To our knowledge, this article is the first study presenting evidence that low concentrations of PM which are not cytotoxic, have an antiapoptotic effect on human bronchial epithelial cells. We report here the cellular effects of PM2.5 from two sites in Paris, sampled in winter and in summer. In order to remove the risk of cell type-specific events, our study was done in parallel on different human bronchial cell lines as well as on primary cells. We show that the four batches of PM2.5 are not cytotoxic on human bronchial cells, at a range of concentration from 1 to 50 μg/cm2. This is supported by data from flow cytometry, with the measurement of the main apoptotic hallmarks, as well as from electron microscopy data. Our results were obtained with a low concentration of PM2.5 unlike previous publications performed with higher doses (e.g. 100 μg/cm2, ). Indeed, the standard dose used here (10 μg/cm2) is a concentration which could mimic a five day exposure of PM2.5 in the tracheobronchial region, considering that PM2.5 mass deposition is 2.3 μg/cm2/24 h . Our results are in agreement with a previous publication where BEAS-2B human bronchial cells were not susceptible to diesel exhaust particles-induced apoptosis  and here, we provided supplementary evidences of a non-toxicological activity of PM2.5 in NHBE primary culture. Moreover, in our studies and those of Sanchez-Perez et al. , the lack of induced-apoptosis triggered by PM at 10 μg/cm2 suggests that a "sub-lethal" concentration could have different impacts on cell fate than at high concentrations.
The originality of this work is that PM2.5 exposure confers a specific decrease in apoptosis induced by A23187, staurosporine and oligomycin as demonstrated in immortalized (16HBE), cancerous (NCI-H292, BEAS-2B) as well as primary normal bronchial epithelial cells (NHBE). In order to characterize the molecular mechanism of the antiapoptotic activity of PM2.5 exposure, first we demonstrated that the reduction of apoptosis is observed prior to proinflammatory cytokines secretion which led us to rule out the involvement of the classical EGFR signaling pathway as well as the proinflammatory cytokines secretion by bronchial epithelial cells. However, PM2.5-antiapoptotic effect in addition to the well-documented inflammatory response might explain the maintenance of a prolonged inflammation state in vivo induced after pollution exposure and might delay repair processes of injured tissues .
To further delineate the mechanism of the antiapoptotic activity, a strategy would be to identify the cellular targets which are in common between staurosporine, A23187 and oligomycin. On one hand, staurosporine and A23187 are known to regulate cellular calcium signaling pathways inducing an endoplasmic reticulum stress which leads to cytoplasmic calcium uptake , mitochondrial Ca2+ overload  and finally ΔΨm drop. Thus, PM2.5 exposure might counteract the Ca2+ uptake induced by these apoptotic inducers. However, this hypothesis is in discrepancy with the fact that the antiapoptotic effects of PM2.5 were not observed when using ionomycin, which is a well-known calcium ionophore, like A23187. Indeed, A23187 and ionomycin, which are both monocarboxylic ionophores, promote a selective increase of cytosolic Ca2+ . But on the contrary to A23187 , a recent study showed that ionomycin did not allow the mitochondrial calcium overload in epimastigote cells of Trypanosoma cruzi . The measurement of cytosolic and mitochondrial calcium uptakes in response to A23187 and ionomycin might allow us to understand why A23187-induced apoptosis is sensitive to PM while ionomycin is not. Moreover, caspases are the main effectors of apoptosis, but A23187, staurosporine and ionomycin can also activate Ca2+-specific proteases, such as calpains [33, 34]. Indeed, our preliminary studies showed that calpains are activated after A23187 treatment of 16HBE and NCI-H292 cells (data not shown). As described for oligomycin, A23187, but not ionomycin, is a specific inhibitor of mitochondrial ATP synthase also known to catalyze the direct exchange of Ca2+/2H+ in liver mitochondria  and to disrupt the mitochondrial transmembrane potential . All these data suggest that ionomycin and A23187 might trigger the apoptotic process by slightly different mechanisms especially at the mitochondrial level. Thus, we hypothesize that PM2.5 could directly reduce apoptosis at the mitochondrial step by maintaining ΔΨm, or via the upregulation of antiapoptotic proteins such as Bcl-2 known to protect from A23187-induced apoptosis [24, 37].
Humans are exposed to a mixture of compounds including organic and inorganic components adsorbed on PM. Evidences suggest that organic compounds such as the polycyclic aromatic hydrocarbons (PAH) can mimic the pro-oxidant  and apoptotic effect of PM . Here, we investigated the role of different organic compounds (PAH, Oex), particles devoid of hydrosoluble components, (wash, CB) and aqueous extracts (Aex) of PM2.5 with respect to cell death. We found that the organic extracts and several heavy PAH, B(a)P in particular, could reproduce the antiapoptotic activity. Moreover, the water-soluble fraction also contributes to the reduction of apoptosis while carbon black, light PAH and endotoxins have no effect. In our study, B(a)P is the compound that protects the most efficiently from apoptosis induced by A23187. This points out a possible link between PM2.5-exposure and the antiapoptotic effect observed herein, as also suggested by Hung et al. . The harmful health impacts of PAH are well-known, like the promotion of cancers. B(a)P-diones, which are photomodified by the sunlight, were also found in air particulate matter. In agreement with our results, a recent work demonstrated that sunlight-exposed B(a)P inhibits apoptosis induced by cell detachment . B(a)P is metabolized by cells, transformed into a reactive intermediate (anti-7,8-dihydrodiol-9,10-epoxy-benzo[a]pyrene, BPDE) that causes DNA damage and mutations in tumor suppressor genes, such as p53 . This toxic metabolite BPDE is also capable to suppress apoptosis of mammary epithelial cells .
The main cellular target of PAH adsorbed on PM is the aryl hydrocarbon receptor (AhR), thus we addressed the question of AhR involvement in the antiapoptotic effect after PM2.5 exposure. We showed here that the activation of AhR by the agonist beta-naphtoflavone improves the antiapoptotic effect. On the contrary, the inhibition of AhR (using a specific inhibitor or RNA silencing) diminished the antiapoptotic effect suggesting that AhR is involved in this process. An additional argument is brought by the absence of antiapoptotic activity when we tested light PAH, which were previously shown to poorly promote AhR activation . AhR is a cytoplasmic ligand-dependent transcription factor which translocates to the nucleus in order to bind specific Xenobiotic Responsive Elements in the promoter of its target genes, leading to the activation of phase I and II metabolizing enzymes and thus contributing to detoxification . But in the absence of ligand, many data suggest other roles than detoxification  and recent evidences suggest that AhR inactivation could modify the expression of numerous genes, including those involved in cell cycle regulation . In accordance with our results, other publications suggest an antiapoptotic activity of AhR by a direct interaction with E2F1 leading to the reduction of E2F1-mediated pro-apoptotic genes expression . This is consistent with the idea that the AhR might modulate cell death at the mitochondrial checkpoint, for instance by upregulating the expression of antiapoptotic bcl-2, bcl-xL, mcl-1 or agr2 genes [48, 49] or by repressing the pro-apoptotic apaf-1 . Moreover, AhR might indirectly regulate apoptosis through the MMP step by increasing the expression of the anti-apototic protein VDAC2  which is known to participate to the permeability transition pore (PTP) and which also bind to and inhibit the apoptotic protein Bak . In the light of our observations, it will be interesting to find out the genes encoding mitochondrial regulators which are modulated by AhR and involved in the protection observed after PM2.5-exposure or B(a)P treatment. It is also important to point out that both A23187 and STS could induce apoptosis via a Ca2+-dependent pathway through mitochondrial PTP opening and that VDAC plays a crucial role in the transport of Ca2+ into this organelle .