Extracellular vesicles as actors in the air pollution related cardiopulmonary diseases.

Many associations were reported between air pollution and daily mortality rates for cardiopulmonary diseases. Humans are exposed to a mixture of oxidizing gases and particles, both anthropogenic and natural. Exposure to air toxics causes or exacerbates cardiovascular damages and respiratory diseases. Numerous studies have identified the induction of oxidative stress and sustained inflammatory response as among the main known underlying pathophysiological mechanisms of air pollutants. More recently, the relationship between these mechanisms of action and the secretion of extracellular vesicles (EVs) by lung cells has been revealed. EVs have been shown to be important mediators of cellular communication in the body. The purpose of this review is to first recall the main air pollutants. Then, the cardiopulmonary diseases caused by exposure to air pollution and the pathophysiological mechanisms are presented before showing, through an exhaustive review of the literature, the involvement of EVs in the toxicity of air pollutants and the initiation of cardiopulmonary diseases.

A prospective pilot study of the T-lymphocyte response to fine particulate matter exposure.

Exposure to air pollution is associated with increased morbidity and mortality. Once the fine atmospheric particulate matter (FP) is inhaled, some of its compounds can pass through the lungs and reach the bloodstream where they can come into contact with immune cells. Exposure to FP particularly affects sensitive populations such as the elderly. Aging affects the immune system, making the elderly more vulnerable. The project aims to determine the effects of FP exposure on human T cells while looking for biomarkers associated with exposure. Blood samples from 95 healthy subjects in three different age groups (20-30, 45-55 and 70-85 years) were collected to determine a potential age effect. T lymphocytes were isolated to be exposed ex vivo for 72 hours to 45 µg/mL of FP collected in Dunkirk and chemically characterized. Overexpression of the CYP1A1, CYP1B1 and CYP2S1 genes was therefore measured after exposure of the T cells to FP. These genes code for enzymes known to be involved in the metabolic activation of organic compounds such as polycyclic aromatic hydrocarbons detected in the FP sample. T-cell profiling allowed us to suggest a mixed T-helper 1/2 profile caused by exposure to FP. With regard to the influence of age, we have observed differences in the expression of certain genes, as well as an increase in interleukin-4 and -13 concentrations in the elderly. These results showed that exposure of T lymphocytes to FP causes effects on both transcriptomic and cytokine secretion levels.

Cellular response and extracellular vesicles characterization of human macrophages exposed to fine atmospheric particulate matter.

Exposure to fine atmospheric Particulate Matter (PM) is one of the major environmental causes involved in the development of inflammatory lung diseases, such as chronic obstructive pulmonary disease (COPD) or asthma. When PM is penetrating in the pulmonary system, alveolar macrophages represent the first line of defense, in particular by triggering a pro-inflammatory response, and also by their ability to recruit infiltrating macrophages from the bone marrow. The aim of this in vitro study was to evaluate the gene expression and cytokine production involved in the toxicological and inflammatory responses of infiltrating macrophages, as well as the Extracellular Vesicles (EVs) production, after their exposure to PM. The ability of these EVs to convey information related to PM exposure from exposed macrophages to pulmonary epithelial cells was also evaluated. Infiltrating macrophages respond to fine particles exposure in a conventional manner, as their exposure to PM induced the expression of Xenobiotic Metabolizing Enzymes (XMEs) such as CYP1A1 and CYP1B1, the enzymes involved in oxidative stress SOD2, NQO1 and HMOX as well as pro-inflammatory cytokines in a dose-dependent manner. Exposure to PM also induced a greater release of EVs in a dose-dependent manner. In addition, the produced EVs were able to induce a pro-inflammatory phenotype on pulmonary epithelial cells, with the induction of the release of IL6 and TNFα proinflammatory cytokines. These results suggest that infiltrating macrophages participate in the pro-inflammatory response induced by PM exposure and that EVs could be involved in this mechanism.

Chemical characterization of fine and ultrafine PM, direct and indirect genotoxicity of PM and their organic extracts on pulmonary cells.

Particulate matter in ambient air constitutes a complex mixture of fine and ultrafine particles composed of various chemical compounds including metals, ions, and organics. A multidisciplinary approach was developed by studying physico-chemical characteristics and mechanisms involved in the toxicity of particulate atmospheric pollution. PM0.3-2.5 and PM2.5 including ultrafine particles were sampled in Dunkerque, a French industrialized seaside city. PM samples were characterized from a chemical and toxicological point of view. Physico-chemical characterization evidenced that PM2.5 comes from several sources: natural ones, such as soil resuspension and marine sea-salt emissions, as well as anthropogenic ones, such as shipping traffic, road traffic, and industrial activities. Human BEAS-2B lung cells were exposed to PM0.3-2.5, or to the Extractable Organic Matter (EOM) of PM0.3-2.5 and PM2.5. These exposures induced several mechanisms of action implied in the genotoxicity, such as oxidative DNA adducts and DNA Damage Response. The toxicity of PM-EOM was higher for the sample including the ultrafine fraction (PM2.5) containing also higher concentrations of polycyclic aromatic hydrocarbons. These results evidenced the major role of organic compounds in the toxicity of PM.

Influence of aging in the modulation of epigenetic biomarkers of carcinogenesis after exposure to air pollution.

BACKGROUND: Classified as carcinogenic to humans by the IARC in 2013, fine air particulate matter (PM2.5) can be inhaled and retained into the lung or reach the systemic circulation. This can cause or exacerbate numerous pathologies to which the elderly are often more sensitive. METHODS: In order to estimate the influence of age on the development of early cellular epigenetic alterations involved in carcinogenesis, peripheral blood mononuclear cells sampled from 90 patients from three age classes (25-30, 50-55 and 75-80 years old) were ex vivo exposed to urban PM2.5. RESULTS: Particles exposure led to variations in telomerase activity and telomeres length in all age groups without any influence of age. Conversely, P16INK4A gene expression increased significantly with age after exposure to PM2.5. Age could enhance MGMT gene expression after exposure to particles, by decreasing the level of promoter methylation in the oldest people. CONCLUSION: Hence, our results demonstrated several tendencies in cells modification depending on age, even if all epigenetic assays were carried out after a limited exposure time allowing only one or two cell cycles. Since lung cancer symptoms appear only at an advanced stage, our results underline the needs for further investigation on the studied biomarkers for early diagnosis of carcinogenesis to improve survival.

Comparative study of diesel and biodiesel exhausts on lung oxidative stress and genotoxicity in rats.

The contribution of diesel exhaust to atmospheric pollution is a major concern for public health, especially in terms of occurrence of lung cancers. The present study aimed at addressing the toxic effects of a repeated exposure to these emissions in an animal study performed under strictly controlled conditions. Rats were repeatedly exposed to the exhaust of diesel engine. Parameters such as the presence of a particle filter or the use of gasoil containing rapeseed methyl ester were investigated. Various biological parameters were monitored in the lungs to assess the toxic and genotoxic effects of the exposure. First, a transcriptomic analysis showed that some pathways related to DNA repair and cell cycle were affected to a limited extent by diesel but even less by biodiesel. In agreement with occurrence of a limited genotoxic stress in the lungs of diesel-exposed animals, small induction of γ-H2AX and acrolein adducts was observed but not of bulky adducts and 8-oxodGuo. Unexpected results were obtained in the study of the effect of the particle filter. Indeed, exhausts collected downstream of the particle filter led to a slightly higher induction of a series of genes than those collected upstream. This result was in agreement with the formation of acrolein adducts and γH2AX. On the contrary, induction of oxidative stress remained very limited since only SOD was found to be induced and only when rats were exposed to biodiesel exhaust collected upstream of the particle filter. Parameters related to telomeres were identical in all groups. In summary, our results point to a limited accumulation of damage in lungs following repeated exposure to diesel exhausts when modern engines and relevant fuels are used. Yet, a few significant effects are still observed, mostly after the particle filter, suggesting a remaining toxicity associated with the gaseous or nano-particular phases.

Smoker extracellular vesicles influence status of human bronchial epithelial cells.

Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for many diseases including cardiovascular disease, chronic obstructive pulmonary disease (COPD), asthma and lung cancer. Evaluation and understanding of tobacco health effects are of major interest worldwide and answer to important societal concerns. Identification of new biomarkers of exposure to tobacco smoke potentially implicated in COPD or lung carcinogenesis would allow a better observation of tobacco exposed population, thanks to screening establishment at reversible stages of pathological processes. In this study, we questioned whether cigarette smoking alters miRNA profiles of Extracellular Vesicles (EVs) present in human Broncho Alveolar Lavages (BALs), which could affect surrounding normal bronchial epithelial cells status. To this aim, BALs were carried out on 10 Smokers and 10 Non-Smokers, and EVs were isolated from the supernatants and characterized. We then compared the amount of 10 microRNAs (miRNAs) present in Smokers versus Non-Smokers BAL EVs and performed statistical analysis to discuss the biological significance by the smoking status and to evaluate BAL EV miRNAs as potential biomarkers of tobacco exposure. Finally, we tested the effects of smokers versus non-smokers EVs on human bronchial epithelial cells (BEAS-2B) to compare their influence on the cells status. Our study shows for the first time in human samples that smoking can alter lung EV profile that can influence surrounding bronchial epithelial cells.

Fine and ultrafine atmospheric particulate matter at a multi-influenced urban site: Physicochemical characterization, mutagenicity and cytotoxicity.

Particulate Matter (PM) air pollution is one of the major concerns for environment and health. Understanding the heterogeneity and complexity of fine and ultrafine PM is a fundamental issue notably for the assessment of PM toxicological effects. The aim of this study was to evaluate mutagenicity and cytotoxicity of a multi-influenced urban site PM, with or without the ultrafine fraction. For this purpose, PM2.5-0.3 (PM with aerodynamic diameter ranging from 0.3 to 2.5 µm) and PM2.5 were collected in Dunkerque, a French coastal industrial city and were extensively characterized for their physico-chemical properties, including inorganic and organic species. In order to identify the possible sources of atmospheric pollution, specific criteria like Carbon Preference Index (CPI) and PAH characteristic ratios were investigated. Mutagenicity assays using Ames test with TA98, TA102 and YG1041 Salmonella strains with or without S9 activation were performed on native PM sample and PM organic extracts and water-soluble fractions. BEAS-2B cell viability and cell proliferation were evaluated measuring lactate dehydrogenase release and mitochondrial dehydrogenase activity after exposure to PM and their extracts. Several contributing sources were identified in PM: soil resuspension, marine emissions including sea-salt or shipping, road traffic and industrial activities, mainly related to steelmaking or petro-chemistry. Mutagenicity of PM was evidenced, especially for PM2.5, including ultrafine fraction, in relation to PAHs content and possibly nitro-aromatics compounds. PM induced cytotoxic effects at relatively high doses, while alteration of proliferation with low PM doses could be related to underlying mechanisms such as genotoxicity.

Air Pollution modifies the association between successful and pathological aging throughout the frailty condition.

The rapid growth in the number of older adults has many implications for public health, including the need to better understand the risks posed by environmental exposures. Aging leads to a decline and deterioration of functional properties at the cellular, tissue and organ level. This loss of functional properties yields to a loss of homeostasis and decreased adaptability to internal and external stress. Frailty is a geriatric syndrome characterized by weakness, weight loss, and low activity that is associated with adverse health outcomes. Frailty manifests as an age-related, biological vulnerability to stressors and decreased physiological reserves. Ambient air pollution exposure affects human health, and elderly people appear to be particularly susceptible to its adverse effects. The aim of this paper is to discuss the role of air pollution in the modulation of several biological mechanisms involved in aging. Evidence is presented on how air pollution can modify the bidirectional association between successful and pathological aging throughout the frailty conditions.

Adipogenic RNAs are transferred in osteoblasts via bone marrow adipocytes-derived extracellular vesicles (EVs).

BACKGROUND: In osteoporosis, bone loss is accompanied by increased marrow adiposity. Given their proximity in the bone marrow and their shared origin, a dialogue between adipocytes and osteoblasts could be a factor in the competition between human Mesenchymal Stem Cells (hMSC) differentiation routes, leading to adipocyte differentiation at the expense of osteoblast differentiation. The adipocyte/osteoblast balance is highly regulated at the level of gene transcription. In our work, we focused on PPARgamma, CEBPalpha and CEBPdelta, as these transcription factors are seen as master regulators of adipogenesis and expressed precociously, and on leptin and adiponectin, considered as adipocyte marker genes. In 2010, our group has demonstrated, thanks to a coculture model, that in the presence of hMSC-derived adipocytes (hMSC-Adi), hMSC-derived osteoblasts (hMSC-Ost) express lesser amounts of osteogenic markers but exhibit the expression of typical adipogenic genes. Nevertheless, the mechanisms underlying this modulation of gene expression are not clarified. Recently, adipocytes were described as releasing extracellular vesicles (EVs), containing and transferring adipocyte specific transcripts, like PPARgamma, leptin and adiponectin. Here, we investigated whether EVs could be the way in which adipocytes transfer adipogenic RNAs in our coculture model. RESULTS: We observed in hMSC-Ost incubated in hAdi-CM an increase in the adipogenic PPARγ, leptin, CEBPα and CEBPδ transcripts as well as the anti-osteoblastic miR-138, miR30c, miR125a, miR-125b, miR-31 miRNAs, probably implicated in the observed osteocalcin (OC) and osteopontin (OP) expression decrease. Moreover, EVs were isolated from conditioned media collected from cultures of hMSC at different stages of adipocyte differentiation and these specific adipogenic transcripts were detected inside. Finally, thanks to interspecies conditioned media exposition, we could highlight for the first time a horizontal transfer of adipogenic transcripts from medullary adipocytes to osteoblasts. CONCLUSIONS: Here, we have shown, for the first time, RNA transfer between hMSC-derived adipocytes and osteoblasts through EVs. Additional studies are needed to clarify if this mechanism has a role in the adipocytic switch driven on osteoblasts by adipocytes inside bone marrow and if EVs could be a target component to regulate the competition between osteoblasts and adipocytes in the prevention or in the therapy of osteoporosis and other osteopenia.

Proinflammatory effects and oxidative stress within human bronchial epithelial cells exposed to atmospheric particulate matter (PM(2.5) and PM(>2.5)) collected from Cotonou, Benin.

After particulate matter (PM) collection in Cotonou (Benin), a complete physicochemical characterization of PM2.5 and PM>2.5 was led. Then, their adverse health effects were evaluated by using in vitro culture of human lung cells. BEAS-2B (bronchial epithelial cells) were intoxicated during short-term exposure at increasing PM concentrations (1.5-96 µg/cm(2)) to determine global cytotoxicity. Hence, cells were exposed to 3 and 12 µg/cm(2) to investigate the potential biological imbalance generated by PM toxicity. Our findings showed the ability of both PM to induce oxidative stress and to cause inflammatory cytokines/chemokines gene expression and secretion. Furthermore, PM were able to induce gene expression of enzymes involved in the xenobiotic metabolism pathway. Strong correlations between gene expression of metabolizing enzymes, proinflammatory responses and cell cycle alteration were found, as well as between proinflammatory responses and cell viability. Stress oxidant parameters were highly correlated with expression and protein secretion of inflammatory mediators.

Ectosomes of polymorphonuclear neutrophils activate multiple signaling pathways in macrophages.

Ectosomes are vesicles shed directly from the cell surface. Human polymorphonuclear neutrophils release ectosomes (PMN-Ect) upon their activation. PMN-Ect expose phosphatidylserine (PS) on the outer leaflet of the plasma membrane, and down-modulate the inflammatory response of human macrophages and dendritic cells exposed to TLR-2 and -4 ligands. This down-modulation is mediated by PS via the engagement and activation of the Mer receptor tyrosine kinase (MerTK). In the present study, we demonstrate that exposure of macrophages to PMN-Ect activates directly 2 additional pathways, an immediate Ca(2+) flux and a rapid release of TGF-ß1. As expected, the Ca(2+) flux was necessary for the activation of TLR-2 pathway with the release of cytokines. However, MerTK blockade with antibodies did not modify the Ca(2+) flux, indicating an independent activation of Ca(2+) by PMN-Ect. Striking was that the rapid release of TGF-ß1 was independent of the MerTK pathway and did not require a Ca(2+) flux. TGF-ß1 was present in cytosolic storage pools, which were depleted after exposure of the macrophages to PMN-Ect, and no increase in TGF-ß1 mRNA could be detected in the 3 first hours when maximal release had occurred. The release of TGF-ß1 by macrophages was seen only for PMN-Ect and not for PS-liposomes or erythrocyte ectosomes, which express PS. However, blocking the PS of PMN-Ect inhibited TGF-ß1 release, suggesting that PS expression was necessary although not sufficient for this release. Interestingly, the effects of PMN-Ect pre-exposure were lasting for 24h with the macrophages being less receptive to TLR-2 activation and TGF-ß1 stores remaining low. In sum, PMN-Ect induce several signaling pathways in resting and stimulated macrophages, which include independently the MerTK pathway, Ca(2+) flux and the release of stored TGF-ß1, and each might influence the immunomodulatory effects of macrophages.

Microparticles (ectosomes) shed by stored human platelets downregulate macrophages and modify the development of dendritic cells.

Microparticles (MP) shed by platelets (PLT) during storage have procoagulant activities, but little is known about their properties to modify inflammation or immunity. In this study, we studied the capacity of MP present in PLT concentrates to alter the function of macrophages and dendritic cells (DC). The size of the purified MP was between 100 and 1000 nm, and they expressed phosphatidylserine; surface proteins of PLT (CD61, CD36, CD47), including complement inhibitors (CD55, CD59), but not CD63; and proteins acquired from plasma (C1q, C3 fragments, factor H). These characteristics suggest that the MP shed by PLT are formed by budding from the cell surface, corresponding to ectosomes. The purified PLT ectosomes (PLT-Ect) reduced the release of TNF-α and IL-10 by macrophages activated with LPS or zymosan A. In addition, PLT-Ect induced the immediate release of TGF-ß from macrophages, a release that was not modified by LPS or zymosan A. Macrophages had a reduced TNF-α release even 24 h after their exposure to PLT-Ect, suggesting that PLT-Ect induced a modification of the differentiation of macrophages. Similarly, the conventional 6-d differentiation of monocytes to immature DC by IL-4 and GM-CSF was modified by the presence of PLT-Ect during the first 2 d. Immature DC expressed less HLA-DP DQ DR and CD80 and lost part of their phagocytic activity, and their LPS-induced maturation was downmodulated when exposed to PLT-Ect. These data indicate that PLT-Ect shed by stored PLT have intrinsic properties that modify macrophage and DC differentiation toward less reactive states.

Ectosomes released by polymorphonuclear neutrophils induce a MerTK-dependent anti-inflammatory pathway in macrophages.

At the earliest stage of activation, human polymorphonuclear neutrophils release vesicles derived directly from the cell surface. These vesicles, called ectosomes (PMN-Ect), expose phosphatidylserine in the outer membrane leaflet. They inhibit the inflammatory response of human monocyte-derived macrophages and dendritic cells to zymosan A (ZymA) and LPS and induce TGF-ß1 release, suggesting a reprogramming toward a tolerogenic phenotype. The receptors and signaling pathways involved have not yet been defined. Here, we demonstrate that PMN-Ect interfered with ZymA activation of macrophages via inhibition of NFκB p65 phosphorylation and NFκB translocation. The MerTK (Mer receptor tyrosine kinase) and PI3K/Akt pathways played a key role in this immunomodulatory effect as shown using specific MerTK-blocking antibodies and PI3K inhibitors LY294002 and wortmannin. As a result, PMN-Ect reduced the transcription of many proinflammatory genes in ZymA-activated macrophages. In sum, PMN-Ect interacted with the macrophages by activation of the MerTK pathway responsible for down-modulation of the proinflammatory signals generated by ZymA.

The proliferating cell nuclear antigen regulates retinoic acid receptor transcriptional activity through direct protein-protein interaction.

Retinoic acid receptors (RARs) interact, in a ligand-dependent fashion, with many coregulators that participate in a wide spectrum of biological responses, ranging from embryonic development to cellular growth control. The transactivating function of these ligand-inducible transcription factors reside mainly, but not exclusively, in their ligand-binding domain (AF2), which recruits or dismiss coregulators in a ligand-dependent fashion. However, little is known about AF2-independent function(s) of RARs. We have isolated the proliferating cell nuclear antigen (PCNA) as a repressor of RAR transcriptional activity, able to interact with an AF2-crippled RAR. The N-terminus of PCNA interacts directly with the DNA-binding domain of RAR, and PCNA is recruited to a retinoid-regulated promoter in intact cells. This interaction affects the transcriptional response to retinoic acid in a promoter-specific manner, conferring an unanticipated role to PCNA in transcriptional regulation. Our findings also suggest a role for RAR as a factor coordinating DNA transcription and repair.

Transcriptional activities of retinoic acid receptors.

Vitamin A derivatives plays a crucial role in embryonic development, as demonstrated by the teratogenic effect of either an excess or a deficiency in vitamin A. Retinoid effects extend however beyond embryonic development, and tissue homeostasis, lipid metabolism, cellular differentiation and proliferation are in part controlled through the retinoid signaling pathway. Retinoids are also therapeutically effective in the treatment of skin diseases (acne, psoriasis and photoaging) and of some cancers. Most of these effects are the consequences of retinoic acid receptors activation, which triggers transcriptional events leading either to transcriptional activation or repression of retinoid-controlled genes. Synthetic molecules are able to mimic part of the biological effects of the natural retinoic acid receptors, all-trans retinoic acid. Therefore, retinoic acid receptors are considered as highly valuable therapeutic targets and limiting unwanted secondary effects due to retinoid treatment requires a molecular knowledge of retinoic acid receptors biology. In this review, we will examine experimental evidence which provide a molecular basis for the pleiotropic effects of retinoids, and emphasize the crucial roles of coregulators of retinoic acid receptors, providing a conceptual framework to identify novel therapeutic targets.

PLZF is a negative regulator of retinoic acid receptor transcriptional activity.

BACKGROUND: Retinoic acid receptors (RARs) are ligand-regulated transcription factors controlling cellular proliferation and differentiation. Receptor-interacting proteins such as corepressors and coactivators play a crucial role in specifying the overall transcriptional activity of the receptor in response to ligand treatment. Little is known however on how receptor activity is controlled by intermediary factors which interact with RARs in a ligand-independent manner. RESULTS: We have identified the promyelocytic leukemia zinc finger protein (PLZF), a transcriptional corepressor, to be a RAR-interacting protein using the yeast two-hybrid assay. We confirmed this interaction by GST-pull down assays and show that the PLZF N-terminal zinc finger domain is necessary and sufficient for PLZF to bind RAR. The RAR ligand binding domain displayed the highest affinity for PLZF, but corepressor and coactivator binding interfaces did not contribute to PLZF recruitment. The interaction was ligand-independent and correlated to a decreased transcriptional activity of the RXR-RAR heterodimer upon overexpression of PLZF. A similar transcriptional interference could be observed with the estrogen receptor alpha and the glucocorticoid receptor. We further show that PLZF is likely to act by preventing RXR-RAR heterodimerization, both in-vitro and in intact cells. CONCLUSION: Thus RAR and PLZF interact physically and functionally. Intriguingly, these two transcription factors play a determining role in hematopoiesis and regionalization of the hindbrain and may, upon chromosomal translocation, form fusion proteins. Our observations therefore define a novel mechanism by which RARs activity may be controlled.