Disclosures for Charles N. Serhan

Transcription

1 IOM NAS Washington June Resolvins & Protectins : SPM in & Organ Protection Charles N. Serhan Director & Prof. Center for Experimental Therapeutics BWH Harvard Medical School Prof. Dept Oral Medicine, Infection and Immunity Harvard School of Dental Medicine New Genus of Pro-Resolving Lipid Mediators from EFA 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 Consultant Major Stockholder Speakers Bureau Honoraria No relevant conflicts of interest to declare No relevant conflicts of interest to declare No relevant conflicts of interest to declare No relevant conflicts of interest to declare No relevant conflicts of interest to declare Scientific Advisory Board Resolvyx Pharmaceuticals Founder, RvD1, PD1 Eyes Vaso-obliteration and neovascularization (Retinopathy) Wound healing (Cornea thermal injury) Resolvins & Protectins Animal Disease Models NPD1/ PD1 Brain Stroke damage and PMN entry into the brain Neural cell survival Stem Cell Cardiovascular Platelet aggregation RvD1, PD1 Kidney Renal ischemic injury Reno protective Anti- Pro-Resolution nano - micrograms Oral Medicine -induced tissue and bone loss (Periodontitis) PD1 & Lungs Airway inflammation (asthma) Serhan et al., Nat Rev Immunol., RvD1, RvD2 GI tract PMN and weight loss Peritonitis Survival (Colitis) RvD1, Pain Neuropathic Inflammatory pain Central & Periperial 21 Serhan et.al.

2 Human Deficiencies (n-3 EFAs) CO DHA C22:6 Neural Protection Severe Depression, postpartum depression Acute coronary syndromes Chronic Alcoholism Nutritional Deficiencies : Nutritional Armor Captain Joseph R. Hibbeln, M.D. Brain Exudate? Genomics Lipidome Metabolomics systems networks Proteomics Lipidomics Lipid Mediator-Informatics Autacoids: Chemical Mediators local acting short lived paracrine / autocrine signals ; 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 Clinical Implications Is pus good or bad for you? 21 Serhan et.al.

3 Decision Paths in Acute : Resolution or Chronic? Injury Cardinal signs Calor (heat) Rubor (redness) Tumor (swelling) Dolor (pain) Loss of function Resolution passive process Acute Abscess formation Infection Chronic Wound Healing Scarring Rheumatoid Arthritis Periodontal Disease Diabetes Cardiovascular diseases Asthma CN Serhan Ann Rev Pathology 29 Human Neutrophils 1st line host defense Protective New concept active process Resolution WAR : Even if Defensive Unwanted Side Effects Rapidly turned on inflammatory challenge Active cellular and biochemical pathways Generation of SPM O 2 radicals Proteases Spatial & Temporal Relationships TNF-a Systems Approach Mapping Resolution Temporal-Differential Analyses of Resolution dermis inner lining Cavity Exudate Acute Temporal Differential TNF-a lower lining Cellular composition Sample collection exudate Differential counting FACS analysis LM-Lipidomics Proteomics Air pouch skin Oral Peritonitis Airway Lung Renal I/R Stroke Solid phase extraction LC-UV-MS-MS Informatics Profiling of lipid mediators 2D- gel electrophoresis LC-nanospray-MS-MS Bioinformatics/ Data analysis Proteins of interest/ Novel proteins CN Serhan Ann Rev Immunology 27

4 Lipid Mediator-Lipidomics Informatics Cells, tissues, and inflammatory exudates unbiased approach Solid phase extraction with IS LC-UV-MS-MS Lipid mediator profiles data mining ELISA LC-UV GC-MS Physical properties Profiling of lipid mediators MRMs Lipidomic databases, Identification and search algorithm 21 Serhan et.al. Ideal Outcome of : Complete Resolution Systems Approach to Mapping Resolution Chronic Chemical mediators: Prostaglandins unresolved Leukotrienes Fibrosis Programmed Resolution Acute Lipid mediator class switching PGE2, D2, LTB4 Resolution Return to homeostasis LX Specialized Pro-Resolving Mediators SPM Resolvins Protectins Lipoxins CO E-series Resolvins D-series Neuroprotectin Protectins Edema PMNs LXA4 CO Monocyte/ Macrophages CO PD1 Activity RvD1 CO sec-min min hrs --days time Families Specialized Pro-Resolving Mediators (SPM): A Novel Genus Endogenous Lipid Mediators Acute phase min to hrs Resolution phase hrs to days prostaglandins Leukotrienes Time Eicosanoid Class Switching Reduce Further PMN Influx Efferocytosis Serhan & Saville Nature Immunology Alpha signals Omega non-fever causing

5 Resolvin E1 : Biosynthesis, Stereochemistry and MOI Exudate PMN (x 1 3 /pouch) Murine Skin Air Pouch 6 TNF 3 Resolving Exudates CO BLT Stop PMN NF-kB activation limit PMN Infiltration CD11b/cd1 adhesion Dendritic cells Migration IL-12 production Resolvin E1 ChemR23 Time hours PBMC - MAPK activation Resolvins: resolution phase interaction products CN Serhan et al JEM X 1 aspirin Macrophages enhances phagocytosis Human Vascular Endothelial Cells Hypoxia OC Aspirin-COX2 P45 microbial Human Leukocytes 5-LO 1-hydroxyEPE Reduction 5-LO O CO 5S-hydroperoxy-1-hydroxy-EPE OC E Series Resolvin Biosynthesis : Temporal 5-LO Function O 5S(6)-epoxy-1hydroxy-EPE Resolvin E2 CO Further enzymatic process Regulates neutrophil infiltration Equipotent as at low dose RvE2 > Pathway Biomarker Resolvin E1 CO Reduces Colitis, Asthma Periodontial Disease Ocular Cell : Chem & Bio Regulates neutrophil infiltration Promotes resolution Regulates dendritic cell function and IL-12 production Protects from osteoclast-mediated bone destruction Anti-Inflammatory Properties Potent actions pg-ng range Microglial cell reduces cytokine expression Aspirin-COX2

6 Protectins Murine Brain, Human Blood and Glial Cells Autacoids that counter inflammation Neuroprotectin D1 / Protectin D1 Anti-Inflammatory in vivo PMN STOP Infiltration air pouch, airway inflammation peritonitis, renal I/R, Liver Protection pm-nm range Glial cells Ligand specific cell signaling Reduces cytokine expression 1R,17S-diHDHA 21 Serhan et.al. Brain & Retina Bazan et al JBC ; PNAS Reduces Stroke Damage & Retinal Injury Corneal Injury & Wound Healing Gronert et al JBC NPD1/ PD1 is Neuroprotective in Transient Focal Stroke Reduces PMN Mediated Damage NPD1/PD1 CO Bazan et al JBC, PNAS NPD1/PD1 T H2 Cell Biosynthesis of Protectin D1 / Neuroprotectin D1 IL-4 Human Neutrophils Human T-cells 15-LOX-1 Murine Exudates Murine Brain 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 CN Serhan Ann. Rev. Path. 2

7 Organ Protection : SPM Accelerate Epithelial Wound Healing Topical Treatment 1μg/tid hrs Saline LXA 4 17S-HDHA (RvD1) NPD1 Re-Epithelialization (% Change) hrs LXA 4 17HDHA NPD1 LTB 4 12HETrE Biomicroscopy 4hrs Post Epithelial Removal Treatment Post Epithelial Removal Gronert et al, J Biol Chem, 25

8 16 5 m/z MS 327 MS/MS 343 m/z 153 on MS/MS 359 m/z 273 on MS/MS 375 MS 31 m/z 195 on MS/MS 333 m/z 233 on MS/MS on MS/MS 349 Fat-1 Transgenic Mice Rich in Endogenous Omega-3 increase Resolvins & Protectins Organ Protection Omega-3 EPA and DHA Relative Intensity 1 Resolvins & Protectins DHA 17HDHA PD1/NPD1 RvD3 14HDHA Time (min) Smith Protection et al tion in Colitis Nature Medicine 27 WT Fat-1 TG Elevated levels of n-3 Rv PD1 decreased vasoobliteration & retinal neovascularization DHA LTB 5 EPA PNAS EPA PGE 3 1 Fat-1 TG WT Time (min) Hudert et al. Proc Natl Acad Sci USA. (26) 13, Relative Intensity Weight loss (percent change) What is pro-resolving? A new bioaction of SPM Endogenous anti-inflammation XX pro-resolution O 2 radicals Proteases 21 Serhan et.al. LXA 4, and PD1 Promote Macrophage Phagocytosis Non-phlogistic Activation of Macrophages 4 IFN-γ F4/ + Gr-1 + macrophage ( % increase) Apoptotic PMN Pg/ml Pg/ml IL-6 (pro-inflammatory) IL-1 (anti-inflammatory) Vehicle LXA 4 PD1 Pg/ml 4 [ 1-1 nm] Vehicle LXA 4 PD1 P<.5, P<.1, P<.1 (versus vehicle) No apparent changes: TNF-α, KC, JE, MIP-2

9 Multi-level Mechanisms of Action BLT1 LTB 4 Receptor cross-talk with GPCRs LXA 4 ALX Receptor cross-talk with growth factor receptors VEGFR PDGFR CTGFR PMN LTB4 functions PMN Monocyte Macrophages HUVEC angiogenesis Mesangial cell proliferation fibrosis RvD1 PD1 Chem R 23 BLT1 ALX GPR 32 PMN Monocyte Macrophages PMN Macrophages PMN LTB4 functions RPE retina proliferation cell death Specialized Pro-Resolving Mediators SPM General Actions 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 Precursors Arachidonic Acid (AA) Eicosapentaenoic Acid (EPA) Docosahexaenoic Acid (DHA) Families Lipoxins E-Series Resolvins D-Series Resolvins Protectins/ Neuroprotectins LXA 4 RvD1 RvD2 PD1/ NPD1 SPM Specific 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-1 production DC IL-12 production DC migration Phosphorylation signals Inhibit NF-κB reporter gene activation Block PMN chemotaxis Mucosal clearance of PMN by CD55 PMN detachment LXA 4 production ROS intracellular Microbial killing Adhesion receptors ROS generation & Pro-Inflammatory cytokines (TNFα, IL-) PMN transmigration PGE 2 production Neovascularization Microglial cell cytokine expression PMN adhesion to NF-κB and COX-2 expression endothelial cells Renal fibrosis Nitric oxide and prostacyclin T-cell migration in endothelial cells TLR-mediated Mφ activation Microbial killing and clearance TNF and IFNγ release Protection of retinal pigment epithelial cells Neuroprotective actions CCR5 expression on T-cells CN Serhan in press 21 Pro-Resolving Lipid Mediators Impact Resolution Indices Arachidonic acid Eicosapentanoic acid EPA Docosahexanoic acid DHA Zymosan-initiated inflammation ψ max = 16.5x1 6, T max ~12 h T 5 ~24 h, Ri ~12 h CO Aspirin-triggered Lipoxin A 4 ATL CO Resolvin E1 CO (Neuro)protectin D1 ψ max, T max PMN number (x1 6 ) T 5 4 Ri Time (h) Time (h) Time (h) Time (h) Resolution Pharmacology Agonists +ATLa (T ) ψ max = 13.2x1 6, T max ~12 h T 5 ~23 h, Ri ~11 h +ATLa (12h) T max ~12 h, T 5 ~22 h, Ri ~1 h + (T ) ψ max = 12.x1 6, T max ~ h T 5 ~2 hr, Ri ~12 h + (12h) T max ~12 h, T 5 ~22 h, Ri ~1 h +PD1 (T ) ψ max = 1.x1 6, T max ~5 h T 5 ~11 h, Ri ~6 h +PD1 (12h) T max ~12 h, T 5 ~22 h, Ri ~1 h p<.5, p<.1 (treatment vs. zymosan); + p<.5 (ATLa vs. ); ++ p<.1 (ATLa vs. PD1). Nature 27

10 cpla2 Self-Limited Evolving Exudate Feedback Control Rolling CN Serhan PMN in press 21 PG n-3 AA LT cpla2 Platelets Adhesion ω-3 EPA, DHA Diapedesis Lipoxins Edema MΦ SPM LXA 4 PD1 Resolvins & Protectins SPM Exudate Transcellular Biosynthesis Apoptotic PMN Complete Phagocytosis Microbial Killing PMN Cytokines & Chemokine LTB 4 Incomplete Phagocytosis Extracellular Release of Collateral ROS, } Tissue hydrolytic Damage enzymes Cellular Debris Cytokine Disposal Microbial Particles Lymphatic Clearance & Removal Specialized Pro-Resolving Mediators Microbial Invasion Injury Novel Microfluidics Chamber & Single Cell with 1 Drop of Blood <5min: Gradient Generator Microscale Actions of Resolvin D1 and Valves DHA [2-3 hours] Chemotaxis Assay Chamber Microscale Valves Gradient Generator 21 Serhan et.al. Human Neutrophil 3

11 Functional RvD1 Stops Decoding PMN Chemotaxis Metabolomics Single-cell Monitoring of PMN Chemotaxis in Microfluidic Chamber Rare & Transient Mediators / Intermediates Chamber Anatomy & Design IL- Microscale Valves Chemotaxis Chamber IL- Gradient Generator RvD1 or DHA Point of Care Medicine DHA -1 min IL- + RvD1 1 nm RvD1 +1 min +5 min 1µm Reporter Cell Systems in 1ul cubic vol Kasuga K, Yang R et al., J Immunol. Biosynthesis of RvD2 Matching of Leukocyte-derived and Synthetic RvD2 DHA 15-LOX 17-HpDHA 5-LOX O 1 CO Relative Intensity (%) Relative Intensity (%) Leukocyte-derived ω-2 PD1 1 RvD2 6 Δ13-trans- RvD1 Δ1-trans- 4 RvD2 RvD Synthetic Relative Intensity (%) Relative Intensity (%) 1 RvD Wavelength (nm) 1 RvD Wavelength (nm) RvD2 34 Wavelength (nm) Resolvin D1 CO CO Resolvin D2 Relative Intensity (%) Co-injection Time (min) Relative Intensity (%) 1 RvD Wavelength (nm) RvD2 Reduces PAF-Stimulated Leukocyte Recruitment Intravital microscopy PAF 1nM After RvD2 1nM Mouse Cremaster Muscle Post-Capillary Venule Prof. M. Perretti et al WHRI London

12 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) 21 Serhan et.al. Formalin-induced 2 nd -phase pain Inhibition of phase-2 pain behavior (%) Morphine NS Dose (ng) Blockade of TNF-mediated ERK activation in dorsal horn neurons Control perk TNF- +TNF- Number of perk+ neurons (fold of control) Control TNF- + TNF- 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

13 Chemical Mediators of Resolution Prostaglandins Leukotrienes Conclusions unresolved Chronic Injury / trauma Microbial infection Acute SPM genus Resolution Host Defense Lipoxins LXs Maresins Specialized pro-resolving mediators Neuroprotectins NPD1/PD1 Resolvin Es Resolvin Ds RvEs RvDs Aspirin-triggered LXs ATL Pus good bonum and laudable et laudabile pus 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.