Systemic sclerosis (SSc) is an autoimmune disease characterized by skin fibrosis and immune cell infiltration. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine with well-established roles in inflammation, yet its specific contribution to SSc pathogenesis remains largely unexplored. This study aims to investigate whether MIF promotes disease progression by modulating macrophage infiltration through the calcium channel transient receptor potential melastatin 2 (TRPM2).

Single-cell RNA sequencing (scRNA-seq) data from skin biopsies of SSc patients and healthy controls, as well as from a bleomycin-induced SSc mouse model, were retrieved from public databases (GEO and ArrayExpress). Differential expression analysis was performed to identify key secreted factors and calcium channels in macrophages. For in vivo experiments, wild-type (WT), MIF knockout (KO), and TRPM2 KO mice were subjected to intradermal injection of bleomycin to induce skin fibrosis. Skin tissues were collected for histological analysis, including H&E and Masson’s trichrome staining to assess dermal thickness and collagen deposition. Immunofluorescence staining was performed to evaluate macrophage infiltration using F4/80 or CD68 antibodies. For in vitro studies, bone marrow-derived macrophages (BMDMs) were isolated from WT and TRPM2 KO mice and cultured in the presence of M-CSF. Cells were treated with recombinant MIF to assess reactive oxygen species (ROS) production using DCFH-DA probe, calcium influx using Fluo-8 AM staining combined with flow cytometry and fluorescence microscopy, cytoskeletal rearrangement by phalloidin staining, and cell migration using Transwell assays. Western blotting and qPCR were performed to detect TRPM2 expression. For endothelial adhesion assays, C166 endothelial cells were pretreated with recombinant MIF, and CMFDA-labeled BMDMs were co-cultured to assess macrophage adhesion under fluorescence microscopy.

Analysis of public single-cell RNA sequencing datasets revealed that MIF was significantly upregulated in skin tissues from both SSc patients and bleomycin-induced SSc mice, with broad expression across multiple cell types, particularly in the dermal compartment. In MIF knockout mice subjected to bleomycin-induced skin fibrosis, histological analysis showed markedly reduced dermal thickness, collagen deposition, and macrophage infiltration. Single-cell transcriptomic profiling of macrophages from SSc patients compared with healthy controls revealed enrichment of pathways related to chemotaxis, cytoskeletal rearrangement, calcium influx, and oxidative stress. Consistent with these findings, previous reports indicated that MIF-mediated cytoskeletal changes in macrophages require calcium influx, but the specific calcium channel involved remained unknown. Screening of calcium channels in macrophages identified TRPM2 as a top candidate, with functional overlap between MIF and TRPM2 signaling. As TRPM2 is a known oxidative stress sensor, we further demonstrated that recombinant MIF stimulation induced reactive oxygen species (ROS) production in bone marrow-derived macrophages (BMDMs). Using Fluo-8-based calcium imaging and flow cytometry, MIF triggered TRPM2-dependent calcium influx, which was abrogated in TRPM2 knockout BMDMs. Moreover, MIF upregulated TRPM2 expression at both the mRNA and protein levels. Functionally, MIF promoted BMDM migration in Transwell assays and induced robust cytoskeletal changes, including increased pseudopodia formation and enhanced actin polymerization; these effects were absent in TRPM2-deficient macrophages. Beyond its direct effects on macrophages, MIF also enhanced macrophage adhesion by acting on endothelial cells. In human data, the MIF receptor CD74 was expressed not only in myeloid cells but also in endothelial cells. Treatment of C166 endothelial cells with MIF upregulated the expression of adhesion molecules and MIF itself. Live-cell tracking revealed that MIF-pretreated C166 cells captured significantly more CMFDA-labeled BMDMs within one hour, with increased fluorescence intensity confirming enhanced adhesion. Finally, in vivo, TRPM2 knockout mice exhibited significantly attenuated skin fibrosis and reduced macrophage infiltration following bleomycin challenge, phenocopying the protective effects observed in MIF-deficient mice.

Our findings identify a previously unrecognized MIF-TRPM2 signaling axis that drives macrophage infiltration and skin fibrosis in SSc. MIF activates TRPM2 through both ROS-dependent channel gating and transcriptional upregulation, leading to sustained calcium influx and macrophage recruitment. This study provides mechanistic insights into the crosstalk between cytokine signaling and ion channels in SSc pathogenesis and highlights TRPM2 as a potential therapeutic target for fibrotic diseases.