ROCK2 signaling is required to induce a subset of T follicular helper cells through opposing effects on STATs in autoimmune settings

Abstract

Rho-associated kinase 2 (ROCK2) determines the balance between human T helper 17 (TH17) cells and regulatory T (Treg) cells. We investigated its role in the generation of T follicular helper (TFH) cells, which help to generate antibody-producing B cells under normal and autoimmune conditions. Inhibiting ROCK2 in normal human T cells or peripheral blood mononuclear cells from patients with active systemic lupus erythematosus (SLE) decreased the number and function of TFH cells induced by activation ex vivo. Moreover, inhibition of ROCK2 activity decreased the abundance of the transcriptional regulator Bcl6 (B cell lymphoma 6) and increased that of Blimp1 by reducing the binding of signal transducer and activator of transcription 3 (STAT3) and increasing that of STAT5 to the promoters of the genes Bcl6 and PRDM1, respectively. In the MRL/lpr murine model of SLE, oral administration of the selective ROCK2 inhibitor KD025 resulted in a twofold reduction in the numbers of TFH cells and antibody-producing plasma cells in the spleen, as well as a decrease in the size of splenic germinal centers, which are the sites of interaction between TFH cells and B cells. KD025-treated mice showed a substantial improvement in both histological and clinical scores compared to those of untreated mice and had reduced amounts of Bcl6 and phosphorylated STAT3, as well as increased STAT5 phosphorylation. Together, these data suggest that ROCK2 signaling plays a critical role in controlling the development of TFH cells induced by autoimmune conditions through reciprocal regulation of STAT3 and STAT5 activation.

Copyright © 2016, American Association for the Advancement of Science.

Figures

Fig. 1. ROCK2 is required for the induction of human Tfh cells through its reciprocal regulation of STAT3 and STAT5 transcriptional activity

(A to H) Human peripheral blood CD4+ T cells were treated with the selective ROCK2 inhibitor KD025 (A, B, and D to H) or were transfected with control or ROCK2-specific siRNAs (C) and then were left unstimulated or were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies in combination with IL-1β and TGF-β (for Th17-type skewing) for 48 hours. (E) Some cells were stimulated under Th17-type skewing conditions for 5 days, treated with or without KD025, and then re-stimulated for 48 hours. (A to C and E) The percentages of CXCR5+PD1+ cells were determined by flow cytometric analysis, whereas cell extracts were analyzed by Western blotting with antibodies against the indicated proteins (F). (D) Treated T cells were co-cultured with autologous B cells for 7 days in the presence of SEB (0.05 ng/ml), and IgG secretion was determined by ELISA. (G and H) ChIP assays were performed with normal rabbit IgG, anti-RNA pII, anti-STAT3, and anti-STAT5 antibodies. Data are means ± SEM of six (B, G, H), five (C, E), or three (D) experiments. Western blots in (F) are from a single experiment and are representative of three independent experiments, whereas data in bar graphs are means ± SEM of three independent experiments. Statistical analysis was performed with the Wilcoxon test. The Bonferroni method was used for controlling for multiplicity (B). *P < 0.05.

Fig. 2. STAT3-dependent, but not STAT4-dependent, human Tfh cells are induced by ROCK2 signaling

(A to C) Naïve CD4+ T cells were stimulated for 4 days with anti-CD3 and anti-CD28 monoclonal antibodies in the presence of IL-1β, TGF-β, and IL-6 in combination with IL-12 (for Th1-type skewing conditions) or IL-23 (for Th17-type skewing conditions) in the presence or absence of 10 µM KD025. (A) The percentages of CXCR5+PD1+ cells were determined by flow cytometry. (B) Top: Cell extracts were analyzed by Western blotting with antibodies against the indicated proteins. Bottom: Densitometric analysis of the indicated band intensities was performed. (C) The amount of secreted IgG was determined by ELISA after culturing the indicated T cells with autologous B cells for 7 days in the presence of SEB (0.05 ng/ml). Data in (A), (C), and the bar chart in (B) are means ± SEM of three independent experiments. Western blots in (B) are from a single experiment and are representative of three independent experiments. Statistical analysis was performed with the Wilcoxon test.

Fig. 3. Treatment of MRL/lpr mice with KD025 decreases the percentage of CXCR5+PD1+ Tfh cells through a STAT3- and STAT5-dependent mechanism

(A to H) MRL/lpr mice (15 per group) were orally treated with vehicle or KD025 (100 mg/kg) once a day or were intraperitoneally treated once a week with cyclophosphamide (50 mg/kg) beginning on week 11 and continuing until week 17. (A) The mean disease score ± SEM was defined by the extent of proteinuria. The concentrations of blood urea nitrogen (B) and anti-dsDNA antibodies (C) in serum were measured at the end of the study. (D to F) Splenocytes were analyzed by flow cytometry to determine the total numbers of cells (D), the percentage of CXCR5+PD1+ cells (E), and the percentage of plasma cells (F). (G) Representative sections of the spleens of the indicated mice at the end of the study were subjected to H&E staining. Arrows indicate GCs. (H) Total spleenocytes were analyzed by Western blotting with antibodies against pSTAT3 (n = 5), Bcl6 (n = 5), and pSTAT5 (n = 4). P values were calculated with the Mann-Whitney test.

Fig. 4. KD025 decreases the abundances of pSTAT3 and Bcl6 while increasing the abundances of pSTAT5 and Blimp1 in PBMCs from patients with active SLE

(A to C) PBMCs from six active SLE patients were treated with the indicated concentrations of KD025 for 1 hour and then were stimulated under Th17-type skewing conditions. (A) The percentage of CXCR5+PD1+ cells was determined by flow cytometric analysis. (B to E) The cells were analyzed by Western blotting with antibodies specific for (B) pSTAT3 (n = 5), (C) Bcl6 (n = 4), (D) pSTAT5 (n = 3), and (E) Blimp1 (n = 3). Statistical analysis was performed with the Wilcoxon test.