Fraxinellone alleviates colitis-related intestinal fibrosis by blocking the circuit between PD-1+ Th17 cells and fibroblasts

Background: Excessive activation of colonic fibroblasts and differentiation of T helper 17 (Th17) cells are the key steps for intestinal fibrogenesis in the process of inflammatory bowel disease (IBD). Although both transforming growth factor-beta (TGF-β)/Mothers Against Decapentaplegic Homolog (SMAD) 3-induced fibroblasts activation and interleukin (IL)-6/signal transducer and activator of transcription (STAT) 3-induced Th17 differentiation have been well studied, the crosstalk between fibroblasts and Th17 cells in the process of intestinal fibrogenesis needs to be unveiled.

Methods: In this study, the activation of colonic fibroblasts was induced with dextran sulfate sodium salt (DSS) and TGF-β in vivo and in vitro respectively. P-SMAD3 and its downstream targets were quantified using RT-PCR, western blot and immunofluorescence. The differentiation of programmed death 1 (PD-1) ⁺ Th17 and activation of fibroblasts were quantified by FACS. PD-1⁺ Th17 cells and fibroblasts were co-cultured and cytokines in the supernatant were tested by ELISA. The anti-fibrosis effects of different chemical compounds were validated in vitro and further confirmed in vivo.

Results: The colonic fibroblasts were successfully activated by DSS and TGF-β in vivo and in vitro respectively, as activation markers of fibroblasts (p-SMAD3 and its downstream targets such as Acta2, Col1a1 and CTGF) were significantly increased. The activated fibroblasts produced more IL-6 compared with their inactivated counterparts in vivo and in vitro. The proinflammatory cytokine IL-6 induced PD-1⁺ Th17 differentiation and TGF-β that in return promoted the activation of colonic fibroblasts. Fraxinellone inhibited TGF-β⁺ PD-1⁺ Th17 cells via deactivating STAT3.

Conclusions: The reciprocal stimulation constructed a circuit of PD-1⁺ Th17 cells and fibroblasts that accelerated the fibrosis process. Fraxinellone was selected as the potential inhibitor of the circuit of PD-1⁺ Th17 cells and fibroblasts in vivo and in vitro. Inhibiting the circuit of PD-1⁺ Th17 cells and fibroblasts could be a promising strategy to alleviate intestinal fibrosis.

Fibroblasts; Intestinal fibrosis; PD-1; Th17.