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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
Wei
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
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