Inhalation of crystalline silica (SiO
) 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 SiO
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 SiO
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 SiO
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 SiO
-exposed MDM and MDM from SSc patients were evaluated
. Our results demonstrated that SiO
significantly decreased EI of human MDM
and mouse alveolar macrophages
. In human MDM, this SiO
-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 SiO
, were reversed by ROCK inhibitors. Moreover, the EI of MDM from SSc patients was similar to the EI of
- SiO
-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 SiO
exposure may contribute to the impairment of efferocytosis capacities of mouse and human macrophages but also of MDM in SiO
-associated autoimmune diseases and fibrotic disorders such as SSc; in this context, the silica/RhoA/ROCK pathway may constitute a relevant therapeutic target.
Macrophages play a central role in the pathogenesis of inflammatory and fibrotic lung diseases. However, alveolar macrophages (AM) are poorly available in humans to perform in vitro studies due to a ...limited access to broncho-alveolar lavage (BAL). In this study, to identify the best alternative in vitro model for human AM, we compared the phenotype of AM obtained from BAL of patients suffering from three lung diseases (lung cancers, sarcoidosis and Systemic Sclerosis (SSc)-associated interstitial lung disease) to human blood monocyte-derived macrophages (MDMs) differentiated with M-CSF or GM-CSF. The expression of eight membrane markers was evaluated by flow cytometry. Globally, AM phenotype was closer to GM-CSF MDMs. However, the expression levels of CD163, CD169, CD204, CD64 and CD36 were significantly higher in SSc-ILD than in lung cancers. Considering the expression of CD204 and CD36, the phenotype of SSc-AM was closer to MDMs, from healthy donors or SSc patients, differentiated by M-CSF rather than GM-CSF. The comparative secretion of IL-6 by SSc-MDMs and SSc-AM is concordant with these phenotypic considerations. Altogether, these results support the M-CSF MDM model as a relevant in vitro alternative to simulate AM in fibrotic disorders such as SSc.
A defect in the apoptotic cell clearance (efferocytosis) by phagocytic cells may participate in autoimmunity and chronic inflammation. The mechanisms leading to the emergence of autoimmunity in ...systemic sclerosis (SSc) are still to be determined. In this study, the efferocytosis capacities of blood monocyte‐derived macrophages (MDM) from patients with SSc were evaluated. Blood monocytes obtained from patients with SSc and healthy donors (HD) were differentiated in vitro into macrophages. The capacities of MDM to engulf CFSE+ apoptotic Jurkat human T lymphocytes were compared between SSc MDM and HD using flow cytometry. The expression of classical engulfing receptors in SSc MDM and HD MDM was also evaluated and their involvement in the modulation of efferocytosis was confirmed using a siRNA approach. The mean phagocytic index (PI) reflecting efferocytosis capacities of SSc MDM (PI = 19.3 ± 3.0; n = 21) was significantly decreased in comparison with the PI of HD MDM (PI = 35.9 ± 3.0; n = 31; P < 0.001). In comparison with HD, SSc MDM exhibited a downregulated expression of scavenger receptor (SR)‐B1, SR‐A1 and integrin β5 (ITGβ5). In HD MDM, the extinction of these receptors was followed by a reduction of efferocytosis only for the repression of ITGβ5, suggesting a possible selective role of this integrin in the impaired efferocytosis observed in SSc. As efferocytosis may be at the crossroads of inflammation, autoimmunity and fibrosis, in showing impaired efferocytosis capacities of blood MDM in SSc, our study offers new pathogenesis considerations for the involvement of macrophages in the autoimmune processes driving this disorder.
Efferocytosis capacities are strongly decreased in monocyte‐derived macrophages from systemic sclerosis (SSc) patients. A downexpression of integrin β5 in proinflammatory and in SSc macrophages may explain a deficient efferocytosis. Impaired efferocytosis of macrophages may play a key role in autoimmune processes in SSc.
Ruxolitinib is a Janus kinase (JAK) 1/2 inhibitor, currently used in the treatment of myeloproliferative neoplasms. It exerts potent anti-inflammatory activity, but the involved molecular and ...cellular mechanisms remain poorly understood. In order to gain insights about this point, ruxolitinib effects towards expression of main inflammatory cytokines were studied in human macrophages, which constitute a key-cell type implicated in inflammation. Analysis of mRNA expression of cytokines (n=84) by PCR array indicated that, among those induced by the pro-inflammatory stimulus lipopolysaccharide (LPS) (n=44), 61.4% (n=27) were repressed by 5μM ruxolitinib. The major inflammatory cytokines, interleukin (IL) 6 and tumor necrosis factor α, were notably down-regulated by ruxolitinib at both the mRNA and protein level. Other repressed cytokines included IL27 and the chemokines CCL2, CXCL9, CXCL10 and CXCL11, but not IL1β. The interferon (IFN) β/JAK/signal transducer and activator of transcription (STAT) pathway, well-activated by LPS in human macrophages as demonstrated by increased secretion of IFNβ, STAT1 phosphorylation, and up-regulation of reference IFNβ-responsive genes, was concomitantly blocked by the JAK inhibitor. Most of cytokines targeted by ruxolitinib were shown to be regulated by IFNβ in a JAK-sensitive manner. In addition, counteracting the IFNβ/JAK/STAT cascade using a blocking monoclonal antibody directed against IFNβ receptor resulted in a similar profile of cytokine repression to that observed in response to the JAK inhibitor. Overall, these data provide evidence for ruxolitinib-mediated repression of inflammatory cytokines in human macrophages through inhibition of the LPS/IFNβ/JAK/STAT signalling pathway, which probably contributes to the anti-inflammatory effects of the JAK inhibitor.
•The JAK inhibitor ruxolitinib represses cytokine induction in LPS-treated macrophages•Most of inflammatory cytokines targeted by ruxolitinib are regulated by IFNβ•Ruxolitinib impairs cytokine expression by inhibiting LPS/TLR4/IFNβ signaling pathway•Cytokine repression likely contributes to the anti-inflammatory effect of ruxolitinib
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