Therapeutic Single-Cell Landscape: Methotrexate Exacerbates Interstitial Lung Disease by Compromising the Stemness of Alveolar Epithelial Cells under Systemic Inflammation

Interstitial lung disease (ILD) poses a serious threat in patients with rheumatoid arthritis (RA). However, the impact of cornerstone drugs, including methotrexate (MTX) and TNF blockade, on RA-associated ILD (RA-ILD) remains controversial. Our study using an SKG mouse model and single-cell transcriptomics revealed that MTX exacerbates pulmonary inflammation by promoting immune cell infiltration, Th17 activation, and fibrosis. In contrast, TNF blockade ameliorates these features and inhibits ILD progression. Analysis of data from a human RA-ILD cohort revealed that patients with ILD progression had persistently higher systemic inflammation than those without progression, particularly among the subgroup undergoing MTX treatment. These findings highlight the need for personalized therapeutic approaches in RA-ILD, given the divergent outcomes of MTX and TNF blockade. At 8-10 weeks of age, male SKG mice received a single intraperitoneal injection of zymosan from Saccharomyces cerevisiae (7.5 mg per 20 g; MilliporeSigma, St. Louis, MO, USA) to induce arthritis and pneumonitis. We then gave them twice-weekly intraperitoneal injections of PBS, MTX (7.5 mg/kg; MilliporeSigma), or TNFα inhibitor (100 μg/kg; R&D Systems, Inc., Minneapolis, MN, USA).