Hypoxia and Inflammation: Insights From High-Altitude Physiology

The Role of Inflammation in High-Altitude Illness

High-altitude illnesses are a common risk for sojourners to high altitude (>2,500 m elevation). Within the first week of exposure, sojourners often present with Acute Mountain Sickness (AMS), which is characterized by headache, nausea, fatigue, and gastrointestinal issues (Roach et al., 2018). AMS typically resolves with acclimatization (Gallagher and Hackett, 2004). However, in serious cases, sojourners may develop severe and potentially fatal illnesses, such as high-altitude pulmonary edema (HAPE), high-altitude pulmonary hypertension (HAPH), or high-altitude cerebral edema (HACE; Gallagher and Hackett, 2004; Mehta et al., 2008; Luks et al., 2017). The incidence and severity of HAPH, HAPE and HACE vary depending on multiple factors, such as ascent time, altitude, and timely recognition and treatment (Gallagher and Hackett, 2004). Despite several decades of examining the physiology of AMS, HAPH, HAPE, and HACE, some questions remain regarding the pathophysiology of these conditions and the extent to which inflammation contributes to their onset and progression.

Inflammation and Erythropoiesis

Erythropoiesis is tightly regulated to maintain homeostatic balance between red blood cell production and degradation. This balance is crucial for optimal oxygen delivery to tissues. An inadequate number of erythrocytes lead to tissue hypoxia, while high erythrocyte concentrations can increase blood viscosity, impair blood flow, and increase risk of thrombosis (Keohane et al., 2013). This is particularly relevant in Chronic Mountain Sickness (CMS), a clinical syndrome commonly presented in high-altitude natives and life-long residents, which frequently coincides with excessive erythrocytosis (Hb ≥ 19 g/dL for women, Hb ≥ 21 g/dL for men) in some high altitude groups (León-Velarde et al., 2005; Oberholzer et al., 2020). The HIF pathway plays a key role in regulating erythropoiesis. HIF activation results in increased transcription of the EPO gene in the kidney and liver, and EPO serum concentration can increase up to several 100-fold in response (Imagawa et al., 1990; Haase, 2013). Additionally, HIF activity plays a role in regulating iron uptake from the gut and mobilizing erythroid progenitor cells in the bone marrow (Nemeth, 2008).

Chronic inflammation typically leads to anemia through several mechanisms: prioritizing myeloid cell production, sequestering iron, and increasing erythroid turnover rate. Inflammation impacts crucial sites of erythrocyte production to redistribute resources toward myeloid cell production and therefore reduces lymphoid and erythroid output. For example, expression of pro-inflammatory IL-1β, interferon gamma (IFNγ), and IL-6 skew multipotential hematopoietic progenitors toward myeloid cell development (Tie et al., 2019). This is an adaptive response since myeloid cells are needed to fight infection; however, this comes at the cost of bone marrow erythropoiesis. Additionally, while iron is an essential component of hemoglobin synthesis, it is also an essential micronutrient to pathogens. During inflammation, increased IL-6 induces hepcidin production, interrupting iron absorption from the gut and blocking iron release from macrophages, leading to hypoferremia (Nemeth et al., 2004; Nemeth, 2008; Zaritsky et al., 2009; Weiss et al., 2019). By decreasing iron availability, the immune system limits pathogen proliferation; however, a consequence of iron sequestration is that this also limits iron availability for red blood cell production.

When steady state erythropoiesis becomes insufficient, or red blood cells are broken down at high rates, a compensatory extramedullary mechanism called stress erythropoiesis is initiated to prevent lethal anemia (Paulson et al., 2011). Stress erythrocytosis produces a burst of new erythrocytes to maintain red cell concentrations until bone marrow erythropoiesis recovers. The mechanism of stress erythropoiesis has been studied extensively in mice, where it occurs in the spleen and liver in response to anemia, hypoxia, or sterile inflammation. Studies in mice revealed that sterile inflammation produced by phenylhydrazine injections lead to decreased erythroid progenitor cells in the bone marrow and increased erythroid progenitor cells in the spleen. It was discovered that bone marrow erythroid progenitor cells and splenic erythroid progenitor cells respond to different factors, indicating that splenic progenitors are distinct from bone marrow progenitors. This splenic stress erythropoiesis response depends on TLR signaling molecules Myd88 and TRIF (Jackson et al., 2010). Furthermore, pro-inflammatory cytokines TNF-α and IL-1β promote the expansion of splenic erythroid progenitors (Liao et al., 2018; Bennett et al., 2019). It is currently unknown if similar mechanisms are responsible for stress erythropoiesis in humans (Kim et al., 2015; Mairbäurl, 2018). Future studies may investigate biomarkers of stress erythropoiesis signaling to determine if this protective mechanism is active during high-altitude exposure or other forms of hypoxic stress.

Inflammatory Pathway Genes Under Selection in High-Altitude Groups

Several studies have identified genes under evolutionary selection in high-altitude native populations. HIF pathway genes, particularly EGLN1 and EPAS1, are consistently highlighted across studies. Additionally, several genes associated with inflammation also show signals of selection but have received less attention (Foll et al., 2014). The inflammatory pathway genes IL6, IL1A, IL1B, NOS1, NOS2, and TNF show signals of evolutionary selection in both Andean and Tibetan high-altitude native populations (Beall, 2007; Bigham and Lee, 2014; Moore, 2017). NOS1 and NOS3 have also been shown to be under positive selection in the Sherpa population (Droma et al., 2006; Zhang et al., 2017). Additional inflammation-related genes have been reported to be under selection in individual groups, including HLA-DQB1, PPARA, and TGFBR3 in Tibetans (Yang et al., 2017), PPARA in Sherpas (Horscroft et al., 2017), BRINP3, DUOX2, and CLC in Andeans (Crawford et al., 2017; Jacovas et al., 2018), and AIMP1 in Ethiopians (Scheinfeldt et al., 2012). Table 1 summarizes inflammation-related genes found to be under evolutionary selection in high altitude human populations.

IL6 is particularly interesting given that its expression has been associated with AMS development. IL-6 is also noted to increase hematopoietic stem cell proliferation under hypoxic conditions, and its effects on red cell production are synergistic with other pro-inflammatory cytokines, including IL-1α and TNF-α (Faquin et al., 1992). Together, this data suggest that IL-6 plays a role in adaptation to chronic hypoxia. It is currently unknown what the precise adaptive IL6 variants are, but it is possible that blunting IL-6 expression in response to chronic hypoxia or chronic intermittent hypoxia may provide an advantage by preventing hypoxia-induced chronic low-grade systemic inflammation. Another important inflammatory gene under selection in both Tibetan and Andean groups is TNF (Moore, 2017), a multifunctional pro-inflammatory cytokine. TNF-α levels have also been found to be elevated in individuals traveling acutely to high altitude (Lundeberg et al., 2018). Like IL6, the adaptive TNF variant is unknown but modulation of TNF expression in response to chronic hypoxia may be important for preventing chronic inflammation at high altitude.

In addition to these genetic changes, epigenetic mechanisms also play a critical role in regulating expression of inflammatory genes and likely influence adaptation of inflammatory pathways to chronic hypoxia (Bayarsaihan, 2011). Epigenetic changes alter gene expression without altering the underlying genetic code. These changes include DNA methylation, histone modifications, and microRNA expression. These epigenetic changes play key roles in environmental adaptation during development and exposure in adulthood (Fernández et al., 2014). There are several HIF-dependent and independent mechanisms by which hypoxia significantly impacts epigenetic modifications (Kim and Park, 2020). For example, HIF induces expression of several histone methyltransferases and demethylases (Batie et al., 2019). Additionally, hydroxylation of histone methyl transferases as well as activity of histone demethylases, are oxygen dependent. DNA methylation patterns are also altered by hypoxia, in part due to the impact on ten-eleven translocation (TET) enzyme expression and activity (Thienpont et al., 2016). The HIF consensus binding site also contains a CpG dinucleotide, indicating that expression of HIF-pathway genes may be dependent on DNA methylation (Wenger et al., 2005; Chen et al., 2020; D’Anna et al., 2020). Given that immune cell phenotypes, including inflammatory macrophage phenotypes, can be regulated by epigenetic modifications, hypoxia has the potential to significantly alter immune cell function via its impact on epigenetics (Davis and Gallagher, 2019). These changes may occur within or across generations of exposure. Future work should investigate if high-altitude acclimatized or adapted groups develop particular patterns of DNA methylation or histone modification, which protect against chronic hypoxia-induced inflammation. Over generations, mutations at loci containing CpG sites could also assist in blunting inflammatory gene expression in the face of chronic hypoxic stress.

Nitric Oxide in Native and Acclimatized High-Altitude Groups

Nitric oxide (NO) is a natural vasodilator that plays a crucial role in regulating vasodilation in vascular smooth muscle. Exhaled NO is also used as marker of airway inflammation (Kharitonov and Barnes, 2001; Birrell et al., 2006). The vasodilatory function of NO protects against pulmonary hypertension at high altitude (Feelisch, 2018). Individuals exposed acutely to high altitude tend to show reduced gas phase NO, which typically returns to baseline levels after a couple days then exceeds baseline levels by 5 days. However, gas phase NO has been demonstrated to remain low in HAPE-sensitive individuals, perhaps, indicating that deficits in pulmonary epithelial NO synthesis contribute to exaggerated pulmonary vasoconstriction and edema (Duplain et al., 2000). Tibetan and Bolivian high-altitude adapted populations also show elevated exhaled NO compared to sea-level residents (Beall et al., 2001, 2012; Erzurum et al., 2007). This phenotype correlates with higher pulmonary blood flow and protection from HAPH. However, He et al. (2017) demonstrate that enhanced NO production may not be unique to high-altitude native populations since individuals of Han Chinese ancestry living at high altitude (3,660–3,700 m) demonstrated an even higher average NO metabolite production than Tibetans at the same altitude (He et al., 2017). Another study reports that the native Sherpa population has a lower level of circulating NO in serum and no differences in circulating NO metabolites in comparison to low-altitude samples (Droma et al., 2006; Horscroft et al., 2017). Interestingly, this finding contrasts the high exhaled NO among Tibetans. This discrepancy in NO substrate bioavailability in Tibetans vs. Sherpas also suggests that these two high-altitude populations may have some distinct adaptation in the nitric oxide pathway (Zhang et al., 2017).

Elevated NO may also be an important adaptation of the immune system to chronic hypoxia-induced inflammation. Under normal physiological conditions, NO plays an anti-inflammatory role and inhibits platelet aggregation and rolling, adherence, and transmigration of leukocytes (Grisham et al., 1998; Coleman, 2001). During inflammatory reactions, the production of the inducible form of nitric oxide synthase in many immune cells, including monocytes, macrophages, and neutrophils leads to very large increases in NO by up to 1,000-fold (Cook and Cattell, 1996; Sharma et al., 2007). NO can then become oxidized to reactive nitrogen oxide species which nitrosate thiol groups in glutathione. This inhibits the activity of many proteins including mitochondrial enzymes and transcription factors. As a result, elevated NO production can impair pathogen function (Sethi and Dikshit, 2000; Bogdan, 2001; Coleman, 2001). At high concentrations, NO also stabilizes HIF by inhibiting HIF PHDs (Taylor and Moncada, 2010). Therefore, NO production during periods of inflammation permit increased expression of HIF-pathway genes as a protective mechanism since tissue inflammation often leads to cellular hypoxia. However, Lüneburg et al. (2016) demonstrated in a rat model that while there is an increase in endothelial nitric oxide synthase (eNOS; NOS3) in chronic hypoxia and chronic intermittent hypoxia conditions, NO bioavailability may be impaired due to increases in asymmetric dimethylarginine (ADMA), a competitive nitric oxide synthase inhibitor that regulates NO production, and hypoxia-induced increase in oxidative stress (Lüneburg et al., 2016). ADMA displaces L-arginine, a NOS substrate, and therefore an increase in ADMA concentrations may result in lower NO substrate bioavailability (Brito et al., 2007). Oxidative stress may lead to superoxide radical production, which may mediate NO degradation and attenuate NO bioavailability (Siques et al., 2014).

Since both NOS1 (nitric oxide synthase 1, neuronal) and NOS2 (nitric oxide synthase 2, inducible) are under selection in multiple high-altitude groups, it is clear that adjusting NO production is a key adaptive phenotype for life at high altitude. While the precise adaptive mechanism is unknown, it likely involves protection against pulmonary hypertension and/or modulation of hypoxia-induced inflammation.