Pharmacologic Resolution of Inflammation as a Novel Therapeutic Approach

Abstracts

Pro-Resolving Lipid Mediators in Clearing Inflammation & Infection
Charles N. Serhan, PhD, DSc (hc), Brigham & Women's Hospital, Harvard Medical School

Uncovering mechanisms controlling inflammation are of paramount importance because persistent and chronic inflammation can impact all organs throughout the body and are involved in many widely occurring diseases. Recent advances in our appreciation of the mechanisms in resolution of acute inflammation (RoI) systematically uncovered several new families of potent pro-resolving autacoids, each biosynthesized from essential polyunsaturated fatty acids in resolving exudates. These include the resolvins (Rv), protectins (PD) and maresins (MaR), collectively termed specialized pro-resolving mediators (SPM) that act in pico-nanogram ranges. SPM are temporally and spatially biosynthesized by resolving- exudates, which proved to evoke potent anti-inflammatory and pro-resolving actions as well as enhance microbial clearance. The potent SPM actions and complete structures are confirmed, which also permitted use of LC-MS-MS-based metabololipidomics to identify SPM in human and murine tissues, isolated human cells types (e.g. apoptotic neutrophils, and macrophage phenotypes) CN Serhan Nature June vol 510, 2014 doi:10.1038/nature13479 . Specific SPM demonstrate potent stereoselective actions that involve specific G-protein-coupled receptors and are not immunosuppressive). This presentation will update advances in SPM mechanisms in RoI, their new sites of formation and actions that opened the door for their role(s) in resolution physiology and pharmacology.
 

G Protein-Coupled Receptors in Resolution of Inflammation: Annexin, Lipoxin A4 and Resolvin D1
Mauro Perretti, PhD, Queen Mary University of London

The recent success with anti-inflammatory therapy in chronic diseases should not make us complacent: as inflammation is critical for survival, the risk:benefit of anti-inflammatory drugs is still narrow. A fresh approach can derive from molecular detailing of the resolution of acute inflammation, a programmed response evoked by mediators with potent tissue-protective properties. Our understanding on how pro-resolving mediators operate led to the identification of specific receptor targets. Our own work has focused on pro-resolving G-protein couple receptors (GPCR) with the dual aim of i) defining their biology and ii) inform therapeutic opportunities. An exemplar is the receptor for Annexin A1, Lipoxin A4 (LXA4) and Resolvin D1, termed FPR2/ALX. What processes are controlled by FPR2/ALX in preclinical disease models? What is best strategy to exploit pro-resolving GPCRs for innovative drug discovery programmes? Using a mouse colony lacking FPR2/ALX orthologues, we identified specific and non-redundant protective properties of FPR2/ALX in settings of sepsis and vascular reperfusion injury. The observed over-exuberant responses in the absence of FPR2/ALX were linked to inadequate LXA4 production, indicating existence of FPR2/ALX-centred protective circuits. For therapeutic innovation, studies with distinct ligands led us to define at least one cell-specific proresolving signature for FPR2/ALX in macrophages, with of a specific signaling response downstream FPR2/ALX homodimerization, yielding IL-10 production, indicating again the existence of complex networks in resolution, with FPR2/ALX at its centre. We predict these efforts will lead to the establishment of novel inflammation therapies under the new branch of ‘Resolution Pharmacology’. Funding: Wellcome Trust (08667/Z/08/Z).
 

Resolving Vascular Injury: Resolvins and Maresins Influence Vascular Remodeling
Michael S. Conte, MD, University of California-San Francisco

The inflammatory response to acute vascular injury plays a central role in the subsequent remodeling response. Protracted inflammation has been associated with aberrant vascular healing leading to failure of common therapeutic procedures such as angioplasty, stenting, and bypass surgery. Mechanisms governing the resolution process in this setting are poorly understood, but may offer new opportunities to regulate vascular injury and restore homeostasis. We have explored the effects of the specialized pro-resolving lipid mediators (SPM) resolvins (Rv, D- and E- series) and maresins (MaR1) on vascular cells, and in models of acute vascular injury.
 
In cultured vascular smooth muscle cells (VSMC), SPM inhibit cell migration in a wound assay and chemotaxis to PDGF. They attenuate TNF-stimulated NFkB activation, monocyte adhesion, gene expression, and generation of reactive oxygen species. In a rabbit model of arterial angioplasty, local delivery of RvD2 attenuated leukocyte recruitment and reduced neointima formation. In a mouse model of carotid ligation, systemic (IP) administration of RvD2 and MaR1 reduced leukocyte recruitment, altered macrophage phenotype (favoring M2 vs M1), and significantly attenuated neointimal hyperplasia. We have measured local levels of SPM and their precursors in arterial tissues and in patients with vascular disease, and demonstrate the expression of some of the known SPM receptors in vascular cells. Taken together our findings indicate that i) biochemical pathways of resolution are operative in vascular injury; ii) SPM have direct effects on vascular cells; and iii) increased availability of SPM may favorably alter the healing response to acute vascular injury.
 

New Mechanisms and Therapeutic Potential of Pro-resolving Mediators in Advanced Atherosclerosis
Gabrielle Fredman, PhD, Columbia University

Persistent inflammation and its failed resolution underlie the pathophysiology of prevalent human diseases, such as atherosclerosis. In this regard, many of the characteristics of clinically dangerous advanced atherosclerotic plaques including 1) imbalances between pro-inflammatory and pro-resolving mediators, 2) thinning of a protective "cap" of subendothelial collagen, 3) oxidative stress, 4) defective clearance of dead cells (efferocytosis), and 5) tissue-damaging necrosis, suggest that this resolution process is defective. The failure of the host to combat these maladaptive processes is likely due to a lack of pro-resolving agonists and/or a defect in their receptors. Hence, enhancing resolution in atherosclerosis by therapeutic administration of pro-resolving mediators is of interest. Here we report the pro-resolving agonists Resolvin D1 (RvD1) and Ac2-26 (that bind the G-protein coupled receptor, FPR2/ALX), decrease oxidative stress and necrosis and stimulate protective fibrous caps. Further, we uncovered a new signaling pathway of how RvD1, via FPR2/ALX, tempers inflammation to facilitate resolution. Together, the therapeutic potential of enhancing resolution in a chronic disease like atherosclerosis could be substantial in that mediators of resolution, unlike anti-inflammatory drugs, have a much greater potential to suppress inflammation and promote tissue repair without compromising host defense.
 

Genetics of Inflammation in the Brain and Alzheimer’s Disease Risk
Rudolph E. Tanzi, PhD, Massachusetts General Hospital

Alzheimer's disease (AD) is strongly influenced by genetic factors such as the APP, PSEN1, and PSEN2, and APOE. Since 2005, we have led the Alzheimer's Genome Project, which in 2008, reported novel AD genes including the spinocerebellar ataxia 1 gene, ATXN1, CD33, and ADAM10. ATXN1 knockout increases BACE1 levels in cortex and hippocampus, but not in cerebellum and brain stem. This increase of BACE1 is concordant with the shift of APP processing into the beta-secretase cleavage pathway along with an increase in Abeta levels and plaque load in the brains of APPswe/PS1deltaE9 mice. CD33 is one of several genes involved in the innate immune system of the brain that have been associated with AD risk. CD33 knock-out led to 1. decreased Abeta load owing to enhanced phagocytosis by microglia and 2. reduced numbers of M1-activated microglia in APP/PS1 and 5XFAD tg mouse brains. Re-sequencing of ADAM10 revealed two rare mutations that tightly co-segregated with AD and impair ADAM10 non-amyloidogenic cleavage of APP in transgenic mice. We also carried out whole genome sequencing on 440 subjects from 452 multiplex NIMH AD families. We then employed a multi-pronged approach to identify highly penetrant functional variants underlying the association of previously reported AD genes that emerged from genome-wide association studies. Our analyses revealed several novel pathogenic variants in the known and GWAS-confirmed AD genes. The elucidation of the genes and functional variants influencing risk for AD should continue to enhance our understanding of AD etiology and pathogenesis. Ultimately, these genes will be used to predict risk for AD and guide novel the development of therapies for the effective treatment and prevention of this terrible disease.
 

Omega-3 Fatty Acids with Antioxidants Increase Amyloid-β Phagocytosis and Attenuate Inflammation in Alzheimer Disease Patients: Role of Resolvin D1
Milan Fiala, MD, David Geffen School of Medicine at UCLA

Alzheimer disease (AD) patients have critical defects of innate immunity related to clearance of amyloid-β  (Aβ) from the brain: (a) Monocytes and macrophages are defective in phagocytosis and degradation of Aβ, and (b) the transcription of inflammatory genes in PBMCs is down regulated in “non-inflammatory” patients and is up regulated in ”inflammatory” patients. We tested Aβ phagocytosis by a flow cytometric test, inflammatory gene transcription by RT PCR, and cognitive status by Minimental State Examination (MMSE) in patients with minor cognitive impairment (MCI) (“early” patients) and advanced AD (”late” patients). The subjects were supplemented by a nutritional drink (Smartfish) with ω-3 fatty acids (ω-3) (1 gm DHA and 1 gm EPA), antioxidants (from pomegranate and chokeberry), vitamin D3 and Resveratrol. On supplementation of “early” patients we found that (a) Aβ phagocytosis by monocytes significantly increased (P=0.04); (b) the “Specialized proresolving mediator” resolvin D1 (RvD1), which stimulates Aβ phagocytosis in vitro, increased in 71% patients; (c) inflammatory gene transcription was up regulated in a non-inflammatory patient and down regulated in an inflammatory patient. The mean MMSE score was 26.1 at the first visit and 26.5 at the last visit after 4 to 12 months of follow-up. These results support a hypothesis that improvement of anti-Aβ immunity by ω-3 supplementation is mediated by specialized proresolving mediators (i.e. RvD1 and other mediators) and is associated with attenuation of cognitive impairment in MCI patients. A clinical trial of the ω-3 drink in prevention of AD is in preparation.
 

Resolvins in Airway Inflammation: An Effective Therapy for Chronic Obstructive Pulmonary Disease?
Patricia J. Sime, MD, University of Rochester School of Medicine

Rationale: Chronic obstructive pulmonary disease (COPD) is a major global health problem predominantly caused by smoking, but also by biomass smoke, and COPD is the third leading cause of death in the United States. Cigarette smoke is a profound inflammatory stimulus and curiously the effects of smoking persist long after smoking cessation. This supports the idea that cigarette smoke interferes with the normal processes that resolve inflammation.
 
Hypothesis: Pro-resolving lipid mediators (PRMs) have profound anti-inflammatory and pro-resolving effects on acute and chronic lung injury. Treatment with PRMs promote resolution: a novel and important therapeutic goal for inflammatory diseases caused by cigarette smoking.
 
Results: Using pre-clinical mouse models of acute and chronic cigarette smoke-incited inflammation, we have demonstrated that PRMs such as resolvin D1 (RvD1) dampen acute neutrophilic inflammation and promote its resolution through M2 pro-resolving macrophages. PRMs also prevent smoke-induced chronic inflammation and emphysema with reductions in apoptosis and oxidative stress. Studies in primary human lung cells revealed that RvD1 inhibits pro-inflammatory signaling by blocking both the MAPK and NF-kB inhibitors through a common regulatory kinase.
 
Conclusions: These studies show for the first time that pro-resolving mediators can be used to prevent inflammation and accelerate resolution/repair of lung injury due to both acute and chronic cigarette smoke exposure. Our results will pave the way for translational development of these exciting new compounds that have the potential to be effective therapies against human diseases of chronic inflammation and smoking. Supported by: NIH T32 HL066988, NIH R01HL110759-01, NIH P30ES01247, NIEHS T32ES007026, PhRMA Foundation, ULITR000042, GM038765
 

Specialized Pro-resolving Mediators in Allergic Airway Responses, Asthma and Acute Lung Injury
Bruce Levy, MD, Brigham & Women's Hospital, Harvard Medical School

Acute lung inflammation is fundamentally important to host defense, but chronic or excessive inflammation can lead to several important diseases. The resolution of inflammation is an active process that is directed, in part, by specialized pro-resolving mediators that are enzymatically derived from polyunsaturated fatty acids. In health, cell-cell interactions at the onset of acute inflammation establish biosynthetic circuits for these pro-resolving mediators, including the omega-3 fatty acid-derived resolvins, protectins and maresins, which serve as agonists to orchestrate a return of the inflamed tissue to homeostasis.  Understanding the cellular and molecular mechanisms for pro-resolving mediators in catabasis is providing new insights into lung tissue responses for resolution of inflammation in health and the pathophysiology of disease; as well as opportunities for therapeutic intervention. E-series and D-series resolvins are enzymatically derived from the essential omega-3 fatty acids eicosapentaenoic acid and docosahexaenoic acid, respectively. Protectin D1 and maresin 1 (MaR1) are also derived from DHA. Relevant to lung inflammation, lipoxin A4 (LXA4), Resolvin E1 (RvE1), resolvin D1 (RvD1) and protectin D1 (PD1) are generated in murine lung. LXA4 and PD1 are generated in human lung.  Receptors for LXA4 and RvD1 (ALX/FPR2) and for RvE1 (CMKLR1) are expressed in lung and are dynamically regulated with lung inflammation. Evidence will be presented for cellular and molecular mechanisms for representative pro-resolving mediators in their protective actions in the regulation of airway inflammation during innate and adaptive immune responses.
 

Suppression of Cell Debris-stimulated Tumor Growth by Resolvin Mediated Clearance
Dipak Panigrahy, MD, Beth Israel Deaconess Medical Center, Harvard Medical School

Chemotherapy and radiotherapy reduce tumor burden by killing tumor cells, but simultaneously generate tumor cell debris that stimulates inflammation promoting tumor growth and progression. This effect is recapitulated in tumor models; co-injection of irradiated, dead cells with tumor cells has been long known to reduce the inoculum of tumor cells needed to produce tumors in rodents (Révész phenomenon). Irradiation and chemotherapy trigger a cytokine storm in the tumor stroma, including the release of the tumor promoting cytokines IL-6 and TNFα. Thus, overcoming the dilemma of debris-induced tumor progression is paramount to prevent tumor recurrence and eventual resistance to cytotoxic drugs, including targeted therapy. Here, we show that tumor cells killed by chemotherapy drastically stimulated tumor growth when co-injected with a sub-optimal inoculum of tumor cells. However, debris-stimulated tumors were suppressed by resolvin (Rv) D1 or RvD2, novel pro-resolving lipid autacoid mediators derived from omega-3 fatty acids. Resolvins specifically inhibited debris-stimulated cancer progression and metastasis by enhancing the endogenous clearance of debris. Resolvin mediated clearance was critically dependent on phagocytosis by macrophages. Furthermore, resolvins counter-regulated the release of cytokines/chemokines, including TNFα, IL-6, IL-8, CCL4, and CCL5 from cell debris-stimulated human macrophages. Our results demonstrate that enhancing endogenous clearance of tumor cell debris represents a novel biological target to complement current cytotoxic cancer therapy and may address the inevitable, intrinsic limitation of current cancer therapy involving cell death.
 

Accelerating Inflammation Resolution to Counteract Chemical Cutaneous and Pulmonary Injury
Satya Achanta, DVM, PhD, Duke University School of Medicine

Inflammation has three temporal phases, initiation, amplification and maintenance, and resolution. Inflammation resolution was thought of as a passive process but recent studies show that resolution is an active process controlled by fatty acid derived specialized pro-resolving mediators. Chemical tissue injuries are often associated with strong and protracted inflammation. These include injuries by exposures to oxidants (inhalation of chlorine gas), electrophiles (smoke inhalation and exposure to tear gas), acids (hydrochloric acid reflux injury, HCl) and skin blistering agents (vesicants, mustard gas). In the past, classical anti-inflammatory strategies were investigated as treatments, interfering with initiation and maintenance of inflammation, with mixed outcomes. We hypothesize that accelerating resolution of inflammation will attenuate the exaggerated inflammatory response following chemical exposures, leading to decreased morbidity and improved recovery. In these studies, pro-resolving agents, Resolvin D1, Resolvin D2, Lipoxin A4, Protectin DX, and 17(R)Resolvin D1 were administered to mice at a dose rate of 2 or 5 µg/kg body weight i.p., following respiratory exposure to chlorine gas or HCl, or cutaneous exposures to a blistering agent (2-Chloroethyl ethyl sulfide) or a tear gas agent (CS). In the cutaneous injury models, pro-resolving agents decreased edema, pro-inflammatory cytokines, and vascular leakage while improving histopathological scores. In the pulmonary injury models, administration of pro-resolving mediators decreased levels of pro-inflammatory cytokines in the lung and serum and reduced deterioration of pulmonary function and tissue integrity. These results support our hypothesis and pave the way for more definitive studies in larger sample sizes and in higher mammalian species.
 

Specialized Proresolving Mediators Differentially Affect Platelet Function
Katie L Lannan, MS, University of Rochester

Antiplatelet therapy is a cornerstone of modern medical practice and is commonplace in preventing myocardial infarction, thrombosis, and stroke. Current antiplatelet therapies, such as aspirin, however often have adverse side effects, including increased risk of bleeding and 4-45% of the human population are “aspirin-resistant”. Platelets are intimately involved in hemostasis and inflammation, and can cause severe problems when excessively or insufficiently activated. A major unmet need in the field of hematology is the development of new agents that safety prevent unwanted platelet activation in patients with underlying cardiovascular disease, without increasing the risk of bleeding. Here, we investigate the potential of endogenously produced, specialized proresolving mediators (SPMs), to be used as novel anti-platelet agents. SPMs are a recently discovered class of lipid-derived molecules that drive the resolution of inflammation, without being overtly immunosuppressive. Our data demonstrate that SPMs differentially regulate platelet hemostatic function, by altering platelet spreading on fibrinogen. Additionally, SPMs suppress release of the proinflammatory and prothrombotic mediators, soluble CD40 ligand and thromboxane B2. Furthermore, we show for the first time that human platelets express the SPM receptors, GPR32 and ALX, which may mediate the beneficial effects of SPMs on platelet function. Taken together, these data support the idea that SPMs differentially regulate platelet function and may represent a novel class of anti-platelet agents. Additionally, SPMs may play an important role in the resolution of inflammation in cardiovascular diseases, such as atherosclerosis.
 

ETC-1002 Modulates Macrophage Immune Response, Attenuates Vascular Inflammation and Prevents Progression of Experimental Atheroma in LDL Receptor-Deficient Mice
Sergey Filippov, MD, PhD, Esperion Therapeutics Inc.

ETC-1002 is a novel non-statin lipid regulating drug that lowers LDL-c and hs‑CRP in patients with hypercholesterolemia. In several long term clinical trials, hs-CRP lowering has been correlated with favorable cardiovascular outcomes. Anti-inflammatory as well as atheroprotective effects of ETC-1002 have been evaluated in vitro and in vivo. In primary human monocyte-derived macrophages treated with ETC-1002, decreased production of pro-inflammatory cytokines and chemokines coincided with increased levels of AMPK phosphorylation and reduced activity of JNK and p38 MAP kinases. Experiments with siRNA-mediated gene silencing revealed that ETC-1002 activates macrophage AMPK and exerts its anti-inflammatory effects via a mechanism linked to the LKB1/AMPK axis. In high-fat high-cholesterol-fed LDL receptor-deficient mice, treatment with ETC-1002 reduced the size of experimental atheroma and reduced the sub-endothelial retention of free and esterified cholesterol. Furthermore, consistent with the anti-inflammatory properties, ETC-1002 lowered expression of MCP-1, VCAM-1, CD68 and CD36 in atherosclerotic lesions. These findings support that ETC-1002 may offer additional clinical benefits by improving vascular inflammation associated with hypercholesterolemia and coronary artery disease.
 

Spontaneous Pro-Resolution Mediators (SPM’s) in Paediatric Cystic Fibrosis (CF) Lung Disease
Fiona Ringholz, MB BChir, National Children’s Research Centre

CF is caused by a mutation in the CFTR and results in airway surface liquid (ASL) dehydration, impaired muco-ciliary clearance, chronic pulmonary infection and inflammation leading to progressive lung destruction. Lipoxin A4 (LXA4) and Resolvin D1 (RvD1) are SPM’s involved in the active resolution of inflammation. We previously reported that LXA4 restores ASL height in CF bronchial epithelium. We sampled the airways of control children and young children with CF at clinical baseline and during CF Pulmonary Exacerbation (CFPE) (PRINCE Study). We studied the physiological effects of RvD1 in CF using primary CF alveolar macrophages and polarized, differentiated NuLi-1 and CuFi-1 cells as a bronchial epithelial model. Ethical approval was granted by Our Lady’s Children’s Hospital, Crumlin. We report an imbalance in LXA4 versus Leukotriene B4 (LTB4) production in CF airways, despite the absence of infection, related to a deficiency of the LXA4 synthetic enzyme 15 LO-2 in CF macrophages. The reported defect in eicosanoid mediator “class switching” from propagation of inflammation to active resolution of inflammation in CF may play a mechanistic role in the failure to resolve inflammation in CF airways. We report that RvD1 restores ASL height and architecture and attenuates TNFα induced IL8 secretion in CuFi-1 cells. Furthermore RvD1 restores phagocytic capacity to CF alveolar macrophages. We present the findings of the PRINCE study; relating SPM production in the airway to the time course of clinical resolution of inflammation during CFPE. Our findings strongly recommend the development of SPM's as candidate therapeutics for CF airways disease.