Myd88 Is Essential to Sustain Mtor Activation Necessary to Promote T Helper 17 Cell Proliferation by Linking IL-1 and IL-23 Signaling

MyD88 is essential to sustain mTOR activation necessary to promote T helper 17 cell proliferation by linking IL-1 and IL-23 signaling

JiHoon Changa,1, Patrick R. Burkettb,c, Christopher M. Borgesd, Vijay K. Kuchroob, Laurence A. Turkae,2, and Cheong-Hee Changa,2

aDepartment of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109; bCenter for Neurologic Diseases and cPulmonary and Critical Care Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; dThe Transplant Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215; and eDepartment of Surgery, Transplantation Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02129

Edited by Richard A. Flavell, Howard Hughes Medical Institute, Yale School of Medicine, New Haven, CT, and approved December 19, 2012 (received for review April 10, 2012) Myeloid differentiation primary response protein 88 (MyD88) is proinflammatory cytokines was shown to be critical in the sup- classically known as an adaptor, linking TLR and IL-1R to down- port of Th17 cell differentiation (8, 11, 12). In particular, an stream signaling pathways in the innate immune system. In inflammatory cytokine, IL-6, favors Th17 cell development by addition to its role in innate immune cells, MyD88 has been shown inhibiting regulatory T cells (13). The role of IL-1 in Th17 cell to play an important role in T cells. How MyD88 regulates helper differentiation has been investigated as well. IL-1 receptor type − − T-cell differentiation remains largely unknown, however. Here 1-deficient (Il1r1 / ) mice showed a lower incidence of EAE and we demonstrate that MyD88 is an important regulator of IL-17- severe defects in the induction of IL-17–producing T cells (14). + producing CD4 T helper cells (Th17) cell proliferation. MyD88-de- IL-1 signaling in T cells was further shown to be involved in early + ficient CD4 T cells showed a defect in Th17 cell differentiation, Th17 cell differentiation by regulating IFN regulatory factor 4 but not in Th1 cell or Th2 cell differentiation. The impaired IL-17 γ γ + and RAR-related orphan receptor t (ROR t) (15). production from MyD88-deficient CD4 T cells is not a result of Although roles for MyD88 in the innate immune system are defective RAR-related orphan receptor γt (RORγt) expression. In- well established, little is known about their potential function in stead, MyD88 is essential for sustaining the mammalian target of the adaptive immune system. Several studies have demonstrated rapamycin (mTOR) activation necessary to promote Th17 cell pro- + important roles of MyD88 in T cells. For instance, T-cell ex- liferation by linking IL-1 and IL-23 signaling. MyD88-deficient CD4 pression of MyD88 is required for resistance to Toxoplasma + T cells showed impaired mTOR activation and, consequently, re- gondii (16); the MyD88-dependent signaling pathway in CD4 duced Th17 cell proliferation. Importantly, the absence of MyD88 T cells has been shown to enhance proliferation and augment in T cells ameliorated disease in the experimental autoimmune en- humoral immune responses (17); and MyD88 is required for T- cephalomyelitis model. Taken together, our results demonstrate cell effector function in the development of inflammatory bowel − − that MyD88 has a dual function in Th17 cells by delivering IL-1 disease (18). Interestingly, although Myd88 / T cells were found signaling during the early differentiation stage and integrating IL- to exhibit decreased IL-17 production (18), how T-cell differen- 23 signaling to the mTOR complex to expand committed Th17 cells. tiation could be regulated by MyD88 was not clear in that study. Here we investigated the molecular mechanism by which + yeloid differentiation primary response protein 88 (MyD88) MyD88 regulates CD4 T-cell differentiation. Our results dem- Mwas originally isolated as a cloned cDNA that was induced in onstrate that MyD88 contributes to Th17 cell differentiation, but M1 myeloblastic leukemia cells on activation with IL-6 (1). The not to Th1 or Th2 cell differentiation. Both IL-1 and IL-23 sig- function of MyD88 was then uncovered, because the C-terminal naling depend on MyD88 and result in up-regulation of IL-23R. portion of MyD88 was found to be similar to the Drosophila Toll MyD88-deficient Th17 cells show reduced IL-23R expression and receptor and the mammalian IL-1 receptor (IL-1R) (2). This mTOR activation, leading to impaired Th17 cell proliferation. conserved region in the cytoplasmic tails of IL-1R and Toll-like Furthermore, MyD88 is crucial for proper Th17 cell differentia- receptor (TLR) is referred to as the Toll/IL-1R (TIR) domain. tion in vivo. Thus, our findings reveal a unique role for the innate MyD88 is now known to play an essential role in the innate im- adaptor MyD88 in the regulation of Th17 cell differentiation. mune response by linking members of the TLR and IL-1R su- perfamily to the downstream activation of NF-κB and MAPKs (3). Results Among cytokines produced by activated innate immune cells, IL- Impaired IL-17A Production in MyD88-Deficient CD4+ T Cells. MyD88- − − 23 has been shown to promote production of the proinflammatory deficient (Myd88 / ) T cells have been shown to exhibit de- + cytokine IL-17 in activated T cells (4). IL-17–producing CD4 creased IL-17 production (18), but the underlying mechanism T helper (Th17) cells were identified after the discovery that IL- 23 is linked to traditionally Th1-associated autoimmune disorders, such as experimental autoimmune encephalitis (EAE). Author contributions: J.C., P.R.B., and C.-H.C. designed research; J.C., P.R.B., and C.M.B. − − − − Il23a / mice, but not Il12a / mice, were shown to be autoim- performed research; V.K.K. and L.A.T. contributed new reagents/analytic tools; J.C., P.R.B., V.K.K., L.A.T., and C.-H.C. analyzed data; and J.C., P.R.B., L.A.T., and C.-H.C. wrote mune-resistant (5). IL-23 is required for Th17 cell-mediated the paper. autoimmune disorders in vivo, but the role of IL-23 in Th17 cell The authors declare no conflict of interest. differentiation remains controversial (6). Previous studies sup- This article is a PNAS Direct Submission. port the idea that IL-23 helps expand or maintain Th17 cells (7– 1Present address: The Transplant Institute, Beth Israel Deaconess Medical Center, Harvard 9). In addition, a recent study reemphasized the importance of Medical School, Boston, MA 02215. IL-23 in the generation of pathogenic Th17 cells, showing that 2To whom correspondence may be addressed. E-mail: [email protected] or heechang@ they can be generated with IL-23, IL-6, and IL-1β (10). umich.edu. β In addition to IL-23, other cytokines, including TGF- , IL-6, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and IL-1, play roles in Th17 cell development. TGF-β with 1073/pnas.1206048110/-/DCSupplemental.

2270–2275 | PNAS | February 5, 2013 | vol. 110 | no. 6 www.pnas.org/cgi/doi/10.1073/pnas.1206048110 Downloaded by guest on September 27, 2021 AB under Th1 cell-polarizing conditions or IL-4 production under Th2 cell-polarizing conditions (Fig. 1A). Given our observation of impaired IL-17A production after T-cell restimulation, we next asked whether the defect started − − + during T-cell differentiation. Myd88 / CD4 T cells already + showed a lower frequency of IL-17A cells after 3 d of Th17 cell differentiation (Fig. S1A), although the defect was less dramatic − − + than that observed after restimulation. Again, Myd88 / CD4 T cells showed no defect in IFN-γ expression under Th1 cell-polarizing conditions (Fig. S1A). In addition, the induction of Il17a − − + mRNA was poorer in Myd88 / CD4 T cells compared with WT under Th17 cell-polarizing conditions; however, Ifng mRNA levels − − + were comparable in WT and Myd88 / CD4 T cells (Fig. S1B). Along with IL-17A, the production of IL-17F, another IL-17 cy- − − + tokine family member, was severely impaired from Myd88 / CD4 T cells after restimulation, and the defect in Il17f mRNA level was C detected based on the early Th17 cell differentiation (Fig. S1C).

RORγt Is Expressed Normally in Myd88−/− Th17 Cells. IL-2 or IL-10 signaling has been shown to modulate Th17 cell generation (19, 20); however, in the present study, impaired IL-17 production in − − + Myd88 / CD4 T cells was not related to increased IL-2 or − − + IL-10 production (Fig. S2). Given that Myd88 / CD4 T cells are defective in IL-17 production under Th17 cell-polarizing conditions but not in IFN-γ or IL-4 production under Th1 or Th2 + Fig. 1. Impaired production of IL-17A in MyD88-deficient CD4 Tcellsis cell-polarizing conditions, respectively, we speculated that the − − + independent of RORγt. (A)Purified Myd88 / or WT CD4 T cells were defect might result from cytokine signaling specific to Th17 cell cultured under Th1-, Th2-, or Th17-polarizing conditions for 5 d, followed differentiation. Among Th17-skewing cytokines, including TGF- by restimulation with plate-bound anti-CD3 for 24 h, as described in β, IL-6, IL-1β, and IL-23, we first focused on IL-1β because of the Materials and Methods. ELISA was performed to detect the amounts of + well-established role of MyD88 as an adaptor in IL-1R signaling IFN-γ, IL-4, and IL-17A in culture supernatants. *P < 0.05. (B)Purified CD4 − − (3). Indeed, the defect in IL-17A production from Myd88 / cells were cultured in the presence of the indicated cytokines for 3 d, and + β Foxp3 and IL-17A expression was analyzed by flow cytometry. (C)RORγt CD4 T cells was less marked in the absence of IL-1 (Fig. S3A). protein levels were analyzed by flow cytometry during Th17 differentia- However, the defect could not be explained solely by the lack of −/− + tion. Results are representative of three experiments (A and B)ortwo IL-1 signaling in Myd88 CD4 T cells, because they still experiments (C). exhibited impaired IL-17A production without IL-1β (Fig. S3A). − − A similar defect in Il17a mRNA expression from Myd88 / + CD4 T cells was observed in the absence of IL-1β (Fig. S3B). has been unclear. To study how MyD88 regulates Th17 cell We then investigated whether TGF-β signaling was altered in + fi − − + differentiation, we enriched CD4 T cells from MyD88-suf cient Myd88 / CD4 T cells by assessing Foxp3 expression as a readout. −/− − − + (i.e., WT) and Myd88 mice and performed in vitro T-cell Compared with WT, Myd88 / CD4 T cells expressed more −/− + differentiation. The Myd88 CD4 T cells showed a substantial Foxp3 after treatment with TGF-β alone (Fig. 1B, Upper). TGF- defect in IL-17A production under Th17 cell-polarizing conditions β–induced Foxp3 has been shown to inhibit Th17 cell differen- (Fig. 1A). MyD88 deficiency did not affect IFN-γ production tiation by antagonizing RORγt (21). Thus, we evaluated whether

Fig. 2. IL-1β and IL-23 contribute ABC synergistically to IL-17 production. + (A)Purified CD4 T cells were cultured under Th17-polarizing conditions for 5 d. Cells were harvested before restimulation (0 h) or after 6 h with plate-bound anti-CD3. The amounts of Il17a, Il17f, Il22, Il21, Il2, and Il10 mRNA were quantified by + qRT-PCR. (B)CD4 T cells were cultured with the indicated cytokines or media (Med) for 3 or 5 d. Culture supernatants were collected, and D IL-17A production was assessed by ELISA. *P < 0.05. NS, not significant. (C and D) Cells were cultured with the indicated cytokines for 3 or 5 d. qRT-PCR was performed to measure the levels of Il17a and Il17f (C)and of Ahr, Batf, Socs3, Runx1, Rora, and Il23r (D). *P < 0.05, WT vs. − − Myd88 / cells. After normalization to Actb, mRNA levels in fresh iso- IMMUNOLOGY + lated CD4 T cells (day 0) were used as a control to compare expression levels. Results are representative of three independent experiments.

Chang et al. PNAS | February 5, 2013 | vol. 110 | no. 6 | 2271 Downloaded by guest on September 27, 2021 −/− + enhanced TGF-β–induced Foxp3 expression in Myd88 CD4 A T cells results in reduced RORγt-directed IL-17 expression. After cells were cultured with various cytokines, both WT and − − + Myd88 / CD4 T cells showed greatly reduced Foxp3 expression under IL-17–producing conditions (TGF-β + IL-6 or TGF-β + IL-6 + IL-1β + IL-23) (Fig. 1B). Importantly, under Th17-polarizing − − + conditions, Myd88 / CD4 T cells showed no signiﬁcant de- crease in Rorc mRNA levels (Fig. S4)orRORγt expression (Fig. 1C). These data suggest that impaired IL-17 production from − − + Myd88 / CD4 T cells is not related to reduced RORγt expression. B

IL-1β and IL-23 Contribute Synergistically to IL-17 Production. Be- cause we observed the greatest impairment in IL-17 production − − + from Myd88 / CD4 T cells after anti-CD3 restimulation (Fig. 1A), we investigated T-cell responses during the later differentiation stage. At 5 d after Th17 cell-polarizing cultures, cells were restimulated with anti-CD3 for 6 h to assess mRNA levels of cytokines. Consistent with our results presented above, genes for IL-17 cytokine family members IL-17A, IL-17F, and IL-22 were − − + poorly expressed from Myd88 / CD4 T cells after anti-CD3 − − + restimulation (Fig. 2A). Importantly, Myd88 / CD4 T cells did not exhibit a global defect in cytokine expression; IL-21, IL-2, and IL-10 expression was actually enhanced (Fig. 2A). Of note, C on day 5 (0 h), mRNA levels of IL-17A, IL-17F, and IL-22 were − − + increased in WT, but not in Myd88 / , CD4 T cells (Fig. 2A). This finding prompted us to examine the later stage of Th17 cell differentiation in depth. IL-17A production was further increased at day 5 in WT, but − − + not in Myd88 / CD4 T cells, when the differentiation was driven by IL-6, TGF-β, IL-1β, and IL-23 (Fig. 2B). The increase – Fig. 3. MyD88-dependent IL-23R expression contributes to IL-17A pro- in IL-17A production during day 3 5 in WT was not observed fi + β β– duction. (A)Puried CD4 T cells were activated and cultured in the without IL-1 and IL-23, suggesting a critical role of IL-1 and presence of the indicated cytokines for 5 d. 5, 20, or 100 ng/mL of IL-23 – IL-23 mediated signaling. At day 5, the amount of Il17a mRNA was added to the culture. Il17a and Il23r mRNA expression was quantified + in WT was increased when both IL-1β and IL-23 were added by qRT-PCR. After normalization to Actb, mRNA levels of CD4 T cells − − + together with TGF-β and IL-6 in the culture (Fig. 2C). Similarly treated with TGF-β + IL-6 were used as a control. (B) Myd88 / CD4 T cells (albeit with different kinetics), Il17f gene expression was main- were transduced with retroviral vectors encoding IRES-Thy1.1 (murine tained in the presence of IL-1β and IL-23 together with TGF-β stem cell virus) or IL-23R-IRES-Thy1.1 (IL-23R) and cultured for 5 d under Th0 or Th17 conditions. IL-17A production was analyzed by flow cytom- and IL-6 (Fig. 2C). − − + etry. (C) Myd88 / CD4 T cells were transduced with retroviruses ex- Along with Rorc, several transcription factors or molecules, pressing NGFR, WT MyD88-NGFR (MyD88), or mutant MyD88 (truncated + including Ahr (22), Batf (23), Socs3 (24), Runx1 (25), Rora (26), TIR domain)-NGFR (TIR) and cultured under Th17 conditions for 5 d. NGFR + and Il23r (13), are known to be involved in Th17 cell differen- CD4 T cells were sorted, and mRNA expression of Il17a and Il23r was tiation. We tested whether the impairment of IL-17 expression in quantified by qRT-PCR. After normalization to Actb, mRNA levels of NGFR- − − + Myd88 / CD4 T cells by IL-1β and IL-23 is caused by differ- transduced CD4+ T cells were used as a control. *P < 0.05. Results are ential expression of these factors. The expression of Ahr, Batf, representative of two independent experiments (A and C)orthreein- − − + and Socs3 mRNA was comparable in WT and Myd88 / CD4 T dependent experiments (B). cells (Fig. 2D). The expression of Runx1 and Rora mRNA did not differ with or without IL-1β and IL-23; however, Il23r expression −/− + − − + from Myd88 CD4 T cells can be restored, at least in part, by was greatly increased at day 5 in WT, but not in Myd88 / ,CD4 T IL-23R expression. cells by IL-1β and IL-23 (Fig. 2D). To further test the role of MyD88-dependent IL-23R expres- − − sion in IL-17 production, we retrovirally transduced Myd88 / MyD88-Dependent IL-23R Expression Enhances IL-17A Production. We + then examined whether IL-23R–mediated signaling requires CD4 T cells with an empty vector, WT MyD88, or TIR. The + MyD88 for Th17 cell differentiation. Unlike WT CD4 T cells, TIR construct lacks the death domain of MyD88, and thus its which showed a synergistic effect on Il17a expression by IL-1β downstream signaling, including IL receptor-associated kinase −/− + (IRAK) interaction and NF-κB activation, is impaired (17). and IL-23, Myd88 CD4 T cells failed to demonstrate the + + same effect even with various combinations of IL-1β and IL-23 Transduced cells (CD4 NGFR ) were sorted, and Il17a and Il23r (Fig. 3A, Left). In parallel studies, Il23r expression was lower from gene expression was assessed. We found that WT MyD88, but not − − + − − + Myd88 / CD4 T cells compared with WT, suggesting a potential TIR, induced the expression of Il17a and Il23r in Myd88 / CD4 role of MyD88 in the induction of IL-23R (Fig. 3A, Right). T cells (Fig. 3C), suggesting that MyD88 is essential for inducing − − + If poor induction of IL-23R expression in Myd88 / CD4 T cells the IL-23R expression necessary for IL-17 production. contributes to low IL-17 production, then overexpression of IL- − − + 23R should restore IL-17 in Myd88 / CD4 T cells. To test this, IL-23–Mediated Signaling Promotes Th17 Cell Expansion by Activating − − + we infected Myd88 / CD4 T cells with retrovirus expressing IL- mTOR. To understand how IL-1 and IL-23 signaling collaborates 23R. IL-23R overexpression had little effect on IL-17A expres- during Th17 cell differentiation, we first analyzed the temporal sion under Th0 cell-polarizing conditions, but increased the expression of their receptors. In line with the observation that + frequency of IL-17A cells under Th17 cell-polarizing conditions IL-1 signaling is critical during early Th17 cell development (15), (Fig. 3B). These findings indicate that impaired IL-17 expression Il1r1 expression was increased during early Th17 cell differenti-

2272 | www.pnas.org/cgi/doi/10.1073/pnas.1206048110 Chang et al. Downloaded by guest on September 27, 2021 A B

Fig. 4. IL-1 and IL-23 signaling sequentially promotes Th17 cell proliferation. (A) The amounts of Il1r1 and Il23r mRNA during Th17 cell differentiation (with TGF-β + IL-6 + IL-1β + IL-23) were quantified by qRT-PCR. *P < 0.05. (B) + CD4 T cells were activated and cultured with the indicated cytokines for 3 d. At the end of day 3, additional cytokines were added to the indicated cultures. On day 5, qRT-PCR was performed to measure the levels of Il17a and Il23r mRNA. (C) Cells were cultured under Th17-polarizing C D conditions for 3 d. The indicated inhibitors ( 0.1 μMrapa- mycin, 5 μM BMS-345541, 10 μM SP600125, and 5 μM SB202190) or DMSO was added at the end of day 3. On day 5, mRNA expression of Il17a was quantified by qRT-PCR. + *P < 0.05, DMSO vs. inhibitors treatment in WT. (D)CD4 T cells from WT (designated +/+)orMyd88−/− (designated −/−) mice were activated and cultured with indicated cytokines. The whole-cell lysates were prepared on day 3 or day 5. P-S6, S6, and GAPDH were detected by immunoblot analysis. (E) Cells were activated and cultured with the indicated cytokines. At day 5, the whole-cell lysates were E F prepared. P-mTOR, P-S6K, P-S6, and β-actin were detected by immunoblot analysis. (F) Cells were activated and cultured with the indicated cytokines for 3 d. At the end of day 3, cells were washed, labeled with carboxyfluorescein diacetate succinimidyl ester, and cultured in T-cell medium for additional 3 d in the presence of same cytokines. Carboxyfluorescein diacetate succinimidyl ester dilution and IL-17A expression were analyzed by flow cytometry. Results are representative of two independent experiments (A and E) or three independent experiments (B, C, D, and F).

ation and diminished after 3 d of culture (Fig. 4A and Fig. S5). 5 when IL-1β and IL-23 were present together with IL-6 and − − + Myd88 / CD4 T cells exhibited no defect in Il1r1 expression; in TGF-β (Fig. 4D). Furthermore, both IL-1β and IL-23 are required contrast, Il23r expression was low until day 3 and increased for activation of the mTOR signaling pathway (Fig. 4E). Because during later Th17 cell differentiation, which was not observed in mTOR activation is linked to cell proliferation, we assessed − − + Myd88 / CD4 T cells (Fig. 4A). Because these two receptors whether IL-1 and IL-23 signaling promotes cell proliferation. WT − − + were expressed during different time periods, we asked whether and Myd88 / CD4 T cells proliferated at a similar rate during IL-1 signaling and IL-23 signaling are sequentially involved in early stages of Th0 or Th17 cell differentiation (Fig. S7). However, Th17 cell differentiation. To test this, we added IL-1β or IL-23 to during later Th17 cell differentiation (day 3–5), MyD88-de- the cultures at different time points. When IL-1 signaling was pendent IL-1β and IL-23 signaling was required for the cell pro- absent during early Th17 cell development, Il17a and Il23r ex- liferation (Fig. 4F). Taken together, these findings indicate that pression was poorly induced (Fig. 4B). Notably, when cells were MyD88 plays a critical role in integrating IL-1 and IL-23 signaling initially cultured with TGF-β, IL-6, and IL-1β, adding IL-23 at for Th17 cell proliferation and expansion. day 3 potently induced Il17a and Il23r expression, similar to when all cytokines were added at the beginning (Fig. 4B; com- MyD88 Is Required for IL-23 Signaling During Later Th17 Cell pare the fifth and seventh groups). Taken together, these results Differentiation. Although both IL-1β and IL-23 have been indicate that IL-1 signaling is essential during early Th17 cell shown to be important for MyD88-mediated Th17 cell pro- development, and that IL-23 is required mainly for later Th17 liferation, whether MyD88 is required for IL-23 signaling is un- cell differentiation. clear. Because early IL-1 signaling is essential for MyD88- We next asked which signaling molecules are involved in the mediated Th17 cell differentiation, we addressed this question later Th17 cell differentiation. To test this, we added specific using inducible deletion of Myd88 during later Th17 cell differ- fl fl inhibitors for mTOR, IKK, JNK, and p38 at day 3 of Th17 cul- entiation. As shown in Fig. 5A, treatment of cells from Myd88 / tures and assessed Il17a expression at day 5. Our results show ERCreT2 mice with 4-hydroxytamoxifen (4-OHT) (27) reduced that blocking NF-κB or MAPK signaling had little effect on Il17a the level of MyD88. To test whether MyD88 is required for IL-23 + expression (Fig. 4C). However, rapamycin treatment reduced signaling during later Th17 cell differentiation, CD4 T cells fl fl fl fl fl fl Il17a expression, suggesting that the mammalian target of from WT (Myd88 / ), Myd88 / ERCreT2, or Myd88 / CD4-Cre rapamycin (mTOR)-mediated signaling pathway is critical for (Myd88ΔT) mice (27) were cultured initially with TGF-β, IL-6, the later Th17 cell differentiation. Moreover, Il23r expression and IL-1β. To avoid hindrance of early IL-1 signaling, 4-OHT was

was reduced by rapamycin treatment (Fig. S6). In fact, phos- added at day 2, followed by IL-23 at day 3. As shown in previously IMMUNOLOGY phorylation of S6 ribosomal protein was sustained in WT at day (Fig. 4B), adding IL-23 to the cultures increased IL-17A

Chang et al. PNAS | February 5, 2013 | vol. 110 | no. 6 | 2273 Downloaded by guest on September 27, 2021 ABCgroups (Table S1). Taken together, these data demonstrate that MyD88 has a crucial role for proper Th17 cell differentiation in vivo. Discussion Many molecules classically known to be critical for the innate immune system also have important functions in adaptive immune responses (28, 29). The present study shows that MyD88 plays an important role in Th17 cell differentiation. In addition, it demonstrates the underlying molecular mechanism by which D MyD88-mediated IL-1 signaling sequentially synergizes with IL- 23 to promote Th17 cell proliferation. IL-23 has a unique function in the maintenance of a T-cell subset producing IL-17 (6). IL-23R was shown to be essential for the terminal differentiation of Th17 cells in vivo (9); however, the molecular mechanism governing expression of the IL-23R remains unclear. IL-23R expression was abolished in the absence of Th17-inducing proinflammatory cytokines such as IL-6 (13). Importantly, it has been suggested that IL-23 induces IL-23R E expression via positive feedback regulation (30, 31). Our results + further show that MyD88-mediated IL-1 signaling in CD4 T cells is a prerequisite for the up-regulation of IL-23R by IL-23. IL-1 and IL-23 are produced mainly by activated antigen-presenting cells. Of interest, IL-1 also up-regulates IL-23 subunit p19 gene expression in antigen-presenting cells (32). Thus, IL-1 and IL-23 signaling seems to be closely regulated through MyD88 in both innate and adaptive immune cells. IL-1 signaling on T cells is known to be important (14, 15). − − The incidence of EAE is significantly lower in Il1r1 / mice Fig. 5. MyD88 is required for IL-23 signaling and Th17 cell differentiation in compared with WT mice, correlated with a failure to induce + fl fl vivo. (A) CD4 T cells from Myd88 / ERCreT2 mice were cultured and Th17 cells (14). The molecular mechanism by which IL-1R treated at day 1 with 4-OHT (1 μM) or untreated. MyD88 and β-actin were contributes to Th17 cell differentiation remains unclear, how- detected by immunoblot analysis from the whole-cell lysates prepared at ever. Chung et al. (15) reported that IL-1 signaling is required day 3 or 5. Densitometry values normalized to β-actin are shown in paren- + for early Th17 cell differentiation by up-regulating RORγt and theses. (B and D)CD4 T cells were activated and cultured initially with TGF- IFN regulatory factor 4 expression; however, as shown in the β + IL-6 or TGF-β + IL-6 + IL-1β. 4-OHT (1 μM) was added to all of the cultures − − present study, RORγt expression is not altered in Myd88 / at day 2, and IL-23 (20 ng/mL) was added to the indicated culture at day 3. + On day 5, IL-17A production in culture supernatants was assessed by ELISA CD4 T cells. This discrepancy might be related to the fact that (B), or qRT-PCR was performed to measure the levels of Il17a and Il23r (D). MyD88 is an adaptor not only for IL-1R, but also for TLR. + *P < 0.05. NS, not significant. (C)CD4 T cells were cultured as indicated in Chung et al. (15) used a coculture system in which dendritic cells B without 4-OHT. IL-17A production was measured by ELISA on day 5. *P < and T cells were cultured in the presence of LPS. Several pre- 0.05. NS, not significant. Results are representative of three independent vious studies have uncovered the critical role of TLR in the – Δ experiments (A) or two independent experiments (B D). (E)WTorMyd88 T adaptive immune system (17, 33, 34). Of note, Reynolds et al. μ ’ + mice were immunized with 100 g of MOG peptide in complete Freund s (35) recently showed that TLR2 signaling in CD4 T cells pro- adjuvant and given 100 ng of pertussis toxin IV on day 0 and 2 post- immunization. Mice were scored (0–5) daily for evidence of clinical disease motes Th17 responses. Thus, in the experimental setup of Chung (n = 16 for WT; n = 13 for Myd88ΔT). Shown are the combined data of three et al. (15), the MyD88-dependent signaling pathway was likely independent experiments, all of which yielded similar results. Error bars activated by TLR even in the absence of IL-1 signaling on T cells. represent SEM. Whether RORγt functions, such as transcriptional activity, are − − + altered in Myd88 / CD4 T cells remains elusive. SIGIRR, a negative regulator for TLR and IL-1R signaling, −/− + production in WT, but not in Myd88 , CD4 T cells (Fig. 5B). has been shown to suppress Th17 cell proliferation (36). Im- Importantly, in the presence of 4-OHT, IL-17A production from portantly, this study demonstrated that Th17 cell effector func- fl/fl + Myd88 ERCreT2 CD4 T cells was significantly reduced tion is linked to mTOR-mediated cell proliferation. When fully − − compared with WT (Fig. 5 B and C). mRNA expression of Il17a differentiated WT and Sigirr / Th17 cells were treated with IL-1, fl fl − − and Il23r genes was also poorly induced in Myd88 / ERCreT2 IL-1–induced mTOR kinase activation was increased in Sigirr / + CD4 T cells compared with WT (Fig. 5D). In addition, MyD88- Th17 cells compared with WT cells (36). In line with this finding, dependent IL-23 signaling was required for activation of mTOR our results show that MyD88-dependent IL-1 and IL-23 signaling signaling pathway (Fig. S8). Taken together, these results indicate is required for the later stage of Th17 cell proliferation by acti- that MyD88 is an important regulator not only of early IL-1, but vating the mTOR signaling pathway. also of later IL-23 signaling for Th17 cell proliferation. There is growing interest in the interplay between host metabolism and the immune system. In particular, mTOR plays an MyD88 Is Crucial for Th17 Cell Differentiation in Vivo. To determine essential role in integrating environmental cues. Several previous whether the proposed role for MyD88 in Th17 cell differentia- studies have shown that mTOR critically regulates the differ- tion is relevant in vivo, EAE was induced in WT and Myd88ΔT entiation of T cells (37–40). mTOR in T cells acts to integrate mice. All WT mice (16 of 16) developed EAE after immuniza- signal 1 (antigen recognition through T-cell receptor) and signal tion with myelin oligodendrocyte glycoprotein (MOG), com- 2, the integrated sum of environmental cues, including CD28, pared with only 6 of 13 Myd88ΔT mice (Table S1). EAE was less IL-2R, amino acids. and skewing cytokines (41). In the present − − + prevalent in Myd88ΔT mice compared with WT mice (Fig. 5E); study, Myd88 / CD4 T cells demonstrated no global defect in however, the disease was of similar onset and severity in the two mTOR activation; however, MyD88 deficiency in T cells could

2274 | www.pnas.org/cgi/doi/10.1073/pnas.1206048110 Chang et al. Downloaded by guest on September 27, 2021 not integrate IL-1 and IL-23 signaling (signal 2) and failed to Materials and Methods fl fl fl fl fl fl generate sustained mTOR activation. This may explain why Myd88-deficient, Myd88 ox/ ox, Myd88 ox/ ox CD4-Cre, Myd88 ox/ ox CreT2, + MyD88 deficiency affects only IL-17 expression, even though and WT mice were used for the experiments. Purified CD4 T cells were mTORC1 was shown to be involved in both Th1 and Th17 activated by plate-bound anti-CD3 and soluble anti-CD28. Tamoxifen-in- responses (40). duced MyD88 deletion was monitored by immunoblot analysis after treating In conclusion, our findings show that MyD88 has a dual the cells with 4-OHT at different time points. Mice were injected with MOG function in Th17 cell differentiation. MyD88 delivers IL-1 sig- peptide and also pertussis toxin on days 0 and 2 after immunization. Full naling during the initial commitment stage and links it to IL-23 materials and methods with associated references, eight figures and one signaling, which expands committed Th17 cells via mTOR ac- table can be found at the extended online supporting information. tivation. Given that MyD88 also plays an important role in activating innate immune responses, MyD88 serves as a good ACKNOWLEDGMENTS. We thank Sang-Won V. Kim and Dan R. Littman (New example linking two arms of the immune systems. Our results York University, New York, NY) for the IL-23R retroviral vectors, Sei Yoshida and Ken Inoki (University of Michigan, Ann Arbor, MI) for helpful comments on have important implications for targeting MyD88-dependent mTOR signaling, and Ryan H. Newton (Beth Israel Deaconess Medical Center) signaling in both innate and adaptive immune cells to treat Th17 for a critical reading of the manuscript. This work was supported by National cell-associated diseases. Institutes of Health Grants AI062789 (to L.A.T.) and AI073677 (to C.-H.C.).

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