Systemic lupus erythematosus (SLE) is a chronic disease and involves virtually every organs and tissues, including kidney, known as lupus nephritis (LN), which is a major cause of morbidity and mortality in SLE patients[32, 33]. The lupus nephritis patients have many representative clinical symptoms, such as increased anti-dsDNA antibody level in serum, infiltration of lymphocytes and immune-complex deposited in the kidney. The IgM and IgG complement-fixing antibodies to dsDNA deposited in the kidney relating to the risk of glomerulonephritis[34].So far, the therapy of SLE is very challenging due to the etiology of the disease remains elusive. It is generally used in combination with a variety of therapies in clinical. Immunosuppressive agents such as azathioprine (AZA), cyclophosphamide (CYC) and chlorambucil were administrated either individually or combining with each other or corticosteroids for treating lupus nephritis[35–38]. However, these agents worked on many immune cells with severe side effects, which limited the application in SLE. Increasing pieces of evidence indicate the critical role of IL-17A and Th17 cells in SLE and other human autoimmune diseases [3–6], and they are an essential role in the pathogenesis of lupus nephritis[8, 9, 39]. In this report, we observed that the levels of anti-dsDNA antibody in serum were significantly decreased after treating TTP for 2 months (Fig. 2a, b), and the deposition of IgM immune-complex in kidney markedly decreased with TTP treatment (Fig. 2d, f). These demonstrated that TTP could ameliorate the clinical manifestation of lupus nephritis in mice, indicating its application in human SLE therapy. RORγt, a central transcription factor in Th17 cells differentiation, had a treating potential for Th17-derived autoimmune inflammation as a therapeutic target[40, 41]. However, RORγt also is mainly expressed in DP thymocytes subpopulations[42, 43], and Rorc-deficient (Rorc-/-) in mice would increase thymocyte apoptosis, alter CD4/CD8 subpopulation in the thymus, leading to the emergence of T lymphoma in the thymus[17, 18, 41, 44, 45]. Generous studies showed that small molecule agents as RORγt inhibitors had an inhibition for the functions of Th17 cells[22, 27, 28, 46], such as SR1001, digoxin, and ML209, but had others side effects in thymus, including DP thymocytes apoptosis, anti-apoptotic genes downregulated and the induction thymic lymphoma [23, 47]. In this study, we found that TTP disturbed the functions of Th17 cells, however, it did not affect the numbers and frequency of CD4 + CD8 + DP thymocytes population, and the total numbers of thymocytes had no significant difference with control group (Fig. 4a, b). Similarly, the subpopulations of T lymphocytes in spleen had no difference in treating TTP after 4-weeks and 8-weeks (data not shown). We also observed no increase in large thymocytes in mice after 8 weeks treated with TTP, indicating no obvious change of thymoma incidence (Fig. 5e). However, it should be to treat for a longer period to confirm this observance about the incidence of lymphoma. Although most evidences about thymocyte development displayed normal, high concentration TTP could increase the speed of apoptosis with the in vitro thymocytes culture (Fig. 5a, b), and proportion of the DNA damaged cell in thymus, which detected by TUNAL assay, had slightly increased after TTP treatment (Fig. 5c, d), indicating low safety risk of TTP on thymocyte development.
RORγt consists of three domains[19, 20]: a highly conserved DNA-binding domain (DBD) to response DNA binding; a conserved ligand-binding domain (LBD) with a AF2 domain to recruit SRC family of co-activators stimulating gene expression[21]; and a hinge domain (HD) link DNA-binding domain with ligand-binding domain. Previously study indicated that mutation of AF2 of RORγt interfered the ability of active target genes and recruitment of coactivators[48]. However, disruption of the functions of LBD and DBD of RORγt inhibits the IL-17A-mediated autoimmune inflammation, as well as the thymocytes' survival[21, 48]. Previously study showed that two amino acid mutations in HD of RORγt were suppressing the functions of Th17 cells but not disrupted thymocyte survival[23]. HD region probably plays a crucial role in discrimination of RORγt function between Th17 differentiation and thymocyte development. In this hypothesis, compound interacted with the HD region of RORγt may specifically affect Th17 differentiation, rather than thymocyte development. In this study, we demonstrated that TTP had a strong affinity with full-length RORγt, rather than the RORγt LBD region, indicating it may bound to HD of RORγt (Fig. 6a, b). Molecular docking computational analysis also demonstrated the best pocked for TTP binding to RORγt located in the HD region. Therefore, the HD of RORγt may be the target region of TTP. It remains to describe the mechanism of TTP regulating RORγt function by interacting with the HD region.
Other T cell subsets also contribute to autoimmune disease pathogenesis. Treg cells, as the major suppressive immune cells, were very important to maintain immunologic self-tolerance as well as prevent autoimmune diseases[49]. Notably, the balance between Treg cells and Th17 cells is crucially involved in autoimmune diseases. However, we didn’t observe TTP affect Treg cells in this study(Fig. 1f, g)indicated Treg were not the targets of TTP. Some autoimmune disease etiologies were also attributed to Th1 cells, including multiple sclerosis, rheumatoid arthritis, and SLE[50]. In this study, TTP also suppressed Th1 differentiation in vitro and decreased the numbers of CD4 + IFN-γ + cells in the LN mouse model (Fig. 1d, 1e and Fig. 3b). These results indicated that Th1 cells could be the alternative pathway of TTP, coordinated with Th17 cells to alleviate LN pathogenesis. More efforts need to conduct for revealing the mechanism of TTP regulating Th1 function. Moreover, the cooperation of manipulating Th1 and Th17 function via TTP remains to describe in the future study.