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American Journal of Physiology - Lung Cellular and Molecular Physiology logoLink to American Journal of Physiology - Lung Cellular and Molecular Physiology
. 2018 Mar 15;314(6):L998–L1009. doi: 10.1152/ajplung.00304.2017

Nintedanib reduces pulmonary fibrosis in a model of rheumatoid arthritis-associated interstitial lung disease

Elizabeth F Redente 1,2,3,✉, Martin A Aguilar 1, Bart P Black 1, Benjamin L Edelman 1, Ali N Bahadur 4, Stephen M Humphries 5, David A Lynch 5, Lutz Wollin 6, David W H Riches 1,2,3,7,8
PMCID: PMC6335004  PMID: 29543042

Abstract

Rheumatoid arthritis (RA)-associated interstitial lung disease (RA-ILD) develops in ~20% of patients with RA. SKG mice, which are genetically prone to development of autoimmune arthritis, develop a pulmonary interstitial pneumonia that resembles human cellular and fibrotic nonspecific interstitial pneumonia. Nintedanib, a tyrosine kinase inhibitor approved for treatment of idiopathic pulmonary fibrosis, has been shown to reduce the decline in lung function. Therefore, we investigated the effect of nintedanib on development of pulmonary fibrosis and joint disease in female SKG mice with arthritis induced by intraperitoneal injection of zymosan (5 mg). Nintedanib (60 mg·kg−1·day−1 via oral gavage) was started 5 or 10 wk after injection of zymosan. Arthritis and lung fibrosis outcome measures were assessed after 6 wk of treatment with nintedanib. A significant reduction in lung collagen levels, determined by measuring hydroxyproline levels and staining for collagen, was observed after 6 wk in nintedanib-treated mice with established arthritis and lung disease. Early intervention with nintedanib significantly reduced development of arthritis based on joint assessment and high-resolution μ-CT. This study impacts the RA and ILD fields by facilitating identification of a therapeutic treatment that may improve both diseases. As this model replicates the characteristics of RA-ILD, the results may be translatable to the human disease.

Keywords: inflammation, interstitial lung disease, rheumatoid arthritis, SKG mice

INTRODUCTION

Rheumatoid arthritis (RA) affects ~1% of the population in developed countries and is more prevalent in women (1). While RA primarily affects the small joints of the hands, recent estimates suggest that over half of all patients with RA will develop some form of extra-articular pulmonary manifestation during their lifetime (5, 8, 20). In addition, ~30% of RA patients who have lung involvement develop RA-associated interstitial lung disease (RA-ILD), a serious diffuse parenchymal lung disease associated with impaired gas exchange, fibrotic injury of the alveolar septae, and reduced life expectancy (6, 10, 26). The most common histological patterns of RA-ILD are usual interstitial pneumonia and nonspecific interstitial pneumonia (21). The appearance of pulmonary disease is a major contributor to morbidity and mortality (38). Little is known about the mechanisms underlying the pathogenesis of RA-ILD or how to treat it. Patients with RA-ILD have been shown to have high titers of rheumatoid factor and anti-cyclic citrullinated peptide antibodies, which have been identified as risk factors for the development of ILD in patients with RA (19, 29, 44, 46). Treatment options for RA-ILD include disease-modifying antirheumatoid drugs, such as methotrexate and leflunomide, and biological anti-TNFα therapies, such as etanercept. For several of these treatments, case report evidence suggests that, while beneficial to joint disease, these treatments may exacerbate pulmonary dysfunction (4, 16, 25, 28, 30). The American College of Rheumatology, therefore, does not recommend the use of methotrexate in patients with RA and established ILD (36). A need remains for prospective randomized trials in patients with RA-ILD to determine the risk factors associated with current therapeutic practices and to evolve treatment options.

To address questions about therapeutic interventions in RA-ILD, we established a model in arthritis-prone female SKG mice (17, 18). The model reproduces many of the manifestations of RA-ILD and includes penetrance of both joint disease (100%) and lung disease (20%). The development of lung histopathological and leukocyte infiltration patterns is similar to cellular nonspecific interstitial pneumonia in humans, while pulmonary fibrosis, along with circulating autoantibodies directed against citrullinated epitopes, develops in a subset of affected mice (18). Given the authenticity of the model, we sought to determine the efficacy of nintedanib, an approved therapy for the ILD in idiopathic pulmonary fibrosis (IPF) (33, 35), in RA-ILD.

Nintedanib is a potent tyrosine kinase inhibitor targeting FGF receptors-1, -2, and -3, PDGF receptor-α/β, VEGF receptors-1, -2, and -3, Flt-3, and non-receptor tyrosine kinases, including Src, Lyn, and Lck (11). The antifibrotic activity of nintedanib has been confirmed in primary lung fibroblasts from patients with IPF and in dermal fibroblasts from patients with systemic sclerosis; nintedanib inhibited growth factor-stimulated migration and proliferation of fibroblasts and attenuated markers of TGFβ-induced transformation to myofibroblasts (12, 13, 40). The in vivo efficacy of nintedanib has been explored in silica-induced lung fibrosis in mice and bleomycin-induced lung fibrosis in mice and rats (39, 40). Nintedanib, administered orally in a preventive or therapeutic approach, exerted significant anti-inflammatory and antifibrotic activity. Nintedanib reduced the number of lymphocytes in bronchoalveolar lavage (BAL) fluid, diminished levels of inflammatory mediators (IL-1β and CXCL1/KC) and the percentage of myeloid dendritic cells in lung tissue, and reduced pulmonary inflammation and granuloma formation (39). Furthermore, nintedanib suppressed transcript levels of fibrosis-related genes (procollagen 1 and TGFβ1) and total collagen levels and reduced the fibrotic score in histomorphometric analyses of fibrotic lungs (39, 40). The ability of nintedanib to inhibit bleomycin-induced skin fibrosis, graft vs. host disease-induced skin fibrosis in mice, and skin fibrosis in tight-skin mice transgenic for the fibrillin-1 gene has also been investigated. Nintedanib reduced fibrotic skin thickening, skin collagen content, and myofibroblast numbers in the skin in all three models (13, 14, 29).

The objective of this study was to investigate the effect of nintedanib on the development of pulmonary fibrosis and joint disease in SKG mice with zymosan-induced arthritis.

MATERIALS AND METHODS

Animals and induction of arthritis and pulmonary disease.

Female SKG mice (37) received a single intraperitoneal injection of zymosan (5 mg; Sigma-Aldrich, St. Louis, MO) at 8–10 wk of age to induce arthritis and ILD, as described previously (43). All studies were approved by the National Jewish Health Institutional Animal Care and Use Committee. The experiments were powered so that 50 mice were injected for each experimental treatment condition to account for the 20% penetrance of lung disease.

Treatment of animals with nintedanib.

Nintedanib treatment (60 mg/kg dissolved in saline, delivered daily by oral gavage for 6 wk) was started 5 or 10 wk after injection of zymosan. The control mice received oral gavage of saline.

Assessment of lung disease.

Lung mechanics and quasi-static compliance (Cst) were assessed using the flexiVent small-animal ventilator (SCIREQ, Montreal, QC, Canada) (17, 31). Changes in lung collagen were quantified by measurement of hydroxyproline levels in the upper right lobe following homogenization in PBS and hydrolysis in an equal volume of 12 N HCl for 8 h at 120°C, as described previously (32). The left lung was inflated with 10% formalin solution and embedded in paraffin, and 5-μm sections were prepared (32). Serial sections from three animals with a hydroxyproline score within 1 SD of the mean per group were stained with hematoxylin and eosin and picrosirius red (PSR) and examined with an upright light microscope (model BX51, Olympus). Lung sections were immunostained for the pan-(myo)fibroblast marker α-smooth muscle actin (α-SMA, 1:1,000 dilution; Sigma Aldrich) and assessed using semiquantitative stereology grid-counting techniques, as previously described (31). Ten animals per group and 10 images per animal were assessed in a blinded manner.

Assessment of joint disease.

Arthritis scores were determined weekly, as described previously (17, 18, 37). Joint swelling was monitored by visual inspection each week and scored as follows: 0, no joint swelling; 0.1, swelling of one finger joint; 0.5, mild swelling of the wrist or ankle; 0.75 moderate swelling of the wrist or ankle; 1.0, severe swelling of the wrist or ankle. Scores for all fingers of fore- and hindpaws, wrists, and ankles were totaled for each mouse. Arthritis was observed and scored by three separate individuals who were blinded to the treatment groups. Hind joints with an average arthritis score equal to the reported mean score [n = 10 mice/group; early treatment group (5–11 wk after zymosan injection)] were scanned at 9 μm using μ-CT (Skyscan 1176, Bruker MicroCT, Kontich, Belgium) and reconstructed into cross-sectional images with an isotropic voxel size of 9 μm. Acquisition parameters for μ-CT were as follows: 50-kV X-ray tube voltage, 500-μA current, and 900-ms exposure time, with 0.5-mm aluminum filter and 0.4° rotation step. Images were thresholded using a consistent value (pixel intensity 75) to identify bone in image volumes. Talus and calcaneus bones were manually segmented from thresholded regions in each volume using open source software (45). Bone volume was computed by multiplication of the number of voxels in each segmentation region by voxel volume.

Isolation of cells by BAL and enzymatic lung dispersal.

BAL was conducted as described elsewhere (7, 31). Briefly, the lungs were lavaged three times with 1 ml of PBS containing 0.6 mM EDTA through the attached endotracheal tube. BAL fluid from 20 samples per treatment group and 10 controls was analyzed for 42 molecules by multiplex ELISA [Rodent MAP 4.0, Ampersand Biosciences, Saranac Lake, NY (https://www.ampersandbio.com/services/rodent-map-4-0-mouse-sample-testing/)]. The lower right lobe was minced with scissors and digested in 1 mg/ml collagenase (Sigma Aldrich) for 30 min at 37°C. The digested lung was passed through an 18-gauge needle and filtered through a 70-μm nylon filter to create a single-cell suspension.

Flow cytometry.

Single-cell suspensions in PBS containing 1% (wt/vol) BSA [fluorescence-activated cell-sorting (FACS) buffer] were incubated with mouse Fc block (BD PharMingen, San Diego, CA) for 30 min and then washed with FACS buffer. Leukocytes were identified (Table 1) with fluorescently tagged monoclonal antibodies directed against CD3, CD4, CD8, B220, CD11c, F4/80, Ly6G, Ly6C, and MHCII (eBiosciences, San Diego, CA) and CD11b (BD PharMingen) in FACS buffer for 1 h. Cells were washed and then fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA). Data were acquired with a flow cytometer (model LSR II, BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR), as described previously (17, 32).

Table 1.

Cell surface markers used in identification and classification of myeloid and lymphoid subsets by flow cytometry

Subsets Cell Surface Markers
Myeloid
Tissue monocytes CD11c−/intCD11b+Ly6C+/−MHCII+/−
Alveolar macrophages CD11c+CD11b−F4/80+
Interstitial/tissue macrophages CD11c−CD11b+F4/80+
Inflammatory macrophages CD11cvarCD11b+F4/80+
CD103+ dendritic cells CD11c+CD11b−MHCII+CD103+
CD11b+ dendritic cells CD11c+CD11b+MHCII+CD103−
Neutrophils CD11c−CD11b+Ly6G+F480−
Lymphocyte
CD4 T cells CD3+CD4+
CD8 T cells CD3+CD8+
B cells CD3−B220+
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Statistics.

Parametric data are means ± SE. Differences between conditions at specific time points were examined using Student’s unpaired t-test; P < 0.05 was considered significant. Nonparametric data are represented as scatter plots and bar graphs of the median and interquartile range and analyzed using the Mann-Whitney t-test; P < 0.05 was considered significant.

RESULTS

Effect of nintedanib on established joint swelling and interstitial pneumonia in SKG mice.

In this model, joint swelling commences 2–3 wk after intraperitoneal injection of zymosan and is detected in >90% of mice (17, 18, 43); interstitial pneumonia develops in ~20% of mice by 10 wk after zymosan injection (17, 18). To account for the low penetrance of lung disease in this model, 50 animals were assessed per treatment group.

To determine the effect of nintedanib on established joint and pulmonary disease, mice received a daily oral gavage of nintedanib or saline beginning 10 wk after zymosan injection (Fig. 1A). There was no significant change in body weight or percent survival in nintedanib- compared with saline-treated mice at week 16 (Fig. 1, B and C). Joint swelling, assessed by visual arthritis score, was first detected 2–3 wk after zymosan injection and gradually increased before peaking in severity 6–10 wk after zymosan injection. Thereafter, the severity of joint swelling declined but remained elevated in mice 16 wk after zymosan injection. Administration of nintedanib starting at week 10 did not increase the resolution of joint swelling compared with saline-treated animals, except for a significant reduction at week 16 (P < 0.001; Fig. 1D).

Fig. 1.

Fig. 1.

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Late treatment with nintedanib did not alter established arthritis in SKG mice. A: daily oral gavage of mice with nintedanib or saline beginning 10 wk after zymosan injection. B and C: there was no significant change in weight or survival in nintedanib- compared with saline-treated mice. D: visual arthritis score was not significantly different in nintedanib- compared with saline-treated mice until week 16. Values are means ± SE; n = 50 mice per treatment group. ***P < 0.001.

We observed a modest, but statistically significant, decline in Cst in zymosan-injected SKG mice compared with saline-injected controls but no significant improvement in Cst in nintedanib-treated animals (Fig. 2A) (17). Analysis of collagen levels in the lungs by measurement of hydroxyproline revealed that daily treatment with nintedanib for 6 wk led to a significant reduction (P < 0.001) in established lung fibrosis. Lung hydroxyproline levels in nintedanib-treated mice were not significantly different from control (non-zymosan-treated) mice (Fig. 2B).

Fig. 2.

Fig. 2.

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Late treatment with nintedanib did not alter lung compliance but significantly reduced collagen levels in the lungs. A: quasi-static compliance was significantly reduced in zymosan-injected, saline-treated mice compared with naïve controls. Treatment with nintedanib did not improve Cst. B: hydroxyproline levels were significantly reduced in nintedanib- compared with saline-treated mice and were not different from naïve controls. Data were obtained at week 16 and are shown as median ± interquartile range (n = 50 mice per treatment group and 10 naïve controls). **P < 0.01; ***P < 0.001.

Lung histology of animals harvested 16 wk after zymosan injection showed a characteristic pattern of patchy subpleural and peribronchovascular inflammatory cell infiltration in nintedanib- and saline-treated mice. Saline-treated animals had increased collagen in the lungs, observed as red in PSR-stained sections, which appeared to form bands between the airways and vasculature running through the parenchyma (Fig. 3). This was reduced in the cohort of mice treated with nintedanib and absent in nondiseased control mice (Fig. 3). Staining and semiquantitation of lung tissue for the pan-(myo)fibroblast marker α-SMA showed a significant reduction of (myo)fibroblasts after nintedanib (P = 0.043) compared with saline treatment (Fig. 4). Percentage of α-SMA staining in mice that received nintedanib was not significantly different from that in naïve control mice (P = 0.757; Fig. 4).

Fig. 3.

Fig. 3.

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Collagen was not increased in lungs harvested from nintedanib-treated mice at week 16. Hematoxylin-eosin (H&E)-stained lung sections from 3 representative animals demonstrate similar patchy areas of inflammation and disease. Picrosirius red (PSR)-stained serial sections show decreased collagen (red) in nintedanib- compared with saline-treated animals. Final magnification ×200, except insets (×300).

Fig. 4.

Fig. 4.

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Late nintedanib treatment significantly reduced the presence of α-smooth muscle actin (SMA)-positive (myo)fibroblasts, as shown by immunostaining (final magnification ×400) and semiquantitative analysis of α-SMA-positive cells. Data were obtained at week 16 and are shown as median ± interquartile range (n = 10 mice per group). *P < 0.01.

Effect of nintedanib on established pulmonary inflammation in SKG mice.

Nintedanib has been shown to decrease lung inflammation in the bleomycin model of pulmonary fibrosis (39). We sought to determine if a similar effect occurred in our SKG model. Similar to the bleomycin model, overall, there was no significant difference in the total number of BAL cells (P = 0.222; data not shown) or alveolar macrophages (P = 0.744) in nintedanib- compared with saline-treated animals (Fig. 5A). However, a small, but significant, elevation in the number of lymphocytes and neutrophils was detected in the nintedanib-treated cohort (P = 0.002 and P = 0.022, respectively) in the SKG model of lung disease (Fig. 5A). Analysis of the pro- and anti-inflammatory cytokine signature in the BAL fluid showed that, of the 42 inflammatory molecules measured, TNF-α, IL-4, and KC were significantly increased, while VCAM-1 was significantly reduced, in nintedanib- compared with saline-treated zymosan-injected mice, (Fig. 5, B–E).

Fig. 5.

Fig. 5.

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Late treatment with nintedanib altered bronchoalveolar lavage (BAL) cell and fluid composition. A: differential counts of BAL cells showed no difference in the number of macrophages (Mac) but a significant increase in the number of lymphocytes (Lymph) and neutrophils (Neuts). B–D: BAL fluid levels of TNF-α, IL-4, and KC were increased in nintedanib- compared with saline-treated mice. E: levels of VCAM-1 were significantly reduced after nintedanib treatment. F: IFN-γ was not significantly changed after nintedanib treatment. Data were obtained at week 16 and are shown as median ± interquartile range (n = 20 mice per treatment group and 10 controls). *P < 0.05; **P < 0.01.

Flow cytometry provided a more in-depth assessment of inflammatory cell subpopulations (Table 1) in digested whole lung tissue (Fig. 6). The total number of nucleated cells isolated from lung tissue of nintedanib- and saline-treated mice at week 16 was not significantly different (Fig. 6A). Similarly, there was no significant difference in numbers of CD4+ T cells, CD8+ T cells, and B220+ B cells in nintedanib- compared with saline-treated mice (Fig. 6, B–D). Likewise, the number of CD11c−CD11b+F4/80+ (tissue/interstitial) macrophages, CD11c+CD11b+F4/80+ (alveolar) macrophages, CD11b+ dendritic cells, CD103+ dendritic cells, Ly6C+ monocytes, and Ly6G+ neutrophils was not significantly different in nintedanib- compared with saline-treated mice at week 16 (Fig. 6, E and G–K). However, the number of CD11cvarCD11b+F4/80+ (inflammatory) macrophages was significantly elevated in nintedanib- compared with saline-treated mice at week 16 (P < 0.05; Fig. 6F).

Fig. 6.

Fig. 6.

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Inflammatory cell subsets in lungs of SKG mice after late treatment with nintedanib. Animals were treated with nintedanib or saline for 6 wk, and lung digests were analyzed by flow cytometry analysis at week 16. A: total cells; B: CD4+ T cells; C: CD8+ T cells; D: B220+ B cells; E: tissue/interstitial macrophages; F: inflammatory macrophages; G: alveolar macrophages; H: monocytes; I: CD11b+ dendritic cells; J: CD103c+ dendritic cells; K: neutrophils. Values are means ± SE; n = 50 mice per treatment group. *P < 0.05.

Effect of nintedanib on development of joint swelling and interstitial pneumonia in SKG mice.

To determine if earlier intervention with nintedanib would alter the development of arthritis and interstitial pneumonia, SKG mice were treated with nintedanib or saline for 6 wk starting 5 wk after zymosan injection (Fig. 7A). As in the experiments described above, joint swelling was scored weekly and the lungs were analyzed in an identical fashion for the same parameters defined in the experiments in which arthritis in lung disease had been established.

Fig. 7.

Fig. 7.

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Early treatment with nintedanib reduced development of arthritis in SKG mice. A: daily oral gavage of mice with nintedanib or saline beginning 5 wk after zymosan injection. B and C: there was no significant change in weight or survival in nintedanib- compared with saline-treated mice. D: visual arthritis score declined significantly beginning 1 wk after nintedanib treatment compared with saline-treated animals. Values are means ± SE; n = 50 mice per treatment group. ***P < 0.001.

Nintedanib had no effect on weight or percent survival compared with saline (Fig. 7, B and C). However, compared with saline treatment, nintedanib led to reversal of joint swelling that became most apparent after the mice had been receiving nintedanib for 1 wk. Thereafter, the arthritis score in nintedanib-treated mice began to decline progressively over the ensuing 5 wk (Fig. 7D). In contrast, the arthritis score progressively increased in saline-treated mice (Fig. 7D). To further assess the effect of nintedanib on the development of joint disease, the hind talus and calcaneus bones were scanned using small-animal μ-CT and analyzed for bone volume and remodeling (Fig. 8). Volume of the talus and calcaneus bones was significantly greater in nintedanib- than vehicle-treated mice (Fig. 8, A and B). Three-dimensional visualization of the talus and calcaneus bones showed differences in the surface of the bone in nintedanib- compared with saline-treated mice (Fig. 8, C and D). The difference was less apparent when nintedanib-treated animals were compared with a control (non-zymosan-injected) animal (Fig. 8, D and E).

Fig. 8.

Fig. 8.

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Early treatment with nintedanib reduced bone loss and remodeling. A and B: volume of hind talus and calcaneus bones was significantly greater after early treatment with nintedanib than saline. Data were obtained at week 10. Values are means ± SE; n = 10 mice per treatment group. *P < 0.05. C and D: bone surface as visualized by 3D reconstruction of talus and calcaneus bones from saline- and nintedanib-treated mice. Bones of saline-treated mice were irregular and deformed, suggesting extensive erosion, while bones of nintedanib-treated mice were almost normal. Representative saline- and nintedanib-treated mice had arthritis scores equal to the reported means of 4.5 and 1.7, respectively, at week 10 (n = 10; Fig. 4D). E: 3D reconstruction of talus and calcaneus bones from a naïve SKG mouse from a similar μ-CT image volume that was acquired at somewhat lower (18-μm) resolution.

Also, at week 16, Cst was slightly, but significantly, decreased in zymosan-injected SKG mice compared with saline-injected control mice (Fig. 9A). Nintedanib treatment did not result in an improvement of Cst (Fig. 9A). The median lung hydroxyproline level in saline-treated mice was similar to that in control mice (Fig. 9B) and to historic data measured at the same time point (17). However, in contrast to the late treatment data, daily treatment with nintedanib for 6 wk had no effect on collagen levels compared with mice receiving saline (P = 0.375). Figure 9B reveals a similar and expected wide scatter in hydroxyproline levels in individual mice.

Fig. 9.

Fig. 9.

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Early treatment with nintedanib during disease development did not improve static lung compliance and did not change collagen levels in the lungs. A: quasi-static compliance was not altered in zymosan-injected, saline- compared with nintedanib-treated mice. B: hydroxyproline levels (a measure of collagen) were not significantly altered among all cohorts of mice. Data were obtained at week 10 and are shown as median ± interquartile range; n = 50 mice per treatment group and 10 naïve controls. *P < 0.05.

Similar to the results obtained in animals harvested 16 wk after zymosan injection, examination of lung histology revealed the expected pattern of patchy inflammatory cell infiltration in the bronchovascular bundles and surrounding tissues as well as in subpleural areas without large areas of excessive collagen deposition (Fig. 10). Gross examination of the sections did not indicate substantial differences between nintedanib- and saline-treated mice. Semiquantitative assessment of immunostaining for (myo)fibroblasts using α-SMA showed an increase in α-SMA-positive cells after zymosan treatment compared with control animals but no significant difference after nintedanib treatment (P = 0.719; Fig. 11).

Fig. 10.

Fig. 10.

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Early treatment with nintedanib did not alter histological appearance of the lungs. Hematoxylin-eosin (H&E)-stained lung sections from 3 representative animals demonstrated similar patchy areas of inflammation and disease. Picrosirius red (PSR)-stained serial sections showed similar amounts of collagen (red) in nintedanib- and saline-treated mice. Final magnification ×200, except insets (×300).

Fig. 11.

Fig. 11.

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Early treatment with nintedanib did not significantly reduce the presence of α-smooth muscle actin (SMA)-positive (myo)fibroblasts, as shown by immunostaining and semiquantitative analysis of α-SMA-positive cells. Data were obtained at week 10 and are shown as median ± interquartile range (n = 10 mice per group). Final magnification ×400.

Effect of nintedanib on development of pulmonary inflammation in SKG mice.

Analysis of total and differential cell counts in BAL fluid showed no significant difference in the total number of recovered BAL cells (P = 0.303; data not shown) or in the number of macrophages, lymphocytes, or neutrophils in nintedanib- compared with saline-treated mice (Fig. 12A). Analysis of the pro- and anti-inflammatory cytokine signature in the BAL fluid showed that nintedanib treatment significantly reduced the levels of IL-1β and KC compared with saline-treated mice (Fig. 12, B–F).

Fig. 12.

Fig. 12.

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Early treatment with nintedanib did not alter bronchoalveloar lavage (BAL) cell counts or fluid composition. A: differential counts of BAL cells showed no difference in the number of macrophages (Mac), lymphocytes (Lymph), or neutrophils (Neuts). B, C, and E: there was no difference in levels of TNF-α, IL-4, or VCAM-1. D and F: KC and IL-1β were significantly reduced in nintedanib- compared with saline-treated mice. Data were obtained at week 10 and are shown as median ± interquartile range (n = 20 mice per treatment group and 10 naïve controls). *P < 0.05.

The total number of cells isolated from lungs of nintedanib- and saline-treated mice was not significantly different (Fig. 13A). The number of CD4+ and CD8+ T cells was also similar between treatment groups (Fig. 13, B and C), although the number of B220+ B cells was modestly elevated in nintedanib-treated mice and approached significance (P = 0.0506; Fig. 13D). Numbers of CD11c−CD11b+F4/80+ (tissue/interstitial) macrophages, CD11CvarCD11b+F4/80+ (inflammatory) macrophages, CD11C+CD11b−F4/80+ (alveolar) macrophages, and CD11b+ dendritic cells were not significantly different between treatment groups (Fig. 13, E–G and I). In contrast, the numbers of CD103+ dendritic cells and Ly6G+ neutrophils were significantly increased in nintedanib- compared with saline-treated mice (Fig. 13, J and K), whereas the number of Ly6C+ monocytes was significantly lower in response to treatment with nintedanib (Fig. 13H).

Fig. 13.

Fig. 13.

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Inflammatory cell subsets in lungs of SKG mice after early treatment with nintedanib. Animals were treated with nintedanib or saline for 6 wk, and lung digests were analyzed by flow cytometry at week 10. A: total cells; B: CD4+ T cells; C: CD8+ T cells; D: B220+ B cells; E: tissue/interstitial macrophages; F: inflammatory macrophages; G: alveolar macrophages; H: monocytes; I: CD11b+ dendritic cells; J: CD103c+ dendritic cells; K: neutrophils. Values are means ± SE; n = 50 mice per treatment group. *P < 0.05; ***P < 0.001.

DISCUSSION

The goal of this study was to conduct a preclinical evaluation of nintedanib, an antifibrotic therapy approved for treatment of IPF, in a well-defined murine model of RA-ILD. Homozygous SKG mice harbor a spontaneous point mutation in the second SH2 domain of ZAP70, resulting in hypomorphic ZAP70 function in T cells (37). Reduced ZAP70 function leads to impaired positive and negative CD4+ T cell selection in the thymus, which results in escape of self-reactive CD4+ T cells into the periphery (37). In mice residing under specific-pathogen-free conditions, intraperitoneal injection of the innate β-glucan agonist zymosan activates this pathway, leading to development of an RA-ILD clinical phenotype (17, 43).

When mice with established lung disease were treated daily with nintedanib for 6 wk starting at week 10, fibrosis (measured by lung hydroxyproline levels) at week 16 was significantly reduced. However, lung compliance, measured as Cst, was not increased by nintedanib treatment. This discordance between decreased hydroxyproline levels and no significant increase in compliance may be due to several factors, including our ability to detect the effects of nintedanib treatment on lung disease due to the heterogeneity of the model system or due to the inflammation that was still present in the lungs. Interestingly, while the total number of inflammatory cells remained unchanged, the proportions of certain subpopulations were altered. The numbers of BAL neutrophils and lymphocytes, along with lung tissue inflammatory macrophages, were increased in nintedanib-treated mice exhibiting reduced fibrosis. Not all inflammation is detrimental, and our previous work in the bleomycin-induced fibrosis model indicates that some types of inflammation may be beneficial (32). In this case, exogenous administration of TNFα accelerated the resolution of bleomycin-induced fibrosis, resulting in a reduction in α-SMA-positive (myo)fibroblasts in the lungs (unpublished data). Similar results were observed at the late time point after nintedanib treatment, when significantly elevated levels of TNFα in the BAL fluid in mice correlated with a significant reduction in hydroxyproline levels and α-SMA-positive (myo)fibroblasts. Whether the response to nintedanib in this study was associated with beneficial changes in inflammation remains unknown, as other inflammatory molecules and cytokines, including IFN-γ, IL-4, KC, and VCAM-1, were also increased in the lungs of nintedanib-treated animals. Decreased fibrosis has been associated with a reduction in an M2/alternative macrophage phenotype (32, 41), and we have shown that, in a model of systemic sclerosis, nintedanib treatment reduces the number and phenotype of M2 macrophages (14). However, because identification of the programming phenotype of macrophages is known to be a complex process and is not limited to the simple M1 and M2 paradigm, a more thorough evaluation of how nintedanib alters macrophage phenotypes may be warranted in future studies (14, 23, 24, 42).

In contrast to the reduced fibrosis in nintedanib-treated mice with established disease, treatment with nintedanib during development of lung disease (5–11 wk after zymosan injection) did not lead to a significant reduction in fibrosis, which is also reflected by no significant difference in Cst. The reasons for this difference are unclear, but several possibilities should be considered. 1) It is possible that nintedanib is more effective in promoting the resolution of established pulmonary fibrosis than in preventing its development in this model. If so, this would distinguish the SKG model from the bleomycin- and silica-induced lung injury and fibrosis models, in which nintedanib has been shown to be effective in reducing both established fibrosis and development of fibrosis (39). 2) It is known that the interstitial pneumonia and fibrotic lung disease observed in SKG mice do not exhibit the high penetrance seen with models involving intratracheal instillation, such as the bleomycin- and silica-induced models. Since we could not identify which mice were in the process of developing lung disease when nintedanib was started, we could not determine if the mice with low levels of fibrosis following nintedanib treatment had lung disease before the start of treatment. This problem is not easily addressed, although, in future studies, μ-CT or mouse survival bronchoscopy and BAL could be used to identify mice with lung disease, thereby allowing stratification of affected mice into treatment and placebo groups. This experimental strategy may also need to be considered in patients with RA, where early high-resolution CT and pulmonary physiology screening are beneficial for identification of asymptomatic preclinical ILD (9). 3) Although nintedanib has been shown to be effective in reducing the development of fibrosis and promoting the resolution and clearance of established fibrotic lung tissues in silica- and bleomycin-induced lung injury and fibrosis (39), pulmonary inflammation and fibrosis develop in SKG mice with slower, but progressive, kinetics. In contrast to the instillation models, the SKG model is an autoimmune model and, therefore, is fundamentally different from the intratracheal instillation models. Furthermore, while a reduction in fibrosis was not observed during the early treatment strategy, the severity of lung disease was not worsened in nintedanib-treated mice, suggesting that although lung disease will continue to progress in saline-treated animals (hydroxyproline level = 25.58 and 32.45 μg/upper right lobe at weeks 10 and 16, respectively), perhaps the progression will plateau in nintedanib-treated animals (hydoxyproline level = 25.12 and 22.8 μg/upper right lobe at weeks 10 and 16, respectively).

The overall effect of nintedanib on lung disease suggests that its use as a treatment in patients with RA-ILD could be beneficial. Because many of the current therapies for RA-ILD, including disease-modifying antirheumatoid and biological drugs, have been reported to cause pulmonary exacerbations, they are a nonideal treatment for this patient population (2–4, 15, 27, 30). While these studies are not conclusive, treatment with nintedanib, a drug that has been shown to slow the decline of forced vital capacity in patients with IPF (34), might be beneficial in patients with RA-ILD.

In addition to the effects on the lungs, we assessed the effects of nintedanib on joint swelling, synovitis, and arthritis development in zymosan-injected SKG mice. In mice treated with nintedanib for 6 wk after the establishment of joint disease, joint swelling was not significantly different from that in mice receiving saline. In contrast, when nintedanib was administered during development of joint disease, joint swelling was progressively reduced. Further analysis of joints by μ-CT revealed a significant prevention of bone volume decline in hind talus and calcaneus bones after nintedanib. These differences were most clearly seen after three-dimensional reconstruction. These data suggest that early treatment of patients with RA may lead to a reduction in arthritis. Clinical trials targeting newly diagnosed patients with RA with or without high-resolution CT will be necessary to determine if nintedanib will prevent the progression of joint and lung disease.

In conclusion, nintedanib was found to have divergent effects in the SKG model of RA-ILD. Treatment during early disease development attenuated joint swelling but had no demonstrable effect on lung function or development of pulmonary fibrosis. However, in mice with established disease, nintedanib reduced pulmonary fibrosis without affecting joint swelling. Nintedanib, therefore, has the potential to prevent lung function decline and attenuate joint swelling, potentially leading to a novel therapeutic benefit for patients with RA-ILD.

GRANTS

This work was supported by Boehringer Ingelheim Pharma Grant DE811138149 and National Heart, Lung, and Blood Institute Grant HL-114754 (to D. W. H. Riches). E. F. Redente was supported by Department of Veterans Affairs Career Development Award-2 1IK2BX002401-01A2.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

E.F.R., L.W., and D.W.R. conceived and designed research; E.F.R., M.A.A., B.P.B., B.E., and A.B. performed experiments; E.F.R., M.A.A., B.P.B., B.E., A.B., S.M.H., L.W., and D.W.R. analyzed data; E.F.R., S.M.H., D.A.L., L.W., and D.W.R. interpreted results of experiments; E.F.R., A.B., and S.M.H. prepared figures; E.F.R. and D.W.R. drafted manuscript; E.F.R., M.A.A., B.P.B., B.E., A.B., S.M.H., D.A.L., L.W., and D.W.R. edited and revised manuscript; E.F.R., M.A.A., B.P.B., B.E., A.B., S.M.H., D.A.L., L.W., and D.W.R. approved final version of manuscript.

REFERENCES

  • 1.Assayag D, Lee JS, King TE Jr. Rheumatoid arthritis associated interstitial lung disease: a review. Medicina (B Aires) 74: 158–165, 2014. [PubMed] [Google Scholar]
  • 2.Barrera P, Van Ede A, Laan RF, Van Riel PL, Boerbooms AM, Van De Putte LB. Methotrexate-related pulmonary complications in patients with rheumatoid arthritis: cluster of five cases in a period of three months. Ann Rheum Dis 53: 479–480, 1994. doi: 10.1136/ard.53.7.479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Cavagna L, Monti S, Grosso V, Boffini N, Scorletti E, Crepaldi G, Caporali R. The multifaceted aspects of interstitial lung disease in rheumatoid arthritis. BioMed Res Int 2013: 759760, 2013. doi: 10.1155/2013/759760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.D’Elia T. Methotrexate-induced pneumonitis: heterogeneity of bronchoalveolar lavage and differences between cancer and rheumatoid arthritis. Inflamm Allergy Drug Targets 13: 25–33, 2014. doi: 10.2174/1871528112666131230013059. [DOI] [PubMed] [Google Scholar]
  • 5.Dawson JK, Fewins HE, Desmond J, Lynch MP, Graham DR. Predictors of progression of HRCT diagnosed fibrosing alveolitis in patients with rheumatoid arthritis. Ann Rheum Dis 61: 517–521, 2002. doi: 10.1136/ard.61.6.517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Doyle TJ, Lee JS, Dellaripa PF, Lederer JA, Matteson EL, Fischer A, Ascherman DP, Glassberg MK, Ryu JH, Danoff SK, Brown KK, Collard HR, Rosas IO. A roadmap to promote clinical and translational research in rheumatoid arthritis-associated interstitial lung disease. Chest 145: 454–463, 2014. doi: 10.1378/chest.13-2408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Frankel SK, Moats-Staats BM, Cool CD, Wynes MW, Stiles AD, Riches DW. Human insulin-like growth factor-IA expression in transgenic mice promotes adenomatous hyperplasia but not pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol 288: L805–L812, 2005. doi: 10.1152/ajplung.00420.2004. [DOI] [PubMed] [Google Scholar]
  • 8.Gabbay E, Tarala R, Will R, Carroll G, Adler B, Cameron D, Lake FR. Interstitial lung disease in recent onset rheumatoid arthritis. Am J Respir Crit Care Med 156: 528–535, 1997. doi: 10.1164/ajrccm.156.2.9609016. [DOI] [PubMed] [Google Scholar]
  • 9.Gochuico BR, Avila NA, Chow CK, Novero LJ, Wu HP, Ren P, MacDonald SD, Travis WD, Stylianou MP, Rosas IO. Progressive preclinical interstitial lung disease in rheumatoid arthritis. Arch Intern Med 168: 159–166, 2008. doi: 10.1001/archinternmed.2007.59. [DOI] [PubMed] [Google Scholar]
  • 10.Habib HM, Eisa AA, Arafat WR, Marie MA. Pulmonary involvement in early rheumatoid arthritis patients. Clin Rheumatol 30: 217–221, 2011. doi: 10.1007/s10067-010-1492-5. [DOI] [PubMed] [Google Scholar]
  • 11.Hilberg F, Roth GJ, Krssak M, Kautschitsch S, Sommergruber W, Tontsch-Grunt U, Garin-Chesa P, Bader G, Zoephel A, Quant J, Heckel A, Rettig WJ. BIBF 1120: triple angiokinase inhibitor with sustained receptor blockade and good antitumor efficacy. Cancer Res 68: 4774–4782, 2008. doi: 10.1158/0008-5472.CAN-07-6307. [DOI] [PubMed] [Google Scholar]
  • 12.Hostettler KE, Zhong J, Papakonstantinou E, Karakiulakis G, Tamm M, Seidel P, Sun Q, Mandal J, Lardinois D, Lambers C, Roth M. Anti-fibrotic effects of nintedanib in lung fibroblasts derived from patients with idiopathic pulmonary fibrosis. Respir Res 15: 157, 2014. doi: 10.1186/s12931-014-0157-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Huang J, Beyer C, Palumbo-Zerr K, Zhang Y, Ramming A, Distler A, Gelse K, Distler O, Schett G, Wollin L, Distler JH. Nintedanib inhibits fibroblast activation and ameliorates fibrosis in preclinical models of systemic sclerosis. Ann Rheum Dis 75: 883–890, 2016. doi: 10.1136/annrheumdis-2014-207109. [DOI] [PubMed] [Google Scholar]
  • 14.Huang J, Maier C, Zhang Y, Soare A, Dees C, Beyer C, Harre U, Chen CW, Distler O, Schett G, Wollin L, Distler JHW. Nintedanib inhibits macrophage activation and ameliorates vascular and fibrotic manifestations in the Fra2 mouse model of systemic sclerosis. Ann Rheum Dis 76: 1941–1948, 2017. doi: 10.1136/annrheumdis-2016-210823. [DOI] [PubMed] [Google Scholar]
  • 15.Hyrich KL, Watson KD, Silman AJ, Symmons DP; British Society for Rheumatology Biologics Register . Predictors of response to anti-TNF-α therapy among patients with rheumatoid arthritis: results from the British Society for Rheumatology Biologics Register. Rheumatology (Oxford) 45: 1558–1565, 2006. doi: 10.1093/rheumatology/kel149. [DOI] [PubMed] [Google Scholar]
  • 16.Inomata M, Nishioka Y, Azuma A. Nintedanib: evidence for its therapeutic potential in idiopathic pulmonary fibrosis. Core Evid 10: 89–98, 2015. doi: 10.2147/CE.S82905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Keith RC, Powers JL, Redente EF, Sergew A, Martin RJ, Gizinski A, Holers VM, Sakaguchi S, Riches DW. A novel model of rheumatoid arthritis-associated interstitial lung disease in SKG mice. Exp Lung Res 38: 55–66, 2012. doi: 10.3109/01902148.2011.636139. [DOI] [PubMed] [Google Scholar]
  • 18.Keith RC, Sokolove J, Edelman BL, Lahey L, Redente EF, Holers VM, Sakaguchi S, Robinson WH, Riches DW. Testosterone is protective in the sexually dimorphic development of arthritis and lung disease in SKG mice. Arthritis Rheum 65: 1487–1493, 2013. doi: 10.1002/art.37943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Kelly CA, Saravanan V, Nisar M, Arthanari S, Woodhead FA, Price-Forbes AN, Dawson J, Sathi N, Ahmad Y, Koduri G, Young A; British Rheumatoid Interstitial Lung (BRILL) Network . Rheumatoid arthritis-related interstitial lung disease: associations, prognostic factors and physiological and radiological characteristics—a large multicentre UK study. Rheumatology (Oxford) 53: 1676–1682, 2014. doi: 10.1093/rheumatology/keu165. [DOI] [PubMed] [Google Scholar]
  • 20.Kim DS. Interstitial lung disease in rheumatoid arthritis: recent advances. Curr Opin Pulm Med 12: 346–353, 2006. doi: 10.1097/01.mcp.0000239552.55326.ee. [DOI] [PubMed] [Google Scholar]
  • 21.Koduri G, Norton S, Young A, Cox N, Davies P, Devlin J, Dixey J, Gough A, Prouse P, Winfield J, Williams P; ERAS (Early Rheumatoid Arthritis Study) . Interstitial lung disease has a poor prognosis in rheumatoid arthritis: results from an inception cohort. Rheumatology (Oxford) 49: 1483–1489, 2010. doi: 10.1093/rheumatology/keq035. [DOI] [PubMed] [Google Scholar]
  • 23.Martinez FO, Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Rep 6: 13, 2014. doi: 10.12703/P6-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.McCubbrey AL, Barthel L, Mohning MP, Redente EF, Mould KJ, Thomas SM, Leach SM, Danhorn T, Gibbings SL, Jakubzick CV, Henson PM, Janssen WJ. Deletion of c-FLIP from CD11bhi macrophages prevents development of bleomycin-induced lung fibrosis. Am J Respir Cell Mol Biol 58: 66–78, 2018. doi: 10.1165/rcmb.2017-0154OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Nakashita T, Ando K, Kaneko N, Takahashi K, Motojima S. Potential risk of TNF inhibitors on the progression of interstitial lung disease in patients with rheumatoid arthritis. BMJ Open 4: e005615, 2014. doi: 10.1136/bmjopen-2014-005615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Olson AL, Swigris JJ, Sprunger DB, Fischer A, Fernandez-Perez ER, Solomon J, Murphy J, Cohen M, Raghu G, Brown KK. Rheumatoid arthritis-interstitial lung disease-associated mortality. Am J Respir Crit Care Med 183: 372–378, 2011. doi: 10.1164/rccm.201004-0622OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Ostör AJ, Chilvers ER, Somerville MF, Lim AY, Lane SE, Crisp AJ, Scott DG. Pulmonary complications of infliximab therapy in patients with rheumatoid arthritis. J Rheumatol 33: 622–628, 2006. [PubMed] [Google Scholar]
  • 28.Perez-Alvarez R, Perez-de-Lis M, Diaz-Lagares C, Pego-Reigosa JM, Retamozo S, Bove A, Brito-Zeron P, Bosch X, Ramos-Casals M. Interstitial lung disease induced or exacerbated by TNF-targeted therapies: analysis of 122 cases. Semin Arthritis Rheum 41: 256–264, 2011. doi: 10.1016/j.semarthrit.2010.11.002. [DOI] [PubMed] [Google Scholar]
  • 29.Perry E, Stenton C, Kelly C, Eggleton P, Hutchinson D, De Soyza A. RA autoantibodies as predictors of rheumatoid arthritis in non-cystic fibrosis bronchiectasis patients. Eur Respir J 44: 1082–1085, 2014. doi: 10.1183/09031936.00064014. [DOI] [PubMed] [Google Scholar]
  • 30.Picchianti Diamanti A, Markovic M, Argento G, Giovagnoli S, Ricci A, Laganà B, D’Amelio R. Therapeutic management of patients with rheumatoid arthritis and associated interstitial lung disease: case report and literature review. Ther Adv Respir Dis 11: 64–72, 2017. doi: 10.1177/1753465816668780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Redente EF, Jacobsen KM, Solomon JJ, Lara AR, Faubel S, Keith RC, Henson PM, Downey GP, Riches DW. Age and sex dimorphisms contribute to the severity of bleomycin-induced lung injury and fibrosis. Am J Physiol Lung Cell Mol Physiol 301: L510–L518, 2011. doi: 10.1152/ajplung.00122.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Redente EF, Keith RC, Janssen W, Henson PM, Ortiz LA, Downey GP, Bratton DL, Riches DW. Tumor necrosis factor-α accelerates the resolution of established pulmonary fibrosis in mice by targeting profibrotic lung macrophages. Am J Respir Cell Mol Biol 50: 825–837, 2014. doi: 10.1165/rcmb.2013-0386OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Richeldi L, Costabel U, Selman M, Kim DS, Hansell DM, Nicholson AG, Brown KK, Flaherty KR, Noble PW, Raghu G, Brun M, Gupta A, Juhel N, Klüglich M, du Bois RM. Efficacy of a tyrosine kinase inhibitor in idiopathic pulmonary fibrosis. N Engl J Med 365: 1079–1087, 2011. doi: 10.1056/NEJMoa1103690. [DOI] [PubMed] [Google Scholar]
  • 34.Richeldi L, Cottin V, du Bois RM, Selman M, Kimura T, Bailes Z, Schlenker-Herceg R, Stowasser S, Brown KK. Nintedanib in patients with idiopathic pulmonary fibrosis: combined evidence from the TOMORROW and INPULSIS® trials. Respir Med 113: 74–79, 2016. doi: 10.1016/j.rmed.2016.02.001. [DOI] [PubMed] [Google Scholar]
  • 35.Richeldi L, du Bois RM, Raghu G, Azuma A, Brown KK, Costabel U, Cottin V, Flaherty KR, Hansell DM, Inoue Y, Kim DS, Kolb M, Nicholson AG, Noble PW, Selman M, Taniguchi H, Brun M, Le Maulf F, Girard M, Stowasser S, Schlenker-Herceg R, Disse B, Collard HR, Investigators IT; INPULSIS Trial Investigators . Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis. N Engl J Med 370: 2071–2082, 2014. doi: 10.1056/NEJMoa1402584. [DOI] [PubMed] [Google Scholar]
  • 36.Saag KG, Teng GG, Patkar NM, Anuntiyo J, Finney C, Curtis JR, Paulus HE, Mudano A, Pisu M, Elkins-Melton M, Outman R, Allison JJ, Suarez Almazor M, Bridges SL Jr, Chatham WW, Hochberg M, MacLean C, Mikuls T, Moreland LW, O’Dell J, Turkiewicz AM, Furst DE; American College of Rheumatology . American College of Rheumatology 2008 recommendations for the use of nonbiologic and biologic disease-modifying antirheumatic drugs in rheumatoid arthritis. Arthritis Rheum 59: 762–784, 2008. doi: 10.1002/art.23721. [DOI] [PubMed] [Google Scholar]
  • 37.Sakaguchi N, Takahashi T, Hata H, Nomura T, Tagami T, Yamazaki S, Sakihama T, Matsutani T, Negishi I, Nakatsuru S, Sakaguchi S. Altered thymic T-cell selection due to a mutation of the ZAP-70 gene causes autoimmune arthritis in mice. Nature 426: 454–460, 2003. doi: 10.1038/nature02119. [DOI] [PubMed] [Google Scholar]
  • 38.Shaw M, Collins BF, Ho LA, Raghu G. Rheumatoid arthritis-associated lung disease. Eur Respir Rev 24: 1–16, 2015. doi: 10.1183/09059180.00008014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Wollin L, Maillet I, Quesniaux V, Holweg A, Ryffel B. Antifibrotic and anti-inflammatory activity of the tyrosine kinase inhibitor nintedanib in experimental models of lung fibrosis. J Pharmacol Exp Ther 349: 209–220, 2014. doi: 10.1124/jpet.113.208223. [DOI] [PubMed] [Google Scholar]
  • 40.Wollin L, Wex E, Pautsch A, Schnapp G, Hostettler KE, Stowasser S, Kolb M. Mode of action of nintedanib in the treatment of idiopathic pulmonary fibrosis. Eur Respir J 45: 1434–1445, 2015. doi: 10.1183/09031936.00174914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Wynn TA, Vannella KM. Macrophages in tissue repair, regeneration, and fibrosis. Immunity 44: 450–462, 2016. doi: 10.1016/j.immuni.2016.02.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Xue J, Schmidt SV, Sander J, Draffehn A, Krebs W, Quester I, De Nardo D, Gohel TD, Emde M, Schmidleithner L, Ganesan H, Nino-Castro A, Mallmann MR, Labzin L, Theis H, Kraut M, Beyer M, Latz E, Freeman TC, Ulas T, Schultze JL. Transcriptome-based network analysis reveals a spectrum model of human macrophage activation. Immunity 40: 274–288, 2014. doi: 10.1016/j.immuni.2014.01.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Yoshitomi H, Sakaguchi N, Kobayashi K, Brown GD, Tagami T, Sakihama T, Hirota K, Tanaka S, Nomura T, Miki I, Gordon S, Akira S, Nakamura T, Sakaguchi S. A role for fungal β-glucans and their receptor dectin-1 in the induction of autoimmune arthritis in genetically susceptible mice. J Exp Med 201: 949–960, 2005. doi: 10.1084/jem.20041758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Ytterberg AJ, Joshua V, Reynisdottir G, Tarasova NK, Rutishauser D, Ossipova E, Haj Hensvold A, Eklund A, Sköld CM, Grunewald J, Malmström V, Jakobsson PJ, Rönnelid J, Padyukov L, Zubarev RA, Klareskog L, Catrina AI. Shared immunological targets in the lungs and joints of patients with rheumatoid arthritis: identification and validation. Ann Rheum Dis 74: 1772–1777, 2015. doi: 10.1136/annrheumdis-2013-204912. [DOI] [PubMed] [Google Scholar]
  • 45.Yushkevich PA, Piven J, Hazlett HC, Smith RG, Ho S, Gee JC, Gerig G. User-guided 3D active contour segmentation of anatomical structures: significantly improved efficiency and reliability. Neuroimage 31: 1116–1128, 2006. doi: 10.1016/j.neuroimage.2006.01.015. [DOI] [PubMed] [Google Scholar]
  • 46.Zhu J, Zhou Y, Chen X, Li J. A metaanalysis of the increased risk of rheumatoid arthritis-related pulmonary disease as a result of serum anticitrullinated protein antibody positivity. J Rheumatol 41: 1282–1289, 2014. doi: 10.3899/jrheum.131341. [DOI] [PubMed] [Google Scholar]

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