Experimental study of the effects of pirfenidone and nintedanib on joint inflammation and pulmonary fibrosis in a rheumatoid arthritis-associated interstitial lung disease mouse model.

Abstract

Background: Rheumatoid arthritis-associated interstitial lung disease (RA-ILD) is a serious pulmonary complication in rheumatoid arthritis (RA) patients, is one of the leading causes of death in RA patients. This study was designed to determine whether pirfenidone and nintedanib can alleviate joint inflammation and pulmonary fibrosis in a mouse model of RA-ILD.

Methods: Male DBA/1 mice were injected with bovine type II collagen (bCII) to establish the RA-ILD model. Pirfenidone (20 mg/kg) and nintedanib (60 mg/kg) were administered, and body weight, joint swelling, pathology of the lungs and knees, macrophage polarization in bronchoalveolar lavage fluid (BALF), and the fluorescence intensity of phosphorylated janus kinase 2/phosphorylated signal transducer and activator of transcription 3 (p-Jak2/p-Stat3) in the lungs and knees were determined. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure mRNA, and western blotting was conducted to detect the protein. Macrophage line RAW264.7 were divided into the following groups: the RAW264.7, RAW264.7 + IL-4/IL-13 (IL-4/IL-13, 60 ng/mL), RAW264.7 + IL-4/IL-13 + pirfenidone (0.5 and 1.0 mmol/L), RAW264.7 + IL-4/IL-13 + nintedanib (0.1 and 0.5 µmol/L). Mouse primary fibroblast-like synovial (FLS) cells were divided into the following groups: the FLS, FLS + transforming growth factor-β1 (TGF-β1; 10 µg/L), FLS + TGF-β1 + pirfenidone (0.5 and 1.0 mmol/L), FLS + TGF-β1 + nintedanib (0.1 and 0.5 µmol/L) groups. Proteins in each group were detected.

Results: The body weights of the mice in the pirfenidone and nintedanib groups were greater than those in the RA-ILD group (P<0.05), the arthritis scores were also significantly lower (P<0.05). The proportion of M2-type macrophages in the BALF of the nintedanib group significantly decreased (P<0.05). Inflammatory cell infiltration in the lung was reduced in the pirfenidone and nintedanib groups; additionally, decreased levels of synovium, collagen, angiogenesis, and bone destruction of the knee joint and a lower synovitis score were observed (P<0.05). Masson staining revealed that collagen deposition in the lungs in the pirfenidone and nintedanib groups was reduced (P<0.05). P-Jak2/p-Stat3 expression in the lungs and knee joints in the pirfenidone and nintedanib groups was low (P<0.001 in the lung and P<0.005 in the knee joint). The mRNA expression of collagen-IV, Stat3, and Jak2 in the lungs was lower in the pirfenidone and nintedanib (P<0.05); the protein expression levels of p-Jak2/Jak2, p-Stat3/Stat3, p-Smad3/Smad3, and TGF-β receptor 2 (TGF-βR2) in the lungs in the pirfenidone and nintedanib groups decreased (P<0.05). P-Jak2/Jak2, p-Stat3/Stat3, TGF-βR2, cluster of differentiation 206 (CD206), and arginase-1 (ARG-1) were lower in the pirfenidone and nintedanib groups of RAW264.7 cells (at all different concentrations, P<0.05). P-JAK2/JAK2, p-Stat3/Stat3, and TGF-βR2 were lower in the pirfenidone and nintedanib groups of FLS cells (at all different concentrations, P<0.05).

Conclusions: Pirfenidone and nintedanib not only reduced the degree of pulmonary fibrosis but also relieved joint symptoms in an RA-ILD mouse model. The mechanisms of action are related to the inhibition of the TGF-β signaling pathway, Jak2/Stat3 signaling pathway, and polarization of macrophages to the M2 phenotype.