The gastrointestinal (GI) tract is lined with an epithelial barrier that functions to selectively allow passage of small molecules into circulation while impeding the translocation of luminal gut microbiota and other antigens into the host circulatory system. Along this barrier, villi in the small intestine as well as crypts in both the small and large intestine maintain several epithelial cell subtypes with functions including nutrient absorption, along with hormone, anti-microbial peptide, and mucus secretion (Panwar et al., 2021). Junctional complexes positioned between adjacent epithelial cells contain tight junctions, adherens junctions, gap junctions and desmosomes (Brooke et al., 2012). These complexes along with the tight junction proteins regulate passage of molecules through paracellular pathways (France and Turner, 2017) and complement the functions of transporters and other mechanisms regulating transcellular transport of compounds from the intestinal lumen.

A healthy GI tract exists under a state of “physiologic hypoxia” characterized by a steep oxygen gradient from the submucosa to the lumen, with luminal oxygen concentrations far below the atmospheric oxygen concentration of ∼21% O₂. (Zheng et al., 2015). This physiologic hypoxia activates transcription factors, known as hypoxia-inducible factors, which regulate pathways involved in maintaining GI epithelial barrier function by modulating tight junction function, inflammatory tone, secretion of mucin and anti-microbial compounds, and activity of epithelial nutrient transporters (Pral et al., 2021). Physiologic hypoxia at the level of the GI tract may be perturbed by acute and chronic stressors that reduce oxygen availability systemically such as high terrestrial altitude (Karl et al., 2018), intense exercise (Hill et al., 2020), and heat stress (Pearce et al., 2013) among others. Inflammation, oxidative stress, metabolic dysfunction, apoptosis and GI barrier damage can result, thereby, initiating a cycle in which translocation of antigens from the gut lumen induce immune and inflammatory responses that exacerbate GI barrier damage. This cycle is thought to contribute to decrements in physical and cognitive performance during acute hypoxic exposures (Ando et al., 2020), and development of disease under conditions of chronic intermittent hypoxia (Behrendt et al., 2022). As such, there is interest in identifying strategies that promote GI barrier resilience to the damaging effects of hypoxia.

Increasing short-chain fatty acid (SCFA) production by the gut microbiota may be one feasible strategy. SCFA are derived from fermentation of undigested carbohydrate by the gut microbiota. Acetate, propionate, and butyrate are the predominant SCFA within the lumen and are commonly found at relative proportions approximating 60:20:20 (acetate/propionate/butyrate) in concentrations that range from 10–40 mM in the small intestine to 50–150 mM in the colon (Schmitt et al., 1977; Cummings et al., 1987). Butyrate, and acetate to a lesser extent, provide an energy source for enterocytes. This promotes epithelial O₂ consumption thereby stabilizing HIF-1α and promoting GI barrier integrity (Pral et al., 2021). Through this and other pathways, SCFA’s regulate intestinal immune function, cellular differentiation, barrier integrity, apoptosis, hormone secretion and cell metabolism (Pearce et al., 2020; Pral et al., 2021).

Favorable effects of SCFA on GI barrier function likely vary according to both the size and composition of the SCFA pool. In support, moderate SCFA concentrations of 40–80 mM ratio have shown positive effects on intestinal barrier function as measured by transepithelial electrical resistance (TEER) in vitro, relative to lower (20 mM) or higher (100–200 mM) concentrations (Chen et al., 2017). In addition, when multiple ratios of acetate, propionate, and butyrate at 40 mM were examined, the largest increase in TEER was found with high butyrate concentrations reaching 50% of the SCFA pool (Chen et al., 2017). That observation may be attributable to butyrate being the preferred energetic substrate for enterocytes. Indeed, butyrate has been shown to enhance epithelial barrier function in several models (Chen et al., 2017; Pearce et al., 2020; Huang et al., 2022) as well as favorably modulate inflammatory pathways within the intestinal epithelium (Yang et al., 2020).

Although SCFA favorably modulate GI barrier function under normal physiologic conditions, whether the same is true when physiologic hypoxia is disrupted by environmental and physiologic stressors that reduce systemic oxygen availability is unclear. Therefore, the objective of this study was to examine the effects of SCFA on changes in GI barrier function, cytokine production and gene expression during hypoxic stress. Two different compositions of SCFA, one reflecting the normal in vivo proportions with a higher ratio of acetate, propionate, and butyrate and the other reflecting a high butyrate ratio were studied. As studying human GI barrier function in vivo is limited by the inaccessibility of the human GI tract, we utilized an intestinal enteroid model that provides a species-specific and physiologically relevant approach to studying mechanisms influencing GI barrier function under controlled environmental conditions and interventions (Taelman et al., 2022). While enteroid models have increasingly been utilized to study the effects of SCFA’s and in vitro fermentation-derived microbial metabolites on the intestinal barrier (Schilderink et al., 2016; Pearce et al., 2020; Pace et al., 2021; Pearce et al., 2022), to our knowledge, these models have not been used to determine the effects of SCFA on intestinal barrier responses to hypoxic stress. We hypothesized that SCFA would improve GI barrier function, reduce the production of pro-inflammatory cytokines, and increase the expression of genes regulating GI barrier function in enteroids exposed to hypoxia and that increasing the proportion of butyrate in the SCFA pool would enhance these benefits.

SCFA’s favorably modulate GI barrier function under normal physiologic conditions, but whether the same is true when challenged by environmental and physiologic stressors that reduce systemic oxygen availability is unclear. Therefore, this study examined whether SCFA’s could provide protection against environmental hypoxia-induced epithelial damage in an in vitro model. One caveat of this study is that duodenal enteroids were used. Though these cells are more sensitive to oxygen changes and have significantly less exposure to SCFA than colonocytes, enterocytes within the ileum are also exposed to microbiota derived SCFA (Parada Venegas et al., 2019a). An additional caveat is that we used standard oxygen concentrations for controls and hypoxia treatments. We recognize that as research continues to advance, studies are moving towards more physiologically relevant conditions (Skovdahl et al., 2021) and models (Kasendra et al., 2018). Herein, we demonstrate that hypoxia exposure decreased enteroid epithelial barrier integrity, after 48 h. Notably, treatment with a high butyrate but not high acetate SCFA mixture concurrent to hypoxic-stress and for 24 h prior to hypoxic-stress attenuated, but did not prevent, hypoxia-induced epithelial barrier dysfunction. Additionally, pre-treatment with SCFA mixtures proportionally high in either butyrate or acetate for 48 h prior to hypoxic-stress transiently enhanced epithelial barrier integrity during hypoxic stress before the benefit was subsequently lost. Collectively observations: 1) confirm that SCFA enhance epithelial barrier integrity in vitro, 2) demonstrate that hypoxia can damage epithelial barrier integrity in vitro, 3) suggest that increasing SCFA production prior to hypoxic stress could transiently strengthen epithelial barrier integrity during hypoxic-stress, and 4) indicate that SCFA-derived benefits on epithelial barrier function during hypoxia might be improved by increasing the proportion of butyrate within the SCFA pool. Whether maintaining SCFA treatments prior to and throughout hypoxic exposures can extend protection epithelial barrier protection beyond the initial 48 h of hypoxic stress should be addressed in future research.

SCFA concentrations (Parada Venegas et al., 2019b) are lower in the small intestine than the large intestine, and the small intestine may be more sensitive to changes in oxygen due to higher basal oxygen concentrations (Konjar et al., 2021). The SCFA butyrate is a primary energy source of intestinal epithelial cells and is utilized via β-oxidation which accounts for ∼75% of oxygen consumption in colonocytes (Roediger, 1980). This process also helps maintain an anaerobic oxygen gradient of <1% in the lumen. After transport into the cells, butyrate enhances oxidative phosphorylation, which consumes oxygen (Kelly et al., 2015). Butyrate has been shown to enhance epithelial barrier function in several models (Chen et al., 2017; Pearce et al., 2020; Huang et al., 2022) as well as modulate inflammatory pathways (Yang et al., 2020).

Results confirmed that SCFA enhance intestinal barrier function in vitro under normoxic conditions, and this is similar to previous research in our laboratory using single SCFA (Pearce et al., 2020). Further, pre-treatment of enteroids with SCFA for 48 h increased TEER dramatically and enabled cells to maintain a high TEER until 72 h of hypoxia treatment, potentially providing some level of protection during hypoxic exposure. However, SCFA, whether provided at physiologic ratios or an enhanced butyrate ratio, and whether provided concurrent to or as a pre-treatment, did not prevent hypoxia-mediated decreases in GI barrier function after 72 h of exposure. It is, however, worth noting that barrier function was maintained up through 48 h of hypoxic exposure, which was especially apparent in the 48 h pre-treatment groups. In contrast, concurrent treatment with SCFA’s during hypoxia does not appear to be long enough to prime an epithelial barrier response as TEER remains stable and then eventually decreases, as opposed to increasing initially.

Protein concentrations and gene expression were measured to identify mechanisms through which SCFA may affect epithelial barrier function during hypoxic stress. Unfortunately, these measurements were taken after 72 h exposure to hypoxia, a time point at which TEER demonstrated few, if any, differences amongst the hypoxia conditions. It is therefore unsurprising that major differences in protein and gene expression were not observed.

One exception was IL-8. Notably, using the same enteroid model, our group has also previously demonstrated an increase in IL-8 due to single SCFA exposure (Pearce et al., 2020) and due to a milleu of fecal metabolites including SCFA (Pearce et al., 2022). In the current study, a significant increase in IL-8 was also observed at the protein level, in addition to a large numeric difference at the gene level with both high butyrate and high acetate treatments. Increased IL-8 has been associated with decreased TEER in previous studies (Ko et al., 2007) as it is an epithelial-produced chemokine which recruits immune cells to the subepithelial area and plays a role in bacterial translocation across the epithelium (Sansonetti et al., 1999). In the current study, we show that IL-8 protein is increased only in treatment groups with the high butyrate ratio under concurrent and 24-h pretreatment. However, with 48 h pre-treatment this trend reverses and IL-8 is increased due to SCFA-treatment alone, regardless of hypoxia. Thus, the current mechanism is unclear. That gene expression patterns differed from protein concentrations may be an effect of timing of sample collection.

Hypoxia and SCFA mixtures also strongly inhibited secretion of the anti-inflammatory cytokine IL-10. This may provide insight into the lack of protection against barrier dysfunction in the hypoxic models. IL-10 plays an important role as a positive regulator of the NFκB pathway and intestinal homeostasis (Papoutsopoulou et al., 2021). In addition, loss of the IL-10 receptor disrupts intestinal cell fate and differentiation (Jenkins et al., 2021).

Hypoxia alone appeared to generally reduce cytokine secretion. SCFA did not appear to affect that reduction except for IL-8 and IL-17, which tended to increase with SCFA treatment and hypoxia in the concurrent and 24 h treatments. SCFA are known to act on immune cells including production of multiple cytokines including TNF-α, IL-6, and IL-10 as well as certain chemokines (Vinolo et al., 2011). In this study BUT appears to be initiating a more pro-inflammatory phenotype without decreasing TEER which could be due to a compensatory response. Research in endothelial cells has shown that hypoxia induces TNF-α which can then in turn upregulate the HIF pathway which may also be a survival mechanism (Jin et al., 2019).

Gene expression results show general downregulated expression patterns for most genes under hypoxic conditions alone, as well as under concurrent treatment and 24 h pre-treatment. Interestingly, several genes were upregulated with 48 h SCFA pre-treatment, especially in the higher butyrate ratio treatment. Upregulated genes were largely related to metabolism. For example, a transporter (SLC5A8) which is known to transport butyrate (Dang et al., 2021) was upregulated in the 48 h BUT pre-treatment groups. SCFA responsive free fatty acid receptor FFAR2 (Pan et al., 2018) was also increased in the same group along with enteroendocrine cell markers CHGA and PYY, and enterocyte enzyme alkaline phosphatase which have all been previously shown to be responsive to butyrate (Pearce et al., 2020). These are likely upregulated due to the 48 h pre-treatment with the high butyrate ratio. It is possible that because only one time point at 72 h was measured that the upregulation of additional genes at earlier time points may have been missed.

Collectively, these observations suggest that a high butyrate ratio may be more effective than a high acetate ratio for attenuating hypoxia induced intestinal barrier dysfunction, but that any benefits are short-lived. Overall, pre-treatment or concurrent treatment with SCFA mixtures were not able to overcome the negative impacts of hypoxia on intestinal function and cells ultimately could not be sustained under hypoxia for 72 h. Possibly, hypoxia may interfere with beneficial effects of SCFA on intestinal barrier function. As mentioned previously this study has several limitations including the type of enteroids used (duodenal vs. colonic) as well as time points collected. Additional studies are needed to elucidate mechanisms and determine whether SCFA may attenuate hypoxia-induced intestinal barrier dysfunction in in vivo models.