Small-molecule RORγt Antagonists: One Stone Kills Two Birds

Abstract

Although small-molecule RORγt antagonists suppressing Th17 cells are widely reported, the effect of these molecules on other RORγt-expressing cells is unknown. A new study reported that RORγt inhibition in CD4⁺CD8⁺ thymocytes resulted in skewed T cell repertoire, contributing to reduction in frequency of self-reactive T cells and resistance to autoimmunity.

Interleukin (IL)-17-producing T helper (Th17) cells are critically involved in many forms of organ-specific autoimmune disorders and chronic inflammations. IL-23 is essential for the development and/or activation of Th17 cells. Therefore, IL-23 and IL-17, as well as their receptors, have been considered as therapeutic targets for treating Th17-mediated diseases¹. In fact, antibodies against IL-23p19, IL-23p40, IL-17A and IL-17RA have shown promising efficacy in treating many autoimmune diseases, including psoriasis, ankylosing spondylitis, and multiple sclerosis, in clinical trials. However, blockage of IL-17A or IL-17RA is ineffective in treating Crohn’s disease. This is possibly because that besides IL-17A, several other effector cytokines, such as IL-17F, IL-22, and GM-CSF, produced by Th17 cells may also play critical roles in inflammation; blockade of one particular cytokine may not be sufficient to repress Th17-mediated inflammation.

Th17 cell differentiation is regulated by a group of important transcription factors, including truncated form of retinoic acid receptor-related orphan nuclear receptor γ (RORγt), signal transducer and activator of transcription 3 (STAT3), Interferon regulatory factor 4 (IRF4), and BATF. Among these critical factors, only RORγt is specifically expressed by Th17 cells among the T helper effector cells and thus is regarded as the master regulator of Th17 cells². Similar to other members of the nuclear hormone receptor family, RORγt has DNA- and ligand-binding domains (DBD and LBD)³. Given that RORγt is the master regulator of Th17 cells, and that its activity could be modulated by cost-effective small molecules, inhibiting RORγt’s transcriptional activity by targeting either domain could be a superior alternative to antibody-mediated cytokine and/or receptor neutralization in treating Th17-related autoimmune diseases.

In addition to Th17 cells, many other mature lymphocytes, including type 3 innate lymphoid cells (ILC3s), CD8⁺ IL-17-producing (Tc17) cells, and subsets of γδT, natural killer T (NKT), regulatory T (Treg) cells, both in human and in mice, express RORγt (Figure 1). Furthermore, during T cell development, CD4⁺CD8⁺ double positive (DP) thymocytes express RORγt, and RORγt promotes the survival of DP thymocytes partly through promoting the expression of anti-apoptotic molecule Bcl-x_L.

Figure 1.

Small-molecule RORγt Antagonists not Only Inhibit the Differentiation and Functions of Th17 cells, but also Regulate Thymopoiesis. Besides Th17 cells, CD8⁺ IL-17-producing (Tc17) cells, type 3 innate lymphoid cells (ILC3s), and subsets of γδT, NKT, regulatory T (Treg) cells also express RORγt. In the thymus, RORγt is expressed by CD4⁺CD8⁺ double positive (DP) thymocytes. Some other lymphoid progenitors such as ILC progenitors may also express RORγt during development. It is well known that pharmacological inhibition of RORγt by allosteric RORγt antagonists suppresses Th17/Tc17 differentiation and function. Small-molecule RORγt antagonists also inhibit IL-17 production by RORγt-expressing γδT cells. In DP thymocytes, these small molecules suppress the expression of RORγt target genes that are involved in cell apoptosis and survival, cell migration and positive selection, which leads to shortened lifespan of DP thymocytes, reduced expression of Bcl-x_L, skewed TCRα V-J usage towards the proximal regions and reduced T cell repertoire. As a result, RORγt inhibition reduces the frequency of “self-reactive” CD4⁺ T cells and renders the host resistant to autoimmune disease induction. However, transient inhibition of RORγt leaves the functions of ILC3s intact. The effect of small-molecule RORγt antagonists on other RORγt-expressing cells requires further investigation. In particular, it is intriguing to know whether RORγt inhibitors can regulate the balance between self-reactive Th17 cells and pTregs.

Several RORγt antagonists or inverse agonists, such as digoxin, SR1001 (targeting both RORγt and RORα), TMP778, GSK805, and MRL-871, have been identified through independent efforts of many groups in drug screening and testing^{3, 4, 5, 6, 7}. MRL-871 binds to a non-canonical site at the LBD of RORγt and induces an aberrant conformational change at the LBD, which blocks the binding of co-activators to RORγt. Therefore, even without altering RORγt DNA binding activity, an allosteric antagonist can still inhibit the transcriptional activity of RORγt. While the impact of RORγt inhibitors on Th17 cells has been extensively studied, their effects on other RORγt-expressing cells are still elusive.

Cua and colleagues recently reported that pharmacological inhibition of RORγt by allosteric RORγt antagonists, MRL-871 or MRL-248 (structurally similar to MRL-871 but with improved in vivo pharmacodynamics) resulted in rapid DP thymocyte apoptosis within 3 days and immediate changes in gene expression within 2 hours of drug treatment⁸. Through RNA-Seq analysis of changes in gene expression in DP cells shortly after RORγt inhibition (2 hr) or with RORγt-overexpression, the authors identified 33 genes, including Bcl-x_L, Plxnd1 and Sla, that are known to be involved in cell apoptosis and survival, cell migration and positive selection, respectively. Therefore, besides maintaining the survival of DP thymocytes, RORγt has a broad role in regulating many other pathways during thymopoiesis. MRL-248 was also very effective in inhibiting IL-23-mediated skin inflammation, although it is not clear whether the induction of RORγt-expressing CD3⁺ T cells was blocked or these RORγt-expressing T cells failed to produce Th17-related effector cytokines with MRL-248 treatment in this model. Nevertheless, small-molecule RORγt inhibitors not only suppress the differentiation and/or functions of IL-17-producing T cells, but also regulate thymopoiesis (Figure 1).

Consistent with the previous reports using RORγt-deficient DP cells⁹, RORγt inhibition also led to a reduced expression of anti-apoptotic factor Bcl-x_L and shortened survival, which is presumably responsible for skewed TCR α-chain rearrangement with J region usage towards the proximal regions. In the absence of functional RORγt, many sequential productive rearrangement events using the distal J regions may not have enough time to occur before the DP thymocytes with functional TCRs can be positively selected by the major histocompatibility complex (MHC). Therefore, RORγt inhibition results in a limited T cell repertoire. These effects of RORγt antagonists on DP thymocytes appear RORγt-specific, since inducible deletion of Rorc (gene encoding RORγt) also led to shortened lifespan of DP thymocytes, reduced expression of Bcl-x_L, skewed TCRα V-J usage towards the proximal regions and reduced T cell repertoire. Skewing of TCRα usage by RORγt inhibition may also interfere the development of other lymphocytes including regulatory T cells and cells with an invariant TCR, including NKT cells and mucosal associated invariant T (MAIT) cells.

Interestingly, RORγt inhibition resulted in reduced frequency of myelin oligodendrocyte glycoprotein (MOG)-specific “self-reactive” CD4⁺ T cells in approximately half of the RORγt antagonist-treated mice⁸. Consequently, RORγt antagonist pre-treatment delayed the autoimmune progression in the experimental autoimmune encephalomyelitis (EAE) model induced by MOG immunization, even though the RORγt antagonist was no longer present during the course of disease induction. Therefore, in addition to suppressing the differentiation and function of Th17 cells, RORγt antagonists can also ameliorate autoimmunity by restricting the number and repertoire of self-reactive thymic emigrants. However, whether this is true for other self-reactive CD4⁺ T cells requires further investigation. Furthermore, although the contraction of T cell repertoire may reduce the risk of some autoimmune diseases, it could potentially increase susceptibility to cancer and some infections.

ILC3s, which also express RORγt, play important roles in host defense and tissue homeostasis. Another recent report indicates that transient inhibition of RORγt does not cause reduction in IL-22 production by ILC3s or impairment of ILC3-mediated protective immunity, suggesting that RORγt inhibition can effectively target Th17-induced inflammatory bowel diseases (IBD) or other chronic inflammations in the gut¹⁰. The effects of RORγt antagonists on RORγt-expressing γδT, NKT and Treg subsets still require further investigation. Th17 cells and peripherally induced Tregs (pTregs) share a developmental program triggered by TGFβ, and there is an intermediate phase of Th17/pTreg differentiation when both Foxp3, the master regulator of Tregs, and RORγt are co-expressed in the same cell. Because of the antagonism between Foxp3 and RORγt, inhibition of RORγt activity by small molecules may divert the differentiation of some newly activated auto-reactive CD4⁺ cells from Th17 cell fate to pTreg fate, which may lead to a permanent cure of autoimmune diseases without a continuous drug treatment. This possibility should be further explored in the future.

In conclusion, the study of Cua and colleagues has shed light on an important effect of RORγt antagonists on thymopoiesis. RORγt inhibition by small molecule inhibitors recapitulates the effect of Rorc deficiency in many aspects, including skewed TCRα rearrangement and reduced T cell repertoire diversity. They also found several previously unknown functions of RORγt in DP thymocytes. In addition, their interesting results provide a novel view of explaining the complex mechanism through which RORγt antagonists may modulate autoimmune diseases. However, it also raises several outstanding issues that need to be addressed in the future. To avoid the complexity of multiple effects of RORγt antagonists on different cell types some of which may be deleterious, one could possibly take advantage of the rapidly developing nanomedicine to deliver RORγt inhibitors by nanoparticles to specific cell types such as Th17 cells in treating autoimmune diseases.