Podoplanin is an inflammatory protein upregulated in Th17 cells in SKG arthritic joints
Highlights
► We improved a Th17 cell differentiation culture and obtained a high purity of Th17 cells. ► Using the Th17 cells, we identified Th17 cell-specific genes by comparative microarray analysis. ► Podoplanin is expressed highly in in vitro differentiated Th17 cells. ► Podoplanin is also expressed highly in joint-infiltrating Th17 cells from SKG arthritic mice.
Introduction
Helper T (Th) cells play critical roles in acquired immune responses. Naïve Th cells differentiate into effector Th cells when stimulated by antigen-MHC complexes on antigen presenting cells, such as dendritic cells, macrophages and B cells. Depending on the type of environmental cytokines, activated Th cells differentiate into functionally distinct subsets, characterized by different cytokine and transcription factor profiles (Bettelli et al., 2008, Dong and Flavell, 2001, Glimcher and Murphy, 2000, Miossec et al., 2009, Mosmann and Coffman, 1989). Along with classical Th1 and Th2 cells, another subset of Th cells, denominated as Th17 cells, has attracted much attention because of their connection with both infection and autoimmune diseases (Korn et al., 2009). The main physiological function of IL-17, the signature cytokine of Th17 cells, is protection from infectious diseases. Indeed, IL-17-deficient mice are highly susceptible to infection by Citrobacter rodentium (Ishigame et al., 2009) and Candida albicans (Huang et al., 2004). Similarly, mice that lack the expression of IL-17RA have severe attenuation in host defense against Klebsiella (Ye et al., 2001) and Candida (Saijo et al., 2010). Th17 cells protect against bacterial and fungal infection through several mechanisms: (1) expression of CCR6 and IL-23R on the cell surface and migration to intestinal lymphoid tissues and inflammatory sites, (2) production of inflammatory cytokines and chemokines, such as IL-17A, IL-17F, IL-22 and CCL20. The induction of Th17 differentiation is induced by IL-6 and TGF-β through upregulation of RORγ, and the Th17 phenotype is maintained and stabilized by IL-23 released from antigen presenting cells, as Th17 cells do not produce IL-6, TGF-β or IL-23 necessary for their own differentiation. (Acosta-Rodriguez et al., 2007, Aggarwal et al., 2003, Ivanov et al., 2006, Wilson et al., 2007, Zhou et al., 2007).
The roles of Th17 cells have become clear over the past decade, based on results from experimental animal models of inflammatory diseases, as well as from clinical studies in human. The pathogenesis of rheumatoid arthritis (RA), like that of several other chronic inflammatory diseases such as Crohn's disease, psoriasis and multiple sclerosis, is linked to the deleterious roles of Th17 cells (Annunziato et al., 2009, Miossec et al., 2009, van den Berg and Miossec, 2009). IL-17-deficient mice show suppression of development of collagen-induced arthritis (Nakae et al., 2003). Moreover, the SKG strain of mice, a mutant on BALB/c background, spontaneously develops Th17-mediated autoimmune arthritis (Hirota et al., 2007a, Sakaguchi et al., 2003). It was shown in this model that CCR6 and CCL20 expressed by Th17 cells and inflamed synovial tissues, respectively, are required for the migration of Th17 cells, leading to self-destructive immune reactions in the joints (Hirota et al., 2007b). Furthermore, adoptive transfer of splenic Th cells of SKG mice into BALB/c mice induces arthritis, whereas that of splenic Th cells of IL-17-deficient SKG mice does not (Hirota et al., 2007a).
In arthritic mice, most cells in the joint tissues express IL-17R, and IL-17 activates synovial cells and inflammatory migrating cells. Activated Th17 cells induce destruction of the extracellular matrix and bone by activation of osteoclasts. The receptor activator of NF-κB ligand (RANKL) on Th17 cells activates the receptor activator of NF-κB (RANK) on osteoclasts, leading to enhanced bone destruction (Miossec et al., 2009).
Moreover, IL-17 levels in the serum and synovial fluid significantly correlate with disease activities of RA patients (Metawi et al., 2011). Indeed, results from a phase I clinical trial show significant beneficial effects of a humanized anti-IL-17 mAb in patients with RA (Genovese et al., 2010, Leonardi et al., 2012, Miossec et al., 2009, Waite and Skokos, 2012). This is underscored by two very recent reports on phase II clinical trials demonstrating the above-mentioned anti-IL-17 mAb, as well as an mAb specific for the IL-17R in the treatment of psoriasis (Genovese et al., 2010, Leonardi et al., 2012, Miossec et al., 2009, Papp et al., 2012, Waite and Skokos, 2012). Taken together, these data confirm that Th17 cells play a critical role in the pathogenesis of RA and other chronic inflammatory disorders. This leads to the assumption that the identification of novel Th17 cell-specific molecules may be of diagnostic or therapeutic value in the treatment of such diseases (Genovese et al., 2010, Leonardi et al., 2012, Miossec et al., 2009, Waite and Skokos, 2012).
In the present study, we have improved the experimental conditions of in vitro differentiation of mouse naïve Th cells, obtained highly differentiated Th populations, and subsequently performed comparative microarray analysis. Thus, we identified podoplanin as a cell surface molecule highly expressed by Th17 cells. We confirmed its expression in the inflamed tissues of experimental animal models of arthritis, autoimmune encephalomyelitis (EAE) and inflammatory bowel disease (IBD). Our results indicate that podoplanin might be a pathogenic Th17 cell marker and useful for diagnosis of autoimmune diseases.
Section snippets
Reagents
Recombinant mouse IL-2, IL-12, IL-4, IL-1β, IL-23 and TNF-α were purchased from R&D Systems. Recombinant mouse IL-6 and human TGF-β, and Abs to IFN-γ FITC (XMG1.2), CD62L FITC (MEL-14), CD25 PE (PC61), IL-17A PE (TC11-18H10), CD44 APC (IM7), IL-4 APC (11B11), and CD4 PEcy7 (RM4-5) were purchased from BD. Abs to Foxp3 PE (150D), CCR6 Alexa Fluor® 647 (29-2L17), CD8a (53–6.7), B220 (RA3-6B2), CD11b (M1/70), Gr1 (RB6-8C5), Ter119 (TER-119) were purchased from Biolegend. Abs to FcγRIIb PE (190909)
Improvement in the culture conditions for Th17 cell differentiation
In order to obtain differentiated Th17 cells, naïve T cells isolated from the spleens of BALB/c mice were stimulated and cultured in the presence of TGF-β and IL-6, according to the conditions reported previously (Bettelli et al., 2006, Mangan et al., 2006).
Initially, a purity of Th17 cells of only 9% was obtained, as shown by intracellular cytokine staining using FCM (Fig. 1A). As the efficiency of Th17 differentiation from naïve Th cells by IL-6 and TGF-β was low, we tried adding other
Discussion
In this study, we set out to identify Th17 cell-specific surface molecules, by differentiating Th1, Th2, Treg and Th17 cells and carrying out a comparative expression microarray analysis. Differentiation of naïve spleen cells under Th1-, Th2- and Treg-polarizing conditions, based on previous studies (Bettelli et al., 2008, Dong and Flavell, 2001, Glimcher and Murphy, 2000, Miossec et al., 2009, Mosmann and Coffman, 1989) yielded bona fide IFN-γ, IL-4 producing and Foxp3-expressing T cell
Acknowledgements
The authors thank Drs. Masatoshi Yanagida, Takahiro Okazawa, Mr. Takehiro Hasegawa, Mses. Megumi Goto and Mina Yabusaki for their discussion; Drs. Hans Yssel and Shigetaka Asano for their kind advices; and Ms. Mizuho Kawate for her assistance. We also thank Translational Research and Informatics (TRI) center for their support of our experiments; Oriental Bioservice, Inc. and Kobe BioMedical Laboratory for support of the animal experiments; Pathotec, Co., Ltd. for histological examination; and
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