0113: ZEB2 and MLCK1 may impair gut barrier integrity in MIF-Deficient SKG Mice

Background/Purpose: Axial Spondyloarthritis (AxSpA) is a chronic inflammatory condition that primarily affects the musculoskeletal system and has multifactorial origins. The majority of AxSpA patients experience gut inflammation, with 60% having microscopic changes and 10% having overt Inflammatory Bowel Disease (IBD). Over the years, researchers have linked many pathways and cell populations to the pathogenesis of gut inflammation in AxSpA. One notable player is the macrophage migration inhibitory factor (MIF), which assumes a crucial role in AxSpA development. In a SpA mouse model (SKG mice), MIF overexpression mirrors key clinical features of AxSpA, while inhibiting or depleting MIF attenuates these symptoms. However, the exact role of MIF in SpA-related gut inflammation is unknown. In this study, we aimed to investigate the effects of MIF on intestinal barrier integrity in SKG mice.

Methods: Eight-week-old SKG mice were used for this four-arm study: SKG control mice (WT), SKG mice treated with curdlan (WT_Curdlan), SKG-MIF Knock Out (KO) mice (MKO), and SKG-MIFKO mice treated with curdlan (MKO_Curdlan). After eight weeks of curdlan/vehicle treatment, 16-week-old mice were euthanized. Formalin-fixed paraffin-embedded (FFPE) blocks of the ileum from these mice (n=2/arm) were used for single-cell RNA sequencing (scRNA-seq) Flex Gene Expression assay to investigate the effects of MIF on the expression of genes related to gut homeostasis. These results were validated by Immunohistochemistry (IHC) or qPCR in WT (n=7) and MKO (n=9) mice. Intestinal permeability was assessed in vivo using the FITC-dextran assay. Mice were administered FITC-dextran orally, and serum fluorescence was measured after 4 hours.

Results: scRNA-seq analysis revealed differential expressions of genes potentially related to gut homeostasis: Zeb2, Mylk, Muc2, Cdh1, Ocln and Cldn7, across the four arms. As compared to WT mice (± curdlan), downregulation of mucin Muc2, adherens junction protein Cdh1 and tight junction proteins Ocln and Cldn7 was observed in MKO mice (± curdlan), which was further confirmed by qPCR and IHC, respectively. These proteins are critical in maintaining gut barrier integrity. Additionally, overexpression of Zeb2 (a transcriptional repressor of Cdh1) and Mylk (responsible for endocytosis of occludin from tight junctions) was observed in MKO mice (± curdlan), further validated by IHC. These findings suggest a potential defect in intestinal epithelial regulation in the absence of MIF. Functional assessment of the intestinal barrier using the FITC-dextran assay demonstrated significantly increased intestinal permeability in MKO mice compared to WT controls (p < 0.05), consistent with the molecular findings.

Conclusion: MIF maintains gut barrier integrity by regulating the expression of ZEB2 and MLCK1. In the absence of MIF, upregulation of ZEB2 and MLCK1 disrupts the expression of Muc2, and function of epithelial tight junction (Cldn7 and Ocln) and adherens junction (Cdh1) markers, contributing to impaired barrier function and increased intestinal permeability in MKO SKG mice. These results highlight the critical role of MIF in maintaining intestinal homeostasis.