Bendik C Brinchmann

Eric Le Ferrec

William H Bisson

Norman Podechard

Henrik S Huitfeldt

Isabelle Gallais

Odile Sergent

Jørn A Holme

Dominique Lagadic- Gossmann

Johan Øvrevik
email: johan.ovrevik@fhi.no

Evidence of Selective Activation of Aryl Hydrocarbon Receptor Nongenomic Calcium Signaling by Pyrene

Keywords: Polycyclic aromatic hydrocarbons, aryl hydrocarbon receptor, calcium, endothelial cells, membrane remodelling

In its classical genomic mode of action, the aryl hydrocarbon receptor (AhR) acts as a ligand activated transcription factor regulating expression of target genes such as CYP1A1 and CYP1B1. Some ligands may also trigger more rapid nongenomic responses through AhR, including calcium signaling (Ca 2+ ). In the present study we observed that pyrene induced a relatively rapid increase in intracellular Ca 2+ -concentrations ([Ca 2+ ] i ) in human microvascular endothelial cells (HMEC-1) and human embryonic kidney cells (HEK293) that was attenuated by AhR-inhibitor treatment and/or transient AhR knockdown by RNAi. In silico molecular docking based on homology models, suggested that pyrene is not able to bind to the human AhR in the agonist conformation. Instead, pyrene docked in in the antagonist conformation of the the AhR PAS-B binding pocket, although the interaction differed from antagonists such as GNF-351 and CH223191. Accordingly, pyrene did not induce CYP1A1 or CYP1B1, but suppressed CYP1-expression by benzo[a]pyrene (B[a]P) in HMEC-1 cells, confirming that pyrene act as an antagonist of AhR-induced gene expression. Use of pharmacological inhibitors and Ca 2+ -free medium indicated that the pyrene-induced AhR nongenomic [Ca 2+ ] i increase was initiated by Ca 2+ -release from intracellular stores followed by a later phase of extracellular Ca 2+influx, consistent with store operated calcium entry (SOCE). These effects was accompanied by an AhR-dependent reduction in ordered membrane lipid domains, as determined by di-4-ANEPPDHQ staining. Addition of cholesterol inhibited both the pyrene-induced [Ca 2+ ] i -increase and alterations in membrane lipid order. In conclusion, we propose that pyrene binds to AhR, act as an antagonist of the canonical genomic AhR/Arnt/CYP1-pathway, reduces ordered membrane lipid domains, and activates AhR nongenomic Ca 2+ -signaling from intracellular stores.

Introduction

The aryl hydrocarbon receptor (AhR), which is a basic helix-loop-helix PAS transcription factor, plays a central role in regulating toxicity from PAHs. In its classical mode of action, ligand-activated AhR translocates to the nucleus and dimerizes with the AhR nuclear translocator (Arnt). The AhR-Arnt complex then binds to so-called dioxin or xenobiotic response elements (DREs or XREs) in the promotor region of target genes. The prototypical genes activated by AhR are the cytochrome P450 enzymes CYP1A1 and CYP1B1, but AhR also regulates a number of other genes including many central proinflammatory mediators [START_REF] Matsumura | The significance of the nongenomic pathway in mediating inflammatory signaling of the dioxin-activated Ah receptor to cause toxic effects[END_REF][START_REF] Tian | Ah receptor and NF-kappaB interactions: mechanisms and physiological implications[END_REF]. Notably, AhR-signaling is highly ligand specific and not restricted to the classical mode of action [START_REF] Denison | Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals[END_REF][START_REF] Guyot | The AhR twist: ligand-dependent AhR signaling and pharmaco-toxicological implications[END_REF]. Nonclassical AhR-signaling involves crosstalk with a number of other transcription factors and signaling molecules independently of Arnt activation [START_REF] Tian | Ah receptor and NF-kappaB interactions: mechanisms and physiological implications[END_REF][START_REF] Denison | Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals[END_REF][START_REF] Guyot | The AhR twist: ligand-dependent AhR signaling and pharmaco-toxicological implications[END_REF]. In addition, it has been suggested that many of the toxic effects of the AhR ligand 2,3,7,8tetrachlorodibenzodioxin (TCDD) could be due to nongenomic AhR-signaling where the receptor rather functions as a signaling molecule in the cytosol [START_REF] Matsumura | The significance of the nongenomic pathway in mediating inflammatory signaling of the dioxin-activated Ah receptor to cause toxic effects[END_REF]. As such, the emerging pattern of AhR signaling is strikingly similar to the signaling from a number of nuclear receptors, including estrogen receptor (ER), which also involves both genomic and nongenomic modes of action [START_REF] Rainville | Membrane-initiated non-genomic signaling by estrogens in the hypothalamus: cross-talk with glucocorticoids with implications for behavior[END_REF].

As for ER-signaling, AhR nongenomic responses appear to involve both activation of protein kinases as well as mediation of calcium (Ca 2+ ) responses within min after exposure, hence preceding the genomic activation of target genes [START_REF] Matsumura | The significance of the nongenomic pathway in mediating inflammatory signaling of the dioxin-activated Ah receptor to cause toxic effects[END_REF][START_REF] Denison | Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals[END_REF][START_REF] Rainville | Membrane-initiated non-genomic signaling by estrogens in the hypothalamus: cross-talk with glucocorticoids with implications for behavior[END_REF]. These early cytosolic responses of the AhR are far less understood than the genomic mode of action and have predominately been investigated for TCDD. It is therefore unclear whether genomic and nongenomic signaling is triggered simultaneously through interaction with the same motifs within the AhR-PAS-B binding pocket, or whether the rank order of ligand potencies could differ for induction of genomic and nongenomic signaling.

Polycyclic aromatic hydrocarbons (PAHs) constitute a central group of AhR ligands originating from incomplete combustion of organic materials. The main toxicological concern has historically been carcinogenic effects, and CYP1A1/CYP1B1is central in the metabolic activation and subsequent mutagenic effects of several PAHs [START_REF] Moorthy | Polycyclic aromatic hydrocarbons: from metabolism to lung cancer[END_REF]. In risk assessment the toxicity of PAHs has been converted into toxicity equivalency factors (TEFs) based on estimates of relative carcinogenicity compared to the known human carcinogen benzo[a]pyrene (B[a]P) [START_REF] Nisbet | Toxic equivalency factors (TEFs) for polycyclic aromatic hydrocarbons (PAHs)[END_REF]. Pyrene is among the most abundant PAHs in outdoor air occurring at concentrations that are orders of magnitudes higher than B[a]P (8), but is not classifiable as carcinogen to humans (IARC group 3 compound), and has a very low TEF value of 0.001 [START_REF] Nisbet | Toxic equivalency factors (TEFs) for polycyclic aromatic hydrocarbons (PAHs)[END_REF]. However, pyrene exposure has been associated with asthma in children [START_REF] Liu | Association of polycyclic aromatic hydrocarbons and asthma among children 6-19 years: NHANES 2001-2008 and NHANES 2011-2012[END_REF] and cardiovascular disease [START_REF] Ochoa-Martinez | Serum adipocyte-fatty acid binding protein (FABP4) levels in women from Mexico exposed to polycyclic aromatic hydrocarbons (PAHs)[END_REF]. Studies in zebrafish embryos suggest that pyrene may induce peripheral vascular defects, cardiotoxicity and neuronal cell death [START_REF] Incardona | Defects in cardiac function precede morphological abnormalities in fish embryos exposed to polycyclic aromatic hydrocarbons[END_REF][START_REF] Incardona | Developmental toxicity of 4-ring polycyclic aromatic hydrocarbons in zebrafish is differentially dependent on AH receptor isoforms and hepatic cytochrome P4501A metabolism[END_REF][START_REF] Zhang | Low-level pyrene exposure causes cardiac toxicity in zebrafish (Danio rerio) embryos[END_REF]. Moreover, pyrene and its derivatives, but not B[a]P or β-naphthoflavone, potentiate pro-inflammatory responses induced by a toll-like receptor-3 ligand [START_REF] Øvrevik | Mechanisms of chemokine responses by polycyclic aromatic hydrocarbons in bronchial epithelial cells: sensitization through toll-like receptor-3 priming[END_REF]. This suggests that pyrene triggers some effects that are distinctly different from B[a]P.

The reported lack of effect on CYP1A1/CYP1B1expression in human cells suggest that pyrene may have limited ability to bind and activate AhR [START_REF] Iwanari | Induction of CYP1A1, CYP1A2, and CYP1B1 mRNAs by nitropolycyclic aromatic hydrocarbons in various human tissuederived cells: chemical-, cytochrome P450 isoform-, and cell-specific differences[END_REF][START_REF] Mayati | Aryl hydrocarbon receptorindependent up-regulation of intracellular calcium concentration by environmental polycyclic aromatic hydrocarbons in human endothelial HMEC-1 cells[END_REF][START_REF] Vondracek | Assessment of the aryl hydrocarbon receptor-mediated activities of polycyclic aromatic hydrocarbons in a human cell-based reporter gene assay[END_REF]. In contrast, it appears that pyrene induce CYPexpression in fish and rats, although with considerable lower potency than B[a]P [START_REF] Barron | Relative potency of PAHs and heterocycles as aryl hydrocarbon receptor agonists in fish[END_REF][START_REF] Elovaara | Polycyclic aromatic hydrocarbon (PAH) metabolizing enzyme activities in human lung, and their inducibility by exposure to naphthalene, phenanthrene, pyrene, chrysene, and benzo(a)pyrene as shown in the rat lung and liver[END_REF][START_REF] Hendon | Molecular and developmental effects of exposure to pyrene in the early life-stages of Cyprinodon variegatus[END_REF], and it has been suggested that developmental toxicity of pyrene in zebrafish is mediated through AhRdependent mechanisms due to qualitative similarities with effects from TCDD [START_REF] Incardona | Defects in cardiac function precede morphological abnormalities in fish embryos exposed to polycyclic aromatic hydrocarbons[END_REF]. Studies from our lab have shown that the nitro-derivative 1-nitropyrene is capable of inducing CYP1A1 expression in rat Hepa1c1c7 cells, but fails to do so in human bronchial epithelial BEAS-2B cells [START_REF] Landvik | Effects of nitrated-polycyclic aromatic hydrocarbons and diesel exhaust particle extracts on cell signalling related to apoptosis: possible implications for their mutagenic and carcinogenic effects[END_REF][START_REF] Øvrevik | Differential effects of nitro-PAHs and amino-PAHs on cytokine and chemokine responses in human bronchial epithelial BEAS-2B cells[END_REF]. This could be due to differences in ligand selectivity and gene regulation between the human AhR and AhR from other species [START_REF] Bisson | Modeling of the aryl hydrocarbon receptor (AhR) ligand binding domain and its utility in virtual ligand screening to predict new AhR ligands[END_REF][START_REF] Flaveny | Differential gene regulation by the human and mouse aryl hydrocarbon receptor[END_REF][START_REF] Flaveny | Ligand selectivity and gene regulation by the human aryl hydrocarbon receptor in transgenic mice[END_REF]. One possibility is that pyrene and pyrene derivatives could bind the human AhR, but fail to induce the classical genomic signaling.

Previous studies from our consortium have shown that pyrene induces a relatively rapid increase in [Ca 2+ ] i in human microvascular endothelial HMEC-1 cells, almost twice the magnitude of calcium responses induced by B[a]P and other PAHs investigated [START_REF] Mayati | Aryl hydrocarbon receptorindependent up-regulation of intracellular calcium concentration by environmental polycyclic aromatic hydrocarbons in human endothelial HMEC-1 cells[END_REF]. In the present study we have explored the mechanism of pyrene-induced increase in [Ca 2+ ] i in HMEC-1 cells. Our results suggest that pyrene is a AhR ligand that may selectively activate nongenomic signaling, triggering increase in [Ca 2+ ] i through store-operated calcium entry (SOCE) in parallel with alteration in membrane organization in HMEC-1 cells. This strongly supports the notion that pyrene may have unique toxicological effects, and shows that AhR nongenomic signaling may be triggered independently of the classical genomic pathway. Applied Biosystems (Foster City, CA, USA). All other reagents were commercial products of the highest purity available.

Materials and methods:

Cell culture

Human endothelial HMEC-1 cells, obtained from ATCC through LGC Standards (Wesel, Germany)

were routinely maintained in MCDB 131 medium containing epidermal growth factor (10 ng/mL), hydrocortisone (1 µg/mL), penicillin (50 unit/mL), and streptomycin (50 µg/mL) and supplemented with 10% FBS, according to the providers' instructions. Experiments were performed on HMEC-1 cells at passage numbers 22-32. Human embryonic kidney HEK293 cells were maintained in Dulbecco's modified Eagle's medium containing penicillin (50 unit/mL), and streptomycin (50 µg/mL) and supplemented with 10% FBS. The HEK293 cells was a gift from the late Hervé Paris, INSERM, Toulouse, France. The passage number of and origin of these cells, that was used in a limited number of experiments are not known. HEK293 cells permanently expressing β1-/β2ADR

were obtained by β1-/β2ADR cDNA transfection using Lipofectamine (Invitrogen) as described elsewhere [START_REF] Mayati | Induction of intracellular calcium concentration by environmental benzo(a)pyrene involves a beta2-adrenergic receptor/adenylyl cyclase/Epac-1/inositol 1,4,5-trisphosphate pathway in endothelial cells[END_REF]. Chemicals were commonly prepared as stock solution in dimethyl sulfoxide (DMSO).

The final concentration of solvent did not exceed 0.2% (v/v); control cultures received similar concentration of DMSO.

Cells used for Ca 2+ measurements or fluorescence measurements of membrane order were grown on glass coverslips to 50-60% confluency and serum starved for a minimum of 12 h prior to exposure.

The glass coverslips were sterilized in ethanol of increasing concentrations from 70-99%, and then coated with serum proteins using pure FBS prior to cell culture. Cells used for q-PCR were cultivated in 6-well plates, grown to near confluence and serum starved for a minimum of 12 h prior to exposure.

Calcium measurements

HMEC-1 were grown on glass lamellas to 50-60% confluency and serum starved for a minimum of ] i were analysed in HMEC-1 cells exposed to all four DEP-OE, by micro-spectrofluorometry using the Ca2+ sensitive probe Fura-2AM, as previously reported [START_REF] Brinchmann | Lipophilic Chemicals from Diesel Exhaust Particles Trigger Calcium Response in Human Endothelial Cells via Aryl Hydrocarbon Receptor Non-Genomic Signalling[END_REF]. Briefly, cells were incubated at 37 °C in cell suspension buffer supplemented with 1.5 µM Fura-2AM and 0.006% pluronic acid. After 30 min loading, cells were washed two times with the buffer before exposure. The [Ca 2+ ] i imaging involved data acquisition every 10 s (emission at 510 nm) at 340-and 380-nm excitation wavelengths using an LEICA DMIRB microscope with an inverse 40x oil objective. We monitored changes in [Ca 2+ ] i using an imaging system equipped with a Retiga 1300I fast-cooled monochromatic digital camera (12-bit) (Qimaging, Burnaby, BC, Canada), and a monochromator for fluorophore excitation. Image acquisition and analysis, involving determination of pixels assigned to each cell, was performed with METAFLUOR software (Universal Imaging, Downingtown, PA, USA). Average pixel value for each cell, was obtained with excitation at both wavelengths and corrected for background. We obtained the ratio by dividing the 340-nm by the 380nm fluorescence image on a pixel-by-pixel base (R=F340 nm/F380 nm. Results are presented as normalized calcium level compared to basal [Ca 2+ ] i measured 3 min prior to exposure. Area under the curve (AUC) was calculated from baseline (1.0).

Determination of structural perturbation of plasma membrane

Plasma membrane-order was visualized by confocal fluorescence microscopy using the membrane order sensitive di-4 ANEPPDHQ probe. This probe displays a fluorescent spectral blue-shift from 620 nm when incorporated into membranes with a low lipid order (in a liquid disordered phase, Ld)

to 560 nm when inserted into membranes with a high lipid order (in a liquid-ordered phase, Lo). After acquisition using confocal fluorescence microscopy of both disordered and ordered-phase fluorescence images, a new image, indicative of membrane lipid order, was obtained by calculating the generalized polarization (GP) value, a ratiometric measurement of fluorescence intensities for each pixel which is correlated to membrane lipid order [START_REF] Owen | Quantitative imaging of membrane lipid order in cells and organisms[END_REF]. Briefly, after each treatment, HMEC-1 cells grown on glass coverslips were washed in phosphate buffer saline (PBS) and then fixed in 4%

paraformaldehyde in PBS at 4 °C. After three washes in PBS, the cells were stained with 5 μM di-4 ANEPPDHQ for 90 min and washed twice in PBS. Cells were then visualized with confocal fluorescence microscope LEICA DMI 6000 CS (Leica Microsystems, Wetzlar, Germany). Under excitation at 488 nm with an argon ion laser, ordered membrane images were acquired with a photomultiplier tube (PMT) range of 500 to 580 nm whereas for disordered membrane images, the PMT range was 620 to 750 nm (magnification x 400). Using Fiji imaging processing software Changes in bulk membrane fluidity were explored by electron paramagnetic resonance (EPR) spectroscopy using paramagnetic reporter groups incorporated into the membrane, as previously described [START_REF] Collin | Cooperative interaction of benzo[a]pyrene and ethanol on plasma membrane remodeling is responsible for enhanced oxidative stress and cell death in primary rat hepatocytes[END_REF]. Membranes were spin labelled by incubating cell suspensions at 37 °C for 15 min with 12-doxyl stearic acid (50 μg/mL). The EPR spectra of labelled samples were acquired at an ambient temperature on a Bruker Elexsys EPR spectrometer operating at 3509 G center field, 20 mW microwave power, 9.86-GHz microwave frequency, 1.77 G modulation amplitude and 100 kHz modulation frequency. The fluidity of the labelled membranes was quantified by calculating the order parameter S, which is inversely related to membrane fluidity.

Immunocytochemical staining and fluorescence microscopy

Cell cultures were fixed with 4% paraformaldehyde in PBS for 10 min, washed in PBS for 10 min, briefly dipped in H2O, and dried. Fixed cells were incubated with rabbit anti-Ahr (Santa Cruz Biotechnology, Dallas, Texas, USA) diluted (1:100) in PBS with 1% BSA for 20 h, followed by 2X10 Mounting Medium (Dako Denmark A/S, Glostrup, Denmark). Hoechst dye (H33254, 0.05ug/ml was added to the last washing buffer. A Leica DMRE fluorescence microscope equipped with A and L5 fluorescence filter cubes were used for observations. An F-view CD camera and Analysis 3.1 software (Olympus, Tokyo, Japan) were used for documentation.

Gene silencing by RNA interference

HMEC-1 cells were transiently transfected with ON-TARGETplus SMARTpool siRNAs against AhR, or ON-TARGETplus non-targeting (NT) control siRNAs (Dharmacon, Chicago, IL; USA).

Briefly described, semi-confluent cells were transfected with 100 nM siRNAs using Dharmafect-1 transfection reagent diluted in antibiotic-free culture medium, as previously described ( 16). Fortyeight h after transfection, cells were exposed to pyrene and analysed for effects on Ca 2+ -signaling.

Gene expression analysis by real-time PCR

Total RNA was isolated using NucleoSpin RNA Plus (Macherey-Nagel; Düren, Germany) and reverse transcribed to cDNA on a PCR System 2400 (PerkinElmer, Waltham, MA, USA) using a High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed using pre-designed TaqMan Gene Expression Assays and TaqMan Universal PCR Master Mix and run on ABI 7500 fast (Applied Biosystems). Gene expression of CYP1A1 (Hs00153120_m1) and CYP1B1 (Hs02382916_s1) were normalized against GAPDH (Hs02758991_g1), and expressed as fold change compared to untreated control as calculated by the

ΔΔCt method (ΔCt = Ct[Gene of Interest] -Ct[18S]; ΔΔCt = ΔCt[Treated] -ΔCt[Control]; Fold change = 2[-ΔΔCt]).

In silico molecular modelling and docking

The homology models of the human AhR-PAS-B in the agonist and antagonist conformation were built and refined as previously described by Bisson and colleagues [START_REF] Perkins | A Structural Switch between Agonist and Antagonist Bound Conformations for a Ligand-Optimized Model of the Human Aryl Hydrocarbon Receptor Ligand Binding Domain[END_REF]. Molecular Docking was run as reported elsewhere [START_REF] Perkins | A Structural Switch between Agonist and Antagonist Bound Conformations for a Ligand-Optimized Model of the Human Aryl Hydrocarbon Receptor Ligand Binding Domain[END_REF]. The ICM program relies on global optimization of the entire flexible ligand in the receptor field and combines large-scale random moves of several types with gradient local minimization and a search history mechanism. In the ICM-VLS (Molsoft ICM) screening procedure, the scoring function should give a good approximation of the binding free energy between a ligand and a receptor, and its usually a function of different energy terms based on a force-field [START_REF] Totrov | Derivation of sensitive discrimination potential for virtual ligand screening[END_REF]. The lower the ICM score, the higher the chance the ligand is a binder.

Statistical analysis

Statistical analyses were performed by ANOVA with Holm-Sidak post-test for multiple comparisons.

Threshold for statistical significance was set at P > 0.05. The gene expression data were analyzed using the ΔCT-values from the q-PCR measurements. All calculations were based on analysis of independent experiments (biological replicates), with exception of the membrane-order measurements by confocal microscopy. Due the inherent variability in di-4-ANEPPDHQ-staining among individual cells, a very high counting-number is needed to obtain statistical significance. The statistical analysis was therefore based on pooled technical replicates from three or more independent experiments. All calculations were performed with GraphPad Prism 6 software (GraphPad Software, Inc., San Diego, CA).

Results

Pyrene triggers a transient increase of intracellular calcium concentration in HMEC-1 cells

We first assessed the effects of pyrene on calcium signaling in human microvascular HMEC-1 cells.

Briefly, cells were incubated with the calcium sensitive fluorescent probe Fura-2-AM, and alterations in calcium signaling were recorded by continuous microscopy imaging. Three min after recordings started, cells were exposed to 1 µM Pyrene. As seen in Figure 1, pyrene induced a relatively rapid and substantial increase in [Ca 2+ ] i in HMEC-1 cells, as compared to DMSO. No visual sign of toxicity was observed during the exposure period (not quantified). Moreover, the pyrene-triggered increase in [Ca 2+ ] i declined towards resting state within one h (Fig 1, insert graph). Thus the observed [Ca 2+ ] iresponse did not seem to result from unspecific cytotoxicity.

Pyrene induces [Ca 2+ ] i increase through AhR-dependent mechanisms

To investigate the potential role of AhR-dependent in regulation Ca 2+ -responses, HMEC-1 cells were pre-incubated with CH223191, a well-known AhR antagonist, prior to pyrene exposure. As shown in figure 2A, CH223191 treatment abrogated the Ca 2+ -response induced by pyrene. Transfection of HMEC-1 cells with siRNA against AhR also attenuated the pyrene-induced [Ca 2+ ] i -response as compared to cells transfected with non-targeting siRNA (Fig. 2B). Moreover, pyrene triggered a similar increase in [Ca 2+ ] i in HEK293 cells that could also be abrogated by CH223191 treatment (Fig. 2C). This suggest that AhR was essential for the pyrene-induced increase in [Ca 2+ ] i , and that this AhR-dependent response was not restricted to HMEC-1 cells.

Pyrene docks in the AhR antagonist conformation in silico

The possible interaction between pyrene and AhR was assessed by in silico molecular docking based on homology models. The AhR agonists TCDD and B[a]P, the AhR antagonists GNF-351 and CH223191, as well as pyrene were first docked into a model of the human AhR-PAS-B binding pocket in the agonist conformation. TCDD docked with the highest score, establishing a dual hydrogen bond interaction with the side chain of Ser 88/Ser 365 and His 14/His 291 (Fig. 3A, Table 1). B[a]P docked with a lower score of -6.8 (Fig. 3B, Table 1), which remains above the threshold value of -5.314, previously calculated with this model, for predicting AhR agonist activity [START_REF] Perkins | A Structural Switch between Agonist and Antagonist Bound Conformations for a Ligand-Optimized Model of the Human Aryl Hydrocarbon Receptor Ligand Binding Domain[END_REF]. Based on this threshold value, both antagonists and pyrene were not predicted being agonists of the human AhR (Table 1).

The AhR antagonists GNF-351 and CH223191, as well as pyrene, were then docked into the human AhR-PAS-B binding pocket in the antagonist conformation. GNF-351 docked with the highest score of -35.14, establishing two hydrogen-bond interactions with the side chain of Gln106/Gln 383 and the backbone carbonyl of Gly 42/Gly 321 (Fig. 4A, Table 2). The antagonist CH223191 docked with a lower score of -25.73, and similarly to GNF-351, favouring a hydrogen bond interaction with the side chain of Gln106/Gln 383 (Fig. 4B, Table 2). Pyrene docked with the lowest score of -19.79 and without non-covalent bindings involved (Fig. 4C, Table 2). Interestingly, the score and the strength of interaction between the ligand and residue Gln 106/ Gln 383, highly correlate with the ranking of AhR binding, going from nanomolar (GNF-351), to low-(CH223191) and high-(pyrene) micromolar range. Moreover, as shown in Figure 4A-B, there is high probability of contact between residues Phe 47/ Phe 324 -Ile 48/ Ile 325 of the AhR and the indole and tolylazo-phenyl groups of GNF-351 and CH223191, respectively. In the case of pyrene, the number of possible contacts between the ligand and the two residues are not significant (Fig. 4C). Thus, the in silico modelling suggest that pyrene may act as a ligand for the AhR. However its docking into the AhR-PAS-B binding pocket differs from both "classical" agonists such as TCDD and B[a]P, as well as the two antagonists GNF-351 and CH223191.

Reciprocal antagonism between pyrene-induced [Ca 2+ ] i and B[a]P-induced CYP1A1/CYP1B1

To further explore the nature of AhR-involvement in pyrene-induced [Ca 2+ ] i , we examined effects of intermediate response considerably lower than pyrene alone, but higher than B[a]P alone (Fig. 5A).

In further agreement with our previous studies [START_REF] Mayati | Aryl hydrocarbon receptorindependent up-regulation of intracellular calcium concentration by environmental polycyclic aromatic hydrocarbons in human endothelial HMEC-1 cells[END_REF], pyrene failed to induce CYP1A1/CYP1B1expression in HMEC-1 cells, while B[a]P induced a statistically significant increase (Fig. 5B). Furthermore, co-exposure with pyrene attenuated B[a]P-induced CYP1A1/CYP1B1expression (Fig. 5B).( 32)Thus, pyrene seemed to be a selective agonist of the AhR nongenomic pathway in HMEC-1 cells, causing an AhR-dependent increase in [Ca 2+ ] i . Moreover, combined exposure to pyrene and B[a]P led to a reciprocal suppression of the individual effects, suggesting that the two PAHs may possess comparable affinities for the AhR ligand binding site.

Pyrene-induced [Ca 2+ ] i increase was attenuated by β-adrenoreceptors

Previous studies from our consortium have shown that B[a]P induced an AhR-independent increase in [Ca 2+ ] i through activation of the β2-adrenergic receptor (β2AR) in HMEC-1 and HEK 293 cells, and that B[a]P interacts directly with the binding-pocket of β2AR (26). Later we also observed that high doses of 1-nitropyrene (10 µM) induced Ca 2+ -responses in bronchial epithelial BEAS-2B cells through a mechanisms that seemed to depend on β2AR [START_REF] Mayati | Calcium signaling and beta2-adrenergic receptors regulate 1-nitropyrene induced CXCL8 responses in BEAS-2B cells[END_REF]. Thus, to explore whether pyrene-induced [Ca 2+ ] i also could be mediated through β2AR we examined [Ca 2+ ] i in HEK wildtype cells (HEK wt ) which express low levels of β-adrenoceptors, and in HEK cells overexpressing either β1AR (HEK β1 ) or β2AR (HEK β2 ). Pyrene induced a strong increase in [Ca 2+ ] i in HEK wt (Fig. 6). Overexpression of β1AR lead to an approximately 50% reduction in pyrene-induced [Ca 2+ ] i , compared to HEK wt, while overexpression of β2AR almost completely abolished the pyrene-induced [Ca 2+ ] i response (Fig. 6).

Furthermore, pre-incubation with the selective β2AR-antagonist ICI-118,551 appeared to increase pyrene-induced [Ca 2+ ] i response in HEK β2 to a level comparable to the effects in HEK wt (preliminary data, not shown). This shows that pyrene induced [Ca 2+ ] i increases through β2AR-independent mechanisms, and that β-adrenoceptor activity rather supressed the pyrene calcium-responses. Again, this underscores that pyrene differ from B[a]P in its mode of action.

Pyrene-induced [Ca 2+ ] i increase involved store-operated Ca 2+ entry

Next, we explored the mechanisms of pyrene Ca 2+ entry. Removing Ca 2+ from the exposure medium caused a partial inhibition of the pyrene-induced [Ca 2+ ] i response. The initial increase in [Ca 2+ ] i was sustained until 5-6 min in absence of extracellular Ca 2+ , but then gradually levelled-off compared to the response in normal medium, and subsequently decreased from around 15 min (Fig. 7A). This suggests that the initial phase of the response was due to Ca 2+ -release from intracellular stores, while later phase depended on influx of extracellular Ca 2+ . SKF 96365 had a similar effect; the initial response was intact, while the late response was partially inhibited (Fig. 8B). SKF 96365 is a storeoperated Ca 2+ entry (SOCE) inhibitor that inhibits Stromal interaction molecule 1 (STIM1), but also blocks transient receptor potential cation (TRPC) channels, voltage-gated Ca 2+ channels and potassium channels. By comparison, 2APB seemed to inhibit both the initial and late response, probably due to its inhibiting effect on inositol trisphosphate-receptor (IP3R) as well as SOCE and TRP channels (Fig. 7C). Surprisingly, BtP2, a potent blocker of SOCE and calcium release-activated calcium (CRAC) channels, abolished the Ca 2+ -response completely (Fig 7D). These result indicate that pyrene may trigger [Ca 2+ ] i through an IP3R-dependent release from endoplasmic reticulum followed by SOCE activation.

Pyrene, but not B[a]P, reduced membrane lipid order in the plasma membrane through an AhR-dependent mechanism

It is known that Ca 2+ -influx can be affected by physico-chemical characteristics of membrane (eg.

membrane order which reflect lipid packing depending on membrane fluidity and local lipid environment thus defining membrane microdomains), and PAHs like B[a]P have been shown to affect membrane fluidity [START_REF] Wiggins | Membrane-protein interactions in mechanosensitive channels[END_REF][START_REF] Gorria | Membrane fluidity changes are associated with benzo[a]pyrene-induced apoptosis in F258 cells: protection by exogenous cholesterol[END_REF]. Moreover, alterations in ordered plasma membrane microdomains, such as caveolae, can affect calcium influx [START_REF] Pani | Lipid rafts/caveolae as microdomains of calcium signaling[END_REF]. Thus we wanted to explore the possibility that pyrene affected the membrane microstructure in HMEC-1. Using the membrane order sensitive dye di-4-ANEPPDHQ, we observed that HMEC-1 cells exposed for 30 min to pyrene (1 µM) decreased global membrane lipid order compared to cells exposed to vehicle control (DMSO) (Fig. 8A andC). By contrast, B[a]P did not affect membrane lipid order in the HMEC-1 cells as determined by di-4-ANEPPDHQ (Fig. 8A). Furthermore, the pyrene-induced reduction in membrane order was reversed by pre-treatment with CH223191 (Fig 8B andC). However, both pyrene and B[a]P, in a larger extent, induced an AhR-dependent increase in bulk membrane fluidity measured by EPR (Fig. 9). Thus, the pyrene-specific effect observed by di-4-ANEPPDHQ staining did not appear to be due to general alteration of membrane fluidity, but more likely restricted to effects on repartition between ordered and disordered membrane. In total, this indicates that pyrene may have induced an AhR-dependent destabilization of ordered plasma membrane microdomains in HMEC-1 cells. However, both pyrene and B[a]P also seemed to elicit more general effects on membrane fluidity through AhR-dependent mechanisms.

Cholesterol, a plasma membrane ordering agent, attenuates pyrene-induced [Ca 2+ ] i increase.

To explore if the membrane order-and the Ca 2+ -responses were linked, we pre-treated HMEC-1 with cholesterol for 30 min prior to pyrene exposure. The addition of cholesterol, which is known to increase membrane lipid order, partially inhibited the pyrene-induced [Ca 2+ ] i -response (Fig. 10). Of interest, cholesterol treatment appeared to suppress both the initial and prolonged [Ca 2+ ] i -increase.

Thus, it seems that cholesterol interacted with both the initial release from intracellular stores as well as the more secondary [Ca 2+ ] i -influx through the plasma membrane. This effect could possibly be related to cholesterol-induced changes in membrane lipid order although it should not be excluded that excess of cholesterol may also have influenced the rate of pyrene-diffusion through the cell membrane.

Discussion

Our present results show that pyrene acts as an antagonist with regard to AhR-genomic responses, but induced a relatively rapid AhR-dependent increase in [Ca 2+ ] i within min after exposure, consistent with activation of AhR nongenomic signaling. In silico molecular modelling confirm that pyrene may dock in the human AhR-PAS-B binding pocket in the antagonist, but not the agonist conformation.

The pyrene-induced [Ca 2+ ] i increase seemed to be initiated by Ca 2+ -release from intracellular stores followed by a later phase of extracellular Ca 2+ -influx. These effects appeared to be closely linked to an AhR-dependent disruption of lipid ordered membrane microdomains. The results show that pyrene induced cellular effects through molecular mechanisms distinctly different from B[a]P, and suggest that pyrene could act as a selective activator of AhR-nongenomic calcium signaling.

Pyrene exposure did not trigger AhR-regulated gene expression when added alone, but reduced the B[a]P-induced CYP1A1/CYP1B1expression by ~50%, indicating that pyrene acts as an antagonist of the classical AhR-genomic signaling. In line with this, in silico molecular modelling indicated that pyrene preferentially docks into AhR-PAS-B binding pocket in the antagonist conformation.

However, pyrene induced an AhR-dependent [Ca 2+ ] i -response, further contrasting B[a]P. B[a]P which has been reported to increase [Ca 2+ ] i by an AhR-independent mechanism [START_REF] Mayati | Aryl hydrocarbon receptorindependent up-regulation of intracellular calcium concentration by environmental polycyclic aromatic hydrocarbons in human endothelial HMEC-1 cells[END_REF][START_REF] Mayati | Induction of intracellular calcium concentration by environmental benzo(a)pyrene involves a beta2-adrenergic receptor/adenylyl cyclase/Epac-1/inositol 1,4,5-trisphosphate pathway in endothelial cells[END_REF], somewhat surprisingly reduced the pyrene induced [Ca 2+ ] i -response by ~50%. Thus with regard to the AhR nongenomic calcium response, pyrene may be considered as an AhR agonist and B[a]P as the antagonist. These differential and mutually suppressive AhR-mediated effects of pyrene and B[a]P are in accordance with the in silico findings discussed below. A possible explanation to the findings is that the two PAHs compete for binding to the PAS-B binding pocket of the human AhR with relatively comparable affinity.

The previously proposed hypothesis regarding AhR function is that the 307-329 (αD-αF) segment of the human AhR behaves as a structural switch between an agonist to an antagonist conformation [START_REF] Perkins | A Structural Switch between Agonist and Antagonist Bound Conformations for a Ligand-Optimized Model of the Human Aryl Hydrocarbon Receptor Ligand Binding Domain[END_REF].

Based also on published AhR-HSP90 interaction studies, Bisson and colleagues [START_REF] Perkins | A Structural Switch between Agonist and Antagonist Bound Conformations for a Ligand-Optimized Model of the Human Aryl Hydrocarbon Receptor Ligand Binding Domain[END_REF] have suggested that AhR-antagonist binding results in a conformation change that either promotes premature dissociation of HSP90, or causes structural changes that inhibit AhR nuclear translocation. In particular, ligand interaction with residues Phe 47/ Phe 324 and Ile 48/ Ile 325 appear to play a critical role in the disruption of AhR-HSP90 interface [START_REF] Perkins | A Structural Switch between Agonist and Antagonist Bound Conformations for a Ligand-Optimized Model of the Human Aryl Hydrocarbon Receptor Ligand Binding Domain[END_REF]. Our present results suggest a high probability of contact between residues Phe 47/Phe 324 -Ile 48/Ile 325 of the AhR and the indole and tolylazophenyl groups of GNF-351 and CH223191, respectively. These antagonists are also capable of inhibiting nuclear translocation and/or AhR/Arnt heterodimerization, in vitro [START_REF] Smith | Identification of a high-affinity ligand that exhibits complete aryl hydrocarbon receptor antagonism[END_REF][START_REF] Zhao | CH223191 is a ligand-selective antagonist of the Ah (Dioxin) receptor[END_REF]. In the case of pyrene, the number of possible contacts between the ligand and the two residues are not significant.

The overall data suggest that pyrene is able to induce a conformational change of the AhR that differs from the one induced upon binding of either a full agonist (TCDD) or a full antagonist (GNF-351).

Thus, AhR-nongenomic calcium signaling may not be restricted to classical agonists of the genomic pathway.

Exposing the HMEC-1 cells in calcium-free medium resulted only in an inhibition of the latter part of the Ca 2+ response. This indicates that the initial response originated from intracellular stores, most likely due to release of Ca 2+ from the endoplasmic reticulum. A similar inhibition-pattern of pyreneinduced [Ca 2+ ] i , was obtained by use of SKF96365 which inhibits SOCE by interacting with STIM1 and blocks TRPC and voltage gated channels, thus preventing extracellular Ca 2+ -entry. By contrast, the commonly used IP3R inhibitor 2APB [START_REF] Maruyama | 2APB, 2-aminoethoxydiphenyl borate, a membrane-penetrable modulator of Ins(1,4,5)P3-induced Ca2+ release[END_REF][START_REF] Saleem | Interactions of antagonists with subtypes of inositol 1,4,5-trisphosphate (IP3) receptor[END_REF], also suppressed the initial phase of pyrene-induced [Ca 2+ ] i in HMEC-1 cells. This thus further supports that the pyrene-induced [Ca 2+ ] i response is initiated by IP3R-mediated release of calcium from endoplasmic reticulum. Notably a high concentration of BTP2 (10 µM), a known SOCE-CRAC and TRPC channel inhibitor [START_REF] Zitt | Potent inhibition of Ca2+ release-activated Ca2+ channels and T-lymphocyte activation by the pyrazole derivative BTP2[END_REF][START_REF] He | A functional link between storeoperated and TRPC channels revealed by the 3,5-bis(trifluoromethyl)pyrazole derivative, BTP2[END_REF], abrogated the entire calcium response. It thus seems that a high dose of BTP2 affects calcium release from ER as well as SOCE-CRAC and TRPCs in our experimental system. Whether this observation could be due to off-target effects of the inhibitor remains unclear. Taken together our results indicate that pyrene triggers a rapid and substantial calcium release from intracellular stores, succeeded by an influx of calcium possibly through SOCE-CRAC and TRPC channels.

Changes in membrane microstructure may affect the activity of membrane bound proteins such as ion channels and receptors and may thus modify cell signaling events including calcium responses.

Interestingly, pyrene induced an AhR-dependent disruption of lipid ordered membrane microdomains, as observed by use of di-4-ANEPPDHQ-staining. It has recently been suggested that di-4-ANEPPDHQ-staining which correlate with lipid order is less sensitive to membrane fluidity modifications than to cholesterol level changes in the membrane [START_REF] Amaro | Laurdan and Di-4-ANEPPDHQ probe different properties of the membrane[END_REF]. This could explain the lack of correlation with membrane fluidity measurements by EPR. Thus, the pyrene-induced AhR-dependent reduction in ΔGP could reflect a disruption in cholesterol-rich microdomains (reduction of their number or lowering their organisation). AhR has been reported to bind directly with caveolin-1 (Cav1) localized in caveolae, cholesterol rich microdomains and invaginations of the plasma membrane, and exposure to the AhR-ligand PCB77 further enhanced the AhR-Cav1 binding in vascular endothelial cells [START_REF] Lim | Coplanar polychlorinated biphenyl-induced CYP1A1 is regulated through caveolae signaling in vascular endothelial cells[END_REF][START_REF] Rey-Barroso | The Dioxin receptor modulates Caveolin-1 mobilization during directional migration: role of cholesterol[END_REF]. AhR-expression also appears to regulate Cav1-localization in ordered microdomains, and that addition of cholesterol may switch the pattern of Cav1 localisation to a AhR-/-phenotype [START_REF] Rey-Barroso | The Dioxin receptor modulates Caveolin-1 mobilization during directional migration: role of cholesterol[END_REF]. Caveolae function as Ca 2+ -signaling microdomains regulating SOCE channels, where Cav1 may act by coupling IP3R with TRPC-channels [START_REF] Pani | Lipid rafts/caveolae as microdomains of calcium signaling[END_REF][START_REF] Adebiyi | Caveolin-1 assembles type 1 inositol 1,4,5trisphosphate receptors and canonical transient receptor potential 3 channels into a functional signaling complex in arterial smooth muscle cells[END_REF]. It is therefore tempting to speculate that the pyrene-induced effects on Ca 2+ -signaling and ordered membrane microdomains is related to this association between AhR and Cav1 within the caveolae. In concordance with this, both Cav1 silencing and Ca 2+ -inhibition appear to attenuate AhR-induced CYP1A1 expression [START_REF] Lim | Coplanar polychlorinated biphenyl-induced CYP1A1 is regulated through caveolae signaling in vascular endothelial cells[END_REF][START_REF] Monteiro | Dioxin-mediated up-regulation of aryl hydrocarbon receptor target genes is dependent on the calcium/calmodulin/CaMKIalpha pathway[END_REF]. Moreover, caveolins also appear to be central in directing the effects of nongenomic estrogen receptor and glucocorticoid receptor signaling by tethering the receptors into the caveolae and dictating the downstream cellular responses [START_REF] Rainville | Membrane-initiated non-genomic signaling by estrogens in the hypothalamus: cross-talk with glucocorticoids with implications for behavior[END_REF][START_REF] Samarasinghe | Nongenomic glucocorticoid receptor action regulates gap junction intercellular communication and neural progenitor cell proliferation[END_REF]. Nongenomic signaling from a range of nuclear receptors may therefore share some common mechanisms.

B[a]P has been shown to trigger an AhR-independent calcium response in HMEC-1 and HEK 293 cells mediated through activation of β2AR [START_REF] Mayati | Induction of intracellular calcium concentration by environmental benzo(a)pyrene involves a beta2-adrenergic receptor/adenylyl cyclase/Epac-1/inositol 1,4,5-trisphosphate pathway in endothelial cells[END_REF]. In contrast, the pyrene-induced [Ca 2+ ] i , was not only AhR-dependent, but also partially suppressed by β1AR-overexpression and almost completely blocked by β2AR-overexpression in HEK293 cells. These results not only fortify the notion that B[a]P and pyrene elicit cellular effects through distinctly different mechanisms, but also suggest that β-adrenoceptors may affect AhR signaling. It has previously been reported that a PAH-mixture containing more than 50% pyrene impaired β2AR function in airway epithelial and smooth muscle cells [START_REF] Factor | Polycyclic aromatic hydrocarbons impair function of beta2adrenergic receptors in airway epithelial and smooth muscle cells[END_REF]. A mutually suppressive crosstalk between AhR and β-adrenoceptor signaling may therefore exist. In light of this, it is interesting to note that also β-adrenoceptors appear to localize in caveolae and interact with caveolins [START_REF] Ostrom | The evolving role of lipid rafts and caveolae in G protein-coupled receptor signaling: implications for molecular pharmacology[END_REF][START_REF] Allen | Caveolin-1 and lipid microdomains regulate Gs trafficking and attenuate Gs/adenylyl cyclase signaling[END_REF].

AhR nongenomic Ca 2+ -signaling has been proposed as a key mechanism regulating proinflammatory effects associated with TCDD poisoning [START_REF] Matsumura | The significance of the nongenomic pathway in mediating inflammatory signaling of the dioxin-activated Ah receptor to cause toxic effects[END_REF]. However, Ca 2+ is one of the main cellular second messengers and plays a central role in intracellular signal transduction and cell homeostasis as such [START_REF] Clapham | Calcium signaling[END_REF][START_REF] Sandow | Calcium and endotheliummediated vasodilator signaling[END_REF]. Thus, AhR-mediated changes in [Ca 2+ ] i may therefore add explanations to several effects linked to AhR-ligands. For instance, [Ca 2+ ] i regulation in endothelial cells is pivotal to vascular signaling pathways, with important implications for regulating blood pressure and flow under physiological as well as pathophysiological conditions [START_REF] Sandow | Calcium and endotheliummediated vasodilator signaling[END_REF]. [Ca 2+ ] i is also involved in regulation of endothelial permeability [START_REF] Tiruppathi | Role of Ca2+ signaling in the regulation of endothelial permeability[END_REF]. Thus, disturbance of normal [Ca 2+ ] i in the vascular endothelium could be of importance to the cardiovascular effects associated with exposure to various AhR ligands [START_REF] Savouret | The aryl hydrocarbon receptor and its xenobiotic ligands: a fundamental trigger for cardiovascular diseases. Nutrition, metabolism, and cardiovascular diseases[END_REF][START_REF] Korashy | The role of aryl hydrocarbon receptor in the pathogenesis of cardiovascular diseases[END_REF][START_REF] Majkova | The role of caveolae in endothelial cell dysfunction with a focus on nutrition and environmental toxicants[END_REF][START_REF] Perkins | Polychlorinated biphenyls and links to cardiovascular disease[END_REF]. Research on PCB-induced activation of nongenomic AhR-responses in the caveolae, have led to the suggestion that these cholesterol-rich microdomains could act as platforms for pro-inflammatory signaling and modulation of the pathology of vascular diseases [START_REF] Majkova | The role of caveolae in endothelial cell dysfunction with a focus on nutrition and environmental toxicants[END_REF]. In contrast to other AhR-agonists, pyrene alone appears to have limited impact on AhR-mediated gene expression. However, results from our laboratory show that pyrene may exacerbate pro-inflammatory responses induced by other agents [START_REF] Øvrevik | Mechanisms of chemokine responses by polycyclic aromatic hydrocarbons in bronchial epithelial cells: sensitization through toll-like receptor-3 priming[END_REF]. The biological or toxicological implications of this apparent selective activation of AhR nongenomic calcium signaling therefore remains to clarify.

In conclusion, our present results show that pyrene act as an antagonist of the canonical genomic AhR/Arnt/CYP1-pathway, and a selective activator of AhR nongenomic signaling in endothelial cells, triggering a SOCE-mediated increase in [Ca 2+ ] i and alterations in plasma membrane microstructure. In silico molecular modelling confirms that pyrene may interact with the PAS-B binding pocket of the human AhR, but suggests that it may induce a conformational change that differs from those induced upon binding of either a full agonist or antagonist. These results support a notion that AhR genomic and nongenomic signaling could be triggered independently and that activation of AhR nongenomic signaling may not be restricted to the classical AhR-agonists.

Tables Table 1. ICM scores into human AHR-PAS-B in the agonist conformation +, not favourable positive score

2. 1

 1 Chemicals Benzo[a]pyrene (B[a]P), pyrene, 2-methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazophenyl)-amide (CH223191), cholesterol, hydrocortisone and EGTA were purchased from Sigma-Aldrich (St. Louis, MO, USA; now Merck). TRIzol® reagent, pluronic acid and Fura-2 acetoxymethylester (Fura-2-AM) were provided by InVitrogen (Carlsbad, CA, USA). 1-[2-(4-Methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole hydrochloride (SKF 96365) and N-[4-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5carboxamide (BtP2) was obtained from TOCRIS (Bristol, UK). 2-Aminoethoxydiphenylborate (2-APB) was purchased from Merck Millipore (Burlington, MA, USA. Di-4ANEPPDHQ, MCDB 131 medium, and L-glutamine (200 mM) were purchased from Thermo Fischer Scientific (Carlsbad, CA, USA). Fetal bovine serum (FBS) by Biochrom AG (Berlin, Germany), and endothelial growth factor by Nerliens Meszansky (Oslo, Norway). Penicillin and streptomycin were from Lonza (Walkersville, MD, USA). All real-time RT-PCR reagents and TaqMan probes/primers were purchased from

(

  ImageJ; National Institutes of Health, Bethesda, USA) and the macro published by Owen et al.[START_REF] Owen | Quantitative imaging of membrane lipid order in cells and organisms[END_REF], GP images were generated according to the following calculation: GP= (I 500-580 -I 620-750 )/ (I 500-580 +I 620-750 ). On each GP image generated (containing usually between 1 to 3 cells), a GP values was measured and normalized by subtraction of the mean of all GP values found for DMSO-treated cells (ΔGP values). Then, ΔGP values of images from at least 3 experiments were used to generate a dot blot. Finally, the global mean of ΔGP values for each condition was calculated from a minimum of 10 images.

  combinatory exposure with B[a]P. B[a]P is a well-known inducer of the prototypical markers of AhR genomic signaling, CYP1A1 and CYP1B1. Thus, HMEC-1 cells were exposed to either pyrene or B[a]P, or the two PAHs in combination for 15 min. In line with previous observations[START_REF] Mayati | Aryl hydrocarbon receptorindependent up-regulation of intracellular calcium concentration by environmental polycyclic aromatic hydrocarbons in human endothelial HMEC-1 cells[END_REF], pyrene induced a considerably stronger [Ca 2+ ] i response, compared to B[a]P (Fig.5A). B[a]P also induced a slower increase in [Ca 2+ ] i in HMEC-1 cells and the effect was only marginally affected by the AhR inhibitor CH223191 (preliminary data, not shown), corroborating previous results obtained by AhR-silencing with siRNA[START_REF] Mayati | Aryl hydrocarbon receptorindependent up-regulation of intracellular calcium concentration by environmental polycyclic aromatic hydrocarbons in human endothelial HMEC-1 cells[END_REF]. Combined exposure to pyrene and B[a]P, resulted in an

FIGURE 1 .

 1 FIGURE 1. Effects of pyrene on [Ca2+ ] i .

FIGURE 2 .

 2 FIGURE 2. Role of AhR in pyrene-induced [Ca 2+ ] i . A: HMEC-1 cells exposed to pyrene (1 µM) in

FIGURE 3 .

 3 FIGURE 3. In silico modelling of ligand docking in the AhR PAS-B agonist conformation.

FIGURE 4 .

 4 FIGURE 4. In silico modelling of ligand docking in the AhR PAS-B antagonist conformation.

FIGURE 5 .

 5 FIGURE 5. Effects of pyrene and B[a]P, alone and in combination, on [Ca2+] i and

FIGURE 6 .FIGURE 7 .

 67 FIGURE 6. Role of β-adrenoceptors in pyrene-induced [Ca 2+ ] i . HEK293 wild type cells (HEK wt )

FIGURE 8 .FIGURE 9 . 1 .

 891 FIGURE 8. Effects of pyrene and B[a]P on membrane lipid order and membrane fluidity in

FIGURE 10 . 1 .

 101 FIGURE 10. Pyrene-induced [Ca 2+ ] i was inhibited by cholesterol in HMEC-1. Three min after

  min washes in PBS, a brief dip in H 2 O, and dried. The cells were then exposed to Alexa488conjugated donkey anti-rabbit IgG (1:200, Jackson Immunoresearch, West Grove, PA, USA) for one h, and washed 2X10 min in PBS, briefly dipped in H 2 O, dried, and mounted with Dako Fluorescent

Table 2 .

 2 ICM scores into human AHR-PAS-B in the antagonist conformation

Acknowledgements

The work was supported by the Research Council of Norway, through the Environmental Exposures and Health Outcomes-program (grant no. 228143), as well as Rennes Métropole (France) which provided additional travel grants. We also wish to thank Inserm and the Université de Rennes 1 for financial support.

Conflict of interest statement

The authors have no conflicts of interest.


Sophie Perinel

Valérie Forest
email: vforest@emse.fr

Mathilde Landraud

Jérémie Pourchez

Françoise Girardot

Serge Riffard

Magalie Stauffert

Jean-Michel Vergnon

Séverine Allegra

Deposition pattern of aerosolized Legionella using an ex vivo human-porcine respiratory model

Keywords: Legionella, lung deposition, aerosolization, broncho-alveolar lavage, ex vivo chimeric model

Legionella are bacteria responsible for severe lung pathologies. However how they enter and are deposited within the respiratory tract remains poorly documented. Data using animal testing led to the establishment of mathematical models allowing the estimation of aerosol dispersion risks. But direct extrapolation to humans is questionable and experimental models more physiologically representative of the inhalation route are welcome. The aim of this study was to develop a model as close as possible to the human anatomy and physiology allowing determining the deposition pattern of aerosolized Legionella while limiting in vivo experiments. To that purpose, we adapted the chimeric respiratory tract model we previously developed. This original model consisted of a replica of the human upper respiratory airways made by additive manufacturing connected to ex vivo porcine lungs ventilated by passive expansion, as for humans in physiological conditions. These experiments didn't imply specific animal sacrifices as pigs were bred for human consumption and lungs were considered as wastes by the slaughterhouse. Fluorescent Legionella were aerosolized and visualized using Cellvizio  Lab (probe-based confocal fluorescence microscope). Legionella were found in the whole respiratory tract. Broncho-alveolar lavages were also performed and the amount of Legionella reaching the thoracic region was quantified by culture and qPCR.

Legionella were found preferentially in the left upper lobe compared to the right lower lobe.

To our knowledge, it is the first time that experiments mimicking so closely human exposure by inhalation are performed while limiting animal experiments and providing a model for further Legionella infectious risk assessment.

Introduction

Legionella are Gram-negative bacteria found in various aquatic environments, especially hot water sources including domestic water systems and showers, cooling towers, decorative fountains, hot tubs, etc. [START_REF] Anses | Légionelles et légionellose | Anses -Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail[END_REF][START_REF] Burillo | Microbiology and Epidemiology of Legionnaire's Disease[END_REF][START_REF] Khodr | Molecular epidemiology, phylogeny and evolution of Legionella[END_REF][START_REF] Phin | Epidemiology and clinical management of Legionnaires' disease[END_REF]. Legionella are usually spread through water droplets in the air. After inhalation by humans, bacteria are conveyed through the tracheo-bronchial tree deep into the alveoli where they infect macrophages and lung epithelial cells, potentially causing pneumonia called legionellosis including Legionnaires' disease and Pontiac fever [START_REF] Burillo | Microbiology and Epidemiology of Legionnaire's Disease[END_REF][START_REF] Khodr | Molecular epidemiology, phylogeny and evolution of Legionella[END_REF][START_REF] Phin | Epidemiology and clinical management of Legionnaires' disease[END_REF]. Legionella pneumophila, especially from the serogroup 1 (Lp1), is the main species responsible for pathologies (about 90% of the cases) [START_REF] Burillo | Microbiology and Epidemiology of Legionnaire's Disease[END_REF][START_REF] Khodr | Molecular epidemiology, phylogeny and evolution of Legionella[END_REF][START_REF] Phin | Epidemiology and clinical management of Legionnaires' disease[END_REF]. The incidence of legionellosis is estimated between 8000 and 18000 new cases each year in the United States and between 1200 and 1500 in France [START_REF] Campèse | Legionnaires' disease in France[END_REF][START_REF] Who | Legionellosis[END_REF].

The monitoring of Legionella consists of their detection in water samples by culture on selective glycine, vancomycine and polymyxine B charcoal yeast extract (GVPC) agar (quantification as colonies forming units (CFU) per liter, as defined by the AFNOR NF T90-431 or ISO 11731-2 standards) or by quantitative PCR (qPCR, as number of genome units (GU) per liter, as defined by the AFNOR NF T90-471 or ISO/TS 12869 standards) [START_REF] Burillo | Microbiology and Epidemiology of Legionnaire's Disease[END_REF][START_REF] Légifrance ; Lück | Arrêté du 1er février 2010 relatif à la surveillance des légionelles dans les installations de production, de stockage et de distribution d'eau chaude sanitaire[END_REF]. However, it is difficult to correlate the number of detected Legionella to an infectious risk probably due to the presence in the water samples of viable but non-culturable (VBNC) Legionella and to a lesser extent because of PCR and culture inhibitors [START_REF] Allegra | Use of flow cytometry to monitor Legionella viability[END_REF]S. Allegra et al., 2011;[START_REF] Allegra | Longitudinal evaluation of the efficacy of heat treatment procedures against Legionella spp. in hospital water systems by using a flow cytometric assay[END_REF].

Regulatory preventive thresholds were established empirically [START_REF] O'brien | Legionnaires' disease: the infective dose paradox[END_REF] based on the observation of legionellosis epidemics when a correlation between patient and environment was possible (less than 16% of the cases) or based on in vivo studies. The way infectious doses are determined depends on the reservoir studied (water or aerosols) and/or on the models used (such as animals). Mathematical models allowing the estimation of aerosol dispersion risks have been described but it is not possible to directly extrapolate their results to humans [START_REF] Armstrong | Legionnaires' disease: evaluation of a quantitative microbial risk assessment model[END_REF][START_REF] Armstrong | Quantitative microbial risk assessment model for Legionnaires' disease: assessment of human exposures for selected spa outbreaks[END_REF]; T. W. Armstrong and Haas, 2007). Therefore, adapted models and methodologies are required to improve Legionella detection and quantification to revisit or refine the infectious doses obtained from animal models. Moreover, inhalation of aerosols should be used, more representative of a physiological route than intratracheal or intraperitoneal injections or instillations. As a matter of fact, to date, how Legionella may enter and be deposited in the respiratory tract upon aerosol generation remains poorly documented [START_REF] Górny | Size distribution of bacterial and fungal bioaerosols in indoor air[END_REF][START_REF] Thomas | Characterization and deposition of respirable large-and small-particle bioaerosols[END_REF].

In this context, the aim of the present study was to propose an experimental model, as close as possible to the human anatomy and physiology, allowing determining the deposition pattern of aerosolized Legionella within the respiratory tract. These data are important to determine a real risk of inhalation exposure by the assessment of the infectious dose reaching the respiratory barrier. One of our main concerns to reach this objective was to limit animal experiments in accordance with the 3R concept introduced by Russell and Burch [START_REF] Russell | The Principles of Humane Experimental Technique[END_REF]. Indeed, we paid attention to replace, reduce and refine our research approach by developing an alternative and very innovative experimental model. To that purpose, we adapted and optimized a chimeric respiratory model we previously developed and validated for other researches in the aerosol therapy field [START_REF] Perinel | Development of an ex vivo human-porcine respiratory model for preclinical studies[END_REF]. It consisted of a complete respiratory tract including a 3D replica of the human upper respiratory airways (nasal cavity, pharynx, sinuses) made by additive manufacturing allowing oro-nasal breathing.

This human replica was connected to ex vivo porcine lungs through a home-made artificial larynx. It is important to mention that lungs did not come from animals bred for scientific research but from animals bred for human consumption. They were provided by a slaughterhouse as in France lungs are not fit for human consumption and are consequently considered as organic wastes. It means that no animals were sacrificed specifically for this study since we used, for scientific research, organic wastes that are currently discarded by slaughterhouse. Lungs were ventilated by passive expansion as in human physiological ventilation allowing to reproduce inhalation exposure to aerosols [START_REF] El Merhie | A new Strategy to Improve Drug Delivery to the Maxillary Sinuses: The Frequency Sweep Acoustic Airflow[END_REF]Le Guellec et al., 2014;Leclerc et al., 2014a;[START_REF] Perinel | Micron-sized and submicron-sized aerosol deposition in a new ex vivo preclinical model[END_REF][START_REF] Perinel | Development of an ex vivo human-porcine respiratory model for preclinical studies[END_REF]. Previously, characterized Legionella aerosols generated upon nebulization using a fluorescent Legionella strain [START_REF] Allegra | Characterization of aerosols containing Legionella generated upon nebulization[END_REF]. This strain was aerosolized in the chimeric model as ventilation was ongoing and then visualized using the Cellvizio  Lab technology (probe-based confocal fluorescence microscope). Broncho-alveolar lavages (BAL) were also performed to quantify the amount of Legionella reaching the thoracic region. To our knowledge, it is the first time that experiments mimicking so closely a real human physiological exposure were performed while limiting animal experiments.

Materials and Methods

Legionella airborne suspensions

A Legionella pneumophila serogroup 1 (Lp1) species (responsible for LD) was used in this study. Bacteria are fluorescent as they were transfected with a GFP-containing plasmid and thus express the green fluorescent protein (GFP) [START_REF] Cormack | FACS-optimized mutants of the green fluorescent protein (GFP)[END_REF][START_REF] Köhler | Expression and use of the green fluorescent protein as a reporter system in Legionella pneumophila[END_REF]. It was provided by Centre National de Référence des Légionelles (CNRL-Lyon) as their 008 strain. In this study, the strain is identified as Lp1-008. Thanks to the GFP, Legionella deposition was followed [START_REF] Köhler | Expression and use of the green fluorescent protein as a reporter system in Legionella pneumophila[END_REF][START_REF] Unal | Dictyostelium discoideum as a model to study host-pathogen interactions[END_REF]. Legionella were plated on BCYE agar (Buffered Charcoal Yeast Extract, SR0110C, Oxoid, France) supplemented with 8 mg/mL chloramphenicol (Sigma Aldrich, France) (BCYE+Chl) to select the bacteria bearing the GFP-containing plasmid.

For the nebulization process, suspensions were prepared from Legionella cultivated in BCYE+Chl for 72h at 37°C under a 5% CO 2 atmosphere to get bacteria with similar morphology to pathogenic Legionella present in aquatic reservoirs [START_REF] Robertson | The many forms of a pleomorphic bacterial pathogen-the developmental network of Legionella pneumophila[END_REF]. 8 mL of calibrated Legionella suspension were prepared in sterile normal saline (0.9% NaCl) water at a final concentration of 2x10 7 CFU/mL. An optical density of 0.2 at 600 nm (Biomate TM3; Avantec, Illkirch, France) was taken as reference for a bacteria suspension containing 10 8 CFU/mL. 5 mL of this suspension were placed in the nebulizer (see the aerosol generation section below) and aerosolized during lung ventilation.

Experimental model

A unique chimeric model was previously developed and well characterized [START_REF] Perinel | Development of an ex vivo human-porcine respiratory model for preclinical studies[END_REF]. It was adapted for this specific research on Legionella bioaerosol exposure by inhalation. As shown by Figure 1, it is composed of a 3D resin replica corresponding to the human upper respiratory airways connected to an ex vivo porcine pulmonary tract placed in a sealed enclosure. It was ventilated artificially by passive expansion, simulating pleural depressions (Super Dimension®, Covidien, Dusseldorf Germany). The anatomical model of the upper respiratory airways includes the entire nasal cavities (nasal fossae, frontal, ethmoid and maxillary sinuses) and the oro-pharynx, allowing an oro-nasal ventilation. It was made by additive manufacturing after a 3D image processing from CT-scan data as described in a previous study (Le [START_REF] Guellec | Validation of anatomical models to study aerosol deposition in human nasal cavities[END_REF]. It was manufactured in transparent water resistant and non-porous resin. Its use was validated for the quantification of aerosol drug deposition in the extra-thoracic regions [START_REF] El Merhie | A new Strategy to Improve Drug Delivery to the Maxillary Sinuses: The Frequency Sweep Acoustic Airflow[END_REF]Le Guellec et al., 2014;Leclerc et al., 2014a). Thus this part of the model represents the extra-thoracic area and was connected to porcine lungs which were provided by a slaughterhouse and used within 24 hours. 6 independent experiments were conducted. Lungs were carefully selected to avoid lesions incompatible with a good functioning of the model and small cuts were fixed by stitches. The artificial laryngeal part was made of plastic tubes with a one-way valve simulating the vocal folds resistance as inhalation was ongoing [START_REF] El Merhie | A new Strategy to Improve Drug Delivery to the Maxillary Sinuses: The Frequency Sweep Acoustic Airflow[END_REF]Le Guellec et al., 2014;Leclerc et al., 2014a). Another one-way valve allowed the path for exhalation as previously described (Leclerc et al., 2014b). An expiratory filter avoided environmental contamination.

Physiological measurements for this model were consistent with the widely accepted human physiology [START_REF] Perinel | Development of an ex vivo human-porcine respiratory model for preclinical studies[END_REF]. Experimental conditions used were biomimetic with a breathing technological process simulating the intrapleural depressions. Indeed, the chimeric model respired artificially by inflation with a negative pressure (passive expansion) applied inside an instrumented sealed enclosure [START_REF] Perinel | Development of an ex vivo human-porcine respiratory model for preclinical studies[END_REF]. The inhalation is due to the depression inside the instrumented sealed enclosure and the exhalation is passive by the return to atmospheric pressure by a pause of the depression generator. Moreover the duration of inhalation and exhalation in the respiratory cycle was chosen with shorter time duration for inhalation. In order to fit the human physiology, an inspiratory-to-expiratory time [I/E] ratio of ½ meaning 1.3 s for inhalation and 2.6 s for exhalation was chosen, leading to a respiratory cycle of 4 s and consequently a respiratory rate of 15/min. These breathing parameters were selected because they correspond to adult male physiology at rest. The extra-thoracic dead space was 21 mL, the tracheal internal diameter 1.7 cm and the tracheal length 7.5 cm [START_REF] Booker | Interpretation and evaluation of pulmonary function tests[END_REF]Criée et al., 2011;[START_REF] Flesch | Lung volumes: measurement, clinical use, and coding[END_REF]. The chimeric model satisfied the major functional and anatomical features necessary to study airborne particle deposition [START_REF] Perinel | Development of an ex vivo human-porcine respiratory model for preclinical studies[END_REF][START_REF] Perinel | Micron-sized and submicron-sized aerosol deposition in a new ex vivo preclinical model[END_REF]. A video showing the lung ventilation is provided as supplementary data.

Aerosol generation

The nebulizer used to aerosolize bacterial suspensions was an e-Flow® Rapid Nebulizer System (PARI GmbH, Stanberg, Germany). The mechanism of aerosol generation was based on a perforated oscillating membrane technology. It consists of the high-frequency vibration of a very thin membrane provided with a multitude of micrometric holes in order to obtain a very fine nebulization through the holes from the liquid contained in the device reservoir [START_REF] Thomas | Characterization and deposition of respirable large-and small-particle bioaerosols[END_REF]. The main advantages of vibrating-mesh nebulizers include consistent aerosol generation efficiency, a high fine particle fraction reaching the peripheral lung and a low residual volume in the reservoir. In addition, inhaled fractions are more important with mesh nebulizers than with jet nebulizers and can provide higher drug doses to patients [START_REF] Ari | Jet, Ultrasonic, and Mesh Nebulizers: An Evaluation of Nebulizers for Better Clinical Outcomes[END_REF]. To secure the experimenters and prevent them from inhaling Legionella during nebulization, the model was placed in a glove box (815-PGB "LA PETITE" GLOVE BOX, Fisher Scientific). Moreover, BCYE agar Petri dishes were placed outside and inside the glove box to evaluate air contamination.

Broncho-alveolar lavages

Broncho-alveolar lavages were performed after Legionella nebulization in the left upper lobe and right lower lobe with 200 mL of sterile normal saline (0.9% NaCl) water (6.5<pH<7.1) for each area according to international guidelines [START_REF] Luyt | Fibreoptic bronchoscopic techniques for diagnosing pneumonia[END_REF]. Ventilation was ongoing during the whole process. The recovered volumes of BAL were slightly different between the assays. In order to compare comparable things, this volume was taken into account in our calculations and the amount of Legionella was finally expressed as CFU/mL of BALF.

Broncho-alveolar lavage fluids analysis

The quantitative assessment of Legionella in broncho-alveolar lavage fluids (BALF) was carried out by culture and qPCR for the 6 experiments. For culture, each initial bacterial suspensions (100 μL) and BALF (500 μL) were plated on two BCYE agar supplemented with or without chloramphenicol and incubated at 37°C for 72h under a 5% CO 2 atmosphere. All BCYE plates were observed under UV at 366 nm to identify and enumerate Legionella colonies (CFU) expressing GFP. For qPCR, the quantification was made as described in our previous study [START_REF] Allegra | Characterization of aerosols containing Legionella generated upon nebulization[END_REF]. Briefly, after a centrifugation at 8000 rpm for 10 min of a 1 mL sample (initial bacterial suspension or BALF), plasmid DNA was extracted (NuCleoSpin Plasmid kit, Macherey-Nagel). qPCR was carried out on a StepOne automate (ThermoFisher Scientific), using the 2X Power SYBR® Green PCR Master Mix (Life Technologies) as follows: initial denaturation for 15 min and a two-step cycle consisting of 15 s denaturation, 1 min annealing and elongation at 60°C. After 45 cycles of amplification, a melting curve program was incorporated to check for any primer dimers or other non-specific amplifications. Each sample was run in duplicate. A standard curve was done with Lp1-008 initial suspensions ranging from 2x10 7 to 2x10 2 CFU/mL to allow the qPCR results expression in CFU (3 independent experiments were carried out). The results were analyzed using StepOne Software v2.3.

Visualization of deposited Legionella by probe-based confocal fluorescence microscope

Because the bacteria are transformed with a GFP-containing plasmid, Legionella reaching the intra-thoracic region can be visualized using the Cellvizio  Lab (Mauna Kea Technologies).

This probe-based confocal fluorescence microscope technology allows the detection of

Legionella on tissue biopsies with a cellular resolution. To assess the presence of fluorescent bacteria and potentially identify areas of privileged deposition, biopsies were performed in the trachea, left lower lobe, right upper lobe and middle lobe. The fluorescence (excitation wavelength = 488 nm) was then determined in each biopsy sample.

Results

Aerodynamic characterization of the Legionella bioaerosol

Aerosols containing Legionella generated upon nebulization were previously extensively characterized [START_REF] Allegra | Characterization of aerosols containing Legionella generated upon nebulization[END_REF]. It was determined that the distribution of airborne Legionella showed a Mass Median Aerodynamic Diameter (MMAD) of 3.4 ± 0.4 µm with a Geometric Standard Deviation (GSD) of 4.1 ± 1.2 µm. It was also evidenced that 0.26-2.5 µm aerosol size range represented 7% of the initial bacterial suspension and among the viable forms, 0.7% of initial viable bacterial suspension could reach the lung alveoli.

Qualitative assessment of airborne Legionella deposition in the thoracic region

After nebulization of fluorescent Legionella, biopsies were performed in various areas of the tracheo-bronchial tree (see Figure 2) and the tissue fluorescence was analyzed using the Cellvizio  Lab technology. The detection was made easier by the fact that porcine lungs, contrary to human lungs, are not auto-fluorescent at the excitation wavelength used. Thus no background noise was recorded and the fluorescence observed necessarily came from Legionella, rendering the signal very specific. Indeed we previously verified in control porcine lungs not submitted to Legionella aerosolization that no fluorescent signal was observed. As shown by Figure 2, Legionella were found in the whole respiratory tract, i.e. fluorescence was detected in the 5 analyzed stages of the tracheo-bronchial tree. In other words, after inhalation Legionella were able to reach the deepest lung structures.

Quantitative assessment of airborne Legionella deposition in the thoracic region

Broncho-alveolar lavages were performed in the left upper lobe and right lower lobe.

Legionella were then quantified in BALF by culture and qPCR. Six independent experiments were performed. Figure 3 shows representative results obtained by culture analysis from experiment 4. BCYE+Chl agar plates were used to select Legionella among the important bacteria of the porcine mucosal flora (Figure 3 C andD). Due to a very important porcine lung bacterial flora in experiment 2, no culture results were obtained for this experiment, even on BCYE+Chl agar plates. As expected, results of culture and qPCR showed no significant difference for the enumeration of Legionella in the initial suspensions aerosolized in the 6 experiments i.e. a median  standard deviation of 2.9x10 7  0.8x10 7 CFU/mL for culture and 2.8x10 7  0.6x10 7 CFU/mL for qPCR. Due to the important inhibition of Legionella growth on BCYE+Chl by porcine lung microflora, only qPCR results were taken into account and shown in Figure 4. A higher concentration of Legionella was systematically enumerated in BALF recovered from the left upper lobe than from the right lower lobe.

Discussion

To the best of our knowledge, it is the first time that an experimental model mimicking so closely human physiological exposure by inhalation is successfully reported, showing the regional distribution of airborne Legionella in the respiratory tract. Indeed, the qualitative and quantitative assessments of the presence of Legionella bacteria in the different lobes of the lungs and in the bronchial tree are possible using a convenient ex vivo model. Moreover, opposite to previous thought and theoretical data, we found a differential quantification of Legionella between lobes.

We developed this model with the objective to fill a gap in the existing experimental respiratory models to better understand the aerosol route of infection during legionellosis. Indeed, since its description in the late 1970s many studies have attempted to better understand the Legionnaires' Disease (LD). To that purpose, in vitro assays and experimental animal models were developed to investigate the pathology mechanisms and to evaluate treatments [START_REF] Baskerville | Experimental transmission of legionnaires' disease by exposure to aerosols of Legionella pneumophila[END_REF].

In vitro models represent the simplest models to study the LD. They can be used for mechanistic studies, to study the bacteria/host interactions or to compare the virulence of different bacterial strains. Cells can be either of animal or human origin. The main advantages of in vitro models are their low cost and the easy supply of large quantities of cells. Although basic, cellular models can be useful as shown by [START_REF] Yan | Infection of murine macrophage cell lines by Legionella pneumophila[END_REF].

In vivo studies on primitive organisms were also performed as alternative experimental models of LD. For instance the soil amoeba Dictyostelium discoideum [START_REF] Hägele | Dictyostelium discoideum: a new host model system for intracellular pathogens of the genus Legionella[END_REF] or the nematode Caenorhabditis elegans [START_REF] Brassinga | The Caenorhabditis elegans model of Legionella infection[END_REF][START_REF] Komura | Caenorhabditis elegans as an alternative model host for legionella pneumophila, and protective effects of Bifidobacterium infantis[END_REF] were found to be simple hosts suitable to study host/bacteria interactions, intracellular pathogenesis of Legionella, contribution of specific genes to the bacterial virulence and host immunity and apoptosis. Although these alternative approaches could indeed provide useful data at a cellular resolution, they are obviously far from a model allowing extrapolation to Legionella inhalation in humans. For this purpose, other animal models were developed.

A variety of species can be experimentally infected with Legionella such as guinea pigs, mice, rats, monkeys, etc. [START_REF] Edelstein | Chapter 34 -The Guinea-pig Model of Legionnaires' Disease[END_REF]. Thus several animal-based infection models of LD have been developed. Nevertheless, guinea pigs were found to be extremely susceptible to Legionella infection and are therefore widely used to produce a disease that simulates human LD in immunocompromised patients [START_REF] Baskerville | Experimental transmission of legionnaires' disease by exposure to aerosols of Legionella pneumophila[END_REF][START_REF] Davis | Legionnaires' pneumonia after aerosol exposure in guinea pigs and rats[END_REF][START_REF] Edelstein | Chapter 34 -The Guinea-pig Model of Legionnaires' Disease[END_REF][START_REF] Fitzgeorge | Aerosol infection of animals with strains of Legionella pneumophila of different virulence: comparison with intraperitoneal and intranasal routes of infection[END_REF]. However, due to the lack of dedicated reagents to study the immune response to Legionella infection in guinea pigs, this model of infection can hardly be used to monitor the disease. Consequently, research on the molecular and cellular aspects of the antibacterial immune response has shifted to mouse infection models [START_REF] Brown | Mouse models of Legionnaires' disease[END_REF].

And indeed, the mouse model of Legionella infection has provided many insights into innate immunity and proved to be a valuable system to elucidate the contribution of inflammatory factors and different immune cells to lung defense [START_REF] Brown | Mouse models of Legionnaires' disease[END_REF]. In addition, mice models have the advantages of being inexpensive, convenient to handle and to generally well tolerate antimicrobial therapy [START_REF] Rouse | Chapter 58 -Animal Models of Gram-negative Bacillary Experimental Pneumonia[END_REF].

Regarding the infection route, different pathways have been assessed and successfully used to produce Legionella pneumonia in experimental animal models: first intratracheal, intranasal, intraperitoneal and more recently via aerosols inhalation [START_REF] Leggett | Chapter 63 -Murine Models of Pneumonia Using Aerosol Inoculation[END_REF]. The issue of exposure is not trivial as it may have different consequences. For instance, Fitzgeorge et al. [START_REF] Fitzgeorge | Aerosol infection of animals with strains of Legionella pneumophila of different virulence: comparison with intraperitoneal and intranasal routes of infection[END_REF] demonstrated that infection of guinea pigs by intranasal instillation of two strains of L. pneumophila did not induce disease while the same preparations administered as an aerosol produced a fatal widespread bronchopneumonia within 3 days. The most obvious difference between these exposure routes was the size of the infective particles and their site of deposition within the respiratory tract. Similarly, aerosol inhalation and intratracheal instillations could be used to establish fatal infection in guinea pigs and less severe infection in rats [START_REF] Edelstein | Chapter 34 -The Guinea-pig Model of Legionnaires' Disease[END_REF]. The intraperitoneal inoculation seems the less relevant way as, in guinea pigs, it produces peritonitis, systemic infection, sepsis and bacteremic pneumonia so, while easy to produce, it is not a good model for LD [START_REF] Edelstein | Chapter 34 -The Guinea-pig Model of Legionnaires' Disease[END_REF]. On the contrary, infection by aerosol inhalation appears to be a successful and reproducible experimental system for the study of LD, and it is particularly relevant to the human disease [START_REF] Baskerville | Experimental transmission of legionnaires' disease by exposure to aerosols of Legionella pneumophila[END_REF]. For living animals, Legionella bioaerosols can be administered via nose-only or whole body exposure systems. However, while the animal-based models of LD provide considerable benefits in improving our understanding of Legionella-induced pneumonia and disease pathogenesis, they have some limitations. The different susceptibility to Legionella between species has already been mentioned and, as another example, Davis et al. showed that after exposure to a Legionellacontaining aerosol, guinea pigs demonstrated illness, fever and 56% mortality, whereas rats showed little illness and 11% mortality [START_REF] Davis | Legionnaires' pneumonia after aerosol exposure in guinea pigs and rats[END_REF]. Another important issue regarding the study of treatment efficacy is that pharmacokinetics of antimicrobial agents given to guinea pigs do not mimic the human situation. In most cases, drug half-lives are shorter in guinea pigs [START_REF] Burillo | Microbiology and Epidemiology of Legionnaire's Disease[END_REF][START_REF] Edelstein | Chapter 34 -The Guinea-pig Model of Legionnaires' Disease[END_REF]. Similarly, some exposure routes are not relevant with respect to what happens in real life. In this context exposure to aerosols seems the most appropriate but it may exhibit technical constraints, for instance Davis et al. exposed guinea pigs and rats to a nose-only inhalation system for 30 minutes [START_REF] Davis | Legionnaires' pneumonia after aerosol exposure in guinea pigs and rats[END_REF]. This exposure time is quite long and can be stressful for animals. This is why we propose here a complementary approach consisting of an experimental model with the objective of limiting animal testing while deepening our knowledge of Legionella infections using the inhalation route, as in physiological conditions.

The aim of this study was to propose a first proof of concept using this original model allowing determining the deposition pattern of Legionella bioaerosol in the respiratory tract.

To mimic as closely as possible physiological conditions, we adapted a model we previously developed for aerosol therapy purposes [START_REF] Perinel | Development of an ex vivo human-porcine respiratory model for preclinical studies[END_REF]. In its first version the model included a plastinated head allowing only nasal ventilation. It was replaced here by a resin replica made by additive manufacturing. This latter allowed a mouth-nose ventilation, thus improving the representation of the physiological spontaneous ventilation in adults.

The major advantage of this model is that it does not imply specific animal sacrifices, respecting the 3R rule and consequently does not raise ethical issues. Indeed, the pigs were dedicated to human consumption and as lungs are considered as wastes they were kindly provided by the slaughterhouse. It implies two other important advantages: an important availability and an inexpensive model. Because the animals are dedicated to human consumption, all the sanitary controls were performed at the slaughterhouse in strict accordance with the French regulation. These controls, carried out in presence of veterinary inspectors, guarantee that animals were healthy.

The model and the experimental setting developed were secure as no contamination was observed (no bacterial growth in the Petri dishes placed outside and inside the glove box).

Besides its safety, this model proved to be efficient. Indeed, it allowed us to determine that airborne Legionella were able to reach the whole respiratory tract and were found in all the stages of the tracheo-bronchial tree.

The inhibition of Legionella growth on plate by lung microbiota was to be expected. Indeed the difficulty to grow Legionella on agar media has already been described [START_REF] Hussong | Viable Legionella pneumophila Not Detectable by Culture on Agar Media[END_REF][START_REF] Légifrance ; Lück | Arrêté du 1er février 2010 relatif à la surveillance des légionelles dans les installations de production, de stockage et de distribution d'eau chaude sanitaire[END_REF]. It is related to the relatively long time of Legionella division compared to other bacteria species, these latter grow faster and fill all the space on the plate. Another explanatory factor is that Legionella enter a viable but not-culturable (VBNC) state. This inhibition is also observed in the clinical analysis of human respiratory samples. The other bacteria present in the porcine lungs do not affect Legionella deposition and viability more than bacteria from the human respiratory mucosa.

We also observed a difference between the deposition of Legionella in the right lower lobe and the left upper lobe. From there, two assumptions could be made to explain this observation: a ventilation gradient between upper and lower zones or an asymmetry of ventilation between left and right lungs. Based on the literature, we can reasonably exclude the first hypothesis. Indeed, it has been demonstrated [START_REF] West | Distribution of blood flow and ventilation-perfusion ratio in the lung, measured with radioactive carbon dioxide[END_REF]) that this gradient is accurate in upright position (due to the gravity) but it disappears in the supine position in favor of an anterior-posterior gradient [START_REF] Kaushik | Probing the regional distribution of pulmonary gas exchange through singlebreath gas-and dissolved-phase 129Xe MR imaging[END_REF]. Consequently it is very unlikely to explain this difference in Legionella deposition by physiology in our experimental model. The second hypothesis could be an asymmetry of ventilation between left and right lungs. Such observation can be made in humans but it has been linked to the increase in density of the left lung due to the heart [START_REF] Tawhai | Supine and prone differences in regional lung density and pleural pressure gradients in the human lung with constant shape[END_REF]. Therefore it cannot be an explanation with our model where the heart was removed before the experimentation. Moreover, a fiberoptic endoscopy was performed before the experiments and did not show any obstruction suggesting homogeneous bronchial tree and no airway obstruction between the right and the left lungs. Further studies are necessary but if they confirm this specific and differential deposition pattern of aerosolized Legionella in lungs, it could be of great interest as, to our knowledge, it is the first time such results are reported. It was quite unexpected and all the more surprising given that in humans the LD is thought to affect the lobes without distinction [START_REF] Mittal | Thoracic Imaging Features of Legionnaire's Disease[END_REF].

It is difficult to further compare our results with those from the literature as, to the best of our knowledge, no such comparable studies were reported. Indeed, we propose an original and innovative model which appears as complementary to previous works.

For the development of further complementary studies we should be aware of the model limitations. First, the model is placed in horizontal position, it does not reproduce the gravity effect human lungs are subjected to. Therefore, the distribution of inhaled bacteria in the lungs may be slightly modified. This horizontal placement is due to technical constraints: in order to protect the experimenters, the experimental system had to be placed under a glove box. However, despite this "non physiological" position, we observed, as discussed before, a differential Legionella deposition between the right lower lobe and the left upper lobe which is consequently not linked to gravity effect. Despite our efforts to reproduce a model as close as possible to the human anatomy and physiology some factors couldn't be taken into account in these first sets of experiments. For instance the nasal and bronchial mucosa, the ciliated cells, the secretion of mucus/surfactant and the body temperature were not reproduced. Nevertheless, we will consider future developments and refinements of the model over time.

We also observed variability in the results. For example, in one case out of the 6 experiments conducted, we noted a lung contamination by the porcine bacterial flora. This is likely due to the fact that pigs can inhale gastric fluids at the time of slaughter (despite the fast for 48 hours before slaughter). This rare event is also possible in humans, for instance in unconscious people or in case of swallowing troubles.

Too few experiments were carried out to draw firm conclusions on the potential infectious dose of Legionella reaching the respiratory tract. But this was beyond the scope of the present study. The first objective of this work was to assess the appropriateness of the model for that kind of research and the feasibility of the techniques. Nevertheless, this preliminary study allowed us to observe that airborne bacteria were viable as it was possible to grow them. So we have a valid model, potentially suitable for Legionella infections studies. But caution should be used and if we actually demonstrated the deposition of Legionella in the tracheobronchial tree, their in situ infectious potential remains to be further examined and characterized.

Conclusions

Despite these limitations, we were able to establish a new model respecting the 3R rule, easy to handle and with physiological similarities to human ventilation. This model allowed us to show that aerosolized Legionella were able to reach the deepest structures of the respiratory tract, we were also able to quantify them in different regions. In conclusion, this study is an interesting proof of concept of a new model and from this starting point and preliminary assays, studies will be designed to exploit this model and further investigate the dose/infection issue in the context of a Legionella infection.

Figure captions Figure 1 -

 captions1 Figure captions

Figure 2 -

 2 Figure 2 -Deposition pattern of fluorescent Legionella within the indicated areas of the tracheo-bronchial tree. Tissue fluorescence was detected with the Cellvizio  Lab technology.

Figure 3 -

 3 Figure 3 -Representative culture results from experiment 4. A) Legionella colonies of

Figure 4 -

 4 Figure 4 -Median levels of Legionella number (CFU/mL, black dash) found in BALF from

Acknowledgements

The authors would like to acknowledge Université Jean Monnet (APR UJM) for the financial support. The Despinasse Viande society (La Talaudière, France) is thanked for providing the porcine respiratory tracts.

Conflicts of interest

None.