MicroRNA-155 drives TH17 immune response and tissue injury in experimental crescentic GN

Abstract

CD4(+) T cells play a pivotal role in the pathogenesis of autoimmune disease, including human and experimental crescentic GN. Micro-RNAs (miRs) have emerged as important regulators of immune cell development, but the impact of miRs on the regulation of the CD4(+) T cell immune response remains to be fully clarified. Here, we report that miR-155 expression is upregulated in the kidneys of patients with ANCA-associated crescentic GN and a murine model of crescentic GN (nephrotoxic nephritis). To elucidate the potential role of miR-155 in T cell-mediated inflammation, nephritis was induced in miR-155(-/-) and wild-type mice. The systemic and renal nephritogenic TH17 immune response decreased markedly in nephritic miR-155(-/-) mice. Consistent with this finding, miR-155-deficient mice developed less severe nephritis, with reduced histologic and functional injury. Adoptive transfer of miR-155(-/-) and wild-type CD4(+) T cells into nephritic recombination activating gene 1-deficient (Rag-1(-/-)) mice showed the T cell-intrinsic importance of miR-155 for the stability of pathogenic TH17 immunity. These findings indicate that miR-155 drives the TH17 immune response and tissue injury in experimental crescentic GN and show that miR-155 is a potential therapeutic target in TH17-mediated diseases.

Figure 1.

miR-155 is upregulated in the kidneys of patients with ANCA-associated and experimental GN. (A) Renal tissue from paraffin-embedded kidney biopsies from patients with ANCA-associated GN or control kidneys was analyzed for RNA expression using Affymetrix RNA Array Human U133 Puls 2.0. Corrected P values>0.05 were considered significant, and fold exchange from controls is given. (B) Quantitative PCR from renal tissue of patients with ANCA-associated GN (n=10) and controls (n=13) for miR-155, miR-301a, and let-7d in relation to RNU48. (C) Renal miR-155 expression levels were correlated to the renal function (GFR). (D) In situ hybridization of cryosections from renal tissue from ANCA-GN and control patients. Detection was performed with digoxigenin-labeled probes. (E) Confocal microscopic images of immunohistochemistry of consecutive renal tissue sections stained for CD45 and ToproIII. (F) miR-155 expression was quantified in the course of murine nephrotoxic nephritis in splenocytes and renal CD4⁺ cells. Bars represent mean ± SEM. **P<0.01.

Figure 2.

miR-155^−/− mice are less susceptible to NTN. (A) PAS staining of renal cortex from miR-155–deficient and wild-type mice 10 days after NTN induction. (B) Crescent formation and tubulointerstitial damage in the different groups (n=10–16 per NTN group). (C) Albumin-to-creatinine ratio as measured by murine albumin-specific ELISA and urinary creatinine on day 8 on NTN induction. (D) Serum creatinine analyzed on day 10 after NTN induction. (E) Renal infiltration of inflammatory cells was quantified for CD3⁺, MAC2⁺, and F4/80⁺. Bars represent mean ± SEM. Con, control. *P<0.05; **P<0.01.

Figure 3.

miR-155 promotes the T_H17 immune response in experimental GN. (A) Renal cortex was analyzed by quantitative PCR for expression of IL-17, IFN-γ, FoxP3, and IL-4 (n=5–6 per NTN group). (B) FACS analysis for intracellular cytokines (IL-17, IFN-γ, and IL-4) in renal T cells (gated on CD45⁺CD3⁺CD4⁺ cells). (C) Quantification of intracellular (IL-17, IFN-γ, and IL-4) and intranuclear (FoxP3) FACS analysis from renal T cells. (D) IL-17 and IFN-γ measurement by ELISA from supernatants of cultured splenocytes from nephritic miR-155–deficient, wild-type, and control mice after stimulation with sheep IgG. (E) Histologic quantification of neutrophils in renal cortex on staining for granulocyte-differentiation antigen (Gr-1)-positive cells. (F) Chemokine expression as measured by quantitative PCR from renal cortex. (G) Crescent formation in miR-155–deficient and wild-type mice after NTN and treatment with anti–IL-17 antibody or isotype control. Animals were treated on days −1, 3, and 7. (H) Mice were treated with miR-155–specific inhibitors (antagomir-155) or nonbinding controls (scramblemir) before NTN induction. On day 10 after NTN, renal CD4⁺ T cells were analyzed for the intracellular cytokines IL-17 and IFN-γ. (I) Quantification of IL-17– and IFN-γ–positive cells. Bars represent mean ± SEM. *P<0.05; **P<0.01; *** P<0.001.

Figure 4.

T cell-intrinsic effect of miR-155 on IL-17/T_H17 immune response in NTN. CD4⁺ splenocytes from miR-155–deficient and wild-type mice were purified using MACS sorting and transferred into Rag-1–deficient mice. Subsequently, NTN was induced. Depicted are (A) crescent formation, (B) tubulointerstitial injury, (C) albuminuria, and (D) serum creatinine levels assessed on day 10 after NTN induction (n=7 per NTN group). (E) FACS analysis of intracellular cytokine staining for IL-17 and IFN-γ of renal CD45⁺CD3⁺CD4⁺ T cells. (F) IL-17 and IFN-γ measurement by quantitative PCR from renal cortex of nephritic RAG-1–deficient mice. Controls are Rag-1–deficient mice without NTN and cell transfer. Bars represent mean ± SEM. *P<0.05; **P<0.01; *** P<0.001.

Figure 5.

Stability of T_H17 cells depends on miR-155. (A) FACS analysis of polarized T cells from miR-155–deficient and wild-type mice. Splenocytes were cultured under T_H0-, T_H1-, and T_H17-polarizing conditions. After 3 days of in vitro stimulation, cells were stained for intracellular cytokines. (B) Quantification of intracellular cytokine staining from FACS analysis (n=5 per group). (C) Congenic CD4⁺ splenocytes from CD45.2⁺ miR-155–deficient and CD45.1⁺ wild-type mice were polarized. Subsequently, competitive transfer of miR-155–deficient and wild-type cells into Rag-1–deficient mice was performed. On day 10 after NTN induction, mice were euthanized, and intracellular IL-17 and IFN-γ were measured by FACS analysis of CD4⁺ T cells from knockout and wild-type (n=8) mice. Bars represent mean ± SEM. *P<0.05.

Figure 6.

Role of B cell miR-155 in experimental GN. Sorted B cells from miR-155–deficient and wild-type mice were transferred with wild-type CD4⁺ T cells into Rag-1^−/− recipients, and subsequently, NTN was induced. Controls are untouched Rag-1^−/− mice (n=7–10 per nephritic group). (A) PAS staining of renal cortex forms the groups as indicated. Depicted are (B) quantification of crescents and tubulointerstitial damage as well as (C) urinary albumin-to-creatinine ratio and serum creatinine. (D) Total IgM and IgG were measured by ELISA. (E) Glomerular antibody depositions of sheep IgG and mouse IgG were assessed by scoring of renal immunohistological staining. (F) Antigen-specific antibody titers from serum were measured by ELISA. (G) Renal cortex was analyzed by quantitative PCR for expression of IL-17, IFN-γ, FoxP3, and IL-4. (H) Quantification of intracellular cytokines (IL-17 and IFN-γ) by FACS analysis from renal CD4⁺ T cells. Bars represent mean ± SEM. Tub Int, tubulointerstitial damage. **P<0.01; *** P<0.001.