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3D cell culture models exposed at the air-liquid interface (ALI) represent a potential alternative to animal experiments for hazard and risk assessment of inhaled compounds. This study compares cocultures composed of either Calu-3, A549 or HBEC3-KT lung epithelial cells, cultured together with THP-1-derived macrophages and EA.hy926 endothelial cells, in terms of barrier capacity and responses to a standard reference sample of fine particulate matter (SRM 2786). High-content imaging analysis revealed a similar cellular composition between the different cell models. The 3D cell cultures with Calu-3 cells showed the greatest barrier capacity, as measured by transepithelial electrical resistance and permeability to Na-fluorescein. Mucus production was detected in 3D cell cultures based on Calu-3 and A549 cells. Exposure to SRM 2786 at ALI increased cytokine release and expression of genes associated with inflammation and xenobiotic metabolism. Moreover, the presence of THP-1-derived macrophages was central to the cytokine responses in all cell models. While the different 3D cell culture models produced qualitatively similar responses, more pronounced pro-inflammatory responses were observed in the basolateral compartment of the A549 and HBEC3-KT models compared to the Calu-3 model, likely due to their reduced barrier capacity and lower retention of secreted mediators in the apical compartment., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest. The following are the supplementary data related to this article. Supplementary data to this article can be found online at https://doi.org/10.1016/j.tiv.2024.105841., (Copyright © 2024. Published by Elsevier Ltd.)

Epidemiological studies have found strong associations between air pollution and respiratory effects including development and/or exacerbation of asthma and chronic obstructive pulmonary disease (COPD) as well as increased occurrence of respiratory infections and lung cancer. It has become increasingly clear that also polycyclic aromatic hydrocarbons (PAHs) may affect processes linked to non-malignant diseases in the airways. The aim of the present paper was to review epidemiological studies on associations between gas phase and particle-bound PAHs in ambient air and non-malignant respiratory diseases or closely related physiological processes, to assess whether PAH-exposure may explain some of the effects associated with air pollution. Based on experimental in vivo and in vitro studies, we also explore possible mechanisms for how different PAHs may contribute to such events. Epidemiological studies show strongest evidence for an association between PAHs and asthma development and respiratory function in children. This is supported by studies on prenatal and postnatal exposure. Exposure to PAHs in adults seems to be linked to respiratory functions, exacerbation of asthma and increased morbidity/mortality of obstructive lung diseases. However, available studies are few and weak. Notably, the PAHs measured in plasma/urine also represent other exposure routes than inhalation. Furthermore, the role of PAHs measured in air is difficult to disentangle from that of other air pollution components originating from combustion processes. Experimental studies show that PAHs may trigger various processes linked to non-malignant respiratory diseases. Physiological- and pathological responses include redox imbalance, oxidative stress, inflammation both from the innate and adaptive immune systems, smooth muscle constriction, epithelial- and endothelial dysfunction and dysregulated lung development. Such biological responses may at the molecular level be initiated by PAH-binding to the aryl hydrocarbon receptor (AhR), but possibly also through interactions with beta-adrenergic receptors. In addition, reactive PAH metabolites or reactive oxygen species (ROS) may interfere directly with ion transporters and enzymes involved in signal transduction. Overall, the reviewed literature shows that respiratory effects of PAH-exposure in ambient air may extend beyond lung cancer. The relative importance of the specific PAHs ability to induce disease may differ between the biological endpoint in question.

Increased use of 1st and 2nd generation biofuels raises concerns about health effects of new emissions. We analyzed cellular and molecular lung effects in Fisher 344 rats exposed to diesel engine exhaust emissions (DEE) from a Euro 5-classified diesel engine running on B7: petrodiesel fuel containing 7% fatty acid methyl esters (FAME), or SHB20 (synthetic hydrocarbon biofuel): petrodiesel fuel containing 7% FAME and 13% hydrogenated vegetable oil. The Fisher 344 rats were exposed for 7 consecutive days (6 h/day) or 28 days (6 h/day, 5 days/week), both with and without diesel particle filter (DPF) treatment of the exhaust in whole body exposure chambers (n = 7/treatment). Histological analysis and analysis of cytokines and immune cell numbers in bronchoalveolar lavage fluid (BALF) did not reveal adverse pulmonary effects after exposure to DEE from B7 or SHB20 fuel. Significantly different gene expression levels for B7 compared to SHB20 indicate disturbed redox signaling (Cat, Hmox1), beta-adrenergic signaling (Adrb2) and xenobiotic metabolism (Cyp1a1). Exhaust filtration induced higher expression of redox genes (Cat, Gpx2) and the chemokine gene Cxcl7 compared to non-filtered exhaust. Exposure time (7 versus 28 days) also resulted in different patterns of lung gene expression. No genotoxic effects in the lungs were observed. Overall, exposure to B7 or SHB20 emissions suggests only minor effects in the lungs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Adsorbed soluble organics seem to be the main drivers of inflammatory responses induced by diesel exhaust particles (DEP). The specific compounds contributing to this process and the cellular mechanisms behind DEP-induced inflammation are not well known. We have assessed pro-inflammatory effects of DEP and various soluble DEP fractions, in human bronchial epithelial cells (BEAS-2B). DEP increased the expression of interleukin (IL)-6 and CXCL8. Silencing of the aryl hydrocarbon receptor (AhR) by siRNA or pretreatment with AhR-antagonists did not attenuate DEP-induced IL-6 and CXCL8 responses. However, the halogenated aromatic hydrocarbon (HAH)-selective AhR antagonist CH223191 caused a considerable reduction in DEP-induced CYP1A1 expression indicating that this response may be due to dioxin or dioxin-like constituents in DEP. Knock-down of protease activated receptor (PAR)-2 attenuated IL-6 responses without affecting CXCL8. Antioxidants did not affect IL-6 expression after 4h DEP-exposure and only partly reduced CXCL8 expression. However, after 24h exposure antioxidant treatment partly suppressed IL-6 protein release and completely blocked CXCL8 release. Furthermore, a heptane-soluble (non-polar) extract of DEP induced both IL-6 and CXCL8 release, whereas a PBS-soluble (highly polar) extract induced only IL-6. Thus, pro-inflammatory responses in DEP-exposed epithelial cells appear to be the result of both reactive oxygen species and receptor signaling, mediated through combinatorial effects between both non-polar and polar constituents adhered to the particle surface., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Diesel exhaust and its particles (DEP) have been under scrutiny for health effects in humans. In the development of these effects inflammation is regarded as a key process. Overall, in vitro studies report similar DEP-induced changes in markers of inflammation, including cytokines and chemokines, as studies in vivo. In vitro studies suggest that soluble extracts of DEP have the greatest impact on the expression and release of proinflammatory markers. Main DEP mediators of effects have still not been identified and are difficult to find, as fuel and engine technology developments lead to continuously altered characteristics of emissions. Involved mechanisms remain somewhat unclear. DEP extracts appear to comprise components that are able to activate various membrane and cytosolic receptors. Through interactions with receptors, ion channels, and phosphorylation enzymes, molecules in the particle extract will trigger various cell signaling pathways that may lead to the release of inflammatory markers directly or indirectly by causing cell death. In vitro studies represent a fast and convenient system which may have implications for technology development. Furthermore, knowledge regarding how particles elicit their effects may contribute to understanding of DEP-induced health effects in vivo, with possible implications for identifying susceptible groups of people and effect biomarkers.

Air pollution in Milan causes health concern due to the high concentrations of particulate matter (PM10 and PM2.5). The aim of this study was to investigate possible seasonal differences in PM10 and PM2.5 chemical composition and their biological effects on pro-inflammatory cytokine release and cytotoxicity. The PM was sampled during winter and summer seasons. The winter PMs had higher levels of PAHs than the summer samples which contained a greater amount of mineral dust elements. The PM toxicity was tested in the human pulmonary epithelial cell lines BEAS-2B and A549. The winter PMs were more cytotoxic than summer samples, whereas the summer PM10 exhibited a higher pro-inflammatory potential, as measured by ELISA. This inflammatory potential seemed partly due to biological components such as bacterial lipopolysaccharides (LPS), as evaluated by the use of Polymixin B. Interestingly, in the BEAS-2B cells the winter PM2.5 reduced proliferation due to a mitotic delay/arrest, while no such effects were observed in the A549 cells. These results underline that the in vitro responsiveness to PM may be cell line dependent and suggest that the PM different properties may trigger different endpoints such as inflammation, perturbation of cell cycle and cell death.

Exposure of human epithelial lung cells to fluorides is known to induce a marked increase in the release of interleukin (IL)-8, a chemokine involved in neutrophil recruitment. In the present study, the involvement of mitogen-activating protein kinases (MAPKs), the role of upstream activation of Src family kinases (SFKs), epidermal growth factor receptor (EGFR) activation and the interrelationships between these pathways in fluoride-induced IL-8 were examined in a human epithelial lung cell line (A549). Sodium fluoride strongly activated MAPK, in particular JNK1/2 and p38. The ERK1/2-inhibitor PD98059, the p38-inhibitor SB202190 and the JNK1/2-inhibitor SP600125 partially inhibited the fluoride-induced IL-8 response. Combinations of these inhibitors reduced the responses nearly to basal levels. Treatment with siRNA against JNK2 also reduced the IL-8 response to fluoride. Furthermore, fluoride activated SFKs, which was abolished by the SFK-inhibitor PP2. PP2 substantially inhibited the increased levels of IL-8, and partially reduced the fluoride-induced activation of ERK1/2, p38 and JNK1/2. Fluoride exposure also led to a phosphorylation of the EGFR, that was partially inhibited by PP2. AG1478, an EGFR-inhibitor, partially reduced the fluoride-induced IL-8 response and the phosphorylation of JNK1/2 and ERK1/2, but less the phosphorylation of p38. The effects of AG1478 were less than that of PP2. In conclusion, our findings suggest that the fluoride-induced IL-8 release involves the combined activation of ERK1/2, JNK1/2 and p38, and that the phosphorylation of these kinases, and in particular JNK1/2 and ERK1/2, partly, is mediated via a SFK-dependent EGFR-linked pathway. SFK-dependent, but EGFR-independent mechanisms seem important, and especially for phosphorylation of p38.

We have investigated the potential of two complex mineral particles (feldspar and mylonite), quartz (Min-U-Sil), and suspended particulate matter (SRM-1648) (SPM) from urban air to induce inflammatory cytokine responses in primary rat alveolar type 2 cells and alveolar macrophages, and the involvement of cellular formation of free radicals in these responses. All particle types induced an increased release of interleukin (IL)-6 and macrophage inflammatory protein (MIP)-2 from type 2 cells. Diphenyleneiodonium chloride (DPI), a selective inhibitor of NADPH-oxidase, reduced the IL-6 and MIP-2 responses to quartz, SPM and mylonite. N-(3-[Aminomethyl] benzyl) acetamidine (1400W), a selective inhibitor of inducible nitric oxide synthase (iNOS), significantly reduced the Il-6 response to SPM and feldspar in the type 2 cells. The macrophages displayed significantly increased TNF-alpha and MIP-2 release upon exposure to quartz or SPM. Here, DPI significantly reduced the tumor necrosis factor (TNF)-alpha and MIP-2 responses to quartz, and the MIP-2 response to SPM. No significant effect of 1400 W was detected in the alveolar macrophages. The role of particle-induced cellular generation of free radicals in lung cytokine responses was further elucidated in mice that lacked either NADPH-oxidase or iNOS as well as in wild-type (wt) mice. All particles were able to elicit increased cytokine levels in the bronchoalveolar lavage (BAL) fluid of the mice, although the levels depended on particle type. The NADPH-oxidase knockout (KO) mice demonstrated a significantly lower IL-6 and MIP-2 responses to SPM compared to their respective wt mice. The iNOS KO mice displayed significantly reduced IL-6, TNF-alpha, and MIP-2 responses to SPM. The overall results indicate the involvement of cellular free-radical formation in the pulmonary cytokine responses to particles of varying composition.

Abstract: Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are found in diesel exhaust and air pollution particles. Along with other PAHs, many nitro-PAHs possess mutagenic and carcinogenic properties, but their effects on pro-inflammatory processes and cell death are less known. In the present study we examined the effects of 1-nitropyrene (1-NP), 3-nitrofluoranthene (3-NF) and 3-nitrobenzanthrone (3-NBA) and their corresponding amino forms, 1-AP, 3-AF and 3-ABA, in human bronchial epithelial BEAS-2B cells. The effects of the different nitro- and amino-PAHs were compared to the well-characterized PAH benzo[a]pyrene (B[a]P). Expression of 17 cytokine and chemokine genes, measured by real-time PCR, showed that 1-NP and 3-NF induced a completely different cytokine/chemokine gene expression pattern to that of their amino analogues. 1-NP/3-NF-induced responses were dominated by maximum effects on CXCL8 (IL-8) and TNF-α expression, while 1-AP-/3-AF-induced responses were dominated by CCL5 (RANTES) and CXCL10 (IP-10) expression. 3-NBA and 3-ABA induced only marginal cytokine/chemokine responses. However, 3-NBA exposure induced considerable DNA damage resulting in accumulation of cells in S-phase and a marked increase in apoptosis. B[a]P was the only compound to induce expression of aryl hydrocarbon receptor (AhR)-regulated genes, such as CYP1A1 and CYP1B1, but did not induce cytokine/chemokine responses in BEAS-2B cells. Importantly, nitro-PAHs and amino-PAHs induced both qualitatively and quantitatively different effects on cytokine/chemokine expression, DNA damage, cell cycle alterations and cytotoxicity. The cytokine/chemokine responses appeared to be triggered, at least partly, through mechanisms separate from the other examined endpoints. These results confirm and extend previous studies indicating that certain nitro-PAHs have a considerable pro-inflammatory potential. [Copyright &y& Elsevier]

Abstract: Airborne particulate matter (PM) has a complex composition, and the relative contribution of different compounds to PM-induced effects is only partly understood. The present study compared the capability of selected components commonly found in PM, to induce pro-inflammatory responses in lung epithelial cells. Ultrafine carbon black (ufCB), ZnCl2, FeSO4, 1-nitropyrene (1-NP), lipopolysaccharide (LPS), and crystalline silica (positive control) were screened for effects on the expression of 84 inflammation-related genes in the bronchial epithelial cell line, BEAS-2B. A total of 22 genes were up-regulated by one or more of the tested compounds, and 5 cytokine and 11 chemokine genes were selected for further studies. After 10h exposure, silica induced significantly increased expression of CCL20, CXCL1/-3/-8/-10/-11, lymphotoxin (LT)β and interleukin (IL)-6; ufCB induced CXCL8/-10 and -11; ZnCl2 induced CCL11/-20/-26, CXCL1/-5/-8/-14 and tumor necrosis factor (TNF)-α; FeSO4 induced a weak up-regulation of CXCL8 and TNF-α; LPS induced CCL20, CXCL1/-5/-8/-10/-11, LTβ and IL-6; and 1-NP induced expression of CCL20, CXCL1/-3/-8, TNF-α and IL-6. Despite obvious differences, all compounds induced response-patterns that correlated relatively well with that of silica, the positive control. The predominant response appeared to be increased gene expression of neutrophil-recruiting CXC-chemokines. CXCL8 was the only gene induced by all tested PM-components, the most up-regulated on average, and also dominating the gene-expression patterns induced by coarse PM. The data show quantitative, and to a certain extent qualitative differences in cytokine/chemokine gene-expression profiles of the compounds tested. However, there were also striking similarities in the response-patterns induced by these physically/chemically widely different compounds. [Copyright &y& Elsevier]