Acute exposure to simulated high-altitude hypoxia alters gut microbiota in mice

Gut microbiota bears adaptive potential to different environments, but little is known regarding its responses to acute high-altitude exposure. This study aimed to evaluate the microbial changes after acute exposure to simulated high-altitude hypoxia. C57BL/6 J mice were divided into hypoxia and normoxia groups. The hypoxia group was exposed to a simulated altitude of 5500 m for 24 h above sea level. The normoxia group was maintained in low altitude of 10 m above sea level. Colonic microbiota was analyzed using 16S rRNA V4 gene sequencing. Compared with the normoxia group, Shannon, Simpson and Akkermansia were significantly increased, while Firmicutes-to-Bacteroidetes ratio and Bifidobacterium were significantly decreased in the hypoxia group. The hypoxia group exhibited lower mobile element containing and higher potentially pathogenic and stress-tolerant phenotypes than those in the normoxia group. Functional analysis indicated that environmental information processing was significantly lower, metabolism, cellular processes and organismal systems were significantly higher in the hypoxia group than those in the normoxia group. In conclusion, acute exposure to simulated high-altitude hypoxia alters gut microbiota diversity and composition, which may provide a potential target to alleviate acute high-altitude diseases.


Introduction
High altitude has substantial meaning not only to the abundant resource, but to the military significance. Hypoxia is one of the main characteristics of high altitude. With increased elevation above sea level, air pressure as well as oxygen pressure decreases. At an altitude of 4,000 m, oxygen content was only ≈ 60% of sea level (Beall 2007).
The oxygen homeostasis is vital for maintaining gastrointestinal health. With altitude increasing, morphologic injuries of villous height, crypt depth, mucosal wall thickness and villous surface area were aggravated (Zhang et al. 2015). Acute hypoxia environment increased bacterial translocation and decreased protein expression of occluding, facilitating the entry of lipopolysaccharide into the blood (Luo et al. 2017). Recent studies highlight that gut microbiota bears adaptive potential to high altitude in human and animals. In a survey of gut microbiota of Tibetans from six regions with altitudes ranging from 2800 to 4500 m, Lan et al. (2017) observed that altitude had a positive correlation to Faecalibacterium, Bacteroides and Bifidobacterium, but negative correlation to Ruminococcaceae, Prevotella and Lachnospiraseae. Comparing the microbiome of Han population living in Chengdu (500 m) and the immigrant Han population living in Lhasa (3600 m) revealed that the latter had a more energy efficient flora (Li et al. 2016). In wild house mice, Suzuki et al (2019) found that anaerobic bacteria were positively correlated with altitude, while facultative anaerobes, microaerophiles and aerotolerant bacteria were negatively correlated with altitude. Chinese rhesus macaques living in Tibet had higher environmental information processing and organismal systems than those in the other geographical populations (Zhao et al. 2018 Therefore, the aims of this study were to evaluate the microbial responses to acute high-altitude hypoxia exposure, and provide basis for future efforts to develop microbiotabased countermeasures that alleviate acute high-altitude diseases.

Animals and experimental design
Twelve-week-old male C57BL/6 J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). With 1 week of acclimatization, mice were randomly divided into 2 groups (ten mice per group): hypoxia group and normoxia group. For the hypoxia group, mice were exposed to a simulated altitude of 5500 m for 24 h above sea level in a hypobaric chamber (Yantai Hongyuan Oxygen Industrial Inc, China). For the normoxia group, mice were maintained in low altitude of 10 m above sea level. Both groups of mice were kept under stable conditions with controlled humidity (40%-60%), a temperature range of 22 °C ± 2 °C, and a 12 h light/dark cycle. AIN-93 M diet and water were given ad libitum. Colonic contents were collected after scarification. The experimental protocols were approved by the Institutional Animal Care and Use Committee of Tianjin Institute of Environmental and Operational Medicine.

Microbiome analysis
Genomic DNA from the colonic contents was extracted using cetyltrimethyl ammonium bromide method. The V4 region of 16S rRNA gene was amplified using primers 515 F and 806R. Sequencing libraries were generated using Ion Plus Fragment Library Kit 48 rxns (Thermofisher, USA). The library was sequenced by Ion S5 TM XL (Thermofisher, USA). Operational taxonomic units that reached 97% similarity were used for alpha diversity estimation. Cluster analysis was preceded by principal component analysis. Linear discriminant analysis effect size was performed to identify specific bacteria. BugBase was used to infer microbiological phenotype. Phylogenetic investigation of communities by reconstruction of unobserved states was used to predict functional change.

Statistical analysis
Data are represented as mean ± standard deviation. Differences between groups were evaluated by Student's t-test (normally distributed) and Mann-Whitney U test (non-normally distributed). Statistical analysis was conducted using PASW statistics 18.0 (SPSS Inc, USA). Significance was defined as P < 0.05.

Body weight, food and water intake, behavioral performance
Body weight, food and water intake were significantly decreased in the hypoxia group than those in the normoxia group ( Fig. 1). Physical activities, such as standing and grooming, were reduced gradually as the hypoxic time extended in the hypoxia group relative to the normoxia group.

Microbiota diversity
Shannon and Simpson were significantly higher in the hypoxia group than those in the normoxia group (Fig. 2). No significant difference was observed in chao1 and abundance-based coverage estimator between the two groups. In the principal component analysis plot, a separation between the hypoxia group and the normoxia group was observed

Microbiota composition
At phyla level, Firmicutes (42.8% in the hypoxia group versus 66.0% in the normoxia group) and Bacteroidetes (42.8% in the hypoxia group versus 13.3% in the normoxia group) were the two most abundant bacterial phyla in both groups (Fig. 4A), Firmicutes-to-Bacteroidetes ratio was significantly lower in the hypoxia group than that in the normoxia group (Fig. 4B). At genus level, Dubosiella, Faecalibaculum and Bifidobacterium were significantly decreased and unidentified_Lachnospiraceae, Akkermansia, Parabacteroides and Bacteroides were significantly increased in the hypoxia group relative to the normoxia group (Fig. 4C).

Microbiota phenotype
Mobile element containing was significantly decreased, whereas potentially pathogenic and stress-tolerant were significantly increased in the hypoxia group relative to the normoxia group (Fig. 5). No significant difference was observed in aerobic, anaerobic and facultatively anaerobic between the two groups.

Discussion
Nowadays, with a growing number of people from low altitude come to high altitude for traveling, mountaineering or strategic reasons, acute high-altitude reaction has become particularly prominent. Although much attention has been  Effect of hypoxia on microbiota phenotype in mice. Data are expressed as mean ± standard deviation. * P < 0.05 versus normoxia. N, normoxia; H, hypoxia paid to respiratory, cardiac and neurological symptoms in acute high-altitude exposure, there is scarcity of reports on gut microflora. In this study, acute exposure to simulated high-altitude hypoxia altered gut microbiota in mice, as indicated by the increase of Shannon, Simpson and Akkermansia, and the decrease of Firmicutes-to-Bacteroidetes ratio and Bifidobacterium.
Among the numerous studies describing disease-associated microbiota, loss of microbiota diversity is a general feature of most dysbiosis. The increase in community diversity, such as Shannon and Simpson, in mice under acute hypoxia exposure observed in our study could reflect a beneficial response to environmental stress. This finding is consistent with individuals who experienced acute mountain sickness (Karl et al. 2018). Moreover, Jiang et al. (2019) reported an elevated microbiome alpha diversity in mice during spaceflight. However, attention should be given to the new perspective that more diversity is not always better (Reese and Dunn 2018). Indeed, alpha diversity estimation revealed higher microbiota diversity in patients with colorectal adenomas (Lu et al. 2016). For men who were HIV-infected, the alpha diversity of the Bacteroidetes phylum was positively correlated with viral load (Nowak et al. 2017). Therefore, the real drivers of microbiome biodiversity in host are worth investigating.
More than 75% gut bacteria are in 1 of 2 phyla: Firmicutes and Bacteroidetes. On the one hand, Firmicutes-to-Bacteroidetes ratio can influence cardiorespiratory fitness. In healthy young adults, Durk et al. (2019) found that maximal oxygen consumption was positively associated with Firmicutes-to-Bacteroidetes ratio. Voluntary exercise increased proportionally to the ΔCt ratio of Firmicutes:Bacteroidetes (Evans et al. 2014). On the other hand, relatively high ratio of Firmicutes to Bacteroidetes is associated with highly efficient energy harvest (Turnbaugh et al. 2006). In youth, Firmicutes-to-Bacteroidetes ratio was positively associated with body mass index, visceral and hepatic fat (Goffredo et al. 2016). Both Tibetans and Chinese Han living at high altitude had a high abundance of Firmicutes and a low abundance of Bacteroidete (Li and Zhao 2015). Similarly, animals living in high altitude, such as Tibetan antelope (Ma et al. 2019), European mouflon and blue sheep , had higher Firmicutes-to-Bacteroidetes ratio than their counterparts living in low altitude. Unlike these residents and animals at high altitude, our results showed a decrease in Firmicutes and an increase in Bacteroidetes after acute hypoxic exposure. This contradiction indicated that Firmicutes-to-Bacteroidetes ratio may be involved in the process of long-term high-altitude adaptation.
At genus level, the lower abundance of Firmicutes was mainly caused by a significant decrease in Dubosiella and Faecalibaculum. The higher abundance of Bacteroidetes was mainly caused by a significant increase in Bacteroides and Parabacteroides. A recent study showed that high-fat diet enlarged Dubosiella and Faecalibaculum ). In addition, Bacteroides and Parabacteroides were negatively associated with obesity (Gong et al. 2019). Our functional analysis also indicated that acute exposure to simulated high-altitude hypoxia upregulated metabolism. In fact, basal metabolic rate was 27% greater than at sea level in men at high altitude (Butterfield et al. 1992). Kong et al. (2019) indicated that the acute-phase response signaling, liver X receptor/retinoid X receptor and farnesoid X receptor/retinoid X receptor pathways were activated in Holstein dairy cows exposed to high-altitude hypoxia.
Bifidobacterium and Akkermansia are considered to be beneficial to the host. In seven mountaineers who took part in German expedition to the Nepalese Himalayas, Kleessen  2005) observed a significant decrease in Bifidobacterium at the high camp. Furthermore, Bifidobacteria deficiency has been identified as a disorder of the ecological barrier after flights in astronauts (Lizko 1991). Consist with these studies, our result also showed a decrease in Bifidobacterium. Unexpectedly, Akkermansia was increased in the hypoxia group. It may be a protective reaction against acute hypoxia exposure. Akkermansia is known to play a vital role in the regulation of energy homeostasis. Gao et al. (2018) found that A. muciniphila treatment promoted the browning of inguinal fat pad, reduced energy efficiency and improved metabolic disorders in the high-fat diet-fed mice. Besides, multiple sclerosis patients also had a higher Akkermansia, and transfer from their fecal microbiota ameliorated disease in recipients by expanding Akkermansia . Likewise, A. muciniphila was significantly increased in IFNγ-deficient mice and restoration of IFNγ level decreased A. muciniphila (Greer et al. 2016).
Intriguingly, acute exposure to simulated high-altitude hypoxia did not result in phenotypic variation in oxygen utilizing, including aerobic, anaerobic and facultatively anaerobic. On the contrary, it was found that the strict anaerobes and obligate anaerobes were increased in large intestine ) and small intestine  under simulated hypobaric hypoxia for 30 days. The difference of exposure time may lead to such discrepancy. In addition, the decrease of mobile element containing and the increase of potentially pathogenic and stress-tolerant shed light on the harmful effects of acute hypoxia. It is consistent with the results obtained from functional analysis, including decreased environmental information processing and increased cellular processes and organismal systems. The underlying mechanisms include promotion of glycolytic capacity and suppression of oxidative metabolism (Murray et al. 2018). Future studies are needed to confirm the phenotypic and functional prediction spectrum of the flora. Moreover, exact details of physiological adaptability in the high-altitude environment remain to be resolved.
A limitation of the study is that only one option for exposure time and altitude was administrated, so the timedependent and altitude-dependent intestinal flora changes have yet to be described. In addition, besides hypoxia, other high-altitude environment variables, such as cold, wind and ultraviolet radiation, were not fully considered. It is an important developing direction to investigate how the gut microbiota respond to high-altitude environment.

Conclusion
In conclusion, acute exposure to simulated high-altitude hypoxia alters gut microbiota diversity and composition. Our findings provide a potential microbiota-based target to alleviate acute high-altitude diseases.