neutrophils in the pathogenesis of sepsis
TRANSCRIPT
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Neutrophils in the
Pathogenesis of Sepsis
John C. Marshall, MD FRCSC
Critical Care Canada Forum
2012
Toronto, Canada
October 29, 2012St. Michael’s Hospital University of Toronto
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Songhui Jia
Maria Jimenez
Yue Li
Zeenat Malam
Jean Parodo
Ravi Taneja
Jennifer Tsang
Bill Watson
Thanks to …
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Neutrophils are a Dynamic Population
• Constitutively apoptotic
• 6 – 8 Hours In vivo
• 24 – 36 Hours In vitro
• 1011 PMN released and die by
apoptosis each day
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Neutrophils kill
bacteria and phagocytose
damaged tissue
Activated
neutrophils also damage
adjacent normal tissues
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Neutrophil Antimicrobial Defenses
Production of reactive
oxygen intermediates
Antimicrobial peptides
• BPI
• Defensins
• Alkaline phosphatase
• Lysozyme
• Lactoferrin
• Elastase
• Cathepsin G
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Neutrophil
extracellular
traps (NETs)
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Time-dependent
Effects of Neutrophil
Depletion in CLP
- Hoesel et al Shock
24:40, 2005
- 24 Hours + 12 Hours
Lo
g C
FU
/ml B
loo
d
0
2
4
6
8Non-specific IgG
Anti-PMN
Time Relative to CLP
*
**
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Duration of In Vitro Culture
0 Hours 18 Hours 24 Hours
Perc
en
t A
po
pto
sis
0
10
20
30
40
50
60
Control
Sepsis
- Taneja, Crit Care Med 32:1460,2004
PMN Apoptosis is Delayed in Sepsis
LPS
*
**
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Microbial Products Host-derived Mediators
Endotoxin Interleukin (IL)-1β
Lipoteichoic acid IL-2
Mannan IL-3
Modulins from CONS IL-4
E. coli verotoxin IL-6
H. pylori surface proteins IL-8
Butyric acid Tumor Necrosis Factor
Propionic acid Interferons
G-CSF, GM-CSF
Respiratory syncytial virus Leptin
PBEF
C5a
Cathelicidins
Physiologic ProcessesTransendothelial migration
Factors Inhibiting Neutrophil Apoptosis
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% A
po
pto
sis
0
10
20
30
40
*
‡‡ ‡
‡ ‡‡
* Control
Herb A
PDTC
CHX
Control LPS
(1mg/ml)GM-CSF
(100U/ml)
Delayed Apoptosis Requires Cellular
Activation and Protein Synthesis- Watson, J.Immunol. 161:957, 1998
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Control Anti-sense Sense Anti-sense
+ IL-1
Perc
en
t A
po
pto
sis
0
10
20
30
40
50
Control
LPS (1ug/ml)
GM-CSF (100U/ml)
Effects of Anti-sense to IL-1 on
LPS and GM-CSF Inhibition of PMN Apoptosis
- Watson, J.Immunol. 161:957, 1998.
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Pre-B Cell Colony
Enhancing
Factor (PBEF)
• 52 kDa secreted protein
• Cytokine-like TATT motifs in mRNA
• Synergizes with IL-7 to induce
pre-B cell colony formation
- Samal; Mol.Cell Biol. 14:1431, 1994
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ConLps
Sepsis Patients
1 2 3 4 5 6 7 8
PBEF mRNA is Highly Expressed
in Sepsis PMN
PBEF
G3PDH
- Jia et al J Clin Invest 113:1318, 2004
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No Oligo Sense Non-sense Anti-sense
Perc
en
t A
po
pto
sis
0
5
10
15
20
25
30
*
PBEF Anti-sense Partially Restores
PMN Apoptosis in Sepsis
- Jia et al J Clin Invest 113:1318:2004
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0
10
20
30
40
50
60
70
Control PBEF S199A S200A
% a
po
pto
sis
Anti-apoptotic effect of Nampt active PBEF on
trauma PMN is inhbited by prior addition of
insulin (20 µU/mL) N=6
no insulin
add ins 2nd
add ins 1st
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Hotchkiss et al Crit.Care Med.
28:3207, 2000
- Imai et al, JAMA 289:2104, 2003
Trauma and Sepsis
Result in Systemic
Epithelial Cell
Apoptosis
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Cytochrome C
CD95/TNFR
Caspase 8
Procaspase 9
APAF-1
Caspase 3Bcl2
Mcl1
Inhibitors of Apoptosis:
Survivin
CIAP1,2
XIAP1, NAIP
DNA, Protein cleavage
SMAC/Diablo
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MDFSRNLYDIGEQLDSEDLASLKFLSLDYIPQRKQEPIK
DALMLFQRLQEKRMLEESNLSFLKELLFRINRLDLLITYL
NTRKEEMERELQTPGRAQISAYRFHFCRMSWAEANSQ
CQTQSVPFWRRVDHLLIRVMLYQISEEVSRSELRSFKF
LLQEEISKCKLDDDMNLLDIFIEMEKRVILGEGKLDILKR
VCAQINKSLLKIINDYEEFSKGEELCGVMTISDSPREQD
SESQTLDKVYQMKSKPRGYCLIINNHNFAKAREKVPKL
HSIRDRNGTHLDAGALTTTFEELHFEIKPHDDCTVEQIY
EILKIYQLMDHSNMDCFICCILSHGDKGIIYGTDGQEAPI
YELTSQFTGLKCPSLAGKPKVFFIQACQGDNYQKGIPV
ETDSEEQPYLEMDLSSPQTRYIPDEADFLLGMATVNNC
VSYRNPAEGTWYIQSLCQSLRERCPRGDDILTILTEVNY
EVSNKDDKKNMGKQMPQPTFTLRKKLVFPSD
Caspase-8
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A549 HEK293
Perc
en
t H
yp
od
iplo
id D
NA
0
10
20
30
40
50
* *
** **
DPI - - + - -Catalase - - - + -
GSH - - - - +
No PMN + PMNWestern:
~ Caspase-8
~pY
Trauma Neutrophils
Induce Caspase-8
Dephosphorylation and
Apoptosis of A549
Lung Epithelial Cells
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pY
pY
SHP-1
Cleavage of caspase-3 and
progression of apoptosis
lyn
PI3 Kinase Activation
PBEF Transcription
IAP Synthesis
CD95/TNFR
FADD
Pro-caspase-8
Caspase-8 Phosphorylation
Caspase-8
Dephosphorylation
p10
p18
The Caspase-8 Survivalsome
IL-1R
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Candida : Neutrophil Ratio
Control 1 : 1 5 : 1 10 : 1
Perc
en
tag
e A
po
pto
sis
0
10
20
30
50
60
70
80
90
Phagocytosis of Candida albicans Induces
Neutrophil Apoptosis In Vitro
40
*
- Rotstein, Shock 2000; 14:278
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Ischemia/reperfusion I/R + E. coli
Intratracheal E. coli Attenuates
Pulmonary Neutrophilia
- Sookhai, Ann Surg 2002; 235:285
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Time (Hours)
0 10 20 30 40 50
Perc
en
t S
urv
ival
0
20
40
60
80
10
0
Sham
I/R + E.coli
I/R + Saline
- Sookhai Ann Surg 2002; 235:285
Intratracheal Heat-killed E. coli Attenuates
Mortality Following Ischemia/Reperfusion
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Conclusions
• Functional neutrophils are critical to
the early response against infection
• Their prolonged activity contributes to
inadvertent organ injury
• Accelerating their involution through
apoptosis may limit this delayed
harm
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Thank you!!