Dasatinib Reduces Lung Inflammation and Fibrosis in Acute Experimental Silicosis

RESEARCH ARTICLE Dasatinib Reduces Lung Inflammation and Fibrosis in Acute Experimental Silicosis Fernanda Ferreira Cruz1, Lucas Felipe Bastos Horta1, Lígia de Albuquerque Maia1, Miquéias Lopes-Pacheco1,2, André Benedito da Silva1,2, Marcelo Marco Morales2, Cassiano Felippe Gonçalves-de-Albuquerque3, Christina Maeda Takiya4, Hugo Caire de Castro-Faria-Neto3, Patricia Rieken Macedo Rocco1* 1 Laboratory of Pulmonary Investigation, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil, 2 Laboratory of Cellular and Molecular Physiology, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil, 3 Laboratory of Immunopharmacology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil, 4 Laboratory of Cellular Pathology, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil * Abstract OPEN ACCESS Citation: Cruz FF, Horta LFB, Maia LdA, LopesPacheco M, Silva ABd, Morales MM, et al. (2016) Dasatinib Reduces Lung Inflammation and Fibrosis in Acute Experimental Silicosis. PLoS ONE 11(1): e0147005. doi:10.1371/journal.pone.0147005 Editor: Thomas H Thatcher, University of Rochester Medical Center, UNITED STATES Received: February 11, 2015 Accepted: December 28, 2015 Published: January 20, 2016 Copyright: © 2016 Cruz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the European Community's Seventh Framework Programme [FP72007-2013] under grant agreement no. HEALTH-F42011-282095 (HCCFN, PRMR), Brazilian Council for Scientific and Technological Development (CNPq) (MMM, CMT, HCCFN, PRMR), the Rio de Janeiro State Research Foundation (FAPERJ) (CNPq) (MMM, CMT, HCCFN, PRMR) and Coordination for the Improvement of Higher Level Personnel (CAPES) (MMM, CMT, HCCFN, PRMR). The funders had no Silicosis is an occupational lung disease with no effective treatment. We hypothesized that dasatinib, a tyrosine kinase inhibitor, might exhibit therapeutic efficacy in silica-induced pulmonary fibrosis. Silicosis was induced in C57BL/6 mice by a single intratracheal administration of silica particles, whereas the control group received saline. After 14 days, when the disease was already established, animals were randomly assigned to receive DMSO or dasatinib (1 mg/kg) by oral gavage, twice daily, for 14 days. On day 28, lung morphofunction, inflammation, and remodeling were investigated. RAW 264.7 cells (a macrophage cell line) were incubated with silica particles, followed by treatment or not with dasatinib, and evaluated for macrophage polarization. On day 28, dasatinib improved lung mechanics, increased M2 macrophage counts in lung parenchyma and granuloma, and was associated with reduction of fraction area of granuloma, fraction area of collapsed alveoli, protein levels of tumor necrosis factor-α, interleukin-1β, transforming growth factor-β, and reduced neutrophils, M1 macrophages, and collagen fiber content in lung tissue and granuloma in silicotic animals. Additionally, dasatinib reduced expression of iNOS and increased expression of arginase and metalloproteinase-9 in silicotic macrophages. Dasatinib was effective at inducing macrophage polarization toward the M2 phenotype and reducing lung inflammation and fibrosis, thus improving lung mechanics in a murine model of acute silicosis. Introduction Silicosis is an occupational disease caused by inhalation of crystalline silica particles, which triggers a persistent inflammatory cascade that leads to progressive lung fibrosis and subsequent respiratory failure due to deterioration of lung function and reduction in gas exchange area [1]. Although therapy for silicosis includes a variety of drugs and non-pharmacological PLOS ONE | DOI:10.1371/journal.pone.0147005 January 20, 2016 1 / 17 Tyrosine Kinase Inhibitor in Silicosis role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. interventions, there is still a pressing need for new therapeutic approaches as no current therapy is able to effectively reduce disease progression or reverse lung fibrosis [2,3]. Dasatinib (DAS; Bristol-Myers Squibb, New York, NY, USA), which has been widely studied for the treatment of cancer [4,5], is a second-generation oral multitarget inhibitor of several tyrosine kinases, including Abl and Bcr-Abl family members, Src and Btk family members, cKit, PDGFR, and Eph receptors [6,7]. Its targets include several receptors associated with the regulation of a wide variety of pathways involved in physiological cell functions and pathophysiological processes such as the recruitment and activation of inflammatory cells [8–14] and fibrosis [15,16]. Thus, inhibition of tyrosine kinases receptors may reduce inflammation and slow the progression of pulmonary fibrosis. We hypothesized that dasatinib would attenuate pulmonary fibrosis, ameliorate inflammatory responses, and improve lung function in experimental acute silicosis. For this purpose, we investigated the potential efficacy and mechanisms of dasatinib in the treatment of silicainduced lung fibrosis. Material and Methods This study was approved by the Ethics Committee of the Health Sciences Centre, Federal University of Rio de Janeiro (CEUA-CCS-019). All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the U.S. National Research Council “Guide for the Care and Use of Laboratory Animals”, and all efforts were made to minimize suffering. Animal Preparation and Experimental Protocol Fifty-two C57BL/6 female mice (weight: 20–25 g, age 8–12 weeks) were assigned to two main groups: control (C) and silicosis (SIL). In group SIL, mice received silica particle suspension (20 mg in 50μL saline, intratracheally [i.t.]), while group C received saline (50 μL, i.t.) using the same protocol. Fourteen days after administration of silica or saline, animals were further randomized to receive dimethyl sulfoxide (DMSO 1% in saline solution, 100 μL, oral gavage) or dasatinib (DAS 1 mg/kg body weight in DMSO 1%, 100 μL, oral gavage) during 14 days (Fig 1). Additionally, another group of animals was treated with saline (SAL, 100 μL) by oral gavage for 14 days. These animals were subsequently compared to those that received DMSO, aiming to evaluate whether DMSO per se might have any effects on lung morphofunction (Tables A-C in S1 File). Lung Mechanics Twenty-four hours after the last dose, the animals were sedated (diazep (...truncated)