The nuclear receptor REV-ERBα modulates Th17 cell-mediated autoimmune disease Christina Changa,b, Chin-San Looa,b, Xuan Zhaoc, Laura A. Soltd, Yuqiong Lianga, Sagar P. Bapata,c,e, Han Choc, Theodore M. Kameneckad, Mathias Leblanca, Annette R. Atkinsc, Ruth T. Yuc, Michael Downesc, Thomas P. Burrisf, Ronald M. Evansc,g,1, and Ye Zhenga,1 aNOMIS Center for Immunobiology and Microbial Pathogenesis, The Salk Institute for Biological Studies, La Jolla, CA 92037; bDivision of Biological Sciences, University of California San Diego, La Jolla, CA 92093; cGene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037; dDepartment of Molecular Therapeutics, The Scripps Research Institute, Jupiter, FL 33458; eMedical Scientist Training Program, University of California San Diego, La Jolla, CA 92093; fCenter for Clinical Pharmacology, Washington University School of Medicine and St. Louis College of Pharmacy, St. Louis, MO 63104; and gHoward Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, CA 92037 Contributed by Ronald M. Evans, July 29, 2019 (sent for review May 9, 2019; reviewed by Ming O. Li and David D. Moore) T helper 17 (Th17) cells produce interleukin-17 (IL-17) cytokines In this study, we show that REV-ERBα is also a key feedback and drive inflammatory responses in autoimmune diseases such as regulator of RORγt in Th17 cells. REV-ERBα is specifically up- multiple sclerosis. The differentiation of Th17 cells is dependent on regulated during Th17 differentiation and plays a dual role in the retinoic acid receptor-related orphan nuclear receptor RORγt. Th17 cells. When expressed at a low level, REV-ERBα promotes Here, we identify REV-ERBα (encoded by Nr1d1), a member of the RORγt expression via the suppression of negative regulator nuclear hormone receptor family, as a transcriptional repressor NFIL3 as reported previously (15, 16). At high expression level, that antagonizes RORγt function in Th17 cells. REV-ERBα binds to REV-ERBα directly competes with RORγt binding to the loci of ROR response elements (RORE) in Th17 cells and inhibits the Th17 signature genes and suppresses Th17 effector function. Ele- expression of RORγt-dependent genes including Il17a and Il17f. vated REV-ERBα activity also ameliorates Th17-driven autoim- Furthermore, elevated REV-ERBα expression or treatment with a mune disease experimental autoimmune encephalomyelitis (EAE). synthetic REV-ERB agonist significantly delays the onset and impedes Our results suggest that modulating REV-ERBα activity could the progression of experimental autoimmune encephalomyelitis provide a way to manipulate Th17 cells in autoimmune diseases. (EAE). These results suggest that modulating REV-ERBα activity may be used to manipulate Th17 cells in autoimmune diseases. Results REV-ERBα Is Highly Expressed during Th17 Cell Differentiation. In an transcriptional repressor | autoimmunity | nuclear receptors effort to identify novel players in the nuclear hormone receptor superfamily that are involved in T cell function, we conducted ex- helper 17 (Th17) cells are the drivers of inflammatory re- pression profiling of nuclear hormone receptors in different T Tsponses in a large number of autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and psoriasis (1, 2). The Significance orphan nuclear receptor RORγt is the lineage-specific tran- scription factor that regulates the differentiation of Th17 cells Th17 cells are a subset of T cells that produce interleukin 17 (3). RORγt expression is induced specifically under Th17 dif- and other proinflammatory cytokines. They are involved in the ferentiation condition. Once expressed, RORγt in turn binds to development of autoimmune diseases such as multiple sclero- the loci of Th17 signature genes Il17a and Il17f and up-regulates sis and rheumatoid arthritis. Previous studies illustrated that γ their expression (4). Several small-molecule RORγt antagonists ROR t is a driver for Th17 cell differentiation. Here, we reveal α γ α γ were identified that can inhibit Th17 cell differentiation and REV-ERB as an antagonist of ROR t. REV-ERB and ROR t effector function (5–8). These findings suggested that RORγt share the same DNA binding motif. REV-ERB can inhibit the expression of RORγt target genes and suppress RORγt-driven inhibitors could be developed for treatment of autoimmune Th17 cell differentiation. Treatment with a synthetic REV-ERB diseases. However, RORγt is also known for its critical role in + + agonist delays the onset and impedes the progression of ex- promoting survival of CD4 CD8 double-positive (DP) thymo- perimental autoimmune encephalomyelitis (EAE), a mouse γ cytes. A recent study showed that ROR t inhibitor treatment model of multiple sclerosis. Taken together, our study suggests leads to not only reduced DP thymocyte numbers but also lim- that modulating REV-ERBα activity may be used to manipulate ited T cell repertoire diversity (9). Therefore, it is still a chal- Th17 cells in autoimmune diseases. lenge to develop a safe strategy to inhibit RORγt activity in Th17 cells in vivo. Author contributions: C.C., M.D., R.M.E., and Y.Z. designed research; C.C., C.-S.L., X.Z., Beyond their critical roles in Th17 cell differentiation, members L.A.S., Y.L., S.P.B., H.C., and T.M.K. performed research; C.C., C.-S.L., L.A.S., M.L., A.R.A., M.D., T.P.B., R.M.E., and Y.Z. analyzed data; and C.C., R.T.Y., M.D., and Y.Z. wrote of the ROR family are known to be key players in the circadian the paper. regulatory machinery, where they function as transcriptional ac- Reviewers: M.O.L., Memorial Sloan Kettering Cancer Center; and D.D.M., Baylor College tivators to turn on the expression of circadian genes (10, 11). In of Medicine. the circadian system, RORs’ transcriptional activity is opposed by The authors declare no conflict of interest. a pair of repressors, REV-ERBα and REV-ERBβ. Like RORs, This open access article is distributed under Creative Commons Attribution-NonCommercial- REV-ERBs are also members of the nuclear hormone receptor NoDerivatives License 4.0 (CC BY-NC-ND). family and play critical roles in circadian and metabolic regula- Data deposition: RNA-Seq data reported in this paper have been deposited in the Na- tions (12). REV-ERBs recognize the same RORE DNA sequence tional Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) data- base (accession no. PRJNA293472). ChIP-Seq data have been deposited in Gene Expression as RORs and function as transcriptional repressors to suppress the Omnibus (GEO), www.ncbi.nlm.nih.gov/geo (accession no. GSE72271). expression of ROR target genes (13, 14). Although the antagonistic 1To whom correspondence may be addressed. Email: [email protected] or [email protected]. relationship between ROR and REV-ERB was well established in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the circadian rhythm system, it is not clear if a similar interaction 1073/pnas.1907563116/-/DCSupplemental. exists in the T cell lineage. Published online August 27, 2019. 18528–18536 | PNAS | September 10, 2019 | vol. 116 | no. 37 www.pnas.org/cgi/doi/10.1073/pnas.1907563116 Downloaded by guest on October 1, 2021 REV-ERBα AB1.0 0.8 RORγt Tbet Foxp3 Gata3 Th1 0.8 Th17 5 0.8 4 0.6 iTreg 4 0.6 0.6 3 0.4 3 0.4 0.4 2 0.2 2 0.2 0.2 1 1 Relative expression 0.0 Day(s) 0.0 Day(s) 0 0.0 0 012 3 4 012 3 4 g 7 g re ve Th0 Th1Th2 iTre Th0 Th1Th2 Th0 Th1Th2 Naive Th17 Naive Th1 iT Nai Th17iTreg C Th1 Th17 Days RORγt REV-ERBα REV-ERBβ 12 34 1234 2.5 1.5 0.6 REV-ERBα Relative expression 2.0 1.0 0.4 REV-ERBβ 1.5 1.0 RORγ 0.5 0.2 0.5 Tbet 0.0 0.0 0.0 7 ve ve Th0 Th1 Th0 Th1Th2 Th0 Th1Th2 Th2Th17iTreg β-Actin Naive Th1 iTreg Nai Th17iTreg Nai 100 D hRORγt hTbet hREV-ERBα E IL-17A MIGR1 REV-ERBα REV-ERBβ 80 γ+ 15 60 3 *** 60 P=0.06 40 Donor 10 40 2 0 0 0 20 %IFN #1 Th1 0 5 20 **** 1 99.8 99.7 99.8 *** INFLAMMATION 50 IMMUNOLOGY AND 0 **** 0 0 40 20 60 8 * 30 *** 41.3 10.5 26.7 15 6 20 40 Relative expression ** Donor 10 4 Th17 10 % IL-17A+ 20 0 #2 5 2 α β 0 0 0 0.26 1.71 0.213 Th1 Th17 Th1 Th17 Th1 Th17 Control IFNγ REV-ERBREV-ERB #2 #1 FGl #2 #2 #1 #1 #2 #2 #2 #1 #2 #1 #1-ERBαREV β βREV BαREV BβREV ERB KEGG Pathway Term Count P value ERBα -ERB -ERBβREV -ERBα -ER - -ERBβ -ERBβ -ER Bα -ER - -ERBα #1 27 7.38E-07 T cell receptor signaling pathway MigR1 Control #1 MigR1 Control #2 REV REV REV REV REV REV REV MigR1 Control #2 MigR1 Control #1 MigR1 Contro MigR1 Control Jak-STAT signaling pathway 28 3.29E-05 Il-17a Pim1 Vav3 Il-17f Spry1 Ccl3 Chemokine signaling pathway 26 0.00334 Il-23r Stat2 Ncf1 5 0.01452 Tgfb3 Stat1 Ccr5 Circadian rhythm signaling Csf2 Il12rb Chemokine Cc14 27 Cytokine-cytokine receptor 0.05854 Ccl20 Il7r interaction Cysltr1 Stat5a Cd40 Ltb4r1 Stat3 Inhba Furin Irf9 Il9 Fam124b Jak2 Fas Vax2 Akt2 Il1r2 Th17 differentiation and function Pik3ca Kit Jak-Stat pathway Tnfsf11 Cry1 Akt3 interaction Arnt1 Myc Il15ra Cd27 Npas2 Il2ra Tnfsf8 Cytokine-cytokine receptor rhythm Bhlhe40 Socs2 Circadian Il2rb Il12rb2 Pik3cg Stat4 log2 fold change Fig. 1. REV-ERBα is up-regulated in Th17 cells and inhibits the expression of Th17 signature genes.