MIF Deficiency Disrupts Epithelial Defense and Metabolic Pathways in the Ileum of SKG Mice

Objectives Spondyloarthritis (SpA) is a chronic inflammatory disorder that primarily affects the musculoskeletal system, with up to 60% of patients developing gut inflammation and 10% experiencing inflammatory bowel disease (IBD). Our laboratory has previously elucidated the role of macrophage migration inhibitory factor (MIF) in SpA, showing that MIF overexpression promotes SpA-like features, while its inhibition alleviates disease severity. Given the strong gut–joint connection, we hypothesized that MIF also regulates intestinal inflammation and epithelial homeostasis. Thus, we aimed to investigate how MIF regulates intestinal epithelial function and metabolism in the SKG mouse model, both under homeostatic and inflammatory (curdlan-induced) conditions, by identifying cell type–specific transcriptional and pathway-level alterations through single-cell RNA sequencing. Methods Single-cell RNA sequencing (Flex Gene Expression) was performed on FFPE ileal samples from SKG wild-type (WT), WT+Curdlan, MIF knockout (MKO), and MKO+Curdlan mice (n=2 per group). UMAP clustering, differential gene expression, and Gene Ontology (GO) analyses were used to assess cellular composition and biological pathways related to immune activation, epithelial integrity, and metabolism. Results Distinct transcriptional and cellular changes were observed across groups. Curdlan treatment induced expansion of neutrophil and macrophage populations in both WT and MKO mice. Compared to WT, MIF deficiency led to reduced enterocyte, goblet cell, and Paneth cell populations, indicating epithelial compromise. Under steady-state conditions, GO analysis revealed that MIF deletion caused loss of pathways associated with bacterial defense, epithelial secretion and transport, and epithelial differentiation, consistent with impaired mucosal defense. Upon curdlan challenge, MIF deficiency further suppressed oxidative phosphorylation, mitochondrial electron transport, and ATP synthesis pathways, indicating metabolic dysfunction in the inflamed gut epithelium. Conclusion These findings highlight MIF as a critical regulator of intestinal epithelial homeostasis. In its absence, mice exhibit loss of antimicrobial defense and epithelial function under basal conditions and marked impairment of mitochondrial and respiratory metabolism upon inflammatory stimulation, collectively predisposing to gut inflammation and barrier breakdown.