Identification of BHLHE40-Expressing CD4⁺ T Cells Producing GM-CSF in Rheumatoid Arthritis

Granulocyte-macrophage colony-stimulating factor (GM-CSF: CSF2) plays a crucial role in the pathogenesis of autoimmune diseases. The basic helix-loop-helix family member e40 (BHLHE40) gene is important for GM-CSF production in CD4⁺ T cells. However, the relationship between the expression of these genes and rheumatoid arthritis (RA) remains unclear, particularly in interleukin-1 (IL-1)-enriched inflammatory sites. Therefore, we investigated the expression of BHLHE40 and CSF2 in CD4⁺ T cells in RA.

We analyzed gene expression using previously deposited and publicly available databases containing peripheral blood (PB) and synovial fluid (SF) from patients with RA and healthy controls (HC). Comprehensive datasets, including single-cell RNA-sequencing (scRNA-seq), RNA-seq, and microarray data, were used for this analysis.

BHLHE40 expression in PB CD4⁺ T cells from HC was higher in central memory, effector memory, Th17, and Th1/17 cells than in naive CD4⁺ T cells. Furthermore, BHLHE40 and CSF2 expression in the CD45RA^-CCR7^{+ /-}CD4⁺ T cell subset was significantly higher in the SF of patients with RA than in those with PB. scRNA-seq revealed that BHLHE40-expressing cells showed higher CSF2 expression than those that did not. Additionally, scRNA-seq showed higher BHLHE40 expression in PB CD4⁺ T cells from RA patients than in those from HC.

We analyzed the gene expressions of BHLHE40, which is crucial for GM-CSF production, IL1R1, which regulates BHLHE40 induction, and CSF2, its resulting product, in CD4⁺ T cells. Their expression levels were compared across RA SF, PB, and HC. Notably, increased expression of these genes was identified in SF.