Background: Rheumatoid arthritis (RA) is a chronic inflammatory joint disease that imposes a massive burden on affected individuals and on society. If left untreated, it can lead to irreversible joint damage. While effective treatment options, particularly with methotrexate (MTX) as a first line of treatment, have been used successfully to reduce this burden, multiple challenges still remain [1]. Due to its slow onset of action, treatment with MTX can take up to 6 months before a clinical decision regarding response is made, which can delay the successful treatment in non-responders [1]. In addition, the precise mode of action of MTX in RA on cellular and molecular level still remains unknown.

Objectives: This study aims to elucidate the complex interplay of diverse anti-inflammatory effects on various pathogenic cell types exhibited by MTX over time.

Methods: To explore this, we utilized single-cell RNA sequencing (scRNA-seq) in conjunction with 40 color immunophenotyping [2] of peripheral blood mononuclear cells (PBMCs) obtained from treatment-naive RA patients and the same patients after 3, 6, and 12 weeks of MTX treatment. All patients responded to treatment at the 12 weeks follow up. We performed variance decomposition analysis and gene co-expression network analysis to define cell-specific gene signatures perturbed by MTX. In addition we profiled age- and sex-matched heathy controls, in order to define an RA-specific single-cell landscape at baseline.

Results: Combining the two technologies we detected perturbations of cell proportions in multiple cell types as early as 3 weeks from the start of MTX treatment in RA patients. Variance decomposition analysis showed a large effect of cell heterogeneity on the sample. Stratifying by cell type showed cell-specific effects of the MTX treatment. Shared as well as unique cell-specific early gene signature modules were identified, suggesting an involvement of pro-inflammatory cellular programs in response to MTX in PBMCs.

Conclusion: The combinination of two distinct methods for cellular profiling allows for deep cellular and molecular phenotyping of cells responding to MTX treatment as well as time-resolved insight in the mechanism of action. This, in turn, can be utilized in biomarker development for early MTX response detection before the onset of clinically detectable responses.

REFERENCES: [1] Aletaha D, Smolen JS. Diagnosis and Management of Rheumatoid Arthritis: A Review. JAMA. 2018 Oct 2;320(13):1360–72.

[2] Park LM, Lannigan J, Jaimes MC. OMIP-069: Forty-Color Full Spectrum Flow Cytometry Panel for Deep Immunophenotyping of Major Cell Subsets in Human Peripheral Blood. Cytometry A. 2020 Oct;97(10):1044-1051.

Acknowledgements: NIL.

Disclosure of Interests: Anela Tosevska: None declared, Teresa Preglej: None declared, Marie Brinkmann: None declared, Philipp Schatzlmaier: None declared, Elisabeth Simader recieved speakers’ bureau from Lilly, support for meeting attendances from Pfizer, Bristol-Myers Squibb, Boehringer-Ingelheim and Astra Zeneca, Daniela Sieghart: None declared, Daniel Aletaha received consulting fees and speakers’ bureau from Abbvie, Amgen, Galapagos, Lilly, Janssen, Merck, Novartis, Pfizer, and Sandoz., grants from Abbvie, Amgen, Galapagos, Lilly, and Sanofi, Philipp Hofer: None declared, Lisa Göschl: None declared, Michael Bonelli received grants from Galapagos and GSK.