Abstract
Rheumatoid arthritis (RA)-associated lung disease is a leading cause of mortality in RA, yet the mechanisms linking lung disease and RA remain unknown. Using an established murine model of RA-associated lung disease combining collagen-induced arthritis (CIA) with organic dust extract (ODE)-induced airway inflammation, differences among lung immune cell populations were analyzed by single cell RNA-sequencing. Additionally, four lung myeloid-derived immune cell populations including macrophages, monocytes/macrophages, monocytes, and neutrophils were isolated by fluorescence cell sorting and gene expression was determined by NanoString analysis. Unsupervised clustering revealed 14 discrete clusters among Sham, CIA, ODE, and CIA+ODE treatment groups: 3 neutrophils (inflammatory, resident/transitional, autoreactive/suppressor), 5 macrophages (airspace, differentiating/recruited, recruited, resident/interstitial, and proliferative airspace), 2 T-cells (differentiating and effector), and a single cluster each of inflammatory monocytes, dendritic cells, B-cells and natural killer cells. Inflammatory monocytes, autoreactive/suppressor neutrophils, and recruited/differentiating macrophages were predominant with arthritis induction (CIA and CIA+ODE). By specific lung cell isolation, several interferon-related and autoimmune genes were disproportionately expressed among CIA and CIA+ODE (e.g. Oasl1, Oas2, Ifit3, Gbp2, Ifi44, and Zbp1), corresponding to RA and RA-associated lung disease. Monocytic myeloid-derived suppressor cells were reduced, while complement genes (e.g. C1s1 and Cfb) were uniquely increased in CIA+ODE mice across cell populations. Recruited and inflammatory macrophages/monocytes and neutrophils expressing interferon-, autoimmune-, and complement-related genes might contribute towards pro-fibrotic inflammatory lung responses following airborne biohazard exposures in setting of autoimmune arthritis and could be predictive and/or targeted to reduce disease burden.
Highlights
Several lung diseases have been associated with rheumatoid arthritis (RA), including interstitial lung disease (ILD), chronic obstructive pulmonary disease (COPD), pulmonary nodules, pleural effusions, bronchiolitis obliterans, and asthma [1,2,3]
Infiltration of recruited CCR2+ inflammatory monocytes was demonstrated in the collagen-induced arthritis (CIA) and organic dust extract (ODE) groups, and these cells were further potentiated in the CIA+ODE group (Fig 1E and 1F)
ScRNA-seq analysis was applied to whole lung immune cells from a mouse model of RA-associated inflammatory lung disease with key findings confirmed in sorted lung cell populations and NanoString analysis
Summary
Several lung diseases have been associated with rheumatoid arthritis (RA), including interstitial lung disease (ILD), chronic obstructive pulmonary disease (COPD), pulmonary nodules, pleural effusions, bronchiolitis obliterans, and asthma [1,2,3]. Evidence of RA-related autoantibodies generated in lung mucosa, even in the absence of articular manifestations of RA [6], as well as increased concentrations of serum anti-citrullinated protein antibody accompanying RA-related lung diseases [1, 4, 7], reinforces the pathogenic links between pulmonary inflammation and autoimmunity leading to the development of RA. We hypothesized that RA-associated lung disease is associated with unique cellular phenotypes and specific novel gene expression of in vivo exposed lungs. Leveraging this novel murine model, single-cell RNA sequencing (scRNA-seq) and unsupervised clustering were applied to lung immune cells among Sham, CIA, ODE, and CIA+ODE treatment groups to explore exposure-related differences in cellular subsets, transcriptional profiles, and associated biologic pathways. In separate complimentary studies to confirm key scRNA-seq findings, lung myeloid-derived cells (i.e. monocytes/macrophages and granulocytes) were isolated and subjected to gene-expression analysis
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