Amélie Héliot
email: amelie.heliot@univ-littoral.fr

Gauthier Trémolet

Yann Landkocz
email: yann.landkocz@univ-littoral.fr

Dorothée Dewaele
email: dorothee.dewaele@univ-littoral.fr

Fabrice Cazier
email: fabrice.cazier@univ-littoral.fr

Frédéric Ledoux
email: frederic.ledoux@univ-littoral.fr

Dominique Courcot
email: dominique.courcot@univ-littoral.fr

Dr Perrine J Martin
email: perrine.martin@univ-littoral.fr

Gauthier Tremolet
email: gauthier.tremolet@univ-littoral.fr

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

Keywords: infiltrating macrophages, extracellular vesicles, PM2.5, lung epithelial cells

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Introduction

World Health Organization (WHO) estimates that exposure to air pollution caused around 7 million deaths worldwide in 2012 [START_REF] Who | Burden of disease from Ambient Air Pollution for 2012[END_REF]. Air pollutants consist of gaseous compounds such as volatile organic compounds, nitrogen oxides and particulate matter (PM). PM with an equivalent aerodynamic diameter below 2.5 µm (PM2.5) is able to penetrate lung until bronchial tubes and alveoli where it can cause the development of respiratory diseases onset such as asthma, Chronic Obstructive Pulmonary Disease (COPD) till lung cancer [START_REF] Li | Function of PM2.5 in the pathogenesis of lung cancer and chronic airway inflammatory diseases[END_REF]. In 2013, PM2.5 has been classified as a Group 1 carcinogen by the International Agency for Research on Cancer [START_REF] Cohen | International Agency for Research on Cancer[END_REF]. PM2.5 is a complex mixture of inorganic (metals, ions… ) and organic compounds (PAHs, dioxins…), living organisms (bacteria, pollen…) whose composition depends on the main contributing sources (urban, industrial, maritime, rural) and of their transport and evolution in the atmosphere [START_REF] Kfoury | PM2.5 source apportionment in a French urban coastal site under steelworks emission influences using constrained non-negative matrix factorization receptor model[END_REF][START_REF] Lepers | Mutagenicity and clastogenicity of native airborne particulate matter samples collected under industrial, urban or rural influence[END_REF].

Macrophages are heterogeneous cells from different origins showing high plasticity as they can change their phenotype depending on their microenvironment. In the lung, they are established during the embryonic development period and maintained in adulthood through longevity and local self-renewal [START_REF] Epelman | Origin and functions of tissue macrophages[END_REF][START_REF] Sheng | Most Tissue-Resident Macrophages Except Microglia Are Derived from Fetal Hematopoietic Stem Cells[END_REF]. These prenatal-derived macrophages are predominant in lung tissues and can differentiate into Alveolar Macrophages (AMs) localized in the airspaces of the lung [START_REF] Fathi | Functional and morphological differences between human alveolar and interstitial macrophages[END_REF]. AMs are the essential immune effector cells in the lung as they are exposed to the harmful outside environment especially through the inhalation of PM2.5. However Goto et al. have shown that the deposition of PM into the lung stimulates the bone marrow and promotes the release of leukocytes into the circulation among which monocytes that are important to control and terminate the inflammatory response (Goto et al., 2004b). These monocytes infiltrate the lung and differentiate into macrophages, which will be called infiltrating macrophages, that are mainly distributed throughout the airways, lung tissues, and alveolar tissues during lung insult [START_REF] Yamasaki | Lung Macrophage Phenotypes and Functional Responses: Role in the Pathogenesis of COPD[END_REF]. After PM exposure, the onset of the inflammatory response usually starts with the release of the early responding cytokines, IL1β and TNFα, and continue with the expression of other cytokines and chemokines (IL6, IL8) [START_REF] Gualtieri | Particulate Matter Physico-Chemical Characterization and in vitro Toxicological Effects[END_REF]. This inflammatory response may be induced by the ability of PM to alter the cells oxidative balance. According to the oxidative stress paradigm, cells respond to low levels of reactive oxygen species (ROS) by increasing the levels antioxidant molecules (such superoxide dismutase (SOD), NAD(P)H quinone dehydrogenase 1 (NQO1), heme oxygenase-1 (HMOX)) thus restoring the intracellular redox homeostasis [START_REF] Gualtieri | Particulate Matter Physico-Chemical Characterization and in vitro Toxicological Effects[END_REF]. In addition, PAHs present on PM, which require metabolic activation to biologically reactive intermediates to elicit their adverse health effects, are metabolized by the CYP superfamily members, and activate their gene expression through the activation of the Aryl hydrocarbon Receptor (AhR) [START_REF] Billet | Ambient particulate matter (PM2.5): Physicochemical characterization and metabolic activation of the organic fraction in human lung epithelial cells (A549)[END_REF]. To our knowledge, no study has focused on these effects of PM2.5 on infiltrating macrophages. It is thereby interesting to assess the cellular response of infiltrating human macrophages exposed to PM2.5 in terms of cytotoxicity, oxidative stress, xenobiotic metabolization and inflammation.

Moreover, recent studies highlight that stimulated or stressed cells possess the ability to release vesicles in the extracellular space. These Extracellular Vesicles (EVs) may have the characteristics of a cell that has just been attacked and are involved in the regulation of several cellular events, with variable phenotypic consequences for recipient cells [START_REF] Neven | Extracellular Vesicles: How the External and Internal Environment Can Shape Cell-To-Cell Communication[END_REF]. They include a variety of nanoscale membranous vesicles (exosomes, microvesicles, microparticles, apoptotic bodies) containing key signaling mediators (miRNAs, proteins, metabolites, lipids) transferred from cells of origin to target cells [START_REF] Jabalee | The Role of Extracellular Vesicles in Cancer: Cargo, Function, and Therapeutic Implications[END_REF]. EVs are known to be critically involved in lung homeostasis as well as inflammation, reflecting crosstalk between AMs and epithelial cells [START_REF] Haggadone | Microenvironmental Influences on Extracellular Vesicle-Mediated Communication in the Lung[END_REF]. They are increasingly recognized as a novel mechanism by which the innate immune response is initiated [START_REF] Lee | Extracellular Vesicle: An Emerging Mediator of Intercellular Crosstalk in Lung Inflammation and Injury[END_REF]. Lung exposure to PM may induce the release of EVs originated from various cellular sources, like endothelial cells, epithelial cells and AMs in the alveolar or bronchial space [START_REF] Benedikter | Extracellular vesicles released in response to respiratory exposures: implications for chronic disease[END_REF]. Up to our knowledge, the effect of fine atmospheric particulate matter on infiltrating human macrophages, the ability of exposed human macrophages to release EVs and the study of the interaction between these released EVs and lung epithelial cells has not been reported yet.

To answer these questions, cellular response of infiltrating human macrophages exposed to PM2.5-0.3 collected in an industrial coastal city was assessed. For this, undifferentiated THP-1 monocyte cells were first used to determine the dose and time of exposure at which these cells respond in terms of metabolic activity, oxidative stress and inflammation but also produce EVs. Then the same experiments were carried out on primary macrophages. Finally, EVs produced in response were characterized and their effects on BEAS-2B lung epithelial cells were evaluated.

Material and Methods

PM sampling and handling

Atmospheric PM was collected continuously over a one year period (March 2014-March 2015), in Dunkerque, Northern France (51°1'36" N; 2°22'18" E), using a high volume sampler (68 m 3 /h) equipped with a 5 stages cascade impactor (model 235, Staplex®, USA). Dunkerque is a coastal city counting roughly 210,000 inhabitants with its suburbs. The study area is characterized by numerous industries mainly in the fields of metallurgy (integrated and electric steelworks, Al and Mn steelmaking) and organic synthesis (refinery, chemicals manufacturing). Briefly, particles were collected directly on plates (without filter) as published elsewhere [START_REF] Borgie | Comparison between ultrafine and fine particulate matter collected in Lebanon: Chemical characterization, in vitro cytotoxic effects and metabolizing enzymes gene expression in human bronchial epithelial cells[END_REF][START_REF] Cazier | Characterisation and seasonal variations of particles in the atmosphere of rural, urban and industrial areas: Organic compounds[END_REF][START_REF] Landkocz | Fine and ultrafine atmospheric particulate matter at a multi-influenced urban site: Physicochemical characterization, mutagenicity and cytotoxicity[END_REF]. Two sampling systems were used in parallel and replaced every 14 days over the one year period. With respect to the cut-off diameter of the impaction plates, atmospheric particles deposited on impaction stages 2 to 5 have an aerodynamic diameter in the 0.3 -2.5 µm size range (PM2.5-0.3). After each of the 14 days sampling period, impaction plates were placed in a laminar flow hood for 48 hours. Then particles were recovered from impaction plates (stages 2-5) using a brush and stored at -18°C in a PTFE vial till further use. At the end of the one year campaign, the 14 days PM2.5-0.3 samples were pooled together and carefully homogenized using two PTFE balls moved by a magnetic stirrer.

The obtained yearly average PM2.5-0.3 sample was then use for chemical characterization and cellular response study.

PM physico-chemical characterization

Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES, iCAP 6300 DUO, ThermoElectron, UK) and ICP-Mass Spectrometry (ICP-MS, Varian® 820-MS, Varian, USA) were used for quantification of the PM inorganic major and trace components (Al, As, Ba, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, P, Pb, Sn, Sr, Ti, V, Zn). Water-soluble ions and cations (F -, Cl -, NO3 -, SO4 2-, Na + , Ca 2+ , Mg 2+ , K + , NH4 + ) were quantified with ion chromatography (model ICS 5000, ThermoElectron, UK). The PM total carbon (TC) content was measured using the CHNS/O analyzer (FLASH, 2000;ThermoElectron, UK). Finally, concentrations of the 16 priority Polycyclic Aromatic Hydrocarbons (PAHs) listed by the United States Environmental Protection Agency (US-EPA) were quantified using gas chromatography-mass spectrometry (GC-MS,1200 TQ, Varian, USA) after PM Soxhlet extraction with dichloromethane. The whole analytical procedure is detailed in .

Human Macrophages culture and PM2

.5-0.3 exposure THP-1 monocytic cell line was purchased from European Collection of Authenticated Cell Cultures (ECACC, Wiltshire, UK; reference: 88081201) and handled following manufacturer's instructions. Briefly THP-1 were seeded at 4 x 10 5 cells/mL in RPMI 1640 medium supplemented with 1% Penicillin-Streptomycin (PS), 0.05mM 2-mercaptoethanol and 10% Heat-Inactivated Fetal Bovine Serum (HI-FBS).

Peripheral Blood Mononuclear Cells (PBMCs) were purified from fresh whole blood (provided by Etablissement Français du Sang, EFS, Lille, France) of 6 healthy donors through Ficoll gradient centrifugation using Lymphoprep (Axis-Shield). Monocytes were then prepared by a 2h adhesion step, and next cultured in RPMI 1640 medium supplemented with 2mM glutamine, 1% PS, and 10% HI-FBS in the presence of 50 ng/mL GM-CSF during 36h to get infiltrating macrophages.

The concentrations of 12.5 and 50 μg/mL of PM2.5-0.3 for THP-1 were determined based on their response to increasing concentrations of PM2.5-0.3 measured by quantifying their release of IL8 (supp. Fig. 1). The minimum dose at which THP-1 releases IL8 at the three times of exposure of 6h, 24h and 48h is 12.5 μg/mL (Lowest Observed Effect Concentration (LOEC). A second PM2.5-0.3 concentration of exposure for THP-1 has been chosen at a value less than 50% of IL8 release (50 µg/mL) in order to identify a potential dose-response effect. Then the concentration of 12.5 µg/mL for primary macrophages has been chosen according to the obtained results with THP-1.

Each cell type was exposed to PM2.5-0.3 as follows. The supernatants were removed and replaced by RPMI medium supplemented with 10% exosome-depleted FBS (Gibco by ThermoFisher Scientific, Illkirch, France), 2.5% Hepes and 1% antibiotics. PM2.5-0.3 was suspended in cell culture medium and dispersed by 5 min sonication just prior to the exposure. Then, PM2.5-0.3 was or not (non-exposed, NE) added at the indicated concentrations to THP-1 cultured for 6h, 24h or 48h and primary macrophages for 48h.

Extracellular vesicles (EVs) isolation and quantification

EVs were isolated by differential centrifugation according to Théry et al. with some modifications [START_REF] Théry | Isolation and characterization of exosomes from cell culture supernatants and biological fluids[END_REF]. Briefly, THP-1 or macrophages supernatants were centrifuged at 500g for 10 min and at 16,500g for 30 min at 4°C to remove cell debris. In addition, the supernatants were filtered with a 0.2μm syringe filter. For the collection of EVs, the supernatants were ultracentrifuged twice at 110 000g for 70 min at 4°C. The EVs were then washed and suspended in Phosphate Buffer Saline (PBS), aliquoted and stored at -80°C until further exposure or extraction.

To determine the size and the concentration changes of EVs obtained in the different conditions, the NTA measurement was performed using the NS300 Nanosight instrument (Malvern Panalitycal, United Kingdom). All NTA analysis was conducted after thawing of the samples. The samples were diluted in PBS at proper ratios to achieve the optimal detectable concentration (about 10 7 particles per mL). For each sample, 1 mL of the diluted sample was injected into the instrument and then the concentration of EVs as well as the mean and median EVs size was given after 4 measurements by the Nanosight dedicated software.

BEAS-2B co-culture with EVs

BEAS-2B is a non-cancerous cell line of human bronchial epithelial cells which was obtained from ECACC (Wiltshire, UK; reference: 95102433). They were cultured in LHC-9 Medium (ThermoFisher Scientific, Illkirch, France) as described previously [START_REF] Héliot | Smoker extracellular vesicles influence status of human bronchial epithelial cells[END_REF]. 250,000 cells were exposed to 50μL of EVs suspension (Supp. Fig. 1) derived from non-exposed or exposed THP-1 or macrophages. After 48h of exposure, cell-free culture supernatants were collected and quickly frozen at -80°C until further study. Adherent cells were washed twice with 500μL of cold PBS (Gibco by ThermoFisher Scientific, Illkirch, France), and quickly frozen at -80 °C. All experiments were performed in triplicate.

Cytotoxicity assay

The cytotoxicity of THP-1 and primary macrophages after PM2.5-0.3 exposure, and of BEAS-2B cells after EVs co-culture, was evaluated as described previously [START_REF] Landkocz | Fine and ultrafine atmospheric particulate matter at a multi-influenced urban site: Physicochemical characterization, mutagenicity and cytotoxicity[END_REF], by measuring the extracellular lactate dehydrogenase (LDH) concentration in the cell culture supernatants (Cytotoxicity Detection Kit LDH, Roche Diagnostics, France). Percentage of cytotoxicity was quantified relatively to 100 % mortality induced by Triton X-100. As a control, increasing concentrations of PM2.5-0.3 were suspended in LDH solution (fixed concentration). Results showed that, at the maximum concentration of PM2.5-0.3 used (50µg/mL), attenuation of absorbance is below 1%. This allows us to neglect the direct effect of PM2.5-0.3 on absorbance measurement values and the artefact related to the adsorption of LDH on PM (Supp. Fig. 2).

Flow cytometry

Phenotypic analysis of primary macrophages was performed using flow cytometry direct immunofluorescence. Cells were first incubated for 30 min in PBS with 5% human autologous serum with FITCconjugated Ab directed against CD14. Isotypic control labeling was performed in parallel. Cells were then analyzed with an Attune flow cytometer (Applied Biosystems, ThermoFisher Scientific, Illkirch, France) using Kaluza software (Beckman Coulter).

RNA isolation and RT-qPCR

THP-1, primary macrophages and BEAS-2B RNA and small RNA were extracted using small and large RNA kit (NucleoSpin® miRNA, Macherey Nagel, Hoerdt, France) respectively, according to manufacturer's instructions. The obtained samples were quickly frozen at -80°C until further study.

Reverse transcription was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, ThermoFisher Scientific, Illkirch, France). mRNAs (i.e IL1β, IL6, IL8, CAT, IL12, TNFα, CYP1A1, CYP1B1, CYP2E1, SOD2, CAT, NRF2, NQO1, HMOX) quantification was performed using TaqMan® Gene Expression Assays (ThermoFisher Scientific, Illkirch, France) (Table 1). Real-time quantitative polymerase chain reaction (qPCR) was carried out using a 7500 Fast Real-Time PCR System (Applied Biosystems, ThermoFisher Scientific, Illkirch, France). The relative changes in gene expression were calculated by the 2 -ΔΔCt method [START_REF] Schmittgen | Analyzing real-time PCR data by the comparative C(T) method[END_REF] and normalized against 18S using the Sequence Detection 7500 Software v2.0.3 (Applied Biosystems, ThermoFisher Scientific, Illkirch, France) (see Table 1 for primer references).

Cytokines and Extracellular Proteins Concentration Measurement

IL-6, IL-1β, IL-8, TNF-α and sFas concentrations in cell-free culture supernatants were determined using MAGPIX® system and the MILLIPLEX® MAP Human Cytokine Panel 1 and Human CD8+ T Cell (Luminex®, EDM Millipore, Billerica, USA), following the manufacturer's instructions.

Protein Extraction and Western blotting

Cells were centrifuged during 10 min at 2,500g and 1mL of M-PER Reagent was added per 100mg of cells.

After stirring for 10 min, the samples were centrifuged again for 15 min at 14,000 g. The supernatant containing the proteins was recovered and Laemmli buffer was added. EVs were directly placed in Laemmli buffer before electrophoresis.

Equal volumes of Novex® Tris-Glycine SDS Sample Buffer (2X) (ThermoFisher Scientific, Illkirch, France) were added to the protein samples, boiled for 2 min, applied to SDS-PAGE, and transferred to Novex™ 4-20% Tris-Glycin Mini Protein Gels (ThermoFisher Scientific, Illkirch, France). After blocking, membranes were incubated with anti-CD63 or anti-CD81 primary antibodies (Novus Biologicals, Littleton, CO, USA) at 4°C overnight, washed with PBS-Tween (0.05% Tween 20), and incubated with a Goat anti-Mouse IgG (H+L) secondary antibody, Horseradish Peroxidase (HRP) conjugated (Life Technologies, ThermoFisher Scientific, Illkirch, France) for 60 min. After washing with PBS-Tween, antigen-antibody reactions were detected with a Chemidoc (Biorad, Marnes la Coquette, France) using the ECL Plus Detection kit (Pierce, ThermoFisher Scientific, Illkirch, France).

For relative quantification of MSR1 protein on total proteins, macrophages proteins were transferred to Mini-PROTEAN TGX Stain Free Gel (Biorad, Marnes la Coquette, France) and detected with a Chemidoc before membrane transfer. The ImageLab Software was used for relative quantification.

Electron microscopy

EVs in PBS were placed on formvar-coated 300 mesh copper grids during 10 min, rinsed 3 times with H2O

and contrasted with 2% phosphotungstic acid (PTA) during 10 seconds. Images were obtained by transmission electron microscopy with a JEOL 7100F electron microscope (JEOL Ltd, Japan).

Statistical analysis and controls

Gene and protein expression as well as protein levels were depicted as median and inter-quartile range and graphically represented by bar-graphs. For relative quantification of gene expression, significance RQ> 2 or RQ <0.5 is reported to the value of unexposed control (NE), normalized to 1 with 18S. For each exposure time and concentration of PM2.5-0.3, cytokines measurement data were compared to those obtained for non exposed (NE) cells. The significance of the differences was calculated by the non-parametric Mann-Whitney U test and is indicated by * when p <0.05. For NTA, the significance of the differences was calculated by the T Student's test and is indicated by * when p <0.01.

Results

PM2.5-0.3 major and trace inorganic species

Major and trace inorganic species as well as Total Carbon (TC) contents are reported in Fig. 1A. TC (14.3%), water-soluble NO3 -(10.0%), SO4 2-(7.5%), Cl -(6.1%), Ca 2+ (4.5%), Na + (4.3%) and NH4 + (1.6%) as well as Fe (3.8%) and Al (2.8%) were the major PM constituents (55% in total). Then five species showed concentrations values between 0.1% and 1%: Mg 2+ (0.65%), K + (0.45%), Mn (0.18%), P (0.14%) and Zn (0.13%). Other species concentrations ranged below 0.1% (Cu, Ti, Ba, Pb, F -, Cr, Sr, Ni and V) and below 0.01% (Sn, Mo, As Co and Cd).

NO3 -and SO4 2-mainly derive from the gas-to-particle conversion of NOX and SO2. NOX are emitted by combustion processes related to the use of fossil fuels in cars (diesel) or industrial processes. SO2 is mainly originated from industrial emissions or related to heavy fuel oil combustion with high sulfur content mainly used in industry or in shipping [START_REF] Ledoux | Influence of ship emissions on NOx, SO2, O3 and PM concentrations in a North-Sea harbor in France[END_REF]. NH4 + results as well from the conversion of the NH3 gas known to be mainly from agricultural origin. However, an industrial contribution cannot be excluded with regard to the presence of an integrated steelworks able to emit particles containing NH4 + in the study area [START_REF] Hleis | Chemical profile identification of fugitive and confined particle emissions from an integrated iron and steelmaking plant[END_REF][START_REF] Kfoury | PM2.5 source apportionment in a French urban coastal site under steelworks emission influences using constrained non-negative matrix factorization receptor model[END_REF]. Na + , Mg 2+ and Cl -are known to be significantly associated with sea-salts [START_REF] Manders | Sea salt concentrations across the European continent[END_REF].

Among the considered metals, Al and Fe had the highest levels. These elements can generally be introduced in the atmosphere as a consequence of soil erosion induced by wind as well as industrial emissions [START_REF] Kfoury | PM2.5 source apportionment in a French urban coastal site under steelworks emission influences using constrained non-negative matrix factorization receptor model[END_REF][START_REF] Oravisjärvi | Source contributions to PM2.5 particles in the urban air of a town situated close to a steel works[END_REF][START_REF] Santacatalina | Impact of fugitive emissions in ambient PM levels and composition: a case study in Southeast Spain[END_REF]. In particular, steelmaking, metallurgy, cement plants are located in the study area and are known to emit such PM components. Ca is as well found in soil dust (aluminosilicates) as in the raw materials used in the latter industrial activities. Cr, Cu, Mn, Ni, Pb, V and Zn are predominantly associated with anthropogenic origins: Ni, V and Co can be considered as tracers of heavy fuel oil combustion, especially used for shipping [START_REF] Mazzei | Characterization of particulate matter sources in an urban environment[END_REF][START_REF] Roche | Etude des concentrations et de la composition des PM10 sur le littoral du Nord de la France -Evaluation des contributions maritimes de l'espace Manche-Mer du Nord[END_REF]; Zn, Pb, Cr and Cu elements are more associated with the wear of mobile vehicle parts such as brakes and tires [START_REF] Gietl | Identification of brake wear particles and derivation of a quantitative tracer for brake dust at a major road[END_REF][START_REF] Thorpe | Sources and properties of non-exhaust particulate matter from road traffic: a review[END_REF]. It should also be noted that, in the Dunkirk study area, Fe, Mn, Zn, Pb, Cr and Cd elements could also be related to the iron and steelmaking activities [START_REF] Kfoury | PM2.5 source apportionment in a French urban coastal site under steelworks emission influences using constrained non-negative matrix factorization receptor model[END_REF].

PM2.5-0.3 Polycyclic Aromatic Hydrocarbon composition

The average concentrations of the 16 US-EPA priority PAHs compounds are given in Fig. 1B. The predominantly detected compounds were dibenz[a,h]anthracene (8.9 μg/g), followed by indeno[1,2,3-cd]pyrene, chrysene and benzo[b]fluoranthene, with very close concentrations values ranging between 5.3 and 6.0 μg/g.

Concentrations of other compounds were below 5 μg/g and below 1µg/g for the 6 lightest PAHs (compounds with 2 and 3 cycles). The main source of dibenz[a,h]anthracene and benzo[b]fluoranthene is road traffic, and more precisely exhaust fumes from diesel engines [START_REF] Duval | Source resolution of polycyclic aromatic hydrocarbons in the Los Angeles atmosphere: application of a chemical species balance method with first order chemical decay[END_REF][START_REF] Ineris | Institut National de l'Environnement Industriel et des Risques. Fiche de données toxicologiques et environnementales des substances chimiques[END_REF]INERIS, , 2004a)).

Dibenz [a,h]anthracene is also found in cigarette smoke or in coke oven emissions (INERIS, 2004a).

Indeno[1,2,3-cd] pyrene results from fossil fuels as well especially after incomplete combustion of wood, coal, gasoline or diesel fuel (INERIS, 2004b). Chrysene can be emitted into the atmosphere during coal and oil distillation and pyrolysis. It is also one of the predominant PAHs in particulate emissions from household waste incinerators, natural gas appliances and domestic heating devices, particularly those using wood (INERIS, 2004c). The predominantly detected compounds can be explained by at least partly the locally based industries.

Indeed, the study area hosts an integrated steel industry (including a coking plant), oil refining (producing marine fuels comparable to heavy fuel oils), a cement factory and a waste incinerator.

Cellular response of THP-1 exposed to PM2.5

The first experiments were done on THP-1 cells. This monocytic cell line was used in a first time to optimize the exposure conditions that the cells will respond in terms of metabolic activity, oxidative stress and inflammation, before switching to primary macrophages. For that, THP-1 were not pre-treated with PMA before exposure to keep a monocytic phenotype and to avoid non-specific activation of THP-1 by PMA as a pro-inflammatory response [START_REF] Lund | The choice of phorbol 12-myristate 13-acetate differentiation protocol influences the response of THP-1 macrophages to a pro-inflammatory stimulus[END_REF]. THP-1 were exposed to PM2.5 at two doses (12.5 and 50 µg/mL) and for three times (6h, 24h and 48h) (see section 2.3 for dose justification). Regarding the cytotoxicity, the measurement of the extracellular LDH did not show any significant effect regardless times and doses of exposure (Fig. 2A). For metabolic activation and oxidative stress induction assessment, relative quantification of expression of genes coding for the Xenobiotic Metabolizing Enzymes (XMEs) CYP1B1, CYP1A1, CYP2E1, and for the Superoxide dismutase 2 (SOD2) which transforms toxic superoxide into hydrogen peroxide and diatomic oxygen, the NAD(P)H quinone dehydrogenase 1 (NQO1), an enzyme involved in the reduction of formation of radical species induced by oxidative stress and heme oxygenase (HMOX), was performed (Fig. 2B). The results showed a significant dose-dependent increase of CYP1A1 and CYP1B1 expression after 6h of exposure, with a tendency to decrease with time for CYP1A1 or to reach a plateau for CYP1B1 at 24h and 48h.

CYP2E1 expression was not significantly modified after PM2.5 exposure. SOD2, NQO1 and HMOX expression was significantly induced proportionally to the doses but not to the time of exposure.

Expression and production of the pro-inflammatory cytokines IL8, IL1β, IL6 and TNFα by THP-1 exposed to PM2.5-0.3 were also quantified (Fig. 2C andD). An early induction of the expression of IL8, IL1β and TNFα was observed decreasing with time and increasing with the doses. For the same cytokines, the release increased with the times and doses of exposure. Concerning IL6, its expression was induced later and its release came only after 48h of exposure to the highest dose of 50 µg/mL. These results demonstrate that undifferentiated THP-1 are able to carry out a metabolic, anti-oxidant, and pro-inflammatory response following PM2.5-0.3 exposure.

3.4 Characterization of extracellular vesicles derived from THP-1 exposed to PM2.5-0.3.

The ability of THP1 to produce EVs after exposure to PM was then evaluated. For that, the EVs produced by the undifferentiated THP-1 after 6h, 24h and 48h of exposure to 12.5 and 50 µg/mL of PM2.5-0.3 were isolated, quantified and characterized. On the one hand, the quantification with the Nanoparticle Tracking Analysis (NTA) of the obtained EVs showed that PM2.5 exposure induced an significant increase of the concentration of EVs produced by THP-1 in particular after 48h of exposure, which corresponds also to an increase of the number of EVs released by cell (Fig. 3A), suggesting that THP-1 intrinsically produce more EVs if exposed for longer and at higher concentrations of PM. On the other hand, NTA measurement indicated that their average size doesn't significantly vary with the time and doses of exposure and ranges between 130 and 200 nm (Fig. 3A).

Electron microscopy confirms the presence of EVs in THP-1 supernatant (Fig. 3B). Finally, it should be noted the presence of exosomes within the EVs produced as evidenced by the western-blot on CD63 and CD81 exosomal proteins (Figure 3C). These results show for the first time that PM2.5 exposure enhances EVs release by undifferentiated THP-1.

Cellular response of infiltrating human macrophages exposed to PM2.5-0.3

Goto et al. have shown that one of the cytokine implicated in infiltrating macrophages recruitment into the lung is GM-CSF (Goto et al., 2004b). Just after isolation from blood and before exposure to PM2.5-0.3, primary macrophages obtained from 6 donors were incubated for 36h with 50 ng/mL GM-CSF to obtain infiltrating cells.

The obtained infiltrating macrophages were then exposed to 12.5µg/mL for 48h as define during the optimization step (section 4), since this was the minimum PM dose at which the THP-1 responded and produced an almost maximal number of EVs. Then as well, metabolic activation, oxidative stress and inflammatory response were studied. Extracellular LDH measurement showed that PM2.5-0.3 exposure did not induce significant cytotoxicity (Fig. 4A) in comparison with non exposed (NE) infiltrating human macrophages. Concerning the gene expression, PM2.5-0.3 induced a significant increase for CYP1A1, CYP1B1, SOD2, HMOX and NQO1 (Fig. 4B), indicating the setting of a metabolic and antioxidant response. As for THP-1, CYP2E1 expression was not modified by PM2.5-0.3 exposure. PM2.5-0.3 was also responsible for a significant enhancement of the proinflammatory cytokines IL8 and IL1β expression, and IL8, IL1β, IL6 and TNFα release (Fig. 4C andD), as well as an increase of the granularity (SSC) of the exposed macrophages in comparison with the non-exposed one (Fig. 5A). Internalization of the particles by the macrophages was also observed (Fig. 5B) and it is correlated with an increase of the Macrophage Scavenger Receptor 1 (MSR1) protein (Fig. 5C). In conclusion of this section, the results show that infiltrating macrophages, such as undifferentiated THP-1, typically respond to PM exposure. This finding makes it possible to consider the EVs produced in response.

Characterization of extracellular vesicles derived from infiltrating macrophages exposed to

PM2.5-0.3.

EVs produced by the macrophages from 6 donors exposed or not to PM2.5-0.3 were isolated as described in Methods (sections 2.3 and 2.4) and pooled to form two average EVs samples deriving from exposed and nonexposed macrophages respectively. NTA characterization of the two obtained EVs samples indicated that PM2.5-0.3 exposure induced an significant increase of the number of EVs released by infiltrating macrophages as observed for THP-1 (Fig. 6A). On the contrary, the quantification revealed that PM2.5-0.3 exposure did not affect size distribution (Fig. 6B). Electron microcopy confirmed the presence of EVs (Fig. 6D). Finally, CD63 and CD81 exosomal proteins were detected by western-blot in EVs derived either from NE macrophages or from macrophages exposed to PM2.5-0.3 (Fig. 6C). These results are in line with those obtained from THP-1 exposed to PM2.5-0.3. To our knowledge and for the first time, this study shows that PM2.5-0.3 is able to induce EVs release from infiltrating human macrophages as well.

Effects of EVs derived from human macrophages exposed to PM2.5-0.3 on lung epithelial cells

One of the objectives of this work was to highlight whether EVs produced by exposed macrophages are able to transfer information from this exposure to unexposed lung cells and to activate typical PM exposure response.

To do this, BEAS-2B cells co-cultured with EVs from exposed and non-exposed macrophages (EVTHP-1/NE and EVTHP-1/PM for EVs derived from non-exposed and exposed THP-1 and EVHM/NE and EVHM/PM for EVs derived from non-exposed and exposed human macrophages) in proportional number as found in supernatants (Supp.

Fig. 3). Surprisingly, an important mortality was observed in BEAS-2B co-cultured with EVs derived from THP-1 regardless their conditions of exposure (data not shown). This cytotoxicity, measured via extracellular LDH activity, was significantly higher (p<0.1) for BEAS-2B co-cultured with EVTHP-1 in comparison with EVHM (Fig. 7A). In an attempt to identify the involved mechanisms, we measured the concentration of soluble Fas (sFas), an inducer of apoptosis generated by alternative mRNA splicing, in the culture supernatants (Fig. 7B). In addition to membrane-anchored Fas, sFas can antagonize cell-surface Fas function, leading to block the Fas-mediated apoptosis [START_REF] Nagata | Fas and Fas ligand: a death factor and its receptor[END_REF]. Here, the results show that, compared to BEAS-2B cells non exposed to EVs, sFas concentration in culture supernatants of BEAS-2B exposed to EVTHP-1 (obtained in all conditions) was three times lower, whereas it did not significantly vary in supernatants of BEAS-2B exposed to EVHM.

Specifically for the BEAS-2B cultured during 48h in the presence of EVHM, the nucleic acids and culture supernatants were isolated. The expression of 12 genes was measured (Table 2) but none of them showed any difference in expression, whether the cells were cultured in the presence of EVHM/NE or EVHM/PM. Interestingly, cytokines quantification showed a significant increase of the secretion for the pro-inflammatory cytokines IL6

and TNFα but not for IL1β (Fig. 7C) while their gene expression do not differ from controls after 48h of exposure. Surprisingly, EVHM/NE significantly induced IL8 secretion by BEAS-2B in comparison with BEAS-2B cultured in the absence of EVs or in the presence of EVHM/PM.

Discussion

Alveolar macrophages are the predominant cells that clean inhaled particulate matter off the lung [START_REF] Miyata | The innate and adaptive immune response induced by alveolar macrophages exposed to ambient particulate matter[END_REF]. In parallel, they produce pro-inflammatory mediators implicated in local as well as systemic inflammatory responses, leading to the recruitment of bone marrow derived macrophages [START_REF] Hiraiwa | Contribution of Lung Macrophages to the Inflammatory Responses Induced by Exposure to Air Pollutants[END_REF][START_REF] Van Eeden | Systemic inflammatory response induced by particulate matter air pollution: the importance of bone-marrow stimulation[END_REF]. Whereas many studies have focused on resident alveolar macrophages response when exposed to PM2.5, none were dealing with the response of infiltrating macrophages.

Here, the PM cytotoxicity and the activation of gene expression of XMEs and enzymes involved in oxidative stress of infiltrating macrophages in response to PM2.5 exposure was firstly assessed. To do that, undifferentiated THP-1 cells (non-treated with PMA) and primary macrophages (obtained by treating primary monocytes with GM-CSF during 36h, to mimic their recruitment by alveolar macrophages towards the lung (Goto et al., 2004b(Goto et al., , 2004a)) were exposed to PM2.5-0.3 (at 0, 12.5 and 50 µ/mL during 6h, 24h and 48h for THP-1 and at 0 and 12.5 µg/mL during 48h for human macrophages). In these conditions, PM exposure was not significantly cytotoxic for both THP-1 and human infiltrating macrophages, compared to non exposed control (Fig. 2A and4A).

Concerning XME expression, previous results have shown that PM2.5-0.3 induces high and dose-dependent increase of CYP1A1 expression in alveolar macrophages, which could be related to the presence of PAHs in PM [START_REF] Abbas | Air pollution particulate matter (PM2.5)-induced gene expression of volatile organic compound and/or polycyclic aromatic hydrocarbon-metabolizing enzymes in an in vitro coculture lung model[END_REF][START_REF] Saint-Georges | Gene expression induction of volatile organic compound and/or polycyclic aromatic hydrocarbonmetabolizing enzymes in isolated human alveolar macrophages in response to airborne particulate matter (PM2.5)[END_REF]. In this study, in undifferentiated THP-1 cells, CYP1A1 expression was significantly induced at 6h and decreased with time. Surprisingly, CYP1B1 expression importantly increased as well, whereas it has been shown that, in PM2.5-0.3 exposed lung epithelial cells, CYP1B1

was much less induced than CYP1A1 (Capucine Lepers et al., 2014). Concerning CYP2E1, while its expression has been shown to be significantly induced in alveolar macrophages by PM2.5-0.3 exposure [START_REF] Abbas | Air pollution particulate matter (PM2.5)-induced gene expression of volatile organic compound and/or polycyclic aromatic hydrocarbon-metabolizing enzymes in an in vitro coculture lung model[END_REF][START_REF] Saint-Georges | Gene expression induction of volatile organic compound and/or polycyclic aromatic hydrocarbonmetabolizing enzymes in isolated human alveolar macrophages in response to airborne particulate matter (PM2.5)[END_REF], in THP-1 monocytic cells, no induction of its expression has been observed. By exposing infiltrating primary macrophages, similar results have been obtained to a lesser extent, with a significant induction of CYP1A1 and CYP1B1 expression (RQ>2) and no significant increase of CYP2E1 expression (RQ<2) (Fig. 4B), suggesting that PAHs associated to the collected PM2.5 (Fig. 1B) are mainly metabolized by the CYP superfamily members CYP1A1 and CYP1B1. In parallel, a role of metals in PM2.5-0.3 on the expression of CYP1A1 cannot be excluded. Indeed, it was demonstrated that metal ions as Hg 2+ , Pb 2+ , and

Cu 2+ were able to induce its gene expression at transcriptional and posttranscriptional levels [START_REF] Korashy | Regulatory mechanisms modulating the expression of cytochrome P450 1A1 gene by heavy metals[END_REF]). On the one hand, an AhR-dependent mechanism contributing to the CYP1A1 induction was involved in the presence of metal ions. The latter did not significantly affect mRNA; however, they decreased the degradation rate of its protein, suggesting a posttranslational regulation of the CYP1A1 transcript by heavy metals. In addition, the authors observed a significant reduction in AhR ligand-mediated induction of CYP1A1 activity along with an increase in HMOX and a decrease in cellular heme content. The same research group also found that an increased of ROS production by the AhR ligands potentially generated in the presence of metals, were accompanied by a decrease in the CYP1A1 catalytic activity [START_REF] Elbekai | Benzo[a]pyrene, 3methylcholanthrene and beta-naphthoflavone induce oxidative stress in hepatoma hepa 1c1c7 Cells by an AHR-dependent pathway[END_REF].

Oxidative stress is a primary effect involved in the PM2.5-0.3 toxicity. It is generally correlated to ROS generated by PAHs and/or metals in PM [START_REF] Cachon | 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[END_REF][START_REF] Dergham | Prooxidant and Proinflammatory Potency of Air Pollution Particulate Matter (PM2.5-0.3) Produced in Rural, Urban, or Industrial Surroundings in Human Bronchial Epithelial Cells (BEAS-2B)[END_REF] (they were detected in the considered PM (Fig. 1A andB)) and associated with the induction of enzymes implicated in quinones detoxification like NQO1 or associated with ROS production like HMOX [START_REF] Jaguin | Exposure to Diesel Exhaust Particle Extracts (DEPe) Impairs Some Polarization Markers and Functions of Human Macrophages through Activation of AhR and Nrf2[END_REF]. This oxidative stress also represents an imbalance between the production and the removal of ROS by the antioxidant enzymes such as SOD family enzymes [START_REF] Michael | Pro-inflammatory effects and oxidative stress in lung macrophages and epithelial cells induced by ambient particulate matter[END_REF]. In this study, SOD2, NQO1 and HMOX expression was significantly induced in primary macrophages (Fig. 4B), dose-dependent in undifferentiated THP-1 (Fig. 2B).

Taken together, these results reveal that human infiltrating macrophages are capable to initiate an antioxidant response after exposure to PM2.5-0.3 and have effective metabolic capacities.

Redox-sensitive signaling cascades induced by PM exposure through oxidative stress can further activate an inflammatory response [START_REF] Gerlofs-Nijland | Particle Induced Toxicity in Relation to Transition Metal and Polycyclic Aromatic Hydrocarbon Contents[END_REF], characterized by pro-inflammatory cytokines production like IL1β, IL6, and TNFα as well as IL8 [START_REF] Dergham | Prooxidant and Proinflammatory Potency of Air Pollution Particulate Matter (PM2.5-0.3) Produced in Rural, Urban, or Industrial Surroundings in Human Bronchial Epithelial Cells (BEAS-2B)[END_REF][START_REF] Miyata | The innate and adaptive immune response induced by alveolar macrophages exposed to ambient particulate matter[END_REF][START_REF] Yan | Oxidative stress and endocytosis are involved in upregulation of interleukin-8 expression in airway cells exposed to PM2.5[END_REF].

In undifferentiated THP-1, in concordance with these previous results, IL8, IL1β and TNFα expression was significantly induced in a dose-dependent manner at the earlier time of 6h (Fig. 2C) and decreased with time.

Their secretion was also classically induced in a dose-and time-dependent manner (Fig. 2D). Similar observations have been done in exposed primary human macrophages (Fig. 4C andD), except for TNFα and IL6

for which 48h could be too late to measure their expression. In a surprising way, IL6 secretion was found to be significantly induced in undifferentiated THP-1 (Fig. 2D) but only at the highest dose of 50 µg/mL of PM and at 48h. All of these results suggest a pro-inflammatory reaction of the infiltrating macrophages in response to the PM2.5-0.3 exposure.

The time of 48h and the dose of 12.5 µg/mL of PM for the exposure of infiltrating primary macrophages have been chosen by taking into account the results obtained for THP-1. At these time and dose, infiltrating macrophages set up (i) a response allowing the metabolization of PAHs, (ii) the antioxidant system and (iii) a pro-inflammatory response. Moreover, primary macrophages presented an increase of their granularity after exposure (Fig. 5A) and even if they were not differentiated, they were able to phagocyte the PM2.5-0.3 (Fig. 5B)

probably through MSR1 receptor found to be increased (Fig. 5C). This is in accordance with previous studies, showing a role for scavenger-type receptors in the uptake of unopsonized environmental particles [START_REF] Hiraiwa | Contribution of Lung Macrophages to the Inflammatory Responses Induced by Exposure to Air Pollutants[END_REF].

Acute and chronic exposure to PM has been associated with increased morbidity and mortality from cardiorespiratory diseases as well as an increase of lung cancer risk [START_REF] Bentayeb | Association between long-term exposure to air pollution and mortality in France: A 25-year follow-up study[END_REF][START_REF] Raaschou-Nielsen | Air pollution and lung cancer incidence in 17 European cohorts: prospective analyses from the European Study of Cohorts for Air Pollution Effects (ESCAPE)[END_REF]. Different mechanisms have been proposed implicating that the exposed cells act as well as on the lung microenvironment and afar through blood circulation. Among the known mechanisms of action through which PM could impact health, EVs are a well suited candidate as they are produced in the pulmonary environment in response to environmental stimuli [START_REF] Neven | Extracellular Vesicles: How the External and Internal Environment Can Shape Cell-To-Cell Communication[END_REF]. Among the studies dealing with EVs, some have shown that the production of EVs is increased in response to PM exposure [START_REF] Bollati | Microvesicleassociated microRNA expression is altered upon particulate matter exposure in healthy workers and in A549 cells[END_REF][START_REF] Bollati | Exposure to Metal-Rich Particulate Matter Modifies the Expression of Candidate MicroRNAs in Peripheral Blood Leukocytes[END_REF][START_REF] Cantone | Extracellular histones mediate the effects of metal-rich air particles on blood coagulation[END_REF][START_REF] Neri | Particulate matter induces prothrombotic microparticle shedding by human mononuclear and endothelial cells[END_REF]. One objective was to determine if infiltrating macrophages are capable to release EVs in response to PM2.5 exposure. To test this hypothesis, EVs were isolated from the supernatants of culture of THP-1 non exposed to PM2.5-0.3 or exposed to 12.5 µg/mL, 50 µg/mL for 6h, 24h and 48h and of infiltrating primary human macrophages non exposed or exposed to 12.5 µg/mL for 48h. It was shown for the first time that THP-1 and infiltrating primary human macrophages exposed to PM2.5-0.3 produce EVs, in a dose-and time-dependent manner as shown by NTA (Fig. 3A and6A). Recently, Soni et al. have highlighted the implication of alveolar macrophages-derived EVs in acute lung injury [START_REF] Soni | Alveolar macrophage-derived microvesicles mediate acute lung injury[END_REF]. Here it can be hypothesized that EVs produced by infiltrating macrophages following PM2.5 exposure have a role in PM2.5-induced injury.

Nevertheless in further studies, it would be essential to ensure that the effects that has been observed in pulmonary epithelial cells is really due to the presence of EVs, or rather due to the presence of free proteins, RNA molecules, or cell membrane fragments into the medium. Other further studies are necessary to characterized these EVs and identify potential exposure biomarkers.

Lung homeostasis is maintained in large part due to the crosstalk between the alveolar macrophages, predominant lung-resident immune cells, and the lung epithelial cells, the major constituents of the respiratory surface [START_REF] Haggadone | Microenvironmental Influences on Extracellular Vesicle-Mediated Communication in the Lung[END_REF]. When exposed to PM2.5, this balance is modified: several studies have shown that macrophages exposed to PM induce the expression of several enzymes like CYP1A1 and NQO1 as well as pro-inflammatory cytokines in co-cultured lung cells [START_REF] Abbas | Air pollution particulate matter (PM2.5)-induced gene expression of volatile organic compound and/or polycyclic aromatic hydrocarbon-metabolizing enzymes in an in vitro coculture lung model[END_REF][START_REF] Wang | Ambient fine particulate matter induce toxicity in lung epithelial-endothelial co-culture models[END_REF],

indicating that PM-exposed macrophages cause pulmonary cells to develop a phenotype of oxidative stress, metabolic activation, and pro-inflammatory response. Based on this statement, the proposed hypothesis is that macrophages released factors could be extracellular vesicles. The expression of genes involved in xenobiotic metabolization, oxidative stress or inflammation in BEAS-2B lung epithelial cells cultured in the presence of EVHM/NE or EVHM/PM was not significantly different (Table 1). However, in the presence of EVHM/PM, the secretion of the two pro-inflammatory cytokines TNFα and IL6 was induced, IL8 secretion was inhibited and no change was observed for IL1β (Fig. 7C). These results suggest that EVs do not act on gene expression but downstream on protein synthesis. It is indeed known that PM2.5 exposure directly introduce the pro-inflammatory cytokines IL6 and IL8 release, and to a lesser extend TNFα, by BEAS-2B, whatever the origin of the particles [START_REF] Cachon | 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[END_REF][START_REF] Dergham | Prooxidant and Proinflammatory Potency of Air Pollution Particulate Matter (PM2.5-0.3) Produced in Rural, Urban, or Industrial Surroundings in Human Bronchial Epithelial Cells (BEAS-2B)[END_REF]. These results were confirmed with our PM2.5-0.3, which induce a high increase of IL6, IL8 and TNFα release and not of IL1β (Supp. Fig. 4). In the case of IL6 and TNFα, EVs could act as enhancers of inflammation during the acute phase of the inflammatory response, whereas, as IL8 mediates the recruitment and activation of immune cells, rather act to inhibit this part of the inflammatory response. To answer these questions, further experiments could be performed, by exposing BEAS-2B with EVs derived from exposed macrophages along with PM2.5-0.3.

To go further in the discussion, it is now well established that one of the main components of EVs is miRNAs. A change in the protein expression could be related to the change in the miRNA profile of EVs obtained after exposure to PM. Assuming that, the quantification of miRNAs in EVs of macrophages exposed or not to PM was performed and no significant change was observed (data not shown). Nevertheless, it was shown that EVHM/PM in culture with BEAS-2B cells induced the release of pro-inflammatory cytokines by pulmonary epithelial cells. It suggests that EVs may be involved in the mechanism of the inflammatory response often associated with PM exposure. Moreover, the type of cells from which the EVs originate is preponderant, in particular the fact that they are cancerous or not. Indeed, very interestingly, EVs from THP-1, whether or not exposed to PM, induce a large cytotoxicity in BEAS-2B compared to EVs from primary macrophages (Fig. 7A).

The fact that the level of sFas in the culture supernatant of BEAS-2B grown in the presence of EVTHP-1 decreases by a factor of 3 compared to BEAS-2B grown in the absence of EVs or in the presence of EVHM suggests that the decrease in sFas could allow Fas to bind to its ligand, which leads to supposed BEAS-2B apoptosis. Coupling these results with post-exposure cytotoxicity measurement, it can be hypothesized that EVs from THP-1, exposed or not to PM2.5-0.3, cause BEAS-2B cell death by inhibiting the secretion of sFas. This difference in effect observed between the EVTHP-1 and the EVHM may be due to the cancerous nature of the THP-1 cell line.

Conclusion

This study shows that, as alveolar macrophages, infiltrating macrophages respond to PM exposure by an increase in the expression of the enzymes involved in the metabolization of xenobiotics, in oxidative stress and inflammatory response, as well an increase of the production of extracellular vesicles. These latter were able to induce on pulmonary epithelial cells an increase in the production of proinflammatory cytokines such as IL6 and TNFα suggesting they could play a role in the maintenance of inflammation induced by PM exposure. Although extensive studies would be needed to confirm these findings, it appears that infiltrating macrophages could exacerbate the PM-induced proinflammatory response on alveolar macrophages and maintain it via the action of extracellular vesicles on pulmonary epithelial cells. Further investigations will be carried out on the effects of PM on BEAS-2B, namely their ability to potentially release more extracellular vesicles in response to PM2.5 exposure and the effects of these EVs on the cellular environment. Significance RQ> 2 or RQ <0.5 reported to the value of unexposed control (NE), normalized to 1 with 18S. D) IL8, IL1β, IL6 and TNFα secretion of primary macrophages exposed 6h, 24h or 48h to 0 (NE), 12.5 or 50 µg/mL to PM2.5-0.3. Results are the medians in pg/mL of duplicates of three independent experiments. Significant differences (*p<0.05) were evaluated by Mann Whitney U test. proteins from primary macrophages from 5 donors non-exposed (NE), or exposed to 12.5 µg/mL of PM2.5-0.3.

List of abbreviations

Declarations

Figure captions

Relative quantification has been performed using stain-free gel of total proteins. Results are the medians in pg/mL of duplicates of three independent experiments. Significant differences (*p<0.05) were evaluated by Mann Whitney U test.

Fig. 1 :

 1 Fig. 1: PM2.5-0.3 composition. A) Major and trace inorganic species concentrations of the PM2.5-0.3 collected in Dunkerque (March 2014 -March 2015). B) Polycyclic Aromatic Hydrocarbon concentrations in the PM2.5-0.3 collected in Dunkerque (March 2014 -March 2015).

Fig. 2 :

 2 Fig. 2: Cellular response of THP-1 exposed to PM2.5-0.3. In each experiment, THP-1 were exposed 6h, 24h or 48h to 0 (NE), 12.5 or 50 µg/mL of PM2.5-0.3. A) LDH cytotoxicity quantification in percentage of 100% mortality. mRNA expression of B) CYP1A1, CYP1B1, CYP2E1, SOD2, NQO1, HMOX and C) IL8, IL1β, IL6 and TNFα was measured by RT-qPCR. Results of 2 -ΔΔCt relative quantification are the medians of duplicates of three independent experiments +/-inter-quartile range. Significance RQ> 2 or RQ <0.5 reported to the value of unexposed control (NE), normalized to 1 with 18S. D) Measurement of IL8, IL1β, IL6 and TNFα secretion of THP-1 exposed 6h, 24h or 48h to 0 (NE), 12.5 or 50 µg/mL to PM2.5-0.3. Results are the medians in pg/mL of duplicates of three independent experiments. Significant differences (*p<0.05) were evaluated by Mann Whitney U test.

Fig. 3 :

 3 Fig. 3: Characterization of extracellular vesicles derived from THP-1 exposed to PM2.5-0.3. EVs were obtained from THP-1 exposed 6h, 24h or 48h to 0 (NE), 12.5 or 50 µg/mL of PM2.5-0.3. A) Graphs representing the amounts and the mean of size of EVs measured by nanoparticle tracking analysis. Significant differences were evaluated by Student's T test (*p<0.01); B) Transmission electronic microscopy images of EVs (bar = 100nm); C) Western-blot using CD63 and CD81 antibodies on EVs obtained from each condition.

Fig. 4 :

 4 Fig. 4: Cellular response of infiltrating macrophages exposed to PM2.5-0.3. In each experiment, primary macrophages from 6 healthy donors were exposed 48h to 0 (NE) or 12.5 µg/mL of PM2.5-0.3.A) LDH cytotoxicity quantification in percentage of 100% mortality. mRNA expression of B) CYP1A1, CYP1B1, CYP2E1, SOD2, NQO1, HMOX and C) IL8, IL1β, IL6 and TNFα was measured by RT-qPCR. Results of 2 -ΔΔCt relative quantification are the medians of duplicates of three independent experiments +/-inter-quartile range.

Fig. 5 :

 5 Fig. 5: Morphology of infiltrating macrophages exposed to PM2.5-0.3. A) Dot plots of macrophages (black dots in the circle) before PM exposure, non-exposed (NE), or exposed to 12.5 µg/mL of PM2.5. Upon PM exposure, the granularity of macrophages increase as evidenced by the shift of SSC-H in comparison to control (NE) macrophages. B) Optical microscopy images of macrophages before PM exposure, non-exposed (NE), or exposed to 12.5 µg/mL of PM2.5 showing particles inside the cells. C) Western-blot using MSR1 antibody on

Fig. 6 :

 6 Fig. 6: Characterization of extracellular vesicles derived from primary macrophages exposed to PM2.5-0.3.EVs were obtained from pooled supernatants of primary macrophages isolated from 6 donors and exposed 48h to 0 (NE) or 12.5 µg/mL of PM2.5-0.3. A) Graph representing the amounts of EVs measured by nanoparticle tracking analysis. Significant differences were evaluated by Student's T test (*p<0.01); B) Graphical representation of size distribution of EVs produced by non exposed (NE) and exposed (12.5 µg/mL) primary macrophages; C) Western-blot using CD63 and CD81 antibodies on EVs obtained from each condition; D) Transmission electronic microscopy images of EVs (bar = 100nm except when indicated).

Fig. 7 :

 7 Fig. 7: Effects of EVs derived from human macrophages exposed to PM2.5-0.3 on lung epithelial cells. In each experiment, BEAS-2B were co-cultured or not (w/o EV) during 48h with EVs isolated from exposed and non-exposed (NE) THP-1 or primary macrophages to PM2.5 at the indicated conditions. A) LDH cytotoxicity quantification in percentage of 100% mortality; B) Quantification sFas secretion by BEAS-2B co-cultured with EVs with ELISA multiplex. Results are the medians in pg/mL of duplicates of three independent experiments. C) Quantification of IL8, IL1β, IL6 and TNFα secretion by BEAS-2B co-cultured with EVs with ELISA multiplex.

Table 1 . mRNA Taqman primer references

 1

	mRNA	Taqman Reference
	18S	Hs99999901_s1
	IL1β	Hs01555410_m1
	IL6	Hs00985639_m1
	IL8	Hs00174109_m1

Table 2 . mRNA expression in BEAS-2B cultured in the presence of EVs from exposed macrophages.

 2

	Results of 2 -ΔΔCt relative quantification are the medians of duplicates of three independent experiments +/-inter-
	quartile range. Significance RQ>2 or RQ<0.5 reported to the value of BEAS-2B cultured in the EVs from non
	exposed macrophages, normalized to 1 with 18S. ND, not detected.

Acknowledgements

We thank Karine Deboudt for providing electron microscopy access and a precious help for acquisition of EVs pictures. We thank Sylvain Billet for his help in writing this manuscript.

Funding

The present work was funded by the French National Research Program for Environmental and Occupational Health of ANSES with the support of the Cancer TMOI of the French National Alliance for Life and Health Sciences (AVIESAN) (2015/1/028), by the Regional Program for Environment, Health and Societies with the support of the Région Hauts- de-France (2014-11426) and by a doctoral fellowship from the French Environment and Energy Management Agency (ADEME).

Ethics approval and consent to participate

Not applicable.

Conflict of Interest

The authors declare that they have no conflict of Interest.