Effects of Hypoxia and High Altitude on Gene Expression, Energetics, and Immune Function
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Environmental stressors, which shape the evolution and ecology of a species, are of major interest to physiological ecologists. The hypoxia encountered at high altitude is a well known abiotic stressor, and how animals cope has long intrigued both physiologists and ecologists. Despite the long interest in high altitude physiology, researchers are continuing to make discoveries in how mammals physiologically acclimatize and adapt to the chronic hypoxia found at high altitude. To add to this growing knowledge, DNA microarray-based mRNA expression profiling was used to to investigate responses to hypoxia. In the first chapter, C57BL/6 strain laboratory mice (Mus domesticus) were subjected to 32 days of hypoxia and liver samples were used for analysis of global gene expression patterns. ANOVA methods identified 580 genes that were statistically significantly differentially expressed in response to chronic hypoxia. Few of these 580 genes had previously been reported to respond to hypoxia. However, many of the 580 genes belonged to functional groups that are important for responding to acute hypoxia (e.g., angiogenesis, glycolysis, lipid metabolism, carbohydrate metabolism, and protein amino acid phosphorylation). Novel to this study were the increased expression of leptin receptor and the differential expression of genes associated with the immune system. In the second chapter, DNA microarray-based mRNA expression profiling was used to compare gene expression of wild populations of house mice (Mus musculus domesticus) living at high and low altitudes. One way and nested ANOVA analyses identified 107 statistically significantly differentially expressed genes, few of which were in common with the laboratory study in the first chapter. When compared with mice at low altitude, mice at high altitude differentially expressed genes with functions associated with the immune system. This effect of altitude on expression of immune genes is consistent with the differential expression of immune genes in hypoxic mice found in the first chapter. Furthermore, EGLN3, a gene responsible for the negative regulation of Hypxoia Inducible Factor (HIF), had decreased expression in high altitude mice. EGLN3 is a homolog to EGLN1 that was recently identified as a gene responsible for evolutionary adaptation in high altitude humans. The third chapter follows up on the differential expression of immune system genes in both chronic hypoxia and high altitude. While there is growing evidence that acute hypoxia has proinflammatory effects, it is was unknown whether or not chronic hypoxia would have the same effect. At least some studies by ecological immunologists have suggested that immune function may be energetically costly. If hypoxia imposes an energetic stress, then an energetic tradeoff hypothesis would predict that the putatively costly immune system should not be up-regulated during this hypoxic stress. I tested (i) whether or not chronic hypoxia affects immune function, and (ii) whether or not hypoxia affects the metabolic cost of immune function. First, flow cytometry was used to monitor the peripheral blood immunophenotype of mice over the course of 36 days of hypoxic exposure. Second, hypoxic and normoxic mice were subjected to an adaptive immune challenge via keyhole limpet hemocyanin (KLH), or to an innate immune challenge via lipopolysaccharide (LPS). The resting metabolic rates of mice in all immune challenge treatments were also measured. Although hypoxia had little effect on the peripheral blood immunophenotype, hypoxic mice challenged with KLH or LPS had enhanced immunological responses in the form of either higher antibody titers or increased TNF-α production, respectively. Initially mice exposed to hypoxia had lower metabolic rates, but this response was transitory and resting metabolic rates were normal by the end of the experiment. Surprisingly, there was no effect of either immune challenge on resting metabolic rate, suggesting that mounting either the acute phase response or a humoral response is not as energetically expensive as previously thought. Taken together, these data indicate that hypoxia has a positive effect on the immune system independent of an energetic relationship. Therefore we propose that the relationship between the immune system and hypoxic stress is governed not by demand for metabolic energy, but by shared transcriptional and hormonal networks that regulate both processes. Further research into the relationship between hypoxic stress and the immune system will help elucidate how the immune system is sensitive to environmental stresses, and how the immune system may be important to the physiological ecology of mammals at high altitude.