The Role of Acetazolamide in Mitigating Inflammation and Innate Immune Activation at High Altitude: a Randomized Cross-over Controlled Trial
Phase: EARLY_PHASE1
Conditions: Hypoxemia, Acute Mountain Sickness (AMS), Inflammation, Immune Response
Sponsor: University of California, Riverside

 
 
 
The Role of Acetazolamide in Mitigating Inflammation and Innate 
Immune Activation at High Altitude 
 
 
Date of document: September 27, 2024 
 
 
 

Title of Project: The role of acetazolamide in mitigating innate immune activation at high 
altitude: a randomized cross-over controlled trial 
Executive Summary: High altitude exposure can lead to an acute pro-inflammatory response 
and activation of innate immune cells.1–6 Our prior work demonstrates that 1 to 3 days at 3800 m 
elevation leads to increased expression of inflammatory damage associated molecular patterns 
(DAMPs), several toll-like receptor pathways involved in innate immune responses, as well as 
increases in pro-inflammatory monocyte and neutrophil subpopulations.1,6,7 Furthermore, our 
data and others indicate that either hypoxia-induced inflammatory responses, or baseline immune 
parameters, likely play a role in the onset and severity of Acute Mountain Sickness (AMS), 
although the precise mechanism is unknown.7 One of the most effective medical treatments for 
AMS is Acetazolamide (AZ).8 The mechanism of action by which AZ alleviates AMS is through 
regulation of blood bicarbonate levels, modification of blood pH, and resulting increases in 
ventilation and improved tissue oxygenation.8–10 However, AZ is also reported to have anti-
inflammatory and immune-modulatory properties in animal models, which have not been 
evaluated as a potential mechanism of action in AMS treatment.11,12 We hypothesize that AZ will 
reduce pro-inflammatory responses and innate immune activation during 3 days of high-altitude 
exposure. To test this hypothesis, we will conduct a randomized, cross-over, placebo-controlled 
trial in which we administer AZ or placebo treatments to N=20 individuals over two visits to 
3800 m elevation. Outcome measures will include the most detailed evaluation of systemic 
inflammatory and immune parameters to date in this field, as well as contributing physiological 
parameters including ventilatory chemoreflex sensitivity, and daytime and nocturnal pulse 
oximetry. This work will advance our knowledge about the pathology and treatment of a key 
wilderness medicine challenge by identifying a potential novel mechanism of AMS onset and 
treatment.  
 

Section I – Summary 
A. Introduction and Background: Acetazolamide (AZ) is a highly effective treatment for acute 
mountain sickness (AMS) at high altitude.8,9 AZ mitigates AMS through its activity as a renal 
carbonic anhydrase inhibitor, which decreases blood pH and stimulates ventilation to improve 
oxygen delivery to tissue.9,10 However, AZ also demonstrates immune modulatory properties, 
with most evidence examining this in lung tissue to investigate the impact on pulmonary 
hypertension in animal models.11,13 Our group and others show that high-altitude exposure 
results in an acute pro-inflammatory response and activation of the innate immune system, which 
some predict contributes to the onset and severity of AMS.1,7 Therefore, the anti-inflammatory 
properties of AZ may play a key role in modulating AMS symptoms independent of ventilatory 
stimulation. However, prior work has examined the impact of AZ on only a select few 
inflammatory mediators, and the impact of AZ on immune function has never been evaluated. 
B. Hypothesis and specific aims: Our goal is to determine if AZ mitigates the pro-inflammatory 
innate immune response during high altitude travel. We hypothesize that AZ improves AMS 
symptoms, in part, by reducing systemic hypoxia-induced inflammation and reducing the 
activation of innate immune cells (monocytes and neutrophils). We will test this hypothesis with 
the following experimental aims: (Aim 1) Determine if AZ treatment during high altitude 
exposure blunts plasma inflammatory cytokine expression and levels of circulating pro-
inflammatory innate immune cell subsets; (Aim 2) Determine if AZ treatment during high 
altitude exposure modulates immune cell function (innate inflammatory responses to bacterial 
and viral stimuli, cell migration, and bacterial killing efficacy).   
C. Study design and methods: 
a. Study Design: This is a double blind, randomized, cross-over, placebo-controlled trial.  
Participants (N=20) will be recruited by word of mouth and advertising, and complete written 

informed consent in the UC Riverside laboratory within 1 month of first ascent (Summer 2025). 
Participants then complete medical history and basic physical screening. At this time, they are 
randomly assigned to a treatment group (i.e. Group A taking AZ on trip 1 and placebo on trip 2), 
maintaining age and sex ratios across groups. Participants will be asked to begin taking their 
assigned treatment as indicated 2 days before each ascent (125 mg twice daily for AZ).14 On the 
morning of ascent, participants arrive at the lab at 6:00 am and fasting peripheral venous blood 
samples will be collected by a licensed phlebotomist or physician. Participants then travel to 
Barcroft Station (3800 m elevation) via vehicle (6 -7 hours). Over three days at high altitude, 
participants complete morning fasting blood draws, morning and evening AMS questionnaires 
(Lake Louise)15, and morning and evening physiological measures (blood pressure, heart rate, 
SpO2). On the second day at high altitude, ventilatory chemoreflex measures will be collected as 
previously described by our group.16 WatchPAT sleep kits will be used to characterize nocturnal 
hypoxic burdens, oxygen desaturation indices, and apnea hypopnea indices as previously 
described by our group.17 Following descent, participants complete 1-week and 1-month follow-
up visits to determine the long-term effects of high-altitude exposure on immune phenotypes.  
100 µl of whole blood will be fixed and stained immediately on-site at each timepoint for 
flow cytometry analysis at UC Riverside within 3 days of sample collection. We will also 
conduct complete blood counts on-site. 20 ml of EDTA-treated blood will be utilized for plasma 
collection and isolation of peripheral blood mononuclear cells by density gradient centrifugation. 
Isolated immune cells will be immediately frozen and stored in liquid nitrogen for transport to 
UC Riverside using a novel protocol optimized by our group for cryogenically storing viable 
cells in remote field locations.7 Upon return to sea level, immune cells will be cultured in the 
presence or absence of several stimulants including bacterial lipopolysaccharide (LPS) and R848 
(Resiquimod, stimulating the antiviral response). Following stimulation, we will collect culture 

supernatants and measure expression of inflammatory factors using Legendplex multi-plex 
cytokine panels as previously described by our group.18 These panels allow simultaneous 
measurement of many key inflammatory mediators including 13 inflammatory cytokines, 12 
markers of vascular inflammation, and 13 anti-viral response factors. We will also conduct 
additional functional assays, including bacterial killing and cell migration assays. Plasma 
cytokine levels will also be measured via Legendplex. All cell culture assays will be conducted 
in triplicate with downstream cytokine profiling in duplicate.  
b. Power calculations: Sample sizes are based on power calculations targeting 80% power to 
detect significant differences across sea level and high altitude at α = 0.05. Calculations were 
performed using pwr in R and are based on previously published data or our own preliminary 
work. A sample size of 14 is required to detect a significant shift from classical to intermediate 
monocyte subsets after 1 day at high altitude with an effect size in our prior studies of 0.46.7 
Additionally, a sample size of 10-36 is required to detect significant correlations between AMS 
score and classical (R2 = 0.79) or non-classical monocytes (R2 = 0.45).  Finally, we previously 
identified elevations in inflammatory response genes after 1-3 days at high altitude with a sample 
size of 17.1 Thus, we predict we are well powered with a sample size of 20 individuals (cross-
over design) to detect significant changes in immune phenotypes at high altitude as well as 
correlations between immune cell distributions and AMS scores. This sample size is also 
consistent with other studies examining the efficacy of AZ for AMS treatment.19 
c. Data analysis plan: Treatments will be assigned by a third party and all study team members 
and participants will be blinded. Data analysis will be conducted by a research team member 
who is blinded to participant treatment, demographic information, and day of sample collection. 
All samples and physiological data will be deidentified and coded. To determine if AZ treatment 
is linked to reduced inflammatory cytokine expression and reduced levels of pro-inflammatory 

innate immune cell subsets (Aim 1), we will first utilize two-way repeated measures ANOVA to 
compare plasma cytokine expression and cell distributions across study days and treatments. To 
determine the impact of AZ treatment on innate immune cell function (Aim 2), we will use a 
three-way ANOVA to examine cell cytokine secretion across study days, treatments, and pre/post 
cell stimulation. Significant main effects will be further evaluated with post-hoc Tukey’s HSD 
tests, and we will verify our data meets all required test assumptions. We will carefully examine 
impacts of sex, age, BMI and other demographic factors on outcomes using mixed linear models. 
WatchPAT data will be analyzed according to the American Academy of Sleep Medicine criteria 
by a qualified sleep technician and ventilatory parameters will be analyzed as described 
previously.16,20–22  To determine if these parameters are associated with our immune and AMS 
outcomes, we will conduct mixed linear models with trip, day, treatment, and sex, as covariates. 
d. Timeframe: This study will take place in the summer of 2025. Participants will be recruited 
in the month prior to study start, and work will be conducted over two expeditions. Participants 
will complete baseline testing within one week of ascent. They will then arrive on the morning of 
ascent for sea-level blood sampling, be driven to Barcroft Station over 6 hours in vans, and will 
remain at the station for 3 nights (4 days) during each visit. The group will then return to sea 
level and a follow-up appointment will be completed 1 week and 1 month following return. The 
second trip will take place 1-2 months after the first. Flow cytometry and multiplex cytokine 
panel tests will be conducted immediately after each trip, and cell functional assays will be 
completed within 2 months of return. Final data analyses will be complete over the following 2 
months in time for an abstract submission to the American Physiology Summit by the deadline in 
Fall 2025 and attendance of the 2026 Winter Conference of the Wilderness Medical Society. The 
manuscript resulting from this work will be submitted for publication in Wilderness & 
Environmental Medicine by Summer 2026.  

e. Facilities: This study will be performed at the UC Riverside School of Medicine and Barcroft 
Station in the White Mountain Research Center. Barcroft Station is located approximately 6 
hours northeast of UC Riverside and our team has extensive experience at this site. The facilities 
house up to 20 participants in dormitory-style lodging, provides three meals per day, and 
contains separate laboratory spaces in for blood sample processing and cell collection. Our 
facilities at UC Riverside include a large, biosafety level 2 laboratory space with separate space 
for studies with human subjects. We have access to all necessary equipment to this work 
including a NovoCyte Quanteon Flow Cytometer, biosafety cabinets and incubators, centrifuges, 
fridges and freezers, and cryogenic storage for cells. 
D. Institutional Review Board approval: Approval for this study has been granted by the UC 
Riverside IRB (project #HS-22-088). An amendment will be required to add the AZ treatment, 
and will be submitted for approval immediately following notice, if we are funded.  
E. Clinical Trial Registration: This study will be registered at ClinicalTrials.gov prior to the 
start of data collection and recruitment. We will work with our UC Riverside Clinical Trial 
Coordinator, Herlinda Bergman, to verify we meet all regulatory requirements.    
F. Significance: This work aligns with the mission of WMS to improve our scientific knowledge 
of human health activities in a wilderness environment. It will provide a novel mechanism by 
which AZ mitigates AMS and may lead to new therapeutic approaches. Furthermore, we will 
generate new knowledge regarding the precise ways in which high altitude exposure influences 
immune function which has implications for understanding the impact of hypoxemia and 
physiological stress on infectious disease susceptibility. This work may also indicate a novel role 
for immune activation in AMS development.  
 

Section II – References 
1. 
Pham K, Frost S, Parikh K, Puvvula N, Oeung B, Heinrich EC. Inflammatory gene 
expression during acute high‐altitude exposure. The Journal of Physiology. 
2022;600(18):4169-4186. doi:10.1113/JP282772 
2.  Pham K, Parikh K, Heinrich EC. Hypoxia and Inflammation: Insights From High-Altitude 
Physiology. Front Physiol. 2021;12:676782. doi:10.3389/fphys.2021.676782 
3.  Facco M, Zilli C, Siviero M, et al. Modulation of Immune Response by the Acute and 
Chronic Exposure to High Altitude: Medicine & Science in Sports & Exercise. 
2005;37(5):768-774. doi:10.1249/01.MSS.0000162688.54089.CE 
4.  Feuerecker M, Crucian BE, Quintens R, et al. Immune sensitization during 1 year in the 
Antarctic high‐altitude Concordia Environment. Allergy. 2019;74(1):64-77. 
doi:10.1111/all.13545 
5.  Meehan R, Duncan U, Neale L, et al. Operation Everest II: Alterations in the immune 
system at high altitudes. J Clin Immunol. 1988;8(5):397-406. doi:10.1007/BF00917156 
6.  Pham K, Vargas A, Frost S, Shah S, Heinrich E. Changes in immune cell populations during 
acclimatization to high altitude. Physiological Reports. in press. 
7.  Vargas A, Mkrtchyan K, Penuelas V, et al. Acute Exposure to High-Altitude Results in 
Changes to Immune Cell Populations. Physiology. 2024;39(S1):2515. 
doi:10.1152/physiol.2024.39.S1.2515 
8.  Forwand SA, Landowne M, Follansbee JN, Hansen JE. Effect of Acetazolamide on Acute 
Mountain Sickness. N Engl J Med. 1968;279(16):839-845. 
doi:10.1056/NEJM196810172791601 
9.  Larson EB. Acute Mountain Sickness and Acetazolamide: Clinical Efficacy and Effect on 
Ventilation. JAMA. 1982;248(3):328. doi:10.1001/jama.1982.03330030034021 
10. Leaf DE, Goldfarb DS. Mechanisms of action of acetazolamide in the prophylaxis and 
treatment of acute mountain sickness. Journal of Applied Physiology. 2007;102(4):1313-
1322. doi:10.1152/japplphysiol.01572.2005 
11. Michael Z, Christou H, Hudalla H, et al. Acetazolamide Modulates Pulmonary 
Inflammation and Ameliorates Severe Experimental Pulmonary Hypertension. Pediatrics. 
2019;144(2_MeetingAbstract):695-695. doi:10.1542/peds.144.2MA7.695 
12. Wang C, Wang R, Xie H, et al. Effect of acetazolamide on cytokines in rats exposed to high 
altitude. Cytokine. 2016;83:110-117. doi:10.1016/j.cyto.2016.04.003 
13. Hudalla H, Michael Z, Christodoulou N, et al. Carbonic Anhydrase Inhibition Ameliorates 
Inflammation and Experimental Pulmonary Hypertension. Am J Respir Cell Mol Biol. 
2019;61(4):512-524. doi:10.1165/rcmb.2018-0232OC 

14. Hackett P, Shilm D. CDC Yellow Book 2024: High Elevation Travel & Altitude Illness. 
https://wwwnc.cdc.gov/travel/yellowbook/2024/environmental-hazards-risks/high-
elevation-travel-and-altitude-illness 
15. Roach RC, Hackett PH, Oelz O, et al. The 2018 Lake Louise Acute Mountain Sickness 
Score. High Altitude Medicine & Biology. 2018;19(1):4-6. doi:10.1089/ham.2017.0164 
16. Frost S, Pham K, Puvvula N, Oeung B, Heinrich EC. Changes in hypoxic and hypercapnic 
ventilatory responses at high altitude measured using rebreathing methods. Journal of 
Applied Physiology. 2024;137(2):364-373. doi:10.1152/japplphysiol.00128.2024 
17. Frost S, Orr J, Oeung B, et al. Improvements in sleep‐disordered breathing during 
acclimatization to 3800 m and the impact on cognitive function. Physiol Rep. 2021;9(9). 
doi:10.14814/phy2.14827 
18. Bergersen KV, Pham K, Li J, et al. Health Disparities in COVID-19: Immune and Vascular 
Changes Are Linked to Disease Severity and Persist in a High-Risk Population in Riverside 
County, California. In Review; 2023. doi:10.21203/rs.3.rs-2800664/v1 
19. Gao D, Wang Y, Zhang R, Zhang Y. Efficacy of Acetazolamide for the Prophylaxis of Acute 
Mountain Sickness: A Systematic Review, Meta-Analysis and Trial Sequential Analysis of 
Randomized Clinical Trials. The American Journal of the Medical Sciences. 
2021;361(5):635-645. doi:10.1016/j.amjms.2020.12.022 
20. Duffin J. Measuring the respiratory chemoreflexes in humans. Respiratory Physiology & 
Neurobiology. 2011;177(2):71-79. doi:10.1016/j.resp.2011.04.009 
21. Duffin J. Measuring the ventilatory response to hypoxia: Measuring ventilatory response to 
hypoxia. The Journal of Physiology. 2007;584(1):285-293. 
doi:10.1113/jphysiol.2007.138883 
22. AASM Manual for the Scoring of Sleep and Associated Events: Rules, Terminology and 
Technical Specifications. Vol Version 3. American Academy of Sleep Medicine; 2023.