Pollen, respiratory viruses, and climate change: Synergistic effects on human health.

In recent years, evidence of the synergistic effects of pollen and viruses on respiratory health has begun to accumulate. Pollen exposure is a known risk factor for the incidence and severity of respiratory viral infections. However, recent evidence suggests that pollen exposure may also inhibit or weaken viral infections. A comprehensive summary has not been made and a consensus on the synergistic health effects has not been reached. It is highly possible that climate change will increase the significance of pollen exposure as a cause of respiratory problems and, at the same time, affect the risk of infectious disease outbreaks. It is important to accurately assess how these two factors affect human health separately and concurrently. In this review article, for the first time, the data from previous studies are combined and reviewed and potential research gaps concerning the synergistic effects of pollen and viral exposure are identified.

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.

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.

50 Hz magnetic field influences caspase-3 activity and cell cycle distribution in ionizing radiation exposed SH-SY5Y neuroblastoma cells.

PURPOSE: Earlier evidence suggests that extremely low frequency magnetic fields (ELF MFs) can modify the effects of carcinogenic agents. However, the studies conducted so far with ionizing radiation as the co-exposure agent are sparse and have provided inconclusive results. We investigated whether 50 Hz MFs alone, or in combination with ionizing radiation alter cell biological variables relevant to cancer and the biological effects of ionizing radiation. MATERIALS AND METHODS: Human SH-SY5Y neuroblastoma cells were sham exposed or exposed to 100 or 500 µT MF for 24 h either before or after ionizing radiation exposure (0, 0.4 or 2 Gy). After the exposures, cells were assayed for viability, clonogenicity, reactive oxygen species, caspase-3 activity, and cell cycle distribution. Cell cycle distribution was assayed with propidium iodide staining followed by flow cytometry analysis and ROS levels were assayed together with cell viability by double staining with DeepRed and Sytox Blue followed by flow cytometry analysis. RESULTS: Increased caspase-3 activity was observed in cells exposed to 500 µT MF before or after ionizing radiation. Furthermore, exposure to the 500 µT MF after the ionizing radiation decreased the percentage of cells in S-phase. No changes in the ROS levels, clonogenicity, or viability of the cells were observed in the MF exposed groups compared to the corresponding sham exposed groups, and no MF effects were observed in cells exposed at 100 µT. CONCLUSIONS: Only the 500 µT magnetic flux density affected SH-SY5Y cells significantly. The effects were small but may nevertheless help to understand how MFs modify the effects of ionizing radiation. The increase in caspase-3 activity may not reflect effects on apoptosis, as no changes were observed in the subG1 phase of the cell cycle. In contrast to some earlier findings, 50 Hz MF exposure after ionizing radiation was not less effective than MF treatment given prior to ionizing radiation.

Common Pollen Modulate Immune Responses against Viral-Like Challenges in Airway Coculture Model.

Recent research indicates that exposure to pollen increases the risk and severity of respiratory infections, while studies also suggest that it may possess a protective function. Our aim was to investigate how exposure to common pollen modifies airway cells' responses to viral- or bacterial-like challenges and vice versa. Cocultured A549 and THP-1 cells were exposed to three doses of four different pollens (Alnus glutinosa, Betula pendula, Phleum pratense, or Ambrosia artemisiifolia) and subsequently to Toll-like receptor (TLR) ligands mimicking bacterial and viral challenges (TLR3, TLR4, TLR7/8). The stimulation experiment was replicated in reverse order. Toxicological and immunological end points were analyzed. When cells were primed with pollen, especially with grass (P. pratense) or weed (A. artemisiifolia), the ability of cells to secrete cytokines in response to bacterial- and viral-like exposure was decreased. In contrast, cells primed with viral ligand TLR7/8 showed greater cytokine responses against pollen than cells exposed to ligands or pollen alone. Our results suggest that pollen exposure potentially weakens immune reactions to bacterial- or viral-like challenges by modulating cytokine production. They also indicate that TLR7/8-mediated viral challenges could elicit exaggerated immune responses against pollen. Both mechanisms could contribute to the acceleration and complication of infections during the pollen season.

Cattle farm dust alters cytokine levels in human airway construct model.

Epidemiological studies have revealed some alterations in systemic immunity that associate with farm exposure and the risk of respiratory diseases, but in vitro studies focusing on immunological responses in the airways are scarce. Our aim was to assess how cowshed dust affects the integrity and inflammation of human airway tissue in vitro. Cowshed dust samples were collected from four different dairy farms. Lung tissue constructs were exposed to dust samples in air-liquid interface. Transepithelial resistance of the tissue, secreted proteins, and a panel of pro-inflammatory cytokines, growth factors, and chemokines were analysed. Cowshed dust stimulation was associated mainly with increased production of IL-13, IL-15, IP-10 and IFN-γ. Some differences between farms were seen. Only one farm dust sample induced a significant change in transepithelial resistance, whereas dust from two of the farms induced the secretion of proteins. The exposure to cowshed dust affected protein and cytokine secretion, but the response profiles were not uniform between farms. The effect on tight junction dynamics was less pronounced, suggesting the relevance of soluble factors in induced responses in the airways. Our results indicate that in addition to farm type, the contribution of cowshed characteristics to dust composition and its immunomodulatory properties should be taken into account.

Toxicity of exhaust emissions from high aromatic and non-aromatic diesel fuels using in vitro ALI exposure system.

The differences in the traffic fuels have been shown to affect exhaust emissions and their toxicity. Especially, the aromatic content of diesel fuel is an important factor considering the emissions, notably particulate matter (PM) concentrations. The ultra-fine particles (UFP, particles with a diameter of <100 nm) are important components of engine emissions and connected to various health effects, such as pulmonary and systematic inflammation, and cardiovascular disorders. Studying the toxicity of the UFPs and how different fuel options can be used for mitigating the emissions and toxicity is crucial. In the present study, emissions from a heavy-duty diesel engine were used to assess the exhaust emission toxicity with a thermophoresis-based in vitro air-liquid interface (ALI) exposure system. The aim of the study was to evaluate the toxicity of engine exhaust and the potential effect of 20 % aromatic fossil diesel and 0 % aromatic renewable diesel fuel on emission toxicity. The results of the present study show that the aromatic content of the fuel increases emission toxicity, which was seen as an increase in genotoxicity, distinct inflammatory responses, and alterations in the cell cycle. The increase in genotoxicity was most likely due to the PM phase of the exhaust, as the exposures with high-efficiency particulate absorbing (HEPA)-filtered exhaust resulted in a negligible increase in genotoxicity. However, the solely gaseous exposures still elicited immunological responses. Overall, the present study shows that decreasing the aromatic content of the fuels could be a significant measure in mitigating traffic exhaust toxicity.

Systematic design of cell membrane coating to improve tumor targeting of nanoparticles.

Cell membrane (CM) coating technology is increasingly being applied in nanomedicine, but the entire coating procedure including adsorption, rupture, and fusion is not completely understood. Previously, we showed that the majority of biomimetic nanoparticles (NPs) were only partially coated, but the mechanism underlying this partial coating remains unclear, which hinders the further improvement of the coating technique. Here, we show that partial coating is an intermediate state due to the adsorption of CM fragments or CM vesicles, the latter of which could eventually be ruptured under external force. Such partial coating is difficult to self-repair to achieve full coating due to the limited membrane fluidity. Building on our understanding of the detailed coating process, we develop a general approach for fixing the partial CM coating: external phospholipid is introduced as a helper to increase CM fluidity, promoting the final fusion of lipid patches. The NPs coated with this approach have a high ratio of full coating (~23%) and exhibit enhanced tumor targeting ability in comparison to the NPs coated traditionally (full coating ratio of ~6%). Our results provide a mechanistic basis for fixing partial CM coating towards enhancing tumor accumulation.

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.

Toxicological and microbiological characterization of cow stable dust.

Exposure to farm environment has been shown to both protect from allergic diseases and increase the risk of respiratory syndromes. Mechanisms have been previously investigated by using farm dust extracts or specific components of dust. The use of authentic farm dust would better reflect the natural exposure. The aim of our study was to highlight the importance of proper assessment of the cow stable dust characteristics before conducting further investigations. For this purpose, we characterized microbiome and size distribution of unprocessed cow stable dust and its toxicological properties, as they have been often overlooked in search of protective factors. Stable dust samples from four Finnish dairy farms were collected by utilizing two different collection methods. Toxicological potential was analysed by stimulating co-cultures of lung epithelial and macrophage-like cells with dust. Size and mass distributions of airborne particles in the stables and bacterial and fungal microbiota of the dust were analysed. Stimulation with dust did not affect viability, but heightened oxidative stress responses and cytokine secretion, and slightly reduced the metabolic activity. There were a few differences in responses between farms, however, the differences were mainly in the intensity and not in the direction of the response. Cellular responses induced by dusts collected by different sampling methods did not differ substantially. Unprocessed stable dust samples showed relatively low direct toxicity but were able to trigger immune responses in studied cell model. This suggest that these dust collection methods could be utilized when investigating e.g. asthma-protective mechanisms.

Cell membrane coating integrity affects the internalization mechanism of biomimetic nanoparticles.

Cell membrane coated nanoparticles (NPs) have recently been recognized as attractive nanomedical tools because of their unique properties such as immune escape, long blood circulation time, specific molecular recognition and cell targeting. However, the integrity of the cell membrane coating on NPs, a key metrics related to the quality of these biomimetic-systems and their resulting biomedical function, has remained largely unexplored. Here, we report a fluorescence quenching assay to probe the integrity of cell membrane coating. In contradiction to the common assumption of perfect coating, we uncover that up to 90% of the biomimetic NPs are only partially coated. Using in vitro homologous targeting studies, we demonstrate that partially coated NPs could still be internalized by the target cells. By combining molecular simulations with experimental analysis, we further identify an endocytic entry mechanism for these NPs. We unravel that NPs with a high coating degree (≥50%) enter the cells individually, whereas the NPs with a low coating degree (<50%) need to aggregate together before internalization. This quantitative method and the fundamental understanding of how cell membrane coated NPs enter the cells will enhance the rational designing of biomimetic nanosystems and pave the way for more effective cancer nanomedicine.

TUBE Project: Transport-Derived Ultrafines and the Brain Effects.

The adverse effects of air pollutants on the respiratory and cardiovascular systems are unquestionable. However, in recent years, indications of effects beyond these organ systems have become more evident. Traffic-related air pollution has been linked with neurological diseases, exacerbated cognitive dysfunction, and Alzheimer's disease. However, the exact air pollutant compositions and exposure scenarios leading to these adverse health effects are not known. Although several components of air pollution may be at play, recent experimental studies point to a key role of ultrafine particles (UFPs). While the importance of UFPs has been recognized, almost nothing is known about the smallest fraction of UFPs, and only >23 nm emissions are regulated in the EU. Moreover, the role of the semivolatile fraction of the emissions has been neglected. The Transport-Derived Ultrafines and the Brain Effects (TUBE) project will increase knowledge on harmful ultrafine air pollutants, as well as semivolatile compounds related to adverse health effects. By including all the major current combustion and emission control technologies, the TUBE project aims to provide new information on the adverse health effects of current traffic, as well as information for decision makers to develop more effective emission legislation. Most importantly, the TUBE project will include adverse health effects beyond the respiratory system; TUBE will assess how air pollution affects the brain and how air pollution particles might be removed from the brain. The purpose of this report is to describe the TUBE project, its background, and its goals.

Cryopreservation affected the levels of immune responses of PBMCs and antigen-presenting cells.

The effect of cryopreservation on antigen-presenting cells (APCs) is understudied. It is important to understand the effects of cryopreservation on these cells as they play a major role in immune responses, and they could be utilized in different clinical applications. In this study, we compared fresh and cryopreserved PBMCs in regards of their general immune responsiveness and, furthermore, the effect of cryopreservation on the circulating APCs among PBMCs. We stimulated fresh and cryopreserved PBMCs (N = 6) with LPS or Poly(I:C).Cytokine production of PBMCs and expression of functional markers CD80 and ILT4 on major types of APCs, dendritic cells (DCs) and monocytes, were analysed. We also analysed whether cryopreservation affects different subtypes of DCs (plasmacytoid and myeloid DCs) differently. Cryopreserved PBMCs produced less cytokines than fresh cells in response to stimulation, but the response profiles were comparable. Cryopreservation had also an effect on the relative proportions of APCs. Stimuli-induced responses were somewhat parallel but weaker than those observed in fresh cells. This study suggests that the use of cryopreserved cells is more suitable in studies that assess general responses to stimuli instead of measuring exact levels of reactions. Thus, the interpretation and comparison of the results of different studies should not be done without considering the differences in cryopreservation techniques and their effects on PBMCs and, more specifically, on APCs.

Thermal dose as a universal tool to evaluate nanoparticle-induced photothermal therapy.

Thermal isoeffect dose (TID) is a widely applied concept to evaluate the safety of medical devices that can expose patients to heat. However, it has rarely been used in photothermal therapy (PTT), where nanoparticles are used as light absorbers. Utilizing TID in an appropriate way would make it feasible to compare the results obtained with different light absorbers as well as clarifying their cellular effects. Herein, we apply TID as a definitive parameter to evaluate the outcomes of a nanoparticle-induced PTT in vitro. We show that cell death measured with an ATP-based viability assay and flow cytometry can be correlated with TID if time-temperature data is available. As an experimental model, black porous silicon nanoparticles were studied as photothermal agents to kill HeLa cancer cells. The results indicate that as the critical TID of 70 min is reached, the cells start to undergo apoptosis independently of the way in which the TID was attained: by long heating at low temperatures or by short heating at high temperatures. Overall, TID is proposed as a valid parameter which could be determined in the PTT studies to allow a straightforward comparison of the published results and the elucidation of the cell death mechanisms.

The lack of natural processes of delivery and neonatal intensive care treatment lead to impaired cytokine responses later in life.

PROBLEM: Birth-related factors and neonatal treatments could affect the maturation of immune system and thus have lasting effects on immune responses. We investigated the effect of obstetric factors other than being born by cesarean section on immune responses later in life. METHOD OF STUDY: Regulatory, inflammatory, TH1 and TH2 cytokines, and a chemokine were analyzed in unstimulated and Toll-like receptors (TLRs) 2-, 3-, and 4-stimulated PBMCs of teenagers born by cesarean delivery (CD; N=79). Data on obstetric factors were collected from patient data archives. RESULTS: Advanced cervical dilatation at the time of CD associated with higher unstimulated production of cytokines compared to adolescents who were delivered before the onset of labor. Neonatal intensive care treatment associated with lower unstimulated production of cytokines. Similar associations were found following TLR stimulations. CONCLUSION: The lack of natural processes of delivery and neonatal intensive care treatment might lead to long-lasting impairment of immune responses.