Background/Purpose: Rheumatoid nodules are the most common cutaneous manifestation in patients with RA, often associated with longstanding and a more severe disease course. Paradoxically, therapy including methotrexate (MTX) and TNF-antagonists, which is effective for synovial inflammation, has been linked to the development of subcutaneous nodules. We employed RNAseq to generate a transcriptome profile of rheumatoid subcutaneous nodules with the aim of identifying pathogenic mechanisms and genes differentially expressed in association with methotrexate therapy.

Methods: Eight subcutaneous nodules were obtained from 8 separate patients with rheumatoid arthritis as defined by ACR criteria, undergoing elective surgery for nodule removal. The patient cohort included 4 patients (3 females) grouped as never/previously (discontinued ≥ 25 months prior) receiving MTX therapy, and 4 patients (3 females) taking MTX (7.5 -20 mg/week) at the time of nodule removal. RNA was extracted using an RNeasy mini kit (Qiagen), including DNase digestion. All nodule RNA passed stringent criteria for purity and integrity (RIN >5.2). Libraries were prepared using a TruSep RNA library preparation kit (Illumina) and sequenced as 125-bp paired end reads on a HiSeq2000 platform. RNAseq read quality was assessed using FastQC, reads were aligned using STAR aligner, and gene count tables and FPKM values obtained using packages within StringTie. To better characterise the cell types responsible for gene expression differences in nodule tissues, we applied CIBERSORT to estimate cell type composition

Results: Immune genes expressed within nodules were identified using the Immunome database as reference. Immune gene set enrichment analysis highlighted cytokine signalling, neutrophil degranulation and IFNγ-signalling pathways as potential candidates for driving inflammation within nodules. Complement and its regulation and IL-4/IL-13 signalling also feature. RNAseq data further identified 10 genes differentially expressed in nodules (FDR < 0.05), associated with MTX therapy. The expression of two genes (ABI3 and EMCN) was validated using digital PCR; both show expression that is down-regulated (~2.3-fold and ~3.9-fold respectively) in association with MTX therapy. CIBERSORT application to estimate cell type composition within nodules was consistent with a macrophage-dominated granuloma, characterised by an inferred large fraction of M2 macrophages as well as M1 macrophages and monocytes. Nodules exposed to MTX harboured an inferred fraction of M0 macrophages (5-26% of leucocytes) that was absent from nodules lacking MTX exposure.

Conclusion: Cytokine signalling, particularly from IFNγ and IL-4/1L-13, are key to inflammatory pathways operating within nodule tissue. Genes differentially expressed within nodules in association with MTX therapy for RA are not directly part of immune/inflammatory mechanisms, implicating diverse pathogenic mechanisms. Nodules from patients with RA receiving MTX contain M0 macrophages suggesting macrophage depolarisation and/or local proliferation associated with MTX therapy.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/transcriptional-profiling-of-the-subcutaneous-rheumatoid-nodule-an-insight-into-pathogenic-mechanisms-and-cellular-content/