MIF promotes Th17 cell differentiation in rheumatoid arthritis through ATF6 signal pathway

Abstract

Rheumatoid arthritis (RA) is a common autoimmune disease that can lead to irreversible joint damage when it occurs, but its pathogenesis has not yet been elucidated. In this study, we explored the roles of macrophage migration inhibitory factor (MIF), endoplasmic reticulum stress (ER stress), and Th17 cells in the pathogenesis of RA. We have preliminarily confirmed that MIF expression in CD4⁺T cells and the proportion of Th17 cells are increased in active RA patients. We also found that ER stress is activated, initiating ATF6 pathway in the UPR. Additionally, using in vitro stimulation and co-immunoprecipitation experiments, we have confirmed the interaction between MIF and ATF6, which enhances protein expression in ATF6 pathway. Subsequently, in the chromatin immunoprecipitation assay, we observed the enrichment of ATF6 subunit on the promoter sequences of the Th17 cell differentiation genes STAT3 and RORC. Additionally, the differentiation of Th17 cells was disrupted by Ceapin-A7 (ATF6 inhibitor). In summary, our results indicate that MIF enhances ATF6 pathway signaling, which promotes the differentiation of Th17 cells. This could be a potential mechanism underlying the pathogenesis of RA, offering a new direction for the clinical treatment of RA.

Keywords: ATF6; ER stress; MIF; Rheumatoid arthritis; Th17 cell.

Fig. 1

Expression of MIF and IL-17A in CD4⁺T cells of active RA patients. (A to C) Compared to healthy controls, MIF expression is increased at both mRNA and protein levels in RA. (D to G) Compared to healthy controls, IL-17A expression is increased at both mRNA and serum levels in RA. (F) Th17 cells were labeled with anti-CD3-PC-cy5.5, CD8-FITC, and IL-17 A-BV421 antibodies for flow cytometric analysis. HC, healthy controls; RA, rheumatoid arthritis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 2

ER stress is activated in CD4⁺T cells of active RA patients. (A, B) RT-PCR detection of ATF6 and BiP mRNA expression. (C) Immunoblotting results show increased expression of ATF6α and BiP in RA compared to HC. (D, E) Quantitative analysis of the protein bands of ATF6α and BiP based on grayscale values, with GAPDH as the reference for statistical graphs. *p < 0.05, **p < 0.01

Fig. 3

Interaction between MIF and ATF6. (A) Protein lysates were extracted from CD4⁺T cells of RA patients and used to isolate bait-prey protein complexes using ATF6 antibodies. (B,C,D,E) PBMCs from healthy controls were treated with TM for 1 h, sorted CD4⁺T cells, and extracted protein lysates for immunoprecipitation experiments. The results suggest that MIF interacts with ATF6 under ER stress conditions

Fig. 4

Elevated MIF enhances ATF6 pathway signaling and promotes Th17 cell differentiation. (A, B) rhMIF was treated with RA patient PBMCs for 6 h, sorted CD4⁺T cells, and extracted protein lysates for immunoblotting experiments. The protein band gray value was quantitatively analyzed and statistically. The protein level of ATF6α increased under rhMIF stimulation. (C, D) rhMIF was treated with RA patient PBMCs for 3 days, and flow cytometry was performed to analyze the proportion of Th17 cells in the control group and the treated group. The results showed that the proportion of Th17 cells increased in the treated group. *p < 0.05, **p < 0.01

Fig. 5

ATF6α regulates the transcription of Th17 cell differentiation genes STAT3 and RORC. (A, B). Compared to the healthy control group, the mRNA expression of STAT3 and RORC in CD4 + T cells of RA patients is upregulated. (C, D, E). At the protein level, the expression of STAT3 and RORC in RA patients is higher than that in the healthy control group. (F, G). Stimulated group, after treating PBMC1h with TM, CD4 + T cells were sorted and lysates were extracted for CHIP. Unstimulated group, no treatment was performed. Compared to the untreated group, the activated ATF6 pathway in the treated group resulted in ATF6α recovering more promoter sequences of STAT3 and RORC. TM, tunicamycin. *ρ < 0.05, **ρ < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 6

Ceapin-A7 inhibits the activation of ATF6 pathway and affects the differentiation of Th17 cells. (A). The expression of ATF6α, STAT3, and RORC in RA patient cells stimulated with rhMIF in the presence of Ceapin-A7. (B, C, D). The expression of ATF6α, STAT3, and RORC is reduced under the influence of Ceapin-A7. (E, F) After treating with Ceapin-A7 for 1 h and stimulating with rhMIF for 3 days, flow cytometry was used to analyze the effect of Ceapin-A7 on Th17 cells. *ρ < 0.05