Abstract
Heatwaves pose a growing risk to public health. While most animal studies use sudden, extreme heat exposure, the systemic and pulmonary impacts of gradual heat exposures, reflective of real-world conditions, remain poorly characterized. This study examined the effects of acute, gradual extreme heat exposure to mice. Adult male and female C57Bl/6 mice were randomly assigned to heat-exposed, control, or pair-fed groups. Heat-exposed mice experienced a controlled 8-hour temperature ramp from 20°C to 38°C, mimicking the daily transition from nighttime lows to daytime highs. Control and pair-fed mice were maintained in parallel at ambient temperature. Multi-omics profiling was performed to assess cytokine levels in lung and serum, cecal microbiome composition, lung transcriptomics, and serum metabolomics. Heat exposure significantly altered the levels of multiple cytokines in serum and lung, including IL-17α, MIP-1α, MIP-1β, IL-1α, IL-12(p40), and RANTES, indicating shifts in mucosal immunity and immune cell recruitment. Random forest analysis identified 20 taxa that distinguished experimental groups, with a reduction in Lactobacillus observed in males. Lung transcriptomic analysis revealed immune-related gene expression changes involving B cell activation pathways. Serum metabolomics revealed significant decreases in ten metabolites across both sexes, identifying disruptions in amino acid and energy metabolism, with enrichment of the "Glycine, Serine, and Threonine Metabolism" KEGG pathway. Integrative network analyses revealed sex-specific correlations among immune genes, cytokines, and bile acid-related metabolites. These findings show that gradual extreme heat exposure triggers sex-specific systemic and pulmonary immunometabolic responses, offering insight into the biological effects of environmental heat stress and its potential health implications.