Background/Purpose: Difficult-to-treat rheumatoid arthritis (D2T-RA) represents a clinically challenging RA subset defined by failure to achieve disease control despite multiple conventional and advanced therapies. The immunopathogenesis of D2T-RA remains poorly understood. Prior studies suggest that elevated pretreatment type I interferon (IFN) signatures are associated with poor responses to tumor necrosis factor inhibitors (TNFi). Although Janus kinase (JAK) inhibitors, which modulate multiple cytokine pathways, are widely used, D2T-RA persists in a subset of patients. This study aimed to elucidate the immunological basis of D2T-RA through transcriptomic comparisons between propensity score–matched D2T and non-D2T patients.

Methods: Peripheral blood samples were obtained from 473 RA patients enrolled at the Department of Rheumatology, Japanese Red Cross Okayama Hospital, between March 2012 and September 2023, prior to initiation of new treatments. Based on EULAR criteria, 14 patients were identified as D2T, and 459 as non-D2T, the latter confirmed by at least two years of follow-up. Propensity score matching (1:1) was performed using age, DAS28-ESR, and the number of prior medications, yielding 13 matched pairs. RNA sequencing was conducted, and differentially expressed genes (DEGs) were identified using the LRM Paired-edgeR approach. Subsequent analyses included hierarchical clustering, Gene Ontology (GO) and pathway enrichment analyses, and weighted gene co-expression network analysis (WGCNA).

Results: At least 11 of 14 D2T patients demonstrated inadequate responses to JAK inhibitors. A total of 2,875 DEGs (P < 0.05) were identified, among which 288 genes with transcripts per million (TPM) ≥ 1 in either group were subjected to further analysis. Hierarchical clustering did not distinctly segregate D2T from non-D2T patients. Of these 288 genes, 114 were upregulated (positive DEGs) and 174 downregulated (negative DEGs) in the D2T group. GO analysis of the positive DEGs revealed enrichment of genes associated with immunity, particularly those involved in T cell function, innate immunity, and Toll-like receptor (TLR) signaling—most notably, the TLR7 pathway. Pathway analysis confirmed significant upregulation of TLR-related pathways. WGCNA identified five modules, one of which was enriched for TLR-associated genes.

Conclusion: This transcriptomic study identified marked upregulation of TLR signaling, especially TLR7-associated pathways, in patients with D2T-RA. These findings suggest that dysregulated TLR signaling may play a central role in the pathogenesis of treatment-refractory RA and offer potential insights for targeted therapeutic strategies.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/upregulated-tlr-signaling-identified-in-difficult-to-treat-ra-a-propensity-score-matched-transcriptome-study/