Small Molecules Targeting ROR?t Treat Autoimmune Diseases By Suppressing Th17 Cell Differentiation

ObjectiveAutoimmune diseases are mediated by Th17 cells,a lymphocyte subpopulation that is characterized by the expression of the transcription factor,retinoic acid receptor-related orphan receptor gamma-t(ROR?t).The current study was set up to discover novel and non-steroidal small-molecule inverse agonists of ROR?t and to determine their effects on autoimmune disease.MethodsStructure-based virtual screening(SBVS)was used to find compounds targeting ROR?t.Flow cytometry was used to detect the Th17 cell differentiation.The dual-luciferase report assay was used to verify the targets of small molecules.Mice with experimental autoimmune uveitis(EAU),experimental autoimmune encephalomyelitis(EAE),or type 1diabetes were injected intraperitoneally with compounds at a concentration of 25 mg / kg.The naive group was modeled only and not administered and the vehicle group was administered intraperitoneally with the same amountof solvent.The effects of the compounds were evaluated by clinical or histological scores.Evans blue staining was used to check the integrity of the retinal barrier.Western blotting was used to detect the expression of the target protein.Immunofluorescence was used to observe the relationship between retinal blood vessels and Th17 cells.ResultsAmong 1.3 million compounds tested,15 candidate compounds were selected using virtual screening.Then CQMU151 and CQMU152 were screened by in vitro tests.They were found to inhibit Th17 cell differentiation without affecting the differentiation of Th1 and Treg lineages(both P = 0.001).These compounds also reduced the severity of EAU(P = 0.01 and 0.013)and functional studies showed that they reduced the number of Th17 cell and the expression of IL-17(Th17),but not IFN-?(Th1)and TGF-?(Treg)in mouse retinas.Further studies showed that these compounds reduced the expression of p-STAT3 by reducing the positive feedback loop of IL-17>IL-6>STAT3.These compounds also reduced the impaired blood retinal barrier function by upregulating the expression of tight junction proteins.These compounds were also found to reduce the severity of EAE and type 1 diabetes.Conclusion1.CQMU151 and CQMU152 can inhibit the differentiation of Th17 cells in vitro by targeting ROR?t without affecting the differentiation ofTh1 and Treg lineages.2.CQMU151 and CQMU152 can inhibit the onset of EAU in vivo by reducing the proportion of Th17 cells and the expression of IL-17,and can maintain the integrity of BRB.3.CQMU151 and CQMU152 can also inhibit EAE and type 1diabetes.