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.

Figure 1: Density of Cit H3+ Cells in RA-ILD, CTD-ILD and, IPF explanted lungs. A) Representative immunofluorescence image of an FFPE section from an RA-ILD explanted lungs to show Cit-H3 (yellow), CD3 (cyan) and CD20 (red). B) Representative density maps of RA-ILD and HC lungs. Maps were generated based on raw cell counts in a 300 µm radius. The blue outline is the tissue based on Hoescht thresholding. The green outline is an area of Cit H3 density with greater than 46 Cit H3+ cells/300 µm radius. C) Percentage of Cit H3 dense areas represented by the Cit H3 dense area (green outline) divided by the total area of the tissue (blue outline). Data are represented as median ±IQR. Kruskal-Wallis test, p values < 0.5 noted above columns.

Figure 2: Proximity of Cit H3+ and CD3+ cells in RA-ILD, CTD-ILD and, IPF explanted lungs. A) Average distances Cit H3 cells and the nearest CD3+ cell for HC, RA-ILD, CTD-ILD and IPF lungs. Data are represented as median ±IQR. Kruskal-Wallis test, p values < 0.5 noted above columns. Correlation of (B) relative abundance of CD3+ or (C) Cit H3+ cells (plotted as percentage of total cells) with average nearest distance between Cit H3+ and CD3+ cells. Simple linear regression, R squared and p values noted in tables below.

Figure 3: Frequency of T cell subsets based on single cell RNA sequencing of CD45+ immune cells from Cit H3 high and Cit H3 low RA-ILD lung explants. Percentages of T peripheral helper cells (Tph), regulatory T cells (Treg), T helper 17 (Th17) cells, T resident memory (TRM) CD8+cells and, cytotoxic CD8+ T cells in Cit H3 high (n=3) and Cit H3 low (n=2) RA-ILD lung explants. Data are represented as median ±IQR. Mann-Whitney test, p values < 0.5 noted above columns.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/identification-of-neutrophil-extracellular-traps-in-explanted-human-lungs-with-rheumatoid-arthritis-related-interstitial-lung-disease/