Inhibition of IL-17 ameliorates systemic lupus erythematosus in Roquinsan/san mice through regulating the balance of TFH cells, GC B cells, Treg and Breg

Systemic lupus erythematosus (SLE) is mediated by a chronic and dysregulated inflammatory response. Interleukin (IL)-17, a proinflammatory cytokine, and T helper (Th)17 cells are associated with chronic autoimmune diseases. We hypothesized that inhibition of IL-17 would decrease the numbers of T cell subsets that function as B-cell helpers, as well as B-cell differentiation into plasma cells and autoantibody expression. The IL-17 level was increased markedly in Roquinsan/san mice. Loss of IL-17 in Roquinsan/san mice improved nephritis by downregulating immunoglobulin (Ig)G, IgG1, and IgG2a production. Formation of germinal centers (GCs), and follicular B- and T-cell differentiation was reduced, whereas the number of regulatory T (Treg) cells and immature B cells was increased, by IL-17 deficiency in Roquinsan/san mice. These results suggest that IL-17 inhibition can ameliorate SLE by inhibiting B-cell differentiation into GCs. Therefore, IL-17–producing Th17 cells show promise as a target for development of novel therapeutics for SLE.

In SLE patients, the population of T follicular helper (Tfh) cells, which play a key role in B-cell differentiation into plasma cells in the germinal centers (GCs), as well as autoAb production, are increased 11,12 . IL-17 is associated with Tfh, GCs, and autoAbs. Indeed, autoAb overproduction is reportedly caused by IL-17 stimulation of peripheral blood mononuclear cells from patients with lupus nephritis 13 . Moreover, IL-17 activates B cells and promotes formation of GCs 14 , as do IL-17-producing Tfh cells 15 .
Roquin was identified as a CCCH-type zinc finger protein and diminish abnormal inducible T cell co-stimulator (ICOS) expression on T cells 16,17 . It has been demonsrated that Roquin deficiency leads to autoimmunity in Roquin san/san mice that are homozygous for a point mutation in Rc3h1, the gene that encodes Roquin 17,18 . Indeed, Roquin san/san mice showed dysregulation of immune response and used as a murine model of SLE 16,19 .
Thus, we hypothesized that IL-17 depletion would ameliorate the lupus-like characteristics of Roquin san/san mice. Thus, we investigated the effect of loss of IL-17 on Tfh cells, GC formation, autoAb production, numbers of IL-17-producing T and B cells, and nephritis in Roquin san/san and Roquin san/san/ IL-17 −/− mice.

Results
AutoAb production and numbers of IL-17-producing T and B cells are increased in Roquin san/san mice. To investigate the potential function of Roquin on immune response, we used mouse genetics to mutant the Rc3h gene ( Supplementary Fig. 1). We observed that IgG, IgG1, and IgG3 levels were increased significantly in Roquin san/san mice compared to C57BL/6 mice (Fig. 1A). Moreover, the expression and production of IL-17 were upregulated significantly in Roquin san/san mice compared to C57BL/6 mice (Fig. 1B). The numbers of IL-17producing CD4 + T cells and CD19 + B cells were increased in Roquin san/san mice (Fig. 1C,D). The frequency of IL-17-producing CD4 + T cells and CD19 + B cells in Roquin san/san mice was increased by LPS treatment (Fig. 1E). These results suggest that the immune response in Roquin san/san mice was enhanced by upregulation of IL-17 expression in T and B cells.
Ig production and nephritis in Roquin san/san/ IL-17 −/− mice. Since we observed the upregulation of IL-17 level in Roquin san/san mice, we hypothesized that IL-17 deficiency can reduce immune inflammatory response and SLE development. We used mouse genetics to mutant the Rc3h gene and IL-17a gene deficiency ( Supplementary Fig. 1). Compared to Roquin san/san mice, serum IgG, IgG1, and IgG3 levels were reduced www.nature.com/scientificreports www.nature.com/scientificreports/ significantly in Roquin san/san /IL-17 −/− mice ( Fig. 2A). Nephritis was attenuated by IL-17 deficiency in Roquin san/san mice (Fig. 2B). Therefore, IL-17 likely plays an important role in dysregulated humoral immunity in SLE.
Treg cell differentiation is promoted by IL-17 deficiency in Roquin san/san mice. The number of Treg cells was increased significantly in Roquin san/san/ IL-17 −/− mice compared to Roquin san/san mice. Moreover, Th2 cell differentiation was increased, but Th17 cell differentiation was reduced significantly, in Roquin san/san/ IL-17 −/− mice compared to Roquin san/san mice (Fig. 3A). The frequency of Treg cells was confirmed by flow cytometry (Fig. 3B). Foxp3 expression in Tfh cells was also increased by IL-17 deficiency in Roquin mice (Fig. 3C). These findings suggest that loss of IL-17 results in an increased Treg population in Roquin san/san mice.

IL-17 deficiency inhibits Tfh cell differentiation in Roquin san/san mice. Tfh cells regulate the differ-
entiation of B cells into plasma cells, which are involved in the pathogenesis of SLE. Therefore, we investigated the effect of IL-17 deficiency in Roquin san/san mice on the numbers of cytokine-producing Tfh cells. The number of CD4 + ICOS + CXCR5 + PD1 + Tfh cells within the GC area was decreased in Roquin san/san /IL-17 −/− mice (Fig. 4A). The numbers of IL-17-, IFN-γ-, and IL-4-producing Tfh cells were determined by confocal microscopy and flow cytometry (Fig. 4B,C). These findings suggest that loss of IL-17 reduces the numbers of cytokine-producing Tfh cells in Roquin san/san mice.  www.nature.com/scientificreports www.nature.com/scientificreports/

IL-17 deficiency suppresses B-cell differentiation and GC formation in Roquin san/san mice. GC
formation and plasma cell activation are hallmarks of SLE. Therefore, we investigated the effect of IL-17 deficiency on B cell differentiation and GC formation in spleen tissue from Roquin san/san mice. GC formation, CD138 + plasma cell, and CD19 + IgD + mature B cell differentiation were reduced in Roquin san/san /IL-17 −/− mice, while formation of CD19 + IgM + immature B cells was increased (Fig. 5A). The numbers of B cells, GC, plasma cells, immature B cells, and mature B cells were determined by flow cytometry (Fig. 5B). These results suggest that IL-17 deficiency in Roquin san/san mice influences B-cell populations and inhibits the differentiation of pathogenic plasma cells and mature B cells.

Effect of IL-17 deficiency on regulatory B cells in the spleen of Roquin san/san mice. The absence
of regulatory B cells (Bregs) exacerbates pathologic inflammatory responses in autoimmune diseases. Therefore, we investigated the numbers of Bregs in IL-17-deficient Roquin san/san mice. The numbers of CD19 + IL-10 + Breg cells and CD19 + CD1d + CD5 + Breg cells were increased in IL-17-deficient Roquin san/san mice (Fig. 6A,B). We have shown the existence of IL-17-producing B cells in Roquin san/san mice. These results suggest that IL-17 + B cells and Breg cells exert opposite effects on the autoimmune response in SLE.

Discussion
Although IL-17 is associated with the pathogenesis of SLE, and Roquin san/san is related to the SLE phenotype, the relationship between IL-17 and Roquin san/san is unclear. SLE is characterized by systemic inflammation and overproduction of proinflammatory cytokines, including IL-17 [6][7][8] . However, little information is recognized about the interaction of IL-17 and Roquin san/san in T and B cells. Our results suggest that IL-17 deficiency in Roquin san/san mice results in an increased number of IL-17-producing T and B cells, improvement of nephritis, and amelioration of the inflammatory response. Moreover, Roquin san/san induced IL-17 expression in T and B cells. To our knowledge, this is the first report of increased IL-17 expression in T and B cells from Roquin san/san mice. On the other hand, loss of IL-17 in Roquin san/san mice ameliorated nephritis that is characteristic of SLE. Notably, IL-17 deficiency reduced the severity of inflammation in Roquin san/san mice. This observation can elucidate IL-17 function related with inappropriate immune inflammation in SLE.
Dysregulation of IL-17 and Tfh cells is related to the pathogenesis of SLE. Differentiation of IL-17-producing Tfh cells was enhanced in BXD2 mice, which may be related to the development of SLE 20 . Moreover, Roquin san/san mice exhibit an accumulation of Tfh cells and develop SLE 21,22 . Although suppression of IFN-γ production by Th1 cells reduces the severity of SLE in Roquin san/san mice 23 , IL-17 expression in Tfh cells was not investigated. In this www.nature.com/scientificreports www.nature.com/scientificreports/ study, IL-17 deficiency in Roquin san/san mice resulted in reduced numbers of IL-17-producing Tfh cells, leading to improvement of nephritis. These results suggest that IL-17 inhibition could be a therapeutic strategy in SLE.
AutoAb production and B-cell activation are related to the pathogenesis of SLE. B-cell activation leads to autoAb secretion 24 . Indeed, SLE is characterized by B-cell differentiation to plasma cells 25 . Moreover, the serum autoAb level is elevated in SLE patients 26 . B cell-targeted therapy involving inhibition of B-cell activation has been proposed 27 . In this study, IL-17 deficiency reduced B-cell differentiation and GC formation in Roquin san/san mice. Serum IgG, IgG1, and IgG3 levels were decreased by IL-17 deficiency in Roquin san/san mice. These results suggest that IL-17 has promise as a target for the development of novel therapeutics in SLE.
Because SLE is an inflammatory autoimmune disease 24 , Tregs and IL-10 are important factors in its treatment. Indeed, the frequency of CD4 + CD25 high FoxP3 + Tregs is decreased in SLE patients 28,29 . IL-10 is produced as an effector molecule by Tregs, and IL-10 receptor expression was reduced in a mouse model of SLE 30,31 . In this study, IL-17 deficiency enhanced Treg differentiation and IL-10 production by effector T cells in Roquin san/san mice. Furthermore, IL-17 deficiency reduced the severity of SLE by increasing the number of Treg cells and the production of IL-10.
The effect of Roquin mutation on IL-17 production has to date been unclear. Our results provide insight into the role of IL-17 in the pathogenesis of SLE: Roquin mutation increased the expression of IL-17 in T and B cells. Thus, IL-17 can be considered a therapeutic target for SLE. Immunohistopathological analysis of kidney. Mouse kidney tissues were fixed in 4% paraformaldehyde, decalcified in ethylenediaminetetraacetic acid (EDTA) bone decalcifier, and embedded in paraffin. Tissues were sectioned at 7 μm thickness, dewaxed using xylene, dehydrated through a gradient of alcohol, and stained