1Bruce D. Levy

Associate Professor of Medicine Pulmonary and Critical Care Medicine

Brigham and Womens Hospital

Harvard Medical School

ESPEN 2012 BarcelonaNew Horizons in Clinical Nutrition

September 9, 2012

Disclosures: Licensed Patents Bayer Healthcare, Resolvyx PharmaceuticalsConsultant: Novartis, Eisai Pharmaceuticals

Resolution of inflammation: Roles for resolvins and protectins

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

BrainEye Brain functions Alzheimers Stroke Depression

Dry eye Macular degeneration

DiabetesCancerArthritis and Gout

GI tractLungs

Skin disorders

Inflammatory Bowel Diseases(Colitis, Crohns disease)

Allergies Asthma

Immune systems

Oral

Tooth loss Periodontal disease

Derek Willougby et al Nature Immunology Reviews 2002

Wave Of Inflammation

InflammationResolution

2Images from C. Fanta and L. Kobzik

Resolution Of Acute Inflammation

Lipoxins ProtectinsResolvins

D-seriesE-series

COOH

OH

OH

OH

COOH

HOOH

RvE1

LXA4

PD1

HO OH

OH

COOH

COOH

HO

HO

OH

RvD1

Specialized Pro-Resolving Mediators

Monocyte/Macrophages

PMNs

Act

ivity

Edema

SPMResolvinsProtectins

timeFamilies Specialized Pro-Resolving Mediators (SPM):

A Novel Genus Endogenous Lipid Mediators

Programmed Resolution

Ideal Outcome of Inflammation : Complete Resolution Systems Approach to Mapping Resolution

Resolution

FibrosisChronic

Inflammation

Acute Inflammation

unresolved

Lipid mediator class switching

Chemical mediators:ProstaglandinsLeukotrienes

PGE2, D2, LTB4 Return to homeostasisLX

sec-min min hrs --days

C20:4 C20:5 C22:6

Pro-Resolving Mediator Signaling Circuits

AA (C20:4)

DHA (C22:6)

EPA (C20:5)

LXA4

17S-RvD117R-RvD1

RvE1

ALX/FPR2

GPR32

CMKLR1

ASA-COX-25-LO

15-LO5-LO

PUFA Mediator Receptor

Key Features in Tissue ResolutionCatabasis

Anti-InflammationBlock PMN recruitmentStop PMN ActivationDecrease vascular permeabilityDampen ROS

RvE1, RvD1, PD1

Pro-ResolutionStimulate endothelial NO and PGI2Epithelial restitutionPromote Mac phagocytosis of apoptotic PMNs, microbes and debris

RvE1, RvD1, PD1

3Acid(pH 1.5, 0.1 N, 50l)

(Time)

HarvestBALTimed intervals

(2, 12, 48, 72h)

PosteriorAnterior

NonNon--Lethal Experimental Model of ALILethal Experimental Model of ALI

Fukunaga et al. J. Immunol. 2005, 174:5033.

Treatment saline EPA RvE1salineHCl - + + +

E.coli + ++ +

*

0

0.5

1.0

1.5

Bacte

rial G

rowth

Index

RvE1 Enhances Bacterial Clearance in a Model of Aspiration Pneumonia

Mean SEM (n>12, each)*, P

4Asthma Pathology

Provided by Robert Padera

5S(6)-epoxy-18hydroxy-EPE

OOH

COOH

5-LOX Leukocytes

HOOC

O(O)HVascular Endothelial Cells

Aspirin: COX2

18R-hydroperoxy-EPEEicosapentanoic AcidEPA

Resolvin E1

OH

COOH

HOOH

Resolvin Es Derived from EPA

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

Serhan et al., J Exp Med. (2000) 192, 1197Arita et al., J Exp Med. (2005) 201, 713

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

RvE1

0 7 2514 1815 16 17 19 20 21

OVA aerosol challenge

Time (d) OVA sensitization Resolution

Vehicle Ri~5 days

*

BALF

cel

ls (

106 )

0.00

0.25

0.50

0.75

1.00

1.25

1.50

*

RvE1 (100ng/d, iv)

Vehicle

*

Lymp

hocy

tes

Macro

phag

es

Eosin

ophil

s

Impact Of RvE1 On Airway Inflammation Resolution

RvE1 Ri~2.5 days

0

1

2

3

4

18 21 25

*

Time (d)

BALF

cel

ls (

106 )

VehicleRvE1

0

RvE1

Tota

l BAL

F ce

lls (

106 )

0.0

0.5

1.0

1.5

2.0

2.5

*

Vehic

le

RvE1

Protocol Day 21n > 8, *P < 0.05 Haworth et al. Nature Immunology. 2008; 9:873.

400

400

Br Br

VV

Br Br

BrBr

Vehicle RvE1

RvE1 Dampens Allergic Airway ResponsesHistopathology and Airway Reactivity

H&E

PAS

Representative of n > 6

0.00

0.25

0.50

0.75

1.00

1.25

1.50

MC

h lo

g ED

200

*

Vehic

leRv

E1

n > 6, *P < 0.05

AHR

Haworth et al. Nature Immunology. 2008; 9:873.

5Resolvin 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

AT-RvD1 Promotes Resolution of Allergic Airway Responses: Ag Clearance

ALX/FPR2 Expression:Epithelial Cells

LeukocytesMacrophages

Journal of Immunology. 2012. in press.

Docosahexanoic AcidDHA

17S-H(p)DHA

LOX Brain, Microglial, retinal cells

COOH

H(O)O

COOH

O

16(17)-epoxy-DHA

17S-HDHA

COOH

H Oreduction

enzymatic epoxydation

enzymatic hydrolysis

second oxygenation

O2

10S,17S-diHDHA

Neuroprotectin D1Protectin D1 (PD1)

Human PBMC TH2-skewed PBMC

15-LOX-1

reduction

Protectins Derived from DHA

COOH

OH

OH

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

Protectin D1 Dampens Airway Inflammation

Lung histopathology

Vehicle

Aerosol challenge of ovalbumin

Allergen-driven leukocyte infiltration

Vehicle Eos Lymphs

Cel

ls in

BAL

F (x

105 )

VehiclePD1 (after aerosol challenge)

J. Immunology 2007, 178:496-502.

6Non-Invasive Technique For Sampling Airway BiomarkersExhaled Breath Condensates

Generation of Protectin D1 in asthma

d4-PGE2 i.s.

J. Immunol. 2007, 178: 496-502.

d4-PGE2 i.s.

Calibration curve r2 = 0.991

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

43Sample size (n)

Trace2.23 +/- 1.55 ngPD1

TraceTrace17(S)-hydroxy-DHA

Asthma ExacerbationHealthy

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

J. Immunol. 2007, 178: 496-502.

In summary, we have presented evidence for:

Resolution of inflammation is an active process that is orchestrated by specific cells and signals, including PUFA-derived lipid mediators.

RvE1, RvD1 and PD1 are natural compounds, enzymatically derived from EPA and DHA that display pro-resolving actions

Pro-resolving mediators (RvE1) enhance host defense

In conclusion, understanding the cellular and molecular mechanisms for pro-resolvingmediators in catabasis is providing new insights into tissue responses for resolution of inflammation in health and the pathophysiology of sepsis and several other diseases ofchronic inflammation with new opportunities for therapeutic intervention.

Inflammation Resolution is a Natural Process

7Acknowledgements

Center for Experimental Therapeutics and Reperfusion InjuryCharles N. Serhan, DirectorSungwhan Oh, Fei Gao

Pulmonary and Critical Care MedicineLevy Lab Troy Carlo, Oliver Haworth, Michael Pfeffer, Anna Planaguma, Hiroyuki Seki, Mohib Uddin, Guangli Zhu, Jennifer Colby, Emiko Ono, Cindy Barnig, Alex de Paula Rogerio,Raja Abdulnour, Cindy Hana Okuma, Jack Timmons

Asthma Research Center - Elliot Israel, Gautham Marigowda, Shamsah KazaniMichael Wechsler, Christopher Fanta

Funding:NHLBI, NIAID, NIGMS