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

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.

Myeloid-derived suppressor cell-derived osteoclasts with bone resorption capacity in the joints of arthritic SKG mice.

Background: Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature myeloid cells with immunosuppressive functions. It is known that MDSCs are expanded at inflammatory sites after migrating from bone marrow (BM) or spleen (Sp). In chronic inflammatory diseases such as rheumatoid arthritis (RA), previous reports indicate that MDSCs are increased in BM and Sp, but detailed analysis of MDSCs in inflamed joints is very limited. Objective: The purpose of this study is to characterize the MDSCs in the joints of mice with autoimmune arthritis. Methods: We sorted CD11b+Gr1+ cells from joints (Jo), bone marrow (BM) and spleen (Sp) of SKG mice with zymosan (Zym)-induced arthritis and investigated differentially expressed genes (DEGs) by microarray analysis. Based on the identified DEGs, we assessed the suppressive function of CD11b+Gr1+ cells from each organ and their ability to differentiate into osteoclasts. Results: We identified MDSCs as CD11b+Gr1+ cells by flow cytometry and morphological analysis. Microarray analysis revealed that Jo-CD11b+Gr1+ cells had different characteristics compared with BM-CD11b+Gr1+ cells or Sp-CD11b+Gr1+ cells. Microarray and qPCR analysis showed that Jo-CD11b+Gr1+ cells strongly expressed immunosuppressive DEGs (Pdl1, Arg1, Egr2 and Egr3). Jo-CD11b+Gr1+ cells significantly suppressed CD4+ T cell proliferation and differentiation in vitro, which confirmed Jo-CD11b+Gr1+ cells as MDSCs. Microarray analysis also revealed that Jo-MDSCs strongly expressed DEGs of the NF-κB non-canonical pathway (Nfkb2 and Relb), which is relevant for osteoclast differentiation. In fact, Jo-MDSCs differentiated into osteoclasts in vitro and they had bone resorptive function. In addition, intra-articular injection of Jo-MDSCs promoted bone destruction. Conclusions: Jo-MDSCs possess a potential to differentiate into osteoclasts which promote bone resorption in inflamed joints, while they are immunosuppressive in vitro.

Nrf2 activation improves experimental rheumatoid arthritis.

Rheumatoid arthritis is a systemic autoimmune disease with pain and functional disorder of joints. Multiple strategies toward treatment of the rheumatoid arthritis are operating, while there are concerns of serious adverse effects of the therapeutic drugs. Here, we show that activation of Nrf2 (Nuclear factor erythroid 2-related factor 2) efficiently improves arthritis of SKG mice, which develop T cell-mediated autoimmune arthritis by zymosan A injection. We found that genetic Nrf2 activation by knockdown of Keap1 (Kelch-like ECH-associated protein 1), a negative regulator of Nrf2, repressed arthritis by inhibiting the expression of pro-inflammatory cytokines and inducing the expression of antioxidant enzymes in SKG mice. In addition, oral administration of CDDO-Im, a representative chemical inducer of Nrf2, had effects of both prevention and treatment toward arthritis of SKG mice in an Nrf2-dependent manner. We also found that Nrf2 activation through myeloid-cell lineage-specific Keap1 disruption did not achieve significant improvement in the arthritis of SKG mice. In contrast, expressions of pro-inflammatory cytokine genes were decreased, and those of antioxidant enzyme genes were increased in fibroblast-like synoviocytes (FLS) isolated from SKG mouse. Our results thus demonstrate that Nrf2 activation exerts marked anti-arthritis effects in the SKG experimental rheumatoid arthritis model mice, supporting the contention that the Nrf2 activation is a new therapeutic strategy for the rheumatoid arthritis.

Treatment with an Anti-CX3CL1 Antibody Suppresses M1 Macrophage Infiltration in Interstitial Lung Disease in SKG Mice.

CX3C Motif Chemokine Ligand 1 (CX3CL1; fractalkine) has been implicated in the pathogenesis of rheumatoid arthritis (RA) and its inhibition was found to attenuate arthritis in mice as well as in a clinical trial. Therefore, we investigated the effects of an anti-CX3CL1 monoclonal antibody (mAb) on immune-mediated interstitial lung disease (ILD) in SKG mice, which exhibit similar pathological and clinical features to human RA-ILD. CX3CL1 and CX3C chemokine receptor 1 (CX3CR1), the receptor for CX3CL1, were both expressed in the fibroblastic foci of lung tissue and the number of bronchoalveolar fluid (BALF) cells was elevated in ILD in SKG mice. No significant changes were observed in lung fibrosis or the number of BALF cells by the treatment with anti-CX3CL1 mAb. However, significantly greater reductions were observed in the number of M1 macrophages than in M2 macrophages in the BALF of treated mice. Furthermore, CX3CR1 expression levels were significantly higher in M1 macrophages than in M2 macrophages. These results suggest the stronger inhibitory effects of the anti-CX3CL1 mAb treatment against the alveolar infiltration of M1 macrophages than M2 macrophages in ILD in SKG mice. Thus, the CX3CL1-CX3CR1 axis may be involved in the infiltration of inflammatory M1 macrophages in RA-ILD.

Effects of dihydrotestosterone on osteoblast activity in curdlan-administered SKG mice and osteoprogenitor cells in patients with ankylosing spondylitis.

BACKGROUND: Ankylosing spondylitis (AS) is characteristically male-predominant, and progressive spinal ankylosis affects male patients more severely; however, the hormonal effects in males with AS are poorly understood. METHODS: In the present study, the regulatory effects of dutasteride, a 5-α reductase inhibitor that blocks the conversion of testosterone to dihydrotestosterone (DHT), were examined in curdlan-administered male SKG mice to determine spinal bone formation, bone metabolism-related markers, and interleukin (IL)-17A cytokine and T cell populations. In addition, the effects of DHT on primary osteoprogenitors from the facet joints of AS patients were assessed based on osteoblast-related parameters. DHT level was measured, and the correlation with modified Stoke Ankylosing Spondylitis Spinal Score (mSASSS) was analyzed in AS patients. RESULTS: In curdlan-administered SKG mice, dutasteride treatment resulted in an increased accumulation of hydroxyapatite in the spine which was positively correlated with serum IL-17A levels. In the analysis of bone metabolism-related molecules, a decrease in sclerostin levels was observed in the sera in the dutasteride group. Continuous exposure to DHT resulted in fewer calcium deposits in AS osteoprogenitors during osteoblast differentiation. DHT-treated AS osteoprogenitors showed decreased osteocalcin and increased DKK1 and SOST1 mRNA expression, supporting the results of the in vivo experiments. Treatment with dutasteride upregulated bone formation in the spine of curdlan-administered SKG mice and DHT treatment downregulated osteoblast differentiation in vitro. CONCLUSIONS: Treatment with dutasteride affected the bone formation in the spine of curdlan-treated SKG mice, and DHT treatment attenuated osteoblast differentiation in vitro. Therefore, contrary to what could be expected if osteoblasts contributed to spinal ankylosis, DHT inhibition might increase rather than decrease the progression of spinal ankylosis despite the higher levels of DHT observed in many AS patients.

The involvement of C5a in the progression of experimental arthritis with Porphyromonas gingivalis infection in SKG mice.

BACKGROUND: Epidemiological evidence to suggest that periodontal disease (PD) is involved in the progression of rheumatoid arthritis (RA) is increasing. The complement system plays a critical role in immune responses. C5a has been implicated in chronic inflammatory diseases, including PD and RA. Porphyromonas gingivalis is the major causative bacteria of PD and can produce C5a. Therefore, it is hypothesized that P. gingivalis infection is involved in the progression of RA by elevating C5a levels. In the present study, P. gingivalis-infected RA model mice were established to investigate the involvement of C5a. METHODS: SKG mice orally infected with P. gingivalis were immunized with intraperitoneal injection of laminarin (LA) to induce arthritis. Arthritis development was assessed by arthritis score (AS), bone destruction on the talus, histology, and serum markers of RA. In order to investigate the effects of serum C5a on bone destruction, osteoclast differentiation of bone marrow mononuclear cells was examined by using serum samples from each group of mice. The relationship between C5a levels and antibody titers to periodontal pathogens in patients with RA was investigated by enzyme-linked immunosorbent assay. RESULTS: P. gingivalis oral infection increased AS, infiltration of inflammatory cells, bone destruction on the talus, and serum markers of RA in mice immunized with LA. The addition of serum from LA-injected mice with the P. gingivalis oral infection promoted osteoclast differentiation, and the addition of a neutralization antibody against C5a suppressed osteoclast differentiation. C5a levels of serum in RA patients with positive P. gingivalis antibody were elevated compared with those in RA patients with negative P. gingivalis antibody. CONCLUSIONS: These results suggest that P. gingivalis infection enhances the progression of RA via C5a.

CP-690550 Treatment Ameliorates Established Disease and Provides Long-Term Therapeutic Effects in an SKG Arthritis Model.

Although pathogenesis of human rheumatoid arthritis (RA) remains unclear, arthritogenic T cells and downstream signaling mediators have been shown to play critical roles. An increasing numbers of therapeutic options have been added for the effective control of RA. Nevertheless, there is still a category of patients that fails treatment and suffers from progressive disease. The recently developed immunosuppressant CP-690550, a small molecule JAK kinase inhibitor, has been implicated as an important candidate treatment modality for autoimmune arthritis. In this study, we evaluated the therapeutic effect of CP-690550 on established arthritis using an SKG arthritis model, a pathophysiologically relevant animal model for human RA. CP-690550 treatment revealed remarkable long-term suppressive effects on SKG arthritis when administered to the well-advanced disease (clinical score 3.5~4.0). The treatment effect lasted at least 3 more weeks after cessation of drug infusion, and suppression of disease was correlated with the reduced pro-inflammatory cytokines, including IL-17, IFN-γ, and IL-6 and increased level of immunoregulatory IL-10.