Background/Purpose: Idiopathic inflammatory myopathies (IIM), or myositis, comprise a group of autoimmune diseases with significant morbidity, yet the pathogenesis of myositis remains incompletely understood. In addition to inflammatory muscle disease, myositis can cause lung, vascular, and skin disease. A previous analysis of subjects with myositis revealed significant elevation of serum IL-9 (p=0.001) relative to healthy volunteers (Saygin et al., 2024). Therefore, we questioned whether IL-9 might directly promote disease pathology by modulating myocyte or immune cell function. We hypothesized that Th9 cells (IL-9+ secreting CD4+ T cells) infiltrate inflamed muscle tissue to promote inflammatory cell recruitment and autoantibody formation in IIM.

Methods: To determine potential cellular targets of high serum IL-9 in myositis, we first re-analyzed GSE220915 and GSE221091, two publicly available RNA-seq datasets on myositis patient muscle biopsies and dermatomyositis (DM) patient sorted immune cells, respectively. We quantified IL-9 receptor (IL-9R) mRNA levels as well as expression of SPI1, the master transcription factor of the Th9 cell lineage. Next, we cultured peripheral blood mononuclear cells (PBMCs) from pre-treatment DM patients and sex and age-matched healthy controls in vitro. Spectral flow cytometry was used to quantify IL-9R expression on B cells, T cells, and monocytes.

Results: In DM patients, immunomodulatory treatment, including oral prednisolone, methotrexate, azathioprine, and cyclophosphamide, reduced expression of SPI1 in CD4+ T cells (Figure 1A, GSE221091). IL9R was expressed in patient muscle biopsy tissue from many subtypes of IIM (Figure 1B, GSE220915). In sorted PBMCs, IL9R mRNA levels were higher in B and T cells from myositis patients compared to monocytes (Figure 1C, GSE221091). Furthermore, not only was IL9R mRNA increased, but IL-9R surface protein expression was significantly increased on circulating B cells, memory T cells, and CD4+ T cells in DM patients compared to healthy controls (Figure 2).

Conclusion: Treatment with immunomodulators reduces SPI1 expression in CD4+ T cells, suggesting involvement of the Th9/IL-9 axis in IIM. DM is also characterized by increased IL-9R surface expression on circulating T and B cells and within inflamed muscle, suggesting they may be key cellular targets of IL-9 signaling in myositis pathogenesis. Future studies will profile human muscle samples (biopsy/RNAscope and scRNA-seq) for Th9 cells and IL-9R-expressing populations. References: Saygin D, Biswas PS, Nouraie SM, Ren D, Moghadam-Kia S, McGeachy MJ, Oddis CV, Dzanko S, Ascherman DP, Aggarwal R. Serum cytokine profiles of adults with idiopathic inflammatory myopathies. Clin Exp Rheumatol. 2024 Feb;42(2):229-236. doi: 10.55563/clinexprheumatol/ipgoev. Epub 2024 Jan 5. PMID: 38179816.

Figure 1. (A) SPI1 expression (RPKM, reads per kilobase per million mapped reads), a marker of Th9 identity, was higher in CD4+ T cells from pre-treatment (Pre-Tx) myositis patients compared to myositis patients currently on medication (On-Tx). (B) IL-9R was expressed in muscle tissue from myositis patients. NT=normal tissue, IMNM=immune-mediated necrotizing myopathy, DM=dermatomyositis, IBM=inclusion-body myositis, AS=anti-synthetase syndrome. (C) IL-9R expression (RPKM) was highest in peripheral blood B cells from myositis patients. *p < 0.05, ***p < 0.001, ****p < 0.0001, Kolmogorov-Smirnov test.

Figure 2. Frequency of IL-9R expression on unstimulated monocytes, B cells, and T cells. p < 0.05, Mann-Whitney.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/immunophenotyping-reveals-upregulated-il-9r-on-circulating-t-and-b-cells-in-dermatomyositis/