The Th17-defining transcription factor RORγt promotes glomerulonephritis

Abstract

Although Th17 responses may contribute to the pathogenesis of glomerulonephritis, whether the key transcription factor in Th17 cell development, RORγt, also promotes glomerulonephritis is unknown. Here, we induced crescentic glomerulonephritis in wild-type and RORγt-deficient (RORγt(-/-)) mice. RORγt(-/-) mice were protected from disease, with reduced histologic and functional injury and decreased leukocyte infiltration. Because RORγt(-/-) mice lack lymph nodes, which may influence the development of nephritis, we performed cell-transfer studies. We reconstituted Rag1(-/-) mice, which lack adaptive immunity but otherwise have normal architecture of the lymphatic system, with splenocytes from naïve wild-type or RORγt(-/-) mice. Mice receiving wild-type splenocytes exhibited high mortality from renal failure after induction of nephritis whereas mice receiving RORγt(-/-) cells were protected. To determine the effect of RORγt deficiency specifically in T helper cells, we isolated naïve CD4(+) T cells from wild-type and RORγt(-/-) mice and transferred them into Rag1(-/-) animals. Recipients of wild-type CD4(+) T cells developed severe glomerulonephritis whereas recipients of RORγt(-/-) cells developed less severe disease. To exclude effects of altered regulatory T cell (Treg) development caused by RORγt deficiency, we transferred naïve CD4(+) T cells depleted of Tregs into Rag1(-/-) mice. Recipients of wild-type, Treg-depleted, CD4(+) T cells developed severe glomerulonephritis whereas recipients of RORγt(-/-), Treg-depleted CD4(+) T cells did not. Taken together, this study demonstrates that RORγt promotes the development of crescentic glomerulonephritis by directing nephritogenic Th17 responses.

Figure 1.

Histologic and functional renal injury is reduced in RORγt^−/− mice with glomerulonephritis. (A) WT mice developed severe glomerulonephritis, whereas RORγt^−/− mice were protected. (PAS stain; top row 400×, bottom row 200× [original magnifications]). (B) Quantification of histologic injury showed protection in RORγt^−/− mice. (C) Functional injury was reduced as demonstrated by less albuminuria and lower serum urea levels in RORγt^−/− mice (n = 10, each group). *P < 0.05, **P < 0.01, ***P < 0.0001.

Figure 2.

Renal leukocyte infiltration is reduced in RORγt^−/− mice with glomerulonephritis. (A) Sections from WT and RORγt^−/− mice stained for CD4⁺ T cells, macrophages, and neutrophils are shown (leukocytes in black, original magnification 400×). (B) Quantifying glomerular CD4⁺ T cells, macrophages, and neutrophils demonstrated fewer leukocytes in kidneys of RORγt^−/− mice (n = 10, each group). *P < 0.05, ***P < 0.0001.

Figure 3.

Th17 responses are reduced in RORγt^−/− mice with glomerulonephritis. (A) IL-17A and IL-17F were produced by splenocytes from WT mice but were not detectable in RORγt^−/− mice. IFNγ and IL-4 production were higher in RORγt^−/− mice than WT mice. (B) Intrarenal IL-17A mRNA was detectable only in WT animals, whereas IFNγ levels were similar in kidneys of both strains. (C) Serum IgG titers specific for the nephritogenic antigen (SG) were not significantly altered in RORγt^−/− mice. Analyses of antigen-specific IgG subtypes showed no differences in IgG1 or IgG2c, whereas IgG3 production was enhanced in RORγt^−/− mice (n = 10, each group). *P < 0.05, ^†P < 0.001.

Figure 4.

Glomerulonephritis is ameliorated in Rag1^−/− mice injected with whole splenocytes from RORγt^−/− mice. (A) Survival was prolonged in Rag1^−/− mice reconstituted with RORγt^−/− splenocytes, whereas most mice injected with WT cells died. (B) Histologic and functional renal injury was reduced in recipients of RORγt^−/− cells (n = 3 WT cells, n = 10 RORγt^−/− cells). *P < 0.05.

Figure 5.

Renal injury and leukocyte infiltration are reduced in Rag1^−/− mice reconstituted with CD4⁺ T cells from RORγt^−/− mice. (A and B) Recipients of cells from WT mice showed severe glomerulonephritis, whereas mice receiving RORγt^−/− T cells were protected. Only mild histologic injury was seen in control Rag1^−/− mice not receiving cells (PAS stain; top row 400×, bottom row 200× [original magnifications]). (C) Albuminuria was lower in mice receiving RORγt^−/− T cells at day 2 of nephritis (with a trend toward a decrease at day 10), whereas serum urea was lower in RORγt^−/− cell recipients (and similar to control mice) compared with the WT cell recipients. (D) Quantification of glomerular CD4⁺ T cells, macrophages, and neutrophils showed fewer leukocytes in the glomeruli of RORγt^−/− cell recipients and non-cell-injected mice (n = 10 WT cells, n = 9 RORγt^−/− cells, n = 4 no cells). *P < 0.05, **P < 0.01, ***P < 0.0001.

Figure 6.

Rag1^−/− mice injected with RORγt^−/− CD4⁺ T cells show altered immune responses and higher proportions of Tregs. (A) Splenic IL-17A production was upregulated in WT cell recipients and reduced in RORγt^−/− cell recipients, IFNγ production was downregulated in RORγt^−/− cell recipients and absent in control mice not injected with cells, and IL-4 was similar in the three groups. (B) Intrarenal IL-17A mRNA was detectable only in WT cell recipients. Renal IFNγ mRNA expression was similar in recipients of WT and RORγt-deficient cells. (C) CD4⁺ T cell activation was reduced in splenocytes of RORγt^−/− mice (% of CD4⁺ cells also CD44^hi). (D) The proportion of Tregs in spleens of RORγt^−/− cell recipients was higher (% of CD4⁺ cells also FoxP3⁺) (n = 10 WT cells, n = 9 RORγt^−/− cells, n = 4 no cells). *P < 0.05, **P < 0.01, ***P < 0.0001.

Figure 7.

Renal injury and leukocyte infiltration are reduced in Rag1^−/− mice injected with Treg-depleted CD4⁺ T cells from naïve RORγt^−/− mice. (A and B) Recipients of cells from WT mice showed severe necrotizing glomerulonephritis, whereas mice receiving RORγt^−/− cells were protected. Only mild histologic injury was seen in control Rag1^−/− mice not receiving cells (PAS stain; top row 400×, bottom row 200× [original magnifications]). (C) Albuminuria was reduced in mice receiving RORγt^−/− cells at days 2 and 9 after the induction of nephritis to levels comparable with non-cell-injected control mice, and serum urea was lower in mice receiving RORγt-deficient cells. (D) RORγt-deficient cell recipients had a trend toward fewer CD4⁺ cells in glomeruli, whereas macrophages and neutrophils were significantly reduced (n = 9 WT cells, n = 6 RORγt^−/− cells, n = 5 no cells). *P < 0.05, **P < 0.01, ***P < 0.0001.

Figure 8.

CD4⁺ T cell activation and Th1 responses are restored in Rag1^−/− mice injected with Treg-depleted CD4⁺ T cells from naïve RORγt^−/− mice. (A) Splenic IL-17A production was upregulated in WT cell recipients and reduced in mice injected with RORγt^−/− cells. IFNγ production was similar in both groups reconstituted with T cells, and IL-4 was similar in all three groups. (B) Renal IL-17A mRNA expression was detectable only in Rag1^−/− mice receiving WT cells. Expression of IFNγ mRNA was similar in both groups of mice that received cells, whereas only low levels were detected in the control group. (C) CD4⁺ T cell activation was similar in WT and RORγt^−/− mice (% of CD4⁺ cells also CD44^hi) (n = 9 WT cells, n = 6 RORγt^−/− cells, n = 5 no cells). *P < 0.05, ***P < 0.0001.