CLIC5B attenuates pro-inflammatory fibroblast migration through inhibition of the CLIC1/CLIC4-PIP5K1 axis

Rheumatoid arthritis (RA) is a chronic autoimmune disease driven by aberrant activation of fibroblast-like synoviocytes (FLS), which promote synovial inflammation, joint destruction, and disease progression through enhanced migration and invasion. While mitochondrial dysfunction and reactive oxygen species (ROS) contribute to FLS pathogenicity, the regulatory mechanisms remain unclear. This study investigates the role of chloride intracellular channel 5 (CLIC5) in RA synovium and FLS function. Bioinformatic analysis of bulk and single-cell RNA sequencing datasets revealed significantly reduced CLIC5 expression in RA synovial tissues compared to healthy or osteoarthritis controls, predominantly in fibroblast subsets. Lower CLIC5 levels correlated negatively with histological inflammation scores (CD3, CD20, CD138), serological markers (CRP, ESR), and disease activity indices (DAS28, joint counts), establishing CLIC5 downregulation as a feature of increased RA severity. Functional experiments in primary human FLS from healthy donors (HFLS-H) and RA patients (HFLS-RA) demonstrated that CLIC5B, the dominant isoform, maintains mitochondrial homeostasis. CLIC5 knockdown in HFLS-H cells induced mitochondrial depolarization and elevated ROS, while CLIC5B overexpression in HFLS-RA cells restored membrane potential and reduced ROS. CLIC5 deficiency promoted ROS-dependent translocation of CLIC1, CLIC4, and CLIC5B to the plasma membrane, enhancing FLS migration and invasion. Mechanistically, CLIC5B attenuated this by inhibiting CLIC1/CLIC4-mediated recruitment of PIP5K1A/C to the membrane, as confirmed by co-immunoprecipitation, immunofluorescence, and rescue assays where CLIC1/CLIC4 overexpression reversed CLIC5B's inhibitory effects. These findings identify CLIC5B as a negative regulator of pro-inflammatory FLS migration via the CLIC1/CLIC4-PIP5K1 axis, highlighting its potential as a therapeutic target for mitigating RA progression.