011 – Proteinase-activated receptor-2 (PAR2) blockade attenuates neuroinflammation in a murine experimental autoimmune encephalomyelitis (EAE) multiple sclerosis model

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011 – Proteinase-activated receptor-2 (PAR2) blockade attenuates neuroinflammation in a murine experimental autoimmune encephalomyelitis (EAE) multiple sclerosis model

Rahil Eftekhari (1, 2) – Benjamin W. Ewanchuk (3) – Koichiro Mihara (1) – Mahmoud Saifedine (1) – Farshid Noorbakhsh (2) – Morley D. Hollenberg (1, 4)
Department of Physiology & Pharmacology and Department of Medicine, Snyder Institute for Chronic Diseases, University of Calgary Cumming School of Medicine, Calgary AB Canada T2N 4N1 (1) – Department of Immunology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran (2) – Department of Biochemistry and Molecular Biology, University of Calgary Cumming School of Medicine, Calgary AB Canada T2N 4N1 (3) – Department of Medicine, Snyder Institute for Chronic Diseases, University of Calgary Cumming School of Medicine, Calgary AB Canada T2N 4N1 (4)


Background: Proteinase-activated receptor 2 (PAR2) is a proteinase-activated G-protein-coupled receptors that is elevated in the central nervous system (CNS) in human multiple sclerosis and in its murine autoimmune encephalomyelitis (EAE) in vivo model, for which EAE is markedly reduced in PAR2-null mice. We therefore hypothesized that a receptor-selective PAR2 antagonist, Pepducin P2pal-18S, would attenuate the progression of EAE in the murine MS/EAE disease model in part by affecting immune cell function that is regulated by PAR2. Methods: To evaluate the impact of PAR2 blockade on EAE progression, P2pal-18S (10mg/kg) was administered on day 0 and day 10 in the murine EAE MS protocol and motor disability was monitored. Spinal cords were isolated and assessed by immunohistochemistry for axon myelination, T cell- and macrophage- specific markers at the peak of disease expression (day 15). Serum was isolated from EAE-affected mice to survey the circulating inflammatory and anti-inflammatory cytokines (Luminex assay). To analyze the effect of P2pal-18S on T cell and macrophage function 1. its reversal of PAR2-stimulated calcium signaling was measured, 2. antiCD3/CD28-activated splenocytes were treated with Pepducin in vitro and proliferation was monitored and 3. the pepducin effect on bone marrow-derived cytokine-mediated macrophage differentiation in vitro was assessed . The impact of P2pal-18S on macrophage differentiation was evaluated by monitoring cytokine-induced M1/M2 phenotype differentiation (semi quantitative PCR). Supernatants were collected from treated splenocytes and bone marrow-derived macrophages to analyze cytokine production (Luminex). Results: EAE mice treated with P2pal-18S exhibited markedly diminished paralysis and clinical scores compared to controls; and CNS T cell and macrophage infiltration and demyelination were decreased. Further, P2pal-18S decreased anti CD3/CD28-triggered lymphocyte proliferation. In addition, P2pal-18S prevented cytokine-induced macrophage M1/M2 differentiation. The decrease of serum GM-CSF in P2pal-18S–treated EAE mice paralleled the decreased production of GM-CSF by pepducin-treated CD3/CD28-activated splenocytes. Conclusions: We conclude that PAR2 plays a key role in EAE/antigen-induced MS-related CNS neuroinflammation and that this GPCR may represent a novel therapeutic target for treating MS and other neuroinflammatory diseases.
Funding: CIHR Canada and Tehran University of Medical Sciences