M694I variant of MEFV drives pathogenesis of familial Mediterranean fever through enhanced Th17 cell differentiation

Abstract Objective To investigate how the M694I variant of MEFV contributes to familial Mediterranean fever (FMF) pathophysiology using knock-in mice, focusing on T helper 17 (Th17) cell responses and the underlying molecular mechanisms. Methods Human MEFV encoding wild-type and M694I knock-in (MefvM694I/M694I) mice were generated using CRISPR/Cas9. Survival and growth trajectories were monitored at 15 weeks of age. Comprehensive immune phenotyping and gene set enrichment analysis (GSEA) were performed using single-cell RNA sequencing (scRNA-seq) of splenocytes with subsequent pathway analysis using the Seurat and fgsea packages. Systemic inflammation was assessed using 25-plex cytokine/chemokine magnetic bead array analysis of the serum samples. CD4+ T-cell subset differentiation into Th1, Th2, Th17 and regulatory T cells (Tregs) was evaluated by flow cytometry. Results Mefv M694I/M694I mice demonstrated significantly reduced survival (P < 0.001) and impaired growth from five weeks onward (P = 0.014). scRNA-seq with GESA revealed enhanced interferon responses in T cells (NES = 1.71, P = 4.6 × 10−5) and TNF-α signalling in monocytes (NES = 1.52, P = 1.4 × 10-³). Serum analysis showed elevated levels of multiple proinflammatory cytokines, including G-CSF, IFN-γ, IL-1α, IL-5, IL-6 and TNF-α (all P < 0.05). Flow cytometry demonstrated selective enhancement of Th17 differentiation (P = 0.047), with no substantial changes in Th1, Th2 or Treg populations. Conclusion The M694I variant drives Th17 cell differentiation and cytokine dysregulation, thereby contributing to the pathogenesis of FMF. This study provides a comprehensive model to explore the mechanisms and therapeutic targets of FMF.