Identification of an IL-27/osteopontin axis in dendritic cells and its modulation by IFN-γ limits IL-17–mediated autoimmune inflammation Gopal Murugaiyan1, Akanksha Mittal1, and Howard L. Weiner2 Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115 Edited* by Michael Sela, Weizmann Institute of Science, Rehovot, Israel, and approved May 17, 2010 (received for review February 24, 2010) Dendritic cells (DCs) play a central role in determining the induc- entiation of CD4+ T cells into functional subsets including IL- tion of T cell responses. IL-27 production by DCs favors induction 17–producing pathogenic Th17 cells and IL-10–producing regu- of IL-10–producing regulatory T cells, whereas osteopontin (OPN) latory Tr1 cells. For example, IL-10 or IL-27 production by DCs promotes pathogenic IL-17 T cell responses. The regulatory mech- favors generation of IL-10–producing Tr1 cells (6, 25), whereas anisms in DCs that control these two cells types are not under- OPN production by DCs promotes IL-17–producing Th17 cells stood well. Here, we show that IFN-γ induces IL-27 while inhi- (16, 17). However, it is unknown how IL-27 and OPN expression biting OPN expression in DCs both in vitro and in vivo and that in DCs are modulated to regulate both IL-10 and IL-17 pro- engagement of IFN-γR expressed by DCs leads to suppression duction by T cells. of IL-17 production while inducing IL-10 from T cells. DCs modi- Here, we show that IFN-γ induces IL-27 while inhibiting OPN − − fied by IFN-γ acquire IL-27–dependent regulatory function, pro- expression in DCs both in vitro and in vivo. IFN-γ / -deficient mote IL-10–mediated T cell tolerance, and suppress autoimmune mice in which EAE is induced have increased serum OPN and inflammation. Thus, our results identify a previously unknown lower IL-27 levels in comparison with WT mice. Engagement of pathway by which IFN-γ limits IL-17–mediated autoimmune in- IFNγR expressed by DCs leads to suppression of IL-17 pro- flammation through differential regulation of OPN and IL-27 ex- duction and induction of IL-10 from T cells. Furthermore, IFN- pression in DCs. γ–modified DCs ameliorate the disease severity of EAE through an IL-27–dependent mechanism. Taken together, our results id- L-27 is a potent antiinflammatory cytokine, which belongs to entify a previously unknown pathway by which IFN-γ limits IL- Ithe IL-12 family and is comprised of an IL-12p40–related pro- 17–mediated autoimmune inflammation through reciprocal DC tein, encoded by the EBV-induced gene 3 (EBI3, also known as modulation of OPN and IL-27. IL27), and a unique IL-12p35–like protein, IL-27p28v (1). Initial animal studies on the biology of IL-27 suggested a role for IL-27 Results in the initiation of the Th1 response (2, 3); however, subsequent IFN-γ Reciprocally Regulates OPN and IL-27 Expression in DCs. To work using mouse models of pathogen-induced and autoimmune study the effect of IFN-γ on DCs, we first examined its effect on inflammation have indicated that IL-27 has broad inhibitory effects the production of both pro- and antiinflammatory cytokines from on Th1, Th2 subsets of T cells and APC function in mice (4, 5). In DCs. We found that IFN-γ stimulation of DCs markedly induced addition, we and others have shown that IL-27 is capable of in- IL-27 levels while inhibiting OPN expression in DCs (Fig. 1 A ducing IL-10–producing regulatory Tr1 cells while inhibiting IL- and B). IFN-γ stimulation did not affect other DC-associated 17–producing Th17 cells both from humans and mice and acts as cytokines including IL-1β, IL-6, IL-12, IL-23, and TGF-β (Fig. a negative feedback mechanism against proinflammatory immune S1). IFN-γ inhibited OPN and induced IL-27 at both mRNA and responses (6–10). protein levels from WT-DCs in a dose-dependent manner. IFN-γ stimulation failed to inhibit OPN or induce IL-27 expression Contrary to the function of IL-27, osteopontin (OPN) is known − − − − to have potent proinflammatory functions. OPN participates in from IFN-γR / DCs. In addition we found that IFN-γR / DCs expressed high levels of OPN in comparison with WT-DCs, a wide range of biological processes (11) and has been linked to − − autoimmune diseases including multiple sclerosis (MS) and its ani- whereas IL-27 levels were low in IFN-γR / DCs compared with mal model experimental autoimmune encephalomyelitis (EAE). WT-DCs (Fig. 1 C and D). Taken together, these results dem- −/− −/− Mice deficient for OPN (Spp1 ,alsoknownasOPN ) show onstrate that IFN-γ reciprocally regulates OPN and IL-27 ex- milder EAE without disease exacerbation or progression com- pression in DCs. paredwithWTmice(12,13).PatientswithMShaveelevated IMMUNOLOGY levels of OPN in their serum and plasma (14, 15). OPN exac- Altered OPN and IL-27 Expression in DCs Differentially Modulate T Cell erbates EAE by skewing T cell differentiation toward IFN-γ– Production of IL-17 and IL-10. To test whether IFN-γ reciprocally producing Th1 cells and IL-17–producing Th17 cells (12, 13, regulated DC expression of OPN and IL-27 in vivo, we induced −/− 16–18). In addition, OPN induces IFN-γ– and IL-17–producing EAE in WT and IFN-γ mice. We found that OPN mRNA T cells in patients with MS (16). expression was markedly increased in DCs at the peak of disease Dendritic cells (DCs) are crucial in the initiation of productive in spleen, lymph node (LN), and CNS. Furthermore, OPN levels −/− antigen-specific T cell responses and the induction of T cell were higher in DCs from IFN-γ mice compared with WT mice tolerance (19, 20). This dual function was initially explained by the existence of specific subpopulations of DCs that preferen- tially trigger T cell priming, whereas other subpopulations were Author contributions: G.M., A.M., and H.L.W. designed research; G.M. and A.M. per- identified to induce T cell tolerance (21–23). The demonstration formed research; G.M. and A.M. analyzed data; and G.M. and A.M. wrote the paper. that a single DC subpopulation can elicit both T cell outcomes, The authors declare no conflict of interest. led to an alternative explanation that the functional status of the *This Direct Submission article had a prearranged editor. DC at the time of antigen presentation, rather than its pheno- 1G.M. and A.M. contributed equally to this work. typic characteristics, is critical for determining T cell responses 2To whom correspondence should be addressed. E-mail: [email protected]. (24). Among the factors linked to the functional status of DCs, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the types of cytokines secreted by DCs can regulate the differ- 1073/pnas.1002099107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1002099107 PNAS | June 22, 2010 | vol. 107 | no. 25 | 11495–11500 Downloaded by guest on October 1, 2021 Spleen LN CNS A P=0.0005 B A 250 1500 P=0.0017 P=0.0005 P=0.0002 P=0.0003 200 1000 150 P=0.003 P=0.009 100 500 50 0 0 C B Spleen LN CNS Naive EAE Naive EAE EAE WT KO WT KO WT KO WT KO WT KO OPN OPN OPN -tub -tub -tub IFN- (ng/ml) IFN- (ng/ml) C Spleen LN CNS P=0.0004 P=0.0006 D 1000 P=0.0001 200 750 150 P=0.004 P=0.002 500 100 250 50 0 0 IFN- (ng/ml) IFN- (ng/ml) Fig. 1. IFN-γ reciprocally regulates OPN and IL-27 expression in DCs. (A and Fig. 2. IFN-γ deficiency alters OPN and IL-27 expression in DCs during EAE. B) Real-time quantitative RT-PCR analysis of mRNA encoding IL-27p28 and (A) Real-time RT-PCR analysis of OPN in DCs isolated from spleen, LN, and OPN in DCs treated with or without mouse rIFN-γ (100 ng/mL). (C and D) − − CNS of naïve and EAE-bearing WT and IFN-γ / mice (n =5–6 per group). (B) Dose-dependent effect of IFN-γ on IL-27 and OPN expression in DCs from − − OPN protein expression was determined by Western blot analysis using anti- WT and IFN-γR / mice. Shown is real-time quantitative RT-PCR analysis of OPN Abs. (C) IL-27 expression is elevated in WT-DCs during the course of mRNA encoding IL-27p28 and OPN in DCs. Data are representative of five EAE. Real-time RT-PCR analysis of IL-27 in DCs isolated from spleen, LN, and independent experiments, and the error bars represent the mean ± SD. − − CNS of naïve and EAE-bearing WT and IFN-γ / mice (n =5–6 per group). − − (Fig. 2A). In accordance with OPN mRNA expression, IFN-γ / DCs and DCs derived from immunized mice from WT and IFN- DCs expressed higher OPN at the protein level as measured by − − γ / mice with MOG-specific TCR transgenic CD4+ T cells (2D2) Western blot (Fig. 2B). To investigate whether IL-27 induction is and measured MOG Ag-specific IL-17 and IL-10 production. We also involved in the inhibitory effects of IFN-γ on EAE de- − − found that T cells cultured with DCs from IFN-γ / mice showed velopment in vivo, we examined IL-27 expression in DCs from − − increased IL-17 levels compared with T cells cultured with WT WT and IFN-γ / mice with EAE.