Background: Difficult-to-treat rheumatoid arthritis (D2T-RA) is a refractory subset of RA characterized by patients who fail to achieve remission despite multiple conventional and advanced therapeutic interventions. While substantial progress has been made in elucidating the pathophysiology of D2T-RA, the underlying immune abnormalities remain incompletely understood. Reports have demonstrated that poor responses to tumor necrosis factor inhibitors (TNFi) are associated with elevated pretreatment type 1 interferon (IFN) signatures [1]. Furthermore, studies suggest that in patients unresponsive to TNFi, tocilizumab (TCZ) is more effective than rituximab (RTX) in synovial tissues with low B-cell lineage expression signatures [2]. These findings highlight the heterogeneity in the underlying pathophysiology of D2T-RA. The introduction of Janus kinase (JAK) inhibitors, which can block multiple cytokine signaling pathways, including interferon, has provided a therapeutic tool capable of intervening in the majority of immune cascades involved in RA pathogenesis. Nevertheless, cases of D2T-RA persist, underscoring the need to elucidate the immunological characteristics of such cases. Doing so may not only pave the way for the development of novel therapeutic targets for D2T-RA but also reveal new RA subtypes.

Objectives: To investigate the immunological mechanisms underlying the pathophysiology of D2T-RA by performing gene expression analyses on blood samples from D2T and non-D2T groups matched by propensity score, identifying potential differences in gene expression patterns between the two groups.

Methods: From March 21, 2012, to September 25, 2023, blood samples were collected from 473 patients diagnosed with RA at the Department of Rheumatology, Japanese Red Cross Okayama Hospital, immediately prior to initiating a new disease-modifying antirheumatic drug (DMARD). On the basis of the EULAR definition of D2T-RA, 14 patients were classified into the D2T group, whereas 459 patients who did not meet the criteria for D2T during a follow-up period of at least two years postdiagnosis were classified into the non-D2T group. Propensity score matching (1:1) was performed using age, DAS28-ESR, and the number of previously used medications at the time of blood sampling as covariates, resulting in 13 matched sample pairs. RNA sequencing was conducted on blood samples to obtain read count data, and differential gene expression analysis was performed via the LRM Paired-edgeR method. Hierarchical clustering analysis, Gene Ontology (GO) enrichment analysis, pathway enrichment analysis, and weighted gene coexpression network analysis (WGCNA) were subsequently conducted on the identified differentially expressed genes (DEGs).

Results: Approximately 70% of the D2T group exhibited inadequate responses to JAK inhibitors. A total of 2,875 DEGs (P < 0.05) were identified through the LRM paired-edgeR method. Among these genes, 288 genes with an average expression level of at least 1 in at least one of the two groups were extracted. Hierarchical clustering analysis of these 288 genes did not clearly segregate the D2T and non-D2T groups. These genes included 114 upregulated genes (positive DEGs) and 174 downregulated genes (negative DEGs) in the D2T group. GO enrichment analysis of the positive DEGs revealed upregulated expression of genes related to immunity, particularly those associated with T cells, innate immunity, and Toll-like receptors (TLRs), especially the TLR7 signaling pathway, in the D2T group. Pathway analysis highlighted significant activation of the TLR signaling pathway. WGCNA of the 288 DEGs revealed five distinct modules. Pathway analysis of the module containing the upregulated genes in the D2T group also revealed increased expression of genes associated with the TLR signaling pathway.

Conclusion: By employing propensity score matching to align background factors between D2T-RA patients and non-D2T-RA patients and comparing their peripheral blood transcriptomes, this study aimed to further elucidate the immunological characteristics of D2T-RA patients. These findings demonstrated significant activation of the TLR signaling pathway, particularly the TLR7-related pathway, in D2T-RA. These results suggest that dysregulated TLR signaling may be central to the refractory pathophysiology of RA and highlight potential new immunological targets for therapeutic intervention.

REFERENCES: [1] Iwasaki, T. et al. Dynamics of Type I and Type II Interferon Signature Determines Responsiveness to Anti-TNF Therapy in Rheumatoid Arthritis. Frontiers in immunology 13 , 901437 (2022). https://doi.org/10.3389/fimmu.2022.901437 .

[2] Humby, F. et al. Rituximab versus tocilizumab in anti-TNF inadequate responder patients with rheumatoid arthritis (R4RA): 16-week outcomes of a stratified, biopsy-driven, multicenter, open-label, phase 4 randomized controlled trial. Lancet 397 , 305-317 (2021). https://doi.org/10.1016/S0140-6736(20)32341-2 .

Acknowledgements: We thank Y. Komori for her valuable assistance in gathering and organizing the data.

Disclosure of Interests: Yoshinori Nishiura: None declared, Yoshiharu Sato: None declared, Yu Nakai: None declared, Moe Tokunaga: None declared, Kenta Shidahara: None declared, Yoshinobu Koyama Abbvie, Abbott, Asahikasei, Ayumi, BMS, Eli Lilly, GSK, Novartis, Taisho and Tanabe Mitsubishi, Asahikasei, Abbvie, Abbott GSK and Novartis.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.