Combined Collagen-Induced Arthritis and Organic Dust-Induced Airway Inflammation to Model Inflammatory Lung Disease in Rheumatoid Arthritis

Abstract

Rheumatoid arthritis (RA) is characterized by extra-articular involvement including lung disease, yet the mechanisms linking the two conditions are poorly understood. The collagen-induced arthritis (CIA) model was combined with the organic dust extract (ODE) airway inflammatory model to assess bone/joint-lung inflammatory outcomes. DBA/1J mice were intranasally treated with saline or ODE daily for 5 weeks. CIA was induced on days 1 and 21. Treatment groups included sham (saline injection/saline inhalation), CIA (CIA/saline), ODE (saline/ODE), and CIA + ODE (CIA/ODE). Arthritis inflammatory scores, bones, bronchoalveolar lavage fluid, lung tissues, and serum were assessed. In DBA/1J male mice, arthritis was increased in CIA + ODE > CIA > ODE versus sham. Micro-computed tomography (µCT) demonstrated that loss of BMD and volume and deterioration of bone microarchitecture was greatest in CIA + ODE. However, ODE-induced airway neutrophil influx and inflammatory cytokine/chemokine levels in lavage fluids were increased in ODE > CIA + ODE versus sham. Activated lung CD11c⁺ CD11b⁺ macrophages were increased in ODE > CIA + ODE > CIA pattern, whereas lung hyaluronan, fibronectin, and amphiregulin levels were greatest in CIA + ODE. Serum autoantibody and inflammatory marker concentrations varied among experimental groups. Compared with male mice, female mice showed less articular and pulmonary disease. The interaction of inhalation-induced airway inflammation and arthritis induction resulted in compartmentalized responses with the greatest degree of arthritis and bone loss in male mice with combined exposures. Data also support suppression of the lung inflammatory response, but increases in extracellular matrix protein deposition/interstitial disease in the setting of arthritis. This coexposure model could be exploited to better understand and treat RA-lung disease. © 2019 American Society for Bone and Mineral Research.

Keywords: AUTOIMMUNITY; COEXPOSURE; INFLAMMATION; INTERSTITIAL LUNG DISEASE; RHEUMATOID ARTHRITIS.

Figure 1.. Organic dust extract (ODE) exposure potentiates collagen-induced arthritis (CIA) and bone/joint disease in male mice.

A panel, Line graph depicts mean with standard error bars of arthritis inflammatory score at respective time points from treatment groups. Statistical difference versus sham denoted as “a” (p<0.05); versus ODE denoted as “b” (p<0.05). N=9 CIA mice and N=10 Sham, ODE, CIA+ODE mice/group from 2 independent studies. B panel, A representative 3D reconstructed image from region of interest of proximal tibia from one mouse per treatment group. Note the loss trabecular bone in the CIA and further loss in the CIA+ODE treatment groups as compared to sham. C panel, Representative image from each treatment group of ankle sections stained for collagen using Masson’s Trichrome and thresholding performed. Green arrows indicate areas of a normal joint tissue with increased presence of collagen. Red arrows indicate areas of articular cartilage loss in the CIA, ODE, and CIA+ODE groups compared to controls. A significant decrease (***p<0.001 vs. sham) in collagen observed when images converted to pixels using ImageJ (D panel).

Figure 2.. ODE-induced airway inflammatory cell influx and mediator release is reduced in the setting of collagen-induced arthritis (CIA) in male mice.

Bar graphs depict mean with standard error bars of cells and mediators from treatment groups. A panel, Total cells, neutrophils, macrophages, and lymphocytes enumerated from bronchoalveolar lavage fluid (BALF). Eosinophils were not detected. B panel, Tumor necrosis factor (TNF)-α, interleukin (IL)-6, murine neutrophils chemoattractants (CXCL1 and CXCL2), and amphiregulin (AREG) levels determined by ELISA. N=9 CIA mice and N=10 Sham, ODE, CIA+ODE mice/group from 2 independent studies. C panel, Numbers of neutrophils (Ly6G⁺), alveolar macrophages (CD11c^hiCD11b^lo), exudative macrophages (CD11c^hiCD11b^hi), B cells (CD19⁺), and B1 (CD19⁺CD11b⁺), B1a (CD19⁺CD11b⁺CD5⁺) and B2 (CD19⁺CD11b^-) B cell subsets from ½ whole lung tissue cells were calculated by multiplying the %cells in respective gate (%CD45⁺ cells, as analyzed by FACS) multiplied by respective total cells for each mouse. N=4 CIA mice. N=5 Sham, ODE, CIA+ODE mice/group. Statistical difference (*p<0.05, **p<0.01, ***p<0.001) versus sham or as indicated.

Figure 3.. ODE-induced lung pathology is altered in the setting of collagen-induced arthritis (CIA) in male mice.

All bar graphs depict mean with standard error bars. A panel, A representative (4–5 μm thick) H&E stained lung section of one mouse per treatment group. B panel, ODE-induced cellular aggregate numbers and semi-quantitative inflammatory scores of alveolar and bronchiolar compartment inflammation. N=9 CIA mice and N=10 Sham, ODE, CIA+ODE mice/group from 2 independent studies. C panel, A representative Masson’s Trichrome (collagen=blue) stained section for each treatment group. D panel, Increased collagen staining in ODE and CIA+ODE when images converted to pixels using imageJ analysis (N=5 images/mouse; 4 mice/group). E panel, Levels of hyaluronan, fibronectin, and amphiregulin (AREG) from left side lung homogenates shown. N=5 mice/group. Statistically significant (*p<0.05, **p<0.01, ***p<0.001) versus saline or as indicated. Images shown at 10× magnification with line scale of 100μm. ODE -induced lung parenchymal cellular aggregates and alveolar compartment inflammation were reduced in the setting of arthritis. However, collagen, lung hyaluronan, fibronectin, and amphiregulin remained elevated with combined CIA+ODE exposures.

Figure 4.. Serum levels of biomarkers, autoreactive IgG antibodies, and immunoglobulins varied by treatment group in male mice.

Bar graphs depict mean with standard error bars. Serum levels of the acute phase reactant protein pentraxin-2, cyclic citrullinated peptide IgG antibody, collagen type II IgG antibody (CII), and anti-MAA IgG antibody, IgG, IgM, and IgA as determined by ELISA. Statistical difference versus sham (*p<5, **p<6, **p<7) and between groups as noted by lines. N=9 CIA mice and N=10 Sham, ODE, CIA=ODE mice/group from 2 independent studies.