REV-ERBβ emerges as a non-redundant brake on TH17-driven inflammation

For years, immunologists and circadian biologists alike assumed that the two REV-ERB nuclear receptors—REV-ERBα and REV-ERBβ—were largely interchangeable molecular timekeepers. Outside the immune system, in tissues such as the liver, adipose tissue, and brain, the pair exhibits similar expression kinetics and overlapping target genes, reinforcing the belief that REV-ERBβ is functionally redundant to its better-studied sibling. But new research published in iScience by Sean Campbell, Sarah A. Mosure, Mohammed Amir, Jonathan Chuck, Qun Lu, and Laura A. Solt at UF Scripps Biomedical Research upends that dogma in one of the body’s most inflammatory cell types: the TH17 cell. Their work demonstrates that REV-ERBβ is not a redundant copy of REV-ERBα but a distinct, cell-intrinsic negative regulator of TH17-mediated pro-inflammatory responses, operating through its own unique transcriptional program and largely independent of the classical co-repressor NCoR1 and the endogenous ligand heme.

TH17 cells are an IL-17A-secreting subset of CD4+ T effector cells that defend against invading extracellular pathogens, maintaining immunity at skin and mucosal barriers under homeostatic conditions. In addition to IL-17A, they produce IL-17F, IL-21, and IL-22, recruiting other pro-inflammatory mediators to promote pathogen clearance. But when their activation goes awry, TH17 cells become key drivers of several autoimmune and chronic inflammatory diseases, including multiple sclerosis, psoriasis, and inflammatory bowel disease. While IL-17A/IL-17R neutralizing antibodies have found success in treating psoriasis, they have had limited success in inflammatory bowel disease, suggesting that targeting factors that broadly regulate TH17 cell function may be more effective. The lineage-defining transcription factor RORγt drives TH17 development, but a complex network of additional transcription factors works in concert to promote or inhibit TH17-mediated inflammation. The Solt laboratory previously identified that REV-ERBα acts as a negative regulator of the TH17 program in vitro and in vivo, and that REV-ERB-selective synthetic ligands mediated protection against TH17-driven diseases in mouse models of multiple sclerosis and ulcerative colitis. However, those ligands targeted both REV-ERBs or REV-ERBα alone, leaving the physiological function of REV-ERBβ in T cell effector responses largely unexplored.

To establish whether REV-ERBβ is required to limit TH17-mediated pro-inflammatory responses in vivo, the team used REV-ERBβ-deficient mice in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Immune phenotyping revealed no overt differences in thymocyte or T cell composition between wild-type and knockout animals under steady-state conditions. But when the researchers immunized both groups with myelin oligodendrocyte glycoprotein peptide (MOG35-55) and monitored disease daily, the knockout mice fared dramatically worse. Mean maximum disease score reached 3.57 in REV-ERBβ-deficient mice relative to 2.05 in wild-type littermates—a statistically significant difference—and the mean day of disease onset was also earlier, at 10.43 versus 11.62 days. Flow cytometric analysis of the central nervous system at the peak of disease revealed an increased frequency and number of CD4+ T cells infiltrating the CNS of knockout animals. The disease phenotype was not attributable to increased frequencies of CD4+CCR6+ cells or decreased frequencies of anti-inflammatory Foxp3+ T regulatory cells, as no differences were observed between genotypes. However, the frequency and number of CD4+RORγt+ cells were elevated in the knockout CNS, and while no statistically significant increase in IL-17A+ T cells was detected, there was a significant increase in the frequency and number of the IL-17A+IFNγ+ population—a double-positive subset whose acquisition of IFNγ has been linked to pathogenicity in several models of chronic disease, including EAE and multiple sclerosis.

Because the EAE model involved global deletion of REV-ERBβ, the excessive neuroinflammation could theoretically have stemmed from direct effects on microglia or other immune cell types rather than T cells themselves. To specifically assess T-cell-mediated effects, the researchers sorted naive CD4+ T cells from wild-type and REV-ERBβ-deficient mice and adoptively transferred them into Rag1-deficient recipients to track the development of colitis, another TH17-driven inflammatory disorder. Over a 12-week experiment, transfer of REV-ERBβ-deficient cells resulted in more severe weight loss and increased colon weight, a function of increased immune cell infiltration leading to exacerbated intestinal inflammation. Colon weight/length ratios—a measure that controls for measurement bias and reflects inflammation and edema—were also higher in mice receiving knockout cells, indicative of more intestinal inflammation. Quantitative real-time PCR of proximal colon tissue revealed significantly increased expression of the pro-inflammatory cytokine genes Il17a, Il17f, Il6, Tnf, and Ifng in mice receiving REV-ERBβ-deficient T cells compared to wild-type recipients. Expression of Il22 was also elevated but did not reach statistical significance, and Cxcl10—a gene activated downstream of IL-17 and IL-22 signaling—was increased, consistent with an overall heightened TH17 pro-inflammatory profile in vivo.

In a shorter-term experiment sacrificed at five to six weeks post-transfer, when T cells are just beginning to enter the colon, mice receiving REV-ERBβ-deficient T cells again showed increased colon weight and higher colon weight/length ratios. Flow cytometry revealed increased infiltration of total immune cells and increased frequency and number of CD3+CD4+ T cells in the mesenteric lymph nodes and colons of knockout-cell recipients, with no differences observed in the spleen. While mice receiving knockout cells did not show increased frequencies of CD4+RORγt+ or RORγt+IL-17A+ TH17 cells, there were increased absolute numbers of both populations in the colons. NanoString experiments on FACS-sorted T cells isolated from the colons confirmed increased expression of pro-inflammatory cytokines and chemokines, including Cxcl10, Ccr9, and Il22. Intriguingly, genes involved in oxidative stress and apoptosis—including Sod1, Bcl2, Casp3, Casp8, and Xiap—were decreased in REV-ERBβ-deficient T cells, suggesting the receptor may regulate apoptosis and cell survival in T cells within the colon. Expression of Il18rap was also downregulated; this receptor acts as a negative regulator of TH17 cell differentiation in the intestine, and its dysfunction is linked to intestinal diseases like Crohn’s disease, inflammatory bowel disease, and celiac disease.

To model REV-ERBβ-dependent T cell functions in vitro, the team tested the requirement for the receptor to limit pro-inflammatory cytokine expression in CD4+ T helper cells using congenic co-cultures. Naive CD4+ T cells from congenic CD45.1 wild-type and CD45.2 REV-ERBβ-deficient mice were differentiated under three different TH17 polarizing conditions: non-pathogenic (TGFβ plus IL-6), pathogenic 1 (TGFβ, IL-6, and IL-23), and pathogenic 2 (IL-6, IL-1β, and IL-23). Loss of REV-ERBβ decreased the ratio of knockout to wild-type cells in all three cultures, suggesting a cell-extrinsic effect on proliferation or survival. Despite the decreased frequency of knockout cells in culture, REV-ERBβ deficiency led to increased IL-17A production under non-pathogenic and pathogenic 1 conditions but not pathogenic 2 conditions, hinting at a potential requirement for TGFβ in the receptor’s regulatory effects. Carboxyfluorescein succinimidyl ester (CFSE) staining revealed no overt defects in proliferation, but a viability dye showed increased non-viable cells in knockout cultures, consistent with the decreased expression of Sod1, Bcl2, and Xiap seen in the NanoString data. These effects appear specific to TH17 cells, as loss of REV-ERBβ did not affect TH1 or inducible T regulatory cell differentiation.

To address whether REV-ERBβ affects TH17 cell function through cell-intrinsic or cell-extrinsic mechanisms in vivo, the researchers performed co-transfer experiments using the T cell transfer model of colitis, mixing equal numbers of congenic CD45.1+ wild-type and CD45.2+ REV-ERBβ-deficient naive CD4+ T cells before injection into Rag1-deficient recipients. After six weeks of expansion, there were decreased REV-ERBβ-deficient cells in the mesenteric lymph nodes and colon lamina propria, corroborating the in vitro data. Upregulation of the chemokine receptor CCR9 and the integrin α4β7 in mesenteric lymph nodes mediates lymphocyte homing to the intestinal lamina propria. While CCR9 expression was increased in REV-ERBβ-deficient T cells in the lymph nodes and colon, the knockout cells did not have an advantage homing to these sites. Despite this, REV-ERBβ-deficient T cells exhibited increased frequency of RORγt+ cells and RORγt+IL-22+ cells in the colons, along with increased frequency of IL-17A+IFNγ+ cells. These results demonstrate that REV-ERBβ is required to control T cell accumulation at inflammatory sites as well as temper downstream TH17 pro-inflammatory responses in a cell-intrinsic manner.

To gain a better understanding of the transcriptional programs controlled by each receptor, the team performed RNA-sequencing on TH17 cells collected at peak mRNA expression of each REV-ERB: time zero (naive) for REV-ERBβ and 24 hours post-activation for REV-ERBα. The results were striking. At both time points, there was very little overlap in transcriptional signatures between REV-ERBβ-deficient and REV-ERBα-deficient T cells. The genes that did overlap fell under metabolic pathways via KEGG analysis. At time zero, REV-ERBβ regulated a large number of pathways relative to REV-ERBα, including genes involved in the cell cycle (Ccnd2, Ccnd3, Ccnb1), whereas REV-ERBα regulated genes involved in circadian rhythm, consistent with its role as a core member of the circadian clock. REV-ERBβ also appeared to regulate genes involved in FoxO, JAK-STAT, and PI3K-Akt signaling pathways at time zero. In TH17 cells at 24 hours, REV-ERBβ regulated genes involved in Polycomb repressive complexes, Notch signaling, regulation of the actin cytoskeleton, and cellular senescence, whereas REV-ERBα regulated genes involved in CD4+ T cell differentiation, including TH1, TH2, and TH17 lineages. RT-qPCR further confirmed that post-activation, REV-ERBα was upregulated similar to RORγt, while REV-ERBβ was downregulated upon TH17 cell differentiation—a divergent expression pattern in contrast to what is observed in other tissues.

Because REV-ERBα’s repressive function is largely instilled by the co-repressor NCoR1, and homology led many to assume REV-ERBβ works similarly, the researchers probed whether this holds true. Mammalian two-hybrid assays in HEK293 cells demonstrated that the REV-ERBβ ligand binding domain can interact with NCoR1 in a dose-dependent manner. However, overlay of publicly available NCoR1 ChIP-seq peaks with differentially expressed genes from naive REV-ERBβ-deficient T cells revealed very little overlap—only 34 shared target genes. The overlapping genes were related more toward overall T cell activation and proliferation (Cd69, Ctse, Ccnd2, Ccnd3, Icos, Il7r, Ptpn22) than TH17 lineage specification. To determine whether REV-ERBβ represses transcription passively by displacing RORs from shared DNA binding sites or actively by recruiting co-repressor proteins, the team generated retroviral constructs encompassing the DNA binding domain alone (βDBD) or all but the DNA binding domain (βΔDBD), along with a heme binding mutation (βHBM, H565F) that reduces heme binding affinity more than 10,000-fold. When overexpressed in TH17 cells, the truncation and deletion constructs were unable to repress Il17a relative to wild-type REV-ERBβ, indicating that active repression requires both DNA binding and cofactor recruitment via the ligand binding domain. Remarkably, the heme binding mutation did not relieve repression at IL-17A, and RNA-sequencing showed it had little effect on transcription, relieving repression of only about 10–15% of wild-type target genes. These data suggest REV-ERBβ functions in a largely heme-independent manner in TH17 cells—a non-canonical signaling mechanism that contrasts sharply with REV-ERBα, whose heme-dependent NCoR1 interaction recruits the histone deacetylase HDAC3 to repress transcription at Il17a.

The findings carry significant therapeutic implications. Several small molecules targeting both REV-ERBα and REV-ERBβ have been developed as alternatives to RORγt modulators, and the Solt laboratory recently developed a ligand selective for REV-ERBα over REV-ERBβ. But the current data suggest that targeting both factors would be ideal to better encompass inhibition of the TH17 program in vivo—both during the initial development of TH17 cells, when REV-ERBβ is more highly expressed, and later post-activation, when REV-ERBα dominates. By this logic, ligands that target both REV-ERBs would have a greater repressive effect than a ligand targeting only a single receptor subtype. The work also raises a compelling paradox: in vivo, REV-ERBβ-deficient TH17 cells accumulate at lesion sites, yet in vitro they exhibit increased cell death. The researchers suggest that in vivo, these cells may reside in a supportive ecosystem, interacting with stromal cells and antigen-presenting cells that provide survival signals absent in culture. Future experiments testing competition, cell death versus proliferation, and accumulation under both conditions would tease out this contradiction. The team acknowledges limitations, including the use of retroviral overexpression rather than endogenous gene editing, the lack of ChIP-grade REV-ERBβ antibodies to map binding sites, and the inability to completely rule out REV-ERBβ’s independence from REV-ERBα using single knockout systems. Nevertheless, the study establishes for the first time that individuality exists between the two REV-ERB receptors in TH17 cells, suggesting they complement each other in a spatiotemporal network rather than overlapping patterns—a discovery that reshapes how scientists think about circadian nuclear receptors in immunity and opens new avenues for treating TH17-driven autoimmune disease.

Subject of Research: Non-redundant negative regulation of TH17-mediated inflammation by the nuclear receptor REV-ERBβ

Article Title: Negative regulation of T H 17-mediated inflammation by the nuclear receptor REV-ERBβ

Article References: Campbell, S., Mosure, S. A., Amir, M., Chuck, J., Lu, Q., & Solt, L. A. (2026). Negative regulation of TH17-mediated inflammation by the nuclear receptor REV-ERBβ. iScience, 29(11), Article 117577. https://doi.org/10.1016/j.isci.2026.117577

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117577

Keywords: REV-ERBβ, REV-ERBα, TH17 cells, autoimmunity, nuclear receptor, IL-17A, EAE, colitis, NCoR1, heme, circadian clock, inflammation