Urban air PM modifies differently immune defense responses against bacterial and viral infections in vitro.

Epidemiological evidence has shown the association between exposure to ambient fine particulate matter (PM) and increased susceptibility to bacterial and viral respiratory infections. However, to date, the underlying mechanisms of immunomodulatory effects of PM remain unclear. Our objective was to explore how exposure to relatively low doses of urban air PM alters innate responses to bacterial and viral stimuli in vitro. We used secondary alveolar epithelial cell line along with monocyte-derived macrophages to replicate innate lung barrier in vitro. Co-cultured cells were first exposed for 24 h to PM2.5-1 (particle aerodynamic diameter between 1 and 2.5 µm) and subsequently for an additional 24 h to lipopolysaccharide (TLR4), polyinosinic-polycytidylic acid (TLR3), and synthetic single-stranded RNA oligoribonucleotides (TLR7/8) to mimic bacterial or viral stimulation. Toxicological endpoints included pro-inflammatory cytokines (IL-8, IL-6, and TNF-α), cellular metabolic activity, and cell cycle phase distribution. We show that cells exposed to PM2.5-1 produced higher levels of pro-inflammatory cytokines following stimulation with bacterial TLR4 ligand than cells exposed to PM2.5-1 or bacterial ligand alone. On the contrary, PM2.5-1 exposure reduced pro-inflammatory responses to viral ligands TLR3 and TLR7/8. Cell cycle analysis indicated that viral ligands induced cell cycle arrest at the G2-M phase. In PM-primed co-cultures, however, they failed to induce the G2-M phase arrest. Contrarily, bacterial stimulation caused a slight increase in cells in the sub-G1 phase but in PM2.5-1 primed co-cultures the effect of bacterial stimulation was masked by PM2.5-1. These findings indicate that PM2.5-1 may alter responses of immune defense differently against bacterial and viral infections. Further studies are required to explain the mechanism of immune modulation caused by PM in altering the susceptibility to respiratory infections.

Inflammatory responses of urban air PM modulated by chemical composition and different air quality situations in Nanjing, China.

The health risks of air pollutants and ambient particulate matter (PM) are widely known. PM composition and toxicity have shown substantial spatiotemporal variability. Yet, the connections between PM composition and toxicological and health effects are vaguely understood. This is a crucial gap in knowledge that needs to be addressed in order to establish air quality guidelines and limit values that consider the chemical composition of PM instead of the current assumption of equal toxicity per inhaled dose. Here, we demonstrate further evidence for varying toxicological effects of urban PM at equal mass concentrations, and estimate how PM composition and emission source characteristics influenced this variation. We exposed a co-culture model mimicking alveolar epithelial cells and macrophages with size-segregated urban ambient PM collected before, during, and after the Nanjing Youth Olympic Games 2014. We measured the release of a set of cytokines, cell cycle alterations, and genotoxicity, and assessed the spatiotemporal variations in these responses by factorial multiple regression analysis. Additionally, we investigated how a previously identified set of emission sources and chemical components affected these variations by mixed model analysis. PM-exposure induced cytokine signaling, most notably by inducing dose-dependent increases of macrophage-regulating GM-CSF and proinflammatory TNFα, IL-6, and IL-1ß concentrations, modest dose-dependent increase for cytoprotective VEGF-A, but very low to no responses for anti-inflammatory IL-10 and immunoregulatory IFNγ, respectively. We observed substantial differences in proinflammatory cytokine production depending on PM sampling period, location, and time of day. The proinflammatory response correlated positively with cell cycle arrest in G1/G0 phase and loss of cellular metabolic activity. Furthermore, PM0.2 caused dose-dependent increases in sub-G1/G0 cells, suggesting increased DNA degradation and apoptosis. Variations in traffic and oil/fuel combustion emissions contributed substantially to the observed spatiotemporal variations of toxicological responses.

Air quality intervention during the Nanjing youth olympic games altered PM sources, chemical composition, and toxicological responses.

Ambient particulate matter (PM) is a leading global environmental health risk. Current air quality regulations are based on airborne mass concentration. However, PM from different sources have distinct chemical compositions and varied toxicity. Connections between emission control measures, air quality, PM composition, and toxicity remain insufficiently elucidated. The current study assessed the composition and toxicity of PM collected in Nanjing, China before, during, and after an air quality intervention for the 2014 Youth Olympic Games. A co-culture model that mimics the alveolar epithelium with the associated macrophages was created using A549 and THP-1 cells. These cells were exposed to size-segregated inhalable PM samples. The composition and toxicity of the PM samples were influenced by several factors including seasonal variation, emission sources, and the air quality intervention. For example, we observed a size-dependent shift in particle mass concentrations during the air quality intervention with an emphasized proportion of smaller particles (PM2.5) present in the air. The roles of industrial and fuel combustion and traffic emissions were magnified during the emission control period. Our analyses revealed that the PM samples demonstrated differential cytotoxic potencies at equal mass concentrations between sampling periods, locations, and time of day, influenced by variations in the predominant emission sources. Coal combustion and industrial emissions were the most important sources affecting the toxicological responses and displayed the least variation in emission contributions between the sampling periods. In conclusion, emission control mitigated cytotoxicity and oxidative stress for particles larger than 0.2 µm, but there was inadequate evidence to determine if it was the key factor reducing the harmful effects of PM0.2.

Toxicological evaluation of exhaust emissions from light-duty vehicles using different fuel alternatives in sub-freezing conditions.

BACKGROUND: Emissions from road traffic are under constant discussion since they pose a major threat to human health despite the increasingly strict emission targets and regulations. Although the new passenger car regulations have been very effective in reducing the particulate matter (PM) emissions, the aged car fleet in some EU countries remains a substantial source of PM emissions. Moreover, toxicity of PM emissions from multiple new types of bio-based fuels remain uncertain and different driving conditions such as the sub-zero running temperature has been shown to affect the emissions. Overall, the current literature and experimental knowledge on the toxicology of these PM emissions and conditions is scarce. METHODS: In the present study, we show that exhaust gas PM from newly regulated passenger cars fueled by different fuels at sub-zero temperatures, induce toxicological responses in vitro. We used exhaust gas volume-based PM doses to give us better insight on the real-life exposure and included one older diesel car to estimate the effect of the new emissions regulations. RESULTS: In cars compliant with the new regulations, gasoline (E10) displayed the highest PM concentrations and toxicological responses, while the higher ethanol blend (E85) resulted in slightly lower exhaust gas PM concentrations and notably lower toxicological responses in comparison. Engines powered by modern diesel and compressed natural gas (CNG) yielded the lowest PM concentrations and toxicological responses. CONCLUSIONS: The present study shows that toxicity of the exhaust gas PM varies depending on the fuels used. Additionally, concentration and toxicity of PM from an older diesel car were vastly higher, compared to contemporary vehicles, indicating the beneficial effects of the new emissions regulations.

Influence of wood species on toxicity of log-wood stove combustion aerosols: a parallel animal and air-liquid interface cell exposure study on spruce and pine smoke.

BACKGROUND: Wood combustion emissions have been studied previously either by in vitro or in vivo models using collected particles, yet most studies have neglected gaseous compounds. Furthermore, a more accurate and holistic view of the toxicity of aerosols can be gained with parallel in vitro and in vivo studies using direct exposure methods. Moreover, modern exposure techniques such as air-liquid interface (ALI) exposures enable better assessment of the toxicity of the applied aerosols than, for example, the previous state-of-the-art submerged cell exposure techniques. METHODS: We used three different ALI exposure systems in parallel to study the toxicological effects of spruce and pine combustion emissions in human alveolar epithelial (A549) and murine macrophage (RAW264.7) cell lines. A whole-body mouse inhalation system was also used to expose C57BL/6 J mice to aerosol emissions. Moreover, gaseous and particulate fractions were studied separately in one of the cell exposure systems. After exposure, the cells and animals were measured for various parameters of cytotoxicity, inflammation, genotoxicity, transcriptome and proteome. RESULTS: We found that diluted (1:15) exposure pine combustion emissions (PM1 mass 7.7 ± 6.5 mg m- 3, 41 mg MJ- 1) contained, on average, more PM and polycyclic aromatic hydrocarbons (PAHs) than spruce (PM1 mass 4.3 ± 5.1 mg m- 3, 26 mg MJ- 1) emissions, which instead showed a higher concentration of inorganic metals in the emission aerosol. Both A549 cells and mice exposed to these emissions showed low levels of inflammation but significantly increased genotoxicity. Gaseous emission compounds produced similar genotoxicity and a higher inflammatory response than the corresponding complete combustion emission in A549 cells. Systems biology approaches supported the findings, but we detected differing responses between in vivo and in vitro experiments. CONCLUSIONS: Comprehensive in vitro and in vivo exposure studies with emission characterization and systems biology approaches revealed further information on the effects of combustion aerosol toxicity than could be achieved with either method alone. Interestingly, in vitro and in vivo exposures showed the opposite order of the highest DNA damage. In vitro measurements also indicated that the gaseous fraction of emission aerosols may be more important in causing adverse toxicological effects. Combustion aerosols of different wood species result in mild but aerosol specific in vitro and in vivo effects.

Integrating farm and air pollution studies in search for immunoregulatory mechanisms operating in protective and high-risk environments.

BACKGROUND: Studies conducted in farm environments suggest that diverse microbial exposure promotes children's lung health. The underlying mechanisms are unclear, and the development of asthma-preventive strategies has been delayed. More comprehensive investigation of the environment-induced immunoregulation is required for better understanding of asthma pathogenesis and prevention. Exposure to air pollution, including particulate matter (PM), is a risk factor for asthma, thus providing an excellent counterpoint for the farm-effect research. Lack of comparable data, however, complicates interpretation of the existing information. We aimed to explore the immunoregulatory effects of cattle farm dust (protective, Finland) and urban air PM (high-risk, China) for the first time using identical research methods. METHODS: We stimulated PBMCs of 4-year-old children (N = 18) with farm dust and size-segregated PM and assessed the expression of immune receptors CD80 and ILT4 on dendritic cells and monocytes as well as cytokine production of PBMCs. Environmental samples were analysed for their composition. RESULTS: Farm dust increased the percentage of cells expressing CD80 and the cytokine production of children's immune cells, whereas PM inhibited the expression of important receptors and the production of soluble mediators. Although PM samples induced parallel immune reactions, the size-fraction determined the strength of the effects. CONCLUSIONS: Our study demonstrates the significance of using the same research framework when disentangling shared and distinctive immune pathways operating in different environments. Observed stimulatory effects of farm dust and inhibitory effects of PM could shape responses towards respiratory pathogens and allergens, and partly explain differences in asthma prevalence between studied environments.

Genotoxic and inflammatory effects of spruce and brown coal briquettes combustion aerosols on lung cells at the air-liquid interface.

Solid fuel usage in residential heating and cooking is one of the largest sources of ambient and indoor air particulate matter, which causes adverse effects on the health of millions of peoples worldwide. Emissions from solid fuel combustion, such as biomass or coal, are detrimental to health, but toxicological responses are largely unknown. In the present study, we compared the toxicological responses regarding cytotoxicity, inflammation and genotoxicity of spruce (SPR) and brown coal briquette (BCB) combustion aerosols on human alveolar epithelial cells (A549) as well as a coculture of A549 and differentiated human monocytic cells (THP-1) into macrophages exposed at the air-liquid interface (ALI). We included both the high emissions from the first hour and moderate emissions from the third hour of the batch combustion experiment in one ALI system, whereas, in the second ALI system, we exposed the cells during the whole 4-hour combustion experiment, including all combustion phases. Physico-chemical properties of the combustion aerosol were analysed both online and offline. Both SPR and BCB combustion aerosols caused mild cytotoxic but notable genotoxic effects in co-cultured A549 cells after one-hour exposure. Inflammatory response analysis revealed BCB combustion aerosols to cause a mild increase in CXCL1 and CXCL8 levels, but in the case of SPR combustion aerosol, a decrease compared to control was observed.

Differences between co-cultures and monocultures in testing the toxicity of particulate matter derived from log wood and pellet combustion.

BACKGROUND: In vitro studies with monocultures of human alveolar cells shed deeper knowledge on the cellular mechanisms by which particulate matter (PM) causes toxicity, but cannot account for mitigating or aggravating effects of cell-cell interactions on PM toxicity. METHODS: We assessed inflammation, oxidative stress as well as cytotoxic and genotoxic effects induced by PM from the combustion of different types of wood logs and softwood pellets in three cell culture setups: two monocultures of either human macrophage-like cells or human alveolar epithelial cells, and a co-culture of these two cell lines. The adverse effects of the PM samples were compared between these setups. RESULTS: We detected clear differences in the endpoints between the mono- and co-cultures. Inflammatory responses were more diverse in the macrophage monoculture and the co-culture compared to the epithelial cells where only an increase of IL-8 was detected. The production of reactive oxygen species was the highest in epithelial cells and macrophages seemed to have protective effects against oxidative stress from the PM samples. With no metabolically active cells at the highest doses, the cytotoxic effects of the PM samples from the wood log combustion were far more pronounced in the macrophages and the co-culture than in the epithelial cells. All samples caused DNA damage in macrophages, whereas only beech and spruce log combustion samples caused DNA damage in epithelial cells. The organic content of the samples was mainly associated with cytotoxicity and DNA damage, while the metal content of the samples correlated with the induction of inflammatory responses. CONCLUSIONS: All of the tested PM samples induce adverse effects and the chemical composition of the samples determines which pathway of toxicity is induced. In vitro testing of the toxicity of combustion-derived PM in monocultures of one cell line, however, is inadequate to account for all the possible pathways of toxicity.

Emissions and atmospheric processes influence the chemical composition and toxicological properties of urban air particulate matter in Nanjing, China.

Ambient inhalable particulate matter (PM) is a serious health concern worldwide, but especially so in China where high PM concentrations affect huge populations. Atmospheric processes and emission sources cause spatial and temporal variations in PM concentration and chemical composition, but their influence on the toxicological characteristics of PM are still inadequately understood. In this study, we report an extensive chemical and toxicological characterization of size-segregated urban air inhalable PM collected in August and October 2013 from Nanjing, and assess the effects of atmospheric processes and likely emission sources. A549 human alveolar epithelial cells were exposed to day- and nighttime PM samples (25, 75, 150, 200, 300 µg/ml) followed by analyses of cytotoxicity, genotoxicity, cell cycle, and inflammatory response. PM10-2.5 and PM0.2 caused the greatest toxicological responses for different endpoints, illustrating that particles with differing size and chemical composition activate distinct toxicological pathways in A549 cells. PM10-2.5 displayed the greatest oxidative stress and genotoxic responses; both were higher for the August samples compared with October. In contrast, PM0.2 and PM2.5-1.0 samples displayed high cytotoxicity and substantially disrupted cell cycle; August samples were more cytotoxic whereas October samples displayed higher cell cycle disruption. Several components associated with combustion, traffic, and industrial emissions displayed strong correlations with these toxicological responses. The lower responses for PM1.0-0.2 compared to PM0.2 and PM2.5-1.0 indicate diminished toxicological effects likely due to aerosol aging and lower proportion of fresh emission particles rich in highly reactive chemical components in the PM1.0-0.2 fraction. Different emission sources and atmospheric processes caused variations in the chemical composition and toxicological responses between PM fractions, sampling campaigns, and day and night. The results indicate different toxicological pathways for coarse-mode particles compared to the smaller particle fractions with typically higher content of combustion-derived components. The variable responses inside PM fractions demonstrate that differences in chemical composition influence the induced toxicological responses.