Abstract
Inhalation of crystalline silica (SiO2) is a risk factor of systemic autoimmune diseases such as systemic sclerosis (SSc) and fibrotic pulmonary disorders such as silicosis. A defect of apoptotic cell clearance (i.e., efferocytosis, a key process in the resolution of inflammation) is reported in macrophages from patients with fibrotic or autoimmune diseases. However, the precise links between SiO2 exposure and efferocytosis impairment remain to be determined. Answering to this question may help to better link innate immunity and fibrosis. In this study, we first aim to determine whether SiO2 might alter efferocytosis capacities of human and mouse macrophages. We secondly explore possible mechanisms explaining efferocytosis impairment, with a specific focus on macrophage polarization and on the RhoA/ROCK pathway, a key regulator of cytoskeleton remodeling and phagocytosis. Human monocyte-derived macrophages (MDM) and C57BL/6J mice exposed to SiO2 and to CFSE-positive apoptotic Jurkat cells were analyzed by flow cytometry to determine their efferocytosis index (EI). The effects of ROCK inhibitors (Y27632 and Fasudil) on EI of SiO2-exposed MDM and MDM from SSc patients were evaluated in vitro. Our results demonstrated that SiO2 significantly decreased EI of human MDM in vitro and mouse alveolar macrophages in vivo. In human MDM, this SiO2-associated impairment of efferocytosis, required the expression of the membrane receptor SR-B1 and was associated with a decreased expression of M2 polarization markers (CD206, CD204, and CD163). F-actin staining, RhoA activation and impairment of efferocytosis, all induced by SiO2, were reversed by ROCK inhibitors. Moreover, the EI of MDM from SSc patients was similar to the EI of in vitro- SiO2-exposed MDM and Y27632 significantly increased SSc MDM efferocytosis capacities, suggesting a likewise activation of the RhoA/ROCK pathway in SSc. Altogether, our results demonstrate that SiO2 exposure may contribute to the impairment of efferocytosis capacities of mouse and human macrophages but also of MDM in SiO2-associated autoimmune diseases and fibrotic disorders such as SSc; in this context, the silica/RhoA/ROCK pathway may constitute a relevant therapeutic target.
Highlights
Exploring the pathogenesis of silica-associated fibrotic and autoimmune disorders may help to decipher the link between fibrotic diseases and autoimmunity
The efferocytosis index (EI) of human monocyte-derived macrophages (MDM) exposed to apoptotic Jurkat cells (M0 + apoJ) was significantly higher (32.4 ± 2.2) than the EI of MDM exposed to non-apoptotic Jurkat cells (M0 + J) (11.1 ± 1.1), confirming the specificity of this efferocytosis assay (Figure 1A)
Considering that the RhoA/Rho-associated protein kinase (ROCK) pathway modulates cytoskeleton in MDM [29] and that RhoA/ROCK inhibitors may shift M1 into M2 M [30,31,32] and could restore efferocytosis reduced by chemical pollutants [33, 34], we explored the role of the RhoA/ROCK pathway in the impairment of efferocytosis induced by SiO2 exposure
Summary
Exploring the pathogenesis of silica-associated fibrotic and autoimmune disorders may help to decipher the link between fibrotic diseases and autoimmunity. The consequences of crystalline silica (SiO2) on health firstly include respiratory disorders, and silicosis, a granulomatous and fibrotic interstitial lung disease [1]. Beyond these direct pulmonary effects, independent epidemiological studies show that exposure to crystalline silica increases the risk of autoimmune connective tissue diseases (CTD) with chronic inflammation such as systemic sclerosis (SSc) or systemic lupus erythematosus (SLE) [2,3,4]. SSc is considered as the main autoimmune disorder associated with silica exposure and, among rheumatic diseases, this chronic inflammatory and fibrotic disease involving lung and skin, has the highest casespecific mortality [5]. The pathogenic links between silica inhalation and the onset of fibrotic autoimmune disorders such as SSc are still to be determined
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