Extracellular Vesicles in Biological Fluids. A Biomarker of Exposure to Cigarette Smoke and Treatment with Chemopreventive drugs

Extracellular vesicles (EVs) are released from cells and enter into body fluids thereby providing a toxicological mechanism of cell-cell communication. The present study aimed at assessing (a) the presence of EVs in mouse body fluids under physiological conditions, (b) the effect of exposure of mice to cigarette smoke for 8 weeks, and (c) modulation of smoke-related alterations by the nonsteroidal anti-inflammatory drug celecoxib, a selective cyclooxygenase-2 inhibitor. To this purpose, ICR (CD-1) mice were either unexposed or exposed to cigarette smoke, either treated or untreated with oral celecoxib. EVs, isolated from bronchoalveolar lavage fluid (BALF), blood serum, and urines, were analyzed by nanoparticle tracking analysis and flow cytometry. EVs baseline concentrations in BALF were remarkably high. Larger EVs were detected in urines. Smoking increased EVs concentrations but only in BALF. Celecoxib remarkably increased EVs concentrations in the blood serum of both male and female smoking mice. The concentration of EVs positive for EpCAM, a mediator of cell-cell adhesion in epithelia playing a role in tumorigenesis, was much higher in urines than in BALF, and celecoxib significantly decreased their concentration. Thus, the effects of smoke on EVs concentrations were well detectable in the extracellular environment of the respiratory tract, where they could behave as delivery carriers to target cells. Celecoxib exerted both protective mechanisms in the urinary tract and adverse systemic effects of likely hepatotoxic origin in smoke-exposed mice. Detection of EVs in body fluids may provide an early diagnostic tool and an end-point exploitable for preventive medicine strategies.

Susceptibility to particle health effects, miRNA and exosomes : rationale and study protocol of the SPHERE study

Despite epidemiological findings showing increased air pollution related cardiovascular diseases (CVD), the knowledge of the involved molecular mechanisms remains moderate or weak. Particulate matter (PM) produces a local strong inflammatory reaction in the pulmonary environment but there is no final evidence that PM physically enters and deposits in blood vessels. Extracellular vesicles (EVs) and their miRNA cargo might be the ideal candidate to mediate the effects of PM, since they could be potentially produced by the respiratory system, reach the systemic circulation and lead to the development of cardiovascular effects.The SPHERE ("Susceptibility to Particle Health Effects, miRNAs and Exosomes") project was granted by ERC-2011-StG 282413, to examine possible molecular mechanisms underlying the effects of PM exposure in relation to health outcomes

Regulatory T cell-derived extracellular vesicles modify dendritic cell function

Regulatory T cells (Treg) are a subpopulation of T cells that maintain tolerance to self and limit other immune responses. They achieve this through different mechanisms including the release of extracellular vesicles (EVs) such as exosomes as shown by us, and others. One of the ways that Treg derived EVs inhibit target cells such as effector T cells is via the transfer of miRNA. Another key target for the immunoregulatory function of Tregs is the dendritic cells (DCs). In this study we demonstrate directly, and for the first time, that miRNAs are transferred from Tregs to DCs via Treg derived EVs. In particular two miRNAs, namely miR-150-5p and miR-142-3p, were increased in DCs following their interaction with Tregs and Treg derived exosomes. One of the consequences for DCs following the acquisition of miRNAs contained in Treg derived EVs was the induction of a tolerogenic phenotype in these cells, with increased IL-10 and decreased IL-6 production being observed following LPS stimulation. Altogether our findings provide data to support the idea that intercellular transfer of miRNAs via EVs may be a novel mechanism by which Tregs regulate DC function and could represent a mechanism to inhibit immune reactions in tissues

Is there a link between air pollution and mental disorders?

Several studies have demonstrated the association between air pollution and different medical conditions including respiratory and cardiovascular diseases. Air pollutants might have a role also in the etiology of mental disorders in the light of their toxicity on central nervous system. Purpose of the present manuscript was to review and summarize available data about an association between psychiatric disorders and air pollution. A research in the main database sources has been conducted to identify relevant papers about the topic. Different air pollutants and in particular PM and nitric oxides have been associated with poor mental health; long exposition to PM2.5 has been associated with an increased risk of new onset of depressive symptoms (Cohen's effect size d: 0.05-0.81), while increased concentration of nitric dioxide in summer with worsening of existing depressive conditions (Cohen's effect size d: 0.05-1.77). However, the interpretation of these finding should take into account the retrospective design of most of studies, different periods of observations, confounding factors such as advanced age or medical comorbidity. Further studies with rigorous methodology are needed to confirm the results of available literature about this topic

A pilot study assessing the effect of air pollution on extracellular vesicles in systemic sclerosis

Introduction: The identification of specific diagnostic and prognostic biomarkers remains an unmet need in Systemic Sclerosis (SSc). Over the past few years, it has been suggested that extracellular vescicles (EVs) and environmental toxicants, such as particulate matter (PM), may have an important role in the pathogenesis of autoimmune diseases. At present, no data are available on the impact of PM exposure on EVs from patients with SSc. Our aim was to evaluate the effects of PM with aerodynamic diameter less than 10 \ub5m (PM10) and 2.5 \ub5m (PM2.5) on EVs in SSc and osteoarthritis (OA). Material and Methods: Plasma EVs were analyzed by Nanosight and flow cytometry after labeling with the following markers: CD14 (monocyte), CD61 (platelet), CD25 (T-reg), human endogenous retrovirus w (HERV-w) and human leukocyte antigen G (HLA-G). Demographic and clinical data were collected for each patient. Plasma EV concentrations were measured in SSc and OA patients and were analyzed by generalized linear regression models. Daily PM concentrations, estimated by the Regional Environmental Protection Agency at municipality resolution, were used to assign short-term exposure (mean of the 7 days preceding the evaluation) to each study subject. Results: 12 consecutive limited cutaneous SSc (11 female, median age 66.8 yrs, median disease duration 12.3 yrs, median mRSS 3.5) and 12 patients with OA (8 female, median age 67.1 yrs, median disease duration 9.3 yrs) were enrolled. In the table below, EV count and MV subtypes are reported with respect to PM2.5 and PM10 exposure both in SSc and OA patients. The increase of PM2.5 led to a decrease of HERV-w+ microvesicles (MV) in both SSc (\u3b2 =-0.10; p=0.01) and OA (\u3b2 =-0.09; p=0.01) and of HLA-G+ (\u3b2 =-0.11; p<0. 01) only in SSc. Similar results were observed analyzing PM10 exposure. Analysis of EV concentration according to their dimensions showed a negative association in the size range of exosomes (63-92nm) in SSc compared to OA (p<0. 05) and in the range of MV (230-510 nm). A positive association between HLA-G+ with ESR (\u3b2 =0.34; p<0.01). Conclusions: In our study, limited cutaneous SSc showed different EV concentrations from controls: SSc tends to have less exosomes and more MV than OA. Moreover, environmental stimuli are confirmed to be able to influence HERV-w+ MV release both in SSc and controls. Finally, in SSc patients PM exposure could significantly alter the release of HLA-G+ MV that has been correlated to the process of self-tolerance maintenance

Nasal microbiota modifies the effects of particulate air pollution on plasma extracellular vesicles

Air pollution exposure has been linked to modifications of both extracellular vesicle (EV) concentration and nasal microbiota structure (NMB), which might act as the respiratory health gatekeeper. This study aimed to assess whether an unbalanced NMB could modify the effect of particulate matter (PM) exposure on plasmatic EV levels. Due to two different NMB taxonomical profiles characterized by a widely different relative abundance of the Moraxella genus, the enrolled population was stratified into Mor\u2010 (balanced NMB) and Mor+ (unbalanced NMB) groups (Moraxella genus\u2019s cut\u2010off 6425% and >25%, respectively). EV features were assessed by nanoparticle tracking analysis (NTA) and flow\u2010cytometry (FC). Multivariable analyses were applied on EV outcomes to evaluate a possible association between PM10 and PM2.5 and plasmatic EV levels. The Mor\u2010 group revealed positive associations between PM levels and plasmatic CD105+ EVs (GMR = 4.39 p = 0.02) as for total EV count (GMR = 1.92 p = 0.02). Conversely, the Mor+ group showed a negative association between exposure and EV outcomes (CD66+ GMR = 0.004 p = 0.01; EpCAM+ GMR = 0.005 p = 0.01). Our findings provide an insight regarding how a balanced NMB may help to counteract PM exposure effects in terms of plasmatic EV concentration. Further research is necessary to understand the relationship between the host and the NMB to disentangle the mechanism exerted by inhaled pollutants in modulating EVs and NMB

Blood-derived extracellular vesicles isolated from healthy donors exposed to air pollution modulate in vitro endothelial cells behavior

The release of Extracellular Vesicles (EVs) into the bloodstream is positively associated with Particulate Matter (PM) exposure, which is involved in endothelial dysfunction and related to increased risk of cardiovascular disease. Obesity modifies the effects of PM exposure on heart rate variability and markers of inflammation, oxidative stress, and acute phase response. We isolated and characterized plasmatic EVs from six healthy donors and confirmed a positive association with PM exposure. We stratified for Body Mass Index (BMI) and observed an increased release of CD61+ (platelets) and CD105+ (endothelium) derived-EVs after high PM level exposure in Normal Weight subjects (NW) and no significant variations in Overweight subjects (OW). We then investigated the ability to activate endothelial primary cells by plasmatic EVs after both high and low PM exposure. NW-high-PM EVs showed an increased endothelial activation, measured as CD105+/CD62e+ (activated endothelium) EVs ratio. On the contrary, cells treated with OW-high-PM EVs showed reduced endothelial activation. These results suggest the ability of NW plasmatic EVs to communicate to endothelial cells and promote the crosstalk between activated endothelium and peripheral cells. However, this capacity was lost in OW subjects. Our findings contribute to elucidate the role of EVs in endothelial activation after PM exposure

The Impact of Air Pollution on Extracellular Vesicles as a Potential Pro-inflammatory Stimulus in Rheumatoid Arthritis

Background/Purpose: Rheumatoid arthritis (RA) is an heterogeneous chronic autoimmune disorder potentially leading to a progressive joint damage with great impact on quality of life. RA pathogenesis is complex and involves environmental factors that trigger disease in genetically susceptible individuals. Extracellular vesicles (EVs) have been described to play an important role in RA pathogenesis and to modulate autoimmune response following environmental exposures, such as air pollution. Our aim was to evaluate the effects of particulate matter (PM) with aerodynamic diameter 64 10 \u3bcm (PM10) and 64 2.5 \u3bcm (PM2.5) on EVs in RA and osteoarthritis (OA) as control. Methods: Plasma EVs were analyzed by Nanosight and flow cytometry: CD14 (monocyte/macrophage), CD61 (platelet), CD25 (T-reg), human endogenous retrovirus w (HERV-w), human leukocyte antigen G (HLAG). Demographic and clinical data were collected for each patient. Plasma EV concentrations were measured in RA and OA patients and were analyzed by generalized linear regression models. Daily PM concentrations, estimated by Regional Environmental Protection Agency at municipality resolution, were used to assign short-term exposure (mean of the 7 days preceding the evaluation) to each patient. Results: 12 consecutive patients with RA (median age 68.1, median disease duration 9.3, 12 female, median DAS28 2.25, 5 positive for rheumatoid factors, 6 positive for anti-citrullinated peptide antibodies) and 12 patients with OA (median age 67.1, median disease duration 9.3, 8 female) were enrolled. Analysis of EVs concentration, according to their dimensions, showed a negative association of exosomes (63-92nm) in RA compared to OA (p< 0.05). The increase of PM2.5 led to a decrease of CD14+ microvesicles (MV) (\u3b2=-0.13; p< 0.01) and CD61+ (\u3b2=-0.08; p=0.05) in RA, and of HERV-w in OA (\u3b2=-0.09; p=0.01). Similar results were observed analyzing PM10 exposure. PM exposure was not observed to modify CD25+ and HLA-G+ MV release both in RA and OA patients (table below). Moreover, we compared plasmatic EVs mean concentration among patients with RA and OA, and we found a significant difference in the two groups in the HERV-w subpopulations (\u3b2AR vs \u3b2OA = 0.044 vs \u2013 0.091; p = 0.011). In RA patients we also observed a significant association between EVs (CD14+ and HLA-G+ MV) and DAS28 (\u3b2CD14+ = 0.03; p = 0.01 and \u3b2HLA-G+ = 0.04; p = 0.02). Finally, we observed a negative association between exosomes and C-reactive protein (CRP) (\u3b2=-1.99; p=0.03), and a positive association between HERV-w and Erythrocyte Sedimentation Rate (ESR) (\u3b2=0.53; p=0.06), and HLA-G+ and ESR (\u3b2=0.29; p=0.01). Conclusion: The results of this pilot study show that PM exposure modulates the release of EVs carrying HLA-G and/or HERV-w in RA patients. This might be interpreted as an attempt of immune system to counteract the perturbation provoked by a pro-inflammatory environmental stimulus. More research is still needed to tie the genetic, epigenetic and environmental factors together and to determine their roles in RA pathogenesis