An in-depth in vitro study decodes hapln1-induced proinflammatory m1 macrophage polarization via FLSs- MΦ crosstalk: integrating bioinformatic analysis to functional study

Abstract Background Secreted by fibroblast-like synoviocytes (FLSs), hyaluronan and proteoglycan link protein 1 (HAPLN1) contributes to a pro-inflammatory reaction in rheumatoid arthritis (RA), according to our previous study. In the current study, we aim to investigate the deeper roles of HAPLN1 in the mechanisms of RA, especially its impact on macrophages (MΦ). Methods Single-cell RNA sequencing (scRNA-seq) data from human- and mouse-derived synovium were obtained from the GEO database to characterize the features of HAPLN1 + FLSs. RA-FLSs were isolated from synovial tissues of RA patients. Gain- and loss-of-function experiments were conducted by transfection of HAPLN1 -overexpression ( HAPLN1 OE ) and small interfering RNA (si- HAPLN1 ) vectors. MΦ derived from the THP-1 cell line were co-cultured with RA-FLSs in a Transwell system or treated with recombinant HAPLN1 (rHAPLN1). MΦ viability was assessed using CCK-8 and EdU assays, while M1 polarization was evaluated via flow cytometry. mRNA expression levels were detected using RNA sequencing (RNA-seq). Inflammatory cytokine expression and pathway activation were assessed by Immunofluorescence, Western blot, and reverse transcription quantitative polymerase chain reaction (RT-qPCR). Results ScRNA-seq analysis revealed elevated HAPLN1 expression in RA-FLSs, along with the upregulation of extracellular matrix-related genes (e.g., COMP , MMP13 ). Cell–cell interaction analysis indicated a strong interaction between HAPLN1 + FLSs and MΦ. HAPLN1 OE RA-FLSs promoted MΦ polarization toward the proinflammatory M1 phenotype under co-culture, marked by increased expression of iNOS, CD80, IL-1β, IL-6, TNF-α, and IL-8 . rHAPLN1-treated MΦ exhibited similarly enhanced viability, an increased M1 polarization ratio, and elevated proinflammatory cytokine levels. In contrast, si- HAPLN1 RA-FLSs reduced M1 polarization, but rHAPLN1 supplementation reversed this effect. RNA-seq and validation assays further suggested that rHAPLN1 facilitates MΦ polarization via PI3K/AKT/NF-κB signaling. Conclusion This study provides the first evidence that RA-FLS-derived HAPLN1 drives M1 polarization through PI3K/AKT/NF-κB signaling, highlighting its pathogenic role in RA and its potential as a therapeutic target.