Uncoupling between Inflammatory and Fibrotic Responses to Silica: Evidence from MyD88 Knockout Mice

et al. (2014) Uncoupling between Inflammatory and Fibrotic Responses to Silica: Evidence from MyD88 Knockout Mice. PLoS ONE 9(7): e99383. doi:10.1371/journal.pone.0099383 Uncoupling between Inflammatory and Fibrotic Responses to Silica: Evidence from MyD88 Knockout Mice Sandra Lo Re 0 Yousof Yakoub 0 Raynal Devosse 0 Francine Uwambayinema 0 Isabelle Couillin 0 Bernard Ryffel 0 Etienne Marbaix 0 Dominique Lison 0 Franc ois Huaux 0 Thomas H. Thatcher, University of Rochester Medical Center, United States of America 0 1 Louvain centre for Toxicology and Applied Pharmacology (LTAP), Institut de Recherche Expe rimentale et Clinique (IREC), Universite catholique de Louvain , Brussels , Belgium , 2 University of Orleans , CNRS, UMR7355, INEM , Transgenose Institute , Orleans , France , 3 de Duve Institute, Universite catholique de Louvain , Brussels , Belgium The exact implication of innate immunity in granuloma formation and irreversible lung fibrosis remains to be determined. In this study, we examined the lung inflammatory and fibrotic responses to silica in MyD88-knockout (KO) mice. In comparison to wild-type (WT) mice, we found that MyD88-KO animals developed attenuated lung inflammation, neutrophil accumulation and IL-1b release in response to silica. Granuloma formation was also less pronounced in MyD88-KO mice after silica. This limited inflammatory response was not accompanied by a concomitant attenuation of lung collagen accumulation after silica. Histological analyses revealed that while pulmonary fibrosis was localized in granulomas in WT animals, it was diffusely distributed throughout the parenchyma in MyD88-KO mice. Robust collagen accumulation was also observed in mice KO for several other components of innate immunity (IL-1R, IL-1, ASC, NALP3, IL-18R, IL-33R, TRIF, and TLR2-3-4,). We additionally show that pulmonary fibrosis in MyD88-KO mice was associated with the accumulation of profibrotic regulatory T lymphocytes (T regs) and pro-fibrotic cytokine expression (TGF-b, IL-10 and PDGF-B), not with T helper (Th) 17 cell influx. Our findings indicate that the activation of MyD88-related innate immunity is central in the establishment of particle-induced lung inflammatory and granuloma responses. The development of lung fibrosis appears uncoupled from inflammation and may be orchestrated by a T reg-associated pathway. - Funding: This work was funded by the Marshall Programme dexcellence Diane convention, by the Fonds de la Recherche Scientifique Medicale (FRSM), by Actions de Recherche Concertees, Communaut e francaise de Belgique, Direction de la Recherche Scientifique (ARC 09/14-021), by the Fondation Contre le Cancer, by the Fonds de la Recherche Scientifique (FNRS; Project PDR T.0119.13 14633768), by the European Commission under FP7-HEALTH-F4-2008 (Contract no. 202047), and by Agence Nationale de securit e sanitaire de lalimentation, de lenvironnement et du travail (ANSES, France). F.H. is a Research Associate with the FNRS, Belgium. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Bernard Ryffel is a PLOS ONE Editorial Board member. This point does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials. Silicosis is a lung disease caused by the inhalation of crystalline silica and is characterized by chronic leukocyte infiltration, fibroblast proliferation, and excessive collagen deposition resulting in the formation of localized silicotic granulomas in the lung [1]. Silicosis reduces lung functions and still remains a prevalent health problem throughout the world, particularly in developing nations [2,3]. It is generally accepted that silica particles activate innate immunity, culminating in the release of pro-inflammatory mediators and growth factors for fibroblasts, which are crucial in driving alveolitis, lung fibrosis, and possibly carcinogenesis [4,5]. Innate immune responses to silica require particle interactions with Fc and MARCO receptors on macrophages or dendritic cells [68]. In response to silica, the processing and secretion of the proinflammatory cytokine Interleukin-1 b (IL-1b) in a caspase-1/ NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-dependent manner initiates a cascade of innate immune responses leading to neutrophilic inflammation and granulomas [913]. Tumor Necrosis Factor a (TNF-a), which promotes the activation of innate immune cells, is also implicated in the pathogenesis of silicosis. Silica-induced lung inflammation and granuloma formation are reduced by the administration of TNF inhibitors or in animals deficient in TNF-a receptors [14 16]. The exacerbation of innate immune responses by repeated LPS exposure also amplifies the granulomatous response to silica in mice [17]. Finally, systemic inhibition of NF-kappa B activation with a pharmacological inhibitor decreases the severity of experimental silicosis [18]. Clinical observations have also led to the assumption that the activation of innate immunity is an important orchestrator of the silicotic process. In humans, several investigators have stressed the close link between innate immune cytokines (IL-1 and TNF-a), chronic inflammation and silicosis [14,19,20]. In conclusion, the prevailing pathogenic paradigm states that silicotic granuloma formation is dependent on the activation of innate immunity. MyD88 links members of the toll-like receptor (TLR), nucleotide-binding oligomerization domain receptor (NLR) and interleukin-1 receptor (IL-1R) superfamily to the downstream activation of NF-kappa B and mitogen-activated protein kinases [21]. To better characterize the innate immune signals involved in the development of particle-induced inflammation, granuloma formation and fibrosis, we determined lung responses to silica in mice KO for MyD88. We demonstrated that MyD88 is crucial for the development of silicotic inflammation and granulomas. However, MyD88-KO mice developed pronounced lung fibrosis even in the absence of progressive inflammation indicating that fibrogenesis is a pathological process, which can also occur independently of inflammatory and innate immune responses. Animals C57BL/6 mice were purchased from Charles River Laboratory (Brussels, Belgium). MyD88-, ASC-, IL-1R1-, IL-1a-, IL-1b-, NALP3-, TLR2/4, TLR3-, TRIF-, IL-23p19-, TCRd- [2228] deficient mice (all on a C57BL/6 background) were obtained from Transgenose (Orleans, France). Mice were maintained in sterile microisolators with sterile rodent feed and acidified water and housed in positive-pressure air-conditioned units (25uC, 50% relative humidity) on a 12-h light/dark cycle. The experimental protocol was approved by the local ethical committee for animal research at the Universite catholique de Louvain (2010/UCL/ MD/034) and conformed to the Belgian and European Community regulations (LA1230312 and CEE nu 86/609). Animal treatmen (...truncated)