resolution of inflammation - az9194.vo.msecnd.netaz9194.vo.msecnd.net/pdfs/100602/009.pdfkumar et...

1

Molecular Mechanisms of Human Disease

Injury, Inflammation, Stem Cells and Tissue Repair

Pulmonary & Critical Care Medicine, Dept. of Internal Medicine

Brigham and Women’s Hospital/Harvard Medical School

Boston, MA USA

Bruce D. Levy

Resolution of Inflammation

2

Outline

Define inflammation resolution at the tissue, cellular

and molecular levels

Chemical mediators of resolution for injury, noxious

stimuli and infection - their biosynthesis and bioactions

Distinguish anti-inflammation from promoting resolution

Translation to human disease

3

Outline

Define inflammation resolution at the tissue,

cellular and molecular levels





4

Cardiovascular diseases

(Atherosclerosis)

Neurological disorders

(Alzheimer’s, Parkinson’s)

Inflammatory bowel diseases

(Colitis, Crohn’s)

Asthma

Cancer

Diabetes

Autoimmune diseases

How does the acute inflammation resolve?

What are the mechanisms/components underlying the resolution process?

Uncontrolled Inflammation Is a Pathological Feature

of Common Diseases

Kumar et al. Robbins Basic Pathology 8/E (Fig. 2-5)

Copyright © 2007 by Saunders, an imprint of Elsevier Inc. All rights

reserved.

5

Resolution Of Acute Inflammation

6

Key Features in Tissue Resolution

Kumar et al. Robbins Basic

Pathology 8/E (Fig. 2-9)

Copyright © 2007 by

Saunders, an imprint of

Elsevier Inc. All rights

reserved.

7

Resolution Defined In Operative and Quantitative Terms

Adapted from Bannenberg G, J. Immunology 2005, 174: 4345-4355 Copyright 2005. The American Association of Immunologists Inc.

8

New Concepts of Resolution of Inflammation

Passive termination of inflammation

Disappearance of local chemotactic stimuli

and pro-inflammatory mediators

InflammationResolution

Previous concept

(passive process):

InflammationResolution

New concept (active process):

Rapidly turn on after acute inflammatory challenge

Active cellular events and biochemical pathways

Generation of anti-inflammatory and pro-resolution mediators

Courtesy of Nan Chiang

9

Resolution Circuits in Inflammation

Host Defense

PMN infiltration

Chemical Mediators

Acute Inflammation

Resolution

Chemical Mediators

“New & Uncharted”

Chronic

Inflammation

Chemical Mediators

Amplification

10

Excessive

“unresolved” Prostaglandins

Leukotrienes

Lipid mediator class switching

PGE2, PGD2

Specialized pro-resolution mediators

Aspirin-triggered

Lipoxins

Lipoxins

LXs

[Neuro]Protectins

PDs

Resolvin Es

RvEs

Resolvin Ds

RvDs

Chronic

Inflammation

Resolution

Fibrosis

Return to homeostasisAcute

Inflammation

Decision Paths in Acute Inflammation: Identification of Specialized Mediators During Resolution

Chronic

Inflammation

Ideal outcome

Omega-3 derived


11

Outline



Chemical mediators of resolution for injury,

noxious stimuli and infection - their biosynthesis

and bioactions



12

AA

Chronic

Inflammation

Pro-inflammatory mediators

Leukotrienes

Prostaglandins

COOH

OH

OH

OH

COOH

HOOH

OH

O F

HO OHCOOCH3

RvE1ATLa PD1

Injury / infection

Host Defense

Resolution of Acute Inflammation Is An Active Process

Polyunsaturated Fatty Acid (PUFA)-derived Lipid Mediators

PMN

Acute

Inflammation

Aspirin-triggered Lipoxins

(ATL)

Resolution

Protective mediators

cell-cell interaction

EPA DHA

Lipoxins

(LXs)

[Neuro]Protectins

(PDs)

Resolvin Es

(RvEs)

Resolvin Ds

(RvDs)

Switching phenotype


13

Eicosanoid Mediators In Inflammation

Phospholipids

Arachidonic Acid

cPLA2

Resolution

Cell-Cell

Interactions

Lipoxins

Transcellular

Biosynthesis

5-Lipoxygenase

LeukotrienesProstaglandins

COX 1 & 2

Initiation

Aspirin Anti-LTs

14

Transcellular Lipoxin & ATL Biosynthesis

COOH

Arachidonic Acid

COOH

COOH COOH

O(O)H

COOH

O

OH

OH

OH OH

OH

HO

HO

15S-H(p)ETE

LXA4 LXB4

15S-Epoxytetraene

PMN

Airway Epithelia

or Monocytes

IL-13

IL-4

15-LO

5-LO

COOHO

LTA4

5-LO

15-LO

12-LO

Platelets

Leukocytes

or

COOH

OH

15R-HETE

COX-II

AspirinEpithelial cells

or Endothelial cells

TNFa

IL-1b

COOH COOH

COOH

O

OH

OH

OH OH

OH

HO

HO

15 epi-LXA4 15 epi-LXB4

15R-Epoxytetraene

Leukocytes

5-LO

15

Aspirin Initiates a Switchin Biosynthesis & Chirality

LXA4OH

COOH

OHHO Switch in chirality

• Enhances bioactivity

• Slow metabolic inactivation

15 epi-LXA4

OH

COOH

OHHO

ASA-triggered

15 epi-Lipoxins

16

Reprinted with permission from Serhan C. Annual Review of Immunology.

Volume 25:101-137 © 2007 by Annual Reviews www.annualreviews.org.

17

PIPP

18

Cardiovascular

Aspirin

Thrombosis

Hypertension

Sudden death

(GISSI study)

Molecular mechanisms of action?

Can EPA and DHA be precursors to generate bioactive mediators?

Polyunsaturated Fatty Acids Are Essential to Health

EPA DHA

Omega-3 PUFA

Milligrams to grams daily

BrainEyeBrain functions

Alzheimer’s

Stroke

Depression

Dry eye

Macular degeneration

DiabetesCancer

Arthritis and Gout

GI tractLungs

Skin disorders

Inflammatory Bowel Diseases

(Colitis, Crohn’s disease)

Allergies

Asthma

Immune systems

Oral

Tooth loss

Periodontal disease

19

5S(6)-epoxy-

18hydroxy-EPE

OOH

COOH

5-LOXLeukocytes

HOOC

O(O)HVascular Endothelial Cells

Aspirin: COX2

18R-hydroperoxy-EPEEicosapentanoic Acid

EPA

Resolvin E1

OH

COOH

HOOH

Resolvin Es Derived from EPA

(5S,12R,18R-trihydroxy-EPE)

© Serhan et al., 2000 J. Exp. Med. 192:1197-1204

© Arita et al., 2005 J Exp. Med. 201:713-722

EPAP450

Aspirin-independent pathway

Healthy Individuals Taking EPA (1g) and Aspirin (160mg)

ng/ml plasma RvE1 18-HEPE EPA

EPA with ASA

(n=6)

0.18 0.06 0.74 0.27 15.95 4.03

EPA w/o ASA (n=3)

0.11 0.02 0.36 0.15 14.20 5.20

20

Resolvin E1: Mechanisms of Action

Resolvin E1

Dendritic cells

Migration

IL-12 production

PBMC

MAPK activation

ChemR23

Epithelium

apical CD55 expression

PMN clearance

Adapted by permission from Macmillan Publishers Ltd.,

Serhan C et al British J Pharmacol. 153:S200-S215, copyright 2008

Route of administration

Diseasemodel Dose, duration

Intravenous Dorsal air pouchPeritonitis

100 ng/mouse, 4 hours100 ng/mouse, 2-24 hours

Intraperitoneal PeritonitisColitisEye retinopathy

10-300 ng/mouse, 2 hours50 µg/kg, 4-12 days10 ng/day, 17 days

Topical Periodontitis 4 µg/tooth, 6 weeks

BLT1 Neutrophils

migration

superoxide anion

21

Resolvin Ds Derived from DHA

Docosahexanoic Acid

DHA

17S-H(p)DHA

LOX

Resolvin D1 Resolvin D2 Resolvin D3 Resolvin D4

Leukocytes

COOH

H(O)O

4S(5)-epoxide intermediate

COOH

HO

HO

OH

HO

OH

COOHOH

COOH

OH

OH

HO

OH

OH

COOH

OH

4S-hydroperoxy,17S-HDHA

7S(8)-epoxide intermediate

7S-hydroperoxy,17S-HDHA

© Serhan et al., 2002 J. Exp. Med. 196:1025-1037

LOX

Neutrophils

Transendothelial migration

Microglia

IL-1 expression

22

Resolvin D1 In Vivo Actions

Disease model Action Dosage (Route) Duration of

post-

exposure

Reference

Skin (Dorsal

Air pouch)

Reduces PMN infiltration 100 ng/mouse

(intrapouch)

4 h Serhan et al. (JEM

2002)

Hong et al. (JBC.

2003)

Peritonitis 100 ng/mouse 2 h Hong et al. (JBC

2003)

0.05-50 µg/kg (IV) 4 h Sun et al. (JBC, 2007)

KidneyIschemia-

reperfusion

Protects in renal perfusion injury

by limiting PMN infiltration

5 µg/mouse (IV, SC) 24-48 h Duffield et al. (JI, 2006)

Eye

(Retinopathy)

Reduces vaso-obliteration by

neovascularization

10 ng/day(IP), from

postnatalday 6 to

day 17

17 days Connor et al. (Nat.

Med., 2007)

Reduces PMN infiltration(IV)

23

Docosahexanoic Acid

DHA

17S-H(p)DHA

LOXBrain,

Microglial, retinal cells

COOH

H(O)O

COOH

O

16(17)-epoxy-DHA

17S-HDHA

COOH

H O

reduction

enzymatic

epoxydation

enzymatic

hydrolysis

second oxygenation

O2

10S,17S-diHDHA

Neuroprotectin D1

Protectin D1 (PD1)

Human PBMC TH2-skewed PBMC

15-LOX-1

reduction

Protectins Derived from DHA

This research was originally published in the Journal of Biological Chemistry. Hong et al. “Novel Docosatrienes and 17S-Resolvins Generated from Docosahexaenoic Acid in Murine Brain, Human Blood, and Glial Cells.” J Biol. Chem. 2003, 278:14677-14687. © The American Society for Biochemistry and Molecular Biology

COOH

OH

OH

Serhan et al. J. Immunol. 2006;176:1848-1859. Copyright

2006. The American Association of Immunologists, Inc.

24

Protectin D1: Potent Stereoselective Actions

COOH

OH

OH

Neuroprotectin D1

Protectin D1 (PD1)

Low nanomolar range

Cell type Bioactions

PMN Transendothelial migration

Macrophage Non-phlogistic phagocytosis of apoptotic PMN

T-cell TNF- and IFN- secretion promotes apoptosis

Microglia IL-1 expression

Epithelia Oxidative stress-induced apoptosis in retinal

pigment epithelia

Route of administration

Disease model Dose, duration

Intravenous PeritonitisAsthmaKidney ischemia-reperfusion

100 ng/mouse, 2 hours2-200 ng/mouse, 4 days5 µg/mouse, 1-2 days

Perfusion Stroke 0.4 µg/mouse, 48 hours

Intraperitoneal PeritonitisColitisEye (retinopathy)

0.1-300 ng/mouse, 2-24 hours50 µg/kg, 4-12 days10 ng/day, 17 days

Topical Eye (wound healing)Periodontitis

1 µg/mouse, 48 hours4 µg/tooth, 6 weeks

Adapted by permission from Macmillan Publishers Ltd., Serhan C et al British J Pharmacol. 153:S200-S215 copyright 2008

Adapted by permission from Macmillan Publishers Ltd., Serhan C et al Nat. Rev. Immunol. 8:349-36 copyright 2008

25

500 l HCl 0.1N, pH = 1.5

5% CO2

Normalize pH to 7.0

with PBS

Exposed to mediators

Responses To Tissue Injury Restitution of Injured Bronchial Epithelia

5min, 37°C

26

NHBE cells

without acid

Transmission Electron

Microscopy :

Morphological features cg

b

gBonnans C et al Am J Pathol 2006, 168:1064-1072

with permission from the American Society for

Investigative Pathology.

27

5 min after acid injury

Bonnans C et al Am J Pathol 2006, 168:1064-1072 with permission from the American Society for Investigative Pathology.

28

2h after acid

injury

Bonnans C et al Am J Pathol 2006, 168:1064-1072



29

6h after acid

injury




30

Acid induced ALX expression

0 2 12 24 72

0

1

2

3

4*

AL

X R

NA

exp

ressio

n(F

old

in

du

cti

on

)

Time (hours)

*

Bonnans C et al Am J Pathol 2006, 168:1064-1072 with

permission from the American Society for Investigative Pathology.

31

LXA4 Stimulated Basal NHBE

Cell Proliferation

0

1

2

3

4

Control 10 100 1000 PGE2 EGF

*

**

*

*

LXA4 (nM)

Basal N

HB

E p

rolif

era

tion

(Fold

induction)


32

LXA4 Inhibited Acid-Induced IL-6 Release

0

20

40

60

80

100

IL-6

(pg/m

l)

Acid LXA4+ PGE2

**

**

(100nM)

LXA4Control


33

LXA4 Inhibited PMN Transmigration Across

Differentiated NHBE

0

20

40

60

80

100

0.1 1 10 100 1000

*

* *

* *

LXA4 (nM)

% in

hib

ition o

f LT

B4-induce

d

PM

N tra

nsm

igra

tion




34

Allergen Sensitization Challenge

Day: 0 7 14 15 16 17 18

OVA: 10 g 10 g 6%

i.p. i.p. Aerosol

+/- LX analog

i.v.

(30 min prior)

On Day 18, determine: 1. Airway Inflammation

- Lipid Profile

- Histology/BAL

2. Airway Reactivity

Responses To Noxious StimuliAnimal Protocol for Allergic Asthma

35

Interleukin-13 in BAL Interleukin-4 in BAL

Veh. ATLaZK-994 Monte.

0

100

200

300

400

500

IL-1

3 (

pg

/ml)

0

20

40

60


IL-4

(p

g/m

l)

8

6

4

2

0


Lym

ph

oc

yte

s x

10

4

Lymphocytes in BAL

0.5 mg/kg, gavage 0.5 mg/kg, gavage0.5 mg/kg, gavage

*

*

**

*

*

*

* P < 0.05 vs vehicle

N = 10 13 6 13 N > 4 all groups N > 4 all groups

ZK-994 and ATLa are orally active in allergic airway inflammation,

modulate Th2 cytokine production and lymphocyte infiltration

3-oxa-ATL efficacy at least equivalent to oral montelukast, modulatory profile broader

OVA-Induced Allergic Airway Inflammation

Levy et al. FASEB J. 21,3877-3884. 2007 © FASEB

36

Control Montelukast ATLa

40x magnification

Representative of n > 4vascular

airways

Impact Of LX Stable Analog and Montelukast On Allergic

Airway Inflammation: OVA


37

Allergen Sensitization Challenge

Day: 0 14 21 23

CRA: 10 g 10 g 4 g 4 g

i.p. nasal i.t. i.t.

On Day 24, determine: 1. Airway Inflammation

- Lipid Profile

- Histology/BAL

2. Airway Reactivity

Animal Protocol for Cockroach Allergen

Sensitization and Airway Challenge

+/- LX analog

(2h prior)

38

CRA-induced Allergic Airway Inflammation

Peribronchial Eosinophilia

control ZK-994

N = 6

*p = <0.05

Peribronchial eosinophilia markedly by ZK-994

Oral ZK-994 modulates lung cytokine/chemokine networks significantly:

- Cytokines: IL-5 (Th2), IFN (Th1)

- Chemokines: C10, RANTES, eotaxin


39

CRA-induced Allergic Airway Inflammation

10 g/ml Blinded


40

0 7 2514 1815 16 17 19 20 21

OVA aerosol

challenge

Time (d) OVA sensitization Resolution

Vehicle Ri~5 days

LXs and Resolution of Allergic Airway Inflammation

LXa Ri~2.5 days

0

1

2

3

4

18 21 25

*

Time (d)

BA

LF

ce

lls (

10

6)

Vehicle

LX analog

0

LX analog

To

tal B

AL

F c

ells (

10

6)

0.0

0.5

1.0

1.5

2.0

2.5

*

Protocol Day 21n > 3, *P < 0.05

*

0.0

100

200

300

400

LX

A4

(pg/m

l B

ALF

)

Day 18 21

LX analog

Adapted by permission from Macmillan Publishers Ltd., Wearsch P et al Nat. Immunol 9:873-881, copyright 2008.

41

Expression of Human ALX in Transgenic Mice

Decreases Pulmonary Inflammation

Non-tg hALX-tg

0

1

2

3

**

*Total Eos Mac Lymph

Non-tghALX-tg

PMN

Ce

lls

(x

10

6)

in B

AL

Seru

m Ig

E (%

Incre

ase)

1

2

P/P P/O O/P O/O

Non-tg

*

hALX-tg

Adapted by permission from Macmillan Publishers Ltd., Levy B et al Nat. Med 8:1018-1023 copyright 2002.

42

Protectin D1 Dampens Airway Inflammation

Lung histopathology

Vehicle

Aerosol challenge of ovalbumin

Allergen-driven leukocyte infiltration

Vehicle Eos Lymphs

Cells in

BA

LF

(x10

5) Vehicle

PD1 (after aerosol challenge)

Adapted from Levy B, J. Immunology 2007, 178:496-502.

Copyright 2007. The American Association of Immunologists Inc.

43

Outline





Distinguish anti-inflammation from promoting

resolution


44

Inhibition of Neutrophils Stimulation of Monocytes/Macrophages

Distinguishing Anti-Inflammation From Pro-Resolution

45

Anti-Inflammation is Distinct From Promoting Resolution

Dual Actions of Lipoxins

Anti-inflammation – (Stop Signals)

Inhibition of PMN recruitment and activation

Inhibition of dendritic cell motility/NK cell cytotoxicity

Block cytokine release from activated T-cells

Pro-resolving – (Go Signals)

Stimulate non-phlogistic recruitment of monocytes

Stimulate uptake of apoptotic PMN by macrophages

Reduce MMP release from fibroblasts

Enhance host defense/bacterial killing

46

Acid(pH 1.5, 0.1 N, 50 l)

(Time)

Harvest

BALTimed intervals(2, 12, 48, 72h)

PosteriorAnterior

Non-Lethal Experimental Model of ALI

Levy B, J. Immunology 2010, 184: 863-843 Copyright 2010. The American Association of Immunologists Inc.

47

The Kinetics of Inflammation and Resolution

After Acid-initiated Acute Lung Injury

Leu

ko

cyte

s i

n B

AL

F (

10

3cells)

20

15

10

5

0

0 2 12 48 72

Time (h) after acid instillation

*

*

500

300

400

0

200

0 2 12 48 72

(h)

*

PMN

Lymphs


48

Involvement of COX In Host Response To

Acid Initiated Lung Injury

Time (h) after acid instillation

0

To

tal le

uko

cyte

s i

n B

AL

F (

10

4cells)

12

8

4

02 12 48

*

vehicle

COX-2 inhibitor

*


49

0

4

8

12

16

Effect of COX Inhibition on Airway Inflammation

48h After Acid Injury

-

-+

-COX-2 inhibitor

ASA

*

-+

*

+

+COX2 -/-

mice

*

Fukunaga K, J. Immunology 2005, 174: 5033-5039 Copyright 2005. The American Association of ImmunologistsInc.

50

COX-2 inhibitor

40

30

20

10

00 2 12 48

Vehicle

*

Time after acid instillation (h)

Impact of COX-2 Inhibition on Airway LXA4

After Acid Initiated Injury (pg/ml)

LX

A4

(pg

/ml B

AL

)

Fukunaga K, J. Immunology 2005, 174: 5033-5039 Copyright 2005. The American Association of Immunologists Inc.

51

OH

COOH

HO

O

PGE2

15-LipoxygenaseStimuli

Cyclooxygenases

5-Lipoxygenase

OHHO

COOH

OH LXA4

CRECREB

P

Eicosanoid ‘CLASS SWITCH ‘ to Resolution

When & Where-------treatment ?

52

Time, January 21, 2002

53

Responses to Bacterial InfectionMurine model of aspiration pneumonia

HCl (pH1, 0.1N, 25 l) E.coli (1-2×105 CFU)

12, 24 or 48h 24h Collect left lungs

→Calculate BGI

Bacterial growth index (BGI):lung CFU

original inoculum instilled

54

*

HCl + + +

Time interval from HCl or

PBS to E. coli (h) 12 24 48

0

1.0

2.0

Bacterial growth index

-

12

Acid injury transiently impairs airway host defense

Mean ± SEM (n>4, each)*, P<0.05 vs HCl (-) Adapted from Seki, H. Immunol .2010, 184:836-843. Copyright

2010. The American Association of Immunologists Inc.

55

Effect of RvE1 in a model of aspiration pneumonia

HCl E.coli

12h 24h

Survival rate

・Calculate BGI

・Measure MPO

・Measure inflammatory mediators

・ Lung histopathology

Collect left lungs

* *

RvE1 (100 ng), EPA (100 ng) or saline i.v.

30min prior to HCl instillation

or

2h after E. coli inoculation

*

56

†

Treatment saline EPA RvE1saline

HCl - + + +

E.coli + ++ +

*

0

0.5

1.0

1.5B

acte

rial G

row

th I

ndex

RvE1 enhances bacterial clearance in a model of

aspiration pneumonia

Mean ± SEM (n>12, each)

*, P<0.05 vs saline / HCl (-) / E. coli (+)†, P<0.05 vs saline / HCl (+) / E. coli (+)

Adapted from Seki, H. Immunol .2010, 184:836-843. Copyright 2010. The American Association of Immunologists Inc.

57

0

400

800

1200

MP

O (

ng/m

l)

*

E.coli ++-

HCl + +-

RvE1 +- -×200

RvE1 (-)

×40

RvE1 (+)

†

RvE1 blocks leukocyte accumulation after

aspiration pneumonia

Mean ± SEM (n>12, each)

*, P<0.05 vs RvE1 (-) / HCl (-) / E. coli (-)

†, P<0.05 vs RvE1 (-) / HCl (+) / E. coli (+) Adapted from Seki, H. Immunol .2010, 184:836-843. Copyright 2010. The American Association of Immunologists Inc.

58

Outline







59

What window into human

pathophysiology can murine

models of asthma provide?

60

Neutrophils In Severe Asthma

Anti-CD15, 1:250 dilution

Hypothesis: Is Severe Asthma a Lipoxin Deficient Condition?

Histology courtesy of K. Haley

61

Lipoxin A4 Generation in Activated Whole Blood

*P<0.05 c/w health, **P<0.05 c/w moderate

Not Severe Severe

*

**

0

1

2

3

Lip

ox

in A

4

(ng

/ml a

cti

va

ted

blo

od

)

Reprinted with permission of the

American Thoracic Society. Copyright ©

American Thoracic Society. Levy, B 2005

“Diminished Lipoxin Biosynthesis in

Severe Asthma.” American Journal of

Respiratory and Critical Care Medicine.

Vol. 172:824-830. The official journal of

the American Thoracic Society.

62

Leukotriene Generation in Activated Whole Blood

*P<0.05 c/w health, **P<0.05 c/w moderate

Non-Asthma

Not Severe

Severe

*

**

LTB4 CysLTs

0

1

2

3

4L

eu

ko

trie

ne

Bio

syn

the

sis

(Fo

ld In

cre

as

e)

Reprinted with permission of the American

Thoracic Society. Copyright © American

Thoracic Society. Levy, B 2005 “Diminished

Lipoxin Biosynthesis in Severe Asthma.”

American Journal of Respiratory and Critical

Care Medicine. Vol. 172:824-830. The official

journal of the American Thoracic Society.

63

Relationship Between Lipoxygenase-Derived Eicosanoids

In Whole Blood and Airflow Obstruction

P=0.003

5-LO Products In Severe Asthma –

Increased LTs

Decreased LXs

Reprinted with permission of the American Thoracic Society. Copyright © American

Thoracic Society. Levy, B 2005 “Diminished Lipoxin Biosynthesis in Severe Asthma.”

American Journal of Respiratory and Critical Care Medicine. Vol. 172:824-830. The

official journal of the American Thoracic Society.

64

LXA4 and 15S-HETE Levels in BALF

Healthy0

100

200

iLXA

4(p

g/m

l BALF)

*

150

50

Not Severe

Asthma

Severe

Asthma

*p < 0.05

LXA

4/CysL

Ts

0

10

20

30

*

Healthy Not Severe

Asthma

Severe

Asthma

n.s.

Reprinted with permission of the American Thoracic Society.

Copyright © American Thoracic Society. Planaguma A, 2008

Airway Lipoxin A4 Generation and Lipoxin A4 Receptor

Expression are Decreased in Severe Asthma. American Journal

of Respiratory and Critical Care Medicine. Vol. 178:574-582. The

official journal of the American Thoracic Society.

65

Lipoxin Biosynthetic Gene Expression in Multiple

Anatomic Compartments

Fold change = 2- CT

5-LO

Blood

-15

-10

-5

0

5

BAL cells

Fold

change

(Severe

to N

ot

Severe

ast

hm

a s

ubje

cts

)

-15

-10

-5

0

5

EBBs

-15

-10

-5

0

5

15-LOA 15-LOB COX-2

Reprinted with permission of the American

Thoracic Society. Copyright © American

Thoracic Society. Planaguma A, 2008

Airway Lipoxin A4 Generation and Lipoxin

A4 Receptor Expression are Decreased in

Severe Asthma. American Journal of

Respiratory and Critical Care Medicine. Vol.

178:574-582. The official journal of the

American Thoracic Society.

66

LXA4 Receptor Gene Expression

in Peripheral Blood

Leukocytes

Healthy5.75

2.75Severe

asthma

Severe

Asthma

Not Severe

Asthma

Healthy0.00

0.25

0.50

0.75

1.00

ALX e

xpre

ssio

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Reprinted with permission of the American Thoracic Society. Copyright © American Thoracic Society. Planaguma A, 2008

Airway Lipoxin A4 Generation and Lipoxin A4 Receptor Expression are Decreased in Severe Asthma. American Journal of

Respiratory and Critical Care Medicine. Vol. 178:574-582. The official journal of the American Thoracic Society.

67

PMN Eosinophils Monocytes

0

5

10

15

20

MF

I-(M

FI is

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on

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l)

Lymphocytes

0

1

2

3

4

5

MF

I-(M

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0

1

2

3

4

5

6

7

8

9

10

11

MF

I-(M

FI is

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P< 0.05

Expression of ALX Receptors in

Peripheral Blood Leukocyte Subsets

0

1

2

3

4

5

6

MF

I-(M

FI is

oty

pe c

on

tro

l)P< 0.05

Reprinted with permission of the American Thoracic Society. Copyright © American Thoracic Society. Planaguma A, 2008

Airway Lipoxin A4 Generation and Lipoxin A4 Receptor Expression are Decreased in Severe Asthma. American Journal of

Respiratory and Critical Care Medicine. Vol. 178:574-582. The official journal of the American Thoracic Society.

68

Summary and Conclusion

Today, we have presented evidence that:

LXA4 levels are decreased in peripheral blood and BAL fluids in severe

compared to not severe asthma subjects

CysLTs and 15-HETE levels are increased in both severe and not severe

asthma subjects

5-LO, 15-LOA, 15-LOB and COX-2 are under distinct regulatory control

that varies by anatomic compartments and asthma severity

In conclusion, our findings indicate that severe asthma is characterized

decreased lipoxin biosynthesis. In conjunction with the decreased LXA4

receptor expression, these data suggest that more severe variants of

asthma may result from a defect in counter-regulatory signaling.

69

Lipoxin Defects In Other Diseases of Chronic Inflammation

Asthma –

Severe Asthma JACI. 2005, 115:55.

Exercise induced Asthma J Asthma. 2008, 45:161.

Cystic Fibrosis – Nature Immunol. 2004, 5:388.

Inflammatory Bowel Disease –

Ulcerative Colitis Prost. & Other Lipid Med. 2006, 79:84

Vasculitis –

Henoch–Schönlein purpura PLEFA. 2009, 80:177.

70

Non-Invasive Technique For Sampling Airway BiomarkersExhaled Breath Condensates

71

Characteristics of Subjects*

Healthy Asthma Exacerbation

Sample size (n) 3 4

Age (yrs) 28 +/- 1 41 +/- 6

M:F 1:2 2:2

Race 3 other 2 Caucasian, 1 African

American, 1 other

Current cigarette smoker 0 2

*EBC was collected during 10 min of tidal breathing from individuals in the BWH emergency

department with acute asthma exacerbation and a control group of healthy subjects.

Plus-minus values are means +/- SD.

Adapted from Levy B J. Immunol. 2007, 178: 496-502. Copyright 2007 The American Association of Immunologists Inc.

72

Generation of Protectin D1 in asthma

d4-PGE2 i.s.


73

d4-PGE2 i.s.

Calibration curve r2 = 0.991

Human EBC 17(S)-Hydroxy-DHA and PD1 Levels

Healthy Asthma Exacerbation

17(S)-hydroxy-DHA Trace Trace

PD1 2.23 +/- 1.55 ng Trace

Sample size (n) 3 4

*Plus-minus values are means +/- SEM.


74

Chronic Inflammation in Disease (Asthma) –A Loss Of Counter-Regulatory Signaling?

75

Resolution of inflammation is an active process that is orchestrated by

specific cells and signals, including PUFA-derived lipid mediators.

Key features in tissue resolution include blocking PMN influx and functions

and enhanced Macrophage-mediated clearance of apoptotic PMN.

Early events in acute inflammation are crucial to timely resolution

pus bonum et laudible

Lipoxins and their bioactive stable analogs are anti-inflammatory and

pro-resolving.

Inflammation Resolution is a Natural Process

76

Outline







78

Acknowledgements

Pulmonary and Critical Care MedicineLevy Lab – Payal Kohli, Caroline Bonnans, Koichi Fukunaga, Oliver Haworth,

Troy Carlo, Anna Planaguma, Guangli ZhuARC - Elliot Israel, Shamsah Kazani, Gautham Marigowda

University of Michigan - PathologyNicholas Lukacs, Aaron Berlin

CETRI - BWHCharles N. Serhan and members

resolution of inflammation - az9194.vo.msecnd.netaz9194.vo.msecnd.net/pdfs/100602/009.pdfkumar et...

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