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An Engineered GM-CSF-CCL2 Fusokine Is a Potent Inhibitor of CCR2-Driven Inflammation As Demonstrated in a Murine Model of Inflammatory Arthritis¹

Moutih Rafei^*, Yamina A. Berchiche^,, Elena Birman^*, Marie-Noëlle Boivin^*, Yoon Kow Young^*, Jian Hui Wu^*, Nikolaus Heveker^, and Jacques Galipeau^2,*

^* The Montreal Center for Experimental Therapeutics in Cancer, Jewish General Hospital, Department of Biochemistry, Université de Montréal, and Research Centre/Hôpital Sainte-Justine, Montréal, Québec, Canada

CCR2 is a chemokine receptor widely expressed by lymphomyeloid cells involved in maladaptive autoimmune ailments. Therefore CCR2 is of great interest as a biological target for immune suppression due to its direct implication in autoimmune diseases such as rheumatoid arthritis. We have generated a novel fusion protein using GM-CSF and an N-terminal truncated version of MCP-1/CCL2 (6–76, GMME1) and investigated its utility as a CCR2-specific immune suppressor. Using BRET studies, we found that distinct to CCL2, GMME1 binding to CCR2 led to altered conformational changes in the CCR2 homodimer and did not induce the recruitment of β-arrestin 2 to the receptor. However, CCR2-dependent calcium mobilization, BAX induction and caspase-3 activation followed by cell death was observed. Using Th17 cells harvested from DBA/1 mice ill with bovine collagen-induced arthritis, we demonstrate that GMME1 is capable of blocking their production of IL-17 in vitro. Upon its delivery to mice symptomatic with inflammatory arthritis, a robust clinical recovery occurred with decreased paw thickness to normal levels and a significant reduction in anti-collagen Ab titer and rheumatoid factor titer, as well as reduction of proinflammatory cytokines levels both intraarticular and systemic. Our data demonstrate that GMME1 is a powerful synthetic suppressor cytokine that coopts CCR2-dependent cellular signaling and blunts the effects of CCR2-expressing lymphomyeloid cells causative of autoimmune arthritis.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Grants MOP-15017 (to J.G.) and HOP-79210 (to N.H.) from the Canadian Institutes for Health Research. M.R. is a recipient of a Fonds de Recherches en Santé du Québec Scholarship. Y.A.B. holds a Canadian Institutes for Health Research Scholarship. N.H. is a Canadian Institute of Health Research Scholarship New Investigator. J.G. is a Fonds de Recherches en Santé du Québec Chercheur-Boursier Sénior.

² Address correspondence and reprint requests to Dr. Jacques Galipeau, Division of Hematology/Oncology, Jewish General Hospital, McGill University, 3755 Cote Ste-Catherine Road, Montreal, Quebéc H3T 1E2, Canada. E-mail address: jacques.galipeau{at}mcgill.ca

³ Abbreviations used in this paper: RA, rheumatoid arthritis; MSC, mesenchymal stromal cell; MMP, matrix metalloproteinase; RF, rheumatoid factor; YFP, yellow fluorescence protein; PI, propidium iodine; HEK, human embryonic kidney; mpCCL2, MMP-processed CCL2; LIX, LPS-treated CXCL5.