0067: Identification of Neutrophil Extracellular Traps in Explanted Human Lungs with Rheumatoid Arthritis-related Interstitial Lung Disease

Background/Purpose: Symptomatic rheumatoid arthritis-related interstitial lung disease (RA-ILD) is prevalent in about 10% of patients with RA and confers a higher mortality compared with RA patients without ILD. Anti-citrullinated protein antibodies (ACPAs) are strongly linked to RA-ILD, suggesting a pathogenic role for citrullinated proteins. Citrullinated histone H3 (Cit H3), a component of neutrophil extracellular traps (NETs), has been identified as a potential biomarker of RA-ILD. Our aim was to quantify and characterize Cit H3 expression in explanted lungs from patients undergoing lung transplant who have RA-ILD compared to patients with other connective tissue disease (CTD)-ILD or idiopathic pulmonary fibrosis (IPF).

Methods: Formalin fixed paraffin embedded explanted lung tissues were obtained from patients with RA-ILD (n=9), other CTD-ILD (n=4) or IPF (n=6). Deceased donor lungs without known pulmonary disease (HC, n=4) were used as controls. Using immunofluorescence, CD3+, CD20+, Cit H3+ and neutrophil elastase+ (NE) cells were quantified. Single cell RNA sequencing was completed on CD45+ immune cells sorted from 5 disaggregated RA-ILD lungs.

Results: Cit H3+ cells were identified in explanted lungs with RA-ILD, other CTD-ILD and IPF. A subset of patients in each group had higher Cit H3 density in lung tissue (Fig 1C). In the RA-ILD samples, a portion of the Cit H3+ cells were also NE+ indicating the presence of NETs. The average distance of Cit H3+ cells to the nearest CD3+ cells was shorter in RA-ILD (37 µm) vs HC (214 µm) (p=0.008) (Fig 2A). The closer proximity of Cit H3+ cells to CD3+ cells in RA-ILD was independent of the number of CD3+ or Cit H3+ cells present (Fig 2B-C). Single cell RNA sequencing from RA-ILD patients with Cit H3 high (n=3) vs Cit H3 low (n=2) samples revealed non-significant trends in T cell subsets (Fig 3). In the Cit H3 high vs low groups, there were higher percentages of T peripheral helper cells (median 13.2% vs 5.1%, p=0.4) and regulatory T cells (median 23.9% vs 7.3%, p=0.2) and lower percentages of helper 17 cells (median 11.8% vs 21.8%, p=0.4), T resident memory CD8+ T cells (median 7.3% vs 24.5%, p=0.2) and cytotoxic CD8+ T cells (median 4.8% vs 11.3%, p=0.4).

Conclusion: These findings suggest a role for NETs in RA-ILD, highlighting the potential interaction of T cells with NETs. Future direction includes single cell TCR sequencing and spatial transcriptomics to further explore the interplay between T cells and NETs and its impact on the pathogenesis of RA-ILD.