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.