Pirfenidone inhibits fibrocyte accumulation in the lungs in bleomycin-induced murine pulmonary fibrosis

Abstract

BackgroundBone marrow-derived fibrocytes reportedly play important roles in the pathogenesis of idiopathic pulmonary fibrosis. Pirfenidone is an anti-fibrotic agent; however, its effects on fibrocytes have not been investigated. The aim of this study was to investigate whether pirfenidone inhibits fibrocyte pool size in the lungs of bleomycin-treated mice.MethodsBleomycin (100 mg/kg) was infused with osmotic pumps into C57BL/6 mice, and pirfenidone (300 mg/kg/day) was orally administered daily for 2 wk. The lungs were removed, and single-cell suspensions were subjected to fluorescence-activated cell sorter (FACS) analysis to detect fibrocytes, which were defined as CD45 and collagen-I double-positive cells. Immunohistochemistry was performed on the lung specimens to quantify fibrocytes. Chemokines in the lung digests were measured with enzyme-linked immunosorbent assay. The effect of pirfenidone on alveolar macrophages was evaluated with bronchoalveolar lavage (BAL). In a therapeutic setting, pirfenidone administration was initiated 10 days after bleomycin treatment. For chemotaxis assay, lung fibrocytes were isolated with immunomagnetic selection (CD45-positive mesenchymal cells) after culture and allowed to migrate toward chemokines in the presence or absence of pirfenidone. Moreover, the effect of pirfenidone on the expression of chemokine receptors on fibrocytes was evaluated.ResultsPirfenidone significantly ameliorated bleomycin-induced pulmonary fibrosis as assessed with quantitative histology and collagen measurement. Fibrocyte pool size in bleomycin-treated mice lungs was attenuated from 26.5% to 13.7% by pirfenidone on FACS analysis. This outcome was also observed in a therapeutic setting. Immunohistochemistry revealed that fibrocytes were significantly decreased by pirfenidone administration compared with those in bleomycin-treated mice (P = 0.0097). Increased chemokine (CC motif) ligand-2 (CCL2) and CCL12 production in bleomycin-treated mouse lungs was significantly attenuated by pirfenidone (P = 0.0003 and P < 0.0001, respectively). Pirfenidone also attenuated macrophage counts stimulated by bleomycin in BAL fluid. Fibrocyte migration toward CCL2 and chemokine (CC motif) receptor-2 expression on fibrocytes was significantly inhibited by pirfenidone in vitro.ConclusionsPirfenidone attenuated the fibrocyte pool size in bleomycin-treated mouse lungs via attenuation of CCL2 and CCL12 production in vivo, and fibrocyte migration was inhibited by pirfenidone in vitro. Fibrocyte inhibition is considered a mechanism of anti-fibrotic action of pirfenidone.

Pirfenidone ameliorates concanavalin A-induced hepatitis in mice via modulation of reactive oxygen species/nuclear factor kappa B signalling pathways

“…The other two groups were Con A + PFD treated where mice were pretreated orally with PFD at two different doses (200, 300 mg/kg, oral) for 5 days. The PFD doses were selected based on previous studies …”

Evaluation of chemokines CXCL8 and CCL2, serotonin, and vascular endothelial growth factor serum concentrations in healthy dogs from seven breeds with variable predisposition for canine idiopathic pulmonary fibrosis

“…In humans, both CXCL8 and CCL2 concentrations were found to be increased in blood (Ziegenhagen et al, 1998a;Suga et al, 1999;Fujiwara et al, 2012) and bronchoalveolar lavage fluid (BALF) (Capelli et al, 2005;Antoniou et al, 2006;Baran et al, 2007) of IPF patients compared with healthy volunteers and correlated with lung function (Capelli et al, 2005;Emad and Emad, 2007;Martina et al, 2009;Vasakova et al, 2009), disease progression (Ziegenhagen et al, 1998b;Totani et al, 2002), and outcome (Shinoda et al, 2009;Richards et al, 2012). Furthermore, several studies suggested an involvement of the chemokine CCL2 in the pathogenesis of IPF, notably through its action on resident pulmonary fibroblast and circulating fibrocytes, promoting the generation of abundant extracellular matrix in the lungs (Gharaee-Kermani et al, 1996;Phillips et al, 2004;Moore et al, 2005;Inomata et al, 2014). Such strong evidence involving CXCL8 in the pathogenesis of the disease are lacking, although this chemokine is thought to act as a pro-fibrotic factor in IPF via the promotion of exacerbated angiogenesis (Strieter et al, 2002;Rosenkilde and Schwartz, 2004;Antoniou et al, 2006;Martina et al, 2009;Cui et al, 2010).…”

Effect of grapefruit juice and food on the pharmacokinetics of pirfenidone in healthy Chinese volunteers: a diet–drug interaction study

“…It has been evaluated in multicenter, randomized, double-blind, placebo-controlled clinical trials conducted in Japan and the USA for the treatment of IPF and has been shown to have an acceptable tolerability and safety profile (Azuma et al, 2005;Kreuter, 2014;Noble et al, 2011). The antifibrotic activity of pirfenidone is believed to result mainly from reduced synthesis and accumulation of collagen in lung tissue and downregulation of the pulmonary growth factor, transforming growth factor-b1 (Inomata et al, 2014).…”