Acidosis regulates immune progression in rheumatoid arthritis by promoting the expression of cytokines and co-stimulatory molecules in synovial fibroblasts.

Abstract

Background: Tissue acidosis is a key characteristic of RA. It remains unclear whether acidosis promotes the formation of the complex adaptive immune landscape mainly characterized by T cell activation in RA by influencing synovial fibroblasts. This study aims to investigate the influence of acidosis on the immune microenvironment of RA by exploring the cytokine secretion and expression of co-stimulatory factors of RA synovial fibroblasts.

Methods: The Bulk RNA-seq dataset (GSE89408, Normal = 23, RA = 150) was utilized for cytokine screening and the immune state assessment based on disease stage. RNA-seq was employed to investigate cytokine and co-stimulatory molecule expression following 6 h of acid stimulation, combined with Bulk RNA-seq data to evaluate contributions to RA. Human cytokine arrays were used to confirm cytokine accumulation in supernatants after 12 h of acid stimulation. Proteomics was applied to explore cellular functional states in RASFs under 6 h of acid stress, with joint RNA-seq analysis elucidating transcription factor activation. Validation of select high-throughput data was performed using qRT-PCR and immune-based assays.

Results: Bulk RNA-seq and RNA-seq identified 56 differentially expressed cytokines at their intersection. Functional enrichment analysis demonstrated that acid stimulation enhanced cytokine secretion and T cell chemotaxis in RA synovial fibroblasts (RASFs). Cytokine array revealed that acid exposure increased the accumulation of growth factors (e.g., FGF, VEGF) by over twofold and promoted the expression of multiple inflammatory and chemotactic factors. Immune state analysis indicated that acid stimulation induced a complex immune landscape by upregulating co-stimulatory and antigen-presenting molecules. Proteomics showed that acid stress enhanced mitochondrial function and triggered metabolic reprogramming in RASFs. Integrated transcriptomic and proteomic analyses revealed that AP1 regulates gene expression in RASFs, with its activation further confirmed by Western blotting and immunofluorescence.