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

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: BHLHE40; GM‐CSF; RNA‐seq; T cells; cytokines.