Particulate matter exposure during smoke inhalation provokes inflammatory immune responses in people exposed to burning biomass including fire fighters and civilians. Persistent occupational exposure to particulate matter represents a unique hazard for firefighters, underpinning a burgeoning research area. This trial will evaluate the effects of sleep deprivation and circadian rhythm disruption on the inflammatory response to woodsmoke associated particulate matter exposure. Participants will undergo 2 experimental trials in a randomized cross-over design. Participants will have either an 8-hour sleep opportunity or a 4-hour sleep opportunity prior to reporting to lab for a 45 minute simulated firefighting trial (wood smoke associated particulate matter filtered to 2.5 um at a concentration of 250 ug/m^3, while exercising at a moderate intensity). The effects of sleep restriction and simulated firefighting will be measured.

Participants: Healthy college age males (n = 15), free from disordered sleep and without recent trans-meridian travel (within the last 2 weeks) will be recruited for this study. Participants will complete chronotype questionnaires (Morningness-Eveningness Questionnaire; MEQ, Munich Chronotype Questionnaire; MCTQ) to establish intermediate chronotypes. This will minimize the effect of circadian preference on the morning exposure to smoke. Participants will subsequently be outfitted with activity monitors (ActiCal) to monitor sleep and physical activity throughout the experimental duration. Participants will be asked to maintain a normal sleep schedule for at least 3 days leading up to the experimental trials and keep a sleep log to verify.

Experimental Trials: Participants will undergo 2 experimental trials in a randomized cross-over design, with at least 1 week washout period between trials; 1) NS-250: Normal Sleep with exposure to woodsmoke at 250 µg/m^3, and RS-250: Restricted Sleep with exposure to woodsmoke at 250 µg/m^3. Participants will have an 8-hour sleep opportunity in their own home during the NS trials (22:00-06:00), and a 4-hour sleep opportunity during the RS trials (00:00-04:00). In all trials, participants will report to the laboratory at 07:30 the morning after the experimental sleep night. PM exposure will occur from 08:00-08:45 while cycling at 70% heart rate reserve (HRR) to simulate the physical demands of firefighting.

Exhaled Breath Condensate (EBC): EBC will be collected using standardized 10 minute collection techniques. In order to preserve sample integrity for potentially labile biomarkers (e.g., oxidative stress), sample pH will be measured immediately prior to aliquoting in multiple cryotubes (500-700µl), flash freezing, and storage at -80 degrees C until further assay. Standardized biomarker panels for oxidative stress and inflammation will be performed using a single thaw approach.

Inflammatory biomarker analysis: Blood will be collected into heparinized vacutainers before (PRE) and immediately following (POST) PM2.5 exposure and spun down for plasma collection. Plasma will be assayed for inflammatory biomarkers (interleukin (IL)-6, tumor necrosis factor (TNF)-α, pentraxin-3, and C-reactive protein (CRP)) using standard enzyme linked immunosorbent assays (ELISA).

Circadian Rhythm Assessment: Throughout the experimental protocol, circadian rhythms will be assessed in two ways; 1) Actigraphy and 2) Clock gene expression in buccal cell swabs. Acticals will be worn throughout to gather sleep variables (timing, duration, quality). Clock gene expression (CLOCK, BMAL1, PER2) will be measured in swabs taken from the cheek at 6 hour intervals (00:00, 06:00, 12:00, 18:00) to assess the effects of sleep deprivation on the molecular circadian rhythm. Cheek swabs will be immediately placed in RNA stabilization buffer until isolation. Swabs taken at 00:00 will be performed with minimal exposure to light to avoid disruption of sleep. These methods have been used previously to assess normal and disrupted sleep.

Inclusion Criteria:

• Healthy
• Male
• 18 - 44 years of age

Exclusion Criteria:

• Preexisting cardiometabolic and/or pulmonary diseases
• Preexisting sleep disorder
• Smoking (current or within last year)

Individual participant data will not be shared with other researchers. Blinded anonymized outcomes data will be shared via publication and presentation of results.

Adverse event data was collected throughout participation in the study, which was roughly three weeks per participant.

The experimental interventions used in this study (mild smoke inhalation, moderate intensity exercise, mild sleep restriction) were not of a life threatening nature and our participants were healthy. As such, none of our participants were at risk for All-Cause Mortality.

Our study was limited by a smaller sample size than we had anticipated.