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Identification of an IL-27/ 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 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 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 , 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 3 (EBI3, also known as modulation of OPN and IL-27. IL27), and a unique IL-12p35–like , 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 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 (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 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 −/− -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 , 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 , 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. We found that expression of DCs (Fig. 3A). When IL-10 secretion was quantified, significant IL-27 was up-regulated in WT-DCs of mice with EAE (Fig. 2C) − − differences were observed between T cells cultured with DCs γ / − − and that DCs from IFN- mice with EAE had much less IL-27 from IFN-γ / mice and WT controls. In accordance with low expression compared with WT mice (Fig. 2C). These results γ−/− γ IL-27 expression, IFN- DCs induced less IL-10 production by demonstrate that IFN- up-regulates the expression of IL-27 T cells whereas T cells cultured with DCs from WT mice show an while inhibiting OPN in DCs to negatively regulate the progres- increase in IL-10 levels (Fig. 3A). We then investigated whether sion of . Consistent with this observation, we the IFN-γ of DCs could alter the balance of IL-10 and γ−/− found that IFN- mice had high serum levels of OPN with little IL-17 production from T cells through differential regulation of or no increase in serum IL-27 levels compared with WT mice (Fig. OPN and IL-27 in DCs. To test this hypothesis, the in vivo isolated γ S2). These results clearly indicate that IFN- differentially mod- DCs from EAE mice were pretreated with IFN-γ and cultured ulates OPN and IL-27 expression in DCs in vivo during the with 2D2 T cells in the presence of MOG. As shown in Fig. 3B, − − course of EAE and, thus, could impact on immune responses IFN-γ–treated DCs both from WT and IFN-γ / mice, which ex- both in the periphery and CNS. press IFNγR, inhibited IL-17 production while increasing IL-10 Polarization of T cells to Th1, Th2, or Th17 phenotypes is production from T cells. However, this effect was not observed a critical feature of cell–mediated and is influenced by when a neutralizing to IL-27 was included in the cul- production of cytokines by DCs. We and others have shown that ture. Taken together, our results demonstrate that IFN-γ signal- DC-expressed OPN induces IL-17 while inhibiting IL-10 pro- ing events in DCs play a major role in negative regulation of Th17 duction from T cells (17, 20). In contrast, IL-27 has been shown development and induction of IL-10 production from T cells. to inhibit IL-17 while inducing IL-10 production from T cells both To directly demonstrate the effect of OPN and IL-27 on T in humans and mice (6–13). Because we observed IFN-γ de- cells, we stimulated CD4+ T cells with rOPN in the presence or ficiency led to altered OPN and IL-27 expression in DCs du- absence of rIL-27. We found that CD4+ T cells activated in the ring EAE, we investigated whether DC-derived OPN and IL-27 presence of OPN produced increased amounts of IL-17 and less had a role in T cell differentiation, specifically T cell production IL-10 (Fig. 3C). However, the addition of IL-27 inhibited OPN- of IL-17 and IL-10. To test this hypothesis, we induced EAE in induced IL-17 while abrogating the inhibitory effect of OPN on − − WT and IFN-γ / mice with oligodendrocyte IL-10 from T cells (Fig. 3C). Because we observed IFN-γ de- (MOG) and, at day 15, postimmunization isolated splenic CD11c+ ficiency leads to increased OPN and low IL-27 levels in DCs − − DCs. Naïve splenic DCs were used as controls. We cocultured naïve during EAE, we next examined whether T cells from IFN-γ /

11496 | www.pnas.org/cgi/doi/10.1073/pnas.1002099107 Murugaiyan et al. Downloaded by guest on October 1, 2021 A 1200 450 A Spleen LN CNS WT-DC (Naive) P=0.0004 360 800 4000 300 P=0.0010 800 P=0.0001 270 IFN- -/-DC (Naive) WT-DC (EAE) 600 3000 180 200 400 IFN- -/-DC(EAE) 400 2000 90 100 0 0 200 1000 0 0 0

WT 600 1000 WT B IFN- -/- IFN- -/- 500 750 Spleen LN CNS 400 B P=0.0039 P=0.0102 P=0.0018 300 500 400 350 300 200 300 250 300 250 100 200 200 200 0 0 150 100 100 100 50 0 0 0

C 300 1500 200 1000 C 700 300 100 500 525 200 WT 350 IFN- -/- 0 0 100 175 0 0

Fig. 4. IFN-γ deficiency differentially regulates IL-17 and IL-10 expression in Fig. 3. Altered OPN and IL-27 expression in DCs differentially modulate T T cells. (A and B) Real-time RT-PCR analysis of IL-17 and IL-10 in spleen, LN, A + − − cell production of IL-17 and IL-10. ( ) Total CD4 T cells isolated from 2D2 and CNS-derived T cells were isolated from WT and IFN-γ / mice with or + – − − mice were cocultured with CD11c DCs isolated from naïve and MOG35 55- without EAE (n = 8 per group). (C) LN cells derived from WT and IFN-γ / γ−/− immunized WT and IFN- mice. Supernatants from cultures were har- mice were activated in vitro with MOG35–55 (20 μg/mL) for 72 h, and cell- vested 72 h after initiation of cultures and assayed by ELISA for IL-17 and free supernatants from cultures were assayed for IL-17 and IL-10. IL-10. (B) IFN-γ treated DCs suppress IL-17 production while inducing IL-10 − − production from 2D2 T cells. WT and IFN-γ / DCs isolated from EAE mice γ were stimulated with IFN- for 12 h and were then cultured with 2D2 mice with MOG and monitored disease progression. We T cells in the presence of MOG peptide for 72 h. After 72 h, IL-17 and IL-10 γ– fi production by CD4+ T cells was determined by ELISA. (C) OPN-induced found that pretreatment with IFN- modi ed DCs markedly IL-17 production or IL-10 inhibition from CD4+ T cells was abrogated by the reduced clinical severity of EAE compared with mice treated with addition of IL-27. CD4+ T cells were activated with anti-CD3 and anti-CD28 control DCs (Fig. 5A). Furthermore, the effect of IFN-γ–modi- mAb (0.3 μg/mL) in the presence of 1 μg/mL mouse rOPN. In some culture fied DCs on reducing the clinical severity of EAE was abrogated conditions, 100 ng/mL of mouse rIL-27 was added together with 1 μg/mL in the mice that received neutralizing IL-27 antibody (Fig. 5A). mouse rOPN. Supernatants from cultures were analyzed for cytokines IL-17 We then analyzed cytokine production in splenocytes of mice and IL-10 by ELISA. Data are representative of three independent experi- γ– ± given IFN- treated DCs or control DCs after ex vivo restim- ments, and the error bars represent the mean SD. ulation with MOG. We found large amounts of IL-17 and small amounts of IL-10 in splenocytes of mice given control DCs (Fig. B γ– mice exhibited changes in IL-17 and IL-10 expression. In ac- 5 ). In contrast, transfer of IFN- treated DCs resulted in less cordance with increased OPN expression in DCs, T cells from secretion of IL-17 and increased synthesis of IL-10 in splenocytes γ−/− of recipient mice (Fig. 5B). Coinjection of IFN-γ–treated DCs IFN- mice had high levels of IL-17 expression in spleen, LN, IMMUNOLOGY and CNS (Fig. 4A). When IL-10 levels were measured, we also with anti-IL-27 antibody resulted in higher secretion of IL-17 and found striking differences between T cells from WT and IFN- less IL-10 production by ex vivo-restimulated splenocytes than by − − γ– γ / mice. In accordance with increased IL-27 levels in DCs, T cells from mice that received IFN- treated DCs. These results γ– cells from WT mice expressed higher levels of IL-10 in com- demonstrate a dominant tolerogenic effect of IFN- treated DCs − − B parison with T cells from IFN-γ / mice in spleen, LN, and CNS (Fig. 5 ). To further examine the contribution of IL-27 to this γ (Fig. 4B). Moreover, we observed higher secretion of IL-17 and tolerogenic effect, we injected IFN- -treated DCs or control DCs −/− γ– fi less IL-10 production by ex vivo-restimulated LN cells from IFN- into WT and IL-27R mice. We found that IFN- modi ed − − γ / mice than by cells from WT mice (Fig. 4C). Taken together, DCs reduced clinical severity only in WT mice. Disruption of IL- our results demonstrate that IFN-γ activation of DCs re- 27 abrogated the tolerogenic effect of IFN-γ–treated DCs, which ciprocally regulates T cell IL-17 and IL-10 production through were not able to limit disease severity (Fig. 5C). In accordance differential induction of OPN and IL-27. with the disease score, disruption of the IL-27 pathway abrogated the disease-modifying effect of DCs by modulating T cell cytokine IFN-γ–Modified DCs Attenuate EAE in an IL-27–dependent Manner. To secretion (Fig. 5D). In the clinical setting of MS, therapeutic in- determine whether IFN-γ–modified DCs have enhanced regula- tervention is often started after the onset of the symptoms. tory function in vivo, we activated DCs with or without mouse Therefore, it is important to investigate whether a treatment recombinant IFN-γ, pulsed them with MOG, and transferred regimen, which is effective EAE prevention, can also reverse these cells into syngeneic naïve mice. We then immunized the established disease. Thus, we tested the efficacy of IFN-γ–mod-

Murugaiyan et al. PNAS | June 22, 2010 | vol. 107 | no. 25 | 11497 Downloaded by guest on October 1, 2021 DC-Unt A DC-IFN- B 3.5 DC-IFN- /anti-IL-27 3.0 2000 1400 DC-Unt 2.5 1050 DC-IFN- 2.0 1000 700 DC-IFN- /anti-IL-27 1.5 1.0 350 0.5 0 0 0.0 0 10 20 30 Days post immunization

DC-Unt WT C DC-IFN- WT D 400 200 DC-Unt IL-27R-/- 4.0 DC-IFN- IL-27R-/- Fig. 5. IFN-γ–modified DCs attenuate EAE in an IL-27– 3.5 300 DC-Unt A 3.0 dependent manner. ( ) DCs were left untreated (DC-Unt) 200 DC-IFN- γ γ 2.5 100 or treated with IFN- (DC-IFN- ) and pulsed with MOG and were then transferred into syngeneic WT mice. As in- 2.0 100 1.5 dicated, some mice were coinjected with neutralizing anti- 1.0 0 0 IL-27 antibody together with IFN-γ-treated DCs. Five days 0.5 later, the DC primed mice were immunized with MOG and 0.0 B 0 10 20 30 monitored for EAE. ( ) LN cells obtained 18 d after im- Days post immunization munization from mice described in (A) above, were restimulated with MOG35–55 (20 μg/mL) for 72 h. Cell-free DC-Unt supernatants were assayed for IL-17 and IL-10 by ELISA. (C) − − E 3.5 DC-IFN- Clinical scores of EAE in WT and IL-27R / mice injected 3.0 DC-IFN- /anti-IL-27 with IFN-γ–treated or –untreated DCs before MOG im- DC-IFN- /IC 2.5 munization. (D) LN cells from IFN-γ-modified DC-treated − − 2.0 and control DC-treated WT and IL-27R / mice were acti- 1.5 DC treatment vated in vitro with MOG35–55 (20 μg/mL) for 72 h, and 1.0 cell-free culture supernatants were assayed for IL-17 and 0.5 IL-10 by ELISA. (E) Clinical scores of EAE in WT mice im- 0.0 munized with MOG and treated with MOG-pulsed DCs 0 10 20 30 modified with IFN-γ or their unmodified counterparts. Ar- Days post immunization row indicates time of DC injection (clinical score of ≥1.5).

ified DCs on EAE after the onset of clinical symptoms. We found WT mice (Fig. 6G). These findings indicate that the reduced that treatment with IFN-γ–modified DCs after the onset of EAE OPN expression observed in DCs from IL-27–treated mice is due (score of ≥1.5) resulted in rapid clinical recovery from EAE (Fig. to the inhibitory effect of IL-27 on OPN. Thus, IL-27–mediated 5E). These results indicate an essential role for IFN-γ in driving resolution of inflammation is mediated both through induction DCs that blunt Th17 responses and halt autoimmune in- of IL-10 and inhibition of IL-17 from CD4+ T cells and by in- flammation through mechanisms involving IL-27 and IL-10. hibition of OPN expression in DCs. Taken together, these results demonstrate that IFN-γ serves a protective role in Th17-medi- IL-27 Treatment Inhibits Clinical Severity of EAE in IFN-γR–Deficient ated CNS inflammation via IL-27 induction. Mice. Our results thus far suggest that IFN-γ suppresses EAE development via induction of IL-27. To further investigate this Discussion observation, we asked whether IL-27 treatment in vivo could Our results identify a previously unknown pathway by which − − reverse the severe EAE phenotype observed in IFNγR / mice. IFN-γ limits IL-17–mediated autoimmune inflammation through − − To test this hypothesis, WT and IFN-γR / mice were adminis- DC modulation of OPN and IL-27. In EAE, it was initially tered recombinant mouse IL-27 after immunization with MOG. thought that CD4+ T cells mediating had a Th1 Consistent with recent studies (10), we found that IL-27 could phenotype characterized by the production of IFN-γ (26). inhibit EAE development in WT mice (Fig. 6A). Strikingly, in- However, this traditional view has been challenged by studies jection of IL-27 suppressed the severe EAE phenotype observed describing more EAE in IFN-γ–deficient animals (27). Evidence − − in IFN-γR / mice compared with PBS controls (Fig. 6A). In now indicates that T cells critical for EAE are characterized − − addition, LN cells from both IL-27-treated WT and IFN-γR / by the production of IL-17. In addition, IL-17 expression has mice produced much less IL-17 and higher IL-10 when been detected in the target tissue in human autoimmune diseases restimulated with MOG antigen (Fig. 6B). Furthermore, we including MS, rheumatoid , and (28). IL-17– found that IL-27 treatment inhibited OPN levels in DCs from deficient mice develop EAE with delayed onset and reduced se- − − both WT and IFN-γR / mice (Fig. 6C). Because we observed verity (29), and anti-IL-17 antibody prevents expres- that IFN-γ–induced IL-27 expression was accompanied by re- sion in the and the subsequent development of EAE (30). duced OPN levels in DCs, we examined whether IL-27 had any Furthermore, T cell infiltration and inflammation in the brain in inhibitory effects on OPN expression in DCs. In accordance with EAE occur only when Th17 cells outnumber Th1 cells (31). − − the in vivo inhibition of OPN, we found that IL-27 significantly IFN-γ plays an important role in Th17 cell biology. IFN-γ / − − inhibited OPN expression in DCs both from WT and IFN-γR / mice immunized with collagen in CFA develop severe arthritis, mice (Fig. 6D). In addition we found that marrow-derived and T cells from such mice secrete increased amounts of IL-17 − − DCs from IL-27R / mice expressed higher levels of OPN than after collagen restimulation in vitro (32). Along the same line, − − DCs from WT mice (Fig. 6E). Moreover, DCs isolated from after mycobacterial infection, IFN-γ / mice had larger numbers − − spleen, LN, and CNS of IL-27R / mice had very high levels of of IL-17–producing T cells than WT mice (33). In addition, OPN compared with WT mice (Fig. 6F), and serum OPN levels a disease-ameliorating effect of IFN-γ has been observed in le- − − were significantly higher in IL-27R / mice in comparison with thal autoimmune myocarditis (34). Nonetheless, although en-

11498 | www.pnas.org/cgi/doi/10.1073/pnas.1002099107 Murugaiyan et al. Downloaded by guest on October 1, 2021 WT-PBS A WT-IL-27 B IFN- R-/--PBS 4.0 IFN- R-/- -IL-27 700 350 3.5 WT-Unt 3.0 525 280 WT-PBSWT-Unt IFN- -/- -Unt 2.5 210 IFN-IFN- R-/--/- -Unt-PBS 350 WT-IL-27 2.0 WT-IL-27 1.5 140IFN- -/- - IL-27 IFN-IFN- R-/--/- --IL-27 IL-27 1.0 175 IL-17 (pg/ml)

IL-10 (pg/ml) 70 0.5 0.0 0 0 0 10 20 30

LN CNS C P=0.0112 P=0.0028 D E Med P=0.0059 P=0.0033 7 350 800 300 IL-2 1200 P=0.0015 280 Fig. 6. IL-27 treatment inhibits clinical severity of EAE in 600 800 γ −/− A 210 200 IFN- R mice. ( ) IL-27 inhibits clinical severity of EAE in 400 −/− −/− 140 400 WT and IFN-γR mice. WT and IFN-γR mice (n =6) 100 70 200 were immunized with MOG peptide emulsified in com- 0 μ 0 0 0 plete freund's adjuvant. Recombinant IL-27 (0.25 g per mouse) was administered by s.c. to immunized WT and IFN- − − γR / mice every other day from day 2 until day 18. (B)LN cells from IL-27-treated and control mice were activated in vitro with MOG35–55 (20 μg/mL) for 72 h, and cell-free culture supernatants were assayed for IL-17 and IL-10 by Spleen LN CNS ELISA. (C) Real-time RT-PCR analysis of OPN in DCs isolated F G P=0.0011 −/− 100 P=0.0068 P=0.0008 P=0.0007 4000 from LN and CNS of WT and IFN-γR mice (n =5–6 per 2000 2500 D 3000 group) treated with or without IL-27. ( ) Real-time RT-PCR 75 2000 1500 analysis of OPN in DCs isolated from spleen of naïve WT 50 1500 2000 − − 1000 and IFN-γR / mice treated with or without rIL-27 (100 ng/ 1000 1000 25 500 mL). (E) Real-time RT-PCR analysis of OPN in DCs isolated 500 − − 0 from spleen of naïve WT and IL-27R / mice. (F) Real-time 0 0 0 RT-PCR analysis of OPN in DCs isolated from spleen, LN, and CNS of naïve and EAE bearing WT and IL-27R−/− mice (n =6 − − per group). (G) ELISA of serum OPN from WT and IL-27R / mice with or without EAE.

dogenous IFN-γ is protective in animal models of arthritis and IL-17 production from T cells was mediated through inhibition of in EAE, the mechanisms that orchestrate the antiinflammatory OPN and induction of IL-27 from DCs that, in turn, leads to effects of IFN-γ in controlling autoimmune inflammation are suppression of IL-17 from T cells. In addition, Th17 generation poorly understood. Here, we show that IFN-γ induces IL-27 was also suppressed by expression of the IFN-αR on DCs while inhibiting OPN expression in DCs and that engagement of and , thus demonstrating the potential similarities IFNγR expressed by DCs leads to suppression of IL-17 pro- between IFN-γ– and IFN-α/β–dependent suppression of the Th17 duction while inducing IL-10 from T cells. In accordance with response (17, 36). − − these in vitro results, DCs from IFN-γ / mice expressed very The nature of specialized DCs that selectively dampen effector high levels of OPN and low levels of IL-27 in comparison with T cell-driven immunity is not well understood. Our results sug- − − DC from WT mice. T cells cultured with IFN-γ / DCs produced gest a dominant function for IFN-γ–modified DCs to dampen higher IL-17 and lower IL-10, whereas T cells cultured with DCs autoimmunity through the induction of IL-10 and/or inhibition from WT mice produced lower IL-17 and higher IL-10. This of IL-17 production from T cells in an IL-27–dependent manner. − − difference in T cell production of IL-17 and IL-10 correlated It has been shown that IL-27R / mice develop exacerbated with the pattern of OPN and IL-27 expression by DCs that are EAE, owing to high IL-17 or low IL-10 production from T cells modulated by IFN-γ. We have reported that OPN-induced IL-17 (8–10). Consistent with these findings, the tolerogenic effect of production by CD4 T cells via β3- receptor and inhibited IFN-γ-modified DCs we observed was completely abrogated in −/−

IL-10 via CD44 receptor (16). We have also shown that OPN- IL-27R mice. The increased expression of IL-27 in WT vs. IMMUNOLOGY induced IL-17 production is associated with an increase in the IFN-γ–deficient mice correlates with increased IL-10 expression IL-17 lineage RORγt expression. IL-27– in T cells during EAE, which suggests that IFN-γ contributes to mediated inhibition of IL-17 has been shown through the down- dampening inflammatory responses and driving the resolution of regulation of RORγt (7). It appears that IL-27 overrides the autoimmune pathology. effect of OPN in activated T cells by down-regulating RORγt An additional mechanism to limit immune responses is the ne- expression and indirectly affects IL-10 via the CD44 receptor. gative feedback of inflammatory process by inflammatory cyto- Thus, our results demonstrate that there is a reciprocal regula- kines. DC expressed OPN promotes induction of IFN-γ in addition tion in the induction of pathogenic cytokine IL-17 and protective to IL-17 from T cells both in humans and mice (19). Our results cytokine IL-10 in the depending on the cytokine demonstrate that OPN induces a negative feedback loop whereby secretion by the . IFN-γ limits IL-17 production by T cells through the induction of Previous studies have shown that the differentiation of Th17 IL-27 and inhibition of OPN from DCs, and that this feedback cells is suppressed by IFN-γ. In vitro, treatment with IFN- loop is perturbed by the absence of IFN-γ (Fig. S3). The role of γ- leads to increased frequency of Th17 cells, IFN-γ in antagonizing the function of IL-17, a critical pathoge- whereas exogenous IFN-γ reduces Th17 populations (35). Our nic cytokine in autoimmune conditions, might be paradoxical for results identify a previously unknown pathway by which IFN-γ IFN-γ, a proinflammatory cytokine. However, it is now recognized limits IL-17–mediated autoimmune inflammation through DC that IFN-γ has regulatory function in limiting inflammation. At the modulation of OPN and IL-27. The negative impact of IFN-γ on height of inflammation during the course of an autoimmune pa-

Murugaiyan et al. PNAS | June 22, 2010 | vol. 107 | no. 25 | 11499 Downloaded by guest on October 1, 2021 − − thology, the immune system is mobilized to restrict excess inflam- from naïve and MOG35-55-immunized WT and IFN-γ / mice. Cocultures mation, and it appears that IFN-γ acts as a master upstream re- were performed at a 1:3 DC/T cell ratio in U-bottom 96-well plates. Cytokines gulator of both inflammatory and regulatory pathways (37, 38). Our were measured by using real-time quantitative RT-PCR, and specific ELISA study identifies an important IFN-γ–dependent self-regulatory pro- and Western blotting were performed with primers and as listed SI Materials and Methods cess that serves to control immune system homeostasis through an in . For EAE induction, mice were injected s.c. with – fi OPN/IL-27 axis in dendritic cells. 100 mg of MOG35 55 peptide emulsi ed in CFA (Difco), supplemented with 5 mg/mL and injected twice i.v. with 200 ng of Materials and Methods pertussis toxin. Clinical assessment of EAE was performed as described in SI Materials and Methods.SeeSI Materials and Methods for full methods. DCs and CD4+ T cells were purified from spleens and LNs by positive selection using CD11C+ and CD4+ microbeads, respectively (Miltenyi Biotec). Bone ACKNOWLEDGMENTS. This work was supported by National Institutes of marrow-derived DCs were generated by using a described method (39). For γ – Health Grants NS038037, AI043458, and NS23132 and the Nancy Davis in vitro assays, DCs were activated with recombinant mouse IFN- for 12 60 Foundation. G.M is supported by a postdoctoral fellowship from National – μ h. Splenocytes or LN cells were activated with MOG 35 55 peptide (20 g/mL) Multiple Sclerosis Society, New York. A.M. is supported by the National + for 60–72 h. For DC-T cell cocultures, total CD4 T cells isolated from MOG- Research Service Award Fellowship Grant F32AI075761 from the National specific TCR-transgenic mice (2D2) were cocultured with CD11c+ DC isolated Institute of and Infectious Diseases.

1. Kastelein RA, Hunter CA, Cua DJ (2007) Discovery and biology of IL-23 and IL-27: 19. Banchereau J, et al. (2000) Immunobiology of dendritic cells. Annu Rev Immunol 18: Related but functionally distinct regulators of inflammation. Annu Rev Immunol 25: 767–811. 221–242. 20. Steinman RM, Hawiger D, Nussenzweig MC (2003) Tolerogenic dendritic cells. Annu 2. Pflanz S, et al. (2002) IL-27, a heterodimeric cytokine composed of EBI3 and p28 Rev Immunol 21:685–711. protein, induces proliferation of naive CD4(+) T cells. Immunity 16:779–790. 21. Huang FP, et al. (2000) A discrete subpopulation of dendritic cells transports apoptotic 3. Takeda A, et al. (2003) Cutting edge: Role of IL-27/WSX-1 signaling for induction of intestinal epithelial cells to T cell areas of mesenteric lymph nodes. J Exp Med 191: T-bet through activation of STAT1 during initial Th1 commitment. J Immunol 170: 435–444. 4886–4890. 22. Munn DH, et al. (2002) Potential regulatory function of human dendritic cells 4. Yoshimoto T, Yoshimoto T, Yasuda K, Mizuguchi J, Nakanishi K (2007) IL-27 suppresses expressing indoleamine 2,3-dioxygenase. Science 297:1867–1870. Th2 cell development and Th2 cytokines production from polarized Th2 cells: A novel 23. Scheinecker C, McHugh R, Shevach EM, Germain RN (2002) Constitutive presentation therapeutic way for Th2–mediated allergic inflammation. JImmunol179:4415–4423. of a natural tissue autoantigen exclusively by dendritic cells in the draining lymph J Exp Med – 5. Wang S, Miyazaki Y, Shinozaki Y, Yoshida H (2007) Augmentation of antigen-presenting node. 196:1079 1090. and Th1-promoting functions of dendritic cells by WSX-1(IL-27R) deficiency. JImmunol 24. Belz GT, et al. (2002) The CD8alpha(+) is responsible for inducing J Exp Med – 179:6421–6428. peripheral self-tolerance to tissue-associated . 196:1099 1104. 6. Awasthi A, et al. (2007) A dominant function for 27 in generating 25. Wakkach A, et al. (2003) Characterization of dendritic cells that induce tolerance and Immunity – –producing anti-inflammatory T cells. Nat Immunol 8:1380–1389. T regulatory 1 cell differentiation in vivo. 18:605 617. Annu Rev Immunol 7. Murugaiyan G, et al. (2009) IL-27 is a key regulator of IL-10 and IL-17 production by 26. Sospedra M, Martin R (2005) Immunology of multiple sclerosis. 23: 683–747. human CD4+ T cells. J Immunol 183:2435–2443. 27. Ferber IA, et al. (1996) Mice with a disrupted IFN-gamma gene are susceptible to the 8. Batten M, et al. (2006) limits autoimmune encephalomyelitis by induction of experimental autoimmune encephalomyelitis (EAE). J Immunol 156:5–7. suppressing the development of –producing T cells. Nat Immunol 7: 28. Bettelli E, Oukka M, Kuchroo VK (2007) T(H)-17 cells in the circle of immunity and 929–936. autoimmunity. Nat Immunol 8:345–350. 9. Stumhofer JS, et al. (2006) Interleukin 27 negatively regulates the development of 29. Komiyama Y, et al. (2006) IL-17 plays an important role in the development of interleukin 17–producing T helper cells during chronic inflammation of the central experimental autoimmune encephalomyelitis. J Immunol 177:566–573. nervous system. Nat Immunol 7:937–945. 30. Park H, et al. (2005) A distinct lineage of CD4 T cells regulates tissue inflammation by 10. Fitzgerald DC, et al. (2007) Suppressive effect of IL-27 on encephalitogenic Th17 cells producing interleukin 17. Nat Immunol 6:1133–1141. and the effector phase of experimental autoimmune encephalomyelitis. J Immunol 31. Stromnes IM, Cerretti LM, Liggitt D, Harris RA, Goverman JM (2008) Differential 179:3268–3275. regulation of central nervous system autoimmunity by T(H)1 and T(H)17 cells. Nat 11. Denhardt DT, Noda M, O’Regan AW, Pavlin D, Berman JS (2001) Osteopontin as Med 14:337–342. a means to cope with environmental insults: regulation of inflammation, tissue 32. Chu CQ, Swart D, Alcorn D, Tocker J, Elkon KB (2007) -gamma regulates J Clin Invest – remodeling, and cell survival. 107:1055 1061. susceptibility to collagen-induced arthritis through suppression of interleukin-17. fl fl 12. Chabas D, et al. (2001) The in uence of the proin ammatory cytokine, osteopontin, Arthritis Rheum 56:1145–1151. Science – on autoimmune demyelinating disease. 294:1731 1735. 33. Cruz A, et al. (2006) Cutting edge: IFN-gamma regulates the induction and expansion of 13. Jansson M, Panoutsakopoulou V, Baker J, Klein L, Cantor H (2002) Cutting edge: IL-17–producing CD4 T cells during mycobacterial infection. J Immunol 177:1416–1420. Attenuated experimental autoimmune encephalomyelitis in eta-1/osteopontin- 34. Afanasyeva M, et al. (2005) Impaired up-regulation of CD25 on CD4+ T cells in IFN- fi J Immunol – de cient mice. 168:2096 2099. gamma knockout mice is associated with progression of myocarditis to heart failure. J 14. Comabella M, et al. (2005) Plasma osteopontin levels in multiple sclerosis. Proc Natl Acad Sci USA 102:180–185. Neuroimmunol – 158:231 239. 35. Harrington LE, et al. (2005) Interleukin 17–producing CD4+ effector T cells develop via 15. Vogt MH, et al. (2004) Osteopontin levels and increased disease activity in relapsing- a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol 6:1123–1132. remitting multiple sclerosis patients. J Neuroimmunol 155:155–160. 36. Guo B, Chang EY, Cheng G (2008) The type I IFN induction pathway constrains Th17– 16. Murugaiyan G, Mittal A, Weiner HL (2008) Increased osteopontin expression in mediated autoimmune inflammation in mice. J Clin Invest 118:1680–1690. dendritic cells amplifies IL-17 production by CD4+ T cells in experimental autoimmune 37. Zhang J (2007) Yin and yang interplay of IFN-gamma in inflammation and autoimmune encephalomyelitis and in multiple sclerosis. J Immunol 181:7480–7488. disease. JClinInvest117:871–873. 17. Shinohara ML, Kim JH, Garcia VA, Cantor H (2008) Engagement of the type I 38. Hu X, Ivashkiv LB (2009) Cross-regulation of signaling pathways by interferon-gamma: interferon receptor on dendritic cells inhibits development: Role of implications for immune responses and autoimmune diseases. Immunity 31:539–550. intracellular osteopontin. Immunity 29:68–78. 39. Murugaiyan G, Agrawal R, Mishra GC, Mitra D, Saha B (2007) Differential CD40/CD40L 18. Steinman L (2009) A molecular trio in relapse and remission in multiple sclerosis. Nat expression results in counteracting antitumor immune responses. J Immunol 178: Rev Immunol 9:440–447. 2047–2055.

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