Results from omic approaches in rat or mouse models exposed to inhaled crystalline silica: a systematic review.

BACKGROUND: Crystalline silica (cSiO2) is a mineral found in rocks; workers from the construction or denim industries are particularly exposed to cSiO2 through inhalation. cSiO2 inhalation increases the risk of silicosis and systemic autoimmune diseases. Inhaled cSiO2 microparticles can reach the alveoli where they induce inflammation, cell death, auto-immunity and fibrosis but the specific molecular pathways involved in these cSiO2 effects remain unclear. This systematic review aims to provide a comprehensive state of the art on omic approaches and exposure models used to study the effects of inhaled cSiO2 in mice and rats and to highlight key results from omic data in rodents also validated in human. METHODS: The protocol of systematic review follows PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. Eligible articles were identified in PubMed, Embase and Web of Science. The search strategy included original articles published after 1990 and written in English which included mouse or rat models exposed to cSiO2 and utilized omic approaches to identify pathways modulated by cSiO2. Data were extracted and quality assessment was based on the SYRCLE's Risk of Bias tool for animal studies. RESULTS: Rats and male rodents were the more used models while female rodents and autoimmune prone models were less studied. Exposure of animals were both acute and chronic and the timing of outcome measurement through omics approaches were homogeneously distributed. Transcriptomic techniques were more commonly performed while proteomic, metabolomic and single-cell omic methods were less utilized. Immunity and inflammation were the main domains modified by cSiO2 exposure in lungs of mice and rats. Less than 20% of the results obtained in rodents were finally verified in humans. CONCLUSION: Omic technics offer new insights on the effects of cSiO2 exposure in mice and rats although the majority of data still need to be validated in humans. Autoimmune prone model should be better characterised and systemic effects of cSiO2 need to be further studied to better understand cSiO2-induced autoimmunity. Single-cell omics should be performed to inform on pathological processes induced by cSiO2 exposure.

Effects of different amosite preparations on macrophages, lung damages, and autoimmunity.

The underlying mechanisms of asbestos-related autoimmunity are poorly understood. As the size, surface reactivity, and free radical activity of asbestos particles are considered crucial regarding the health effects, this study aims to compare the effects of exposure to pristine amosite (pAmo) or milled amosite (mAmo) particles on lung damage, autoimmunity, and macrophage phenotype. Four months after lung exposure to 0.1 mg of amosite, BAL levels of lactate dehydrogenase, protein, free DNA, CCL2, TGF-ß1, TIMP-1, and immunoglobulin A of pAmo-exposed C57Bl/6 mice were increased when compared to fluids from control- and mAmo-exposed mice. Effects in pAmo-exposed mice were associated with lung fibrosis and autoimmunity including anti-double-strand DNA autoantibody production. mAmo or pAmo at 20 µg/cm2 induced a pro-inflammatory phenotype characterized by a significant increase in TNFα and IL-6 secretion on human monocyte-derived macrophages (MDMs). mAmo and pAmo exposure induced a decrease in the efferocytosis capacities of MDMs, whereas macrophage abilities to phagocyte fluorescent beads were unchanged when compared to control MDMs. mAmo induced IL-6 secretion and reduced the percentage of MDMs expressing MHCII and CD86 markers involved in antigen and T-lymphocyte stimulation. By contrast, pAmo but not mAmo activated the NLRP3 inflammasome, as evaluated through quantification of caspase-1 activity and IL-1ß secretion. Our results demonstrated that long-term exposure to pAmo may induce significant lung damage and autoimmune effects, probably through an alteration of macrophage phenotype, supporting in vivo the higher toxicity of entire amosite (pAmo) with respect to grinded amosite. However, considering their impact on efferocytosis and co-stimulation markers, mAmo effects should not be neglected. KEY MESSAGES: Lung fibrosis and autoimmunity induced by amosite particles depend on their physicochemical characteristics (size and surface) Inhalation exposure of mice to pristine amosite fibers is associated with lung fibrosis and autoimmunity Anti-dsDNA antibody is a marker of autoimmunity in mice exposed to pristine amosite fibers Activation of lung mucosa-associated lymphoid tissue, characterized by IgA production, after exposure to pristine amosite fibers Pristine and milled amosite particle exposure reduced the efferocytosis capacity of human-derived macrophages.

Distinct Pro-Inflammatory Mechanisms Elicited by Short and Long Amosite Asbestos Fibers in Macrophages.

While exposure to long amphibolic asbestos fibers (L > 10 µm) results in the development of severe diseases including inflammation, fibrosis, and mesothelioma, the pathogenic activity associated with short fibers (L < 5 µm) is less clear. By exposing murine macrophages to short (SFA) or long (LFA) fibers of amosite asbestos different in size and surface chemistry, we observed that SFA internalization resulted in pyroptotic-related immunogenic cell death (ICD) characterized by the release of the pro-inflammatory damage signal (DAMP) IL-1α after inflammasome activation and gasdermin D (GSDMD)-pore formation. In contrast, macrophage responses to non-internalizable LFA were associated with tumor necrosis factor alpha (TNF-α) release, caspase-3 and -7 activation, and apoptosis. SFA effects exclusively resulted from Toll-like receptor 4 (TLR4), a pattern-recognition receptor (PRR) recognized for its ability to sense particles, while the response to LFA was elicited by a multifactorial ignition system involving the macrophage receptor with collagenous structure (SR-A6 or MARCO), reactive oxygen species (ROS) cascade, and TLR4. Our findings indicate that asbestos fiber size and surface features play major roles in modulating ICD and inflammatory pathways. They also suggest that SFA are biologically reactive in vitro and, therefore, their inflammatory and toxic effects in vivo should not be underestimated.

Effects of Ruxolitinib on fibrosis in preclinical models of systemic sclerosis.

Systemic sclerosis (SSc) is an autoimmune fibrotic disorder notably characterized by the production of antinuclear autoantibodies, which have been linked to an excess of apoptotic cells, normally eliminated by a macrophagic efferocytosis. As interferon (IFN) signature and phosphorylation of JAK-STAT proteins are hallmarks of SSc tissues, we tested the hypothesis that a JAK inhibitor, ruxolitinib, targeting the IFN signaling, could improve efferocytosis of IFN-exposed human macrophages in vitro as well as skin and lung fibrosis. In vivo, BLM- and HOCl-induced skin thickness and fibrosis is associated with an increase of caspase-3 positive dermal cells and a significant increase of IFN-stimulated genes expression. In BLM-SSc model, ruxolitinib prevented dermal thickness, fibrosis and significantly decreased the number of cleaved caspase-3 cells in the dermis. Ruxolitinib also improved lung architecture and fibrosis although IFN signature was not entirely decreased by ruxolitinib. In vitro, ruxolitinib improves efferocytosis capacity of human monocyte-differentiated macrophages exposed to IFN-γ or IFN-ß. In human fibroblasts derived from lung (HLF) biopsies isolated from patients with idiopathic pulmonary fibrosis, the reduced mRNA expression of typical TGF-ß-activated markers by ruxolitinib was associated with a decrease of the phosphorylation of SMAD2 /3 and STAT3. Our finding supports the anti-fibrotic properties of ruxolitinib in a systemic SSc mouse model and in vitro in human lung fibroblasts.

Contribution of monocytes and macrophages to the pathogenesis of systemic sclerosis: recent insights and therapeutic implications.

PURPOSE OF REVIEW: To discuss recent studies addressing the role of monocytes and macrophages in the pathogenesis of systemic sclerosis (SSc) based on human and mouse models. RECENT FINDINGS: Studies indicate that monocyte adhesion could be increased in SSc secondary to an interferon-dependent loss of CD52, and chemotaxis up-regulated through the CCR3/CCL24 pathway. Beyond the conventional M1/M2 paradigm of macrophage subpopulations, new subpopulations of macrophages have been recently described in skin and lung biopsies from SSc patients. Notably, single-cell ribonucleic acid sequencing has provided evidence for SPP1+ lung macrophages or FCGR3A+ skin macrophages in SSc. Impaired pro-resolving capacities of macrophages such as efferocytosis, i.e. the ability to phagocyte apoptotic cells, could also participate in the inflammatory and autoimmune features in SSc. SUMMARY: Through their potential pro-fibrotic and pro-inflammatory properties, macrophages are at the cross-road of key SSc pathogenic processes and associated manifestations. Investigative drugs targeting macrophage polarization, such as pan-janus kinase inhibitors (tofacitinib or ruxolitinib) impacting both M1 and M2 activations, or Romilkimab inhibiting IL-4 and IL-13, have shown promising results in preclinical models or phase I/II clinical trials in SSc and other fibro-inflammatory disorders. Macrophage-based cellular therapy may also represent an innovative approach for the treatment of SSc, as initial training of macrophages may modulate the severity of fibrotic and autoimmune manifestations of the disease.

Interactions of janus kinase inhibitors with drug transporters and consequences for pharmacokinetics and toxicity.

Introduction: Janus kinase inhibitors (JAKinibs) constitute an emerging and promising pharmacological class of anti-inflammatory or anti-cancer drugs, used notably for the treatment of rheumatoid arthritis and some myeloproliferative neoplasms.Areas covered: This review provides an overview of the interactions between marketed JAKinibs and major uptake and efflux drug transporters. Consequences regarding pharmacokinetics, drug-drug interactions and toxicity are summarized.Expert opinion: JAKinibs interact in vitro with transporters in various ways, as inhibitors or as substrates of transporters or as regulators of transporter expression. This may theoretically result in drug-drug interactions (DDIs), with JAKinibs acting as perpetrators or as victims, or in toxicity, via impairment of thiamine transport. Clinical significance in terms of DDIs for JAKinib-transporter interactions remains however poorly documented. In this context, the in vivo unbound concentration of JAKinibs is likely a key parameter to consider for evaluating the clinical relevance of JAKinibs-mediated transporter inhibition. Additionally, the interplay with drug metabolism as well as possible interactions with transporters of emerging importance and time-dependent inhibition have to be taken into account. The role drug transporters may play in controlling cellular JAKinib concentrations and efficacy in target cells is also an issue of interest.

Association of silica exposure with chest HRCT and clinical characteristics in systemic sclerosis.

CONTEXT: Thoracic lymphadenopathy (LA) has been identified as a key prognostic factor in interstitial lung disease (ILD) of all-cause. Crystalline silica is a risk factor of systemic sclerosis (SSc). The association of a history of crystalline silica exposure with chest high-resolution computed tomography (HRCT) features and thoracic LA are still to be determined in SSc patients. OBJECTIVES: We performed an observational study to assess the association of lifetime exposure to silica, with chest HRCT characteristics in a population of SSc patients fulfilling the 2013 ACR/EULAR classification criteria for SSc. METHODS: A specific questionnaire based on a multidisciplinary approach was used to assess occupational and non-occupational exposure to silica in 100 consecutive SSc patients. Clinical characteristics and chest HRCT at diagnosis and at the latest visit were evaluated to assess the association of silica exposure with disease characteristics. RESULTS: 16% of the overall population and 58% of men had an occupation with specific high silica exposure. A higher silica exposure score was associated with the combination of mediastinal and hilar LA on HRCT (OR=8.09, 95%CI=2.01-32.52, P = 0.002). More than 12% of the patients had a combination of mediastinal and hilar LA on HRCT. This marker of silica exposure was predictive of worsening of pulmonary involvement in univariate analysis (OR=5.86, 95%CI=1.64-20.89, P = 0.007) and multivariate analysis (OR=4.57, 95%CI =1.12-18.60, P = 0.034). CONCLUSIONS: In patients with SSc, the combination of mediastinal and hilar LA on HRCT was associated with exposure to silica and was also significantly associated with a more severe evolution of ILD.

Nitrogen Dioxide Inhalation Exposures Induce Cardiac Mitochondrial Reactive Oxygen Species Production, Impair Mitochondrial Function and Promote Coronary Endothelial Dysfunction.

Traffic air pollution is a major health problem and is recognized as an important risk factor for cardiovascular (CV) diseases. In a previous experimental study, we showed that diesel exhaust (DE) exposures induced cardiac mitochondrial and CV dysfunctions associated with the gaseous phase. Here, we hypothesized that NO2 exposures to levels close to those found in DE induce a mitochondrial reactive oxygen species (ROS) production, which contribute to an endothelial dysfunction, an early indicator for numerous CV diseases. For this, we studied the effects of NO2 on ROS production and its impacts on the mitochondrial, coronary endothelial and cardiac functions, after acute (one single exposure) and repeated (three h/day, five days/week for three weeks) exposures in Wistar rats. Acute NO2 exposure induced an early but reversible mitochondrial ROS production. This event was isolated since neither mitochondrial function nor endothelial function were impaired, whereas cardiac function assessment showed a reversible left ventricular dysfunction. Conversely, after three weeks of exposure this alteration was accompanied by a cardiac mitochondrial dysfunction highlighted by an alteration of adenosine triphosphate (ATP) synthesis and oxidative phosphorylation and an increase in mitochondrial ROS production. Moreover, repeated NO2 exposures promoted endothelial dysfunction of the coronary arteries, as shown by reduced acetylcholine-induced vasodilatation, which was due, at least partially, to a superoxide-dependent decrease of nitric oxide (NO) bioavailability. This study shows that NO2 exposures impair cardiac mitochondrial function, which, in conjunction with coronary endothelial dysfunction, contributes to cardiac dysfunction. Together, these results clearly identify NO2 as a probable risk factor in ischemic heart diseases.

Comparative study on gene expression profile in rat lung after repeated exposure to diesel and biodiesel exhausts upstream and downstream of a particle filter.

Biodiesel is considered as a valuable and less toxic alternative to diesel. However, cellular and molecular effects of repeated exposure to biodiesel emissions from a recent engine equipped with a diesel particle filter (DPF) remain to be characterized. To gain insights about this point, the lung transcriptional signatures were analyzed for rats (n = 6 per group) exposed to filtered air, 30% rapeseed biodiesel (B30) blend or reference diesel (RF0), upstream and downstream a DPF, for 3 weeks (3 h/day, 5 days/week). Genomic analysis revealed a modest regulation of gene expression level (lower than a 2-fold) by both fuels and a higher number of genes regulated downstream the DPF than upstream, in response to either RF0 or to B30 exhaust emissions. The presence of DPF was found to notably impact the lung gene signature of rats exposed to B30. The number of genes regulated in common by both fuels was low, which is likely due to differences in concentrations of regulated pollutants in exhausts, notably for compound organic volatiles, polycyclic aromatic hydrocarbons, NO or NOx. Nevertheless, we have identified some pathways that were activated for both exhaust emissions, such as integrin-, IGF-1- and Rac-signaling pathways, likely reflecting the effects of gas phase products. By contrast, some canonical pathways relative to "oxidative phosphorylation" and "mitochondrial dysfunction" appear as specific to B30 exhaust emission; the repression of transcripts of mitochondrial respiratory chain in lung of rats exposed to B30 downstream of DPF supports the perturbation of mitochondria function. This study done with a recent diesel engine (compliant with the European IV emission standard) and commercially-available fuels reveals that the diesel blend composition and the presence of an after treatment system may modify lung gene signature of rats repeatedly exposed to exhaust emissions, however in a rather modest manner.

Combined anti-fibrotic and anti-inflammatory properties of JAK-inhibitors on macrophages in vitro and in vivo: Perspectives for scleroderma-associated interstitial lung disease.

Janus kinase (JAK) inhibitors (also termed Jakinibs) constitute a family of small drugs that target various isoforms of JAKs (JAK1, JAK2, JAK3 and/or tyrosine kinase 2 (Tyk2)). They exert anti-inflammatory properties linked, in part, to the modulation of the activation state of pro-inflammatory M1 macrophages. The exact impact of JAK inhibitors on a wider spectrum of activation states of macrophages is however still to be determined, especially in the context of disorders involving concomitant activation of pro-inflammatory M1 macrophages and profibrotic M2 macrophages. This is especially the case in autoimmune pulmonary fibrosis like scleroderma-associated interstitial lung disease (ILD), in which M1 and M2 macrophages play a key pathogenic role. In this study, we directly compared the anti-inflammatory and anti-fibrotic effects of three JAK inhibitors (ruxolitinib (JAK2/1 inhibitor); tofacitinib (JAK3/2 inhibitor) and itacitinib (JAK1 inhibitor)) on five different activation states of primary human monocyte-derived macrophages (MDM). These three JAK inhibitors exert anti-inflammatory properties towards macrophages, as demonstrated by the down-expression of key polarization markers (CD86, MHCII, TLR4) and the limited secretion of key pro-inflammatory cytokines (CXCL10, IL-6 and TNFα) in M1 macrophages activated by IFNγ and LPS or by IFNγ alone. We also highlighted that these JAK inhibitors can limit M2a activation of macrophages induced by IL-4 and IL-13, as notably demonstrated by the down-regulation of the M2a associated surface marker CD206 and of the secretion of CCL18. Moreover, these JAK inhibitors reduced the expression of markers such as CXCL13, MARCO and SOCS3 in alternatively activated macrophages induced by IL-10 and dexamethasone (M2c + dex) or IL-10 alone (M2c MDM). For all polarization states, Jakinibs with inhibitory properties over JAK2 had the highest effects, at both 1 µM or 0.1 µM. Based on these in vitro results, we also explored the effects of JAK2/1 inhibition by ruxolitinib in vivo, on mouse macrophages in a model of HOCl-induced ILD, that mimics scleroderma-associated ILD. In this model, we showed that ruxolitinib significantly prevented the upregulation of pro-inflammatory M1 markers (TNFα, CXCL10, NOS2) and pro-fibrotic M2 markers (Arg1 and Chi3L3). These results were associated with an improvement of skin and pulmonary involvement. Overall, our results suggest that the combined anti-inflammatory and anti-fibrotic properties of JAK2/1 inhibitors could be relevant to target lung macrophages in autoimmune and inflammatory pulmonary disorders that have no efficient disease modifying drugs to date.

Crystalline Silica Impairs Efferocytosis Abilities of Human and Mouse Macrophages: Implication for Silica-Associated Systemic Sclerosis.

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.

TNF-α and IL-10 Control CXCL13 Expression in Human Macrophages.

The chemokine CXCL13 controls the normal organization of secondary lymphoid tissues and the neogenesis of ectopic lymphoid structures in nonlymphoid organs, particularly the lungs. The progression and severity of idiopathic pulmonary fibrosis (IPF), a fatal and irreversible interstitial lung disease, is predicted by the circulating blood concentrations of CXCL13. Although CXCL13 is produced by pulmonary tissues, it has not been determined which cells are involved. This study examines CXCL13 production by lung tissue macrophages from patients with IPF and the signaling pathways controlling CXCL13 gene expression in human alveolar macrophages (AM) and monocyte-derived macrophages (MoDM). CXCL13 is found in CD68- and CD206-positive AM from patients with IPF, and the CXCL13 gene is induced in these macrophages and MoDM when they are stimulated with LPS. We found that TNF-α and IL-10 control optimal CXCL13 gene expression in MoDM and possibly in AM by activating the NF-κB and JAK/STAT pathways, respectively. We also found that blood TNF-α and CXCL13 concentrations are significantly correlated in patients with IPF, suggesting that TNF-α contributes to CXCL13 production in humans. In conclusion, the results of this study demonstrate that AM from patients with IPF produces CXCL13 and that the NF-κB and JAK/STAT pathways are required to induce the expression of this major chemokine.

Potent repression of C-reactive protein (CRP) expression by the JAK1/2 inhibitor ruxolitinib in inflammatory human hepatocytes.

OBJECTIVE AND DESIGN: To determine whether inflammatory hepatocytes may constitute primary targets for ruxolitinib, a Janus kinase (JAK) inhibitor, its effects towards expression of hepatic acute-phase proteins, especially C-reactive protein (CRP), were assessed. MATERIALS: Ruxolitinib effects were analysed in primary human hepatocytes and human hepatoma HepaRG cells exposed to various inflammatory stimuli. RESULTS: Ruxolitinib was found to fully inhibit lipopolysaccharide (LPS)-induced CRP secretion and mRNA expression, at concentrations (IC50 = 12.9 nM) achievable in human blood. It similarly repressed CRP up-regulation due to several Toll-like receptor agonists or pro-inflammatory cytokines [interleukin (IL) 1ß, IL6 and tumour necrosis factor α] and counteracted LPS-mediated induction of serum amyloid A, fibrinogen, haptoglobin and serpin. Ruxolitinib was additionally found to block the activation of the IL6/JAK/signal transducer and activator of transcription (STAT) pathway triggered by LPS and whose inhibition by the neutralizing anti-IL6 receptor antibody tocilizumab prevented CRP induction. CONCLUSION: Ruxolitinib can potently repress induction of CRP in inflammatory human hepatocytes, most likely through targeting the IL6/JAK/STAT signalling cascade. Hepatic production of acute-phase proteins during liver inflammation may, therefore, constitute a target for ruxolitinib.

Alteration of human macrophage phenotypes by the anti-fibrotic drug nintedanib.

The tyrosine kinase inhibitor, Nintedanib (NTD), has been approved for the treatment of idiopathic pulmonary fibrosis (IPF). In cell-free systems, NTD was recently shown to inhibit kinase activity of the human recombinant colony-stimulating factor 1 (CSF1) receptor (CSF1R) which mediates major functions of pulmonary macrophages. In the present study, we have investigated the effects of NTD on the phenotype of human monocyte-derived macrophages controlled by CSF1 in order to identify its anti-inflammatory properties via CSF1R inhibition. NTD (0.01 to 1 µM) prevented the CSF1-induced phosphorylation of CSF1R and activation of the downstream signaling pathways. NTD, like the CSF1R inhibitor GW2580, significantly decreased the adhesion of macrophages and production of the chemokine ligand (CCL) 2. NTD also altered the polarization of macrophages to classical M1 and alternative M2a macrophages. It reduced the secretion of several pro-inflammatory and/or pro-fibrotic cytokines (IL-1ß, IL-8, IL-10 and CXCL13) by M1 macrophages but did not prevent the expression of M1 markers. While NTD (50-200 nM) partially blocked the synthesis of M2a markers (CD11b, CD200R, CD206, and CD209), it did not reduce synthesis of the M2a pro-fibrotic cytokines CCL22 and PDGF-BB, and increased CCL18 release when used at its highest concentration (1 µM). The effects of NTD on macrophage polarization only was partially mimicked by GW2580, suggesting that the drug inhibits other molecules in addition to CSF1R. In conclusion, NTD alters the CSF1-controlled phenotype of human macrophages mainly by blocking the activation of CSF1R that thus constitutes a new molecular target of NTD, at least in vitro.

Induction of multidrug resistance-associated protein 3 expression by diesel exhaust particle extract in human bronchial epithelial BEAS-2B cells.

Diesel exhaust particles (DEPs) are common environmental air pollutants known to impair expression and activity of drug detoxifying proteins, including hepatic ATP-binding cassette (ABC) drug transporters. The present study was designed to determine whether organic DEP extract (DEPe) may also target ABC drug transporters in bronchial cells. DEPe (10 µg/mL) was demonstrated to induce mRNA and protein expression of the multidrug resistance-associated protein (MRP) 3 in cultured bronchial epithelial BEAS-2B cells, whereas mRNA levels of other MRPs, multidrug resistance gene 1 or breast cancer resistance protein were unchanged, reduced or not detected. DEPe also increased MRP3 mRNA expression in normal human bronchial epithelial cells. Inhibition of the aryl hydrocarbon receptor (AhR) pathway by AhR antagonist or AhR silencing, as well as the silencing of nuclear-factor-E2-related factor 2 (Nrf2) repressed DEPe-mediated MRP3 induction. This underlines the implication of the AhR and Nrf2 signaling cascades in DEPe-mediated MRP3 regulation. DEPe was additionally demonstrated to directly inhibit MRP activity in BEAS-2B cells, in a concentration-dependent manner. Taken together, these data indicate that DEPs may impair expression and activity of MRPs, notably MRP3, in human bronchial cells, which may have consequences in terms of lung barrier and toxicity for humans exposed to diesel pollution.

A novel Staphylococcus aureus cis-trans type I toxin-antitoxin module with dual effects on bacteria and host cells.

Bacterial type I toxin-antitoxin (TA) systems are widespread, and consist of a stable toxic peptide whose expression is monitored by a labile RNA antitoxin. We characterized Staphylococcus aureus SprA2/SprA2AS module, which shares nucleotide similarities with the SprA1/SprA1AS TA system. We demonstrated that SprA2/SprA2AS encodes a functional type I TA system, with the cis-encoded SprA2AS antitoxin acting in trans to prevent ribosomal loading onto SprA2 RNA. We proved that both TA systems are distinct, with no cross-regulation between the antitoxins in vitro or in vivo. SprA2 expresses PepA2, a toxic peptide which internally triggers bacterial death. Conversely, although PepA2 does not affect bacteria when it is present in the extracellular medium, it is highly toxic to other host cells such as polymorphonuclear neutrophils and erythrocytes. Finally, we showed that SprA2AS expression is lowered during osmotic shock and stringent response, which indicates that the system responds to specific triggers. Therefore, the SprA2/SprA2AS module is not redundant with SprA1/SprA1AS, and its PepA2 peptide exhibits an original dual mode of action against bacteria and host cells. This suggests an altruistic behavior for S. aureus in which clones producing PepA2 in vivo shall die as they induce cytotoxicity, thereby promoting the success of the community.