Ambient Air Pollution Exposure (AMBIEX)

We will investigate the toxicity of real-world complete ambient air in human lung bronchi and olfactory mucosa (a proxy to brain effects) tissues from healthy and diseased donors (asthma, Alzheimer´s disease). The cells will be grown at the air-liquid interface (ALI = cells are grown on a membrane with their basal surfaces in contact with media, and the top of the cellular layer is exposed to the air) in our exposure system in the field conditions of localities differing in air pollution levels.

• Direct exposure to a complete mixture of ambient air pollutants
• Models consisting of combinations of relevant cell types grown at the air-liquid interface
• No monocultures/monocellular cultures
• No animal testing
• No particulate matter separated from the ambient air using filters
• No organic or water-based extracts from particulate matter

Reference numbers are mentioned in square brackets in the text. A list of references can be found at the bottom of the page.

Exposure to air pollutants is associated with elevated asthma, chronic obstructive pulmonary disease or lung cancer mortality [23]. It also affects other organs in the organism, including the central nervous system, resulting in an increased risk of, e.g. Alzheimer’s disease [24].

The project aims to investigate the biological response of human lung and olfactory cells (a proxy to brain effects) exposed to real-world ambient air at the air-liquid interface and assess differences in the response between samples collected from healthy and diseased (asthma and Alzheimer’s disease) individuals in the field conditions at four localities of the Czech Republic differing in concentrations of air pollutants (characterised by concentrations of PM, ozone, nitric oxides, VOC and PAHs):

• consistently high pollution levels from several sources (Ostrava region)
• pollution associated with industrial production (car manufacturing, North-Bohemian area)
• traffic-related pollution (Prague)
• a background station with low levels of pollutants (Bohemian-Moravian Highlands)

Specifically, to investigate the impacts on the pulmonary system, we propose to use a 3D model of human upper airway epithelium (MucilAir^TM, Epithelix Sàrl, Geneva, Switzerland) reconstituted from tissues of 8 healthy and 8 asthmatic individual donors. The model represents a fully differentiated and functional respiratory epithelium consisting of basal, goblet and ciliated cells [26].

Since olfactory mucosa is considered one of the most feasible routes of air pollutants entry into the brain [27], we will further study the effects of air pollution exposure in human olfactory mucosa cells (hOM) obtained from 8 healthy subjects and 8 Alzheimer’s disease (AD) patients (provided by a collaborative institution – Prof. Katja Kanninen, University of Eastern Finland). These cultures contain fibroblasts/stromal-like cells, globose basal cells and myofibroblasts-like cells obtained from a biopsy from the nasal septum and further processed as previously described [28].

We will expose MucilAirTM tissues derived from 8 asthmatic and 8 healthy subjects and hOM from 8 AD and 8 healthy controls to ambient air for up to 6 hours/day for 5 consecutive days. As a clean air control, synthetic air will be used.

Because adverse response to air pollutants in diseased subjects has been shown to differ from that in healthy individuals [31], simultaneous analysis of both types of samples will improve the quality and general validity of our data.

1. We will compare the differences between the reactions of healthy and diseased donors and between clean air and ambient air exposure.
2. Additionally, investigating the response in the samples originating from several independent subjects will simulate inter-individual differences among individuals in a human population. The donors of the tissues will be matched by gender, age and smoking status.

• Parameters of cytotoxicity will be evaluated with the aim of identifying optimal treatment conditions (time of exposure/day; number of days of exposure) for further use in the project.
• Oxidative stress and inflammation are believed to play a major role in the etiology of asthma, as well as AD [29,30], and are linked to exposure to air pollutants. We will thus focus on analysing selected markers of oxidative stress and immunomodulatory molecules.
• Also, the whole genome expression of mRNA and miRNA will be analysed by next-generation sequencing on the Illumina platform. We will specifically focus on the identification of mRNA-miRNA interactions and their impact on mechanisms of biological response and on the characterisation of inter-individual differences in the response.

We will use our previously developed exposure system within the project MUCILTOX [25] which will be modified for application in the field conditions to be suitable for direct exposure to complex ambient air pollutants.

Mechanisms of toxicity of gasoline engine emissions in 3D tissue cultures and a model bronchial epithelial cell line (project no. 18-04719S) were funded by the Czech Science Foundation, which specifically focused on the effects of gasoline engine emissions tested in laboratory conditions.

In MUCILTOX, we directed our experiments towards the application of 3D models grown at the ALI [26][43–45]. We developed the exposure system that allows real-time ALI exposure of a cell model of choice to complete engine emissions in laboratory conditions [25]. Using this system, we investigated the biological effects of exposure to MucilAirTM and a standard cell monolayer (BEAS-2B) to complete ordinary and alternative gasoline engine emissions [46,47]. We observed significant changes for inflammatory markers in the 3D model exposed to complete emissions from standard gasoline fuel [48].

1. The toxic response will significantly differ between cell models derived from healthy and diseased
   subjects. The response will further depend on the origin of the models (bronchi vs. olfactory
   mucosa).
2. The intensity of the response will be more pronounced in the cell models exposed in the
   localities with elevated concentrations of air pollutants.
3. Ambient air pollution in the industrial region will induce higher levels of lipid peroxidation and
   inflammation markers than pollutants from other localities.
4. The type of ambient air pollution (mixed vs industrial sources vs traffic) will affect the pattern of
   mRNA and miRNA expression.
5. Substantial inter-individual differences will be observed for investigated parameters. They will be
   more pronounced for gene expression data and in diseased subjects.

Among other impacts, it contributes to increased incidence of pulmonary, cardiovascular and neurological/neurodegenerative disorders, and it is also responsible for shortened life expectancy [1–5]. In 2015, outdoor air pollution was classified by the International Agency for Research on Cancer (IARC) as carcinogenic to humans (Group 1) [6].

Air pollutants originate both from natural sources (e.g. volcano eruptions, forest fires) and human activities (road traffic, residential heating, industrial production), and their concentrations are particularly high in densely populated areas (e.g. large cities).

Polluted air contains a complex mixture consisting of gaseous pollutants [ozone, SO2, CO, NOx, volatile organic compounds (VOC)], particulate matter (PM) of various sizes and combinations bound to it, including e.g. polycyclic aromatic hydrocarbons (PAHs) and metals. Depending on the aerodynamic diameter, PM is divided into coarse (PM10, ≤ 10 μm), fine (PM2.5, ≤ 2.5 μm) and ultrafine (PM0.1, ≤ 0.1 μm) fractions.

The toxicity of polluted air arises from a complex mixture of various components. For a comprehensive evaluation of the biological effects of air pollutants is a crucial application of exposure experiments that involve a complete mixture of ambient air pollutants (complex gaseous pollutants, PM with adsorbed compounds in the form they exist in the atmosphere).

Human population studies may provide the ultimate information on the effects of air pollutants on human health in real-world settings. However, such research is usually costly, logistically and ethically challenging [2]. Thus, alternative approaches based on exposure of laboratory animals to urban air or road traffic pollutants have been used [15–17]. Although easier to perform, these studies still require specific conditions and differences between experimental animals and human organisms to further complicate the extrapolation of animal data to humans.

Despite this fact, studies investigating in vitro toxicity of ambient air are often limited to the exposure of cell cultures to organic or water-based extracts from PM or PM separated from the ambient air using filters or other methods [14].

In the lungs, fine particles penetrate into alveoli and can interfere with the gas exchange between inhaled air and blood [7]. At the same time, the ultrafine fraction enters individual cells, including subcellular structures and can be distributed to distant tissues and organs via the bloodstream. The size of the particles, along with the chemical composition of the complex mixture, determines possible adverse health effects of air pollutants on human health.

In the organism, the effects mediated by PM are linked to both the physical properties of PM and the chemical composition of the compounds adsorbed to it. The presence of particles themselves contributes to the activation of the immune system, potentially resulting in the production of reactive oxygen species (ROS) by the immune cells. ROS cause oxidative damage to macromolecules, including nucleic acids, inducing mutations and thus increasing the risk of tumorigenesis [9]. Additionally, ROS act as signalling molecules affecting the expression of genes involved in metabolic regulation or stress response [10]. Some of the compounds bound to PM, notably polycyclic aromatic hydrocarbons (PAHs), are possibly carcinogenic or carcinogenic to humans. PAH reactive intermediates may also be metabolised into o-quinones that enter redox cycling and cause oxidative stress by the formation of ROS [11]. The presence of transition metals in PM further contributes to ROS generation and oxidative damage of macromolecules [12]. Although the effect of gaseous components of air pollution on human health is less significant than that of PM [13], they further potentiate pro-oxidant properties and overall carcinogenicity of polluted air.

General Objectives

To identify the biological effects of exposure, we will perform a comprehensive toxicological assessment of a panel of relevant biomarkers, including parameters of cytotoxicity, markers of oxidative stress and inflammatory response, as well as global mRNA and miRNA expression analysis.

• To modify and optimise a system for exposure of cell models grown at the air-liquid interface to ambient air pollution.
• To investigate the biological response of a human bronchi and olfactory mucosa model to exposure to real-world ambient air with different concentrations of air pollutants.
• To assess differences in the toxic response between cell models obtained from healthy individuals and those suffering from asthma or Alzheimer’s disease.

Specific Objectives

• To expose the studied cell models to ambient air pollution and the synthetic air at four localities differing in air pollution levels.
• To assess basic markers of toxicity (cytotoxicity, TEER, mucin production), ROS generation, lipid peroxidation induction and inflammatory mediators production in the studied cells.
• To perform a comprehensive analysis of global mRNA and miRNA expression and their interactions to investigate the impacts of exposure to genome and epigenome.
• To determine differences in the response to ambient air exposure between healthy subjects and those suffering from asthma and Alzheimer’s disease.
• To evaluate inter-individual differences in response to air pollutants for both the healthy and diseased subjects and associate them with concentrations of pollutants in the ambient air.

For further experiments, tissue culture media and cell lysates will be collected and stored at -80 °C.

• For cytotoxicity evaluation, the Lactate dehydrogenase and Adenylate kinase assays will be used.
• The tissue integrity of MucilAir^TM cultures will be evaluated by Transepithelial electrical resistance (TEER) measurement.
• Mucin production, a parameter of negative effects of exposure to ambient air, will be assessed in MucilAir^TM cultures by the Sandwich enzyme-linked lectin assay.
• Production of extracellular reactive oxygen species (ROS) will be assayed by Acridan Lumigen PS-3 Assay.
• Markers of lipid oxidation, 22 selected metabolites of arachidonic and linoleic acid, will be measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) by an external collaborator.
• A panel of 25 selected inflammatory markers (cytokines, chemokines and growth factors, including, e.g. IL-1, IL-6, IL-7, IL-8, TNFα, MCP-1, RANTES, GM-CSF, EGF or LIF) will be assessed by a multiplex immunoassay in a service laboratory (Thermo Fisher Scientific).
• Whole genome expression of mRNA and miRNA will be analysed by next-generation sequencing on the Illumina platform. We will specifically focus on identification of mRNA-miRNA interactions and their impact on mechanisms of biological response and on the characterisation of inter-individual differences in the response.

In our previous work, we studied in vitro effects of air pollutants using standard cell monolayers (model lung cell lines: alveolar epithelial cells A549; bronchial epithelial cells BEAS-2B; embryonic lung fibroblasts HEL), incubated in submerged conditions and treated with organic extracts from PM originating from ambient air pollution and/or engine exhaust [32–42]. Recently, we directed our experiments towards the application of 3D models grown at the air-liquid interface ALI (cells are grown on a membrane with their basal surfaces in contact with media, and the top of the cellular layer is exposed to the air [26][43–45] and developed the exposure system that allows real-time ALI exposure of a cell model of choice to complete engine emissions in laboratory conditions [25].

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