Lung disease in relation to unique monocyte-macrophage subpopulations induced by combined inhalant endotoxin and collagen-induced arthritis

Lung disease is the most overrepresented cause of death in rheumatoid arthritis (RA). Animal studies have demonstrated potentiated autoimmunity, arthritis, and profibrotic/inflammatory lung disease with a combination of airborne exposures and collagen-induced arthritis (CIA), a model that recapitulates features of RA-associated interstitial lung disease (RA-ILD). As patients with RA-ILD demonstrate unique circulating monocyte subpopulations, this study aims to characterize lung infiltrating monocytes/macrophages in a mouse model of RA-ILD and determine whether reducing these cells mitigates the development of lung disease. Autoimmune-prone DBA/1J mice received intranasal inhalation of lipopolysaccharide (LPS) daily for up to 5 weeks and CIA induction. Experimental groups included Sham (saline injection/saline inhalation), CIA (CIA/saline), LPS (saline/LPS), and CIA+LPS (CIA/LPS). Lung disease was assessed by longitudinal imaging, lung function measurements, bronchoalveolar lavage fluid, lung tissues, and lung histopathology. Cell subpopulations were analyzed by single cell RNA-sequencing. Unsupervised clustering revealed 16 discrete clusters among the experimental groups with 2 robust clusters characterized as infiltrating inflammatory monocytes/macrophages for both CIA+LPS and CIA. The interaction of inhalation-induced airway inflammation and autoimmune arthritis results in lung disease associated with uniquely activated infiltrating inflammatory monocytes-macrophages that mediate adverse lung consequences. Whereas the induced monocyte/M? immunophenotype is more aligned to CIA than endotoxin exposure, co-exposure modeling renders unique features that potentially inform the pathogenesis and treatment of RA-ILD. Overall design: An established 5-week exposure model was utilized whereby mice were randomized to 1 of 4 experimental groups including Sham (saline injection/saline inhalation), LPS alone (saline injection/LPS inhalation), collagen-induced arthritis (CIA) alone (CIA induction/saline inhalation), and CIA+LPS co-exposure (CIA injection/LPS inhalation). DBA/1J mice (age 6-8 weeks) were purchased from Envigo and allowed to acclimate for one week prior to initiation of experiments. Male mice were utilized for all studies because we have previously demonstrated that female mice were profoundly less susceptible to development of lung disease in the setting of arthritis induction. CIA was induced as per the Chondrex protocol (Chondrex, Inc, Redmond, WA). Airway inflammation was induced using an established model of repetitive intranasal LPS inhalation. Under light sedation with isoflurane, mice received 100 ng of LPS from gram-negative Escherichia coli (O55:B5; Sigma, St. Louis, MO) in 50 µl of sterile saline or saline alone daily for up to 5 weeks (weekends excluded). Animals were euthanized 1 day after the final LPS exposure by isoflurane. 4-5 mice per treatment group were euthanized with whole lungs harvested after removal of blood from the pulmonary vasculature. Lungs were dissociated with gentleMACS Dissociator (Miltenyi Biotech, Gaithersburg (MD)) in a digestion solution (collagenase I, 0.2 µg/µl + DNase I, 75 U/ml + heparin, 1.5 U/ml, in Dulbecco's Modified Eagle's Media; DMEM) and incubated for 30 minutes at 37°C in a shaking incubator. Digestion solution activity was neutralized with PBS containing 4 mM EDTA. Red blood cells were lysed with 1 ml ammonium-chloride-potassium (ACK) lysis buffer (Quality Biological, Gaithersburg, MD) for 1 minute and neutralized with ice-cold DMEM (Gibco). Sample were then pooled by treatment group.