Wildfire smoke (WFS) is the leading climate-related risk in Canada and the main source of harmful air pollution. While short-term breathing problems caused by smoke are well known, there is limited knowledge on how repeated exposure contributes to long-term lung disease. This study is a controlled human exposure to varying concentrations of WFS in a safe setting. By comparing the effects of different concentrations, this research will improve understanding of health impacts, identify who may be most vulnerable to exposures, and explore biological changes that could lead to chronic illness.

PURPOSE: To use responses to controlled human exposures to woodsmoke, as a model of wildfire smoke (WFS), to quantify risk of, and add biological plausibility to, the supposition that WFS-related particulate matter with a diameter of 2.5 micrometers or less (PM2.5) contributes to the development of neurological and chronic lung diseases.

Study Exposure Arms:

Arm A: 3 consecutive days with 2h woodsmoke exposures at 35 µg/m3 of PM2.5 Arm B: 3 consecutive days with 2h woodsmoke exposures at 105 µg/m3 of PM2.5 Arm C: 2 consecutive days of Filtered Air (FA), followed by 1 day with a 2h woodsmoke exposure at 315 µg/m3 of PM2.5

HYPOTHESIS, JUSTIFICATIONS, AND OBJECTIVES Aim 1A: Enhance plausibility for, and quantify risk of, WFS contributions to chronic lung disease.

Hypothesis 1A: The investigators will determine mechanisms that plausibly link WFS exposure to the development of chronic disease, by connecting WFS exposures to lung function decline, asthma, chronic obstructive pulmonary disease (COPD), or lung cancer.

To assess the plausibility of linking WFS exposure to Chronic Lung Diseases such as asthma, COPD, and lung cancer.

To evaluate the biological impacts of WFS on the airways.

• Specifically, what are the effects of exposure concentration, intensity, and time on inflammatory and immunomodulatory responses to WFS exposure?
• Several biological pathways may link WFS exposures with the development of chronic respiratory diseases such as asthma, COPD, and lung cancer. These include effects of oxidative stress and inflammation on epithelial barrier integrity, which may facilitate contact with immune cells, allergen sensitization, increased infection susceptibility, and tissue remodelling that impairs lung function. Epigenetics, notably DNA methylation (DNAm), is another potential mechanism linking WFS to chronic disease, and the airway microbiome may also contribute through several pathways. However, WFS differs from general urban and traffic-related air pollution (TRAP), so effects of TRAP exposure demonstrated to date cannot be assumed to apply to WFS.

Aim 1B: Use a controlled human exposure study to identify, across different intensities of WFS exposure:

1.1 Neurocognitive changes relevant to the development of chronic neurological diseases 1.2 Temporal patterns of onset and resolution in relevant circulating neuro-inflammatory markers 1.3 Sex, age, and genetics as effect-modifying susceptibility factors for the above phenomena

Hypothesis 1B: The investigators will demonstrate disturbances in the brain default mode network (DMN), and intensity-dependent increases in circulating neuro-inflammatory markers, particularly in older individuals and those with genotypes conferring deficient anti-oxidant metabolism or those conferring risk of dementia.

RESEARCH DESIGN A randomized, double-blinded, crossover-controlled human exposure study.

STATISTICAL ANALYSIS Data will be analysed in R using generalized and linear mixed-effects models. Models will run with exposure as a fixed effect and participant ID as a random effect.

Inclusion Criteria:

• Healthy Adults between the ages of 19 to 80 (12 of each biological sex assigned at birth).

Exclusion Criteria:

1. Current smoker (within six months before screening; potential to confound exposure effects).
2. History or current diagnosis of any respiratory conditions (including, but not limited to asthma or chronic obstructive pulmonary disease (COPD), asthma/COPD overlap) or other medical conditions that the study physician determines may impact participant safety.
3. Any comorbidities or other concerns identified by the study physician which may impact study participation.
4. For participants of child-bearing potential: Current pregnancy, or plans to become pregnant during study enrolment.

Blinding of exposures will be performed by the air pollution exposure laboratory (APEL) engineer. Participant coordination and testing will be conducted by a researcher without knowledge of which study arm a given participant is undergoing. The study investigator will be blinded to which study arm participants are undergoing. All assays will be performed by personnel who do not know the exposure conditions of individual samples.