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
Chemical mediators of resolution for injury, noxious
stimuli and infection - their biosynthesis and bioactions
Distinguish anti-inflammation from promoting resolution
Translation to human disease
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
Courtesy of Nan Chiang
11
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
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
Courtesy of Nan Chiang
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
with permission from the American Society for
Investigative Pathology.
29
6h after acid
injury
Bonnans C et al Am J Pathol 2006, 168:1064-1072
with permission from the American Society for
Investigative Pathology.
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)
Bonnans C et al Am J Pathol 2006, 168:1064-1072 with permission from the American Society for Investigative Pathology.
32
LXA4 Inhibited Acid-Induced IL-6 Release
0
20
40
60
80
100
IL-6
(pg/m
l)
Acid LXA4+ PGE2
**
**
(100nM)
LXA4Control
Bonnans C et al Am J Pathol 2006, 168:1064-1072 with permission from the American Society for Investigative Pathology.
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
Bonnans C et al Am J Pathol 2006, 168:1064-1072
with permission from the American Society for
Investigative Pathology.
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
Veh. ATLaZK-994 Monte.
IL-4
(p
g/m
l)
8
6
4
2
0
Veh. ATLaZK-994 Monte.
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
Levy et al. FASEB J. 21,3877-3884. 2007 © FASEB
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
Levy et al. FASEB J. 21,3877-3884. 2007 © FASEB
39
CRA-induced Allergic Airway Inflammation
10 g/ml Blinded
Levy et al. FASEB J. 21,3877-3884. 2007 © FASEB
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
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
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
Levy B, J. Immunology 2010, 184: 863-843 Copyright 2010. The American Association of Immunologists Inc.
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
*
Levy B, J. Immunology 2010, 184: 863-843 Copyright 2010. The American Association of Immunologists Inc.
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
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
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
n(R
ela
tive t
o H
ealt
hy s
ubje
cts
)
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
oty
pe c
on
tro
l)
Lymphocytes
0
1
2
3
4
5
MF
I-(M
FI is
oty
pe c
on
tro
l)
0
1
2
3
4
5
6
7
8
9
10
11
MF
I-(M
FI is
oty
pe c
on
tro
l)
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.
Adapted from Levy B J. Immunol. 2007, 178: 496-502. Copyright 2007 The American Association of Immunologists Inc.
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.
Adapted from Levy B J. Immunol. 2007, 178: 496-502. Copyright 2007 The American Association of Immunologists Inc.
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
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
77
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