An Inflammation Loop Orchestrated by S100A9 and Calprotectin Is Critical for Development of Arthritis Annabelle Cesaro, Nadia Anceriz, Audrey Plante, Nathalie Page´ , Me´ lanie R. Tardif, Philippe A. Tessier* Centre de recherche en Infectiologie, Centre de recherche du Centre Hospitalier Universitaire de Quebec, and Faculty of Medecine, Universite´ Laval, Quebec, Canada Abstract Objective: The S100A9 and S100A8 proteins are highly expressed by neutrophils and monocytes and are part of a group of damage-associated molecular pattern molecules that trigger inflammatory responses. Sera and synovial fluids of patients with rheumatoid arthritis (RA) contain high concentrations of S100A8/A9 that correlate with disease activity. Methods: In this study, we investigated the importance of S100A9 in RA by using neutralizing antibodies in a murine lipopolysaccharide-synchronized collagen-induced arthritis model. We also used an in vitro model of stimulation of human immune cells to decipher the role played by S100A9 in leukocyte migration and pro-inflammatory cytokine secretion. Results: Treatment with anti-S100A9 antibodies improved the clinical score by 50%, diminished immune cell infiltration, reduced inflammatory cytokines, both in serum and in the joints, and preserved bone/collagen integrity. Stimulation of neutrophils with S100A9 protein led to the enhancement of neutrophil transendothelial migration. S100A9 protein also induced the secretion by monocytes of proinflammatory cytokines like TNFa, IL-1b and IL-6, and of chemokines like MIP-1a and MCP-1. Conclusion: The effects of anti-S100A9 treatment are likely direct consequences of inhibiting the S100A9-mediated promotion of neutrophil transmigration and secretion of pro-inflammatory cytokines from monocytes. Collectively, our results show that treatment with anti-S100A9 may inhibit amplification of the immune response and help preserve tissue integrity. Therefore, S100A9 is a promising potential therapeutic target for inflammatory diseases like rheumatoid arthritis for which alternative therapeutic strategies are needed. Citation: Cesaro A, Anceriz N, Plante A, Page´ N, Tardif MR, et al. (2012) An Inflammation Loop Orchestrated by S100A9 and Calprotectin Is Critical for Development of Arthritis. PLoS ONE 7(9): e45478. doi:10.1371/journal.pone.0045478 Editor: Pierre Bobe´, Institut Jacques Monod, France Received February 25, 2012; Accepted August 21, 2012; Published September 18, 2012 Copyright: 2012 Cesaro et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants to P.A.T. from the Canadian Institutes of Health Research (grant number 84226). P.A.T. holds a scholarship from the Fonds de la Recherche en Sante´ du Que´bec. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: P.A.T. is the founder of InflammatoRx Inc., which is developing anti-S100A9 antibodies for commercial purposes. The other authors have declared that no competing interests exists. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: philippe.tessier@crchul.ulaval.ca Introduction Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease characterized by a massive infiltration of immune cells into the synovial lining, initiating local inflammation and ultimately leading to cartilage/bone destruction. Although the causes of RA remain unknown, multiple pro-inflammatory mediators actively participate in the progression and severity of the disease. These molecules include cytokines and chemokines like tumor necrosis factor-alpha (TNFa), interleukin (IL)-6, and macrophage inflam- matory protein (MIP)-1a [1,2], which trigger a feed-forward loop sustaining inflammation in synovial joints. Inhibitors of some of these cytokines, like TNFa or IL-6, are routinely used clinically to treat RA. While these treatments are effective, various side effects have been reported [3,4,5]. Moreover, approximately 30% of patients do not respond efficiently to the treatment, while others become resistant over time [6]. S100A9 and S100A8 are small calcium-binding proteins recognized as damage-associated molecular pattern (DAMP) molecules [7] upon their release in the extracellular environment. They are increased in the serum and upregulated the synovium of RA patients and their levels correlate with disease severity [7,8,9,10]. This increase is observed not only in RA but also in other inflammatory diseases like inflammatory bowel disease [11,12] and gout [13,14]. S100A8 and S100A9 are primarily expressed in innate immune cells, particularly in neutrophils, constituting approximately 40% of the cytosolic proteins in these cells. They are also expressed, albeit to a lesser extent, in monocytes [15,16]. They can form homo- or hetero-complexes, the latter known as calprotectin. Both forms are abundantly released by neutrophils and monocytes under stress or inflam- matory conditions [17,18]. Signaling pathways that are induced upon sensing these molecules trigger inflammatory responses such as chemotaxis [14,19], phagocyte migration [20], and modulation of various macrophage functions. Based on their involvement in inflammatory processes and abundant levels in numerous pathologies, it is likely that S100A9 and calprotectin play a pivotal role in the pathophysiology of various inflamma- tory disorders. In this study, we used a combination of in vivo and in vitro experiments to gain new insights into the proinflammatory PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e45478