Discussion
Foxp3+ regulatory T cell are required to prevent autoimmunity against self-antigens and prevent tissue destruction resulting from excessive immune response. Treg cells differentiate into distinct subsets to inhibit the immune response caused by distinct T helper cell subsets. RORγt+Foxp3+Treg cells have been identified as Tr17 cells, because they regulate Th17 cell-mediated immune response in EAE15. By analyzing T cell-specific RORγt transgenic mice, we also discovered the potential of RORγt+Foxp3+Tr17-like cells to suppress the development of CIA16. In this study, we observed significant exacerbation of CIA in Foxp3creRORγtfl/fl mice accompanied by the disappearance of Tr17 cells, and thus it was suggested that Tr17 cells might be a specific regulatory cell subset in the development of autoimmune arthritis.
How do Tr17 cells regulate the pathogenesis of autoimmune arthritis? We propose the following two possibilities: 1) enhancement of suppressive function as Treg cells; and 2) preferential migration of Tr17 cells into inflamed joints.
First, we examined the regulatory function of Tr17 cells and found that suppressor molecules such as CTLA-4, GITR, and IL-10 were significantly up-regulated in Tr17 cells compared to RORγt negative Treg cells after CIA induction. CTLA-4 expression on Treg cells is required to suppress CD4+T and CD8+T cells via the blockade between CD28 on T cells and CD80/86 on dendritic cells. It is also required to prevent the differentiation from naive T cells to Th-subsets and induce T cells anergy22,23,24. It was also reported that CIA was exacerbated in CTLA-4 KO mice25. GITR, which is a potential Treg cell marker, controls their suppressive phenotype26, and regulates autoreactive CD4+T cell activation in EAE27. IL-10 is an anti-inflammatory cytokine and plays a crucial role in preventing inflammatory and autoimmune pathologies28. A previous study reported that IL-10-/- mice showed CIA deterioration29 and another study found that Th17 cells express IL-10 receptor and are directly regulated by IL-1030. It was also pointed out that immune response of Th17 cells were specifically suppressed by Tr17 cells via IL-10. Consequently, enhanced expression of suppressor molecules in Tr17 cells might play an essential role in regulation against immune response especially by arthritogenic T cells in autoimmune arthritis.
We analyzed the second possibility according to the preferential migration of Tr17 cells into inflamed joints. Treg cells can undergo stimulus-specific differentiation, which is regulated by transcription factors associated with the differentiation of conventional CD4+ T cells31. Moreover, differentiated Treg cells have unique migratory properties matched to the stimulus that elicited the initial response. CCR6 is one of the chemokine receptors which is expressed on various immune cells and is required for their trafficking via chemokine ligand CCL209. Past studies have revealed that CCR6 expressed in RORγt-positive Treg cells regulates their recruitment and inhibition of Th17 cell-mediating inflammation15,32. Interestingly, CCR6 expression was regulated by RORγt, and also contributed to the recruitment of arthritogenic Th17 cells to the inflamed joints9. Our study showed that Tr17 cells accumulated in inflamed joints after the onset of CIA, and that CCR6 was highly expressed in Tr17 cells compared to RORγt-negative Treg cells. Moreover, Treg cell-specific deletion of RORγt Foxp3creRORγtfl/fl cKO mice resulted in reduction of CCR6-expressing Treg cells. Collectively, we speculated that Tr17 cells might preferentially infiltrate into inflamed joints and regulate arthritogenic Th17-mediated inflammation resulting in suppression of the prolongation of autoimmune arthritis.
Why do Tr17 cells maintain their suppressive capacity despite the expression of RORγt, the master transcription factor in the differentiation of Th17 cells? Past reports have pointed out that RORγt-positive Treg cells had almost the same pattern of methylations status of the Treg-specific demethylated region (TSDR) and other Treg-associated epigenetic loci indicates as RORγt-negative Treg cells33 and that Tr17 cells highly expressed Blimp-1, which is master transcriptional factor of IL-10 and regulates IL-17 expression, and that Tr17 cells have a role on preservation of stability of Treg cells at inflammation sites20,21,34,35. In the current study, Foxp3 expression in Tr17 cells was comparable to RORγt-negative Treg cells. In addition, Tr17 cells highly expressed Blimp-1 compared to RORγt-negative Treg cells. Accordingly, we speculated that RORγt-expressing Tr17 cells maintained their suppressive function through high expression of Blimp-1 in inflammation.
This study has some limitations. First, we could not clarify the exact mechanism of how Tr17 cells regulate Th17 cell-mediated inflammatory response in vivo and in vitro. To investigate this, it is essential that RORγt-positive cells are isolated with a high degree of purity, and experiments such as adoptive cell transfer of RORγt-positive Tr17 cells into RORγt Foxp3creRORγtfl/fl cKO mice and in vitro suppression assay against RORγt-positive Th17 cells are performed. The second limitation is that little is known concerning Tr17 cells in the patients with RA. Further analysis should be performed to elucidate the precise role of Treg cells in the development of RA.
In conclusion, our experiments show that Tr17 cells increase during the clinical course of CIA and accumulate in inflamed joints. Tr17 cells express CD25, CTLA4, and GITR molecules and over-produced IL-10 via Blimp-1 up-regulation Moreover, Tr17-enriched CCR6+Treg cells significantly suppress cell proliferation. Taken together, our study demonstrates that Tr17 cells play a crucial role in the regulation of autoimmune arthritis.