Local inhibition of TGF-β1 signaling improves Th17/Treg balance but not joint pathology during experimental arthritis

Abstract

TGF-β1 is an important growth factor to promote the differentiation of T helper 17 (Th17) and regulatory T cells (Treg). The potential of TGF-β1 as therapeutic target in T cell-mediated diseases like rheumatoid arthritis (RA) is unclear. We investigated the effect of TGF-β1 inhibition on murine Th17 differentiation in vitro, on human RA synovial explants ex vivo, and on the development of experimental arthritis in vivo. Murine splenocytes were differentiated into Th17 cells, and the effect of the TGF-βRI inhibitor SB-505124 was studied. Synovial biopsies were cultured in the presence or absence of SB-505124. Experimental arthritis was induced in C57Bl6 mice and treated daily with SB-505124. Flow cytometry analysis was performed to measure different T cell subsets. Histological sections were analysed to determine joint inflammation and destruction. SB-505124 potently reduced murine Th17 differentiation by decreasing Il17a and Rorc gene expression and IL-17 protein production. SB-505124 significantly suppressed IL-6 production by synovial explants. In vivo, SB-505124 reduced Th17 numbers, while increased numbers of Tregs were observed. Despite this skewed Th17/Treg balance, SB-505124 treatment did not result in suppression of joint inflammation and destruction. Blocking TGF-β1 signalling suppresses Th17 differentiation and improves the Th17/Treg balance. However, local SB-505124 treatment does not suppress experimental arthritis.

Figure 1

TGFB-RI inhibitor SB-505124 prevents TGF-β1-induced expression of Th17 genes and proteins. Splenocytes were differentiated into Th17 cells in the presence of αCD3 and αCD28 with either αIL-2 alone, Th17 cocktail (IL-1, IL-6, IL-23) with or without the addition of increased concentrations of TGF-β1 and SB-505124 (5 µM) for five days of culture. Cytokine levels in supernatant were measured using Luminex (A,B,F,G). CD4 + IL-17 + T cells cultured with increased concentrations of TGF-β1 were determined by flowcytometry (C). Gene expression was determined by QPCR (D,E). n = 3 mice/group (ABC) and n = 3 biological replicates from one mouse (DEFG), values are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 calculated using one-way ANOVA followed by Bonferroni post-test.

Figure 2

Inhibiting TGF-β1 signaling using SB-505124 significantly reduced IL-6 production by human RA synovial explants. Synovium biopsy punches were collected from synovium collected during joint replacement surgery. Explants were cultured for 24 h in the presence or absence of 5 µM SB-505124 and cytokine levels in the supernatants were measured by Luminex. Graphs show results for IL-6 (A), IL-10 (B), and TNFα (C). N = 9 donors, mean of 2–6 biopsies per donor per condition. **P < 0.01, as determined by paired Student's t-test.

Figure 3

Intra-articular injection of SB-505124 does not induce joint inflammation and cartilage PG depletion. Mice were daily injected with 75 nmol SB-505124 or 20% DMSO as vehicle control from day 0–4, and sacrificed after 7 days. Knee joints were subsequently isolated for histological analysis of inflammation (HE stain, original magnification ×100) (A) and cartilage PG depletion (SafO stain, original magnification ×100). (B) Representative pictures of n = 4 joints per group.

Figure 4

Intra-articular injection of SB-505124 enhances Tregs and suppresses Th17 cell levels in arthritic mice. Cells from draining lymph nodes (n = 8 mice/group) were stimulated for 4 h with PMA/ionomycin and analyzed by flow cytometry. The percentage of CD3 + CD4 + T cells was decreased in the SB-505124 injected mice (A). Levels of Th17 cells were decreased by SB-505124 treatment (B), whereas FOXP3 + T cells (Tregs) were increased. (B) Levels of IL-17 + T cells were similar in vehicle and SB-505124 injected mice. ** P < 0.01, * P < 0.05 by Student’s t-test. Values are mean +/- SEM.

Figure 5

Intra-articular injections of SB-505124 do not decrease joint inflammation and PG depletion during experimental arthritis. Mice were daily injected i.a. with vehicle or SB-505124 for four days. Total knee joints were isolated for histopathologic analysis (n = 8 mice/group). Joint inflammation (H&E stain, original magnification ×100) and PG depletion (Safranin O staining, original magnification ×100) on day 4 (A) and day 7 (B) were analyzed on histological slides. Values are mean ± SEM. ** P < 0.01, *** P < 0.001 by one-way ANOVA and Bonferroni’s multiple comparison test (B) for all treatment groups.