IOM NAS Washington June 24 2010

Charles N. Serhan Director & Prof. Center for Experimental Therapeutics

BWH

Prof. Dept Oral Medicine, Infection and Immunity Harvard School of Dental Medicine

Harvard Medical School

New Genus of Pro-Resolving Lipid Mediators from EFA

Resolvins & Protectins : SPM in Inflammation & Organ Protection

Disclosures for Charles N. Serhan

In compliance with ACCME policy, Harvard Medical School requires the following disclosures to the session audience:

Research Support/P.I.NIH : NIGMS, NIDCR, NIDDK, BayerHeathCare

NIH Center Grant

Employee No relevant conflicts of interest to declare

Consultant No relevant conflicts of interest to declare

Major Stockholder No relevant conflicts of interest to declare

Speakers Bureau No relevant conflicts of interest to declare

Honoraria No relevant conflicts of interest to declare

Scientific Advisory Board Resolvyx Pharmaceuticals

Founder

Resolvyxy Pharmaceuticals

nano - micrograms

Anti-Inflammation

Pro-Resolution

Resolvins & Protectins

Animal Disease Models

RvE1, RvD1, PD1

Eyes↓ Vaso-obliteration and

neovascularization (Retinopathy)

↑ Wound healing (Cornea thermal injury)

RvE1

Cardiovascular↓ Platelet aggregation

RvE1, RvD1, RvD2

GI tract↓ PMN and weight loss

↓ Peritonitis

↑ Survival (Colitis)

RvD1, PD1

Kidney↓ Renal ischemic injury

Reno protective

Serhan et al.,

Nat Rev Immunol.

© 2010 Serhan et.al.

NPD1/ PD1

Brain↓ Stroke damage and PMN

entry into the brain

↑ Neural cell survival Stem Cell

RvE1

Oral Medicine↓ Inflammation-induced tissue

and bone loss (Periodontitis)

PD1 & RvE1

Lungs↓ Airway inflammation (asthma)

RvD1, RvE1

Pain Neuropathic↓ Inflammatory pain

Central & Periperial

Human Deficiencies (n-3 EFAs)

Nutritional Deficiencies : Nutritional Armor Captain Joseph R. Hibbeln, M.D.

DHA

• Neural Protection

•Severe Depression, postpartum depression

• Acute coronary syndromes

• Chronic Alcoholism

COOH

Brain ≈ Exudate?

C22:6

Autacoids: Chemical Mediators

local acting short lived paracrine / autocrine signals ;

Lipid Mediator-Informatics

AACC

Proteomics

Metabolomics systems networks

Lipidomics

Lipidome

Genomics

Today’s Outline

• Structural Elucidation of Novel Specialized Pro-

Resolving Chemical Mediators (SPM)

Pro-Resolving Actions : Resolvins & Protectins

•Biosynthesis and Actions of Resolvins & Protectins

in Disease Models

•MOA of Resolvins & Protectins

•Gaps in knowledege

© 2010 Serhan et.al.

Clinical Implications Is pus “good or bad” for you ?

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passive process

Decision Paths in Acute Inflammation: Resolution or Chronic Inflammation ?

Injury

Infection

Acute

Inflammation

Abscess formation

Wound Healing Scarring

CN Serhan Ann Rev Pathology 2009

Cardinal signs Calor (heat)

Rubor (redness)

Tumor (swelling)

Dolor (pain)

Loss of function

Resolution

Chronic Inflammation

Rheumatoid Arthritis Periodontal Disease

DiabetesCardiovascular diseasesAsthma

Proteases

O2 radicalsWAR : Even if Defensive Unwanted Side

Effects

Human Neutrophils1st line host defense

Protective

Inflammation

ResolutionNew concept active process

Rapidly turned on inflammatory challenge

Active cellular and biochemical pathways

Generation of SPM

CN Serhan Ann Rev Immunology 2007

Sample collection

exudateCellular composition

Differential countingFACS analysis

LM-Lipidomics Proteomics

Acute Inflammation

Temporal Differential

Solid phase extraction

LC-UV-MS-MS

Informatics

Profiling of lipid mediators

2D- gel electrophoresis

Bioinformatics/Data analysis

Proteins of interest/Novel proteins

LC-nanospray-MS-MS

Air pouch skinOral InflammationPeritonitisAirway LungRenal I/R Stroke

TNF-a

dermis

inner lining

lower lining

Cavity

Exuda

te

Systems Approach Mapping ResolutionTemporal-Differential Analyses of Resolution

SSpatial & Temporal Relationships

TNF-a

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Lipid Mediator-Lipidomics Informatics

GC-MSLC-UV

Solid phaseextraction

with IS

Lipid mediator profiles“data mining”

Profiling of

lipid mediators

MRMsELISA

LC-UV-MS-MS

Physical properties

Cells, tissues, and inflammatory

exudates

Lipidomic databases,Identification and search

algorithm

© 2010 Serhan et.al.

unbiased approach

Neuroprotectin

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

Activity

Edema

SPMResolvins Protectins

time

OHOHOH

OHOHOH

COO

HO

OH

RvD1

HOOH

Families 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

Eicosanoid Class Switching

Resolution phase hrs to days

Acute phase min to hrs

prostaglandins

Leukotrienes

Time

Alpha signals Omega

prostaglandins

Leukotrienes

Serhan & Saville Nature Immunology

Reduce FurtherPMN Influx

non-fever causing

Efferocytosis

Stop PMN ↓ NF-kB activation↓ limit PMN Infiltration ↓ CD11b/cd18 adhesion

8

Resolvin E1 : Biosynthesis,

Stereochemistry and MOI

Resolvin E1

OH

COOH

HO

OH

Murine Skin Air Pouch

Time hours

Exu

date

PM

N(x

10

3/p

ou

ch

)

0

600

300

TNF

2 4 6

Resolving Exudates

CN Serhan et al JEM

Dendritic cells↓ Migration↓ IL-12 production

PBMC- MAPK activation

ChemR23

BLT

Resolvins: resolution phase interaction products

X 1000 aspirin

Macrophages enhances phagocytosis

COOH

OH

HO

Resolvin E2

Reduction

Regulates neutrophil infiltrationEquipotent as RvE1 at low dose RvE2 > RvE1 Pathway Biomarker

18-hydroxyEPE

HOOC

OH

5S-hydroperoxy-18-hydroxy-EPE

COOH

OOH

HO

5-LO

Regulates neutrophil infiltration Promotes resolution Regulates dendritic cell function and IL-12 production

Protects from osteoclast-mediated bone destruction

HOOC

OH O

5-LO

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

COOH

HOOH

OH

Further enzymatic process

Resolvin E1

Cell : Chem & Bio

E Series Resolvin Biosynthesis : Temporal 5-LO Function

Human Vascular Endothelial Cells Human

LeukocytesHypoxia

Aspirin-COX2 P450microbial

Reduces Colitis, Asthma

Periodontial Disease Ocular Inflammation

Anti-Inflammatory Properties

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Aspirin-COX2

Microglial cell reduces cytokine expression

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Potent actions pg-ng range

Anti-Inflammatory in vivo PMN “STOP” Infiltration

air pouch, airway inflammation

peritonitis, renal I/R, Liver Protection

Glial cells

Ligand specific cell signaling

Reduces cytokine expression

Protectins Murine Brain, Human Blood and Glial Cells

Autacoids that counter inflammation

Neuroprotectin D1 / Protectin D1

Brain & Retina Bazan et al JBC ; PNAS

Reduces Stroke Damage & Retinal InjuryCorneal Injury & Wound Healing Gronert et al JBC

10R,17S-diHDHA

pM-nM range

© 2010 Serhan et.al.

NPD1/ PD1 is Neuroprotective in Transient Focal Stroke Reduces PMN Mediated Damage

Bazan et al JBC , PNAS

COOH

HO

OH

NPD1/PD1

NPD1/PD1

IL-4

TH2 Cell 15-LOX-1

Immune Activities

• Blocks T-cell and PMN migration

• Inhibits TNF & INF secretion

• Promotes T-Cell apoptosis mediated by raft clustering

• Reduces peritonitis and airway inflammation, etc.

• Upregulates CCR5 expression on apoptotic PMN, as scavenger for CCL3 and CCL5

Brain Ischemia/Reperfusion

• Neuroprotective actions in ischemia/reperfusion injury

• Dampens NFκB expression and COX-2 induction

• Reduces PMN influx

Kidney Ischemia Injury

• Mitigates acute kidney injury

• Reduces leukocytes infiltration

• Blocks TLR mediated activation of macrophage

• Anti-fibrotic action

Biosynthesis of Protectin D1 / Neuroprotectin D1

• Human Neutrophils • Human T-cells • Murine Exudates• Murine Brain

CN Serhan Ann. Rev. Path. 2008

Saline

LXA4

17S-HDHA (RvD1)

NPD1

Biomicroscopy 48hrs Post Epithelial Removal

12HETrELTB4LXA4 17HDHA NPD1

Treatment Post Epithelial Removal

0

20

40

60

80

100

-20

0

20

40

60

80

100

120

140

*

*

*

*

**

24 hrs

Re

-Ep

ith

eli

ali

za

tio

n (

% C

ha

ng

e)

48 hrs

Organ Protection : SPM Accelerate Epithelial Wound Healing

Gronert et al, J Biol Chem, 2005

Topical Treatment 1μg/tid

8 16Time (min)

0

100

240 32

m/z 273 on MS/MS 375

m/z 153 on MS/MS 359

MS/MS 343

MS 327

RvD3

PD1/NPD1

17HDHA

14HDHA

DHA

Re

lati

ve

In

ten

sit

y

m/z 195 on MS/MS 333

8 16Time (min)

0

100

240 32

0

5 m/z 291 on MS/MS 349

m/z 233 on MS/MS 349

MS 301

RvE1PGE3

LTB5

EPA

Re

lati

ve

In

ten

sit

y

Resolvins & Protectins

Fat-1 Transgenic Mice Rich in Endogenous Omega-3 increase Resolvins & Protectins Organ Protection

Omega-3 EPA and DHA

EPA

Hudert et al. Proc Natl Acad Sci USA. (2006) 103, 11276

Protection in Colitis

Fat-1 TGWT

Wei

ght l

oss

(per

cent

cha

nge)

Fat-1 TG

WT

PNAS

DHA

9

P tProtecProtecti ition ition i C lin Colin Colititistis

Fat-1 TGWT

Smith et al Nature Medicine 2007

Elevated levels of n-3 Rv PD1

decreased vaso-obliteration & retinal neovascularization

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ghtl

Wei

ghtl

Wei

ght l

gos

s(p

eros

s(p

eros

s (p

er(p

cent

cha

cent

cha

cent

cha

nge)

nge)

nge)g)

Fat-1 TG

WT

Proteases

O2 radicals

What is pro-resolving ? A new bioaction of SPM

© 2010 Serhan et.al.

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Endogenous anti-inflammation XX pro-resolution

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Non-phlogistic Activation of Macrophages

IL-10 (anti-inflammatory)

Pg

/ml

0

4

8

12

16

LXA4 RvE1 PD1Vehicle

*

0

10

20

30

40

50 IL-6 (pro-inflammatory)

Pg

/ml

** ***

**

IFN-γ

Pg

/ml

0

10

20

30

40

***

******

No apparent changes: TNF-α , KC, JE, MIP-2

LXA4, RvE1 and PD1 Promote Macrophage Phagocytosis

F

4/8

0+

Gr-

1+

ma

cro

ph

ag

e (

% i

nc

rea

se

)

LXA4 RvE1 PD1Vehicle

Apoptotic PMN

**

*

*

0

20

40

60

[ 10 -100 nM]

* P<0.05, **P<0.01, ***P<0.001 (versus vehicle)

Multi-level Mechanisms of Action

↓ PMN

LXA4

↑ MonocyteMacrophages

ALX

Receptor cross-talk

with growth factor

receptors

HUVEC ↓ angiogenesis

VEGFR

Mesangial cell↓ proliferation ↓ fibrosis

PDGFRCTGFR

Receptor c

ross-ta

lk

with G

PCRs

PMN↓ LTB4 functions

BLT1

LTB4

↓ PMN

RvD1

↑ Macrophages

ALX

GPR 32

PMN↓ LTB4 functions

PD1

RPE retina↓ proliferation↓ cell death

↓ PMN

RvE1

↑ MonocyteMacrophages

Chem R 23

BLT1

Specialized Pro-Resolving Mediators

• Stop PMN transmigration and chemotaxis, brake eosinophils

• Block prostaglandins and leukotrienes

• Reduce cytokine release and function (TNFα)

• Non-phlogistic monocyte recruitment

• Uptake and removal of apoptotic PMN and microbial particles by macrophages

• Enhance anti-microbial defense mechanisms and clearance at mucosal surfaces

SPMGeneral Actions

↓PMN-mediated tissue damage↓Pain Signals↓Angiogenesis and cell proliferation↓I/R Injury↓ROS extracellular release↓Adhesion↓Pro-inflammatory cytokines (TNFα, IL-12)

↓DC-lymphocyte interactions (immune synapse)↑Phagocytosis and IL-10

production

SPMSpecific Actions

LXA4

↓DC IL-12 production↓DC migration↓Phosphorylation signalsInhibit NF-κB reporter

gene activationBlock PMN chemotaxis↑Mucosal clearance of

PMN by CD55↑PMN detachment

↑LXA4 production

↑ROS intracellular

↑Microbial killing

RvE1

Arachidonic Acid(AA)

LipoxinsE-Series

Resolvins

D-SeriesResolvins

Protectins/Neuroprotectins

Eicosapentaenoic Acid(EPA)

Docosahexaenoic Acid(DHA)

Families

Precursors

↓PMN adhesion to endothelial cells↑Nitric oxide and prostacyclin

in endothelial cells

↑Microbial killing and clearance

↓Adhesion receptors↓ROS generation & Pro-Inflammatory cytokines (TNFα, IL-8)

↓PMN transmigration↓PGE2 production

↓Neovascularization↓Microglial cell cytokine expression

RvD1

↓NF-κB and COX-2 expression↓Renal fibrosis↓T-cell migration↓TLR-mediated Mφ activation

↓TNF and IFNγ release

↑Protection of retinal

pigment epithelial cells↑Neuroprotective actions

↑CCR5 expression on T-cells

PD1/NPD1

RvD2

CN Serhan in press 2010

Zymosan-initiated

inflammation

ψ max = 16.5x106, Tmax ~12 h

T50 ~24 h, Ri ~12 h

PM

N n

um

be

r (x

10

6)

Time (h)

4

8

12

16

0 4 8 12 16 20 24

ψ max, Tmax

Ri

T50

+RvE1 (T0)

ψ max = 12.0x106, Tmax ~8 h

T50 ~20 hr, Ri ~12 h

+RvE1 (12h)

Tmax ~12 h, T50 ~22 h, Ri ~10 h

Eicosapentanoic acidEPA

Resolvin E1

OH

COOH

HOOH

Time (h)

4

8

12

16

0 4 8 12 16 20 24

*

+

Docosahexanoic acid DHA

+PD1 (T0)

ψ max = 10.0x106, Tmax ~5 h

T50 ~11 h, Ri ~6 h

+PD1 (12h)

Tmax ~12 h, T50 ~22 h, Ri ~10 h

(Neuro)protectin D1

COOH

OH

OH

Time (h)

4

8

12

16

0 4 8 12 16 20 24

****

++

+ATLa (T0)

ψ max = 13.2x106, Tmax ~12 h

T50 ~23 h, Ri ~11 h

+ATLa (12h)

Tmax ~12 h, T50 ~22 h, Ri ~10 h

Arachidonic acid

Aspirin-triggered Lipoxin A4

ATL

COOH

OH

OHHO

Time (h)

4

8

12

16

0 4 8 12 16 20 24

*

***

+

++

Pro-Resolving Lipid Mediators Impact Resolution Indices

*p<0.05, ***p<0.001 (treatment vs. zymosan); +p<0.05 (ATLa vs. RvE1); ++p<0.01 (ATLa vs. PD1).

Resolution Pharmacology Agonists

Nature 2007

cPLA2

PMN

SpecializedPro-Resolving

Mediators

Cytokines & Chemokine

Self-Limited Evolving Exudate

MΦ

Apoptotic PMN

CytokineDisposal

CellularDebris

Lymphatic Clearance& Removal

SPM

ExudateTranscellularBiosynthesis

Resolvins&

Protectins

ω-3

EPA, DHA

cPLAA2A2

AAPG

LT

PMN

Platelets

Lipoxins

Diapedesis

Adhesion

Rolling

InjuryMicrobial Invasion

CollateralTissue

Damage}Extracellular

Release ofROS, hydrolytic enzymes

IncompletePhagocytosis

MicrobialParticles

LTB4

CompletePhagocytosisMicrobialKilling

Edema

LXA4

RvE1PD1

FeedbackControl

SPM

CN Serhan in press 2010 n-3

Microscale Valves

Chemotaxis Assay Chamber

Microscale Valves

Gradient Generator

Gradient Generator

Novel Microfluidics Chamber & Single Cell with 1 Drop of Blood <5min: Actions of Resolvin D1 and DHA [2-3 hours]

© 2010 Serhan et.al.

3030

Human Neutrophil

-1 min

+1 min +5 min

IL-8

10µm

IL-8 + RvD1 10 nM

RvD1 Stops PMN Chemotaxis

Single-cell Monitoring of PMN Chemotaxis in Microfluidic Chamber

Kasuga K, Yang R et al., J Immunol.

RvD1 or DHA

DHA

RvD1

IL-8

Reporter Cell Systems in 1ul cubic vol

R D1 St PMN Ch t i

r• Functional Decoding Metabolomics

� Rare & Transient Mediators / Intermediates

Chamber Anatomy & Design

Point of Care Medicine

Chemotaxis Chamber

Microscale Valves

Gradient Generator

HO

OH

COOHOH

COOH

HO

HO

OH

Resolvin D1 Resolvin D2

Biosynthesis of RvD2

6.0 8.0 10.0 12.0

Re

lative

In

ten

sity (

%)

100

50

0

100

50

0

Re

lative

In

ten

sity (

%)

260 300 340

340300260

Wavelength (nm)

Wavelength (nm)

Re

lative

In

ten

sity (

%)

Re

lative

In

ten

sity (

%)

Re

lative

In

ten

sity (

%)

100

50

0260 300 340

Wavelength (nm)

ω-2 OH PD1

80

60

40

20

0

80

80

60

60

40

40

20

20

0

0

100

100

100

Δ10-trans-RvD2

RvD2

RvD1

Δ13-trans-RvD1

100

50Re

lative

In

ten

sity (

%)

260 300 340Wavelength (nm)

0

RvD2

RvD2

RvD1

RvD2

Leukocyte-derived

Co-injection

Synthetic

Time (min)

Matching of Leukocyte-derived and Synthetic RvD2

15-LOX

DHA

17-HpDHA

5 4

5-LOX

O

COOH

HO

7 6 3 21

8

RvD2 Reduces PAF-Stimulated Leukocyte Recruitment Intravital microscopy

PAF 100nM After RvD2 1nM

Mouse Cremaster Muscle Post-Capillary Venule

Prof. M. Perretti et alWHRI London

Key Concepts & Points

• Resolution is an active not passive

• Resolvins & SPM genus

are Anti-inflammatory and Pro-resolving

•Organ Protective : Renal , Lung & Neural Tissues

• SPM are not immunosuppressive

stimulate containment : Sepsis (CPL)© 2010 Serhan et.al.

Num

ber

of pE

RK

+ n

euro

ns

(f

old

of contr

ol)

*�

0

1

2

3

4

5

6

Con

trol

RvE

1 TN

F-

RvE

1 +

TNF-

�

*�

Control

TNF-�

pERK

RvE1+TNF-�

Blockade of TNF-mediated

ERK activation in dorsal horn neurons

Contro

TNF-

RvE1+

ERK

0

20

40

60

80

100

1 10 102 103 104

Dose (ng)

In

hib

itio

n o

f p

ha

se

-2

pa

in b

eh

avio

r (

%)

RvE1

Morphine

NS-398

0.1

Formalin-induced 2nd-phase pain

GAPS in Current Knowledge

1) Are EFA n-3 ( DHA , EPA ) vitamins ? Can we consider the “Green Pro-drugs” ?

2) What is the relationship between serum DHA/EPA to local resolvin & protectin ( SPM ) levels & actions?

3) What are the physiologic sources of DHA ? Edema

4) Relevant Biomarkers : SPM receptor expression pathway metabolome pathway markers 17-HDHA serum 14-HDHA, RvE2

Pus bonum et laudabile “good and laudable pus”

Chemical Mediators of Resolution

• SPM deficiencies in production and/or actions may allow local inflammation to go on beyond its normal self-limiting process.

• If local tissue injury in Brain Trauma is indeed a non-resolving form of local inflammation than these results may have implications for nutrition & treatment .

SPM genus

Conclusions

Chronic Inflammation

Host Defense

Acute Inflammation ResolutionInjury / trauma

Microbial infection

“unresolved”

Lipoxins

LXs

Neuroprotectins

NPD1/PD1

Aspirin-triggered LXs

ATL

Resolvin Es

RvEs

Resolvin Ds

RvDs

Specialized pro-resolving mediators

ProstaglandinsLeukotrienes

Maresins