data
analysis and the usage of data for further analysis. C.H.L. performed the
western blots and in silico analyses for the tryptic peptides detected and
identified. Y.K.C. did the gel mobility shift experiments. R.S. contributed
to parts on thrombin and plasmin interactions with DNA. C.H.L., S.A. and
A.S. wrote and reviewed the manuscript. All authors reviewed the
manuscript.
3.8 Conflict of interest
The authors declare no conflict of interest.
146
3.9 Supplementary Information
Supplementary Figure S3.1: GKY25 binds NETs / DNA. (A) Immunofluorescence
of PMA-induced NETs for 3 hrs at 37oC before addition of 5 μM TAMRA-labelled
GKY25 for 1 hr at 37oC. Scale bar = 10 µM. (B-E) Indicated concentrations of GKY25
were first incubated with approximately 2 μg/ml purified DNA for 30 min, followed by
the addition of (B,D) buffer or (C,E) 0.2 U/ml MNase for another 15 min, and a final
incubation with 0.25 mg/ml Proteinase K for 90 min, as indicated. (B,C) A total of 100
ng of DNA was used for gel electrophoresis. (D,E) Fluorescence intensity of 50 nM
Sytox Green with 1 µg/ml DNA, with or without GKY25 as above, was measured. n =
3 independent experiments. Two-way ANOVA.
147
Supplementary Figure S3.2: Data
processing workflow for the
identification of NET-associated
proteins. Peptides spectra were
subjected to Mascot search with the
specified criteria and FDR for
identification. The identified proteins
were first assessed for their presence
in at least 1 treatment condition (i.e.
untreated, thrombin- or plasmin-
treated), followed by whether they
were identified in at least 2 donors
within the same treatment condition.
This initial screening further rules out
proteins that might have been non-
specifically identified. Then, emPAI
values of -DNase samples were
subtracted from +DNase samples to
further filter out proteins that might
have been constitutively released
from cell membrane / debris or wells
during DNase I treatment. If the
emPAI score is equal or less than 0,
the protein is not considered as NET-
bound. Positive emPAI scores (>0)
from at least 2 donors were then
considered as NET-associated and
used for further analyses.
148
Supplementary Figure S3.3: Correlation analysis plots for reproducibility
assessment. Linear correlation plots were generated when comparing data between
the 3 donors and their respective treatments – untreated (left column), thrombin-
treated (middle column) and plasmin-treated (right column). Pearson’s correlation
coefficient (r2) was applied.
149
Supplementary Figure S3.4: Comparison between +DNase and -DNase samples.
The emPAI scores for +DNase and -DNase samples of (A) thrombin-treated and (B)
plasmin-treated samples were plotted to show that there was a general increase in
detection following DNase I treatments.
Supplementary Figure S3.5: hierarchical clusters of NET-associated proteins.
Segregated view of the hierarchical clusters – (A) proteins enriched in untreated
samples (cluster C3 and C5), (B) proteins enriched in thrombin-treated samples
(cluster C1, C2 and C4) and (C) proteins enriched in plasmin-samples (cluster C6).
Enriched proteins are displayed in red, lowered proteins are displayed in blue and the
intermediate values are displayed in the shades of red and blue.
150
Supplementary Table 3.1: Tryptic peptides of neutrophil elastase (ELANE).
Summed peptide scores for each peptide from the respective samples are presented.
The value = 0 denotes that the peptide was not identified.
151
– CHAPTER 4 –
Significance, future work and conclusion
152
153
4 Significance, future work and conclusions
We have shown in this work that neutrophils can be modulated by
thrombin and its derived HDPs, which may consequently affect
downstream immune and homeostatic activities. Using TCPs as a novel
immunomodulator, we demonstrate the possibility of down-modulating
neutrophil functions by attenuating its physiological recruitment,
chemotaxis and functional responses. On NETs, we provide proof-of-
concept evidence that non-neutrophil derived proteins not only can bind
to NETs, they can also influence proteomic changes on the NETome.
4.1 TCPs: balance between therapy, immunomodulation and host
evasion
With respect to TCPs, studies on the prototypic GKY25 have provided
evidence on its potential as a novel anti-infective and immunomodulator.
Like other cationic HDPs, we together with others have shown that
GKY25 can effectively inhibit LPS-elicited inflammatory responses by
innate immune cells in vitro and in vivo87,181,182,215,216. In addition, when
infected with P. aeruginosa, mice treated with GKY25 had increased
survivability87,181, probably by the killing of the bacteria or modulating the
inflammatory responses, or both. In addition, GKY25 can modulate innate
immune cells independently, such as the possible attenuation of
neutrophil recruitment and functional response216. These studies highlight
the potential in using GKY25 as a therapeutic for such inflammatory
conditions.
154
Using GKY25 as a therapeutic though has its challenge. Till date, not
many HDPs or AMPs are available in the market, while some are clinically
trialled302. The most relevant example is polymyxin B and polymyxin E
(also known as colistin) which are used as the last line-of-defence against
multi-drug resistant (MDR) Gram negative bacterial infections. While
reviews in the late 2000s found that the clinical usage of polymyxins for
such cases were largely effective303,304, concerns were also raised
regarding their nephrotoxic and neurotoxic effects following intravenous
administrations304,305. This is of particular relevance as we have shown
that GKY25 can be cytotoxic at higher concentrations in vitro216, indicating
that dosing regimens must be studied in detail if there is any prospective
use of GKY25 for systemic applications.
Several AMPs are now clinically trialled with the objective to control
infections. Of interest are those indicated for wound treatments, such as
Pexiganan in phase III trials for infected diabetic foot ulcers and LL37 in
phase I and II trials for chronic venous leg ulcers. Lytixar, a synthetic
peptidomimetic, is also trialled for uncomplicated Gram-positive bacterial
skin infections and impetigo, a skin condition commonly caused by Gram-
positive bacteria such as S. aureus. As observed, many of these clinically
trialled AMPs’ are restricted to topical applications, hence bypassing the
circulatory, hepatic and renal systems which will significantly reduce their
half-lives through proteolysis by blood proteases or clearance from the
system. Even topical applications of AMPs, especially in wounds, are
subjected to the highly-proteolytic environments166,262. Hence,
155
overcoming this limitation remains high on the agenda for peptide
research and applications302.
To this end, strategies have been developed to improve AMPs’ stability
or reduce their susceptibility to enzymatic digestions. For example,
cyclization of the AMPs, replacement of L-amino acids with D-amino acids
and N- or C-terminal modifications have all been shown to confer AMPs’
increased resistance to proteolysis. Novel modes of delivery such as bio-
compatible / -degradable nanocarriers have also shown to be effective in
vitro and in preclinical models, thus preceding interests for wider
investigations302. Peptide hydrocarbon stapling is also a viable way to
reduce their proteolysis, especially for longer-chained peptides306. More
extensive investigations on these methods will surely present insights and
opportunities on the therapeutic applications of AMPs.
With respect to GKY25, as a relatively new player as a novel anti-
infective, it has been shown to exhibit effective anti-bacterial, anti-
endotoxic and immunomodulatory potentials87,181,182,215,216. However,
more can and must be done to assess its stability and feasibility of use.
Addressing the said challenges in using AMPs as therapeutics will greatly
enhance GKY25’s use as treatments in wounds and various inflammatory
conditions. Compounded by the fact that antibiotic resistance is becoming
more prevalent, AMPs offer attractive alternatives for their broad-
spectrum antimicrobial effects, specificity for bacteria versus mammalian
cells, low immunogenicity and low chance of bacteria developing
156
resistance against them302. In the case of GKY25, its ability to exert
effective antimicrobial pressure and downplay immune response and
inflammation might facilitate effective healing. Their immunomodulatory
potentials can also be extradited for use as prophylactics, such as before
surgical procedures where bacterial infections or endotoxins
dissemination is likely.
Separately, we have preliminarily assessed 2 truncated TCPs’
immunomodulatory effects on PMNs. We show that FYT21, the
predominant TCP found in P. aeruginosa-infected wounds, effectively
inhibits LPS and induces shedding of the surface adhesion molecule
CD62L. Contrarily, HVF18, the TCP which can be derived following
thrombin proteolysis with neutrophil elastase, did not have any LPS
inhibitory or neutrophil modulatory effects at physiologically-relevant
concentrations. Recent report shows that supplementation of elastase-
deficient P. aeruginosa conditioned medium with P. aeruginosa elastase
reduced blood inflammatory cytokines when injected in mice, suggesting
that FYT21 might be produced in the presence of the elastase to
attenuate said responses88. However, there was no evidence on the
survivability of the bacteria in the absence of elastase or FYT21 in the
context of wound infections or other infective conditions, hence such
evasive strategy remains speculative. Nevertheless, there are evidence
to support the claim that proteases produced by P. aeruginosa are
virulence factors307. Therefore, studies on the host-pathogen interactions
with the bacteria, the relevance of the elastase and/or FYT21 produced
157
in relation to the other virulence factors, and thus how we can target them
will greatly improve our understanding and development of new
therapeutic strategies against P. aeruginosa-mediated complications.
FYT21 or P. aeruginosa elastase, as a virulence factor per se, can add a
layer of complexity in the application of GKY25 for intended conditions,
especially if P. aeruginosa is involved. Certain considerations such as
whether the doses applied can eradicate the infecting bacteria sufficiently,
the peptide’s stability against the bacteria’s elastase, and maintenance of
its immunomodulatory effects must be addressed. For example, the
untimely down-regulation of immune responses despite insufficient
clearance of the bacteria might worsen the condition, thus complicating
such conditions. Hence, as mentioned, understanding the host-pathogen
interactions and perhaps in relation to GKY25 can certainly provide
valuable information in assessing its feasibility of use under various
conditions.
4.2 NETs: trapping biomarkers?
Conceptually, we have provided evidence on the possible regulation on
the proteome of NETs (or NETome) by external factors, such as the
proteases thrombin and plasmin. Most significantly, major NET proteins
such as histones, ELANE, MPO and azurocidin were reduced in the
presence of either protease267. The immediate implication is the relevance
of previous studies with respect to NET-mediated antibacterial properties,
NET quantification methods based on its enzymatic activities and the
158
physiological relevance of NETs. Pertaining to the first concern, the fact
that NETs are decorated with a myriad of antimicrobial proteins makes
the idea that NETs are therefore antimicrobial tempting. However,
whether NETs are microbicidal or merely trap microbes to prevent their
growth and dissemination is still being debated123,143. Nevertheless,
Brinkman et al (2004) report that histones contributed to the antimicrobial
properties of NETs101. Subsequently, studies attempting to identify or
attribute certain proteins or peptides for NET-mediated antimicrobial
effects surfaced, describing direct or indirect (such as in
phagolysosomes) antimicrobial effects138,139,265,308. Based on our study
where many of these major NET-associated antimicrobial proteins were
reduced in the presence of physiological concentrations of thrombin or
plasmin267, we question the relevance of in vitro methods to assess NET-
mediated antimicrobial properties. This is especially so as majority of such
studies were performed in culture medium or buffer alone in the absence
of serum. Therefore, more extensive studies accounting for the related
physiological and inflammatory settings are mandated to accurately
elucidate the antimicrobial effects of NETs.
There are now several methods to quantify NETs, either based on DNA
content, fluorescence image analysis, flow cytometry, NET-protein
complexes and enzymatic activity of NET-associated proteins309-311. With
respect to methods targeting NET-associated proteins, either by
immunostaining or assessing their enzymatic activities, it must be noted
now that they can be dynamically regulated by external factors267,
159
particularly when we consider in vivo settings. Hence, when using such
quantification methods for ex vivo samples, one must scrutinise and
consider its limitations before drawing any conclusions. Complementary
methods may be warranted for more accurate determination of NETs or
NETosis.
On the other hand, knowing that the NETome can be dynamically
modulated presents new opportunities to translate them into clinical
applications in the form of biomarkers. For example, ELANE has been
implicated in the worsening of the disease and is a therapeutic target for
CF312,313, yet there is evidence to show that exogenous ELANE can
synergise with DNase to digest the DNA / NETs to solubilise the
sputum314, hence the pathogenicity of ELANE remains to be further
investigated. Additionally, it is note-worthy that ELANE’s activity is
reduced when bound to DNA315. Furthermore, auto-proteolysed ELANE
was also identified in the sputum of CF patients299, suggesting some
forms of regulation involved which might affect the protease’s
pathogenicity or pro-resolving effects. Intriguingly, although not
conclusive, our study suggests that ELANE might be regulated in a more
complex manner, possibly through auto-proteolysis as well267. Therefore,
with sufficient and relevant investigations on the activity and binding
status of ELANE, this information can be useful to assess the patients’
conditions or predict treatment effectivity. Likewise, for other conditions
where NETs are involved, understanding the proteomic changes and their
160
physiological implications can enrich our knowledge in targeting NETs for
various diseases.
In relation to wounds, the proteome of NETs might be regulated
differently. For example, even in the study of the NETome induced by P.
aeruginosa, bacterial products which could potentially bind directly or
indirectly to NETs were excluded from analysis275. In the case of wounds
where they are constantly exposed to bacterial assaults, knowledge on
whether bacteria or bacterial products can interact with NETs will be
useful to understand the infective states of wounds. Furthermore, the
highly complex proteolytic environment possibly further contributed by
pathogens offers a wealth of opportunities to discover novel
biomarkers166,262. For example, P. aeruginosa elastase can proteolyse
thrombin to form the 21-amino acid C-terminal peptide FYT2188.
Furthermore, we show that the prototypic TCP GKY25 and thrombin can
bind to NETs (Supp. Fig. S3.1 and Fig. 3.3), indicating that the
endogenous truncations HVF18 and FYT21 could also associate with
NETs. Hence, proteo- / peptidomics analyses of the NETome might reveal
unique patterns depending on the infective states and possibly healing
stages of different wounds.
To this end, there are existing studies now to compare and assess wound
states via the proteomics approach262,263,316. Several factors are enriched
or specific for certain types or stages of wounds (e.g. healing vs. non-
healing), such as the enrichment of thrombin and SerpinA1 in healing
161
wounds and significant abundance of neutrophil-derived proteins in non-
healing ulcers263. With these data, the present challenge is to define a set
of proteins which is dysregulated when comparing non-healing wounds
against ‘healthy’ and healing wounds. This is an important process as
biological samples are complex, protein-rich materials and thus,
proteomics analysis is not straight-forward. Identification of specific
proteins that are dysregulated in non-healing wounds can aid in the
creation of devices or assays to rapidly examine and identify specific
proteins from the collected biological samples. Here, the potential of NETs
to ‘trap’ biomarkers may hence help to concentrate these proteins on a
relatively easy-to-isolate biological material. Henceforth, proteomic
assessment of the isolated NETs from different types of wounds can be
conducted.
The caveat with using NETs as a scavenging mesh for biomarkers is
whether sufficient NETs can be isolated from wound exudates or wound
beds. Then, the amounts of NETs produced and are present in wounds
must be investigated to assess the feasibility of such approaches. There
is also the limitation of missing the non-NET binding proteins which of
course can provide important information as well. Therefore, studies to
address these clinical and experimental issues must be conducted.
Nevertheless, examination on the NETome, particularly those isolated
from wounds or other inflammatory conditions, can add on to the current
knowledge pertaining to their functions in health and diseases, as well as
their relevance as diagnostic or prognostic markers.
162
4.3 Concluding remarks
These studies exhibit the multifaceted functions of host-derived products
and a wealth of information which can potentially be utilised to study host-
pathogen interactions, design therapeutics and discover biomarkers. With
a focus on wounds and innate immunity, we present evidence on the
complex interactions between and within systems. Nonetheless, further
studies can be done to more comprehensively define and assess their
uses in the development of therapeutics or discovery of biomarkers.
These studies therefore provide fundamental indications on which future
hypotheses and investigations can base on.
163
164
165
5 References
1 Kolarsick, P. A. J., Kolarsick, M. A. & Goodwin, C. Anatomy and Physiology of the Skin. Journal of the Dermatology Nurses' Association 3, 203-213, doi:10.1097/JDN.0b013e3182274a98 (2011).
2 Albanna, M. Z. & Holmes, J. H. Skin tissue engineering and regenerative medicine. (Elsevier/AP, Academic Press is an imprint of Elsevier, 2016).
3 Leaper, D. J. Traumatic and surgical wounds. BMJ 332, 532-535, doi:10.1136/bmj.332.7540.532 (2006).
4 Belin, R. & Carrington, S. Management of pedal puncture wounds. Clin Podiatr Med Surg 29, 451-458, doi:10.1016/j.cpm.2012.01.009 (2012).
5 Agale, S. V. Chronic Leg Ulcers: Epidemiology, Aetiopathogenesis, and Management. Ulcers 2013, 1-9, doi:10.1155/2013/413604 (2013).
6 Church, D., Elsayed, S., Reid, O., Winston, B. & Lindsay, R. Burn wound infections. Clin Microbiol Rev 19, 403-434, doi:10.1128/CMR.19.2.403-434.2006 (2006).
7 Schaefer, T. J. & Szymanski, K. D. in StatPearls (2018). 8 Sen, C. K. et al. Human skin wounds: a major and snowballing threat to
public health and the economy. Wound Repair Regen 17, 763-771, doi:10.1111/j.1524-475X.2009.00543.x (2009).
9 Manorama, A. A., Baek, S., Vorro, J., Sikorskii, A. & Bush, T. R. Blood perfusion and transcutaneous oxygen level characterizations in human skin with changes in normal and shear loads--implications for pressure ulcer formation. Clin Biomech (Bristol, Avon) 25, 823-828, doi:10.1016/j.clinbiomech.2010.06.003 (2010).
10 Grey, J. E., Harding, K. G. & Enoch, S. Venous and arterial leg ulcers. BMJ 332, 347-350, doi:10.1136/bmj.332.7537.347 (2006).
11 Irving, G. & Hargreaves, S. Venous and Arterial Leg Ulceration. InnovAiT: Education and inspiration for general practice 2, 415-422, doi:10.1093/innovait/inn183 (2009).
12 Pendsey, S. P. Understanding diabetic foot. Int J Diabetes Dev Ctries 30, 75-79, doi:10.4103/0973-3930.62596 (2010).
13 Wan He, D. G., Paul Kowal. An Aging World: 2015. U.S. Census Bureau, International Population Reports P95/16-1 (2016).
14 World Health Organization: Obesity and overweight, 2017). 15 Kelly, T., Yang, W., Chen, C. S., Reynolds, K. & He, J. Global burden of
obesity in 2005 and projections to 2030. Int J Obes (Lond) 32, 1431-1437, doi:10.1038/ijo.2008.102 (2008).
16 Ban, K. A. et al. American College of Surgeons and Surgical Infection Society: Surgical Site Infection Guidelines, 2016 Update. J Am Coll Surg 224, 59-74, doi:10.1016/j.jamcollsurg.2016.10.029 (2017).
17 Velnar, T., Bailey, T. & Smrkolj, V. The wound healing process: an overview of the cellular and molecular mechanisms. J Int Med Res 37, 1528-1542, doi:10.1177/147323000903700531 (2009).
18 Gurtner, G. C., Werner, S., Barrandon, Y. & Longaker, M. T. Wound repair and regeneration. Nature 453, 314-321, doi:10.1038/nature07039 (2008).
19 Wolberg, A. S. & Campbell, R. A. Thrombin generation, fibrin clot formation and hemostasis. Transfus Apher Sci 38, 15-23, doi:10.1016/j.transci.2007.12.005 (2008).
166
20 de Oliveira, S., Rosowski, E. E. & Huttenlocher, A. Neutrophil migration in infection and wound repair: going forward in reverse. Nat Rev Immunol 16, 378-391, doi:10.1038/nri.2016.49 (2016).
21 Nauseef, W. M. & Borregaard, N. Neutrophils at work. Nat Immunol 15, 602-611, doi:10.1038/ni.2921 (2014).
22 Wynn, T. A. & Vannella, K. M. Macrophages in Tissue Repair, Regeneration, and Fibrosis. Immunity 44, 450-462, doi:10.1016/j.immuni.2016.02.015 (2016).
23 Landen, N. X., Li, D. & Stahle, M. Transition from inflammation to proliferation: a critical step during wound healing. Cell Mol Life Sci 73, 3861-3885, doi:10.1007/s00018-016-2268-0 (2016).
24 Minutti, C. M., Knipper, J. A., Allen, J. E. & Zaiss, D. M. Tissue-specific contribution of macrophages to wound healing. Semin Cell Dev Biol 61, 3-11, doi:10.1016/j.semcdb.2016.08.006 (2017).
25 Kolaczkowska, E. & Kubes, P. Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol 13, 159-175, doi:10.1038/nri3399 (2013).
26 Arinobu, Y. et al. Reciprocal activation of GATA-1 and PU.1 marks initial specification of hematopoietic stem cells into myeloerythroid and myelolymphoid lineages. Cell Stem Cell 1, 416-427, doi:10.1016/j.stem.2007.07.004 (2007).
27 Radomska, H. S. et al. CCAAT/enhancer binding protein alpha is a regulatory switch sufficient for induction of granulocytic development from bipotential myeloid progenitors. Mol Cell Biol 18, 4301-4314 (1998).
28 Dale, D. C. et al. Long-Term Effects of G-CSF Therapy in Cyclic Neutropenia. N Engl J Med 377, 2290-2292, doi:10.1056/NEJMc1709258 (2017).
29 Borregaard, N. & Cowland, J. B. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 89, 3503-3521 (1997).
30 Lominadze, G. et al. Proteomic analysis of human neutrophil granules. Mol Cell Proteomics 4, 1503-1521, doi:10.1074/mcp.M500143-MCP200 (2005).
31 Semerad, C. L., Liu, F., Gregory, A. D., Stumpf, K. & Link, D. C. G-CSF is an essential regulator of neutrophil trafficking from the bone marrow to the blood. Immunity 17, 413-423 (2002).
32 Wengner, A. M., Pitchford, S. C., Furze, R. C. & Rankin, S. M. The coordinated action of G-CSF and ELR + CXC chemokines in neutrophil mobilization during acute inflammation. Blood 111, 42-49, doi:10.1182/blood-2007-07-099648 (2008).
33 Martin, C. et al. Chemokines acting via CXCR2 and CXCR4 control the release of neutrophils from the bone marrow and their return following senescence. Immunity 19, 583-593 (2003).
34 Eash, K. J., Means, J. M., White, D. W. & Link, D. C. CXCR4 is a key regulator of neutrophil release from the bone marrow under basal and stress granulopoiesis conditions. Blood 113, 4711-4719, doi:10.1182/blood-2008-09-177287 (2009).
35 Eash, K. J., Greenbaum, A. M., Gopalan, P. K. & Link, D. C. CXCR2 and CXCR4 antagonistically regulate neutrophil trafficking from murine bone marrow. J Clin Invest 120, 2423-2431, doi:10.1172/JCI41649 (2010).
36 Suratt, B. T. et al. Role of the CXCR4/SDF-1 chemokine axis in circulating neutrophil homeostasis. Blood 104, 565-571, doi:10.1182/blood-2003-10-3638 (2004).
167
37 Theilgaard-Monch, K. et al. The transcriptional program of terminal granulocytic differentiation. Blood 105, 1785-1796, doi:10.1182/blood-2004-08-3346 (2005).
38 Simon, S. I., Hu, Y., Vestweber, D. & Smith, C. W. Neutrophil tethering on E-selectin activates beta 2 integrin binding to ICAM-1 through a mitogen-activated protein kinase signal transduction pathway. J Immunol 164, 4348-4358 (2000).
39 Faurschou, M. & Borregaard, N. Neutrophil granules and secretory vesicles in inflammation. Microbes and Infection 5, 1317-1327, doi:10.1016/j.micinf.2003.09.008 (2003).
40 McHale, J. F., Harari, O. A., Marshall, D. & Haskard, D. O. TNF-alpha and IL-1 sequentially induce endothelial ICAM-1 and VCAM-1 expression in MRL/lpr lupus-prone mice. J Immunol 163, 3993-4000 (1999).
41 Sun, S. et al. Mitochondrial DAMPs increase endothelial permeability through neutrophil dependent and independent pathways. PLoS One 8, e59989, doi:10.1371/journal.pone.0059989 (2013).
42 Fiuza, C. et al. Inflammation-promoting activity of HMGB1 on human microvascular endothelial cells. Blood 101, 2652-2660, doi:10.1182/blood-2002-05-1300 (2003).
43 Yan, W. et al. Role of p38 MAPK in ICAM-1 expression of vascular endothelial cells induced by lipopolysaccharide. Shock 17, 433-438 (2002).
44 Middleton, J. et al. Transcytosis and surface presentation of IL-8 by venular endothelial cells. Cell 91, 385-395 (1997).
45 Lundahl, J., Jacobson, S. H. & Paulsson, J. M. IL-8 from local subcutaneous wounds regulates CD11b activation. Scand J Immunol 75, 419-425, doi:10.1111/j.1365-3083.2012.02679.x (2012).
46 DiVietro, J. A. et al. Immobilized IL-8 triggers progressive activation of neutrophils rolling in vitro on P-selectin and intercellular adhesion molecule-1. J Immunol 167, 4017-4025 (2001).
47 Borregaard, N. et al. Changes in subcellular localization and surface expression of L-selectin, alkaline phosphatase, and Mac-1 in human neutrophils during stimulation with inflammatory mediators. J Leukoc Biol 56, 80-87 (1994).
48 Steeber, D. A. et al. Leukocyte entry into sites of inflammation requires overlapping interactions between the L-selectin and ICAM-1 pathways. J Immunol 163, 2176-2186 (1999).
49 Boueiz, A. & Hassoun, P. M. Regulation of endothelial barrier function by reactive oxygen and nitrogen species. Microvasc Res 77, 26-34, doi:10.1016/j.mvr.2008.10.005 (2009).
50 Royall, J. A. et al. Tumor necrosis factor and interleukin 1 alpha increase vascular endothelial permeability. Am J Physiol 257, L399-410, doi:10.1152/ajplung.1989.257.6.L399 (1989).
51 Wolfson, R. K., Chiang, E. T. & Garcia, J. G. HMGB1 induces human lung endothelial cell cytoskeletal rearrangement and barrier disruption. Microvasc Res 81, 189-197, doi:10.1016/j.mvr.2010.11.010 (2011).
52 Nooteboom, A., Hendriks, T., Otteholler, I. & van der Linden, C. J. Permeability characteristics of human endothelial monolayers seeded on different extracellular matrix proteins. Mediators Inflamm 9, 235-241, doi:10.1080/09629350020025755 (2000).
168
53 Zhang, H. & Sun, G. Y. LPS induces permeability injury in lung microvascular endothelium via AT(1) receptor. Arch Biochem Biophys 441, 75-83, doi:10.1016/j.abb.2005.06.022 (2005).
54 Uhlig, S. et al. Differential regulation of lung endothelial permeability in vitro and in situ. Cell Physiol Biochem 34, 1-19, doi:10.1159/000362980 (2014).
55 Schulman, C. I., Wright, J. K., Nwariaku, F., Sarosi, G. & Turnage, R. H. The effect of tumor necrosis factor-alpha on microvascular permeability in an isolated, perfused lung. Shock 18, 75-81 (2002).
56 Li, J. L. et al. Neutrophils Self-Regulate Immune Complex-Mediated Cutaneous Inflammation through CXCL2. J Invest Dermatol 136, 416-424, doi:10.1038/JID.2015.410 (2016).
57 Fujishima, S. et al. Regulation of neutrophil interleukin 8 gene expression and protein secretion by LPS, TNF-alpha, and IL-1 beta. J Cell Physiol 154, 478-485, doi:10.1002/jcp.1041540305 (1993).
58 Afonso, P. V. et al. LTB4 is a signal-relay molecule during neutrophil chemotaxis. Dev Cell 22, 1079-1091, doi:10.1016/j.devcel.2012.02.003 (2012).
59 Chou, R. C. et al. Lipid-cytokine-chemokine cascade drives neutrophil recruitment in a murine model of inflammatory arthritis. Immunity 33, 266-278, doi:10.1016/j.immuni.2010.07.018 (2010).
60 Ramos, C. D. et al. Neutrophil recruitment in immunized mice depends on MIP-2 inducing the sequential release of MIP-1alpha, TNF-alpha and LTB(4). Eur J Immunol 36, 2025-2034, doi:10.1002/eji.200636057 (2006).
61 Lammermann, T. et al. Neutrophil swarms require LTB4 and integrins at sites of cell death in vivo. Nature 498, 371-375, doi:10.1038/nature12175 (2013).
62 Borregaard, N., Sorensen, O. E. & Theilgaard-Monch, K. Neutrophil granules: a library of innate immunity proteins. Trends Immunol 28, 340-345, doi:10.1016/j.it.2007.06.002 (2007).
63 Sengelov, H., Kjeldsen, L. & Borregaard, N. Control of exocytosis in early neutrophil activation. J Immunol 150, 1535-1543 (1993).
64 Sengelov, H. et al. Mobilization of granules and secretory vesicles during in vivo exudation of human neutrophils. J Immunol 154, 4157-4165 (1995).
65 Mollinedo, F., Martin-Martin, B., Calafat, J., Nabokina, S. M. & Lazo, P. A. Role of vesicle-associated membrane protein-2, through Q-soluble N-ethylmaleimide-sensitive factor attachment protein receptor/R-soluble N-ethylmaleimide-sensitive factor attachment protein receptor interaction, in the exocytosis of specific and tertiary granules of human neutrophils. J Immunol 170, 1034-1042 (2003).
66 Mollinedo, F. et al. Combinatorial SNARE complexes modulate the secretion of cytoplasmic granules in human neutrophils. J Immunol 177, 2831-2841 (2006).
67 Schroeder, H. W., Jr. & Cavacini, L. Structure and function of immunoglobulins. J Allergy Clin Immunol 125, S41-52, doi:10.1016/j.jaci.2009.09.046 (2010).
68 Ghiran, I. et al. Complement receptor 1/CD35 is a receptor for mannan-binding lectin. J Exp Med 192, 1797-1808 (2000).
69 Futosi, K., Fodor, S. & Mocsai, A. Neutrophil cell surface receptors and their intracellular signal transduction pathways. Int Immunopharmacol 17, 638-650, doi:10.1016/j.intimp.2013.06.034 (2013).
169
70 Dunkelberger, J. R. & Song, W. C. Complement and its role in innate and adaptive immune responses. Cell Res 20, 34-50, doi:10.1038/cr.2009.139 (2010).
71 Gordon, S. Phagocytosis: An Immunobiologic Process. Immunity 44, 463-475, doi:10.1016/j.immuni.2016.02.026 (2016).
72 Lee, W. L., Harrison, R. E. & Grinstein, S. Phagocytosis by neutrophils. Microbes Infect 5, 1299-1306 (2003).
73 Johnson, M. B. & Criss, A. K. Neisseria gonorrhoeae phagosomes delay fusion with primary granules to enhance bacterial survival inside human neutrophils. Cell Microbiol 15, 1323-1340, doi:10.1111/cmi.12117 (2013).
74 Goren, M. B., D'Arcy Hart, P., Young, M. R. & Armstrong, J. A. Prevention of phagosome-lysosome fusion in cultured macrophages by sulfatides of Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 73, 2510-2514 (1976).
75 Mayadas, T. N., Cullere, X. & Lowell, C. A. The multifaceted functions of neutrophils. Annu Rev Pathol 9, 181-218, doi:10.1146/annurev-pathol-020712-164023 (2014).
76 Jankowski, A., Scott, C. C. & Grinstein, S. Determinants of the phagosomal pH in neutrophils. J Biol Chem 277, 6059-6066, doi:10.1074/jbc.M110059200 (2002).
77 Perskvist, N., Roberg, K., Kulyte, A. & Stendahl, O. Rab5a GTPase regulates fusion between pathogen-containing phagosomes and cytoplasmic organelles in human neutrophils. J Cell Sci 115, 1321-1330 (2002).
78 Rada, B. & Leto, T. L. Oxidative innate immune defenses by Nox/Duox family NADPH oxidases. Contrib Microbiol 15, 164-187, doi:10.1159/000136357 (2008).
79 Belaaouaj, A., Kim, K. S. & Shapiro, S. D. Degradation of outer membrane protein A in Escherichia coli killing by neutrophil elastase. Science 289, 1185-1188 (2000).
80 Belaaouaj, A. et al. Mice lacking neutrophil elastase reveal impaired host defense against gram negative bacterial sepsis. Nat Med 4, 615-618 (1998).
81 Standish, A. J. & Weiser, J. N. Human neutrophils kill Streptococcus pneumoniae via serine proteases. J Immunol 183, 2602-2609, doi:10.4049/jimmunol.0900688 (2009).
82 Bangalore, N., Travis, J., Onunka, V. C., Pohl, J. & Shafer, W. M. Identification of the primary antimicrobial domains in human neutrophil cathepsin G. J Biol Chem 265, 13584-13588 (1990).
83 Shafer, W. M. et al. Tailoring an antibacterial peptide of human lysosomal cathepsin G to enhance its broad-spectrum action against antibiotic-resistant bacterial pathogens. Curr Pharm Des 8, 695-702 (2002).
84 Shafer, W. M., Hubalek, F., Huang, M. & Pohl, J. Bactericidal activity of a synthetic peptide (CG 117-136) of human lysosomal cathepsin G is dependent on arginine content. Infect Immun 64, 4842-4845 (1996).
85 Sorensen, O. E. et al. Human cathelicidin, hCAP-18, is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3. Blood 97, 3951-3959 (2001).
86 Nordahl, E. A. et al. Activation of the complement system generates antibacterial peptides. Proc Natl Acad Sci U S A 101, 16879-16884, doi:10.1073/pnas.0406678101 (2004).
170
87 Papareddy, P. et al. Proteolysis of human thrombin generates novel host defense peptides. PLoS Pathog 6, e1000857, doi:10.1371/journal.ppat.1000857 (2010).
88 van der Plas, M. J. et al. Pseudomonas aeruginosa elastase cleaves a C-terminal peptide from human thrombin that inhibits host inflammatory responses. Nat Commun 7, 11567, doi:10.1038/ncomms11567 (2016).
89 Papareddy, P. et al. Tissue factor pathway inhibitor 2 is found in skin and its C-terminal region encodes for antibacterial activity. PLoS One 7, e52772, doi:10.1371/journal.pone.0052772 (2012).
90 Stapels, D. A., Geisbrecht, B. V. & Rooijakkers, S. H. Neutrophil serine proteases in antibacterial defense. Curr Opin Microbiol 23, 42-48, doi:10.1016/j.mib.2014.11.002 (2015).
91 Nguyen, G. T., Green, E. R. & Mecsas, J. Neutrophils to the ROScue: Mechanisms of NADPH Oxidase Activation and Bacterial Resistance. Front Cell Infect Microbiol 7, 373, doi:10.3389/fcimb.2017.00373 (2017).
92 Abdel-Latif, D. et al. Rac2 is critical for neutrophil primary granule exocytosis. Blood 104, 832-839, doi:10.1182/blood-2003-07-2624 (2004).
93 Mitchell, T., Lo, A., Logan, M. R., Lacy, P. & Eitzen, G. Primary granule exocytosis in human neutrophils is regulated by Rac-dependent actin remodeling. Am J Physiol Cell Physiol 295, C1354-1365, doi:10.1152/ajpcell.00239.2008 (2008).
94 Nordenfelt, P., Winberg, M. E., Lonnbro, P., Rasmusson, B. & Tapper, H. Different requirements for early and late phases of azurophilic granule-phagosome fusion. Traffic 10, 1881-1893, doi:10.1111/j.1600-0854.2009.00986.x (2009).
95 Bennison LR, M. C., Summers RJ, Minnis AMB, Sussman G & McGuiness W. The pH of wounds during healing and infection: a descriptive literature review. Wound Practice and Research 25, 63-69 (2017).
96 Stein, J. M. & Luzio, J. P. Ectocytosis caused by sublytic autologous complement attack on human neutrophils. The sorting of endogenous plasma-membrane proteins and lipids into shed vesicles. Biochem J 274 ( Pt 2), 381-386 (1991).
97 Lorincz, A. M. et al. Functionally and morphologically distinct populations of extracellular vesicles produced by human neutrophilic granulocytes. J Leukoc Biol 98, 583-589, doi:10.1189/jlb.3VMA1014-514R (2015).
98 Timar, C. I. et al. Antibacterial effect of microvesicles released from human neutrophilic granulocytes. Blood 121, 510-518, doi:10.1182/blood-2012-05-431114 (2013).
99 Hess, C., Sadallah, S., Hefti, A., Landmann, R. & Schifferli, J. A. Ectosomes released by human neutrophils are specialized functional units. J Immunol 163, 4564-4573 (1999).
100 Dalli, J. et al. Heterogeneity in neutrophil microparticles reveals distinct proteome and functional properties. Mol Cell Proteomics 12, 2205-2219, doi:10.1074/mcp.M113.028589 (2013).
101 Brinkmann, V. et al. Neutrophil extracellular traps kill bacteria. Science 303, 1532-1535, doi:10.1126/science.1092385 (2004).
102 Hosseinzadeh, A., Messer, P. K. & Urban, C. F. Stable Redox-Cycling Nitroxide Tempol Inhibits NET Formation. Front Immunol 3, 391, doi:10.3389/fimmu.2012.00391 (2012).
171
103 Barrientos, L. et al. An improved strategy to recover large fragments of functional human neutrophil extracellular traps. Front Immunol 4, 166, doi:10.3389/fimmu.2013.00166 (2013).
104 Pilsczek, F. H. et al. A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus. J Immunol 185, 7413-7425, doi:10.4049/jimmunol.1000675 (2010).
105 Halverson, T. W., Wilton, M., Poon, K. K., Petri, B. & Lewenza, S. DNA is an antimicrobial component of neutrophil extracellular traps. PLoS Pathog 11, e1004593, doi:10.1371/journal.ppat.1004593 (2015).
106 Braian, C., Hogea, V. & Stendahl, O. Mycobacterium tuberculosis- induced neutrophil extracellular traps activate human macrophages. J Innate Immun 5, 591-602, doi:10.1159/000348676 (2013).
107 Floyd, M. et al. Swimming Motility Mediates the Formation of Neutrophil Extracellular Traps Induced by Flagellated Pseudomonas aeruginosa. PLoS Pathog 12, e1005987, doi:10.1371/journal.ppat.1005987 (2016).
108 Johnson, C. J., Kernien, J. F., Hoyer, A. R. & Nett, J. E. Mechanisms involved in the triggering of neutrophil extracellular traps (NETs) by Candida glabrata during planktonic and biofilm growth. Sci Rep 7, 13065, doi:10.1038/s41598-017-13588-6 (2017).
109 Saitoh, T. et al. Neutrophil extracellular traps mediate a host defense response to human immunodeficiency virus-1. Cell Host Microbe 12, 109-116, doi:10.1016/j.chom.2012.05.015 (2012).
110 Guimaraes-Costa, A. B. et al. Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps. Proc Natl Acad Sci U S A 106, 6748-6753, doi:10.1073/pnas.0900226106 (2009).
111 Neumann, A. et al. The antimicrobial peptide LL-37 facilitates the formation of neutrophil extracellular traps. Biochem J 464, 3-11, doi:10.1042/BJ20140778 (2014).
112 Chatfield, S. M. et al. Monosodium Urate Crystals Generate Nuclease-Resistant Neutrophil Extracellular Traps via a Distinct Molecular Pathway. J Immunol 200, 1802-1816, doi:10.4049/jimmunol.1701382 (2018).
113 Azevedo, E. P. et al. Amyloid fibrils trigger the release of neutrophil extracellular traps (NETs), causing fibril fragmentation by NET-associated elastase. J Biol Chem 287, 37206-37218, doi:10.1074/jbc.M112.369942 (2012).
114 Aleyd, E., Al, M., Tuk, C. W., van der Laken, C. J. & van Egmond, M. IgA Complexes in Plasma and Synovial Fluid of Patients with Rheumatoid Arthritis Induce Neutrophil Extracellular Traps via FcalphaRI. J Immunol 197, 4552-4559, doi:10.4049/jimmunol.1502353 (2016).
115 Behnen, M. et al. Immobilized immune complexes induce neutrophil extracellular trap release by human neutrophil granulocytes via FcgammaRIIIB and Mac-1. J Immunol 193, 1954-1965, doi:10.4049/jimmunol.1400478 (2014).
116 Kaufman, T. et al. Nucleosomes and neutrophil extracellular traps in septic and burn patients. Clin Immunol 183, 254-262, doi:10.1016/j.clim.2017.08.014 (2017).
117 Fadini, G. P. et al. NETosis Delays Diabetic Wound Healing in Mice and Humans. Diabetes 65, 1061-1071, doi:10.2337/db15-0863 (2016).
118 Manzenreiter, R. et al. Ultrastructural characterization of cystic fibrosis sputum using atomic force and scanning electron microscopy. J Cyst Fibros 11, 84-92, doi:10.1016/j.jcf.2011.09.008 (2012).
172
119 Czaikoski, P. G. et al. Neutrophil Extracellular Traps Induce Organ Damage during Experimental and Clinical Sepsis. PLoS One 11, e0148142, doi:10.1371/journal.pone.0148142 (2016).
120 Kessenbrock, K. et al. Netting neutrophils in autoimmune small-vessel vasculitis. Nat Med 15, 623-625, doi:10.1038/nm.1959 (2009).
121 Lande, R. et al. Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus. Sci Transl Med 3, 73ra19, doi:10.1126/scitranslmed.3001180 (2011).
122 Logters, T. et al. Diagnostic accuracy of neutrophil-derived circulating free DNA (cf-DNA/NETs) for septic arthritis. J Orthop Res 27, 1401-1407, doi:10.1002/jor.20911 (2009).
123 Yipp, B. G. & Kubes, P. NETosis: how vital is it? Blood 122, 2784-2794, doi:10.1182/blood-2013-04-457671 (2013).
124 Papayannopoulos, V., Metzler, K. D., Hakkim, A. & Zychlinsky, A. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps. J Cell Biol 191, 677-691, doi:10.1083/jcb.201006052 (2010).
125 Sorensen, O. E. et al. Papillon-Lefevre syndrome patient reveals species-dependent requirements for neutrophil defenses. J Clin Invest 124, 4539-4548, doi:10.1172/JCI76009 (2014).
126 Farley, K., Stolley, J. M., Zhao, P., Cooley, J. & Remold-O'Donnell, E. A serpinB1 regulatory mechanism is essential for restricting neutrophil extracellular trap generation. J Immunol 189, 4574-4581, doi:10.4049/jimmunol.1201167 (2012).
127 Wang, Y. et al. Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol 184, 205-213, doi:10.1083/jcb.200806072 (2009).
128 Li, P. et al. PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps. J Exp Med 207, 1853-1862, doi:10.1084/jem.20100239 (2010).
129 Konig, M. F. & Andrade, F. A Critical Reappraisal of Neutrophil Extracellular Traps and NETosis Mimics Based on Differential Requirements for Protein Citrullination. Front Immunol 7, 461, doi:10.3389/fimmu.2016.00461 (2016).
130 Yipp, B. G. et al. Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo. Nat Med 18, 1386-1393, doi:10.1038/nm.2847 (2012).
131 Byrd, A. S., O'Brien, X. M., Johnson, C. M., Lavigne, L. M. & Reichner, J. S. An extracellular matrix-based mechanism of rapid neutrophil extracellular trap formation in response to Candida albicans. J Immunol 190, 4136-4148, doi:10.4049/jimmunol.1202671 (2013).
132 Douda, D. N., Khan, M. A., Grasemann, H. & Palaniyar, N. SK3 channel and mitochondrial ROS mediate NADPH oxidase-independent NETosis induced by calcium influx. Proc Natl Acad Sci U S A 112, 2817-2822, doi:10.1073/pnas.1414055112 (2015).
133 Arita, K. et al. Structural basis for Ca(2+)-induced activation of human PAD4. Nat Struct Mol Biol 11, 777-783, doi:10.1038/nsmb799 (2004).
134 Guiducci, E. et al. Candida albicans-Induced NETosis Is Independent of Peptidylarginine Deiminase 4. Front Immunol 9, 1573, doi:10.3389/fimmu.2018.01573 (2018).
135 Pang, L., Hayes, C. P., Buac, K., Yoo, D. G. & Rada, B. Pseudogout-associated inflammatory calcium pyrophosphate dihydrate microcrystals
173
induce formation of neutrophil extracellular traps. J Immunol 190, 6488-6500, doi:10.4049/jimmunol.1203215 (2013).
136 Young, R. L. et al. Neutrophil extracellular trap (NET)-mediated killing of Pseudomonas aeruginosa: evidence of acquired resistance within the CF airway, independent of CFTR. PLoS One 6, e23637, doi:10.1371/journal.pone.0023637 (2011).
137 Juneau, R. A., Stevens, J. S., Apicella, M. A. & Criss, A. K. A thermonuclease of Neisseria gonorrhoeae enhances bacterial escape from killing by neutrophil extracellular traps. J Infect Dis 212, 316-324, doi:10.1093/infdis/jiv031 (2015).
138 Lauth, X. et al. M1 protein allows Group A streptococcal survival in phagocyte extracellular traps through cathelicidin inhibition. J Innate Immun 1, 202-214, doi:10.1159/000203645 (2009).
139 Urban, C. F. et al. Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans. PLoS Pathog 5, e1000639, doi:10.1371/journal.ppat.1000639 (2009).
140 Berends, E. T. et al. Nuclease expression by Staphylococcus aureus facilitates escape from neutrophil extracellular traps. J Innate Immun 2, 576-586, doi:10.1159/000319909 (2010).
141 de Buhr, N., Neumann, A., Jerjomiceva, N., von Kockritz-Blickwede, M. & Baums, C. G. Streptococcus suis DNase SsnA contributes to degradation of neutrophil extracellular traps (NETs) and evasion of NET-mediated antimicrobial activity. Microbiology 160, 385-395, doi:10.1099/mic.0.072199-0 (2014).
142 Lappann, M. et al. In vitro resistance mechanisms of Neisseria meningitidis against neutrophil extracellular traps. Mol Microbiol 89, 433-449, doi:10.1111/mmi.12288 (2013).
143 Menegazzi, R., Decleva, E. & Dri, P. Killing by neutrophil extracellular traps: fact or folklore? Blood 119, 1214-1216, doi:10.1182/blood-2011-07-364604 (2012).
144 Azzouz, L. et al. Relative antibacterial functions of complement and NETs: NETs trap and complement effectively kills bacteria. Mol Immunol 97, 71-81, doi:10.1016/j.molimm.2018.02.019 (2018).
145 Wang, J. Neutrophils in tissue injury and repair. Cell Tissue Res 371, 531-539, doi:10.1007/s00441-017-2785-7 (2018).
146 Dovi, J. V., He, L. K. & DiPietro, L. A. Accelerated wound closure in neutrophil-depleted mice. J Leukoc Biol 73, 448-455 (2003).
147 Martin, P. et al. Wound healing in the PU.1 null mouse--tissue repair is not dependent on inflammatory cells. Curr Biol 13, 1122-1128 (2003).
148 Fadok, V. A. et al. Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF. J Clin Invest 101, 890-898, doi:10.1172/JCI1112 (1998).
149 Farrera, C. & Fadeel, B. Macrophage clearance of neutrophil extracellular traps is a silent process. J Immunol 191, 2647-2656, doi:10.4049/jimmunol.1300436 (2013).
150 Zheng, L., He, M., Long, M., Blomgran, R. & Stendahl, O. Pathogen-induced apoptotic neutrophils express heat shock proteins and elicit activation of human macrophages. J Immunol 173, 6319-6326 (2004).
151 Persson, Y. A., Blomgran-Julinder, R., Rahman, S., Zheng, L. & Stendahl, O. Mycobacterium tuberculosis-induced apoptotic neutrophils trigger a pro-inflammatory response in macrophages through release of heat
174
shock protein 72, acting in synergy with the bacteria. Microbes Infect 10, 233-240, doi:10.1016/j.micinf.2007.11.007 (2008).
152 Martinez, F. O. & Gordon, S. The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Rep 6, 13, doi:10.12703/P6-13 (2014).
153 Lewkowicz, N. et al. Induction of human IL-10-producing neutrophils by LPS-stimulated Treg cells and IL-10. Mucosal Immunol 9, 364-378, doi:10.1038/mi.2015.66 (2016).
154 Fridlender, Z. G. et al. Polarization of tumor-associated neutrophil phenotype by TGF-beta: "N1" versus "N2" TAN. Cancer Cell 16, 183-194, doi:10.1016/j.ccr.2009.06.017 (2009).
155 Ma, Y. et al. Temporal neutrophil polarization following myocardial infarction. Cardiovasc Res 110, 51-61, doi:10.1093/cvr/cvw024 (2016).
156 Buckley, C. D., Gilroy, D. W. & Serhan, C. N. Proresolving lipid mediators and mechanisms in the resolution of acute inflammation. Immunity 40, 315-327, doi:10.1016/j.immuni.2014.02.009 (2014).
157 Clish, C. B., Levy, B. D., Chiang, N., Tai, H. H. & Serhan, C. N. Oxidoreductases in lipoxin A4 metabolic inactivation: a novel role for 15-onoprostaglandin 13-reductase/leukotriene B4 12-hydroxydehydrogenase in inflammation. J Biol Chem 275, 25372-25380, doi:10.1074/jbc.M002863200 (2000).
158 Fullerton, J. N., O'Brien, A. J. & Gilroy, D. W. Lipid mediators in immune dysfunction after severe inflammation. Trends Immunol 35, 12-21, doi:10.1016/j.it.2013.10.008 (2014).
159 Reis, M. B. et al. Lipoxin A4 encapsulated in PLGA microparticles accelerates wound healing of skin ulcers. PLoS One 12, e0182381, doi:10.1371/journal.pone.0182381 (2017).
160 Brubaker, A. L., Rendon, J. L., Ramirez, L., Choudhry, M. A. & Kovacs, E. J. Reduced neutrophil chemotaxis and infiltration contributes to delayed resolution of cutaneous wound infection with advanced age. J Immunol 190, 1746-1757, doi:10.4049/jimmunol.1201213 (2013).
161 Nishio, N., Okawa, Y., Sakurai, H. & Isobe, K. Neutrophil depletion delays wound repair in aged mice. Age (Dordr) 30, 11-19, doi:10.1007/s11357-007-9043-y (2008).
162 Eckert, J. W., Abramson, S. L., Starke, J. & Brandt, M. L. The surgical implications of chronic granulomatous disease. Am J Surg 169, 320-323, doi:10.1016/S0002-9610(99)80167-6 (1995).
163 De Ravin, S. S. et al. Chronic granulomatous disease as a risk factor for autoimmune disease. J Allergy Clin Immunol 122, 1097-1103, doi:10.1016/j.jaci.2008.07.050 (2008).
164 Yager, D. R., Zhang, L. Y., Liang, H. X., Diegelmann, R. F. & Cohen, I. K. Wound fluids from human pressure ulcers contain elevated matrix metalloproteinase levels and activity compared to surgical wound fluids. J Invest Dermatol 107, 743-748 (1996).
165 Nwomeh, B. C., Liang, H. X., Cohen, I. K. & Yager, D. R. MMP-8 is the predominant collagenase in healing wounds and nonhealing ulcers. J Surg Res 81, 189-195, doi:10.1006/jsre.1998.5495 (1999).
166 Moor, A. N., Vachon, D. J. & Gould, L. J. Proteolytic activity in wound fluids and tissues derived from chronic venous leg ulcers. Wound Repair Regen 17, 832-839, doi:10.1111/j.1524-475X.2009.00547.x (2009).
167 Korkmaz, H. I. et al. Neutrophil extracellular traps coincide with a pro-coagulant status of microcirculatory endothelium in burn wounds. Wound Repair Regen 25, 609-617, doi:10.1111/wrr.12560 (2017).
175
168 Gould, T. J. et al. Neutrophil extracellular traps promote thrombin generation through platelet-dependent and platelet-independent mechanisms. Arterioscler Thromb Vasc Biol 34, 1977-1984, doi:10.1161/ATVBAHA.114.304114 (2014).
169 Pannucci, C. J., Osborne, N. H. & Wahl, W. L. Venous thromboembolism in thermally injured patients: analysis of the National Burn Repository. J Burn Care Res 32, 6-12, doi:10.1097/BCR.0b013e318204b2ff (2011).
170 Nguyen, K. T. et al. Deficient cytokine expression and neutrophil oxidative burst contribute to impaired cutaneous wound healing in diabetic, biofilm-containing chronic wounds. Wound Repair Regen 21, 833-841, doi:10.1111/wrr.12109 (2013).
171 Joshi, M. B. et al. High glucose modulates IL-6 mediated immune homeostasis through impeding neutrophil extracellular trap formation. FEBS Lett 587, 2241-2246, doi:10.1016/j.febslet.2013.05.053 (2013).
172 Wong, S. L. et al. Diabetes primes neutrophils to undergo NETosis, which impairs wound healing. Nat Med 21, 815-819, doi:10.1038/nm.3887 (2015).
173 Ayilavarapu, S. et al. Diabetes-induced oxidative stress is mediated by Ca2+-independent phospholipase A2 in neutrophils. J Immunol 184, 1507-1515, doi:10.4049/jimmunol.0901219 (2010).
174 Liu, S. et al. Neutrophil extracellular traps are indirectly triggered by lipopolysaccharide and contribute to acute lung injury. Sci Rep 6, 37252, doi:10.1038/srep37252 (2016).
175 Hoppenbrouwers, T. et al. In vitro induction of NETosis: Comprehensive live imaging comparison and systematic review. PLoS One 12, e0176472, doi:10.1371/journal.pone.0176472 (2017).
176 Riyapa, D. et al. Neutrophil extracellular traps exhibit antibacterial activity against burkholderia pseudomallei and are influenced by bacterial and host factors. Infect Immun 80, 3921-3929, doi:10.1128/IAI.00806-12 (2012).
177 Ocana, A., Nieto-Jimenez, C., Pandiella, A. & Templeton, A. J. Neutrophils in cancer: prognostic role and therapeutic strategies. Mol Cancer 16, 137, doi:10.1186/s12943-017-0707-7 (2017).
178 Satish, L. Chemokines as Therapeutic Targets to Improve Healing Efficiency of Chronic Wounds. Adv Wound Care (New Rochelle) 4, 651-659, doi:10.1089/wound.2014.0602 (2015).
179 Marteyn, B. S., Burgel, P. R., Meijer, L. & Witko-Sarsat, V. Harnessing Neutrophil Survival Mechanisms during Chronic Infection by Pseudomonas aeruginosa: Novel Therapeutic Targets to Dampen Inflammation in Cystic Fibrosis. Front Cell Infect Microbiol 7, 243, doi:10.3389/fcimb.2017.00243 (2017).
180 Yang, C., Chilvers, M., Montgomery, M. & Nolan, S. J. Dornase alfa for cystic fibrosis. Cochrane Database Syst Rev 4, CD001127, doi:10.1002/14651858.CD001127.pub3 (2016).
181 Kalle, M. et al. Host defense peptides of thrombin modulate inflammation and coagulation in endotoxin-mediated shock and Pseudomonas aeruginosa sepsis. PLoS One 7, e51313, doi:10.1371/journal.pone.0051313 (2012).
182 Hansen, F. C. et al. The Thrombin-Derived Host Defense Peptide GKY25 Inhibits Endotoxin-Induced Responses through Interactions with Lipopolysaccharide and Macrophages/Monocytes. J Immunol 194, 5397-5406, doi:10.4049/jimmunol.1403009 (2015).
176
183 Jorch, S. K. & Kubes, P. An emerging role for neutrophil extracellular traps in noninfectious disease. Nat Med 23, 279-287, doi:10.1038/nm.4294 (2017).
184 Pasupuleti, M., Schmidtchen, A. & Malmsten, M. Antimicrobial peptides: key components of the innate immune system. Crit Rev Biotechnol 32, 143-171, doi:10.3109/07388551.2011.594423 (2012).
185 Zasloff, M. Antimicrobial peptides of multicellular organisms. Nature 415, 389-395, doi:10.1038/415389a (2002).
186 Brogden, K. A. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 3, 238-250, doi:10.1038/nrmicro1098 (2005).
187 Hancock, R. E. & Sahl, H. G. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat Biotechnol 24, 1551-1557, doi:10.1038/nbt1267 (2006).
188 Nguyen, L. T., Haney, E. F. & Vogel, H. J. The expanding scope of antimicrobial peptide structures and their modes of action. Trends Biotechnol 29, 464-472, doi:10.1016/j.tibtech.2011.05.001 (2011).
189 Ehrenstein, G. & Lecar, H. Electrically gated ionic channels in lipid bilayers. Q Rev Biophys 10, 1-34 (1977).
190 Nakatsuji, T. & Gallo, R. L. Antimicrobial peptides: old molecules with new ideas. J Invest Dermatol 132, 887-895, doi:10.1038/jid.2011.387 (2012).
191 Braff, M. H., Zaiou, M., Fierer, J., Nizet, V. & Gallo, R. L. Keratinocyte production of cathelicidin provides direct activity against bacterial skin pathogens. Infect Immun 73, 6771-6781, doi:10.1128/IAI.73.10.6771-6781.2005 (2005).
192 Howell, M. D. et al. Selective Killing of Vaccinia Virus by LL-37: Implications for Eczema Vaccinatum. The Journal of Immunology 172, 1763-1767, doi:10.4049/jimmunol.172.3.1763 (2004).
193 Ong, P. Y. et al. Endogenous antimicrobial peptides and skin infections in atopic dermatitis. N Engl J Med 347, 1151-1160, doi:10.1056/NEJMoa021481 (2002).
194 Sieprawska-Lupa, M. et al. Degradation of human antimicrobial peptide LL-37 by Staphylococcus aureus-derived proteinases. Antimicrob Agents Chemother 48, 4673-4679, doi:10.1128/AAC.48.12.4673-4679.2004 (2004).
195 Abtin, A. et al. Flagellin is the principal inducer of the antimicrobial peptide S100A7c (psoriasin) in human epidermal keratinocytes exposed to Escherichia coli. FASEB J 22, 2168-2176, doi:10.1096/fj.07-104117 (2008).
196 Sumikawa, Y. et al. Induction of beta-defensin 3 in keratinocytes stimulated by bacterial lipopeptides through toll-like receptor 2. Microbes Infect 8, 1513-1521, doi:10.1016/j.micinf.2006.01.008 (2006).
197 Wang, Z. et al. Skin mast cells protect mice against vaccinia virus by triggering mast cell receptor S1PR2 and releasing antimicrobial peptides. J Immunol 188, 345-357, doi:10.4049/jimmunol.1101703 (2012).
198 Tang, X., Basavarajappa, D., Haeggstrom, J. Z. & Wan, M. P2X7 Receptor Regulates Internalization of Antimicrobial Peptide LL-37 by Human Macrophages That Promotes Intracellular Pathogen Clearance. J Immunol 195, 1191-1201, doi:10.4049/jimmunol.1402845 (2015).
199 Easton, D. M., Nijnik, A., Mayer, M. L. & Hancock, R. E. Potential of immunomodulatory host defense peptides as novel anti-infectives.
177
Trends Biotechnol 27, 582-590, doi:10.1016/j.tibtech.2009.07.004 (2009).
200 De, Y. et al. LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells. J Exp Med 192, 1069-1074 (2000).
201 Zhang, Z. et al. Evidence that cathelicidin peptide LL-37 may act as a functional ligand for CXCR2 on human neutrophils. Eur J Immunol 39, 3181-3194, doi:10.1002/eji.200939496 (2009).
202 Rohrl, J., Yang, D., Oppenheim, J. J. & Hehlgans, T. Human beta-defensin 2 and 3 and their mouse orthologs induce chemotaxis through interaction with CCR2. J Immunol 184, 6688-6694, doi:10.4049/jimmunol.0903984 (2010).
203 Barlow, P. G. et al. The human cationic host defense peptide LL-37 mediates contrasting effects on apoptotic pathways in different primary cells of the innate immune system. J Leukoc Biol 80, 509-520, doi:10.1189/jlb.1005560 (2006).
204 Alalwani, S. M. et al. The antimicrobial peptide LL-37 modulates the inflammatory and host defense response of human neutrophils. Eur J Immunol 40, 1118-1126, doi:10.1002/eji.200939275 (2010).
205 Zheng, Y. et al. Cathelicidin LL-37 induces the generation of reactive oxygen species and release of human alpha-defensins from neutrophils. Br J Dermatol 157, 1124-1131, doi:10.1111/j.1365-2133.2007.08196.x (2007).
206 Neumann, A. et al. Novel role of the antimicrobial peptide LL-37 in the protection of neutrophil extracellular traps against degradation by bacterial nucleases. J Innate Immun 6, 860-868, doi:10.1159/000363699 (2014).
207 Schuerholz, T., Brandenburg, K. & Marx, G. Antimicrobial peptides and their potential application in inflammation and sepsis. Crit Care 16, 207, doi:10.1186/cc11220 (2012).
208 Lai, Y. & Gallo, R. L. AMPed up immunity: how antimicrobial peptides have multiple roles in immune defense. Trends Immunol 30, 131-141, doi:10.1016/j.it.2008.12.003 (2009).
209 Bjorstad, A., Fu, H., Karlsson, A., Dahlgren, C. & Bylund, J. Interleukin-8-derived peptide has antibacterial activity. Antimicrob Agents Chemother 49, 3889-3895, doi:10.1128/AAC.49.9.3889-3895.2005 (2005).
210 Dai, C. et al. CXCL14 displays antimicrobial activity against respiratory tract bacteria and contributes to clearance of Streptococcus pneumoniae pulmonary infection. J Immunol 194, 5980-5989, doi:10.4049/jimmunol.1402634 (2015).
211 Yang, D. Many chemokines including CCL20/MIP-3 display antimicrobial activity. Journal of Leukocyte Biology 74, 448-455, doi:10.1189/jlb.0103024 (2003).
212 E, M. C. C., Dean, S. N., Propst, C. N., Bishop, B. M. & van Hoek, M. L. Komodo dragon-inspired synthetic peptide DRGN-1 promotes wound-healing of a mixed-biofilm infected wound. NPJ Biofilms Microbiomes 3, 9, doi:10.1038/s41522-017-0017-2 (2017).
213 Lam, S. J. et al. Combating multidrug-resistant Gram-negative bacteria with structurally nanoengineered antimicrobial peptide polymers. Nat Microbiol 1, 16162, doi:10.1038/nmicrobiol.2016.162 (2016).
178
214 Kasetty, G. et al. Structure-activity studies and therapeutic potential of host defense peptides of human thrombin. Antimicrob Agents Chemother 55, 2880-2890, doi:10.1128/AAC.01515-10 (2011).
215 Hansen, F. C., Stromdahl, A. C., Morgelin, M., Schmidtchen, A. & van der Plas, M. J. A. Thrombin-Derived Host-Defense Peptides Modulate Monocyte/Macrophage Inflammatory Responses to Gram-Negative Bacteria. Front Immunol 8, 843, doi:10.3389/fimmu.2017.00843 (2017).
216 Lim, C. H. et al. Thrombin-derived host defence peptide modulates neutrophil rolling and migration in vitro and functional response in vivo. Sci Rep 7, 11201, doi:10.1038/s41598-017-11464-x (2017).
217 Hou, H. W. et al. Rapid and label-free microfluidic neutrophil purification and phenotyping in diabetes mellitus. Sci Rep 6, 29410, doi:10.1038/srep29410 (2016).
218 Ciornei, C. D., Sigurdardottir, T., Schmidtchen, A. & Bodelsson, M. Antimicrobial and chemoattractant activity, lipopolysaccharide neutralization, cytotoxicity, and inhibition by serum of analogs of human cathelicidin LL-37. Antimicrob Agents Chemother 49, 2845-2850, doi:10.1128/AAC.49.7.2845-2850.2005 (2005).
219 Ducker, T. P. & Skubitz, K. M. Subcellular localization of CD66, CD67, and NCA in human neutrophils. J Leukoc Biol 52, 11-16 (1992).
220 Hayashi, F., Means, T. K. & Luster, A. D. Toll-like receptors stimulate human neutrophil function. Blood 102, 2660-2669, doi:10.1182/blood-2003-04-1078 (2003).
221 Singh, A., Zarember, K. A., Kuhns, D. B. & Gallin, J. I. Impaired priming and activation of the neutrophil NADPH oxidase in patients with IRAK4 or NEMO deficiency. J Immunol 182, 6410-6417, doi:10.4049/jimmunol.0802512 (2009).
222 DeLeo, F. R. et al. Neutrophils exposed to bacterial lipopolysaccharide upregulate NADPH oxidase assembly. J Clin Invest 101, 455-463, doi:10.1172/JCI949 (1998).
223 Pacquelet, S. et al. Cross-talk between IRAK-4 and the NADPH oxidase. Biochem J 403, 451-461, doi:10.1042/BJ20061184 (2007).
224 Li, Y., Brazzell, J., Herrera, A. & Walcheck, B. ADAM17 deficiency by mature neutrophils has differential effects on L-selectin shedding. Blood 108, 2275-2279, doi:10.1182/blood-2006-02-005827 (2006).
225 Wang, Y., Zhang, A. C., Ni, Z., Herrera, A. & Walcheck, B. ADAM17 activity and other mechanisms of soluble L-selectin production during death receptor-induced leukocyte apoptosis. J Immunol 184, 4447-4454, doi:10.4049/jimmunol.0902925 (2010).
226 Gomez-Gaviro, M. et al. Expression and regulation of the metalloproteinase ADAM-8 during human neutrophil pathophysiological activation and its catalytic activity on L-selectin shedding. J Immunol 178, 8053-8063 (2007).
227 Yang, D. et al. Defensin participation in innate and adaptive immunity. Curr Pharm Des 13, 3131-3139 (2007).
228 Butenas, S. & Mann, K. G. Blood coagulation. Biochemistry (Mosc) 67, 3-12 (2002).
229 Skubitz, K. M., Campbell, K. D. & Skubitz, A. P. CD66a, CD66b, CD66c, and CD66d each independently stimulate neutrophils. J Leukoc Biol 60, 106-117 (1996).
230 Jutila, M. A., Rott, L., Berg, E. L. & Butcher, E. C. Function and regulation of the neutrophil MEL-14 antigen in vivo: comparison with LFA-1 and MAC-1. J Immunol 143, 3318-3324 (1989).
179
231 Tedder, T. F., Steeber, D. A. & Pizcueta, P. L-selectin-deficient mice have impaired leukocyte recruitment into inflammatory sites. J Exp Med 181, 2259-2264 (1995).
232 Venturi, G. M. et al. Leukocyte migration is regulated by L-selectin endoproteolytic release. Immunity 19, 713-724 (2003).
233 Hickey, M. J. et al. L-Selectin Facilitates Emigration and Extravascular Locomotion of Leukocytes During Acute Inflammatory Responses In Vivo. The Journal of Immunology 165, 7164-7170, doi:10.4049/jimmunol.165.12.7164 (2000).
234 Kamp, V. M. et al. Human suppressive neutrophils CD16bright/CD62Ldim exhibit decreased adhesion. J Leukoc Biol 92, 1011-1020, doi:10.1189/jlb.0612273 (2012).
235 Subramanian, H. et al. Signaling through L-selectin mediates enhanced chemotaxis of lymphocyte subsets to secondary lymphoid tissue chemokine. J Immunol 188, 3223-3236, doi:10.4049/jimmunol.1101032 (2012).
236 Steeber, D. A., Engel, P., Miller, A. S., Sheetz, M. P. & Tedder, T. F. Ligation of L-selectin through conserved regions within the lectin domain activates signal transduction pathways and integrin function in human, mouse, and rat leukocytes. J Immunol 159, 952-963 (1997).
237 Simon, S. I. et al. L-selectin (CD62L) cross-linking signals neutrophil adhesive functions via the Mac-1 (CD11b/CD18) beta 2-integrin. J Immunol 155, 1502-1514 (1995).
238 Davenpeck, K. L., Brummet, M. E., Hudson, S. A., Mayer, R. J. & Bochner, B. S. Activation of Human Leukocytes Reduces Surface P-Selectin Glycoprotein Ligand-1 (PSGL-1, CD162) and Adhesion to P-Selectin In Vitro. The Journal of Immunology 165, 2764-2772, doi:10.4049/jimmunol.165.5.2764 (2000).
239 Norman, K. E., Moore, K. L., McEver, R. P. & Ley, K. Leukocyte rolling in vivo is mediated by P-selectin glycoprotein ligand-1. Blood 86, 4417-4421 (1995).
240 Kahn, J., Walcheck, B., Migaki, G. I., Jutila, M. A. & Kishimoto, T. K. Calmodulin regulates L-selectin adhesion molecule expression and function through a protease-dependent mechanism. Cell 92, 809-818 (1998).
241 Gifford, J. L., Ishida, H. & Vogel, H. J. Structural insights into calmodulin-regulated L-selectin ectodomain shedding. J Biol Chem 287, 26513-26527, doi:10.1074/jbc.M112.373373 (2012).
242 Lee, D., Schultz, J. B., Knauf, P. A. & King, M. R. Mechanical shedding of L-selectin from the neutrophil surface during rolling on sialyl Lewis x under flow. J Biol Chem 282, 4812-4820, doi:10.1074/jbc.M609994200 (2007).
243 Stoddart, J. H., Jr., Jasuja, R. R., Sikorski, M. A., von Andrian, U. H. & Mier, J. W. Protease-resistant L-selectin mutants. Down-modulation by cross-linking but not cellular activation. J Immunol 157, 5653-5659 (1996).
244 Palecanda, A., Walcheck, B., Bishop, D. K. & Jutila, M. A. Rapid activation-independent shedding of leukocyte L-selectin induced by cross-linking of the surface antigen. Eur J Immunol 22, 1279-1286, doi:10.1002/eji.1830220524 (1992).
245 Gasser, O. et al. Characterisation and properties of ectosomes released by human polymorphonuclear neutrophils. Experimental cell research 285, 243-257 (2003).
180
246 Headland, S. E., Jones, H. R., D'Sa, A. S., Perretti, M. & Norling, L. V. Cutting-edge analysis of extracellular microparticles using ImageStream(X) imaging flow cytometry. Sci Rep 4, 5237, doi:10.1038/srep05237 (2014).
247 Honda, F. et al. The kinase Btk negatively regulates the production of reactive oxygen species and stimulation-induced apoptosis in human neutrophils. Nat Immunol 13, 369-378, doi:10.1038/ni.2234 (2012).
248 Bohmer, R. H., Trinkle, L. S. & Staneck, J. L. Dose effects of LPS on neutrophils in a whole blood flow cytometric assay of phagocytosis and oxidative burst. Cytometry 13, 525-531, doi:10.1002/cyto.990130512 (1992).
249 Gomes, N. E. et al. Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood. Brazilian Journal of Medical and Biological Research 43, 853-858, doi:10.1590/s0100-879x2010007500078 (2010).
250 McMillan, S. J., Sharma, R. S., Richards, H. E., Hegde, V. & Crocker, P. R. Siglec-E promotes beta2-integrin-dependent NADPH oxidase activation to suppress neutrophil recruitment to the lung. J Biol Chem 289, 20370-20376, doi:10.1074/jbc.M114.574624 (2014).
251 Kielland, A. et al. In vivo imaging of reactive oxygen and nitrogen species in inflammation using the luminescent probe L-012. Free Radic Biol Med 47, 760-766, doi:10.1016/j.freeradbiomed.2009.06.013 (2009).
252 Jersmann, H. P., Rathjen, D. A. & Ferrante, A. Enhancement of lipopolysaccharide-induced neutrophil oxygen radical production by tumor necrosis factor alpha. Infect Immun 66, 1744-1747 (1998).
253 McLean, L. R., Hagaman, K. A., Owen, T. J. & Krstenansky, J. L. Minimal peptide length for interaction of amphipathic alpha-helical peptides with phosphatidylcholine liposomes. Biochemistry 30, 31-37 (1991).
254 Lecomte, J. T. J. & Moor, C. D. Helix Formation in Apocytochrome bs: The Role of a Neutral Histidine at the N-Cap Position. J. Am. Chem. Soc. 113, 9663-9665, doi:10.1021/ja00025a037 (1991).
255 Armstrong, K. M. & Baldwin, R. L. Charged histidine affects alpha-helix stability at all positions in the helix by interacting with the backbone charges. Proc Natl Acad Sci U S A 90, 11337-11340 (1993).
256 Holdbrook, D. A. et al. Influence of pH on the activity of thrombin-derived antimicrobial peptides. Biochim Biophys Acta, doi:10.1016/j.bbamem.2018.06.002 (2018).
257 Kuang, Z. et al. Pseudomonas aeruginosa elastase provides an escape from phagocytosis by degrading the pulmonary surfactant protein-A. PLoS One 6, e27091, doi:10.1371/journal.pone.0027091 (2011).
258 Parmely, M., Gale, A., Clabaugh, M., Horvat, R. & Zhou, W. W. Proteolytic inactivation of cytokines by Pseudomonas aeruginosa. Infect Immun 58, 3009-3014 (1990).
259 Branzk, N. & Papayannopoulos, V. Molecular mechanisms regulating NETosis in infection and disease. Semin Immunopathol 35, 513-530, doi:10.1007/s00281-013-0384-6 (2013).
260 O'Donoghue, A. J. et al. Global substrate profiling of proteases in human neutrophil extracellular traps reveals consensus motif predominantly contributed by elastase. PLoS One 8, e75141, doi:10.1371/journal.pone.0075141 (2013).
261 Leffler, J. et al. Neutrophil extracellular traps that are not degraded in systemic lupus erythematosus activate complement exacerbating the
181
disease. J Immunol 188, 3522-3531, doi:10.4049/jimmunol.1102404 (2012).
262 Sabino, F. et al. In vivo assessment of protease dynamics in cutaneous wound healing by degradomics analysis of porcine wound exudates. Mol Cell Proteomics 14, 354-370, doi:10.1074/mcp.M114.043414 (2015).
263 Eming, S. A. et al. Differential proteomic analysis distinguishes tissue repair biomarker signatures in wound exudates obtained from normal healing and chronic wounds. J Proteome Res 9, 4758-4766, doi:10.1021/pr100456d (2010).
264 Schauer, C. et al. Aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines. Nat Med 20, 511-517, doi:10.1038/nm.3547 (2014).
265 Parker, H., Albrett, A. M., Kettle, A. J. & Winterbourn, C. C. Myeloperoxidase associated with neutrophil extracellular traps is active and mediates bacterial killing in the presence of hydrogen peroxide. J Leukoc Biol 91, 369-376, doi:10.1189/jlb.0711387 (2012).
266 Rittirsch, D., Flierl, M. A. & Ward, P. A. Harmful molecular mechanisms in sepsis. Nat Rev Immunol 8, 776-787, doi:10.1038/nri2402 (2008).
267 Lim, C. H. et al. Thrombin and Plasmin Alter the Proteome of Neutrophil Extracellular Traps. Frontiers in Immunology 9, doi:10.3389/fimmu.2018.01554 (2018).
268 Ishihama, Y. et al. Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol Cell Proteomics 4, 1265-1272, doi:10.1074/mcp.M500061-MCP200 (2005).
269 Reich, M. et al. GenePattern 2.0. Nat Genet 38, 500-501, doi:10.1038/ng0506-500 (2006).
270 Manguy, J. et al. Peptigram: A Web-Based Application for Peptidomics Data Visualization. J Proteome Res 16, 712-719, doi:10.1021/acs.jproteome.6b00751 (2017).
271 Cederholm-Williams, S. A. Concentration of plasminogen and antiplasmin in plasma and serum. J Clin Pathol 34, 979-981 (1981).
272 Macaya, R. F., Schultze, P., Smith, F. W., Roe, J. A. & Feigon, J. Thrombin-binding DNA aptamer forms a unimolecular quadruplex structure in solution. Proc Natl Acad Sci U S A 90, 3745-3749 (1993).
273 Komissarov, A. A., Florova, G. & Idell, S. Effects of extracellular DNA on plasminogen activation and fibrinolysis. J Biol Chem 286, 41949-41962, doi:10.1074/jbc.M111.301218 (2011).
274 Herren, T., Burke, T. A., Jardi, M., Felez, J. & Plow, E. F. Regulation of plasminogen binding to neutrophils. Blood 97, 1070-1078 (2001).
275 Dwyer, M. et al. Cystic fibrosis sputum DNA has NETosis characteristics and neutrophil extracellular trap release is regulated by macrophage migration-inhibitory factor. J Innate Immun 6, 765-779, doi:10.1159/000363242 (2014).
276 Missen, M. A., Haylock, D., Whitty, G., Medcalf, R. L. & Coughlin, P. B. Stage specific gene expression of serpins and their cognate proteases during myeloid differentiation. Br J Haematol 135, 715-724, doi:10.1111/j.1365-2141.2006.06360.x (2006).
277 Matamala, N. et al. Identification of Novel Short C-Terminal Transcripts of Human SERPINA1 Gene. PLoS One 12, e0170533, doi:10.1371/journal.pone.0170533 (2017).
182
278 Le Bonniec, B. F., Guinto, E. R. & Stone, S. R. Identification of thrombin residues that modulate its interactions with antithrombin III and alpha 1-antitrypsin. Biochemistry 34, 12241-12248 (1995).
279 Cooley, J., Takayama, T. K., Shapiro, S. D., Schechter, N. M. & Remold-O'Donnell, E. The serpin MNEI inhibits elastase-like and chymotrypsin-like serine proteases through efficient reactions at two active sites. Biochemistry 40, 15762-15770 (2001).
280 Venge, P., Eriksson, A. K., Douhan-Hakansson, L. & Pauksen, K. Human Neutrophil Lipocalin in Activated Whole Blood Is a Specific and Rapid Diagnostic Biomarker of Bacterial Infections in the Respiratory Tract. Clin Vaccine Immunol 24, doi:10.1128/CVI.00064-17 (2017).
281 Monteseirin, J. et al. Neutrophils as a novel source of eosinophil cationic protein in IgE-mediated processes. J Immunol 179, 2634-2641 (2007).
282 Lubbers, R., van Essen, M. F., van Kooten, C. & Trouw, L. A. Production of complement components by cells of the immune system. Clin Exp Immunol 188, 183-194, doi:10.1111/cei.12952 (2017).
283 Bode, W. et al. The refined 1.9 A crystal structure of human alpha-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J 8, 3467-3475 (1989).
284 Padmanabhan, K., Padmanabhan, K. P., Ferrara, J. D., Sadler, J. E. & Tulinsky, A. The structure of alpha-thrombin inhibited by a 15-mer single-stranded DNA aptamer. J Biol Chem 268, 17651-17654 (1993).
285 Krem, M. M., Rose, T. & Di Cera, E. The C-terminal sequence encodes function in serine proteases. J Biol Chem 274, 28063-28066 (1999).
286 Hakkim, A. et al. Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis. Proc Natl Acad Sci U S A 107, 9813-9818, doi:10.1073/pnas.0909927107 (2010).
287 Zhang, S. et al. Enhanced formation and impaired degradation of neutrophil extracellular traps in dermatomyositis and polymyositis: a potential contributor to interstitial lung disease complications. Clin Exp Immunol 177, 134-141, doi:10.1111/cei.12319 (2014).
288 Parker, H., Dragunow, M., Hampton, M. B., Kettle, A. J. & Winterbourn, C. C. Requirements for NADPH oxidase and myeloperoxidase in neutrophil extracellular trap formation differ depending on the stimulus. J Leukoc Biol 92, 841-849, doi:10.1189/jlb.1211601 (2012).
289 Clark, S. R. et al. Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat Med 13, 463-469, doi:10.1038/nm1565 (2007).
290 Carestia, A. et al. Mediators and molecular pathways involved in the regulation of neutrophil extracellular trap formation mediated by activated platelets. J Leukoc Biol 99, 153-162, doi:10.1189/jlb.3A0415-161R (2016).
291 Bizios, R., Lai, L., Fenton, J. W., 2nd & Malik, A. B. Thrombin-induced chemotaxis and aggregation of neutrophils. J Cell Physiol 128, 485-490, doi:10.1002/jcp.1041280318 (1986).
292 Ryan, T. J., Lai, L. & Malik, A. B. Plasmin generation induces neutrophil aggregation: dependence on the catalytic and lysine binding sites. J Cell Physiol 151, 255-261, doi:10.1002/jcp.1041510206 (1992).
293 Lo, S. K., Ryan, T. J., Gilboa, N., Lai, L. & Malik, A. B. Role of catalytic and lysine-binding sites in plasmin-induced neutrophil adherence to endothelium. J Clin Invest 84, 793-801, doi:10.1172/JCI114238 (1989).
183
294 Montrucchio, G. et al. Plasmin promotes an endothelium-dependent adhesion of neutrophils. Involvement of platelet activating factor and P-selectin. Circulation 93, 2152-2160 (1996).
295 Hoeksema, M., van Eijk, M., Haagsman, H. P. & Hartshorn, K. L. Histones as mediators of host defense, inflammation and thrombosis. Future Microbiol 11, 441-453, doi:10.2217/fmb.15.151 (2016).
296 Fuchs, T. A. et al. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci U S A 107, 15880-15885, doi:10.1073/pnas.1005743107 (2010).
297 McDonald, B. et al. Platelets and neutrophil extracellular traps collaborate to promote intravascular coagulation during sepsis in mice. Blood 129, 1357-1367, doi:10.1182/blood-2016-09-741298 (2017).
298 Longstaff, C. et al. Mechanical stability and fibrinolytic resistance of clots containing fibrin, DNA, and histones. J Biol Chem 288, 6946-6956, doi:10.1074/jbc.M112.404301 (2013).
299 Dau, T., Sarker, R. S., Yildirim, A. O., Eickelberg, O. & Jenne, D. E. Autoprocessing of neutrophil elastase near its active site reduces the efficiency of natural and synthetic elastase inhibitors. Nat Commun 6, 6722, doi:10.1038/ncomms7722 (2015).
300 Hansen, G. et al. Unexpected active-site flexibility in the structure of human neutrophil elastase in complex with a new dihydropyrimidone inhibitor. J Mol Biol 409, 681-691, doi:10.1016/j.jmb.2011.04.047 (2011).
301 Mohanty, T. et al. A novel mechanism for NETosis provides antimicrobial defense at the oral mucosa. Blood 126, 2128-2137, doi:10.1182/blood-2015-04-641142 (2015).
302 Mahlapuu, M., Hakansson, J., Ringstad, L. & Bjorn, C. Antimicrobial Peptides: An Emerging Category of Therapeutic Agents. Front Cell Infect Microbiol 6, 194, doi:10.3389/fcimb.2016.00194 (2016).
303 Zavascki, A. P., Goldani, L. Z., Li, J. & Nation, R. L. Polymyxin B for the treatment of multidrug-resistant pathogens: a critical review. J Antimicrob Chemother 60, 1206-1215, doi:10.1093/jac/dkm357 (2007).
304 Landman, D., Georgescu, C., Martin, D. A. & Quale, J. Polymyxins revisited. Clin Microbiol Rev 21, 449-465, doi:10.1128/CMR.00006-08 (2008).
305 Zavascki, A. P. & Nation, R. L. Nephrotoxicity of Polymyxins: Is There Any Difference between Colistimethate and Polymyxin B? Antimicrob Agents Chemother 61, doi:10.1128/AAC.02319-16 (2017).
306 Bird, G. H. et al. Hydrocarbon double-stapling remedies the proteolytic instability of a lengthy peptide therapeutic. Proc Natl Acad Sci U S A 107, 14093-14098, doi:10.1073/pnas.1002713107 (2010).
307 Gellatly, S. L. & Hancock, R. E. Pseudomonas aeruginosa: new insights into pathogenesis and host defenses. Pathog Dis 67, 159-173, doi:10.1111/2049-632X.12033 (2013).
308 Stephan, A. et al. LL37:DNA complexes provide antimicrobial activity against intracellular bacteria in human macrophages. Immunology 148, 420-432, doi:10.1111/imm.12620 (2016).
309 Carmona-Rivera, C. & Kaplan, M. J. Induction and Quantification of NETosis. Curr Protoc Immunol 115, 14 41 11-14 41 14, doi:10.1002/cpim.16 (2016).
310 Lee, K. H. et al. Quantification of NETs-associated markers by flow cytometry and serum assays in patients with thrombosis and sepsis. Int J Lab Hematol 40, 392-399, doi:10.1111/ijlh.12800 (2018).
184
311 Yizengaw, E. et al. Visceral Leishmaniasis Patients Display Altered Composition and Maturity of Neutrophils as well as Impaired Neutrophil Effector Functions. Front Immunol 7, 517, doi:10.3389/fimmu.2016.00517 (2016).
312 Wagner, C. J., Schultz, C. & Mall, M. A. Neutrophil elastase and matrix metalloproteinase 12 in cystic fibrosis lung disease. Mol Cell Pediatr 3, 25, doi:10.1186/s40348-016-0053-7 (2016).
313 Kelly, E., Greene, C. M. & McElvaney, N. G. Targeting neutrophil elastase in cystic fibrosis. Expert Opin Ther Targets 12, 145-157, doi:10.1517/14728222.12.2.145 (2008).
314 Papayannopoulos, V., Staab, D. & Zychlinsky, A. Neutrophil elastase enhances sputum solubilization in cystic fibrosis patients receiving DNase therapy. PLoS One 6, e28526, doi:10.1371/journal.pone.0028526 (2011).
315 Belorgey, D. & Bieth, J. G. DNA binds neutrophil elastase and mucus proteinase inhibitor and impairs their functional activity. FEBS Lett 361, 265-268 (1995).
316 Kalkhof, S. et al. Proteomics and metabolomics for in situ monitoring of wound healing. Biomed Res Int 2014, 934848, doi:10.1155/2014/934848 (2014).