Background: Immunomodulating treatment in rheumatoid arthritis (RA) prevent aberrant proliferation and activation of CD4+T cells by various mechanisms. Yet, drug choice remains empirical. In RA, unrepaired DNA damage promote the arthritogenic potential of effector T cells [1]. DNA damage response (DDR) combines cell cycle check points, DNA repair and DNA damage tolerance. It is controlled epigenetically by the Switch/Sucrose NonFermentable complex (SWI/SNF or BRG1) at chromatin marked by histone H3 tail modifications. DDR has a strong IFN adherence [2].

Objectives: This study investigates if the molecular signature of DNA damage is connected to high BRG1 expression in CD4+ cells of RA patients and how it is affected by immunomodulating drugs.

Methods: Transcriptome of BRG1 ^hi CD4+ cells of RA patients (n=24) were identified by RNAseq followed by DESeq2 analysis. RA treatment effect was identified in paired CD4+ T cells of patients pre- and post-treatment with abatacept (n=14, GSE121827, 24 weeks), tocilizumab (n=6, GSE113156, 24 weeks), and methotrexate (MTX, n=28, GSE176440, 12 weeks), by RNAseq. Treatment responsive differentially expressed genes (trDEG) were identified by DESeq2.

DNA sequences binding H3K4me3, H3K27me3 and H3K27ac were obtained through immunoprecipitation and sequencing (ChIPseq). ChIPseq overlap indicating bivalent chromatin regions (BvCR) was identified by R package ChIPPeakAnno. Tag normalization enabled comparison between different ChIPseq to identify the dominant H3 modification. Genes connected to overlapped ChIPseq peaks (BvCR-dependent) within cis -regulatory elements were retrieved from GeneHancer database. Annotation to DDR pathway was done using Enrichr. Weighted gene correlation network analysis was performed using R package WGCNA. To identify IFN-sensitive genes, CD4+ cells sorted from blood (n=4) were cultured with IFNγ (50ng/mL) for 72h. Transcriptome was analysed by RNAseq followed by DESeq2.

Results: Analysis of BRG1 ^hi CD4+ cells revealed 8358 DEG compared to BRG1 ^lo CD4+ cells, which had accumulation of immune checkpoint proteins PD1, PD-L1 and CTLA4, and chemokine receptors CCR1, CCR5 and CD69, indicating a PD1+CD4+ cell phenotype promoting RA pathogenesis. The DDR pathway was significantly enriched in DEG of BRG1 ^hi CD4+ cells (FDR=1.5e-23) and included the canonical SWI complex (ARID1A/B, SMARCA2), PBAF complex (PBRM1, ARID2) and the common subunits (BRG1, SMARCC1/C2, SMARCD1/D3). DNA damage marker γH2AX and classic DNA repair proteins FANCI, MSH2, MSH6, and MRE11 were significantly upregulated in BRG1 ^hi CD4+ cells, while ATM was repressed.

Immunomodulating treatment affected 40% of BRG1 ^hi DEG. Among those, MTX reduced expression of PD1 (log2FC=-0.35, p=4.2e-4) and increased CD86 (log2FC=0.63, p=0.054), while abatacept and tocilizumab had no effect on PD1 expression.

One-fourth of trDEG were BvCR-dependent and 40% (682 genes) were IFNγ−sensitive, with a majority regulated by H3K4me3-dominated BvCR. MTX affected 71% of these IFNγ-sensitive genes (484 genes) making a significant impact in epigenetic processes in RA CD4 cells. Among the DDR pathway trDEG were numerous SWI complex subunits, while BRG1 expression was unaffected. WGCNA of IFNγ-sensitive genes in BRG1 ^hi cells (682 genes) showed downregulation of chromatin remodeling (GO:0006338, ARID1B, CHD7, SATB1) and upregulation of cell cycle phase transition (GO:0044772, CDKN1A, WEE1, MYC).

In the trDEG under BvCR control in BRG1 ^hi cells, H3K4me3-BvCR regulated 15 genes annotated to apoptotic DNA damage signaling (GO:0042771, DYRK2, PML). Genes controlled by H3K4me3-BvCR were mostly downregulated by immunomodulation, with MTX having the largest effect.

Conclusion: BRG1 ^hi CD4 ⁺ cells embody the activated DNA damage response and promote RA pathogenesis. MTX treatment affected the DNA damage response controlled by IFN signaling in CD4+T cells through the SWI/BRG1 complex. This makes a significant impact in epigenetic processes in RA CD4 cells and implies that high expression of BRG1 in RA recognize MTX-responsive RA patients.

REFERENCES: [1] Li, Y. et al. Immunity 45 , 903–916 (2016).

[2] Begg, K. A. G., et al. DNA Repair 133 , 103609 (2024).

Acknowledgements: NIL.

Disclosure of Interests: None declared.