Ambient Air Pollution Exposure (AMBIEX)

The impact of real-world ambient air pollution exposure on human lung and olfactory cells grown at the air-liquid interface.

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Air Pollution and Human Health

Air pollution is one of the most significant environmental threats to human health. Long-term exposure to polluted air has been associated with an increased incidence of respiratory, cardiovascular, neurological and neurodegenerative diseases, while also contributing to reduced life expectancy [1–5]. In 2015, the International Agency for Research on Cancer (IARC) classified outdoor air pollution as carcinogenic to humans (Group 1) [6].
Air pollutants originate from both natural sources, such as volcanic eruptions and wildfires, and human activities including road traffic, residential heating and industrial production. As a result, pollutant concentrations are generally highest in densely populated urban areas.

Ambient air is a complex mixture of gaseous pollutants, including ozone (O₃), sulfur dioxide (SO₂), carbon monoxide (CO), nitrogen oxides (NOₓ) and volatile organic compounds (VOCs), together with airborne particulate matter (PM) of different sizes and numerous chemicals, such as polycyclic aromatic hydrocarbons (PAHs) and metals, adsorbed onto particle surfaces. According to their aerodynamic diameter, particulate matter is classified into coarse particles (PM₁₀; ≤10 μm), fine particles (PM₂.₅; ≤2.5 μm) and ultrafine particles (PM₀.₁; ≤0.1 μm).

The adverse health effects of air pollution do not result from a single pollutant but from the combined action of this complex mixture. Therefore, understanding the biological effects of air pollution requires experimental approaches that investigate exposure to real-world ambient air, rather than to isolated pollutants or particulate matter extracts alone.

How Air Pollution Affects the Human Body

Once inhaled, fine particulate matter (PM₂.₅) can penetrate deep into the lungs and reach the alveoli, where it may interfere with the exchange of oxygen and carbon dioxide between the air and the bloodstream [7]. Even smaller ultrafine particles (PM₀.₁) are capable of entering individual cells, including their subcellular structures, and may subsequently be transported via the bloodstream to distant tissues and organs.

The biological effects of air pollution depend not only on particle size but also on the chemical composition of the complex pollutant mixture. The toxicity of particulate matter is therefore determined by both its physical properties and the diverse chemicals adsorbed onto the particle surface.

The presence of airborne particles activates the immune system, stimulating the production of reactive oxygen species (ROS). Although ROS play an important role in normal cellular signaling, their excessive production results in oxidative stress, leading to damage of essential cellular macromolecules, including DNA. Such damage may induce mutations and increase the risk of cancer development [8]. In addition, ROS regulate the expression of numerous genes involved in cellular metabolism, inflammation and stress responses [9].

Many chemicals associated with particulate matter also contribute to its toxicity. Among the most important are PAHs, several of which are classified as known or probable human carcinogens. During their metabolism, PAHs can be converted into reactive intermediates and o-quinones that undergo redox cycling, generating additional reactive oxygen species and amplifying oxidative stress [10]. Transition metals adsorbed onto particulate matter further promote ROS formation and oxidative damage to cellular components [11].

Although the health effects of gaseous pollutants are generally less pronounced than those of particulate matter [12], they substantially enhance the overall oxidative potential and toxicity of ambient air. Since humans are continuously exposed to the complete mixture of airborne pollutants, studying their combined biological effects is essential for understanding how air pollution contributes to disease development.

Impacts of air pollution on human body


Why AMBIEX?

Human population studies provide the most reliable evidence on the health effects of air pollution. However, they are expensive, time-consuming and often limited by logistical and ethical constraints [2].

Experimental studies using laboratory animals have therefore become an important alternative for investigating the biological effects of urban air pollution and traffic-related emissions [13–15]. While these models offer better experimental control, they also have important limitations. Differences between animal physiology and the human body make it difficult to directly translate experimental findings into human health outcomes.

Similarly, many in vitro studies have relied on exposing cultured cells to organic or aqueous extracts of particulate matter, or to particles collected on filters [16]. Although these approaches have provided valuable insights, they do not accurately reproduce real-life human exposure, where the respiratory system encounters a complex mixture of airborne particles and gaseous pollutants simultaneously. Furthermore, relatively few studies have employed advanced human tissue models cultured at the air–liquid interface (ALI), which closely mimic the physiology of the human respiratory tract.

The AMBIEX project (The Impact of Real-World Ambient Air Pollution Exposure on Human Lung and Olfactory Cells Grown at the Air–Liquid Interface; Czech Science Foundation, Project No. 22-10279S) was designed to overcome these limitations.

Our objective was to investigate the biological effects of real-world ambient air using advanced human tissue models representing both the bronchial epithelium and the olfactory mucosa. These models were prepared from cells obtained from healthy donors as well as from patients with asthma or Alzheimer’s disease, enabling us to compare how pre-existing disease influences the biological response to air pollution.

The cells were cultured at the ALI, where their basal surface remains in contact with culture medium while the apical surface is directly exposed to air. This culture system closely reproduces the physiological conditions of the human respiratory tract and allows direct exposure to ambient air without the need for particle extraction or artificial sample preparation.

Field exposure experiments were performed using a portable exposure system deployed at four locations representing distinct pollution environments. These sites differed in both the concentration and composition of ambient air pollutants, including PM, ozone, NOₓ, VOCs and PAHs.

Exposure Sites

  • Urban locality (Ostrava region) – consistently high levels of air pollution originating from multiple sources.
  • Industrial locality (Northeastern Bohemia) – pollution dominated by industrial activities, primarily automotive manufacturing and wood-processing industries.
  • Traffic locality (Prague) – air pollution predominantly associated with road traffic.
  • Background locality (Bohemian–Moravian Highlands) – a rural reference site with low concentrations of air pollutants.

Advantages of Our Approach

Compared with conventional laboratory studies, the AMBIEX project offers several important advantages:

  • Direct exposure to real-world ambient air, preserving the complete mixture of airborne pollutants.
  • Exposure to both particulate and gaseous pollutants simultaneously, reflecting real human exposure conditions.
  • Advanced three-dimensional human tissue models cultured at the ALI.
  • Complex multicellular tissue models rather than conventional monocultures.
  • No animal experimentation.
  • No particle collection using filters prior to exposure.
  • No organic or aqueous extracts of particulate matter.

Localities in which the exposure was performed


Human Airway Epithelium Model

The respiratory component of the study was based on the MucilAir™ three-dimensional model of human airway epithelium (Epithelix Sàrl, Geneva, Switzerland). The model was reconstructed from primary cells obtained from both healthy donors and individuals with asthma [17].

Unlike conventional monolayer cell cultures, MucilAir™ represents a fully differentiated, functional airway epithelium composed of basal, goblet and ciliated cells. These cell types closely resemble the structure and function of the human bronchial epithelium in vivo, making the model highly suitable for studying the biological effects of inhaled pollutants.

Human Olfactory Mucosa Model

The olfactory mucosa represents one of the most important pathways through which ultrafine particles may reach the central nervous system [18]. Increasing evidence suggests that airborne pollutants can bypass the blood–brain barrier by travelling directly along the olfactory nerve, potentially contributing to the development of neurodegenerative diseases.

To investigate this pathway, we used primary cultures of human olfactory mucosa (hOM) obtained from healthy donors and patients with Alzheimer’s disease through our collaboration with Professor Katja Kanninen (University of Eastern Finland).

The cultures consisted of several cell populations, including stromal (fibroblast-like) cells, globose basal cells and myofibroblast-like cells isolated from biopsies of the nasal septum. This multicellular composition provides a biologically relevant model for investigating the effects of ambient air pollution on the olfactory mucosa [19].

Air–Liquid Interface Exposure

Both tissue models were cultured at the ALI, where the basal side of the tissue remained in contact with nutrient medium while the apical surface was directly exposed to ambient air. This culture technique closely reproduces the physiological environment of the human respiratory tract and allows direct exposure to airborne pollutants without prior particle collection or extraction.

Cells were exposed to real-world ambient air for up to six hours per day over five consecutive days. Parallel control cultures were maintained under identical conditions but exposed to synthetic clean air, allowing us to distinguish biological responses specifically induced by ambient air pollution.

Why these models?

By combining two complementary human tissue models, AMBIEX was able to investigate different aspects of air pollution toxicity:

  • Human airway epithelium — to study respiratory toxicity, epithelial integrity and inflammatory responses.
  • Human olfactory mucosa — to investigate potential links between air pollution and neurodegenerative diseases.
  • Healthy and diseased donors — to determine how pre-existing disease influences susceptibility to ambient air pollution.
  • Air–liquid interface culture — to mimic human inhalation exposure as closely as possible.

Development of the Exposure System

One of the key innovations of the AMBIEX project was the development of a portable exposure system capable of exposing human tissue models directly to real-world ambient air under field conditions. This technology enables realistic simulation of human inhalation exposure while maintaining the highly controlled environment required for advanced in vitro experiments.

From MUCILTOX to AMBIEX

The exposure system used in AMBIEX builds upon technology originally developed during our previous Czech Science Foundation project MUCILTOX (Mechanisms of Toxicity of Gasoline Engine Emissions in 3D Tissue Cultures and a Model Bronchial Epithelial Cell Line; Project No. 18-04719S) [20].

In MUCILTOX, we developed an experimental platform that allowed three-dimensional human airway models cultured at the air–liquid interface (ALI) to be exposed directly to complete gasoline engine emissions under laboratory conditions [17,21–23]. Unlike conventional exposure systems based on particle extracts or individual pollutants, the MUCILTOX platform delivered the complete emission mixture to the tissue models in real time.

Using this approach, we demonstrated that complete engine emissions induced pronounced inflammatory responses in the MucilAir™ airway model and provided biologically more relevant information than experiments based on isolated particulate matter or chemical extracts [24-26]. These findings formed the foundation for the AMBIEX project.

From the Laboratory to the Field

For AMBIEX, the original laboratory system was redesigned to create a compact, field-deployable exposure platform.

The new system is portable, easy to transport and can be installed at virtually any location. Most importantly, it enables direct exposure of human tissue models to real-world ambient air, preserving the complete mixture of particulate and gaseous pollutants without filtration, extraction or artificial manipulation.

This capability represents one of the major methodological advances of the project, allowing controlled biological experiments to be performed directly in environments with different types and levels of air pollution.

Why Compare Healthy and Diseased Donors?

Previous studies have shown that susceptibility to air pollution differs considerably between healthy individuals and people with pre-existing diseases [27]. Understanding these differences is essential for identifying vulnerable populations and for improving health risk assessment.

For this reason, AMBIEX simultaneously investigated tissue models derived from:

  • healthy donors,
  • patients with asthma, and
  • patients with Alzheimer’s disease.

Comparing these groups allowed us to determine how pre-existing disease influences the biological response to ambient air pollution and to identify molecular pathways associated with increased susceptibility.

A Platform for Real-World Air Pollution Research

The portable exposure system developed within AMBIEX provides several unique advantages over conventional laboratory exposure methods:

  • Direct exposure to ambient air under real environmental conditions.
  • Simultaneous exposure to particulate and gaseous pollutants.
  • Physiological air–liquid interface culture conditions.
  • Controlled temperature, humidity and carbon dioxide concentration throughout the experiment.
  • Compatibility with advanced three-dimensional human tissue models.
  • Easy deployment at locations with different sources and levels of air pollution.

The system therefore bridges the gap between highly controlled laboratory experiments and real-world human exposure, providing a platform for investigating the biological effects of ambient air pollution.

The container in which the exposure system was deployed in the Background locality


Research Hypotheses

The AMBIEX project was built upon several scientific hypotheses concerning the biological effects of real-world ambient air pollution.

Different Individuals Respond Differently to Air Pollution

We hypothesized that tissue models derived from healthy donors would respond differently to ambient air pollution than models established from patients with asthma or Alzheimer’s disease. We also expected biological responses to differ between the bronchial epithelium and the olfactory mucosa, reflecting their distinct physiological functions and exposure pathways.

Air Pollution Intensity Determines Biological Response

We expected that tissue models exposed at locations with higher concentrations of air pollutants would exhibit stronger biological responses than those exposed at cleaner sites. In particular, we anticipated that highly polluted environments would induce more pronounced inflammatory and molecular changes.

Pollution Sources Matter

Ambient air pollution varies considerably depending on its source. We therefore hypothesized that pollution originating predominantly from industrial activities, road traffic or mixed urban sources would produce distinct biological responses. These differences were expected to be reflected in both inflammatory processes and changes in gene expression.

Individual Susceptibility Influences Cellular Response

Finally, we expected substantial inter-individual variability in cellular responses to ambient air pollution. We hypothesized that these differences would be particularly evident at the transcriptomic level and would be more pronounced in tissue models derived from donors with pre-existing disease.

Project Objectives

The overall objective of AMBIEX was to investigate the biological effects of real-world ambient air pollution using advanced human tissue models exposed directly to outdoor air under controlled experimental conditions.

To achieve this goal, we performed a comprehensive toxicological and molecular assessment that included biomarkers of cytotoxicity, oxidative stress, inflammation, and genome-wide changes in gene expression.

The project had three principal objectives:

Develop an Advanced Field Exposure Platform

To further develop and optimize a portable exposure system capable of exposing human tissue models cultured at the air–liquid interface directly to ambient air under field conditions.

Investigate Biological Responses to Ambient Air Pollution

To characterize the biological effects of real-world ambient air pollution on human bronchial epithelium and olfactory mucosa models exposed at locations representing different pollution environments.

Identify Factors Determining Susceptibility

To determine how tissue origin, disease status and individual variability influence cellular responses to ambient air pollution, with particular emphasis on asthma and Alzheimer’s disease.

Specific Objectives

The project addressed these objectives through an experimental program that included:

  • direct exposure of human tissue models to ambient air and synthetic clean air at four locations representing different pollution environments;
  • assessment of cytotoxicity, epithelial integrity (TEER), mucin production, oxidative stress, lipid peroxidation and inflammatory mediators;
  • genome-wide transcriptomic and microRNA profiling to investigate molecular mechanisms underlying exposure responses;
  • comparison of responses between healthy donors and donors with asthma or Alzheimer’s disease;
  • evaluation of inter-individual variability in relation to both donor characteristics and local air pollution levels.

Experimental details

Exposure Chamber

The exposure system enabled advanced human tissue models to be exposed directly to real-world ambient air under field conditions. Before reaching the tissue models, both ambient air and the synthetic clean air used as the control were conditioned to physiological culture conditions (37 °C, 85–90% relative humidity and 5% CO₂). The airflow was then evenly distributed among multiple exposure chambers, ensuring identical exposure conditions for all tissue samples while preserving the natural particle size distribution of ambient air.

The exposure system was validated to quantify particle losses during transport and to determine the fraction of airborne particles deposited onto the tissue surface. These measurements ensured that the delivered dose accurately reflected real exposure conditions.

Detailed view of the Toxicology Incubator

Exposure chamber


24-well plate with inserts


An insert in 24-well plate containing cells on porous membrane (ALI system)


A modified cell incubator with exposure boxes


Biological Endpoints

Following each exposure experiment, culture media and cell lysates were collected and stored at −80 °C for subsequent laboratory analyses.

The following biological endpoints were evaluated to characterize the cellular response to ambient air pollution.

Cell Viability and Tissue Integrity

  • cytotoxicity using lactate dehydrogenase (LDH) and adenylate kinase (AK) assays,
  • epithelial barrier integrity by measuring transepithelial electrical resistance (TEER),
  • mucin production as an indicator of normal airway epithelial function.

Oxidative Stres

  • production of reactive oxygen species,
  • lipid peroxidation by measuring 15-F₂t-isoprostane concentrations.

Inflammatory Response

The inflammatory response was characterized using a multiplex immunoassay measuring a panel of 25 cytokines, chemokines and growth factors, including IL-1, IL-6, IL-8, TNFα, MCP-1, RANTES, GM-CSF and EGF.

Genome-wide Molecular Profiling

To investigate the molecular mechanisms underlying cellular responses to ambient air pollution, we performed next-generation sequencing of both messenger RNA (mRNA) and microRNA (miRNA). Genome-wide transcriptomic profiling allowed us to identify biological pathways altered by exposure and to explore how these changes differ between healthy and diseased donors.

Project outputs

Publicity


Research papers

Real-world outdoor air exposure effects in a model of the human airway epithelium – A comparison of healthy and asthmatic individuals using a mobile laboratory setting [28]

Portable emissions toxicity system: Evaluating the toxicity of emissions or polluted air by exposure of cell cultures at air-liquid interface in a compact field-deployable setup [29]

Transcriptomic and epigenomic profiling reveals altered responses to diesel emissions in Alzheimer’s disease both in vitro and in population-based data [30]

Molecular alterations in human olfactory mucosal cells from healthy individuals and individuals with Alzheimer’s disease induced by real-world ambient air [31]

Real-world traffic-polluted air and its impact on a 3D model of the human airway epithelium [32]

Transcriptional responses to real-world ambient air pollution in healthy and asthmatic human 3D airway models grown at the air-liquid interface [33]

Key Results

A Portable System for Real-World Air Pollution Research

A portable field-deployable exposure system capable of exposing human tissue models directly to ambient air under controlled experimental conditions was successfully developed. This enabled biologically relevant experiments to be performed at locations with different sources and levels of air pollution.

Air Pollution Can Cause Rapid Cellular Damage

Exposure experiments performed at the Traffic locality revealed exceptionally high pollutant concentrations that induced early cell death in the tissue models. As a consequence, several experiments had to be repeated using shorter exposure periods [28,29].

Oxidative Stress Was Not the Dominant Response

Although oxidative stress is widely recognized as one of the principal mechanisms of air pollution toxicity, lipid peroxidation was not significantly increased in either tissue model at any of the exposure sites [31,33].

Industrial Air Pollution Strongly Influenced Immune Responses

The most pronounced changes in inflammatory signaling were observed in airway tissue models exposed at the Industrial locality [33]. Altered production of cytokines and other immune mediators indicates that long-term exposure to industrial air pollution may contribute to chronic inflammation and increase susceptibility to respiratory diseases.

Air Pollution Alters Gene Expression

Genome-wide transcriptomic analysis demonstrated that ambient air pollution affects numerous biological pathways involved in tissue homeostasis and cellular function. In tissue models derived from healthy donors, exposure at the Industrial locality altered pathways associated with cell adhesion, extracellular matrix organization, focal adhesion, and PI3K–Akt signaling. Together, these changes suggest active tissue remodeling and altered communication between cells and their surrounding microenvironment.

In contrast, airway models derived from patients with asthma exhibited reduced activity of pathways involved in epithelial repair, secretory function and cell proliferation, indicating impaired tissue regeneration and an enhanced stress response [33].

Interestingly, a remarkably similar molecular profile was observed in healthy donor samples exposed at the Traffic locality, most likely reflecting the exceptionally high pollution levels measured at this site [31].

Evidence of Neurodegenerative Disease-Related Processes

The olfactory mucosa model revealed that ambient air pollution may affect biological pathways linked to neurodegenerative diseases. In tissue models derived from healthy donors, exposure at the Industrial locality altered molecular processes associated with neurodegeneration. In cultures established from patients with Alzheimer’s disease, the predominant response involved modulation of inflammatory pathways. These findings support the evidence suggesting that long-term exposure to air pollution may contribute to the development and progression of neurodegenerative disorders [31].

Images from the field campaigns

Industrial locality

+6

Traffic locality

+3

Background locality

+9

Urban locality

Project Partners

The Institute of Experimental Medicine of the CAS

The Institute of Experimental Medicine CAS coordinated the biological part of the AMBIEX project and carried out comprehensive toxicological and molecular analyses of human tissue models exposed to real-world ambient air.

Its main responsibilities included:

  • evaluation of cytotoxicity, epithelial integrity (TEER) and mucin production;
  • assessment of oxidative stress through measurements of ROS production and lipid peroxidation;
  • quantification of inflammatory mediators, including selected cytokines, chemokines and growth factors;
  • genome-wide analysis of mRNA and miRNA expression to identify molecular pathways affected by ambient air pollution.

Technical University of Liberec

The Technical University of Liberec (TUL) was responsible for the development and operation of the portable exposure system used during the field campaigns.

Its main responsibilities included:

  • design and construction of a compact field-deployable exposure chamber;
  • monitoring of airborne particle size distributions throughout the exposure experiments;
  • collection of particulate matter samples for subsequent analysis of PAHs;
  • measurement of key gaseous air pollutants, including O₃, NOₓ and VOCs;
  • management of power supply and thermal stability of the exposure system;
  • continuous monitoring of air pollution levels and meteorological conditions during field exposures.Technical University of Liberec

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