Heat stress negatively impacts human health, as well as animal agriculture (Pearce et al., 2013b; Ghulam Mohyuddin et al., 2022; Rosinger, 2023). Heatwaves and heat-stroke can kill humans and animals, and there are still no standard treatments available aside from cooling and hydration therapies (Hutchins et al., 2022; Wood et al., 2022). Combined with reduced feed intake, HS markedly impacts animal gastrointestinal tract integrity. Previous studies have shown that HS reduces intestinal integrity (Pearce et al., 2013a; Vásquez et al., 2022) in pigs, however the mechanisms underlying the cause are still not fully elucidated. Heat stress has been shown to cause intestinal ischemia/hypoxia as blood is partitioned to the periphery to aid in cooling. The result of this redistribution of blood flow is a lack of blood flow to internal organs, including the gastrointestinal tract. Decrements in intestinal integrity under HS have been observed previously by our group when core temperatures reach 41–41.8°C (Pearce et al., 2013a; Pearce et al., 2014; Pearce et al., 2015) for between 6 and 24 hours. Evidence of hypoxia has also been shown in cases of environmental hyperthermia (Pearce et al., 2013a). The gastrointestinal tract requires an extremely balanced gradient of oxygen from the luminal to the mucosal surface, thus the GI tract is one of the most sensitive organs to changes in oxygenation (Konjar et al., 2021; Lian et al., 2021). Heat shock protein expression has been shown to be a key regulator of the hyperthermic response (Hu et al., 2022). Hypoxia and oxidative stress have been separately proven to induce alterations in tight junction complexes, which results in epithelial dysregulation and increased permeability. There is a clear link between environmental hyperthermia and intestinal permeability, although the role of hypoxia is still not fully understood. However, it is known that the main transcription factor involved in the hypoxic response (hypoxia-inducible factor 1, or HIF-1α) can interact with tight junction proteins including claudins (Della Rocca et al., 2022).

Few studies have examined the combined effects of HS and hypoxia in vitro, and these have been done in human cell lines under acute conditions and normal human body temperatures for controls (Lian et al., 2021; Lian et al., 2023). The current knowledge base is still quite new. The IPEC-J2 cell line, which is a non-transformed small intestinal cell line derived from neonatal pigs, has been shown to be a good model to study nutrition (Rhoads et al., 1994; Yan and Ajuwon, 2017), bacterial invasion (Duan et al., 2022), viral infection (Guo et al., 2022), toxins (Li et al., 2022), and environmental factors such as HS (Cao et al., 2016) and hypoxia (Chapman et al., 2012).

Mechanisms underlying barrier dysfunction and the reduced integrity of tight junctional complexes due to HS are not fully understood; however, several pathways have been implicated. It is also known that hypoxia and HS-related transcription factors regulate the intestinal barrier and inflammatory signaling pathway. Therefore, our objective was to use an agriculturally relevant in vitro intestinal epithelial model to examine the effects of hypoxia and high ambient heat (i.e., HS) on barrier integrity, as well as tight junction protein localization and internalization. We hypothesized that hypoxia is a critical factor and important step in the pathway and hypoxia alone would result in tight junction protein remodeling.

To assess changes in IPEC barrier integrity, Transwell™ TEER was analyzed as a measure of paracellular permeability between adjacent epithelial cells. It was measured as the change from the baseline of the control (TN NX), which was set to 100%, with a higher number indicating better barrier integrity and lower permeability. Unexpectedly, HS alone (HS NX) increased TEER by more than 100% over 24 hours, whereas hypoxia alone (TN HX) and HS combined with hypoxia (HS HX) reduced TEER by approximately 50% (p<0.05; Figure 1A ). Substantial changes in TEER were observed starting after approximately 12 hours of treatment. These results indicate that hypoxia was required to reduce TEER. FITC-dextran, another marker for paracellular permeability was increased by 133% in the TN HX treatment compared with TN NX control and HS NX treatment (p<0.05; Figure 1B ). FITC-dextran was highest in the HS HX group, increasing by more than 500% compared with the TN NX controls and by nearly 200% compared with the TN HX treatment (p<0.05; Figure 1B ). Similar to the findings concerning TEER, hypoxia was required to increase permeability using the FITC-dextran marker. Interestingly, one of the major mediators of the intestinal inflammatory response, basolateral chemokine IL-8, was reduced by more than 70% in both HS treatments (p<0.05; Figure 1C ). There was a tendency for IL-8 to be increased due to hypoxia alone (p<0.10; Figure 1C ). These results indicate a dampened IL-8 response to HS.

The immunofluorescence distribution and structure of CLDN4 were significantly affected by the heat stress and hypoxia treatments. This included areas of redistribution of CLDN4 (white arrows; Figure 2A ). CLDN4 appeared to redistribute from the membrane to the cytosol and formed cluster-like structures. In addition, total relative fluorescence (CTCF) was only significantly increased by 34% in the HS HX group relative to the CON group (p<0.05; Figure 2B ). Although not different from the CON group, the TN HX group had significantly less CLDN4 than the HS NX group (30% decrease; p<0.05). The distribution and structure of the tight junction protein ZO-1 was largely unchanged due to treatment; however, there were some areas in which ZO-1 appears to cluster like CLDN4 (white arrows; Figure 2A ). However, the total amount of ZO-1 was significantly altered. Compared with the CON group, TN HX-treated cells experienced a 31% decrease in ZO-1 fluorescence (p<0.05; Figure 2B ). ZO-1 in both HS groups was significantly increased by nearly 20% compared with the CON group (p<0.05; Figure 2B ). EEA1 was largely not present in the CON group but was present in significant amounts in the HS HX treatment ( Figure 2A ). However, total fluorescence was increased in both HS-treated groups compared with the CON and TN HX groups (p<0.05; Figure 2B ). Total fluorescence of DAPI was not different between treatments (p>0.05; data not shown). EEA1 demonstrated high co-localization with CLDN4 with a Pearson’s r-value of 0.74, which was significant (p < 0.05; Supplemental Figure 1 ).

Protein expression of two major heat shock proteins and cell chaperones, HSP70 and HSP90, was measured in cell lysate to confirm heat shock response in treated cells. HSP70 levels decreased by 40% (p<0.05; Figure 3A ) due to hypoxia treatment but, as expected, increased by more than 150% due to heat exposure. HSP90 levels were not decreased due to hypoxia treatment alone (p>0.05; Figure 3B ) but increased by more than 200% due to heat exposure. Representative blot images are shown in Figure 3C .

Changes in intestinal integrity can occur due to alterations in the amount and localization of tight junction protein at the plasma membrane of intestinal epithelial cells. The reduction of membrane-associated tight junction proteins and the consequential internalization of these proteins into the early endosome may result in the decreased polarization and integrity of the epithelial barrier. However, few studies have assessed this under heat stress or hypoxia conditions in pig models. Previous studies have shown that HS reduces intestinal integrity (Pearce et al., 2013a); however, it is largely unknown if the ambient heat or the associated intestinal hypoxia results in tight junction protein remodeling. Heat stress causes intestinal ischemia/hypoxia as blood is partitioned to the periphery, and there has been evidence of hypoxia in cases of environmental hyperthermia (Pearce et al., 2013a). Thus, in this agriculturally relevant in vitro model, our data suggest that hypoxia may be a driving factor in HS-induced barrier dysfunction. The IPEC-J2 cell line used herein is a non-transformed small intestinal cell line derived from neonatal pigs and has been shown to be a good model for studying nutrition (Rhoads et al., 1994; Yan and Ajuwon, 2017), bacterial invasion (Duan et al., 2022), viral infection (Guo et al., 2022), toxins (Li et al., 2022), and environmental factors such as HS (Cao et al., 2016) and hypoxia (Chapman et al., 2012). Under standard cell culture conditions, cells are typically grown at 37°C. However, pigs’ normal body temperature ranges from approximately 38.5°C to 39.7°C, which is higher than that of humans (Morales et al., 2016). Therefore, we propagated the IPEC at 38°C for our thermal neutral condition to mimic the source tissue. Cell viability and polarization was not affected by this warmer thermoneutral culturing condition (data not shown).

Under in vitro conditions, HS has been shown to upregulate the tight junction protein zonula-occluden-1 (ZO-1), as well as inflammatory genes IL-6, ICAM-1, and TGF-β; however, functional integrity such as macromolecular transport was not measured (Cao et al., 2016). Separately, Chapman et al. (2012) reported that hypoxia treatment of IPEC-J2 cells caused a large reduction in TEER, a functional measure of monolayer barrier function, after 12 hours. At the same time, inulin flux (a marker of paracellular macromolecular flux) was greatly increased by hypoxia treatment alone. These hypoxia functional data were also accompanied by changes in the tight junction protein distribution of ZO-1 (Chapman et al., 2012). In the current study, we reported large reductions in IPEC-J2 intestinal barrier function, as observed by TEER and FITC-dextran transport, in a short period of time in both the hypoxia alone and HS HX treatments. Intriguingly, HS alone increased TEER in IPEC-J2 cells, and this result was similar across multiple time points and experimental replications. Other cell culture models have been utilized to study the effects of HS, including IEC-6 (He et al., 2016), SW480 (Yi et al., 2017), Caco2 (Dokladny et al., 2008), and HT-29 (Lian et al., 2021). Of these models, all have reported decreases in intestinal integrity due to elevated culturing temperature conditions, which is in contrast to our current model. The discrepancies in barrier TEER under elevated culturing temperatures could also be attributed to our TN controls being cultured at a starting temperature 1–2°C higher than the standard to mimic normal porcine physiology. It is possible that this may have caused some adaptation or acclimation to heat in the cells; however, a robust HSP response was still observed.

The epithelial barrier between cells is created by a series of junctional proteins, including tight junctions (TJs), gap junctions, adherens junctions, and desmosomes. Epithelial TJs regulate the polarity and movement of molecules between adjacent cells. Claudins are a family of transmembrane TJ proteins that can be classified as either “tight” or “leaky” based on what they allow through. Claudin 4 is considered a tight claudin and is involved in regulating barrier function. Zonula occludens are another type of TJ protein that are cytoplasmic. ZO-1 helps to stabilize claudin strands and the actin cytoskeleton (Garcia-Hernandez et al., 2017).

Polarized epithelial cells are capable of rapidly remodeling and reshaping paracellular pore spaces through diffusion, endocytosis, and the internalization of tight junction proteins (Stamatovic et al., 2017). This internalization of tight junction proteins is interconnected to membrane-bound endosome compartments. Protein expression via immunofluorescence of tight junction proteins also appeared to redistribute to the cytosol from the membrane, especially CLDN4. This occurred under all treatments; however, the most noticeable changes were in the HS HX cells where large, internalized clusters of CLDN4 were found. This contrasts with the controls, which exhibited the typical honeycomb structure of tight junctions. Interestingly, total fluorescence did not correlate entirely with functional data or redistribution observations. Specifically, total fluorescence was decreased under hypoxia alone but increased under HS alone and HS HX, although functional data were similar between the hypoxia and HS HX treatments. This could be due to several factors, including the methods utilized, timing of protein translation and function, and proteins analyzed, as functional data is representative of the entire junctional complex. CLDN4 also appeared to also co-localize with EEA1. Endocytosis is a process by which substances are taken in by a cell, and during this process vesicles are formed containing the ingested material (Nighot and Ma, 2021). Early endosome antigen 1 (EEA1) localizes specifically to early endosomes; it is involved in endosomal trafficking and has been described as a central sorting station (Barone and Zimmer, 2016). Endocytosis is important for the regulation of tight junction complexes and allows cells to adapt to various stressors. Specifically, it has emerged as an important process for tight junction remodeling and endocytic removal (Li and Ajuwon, 2021; Nighot and Ma, 2021). EEA1 has previously been shown to co-localize with TJ proteins (Matsuda et al., 2004; Li et al., 2022). Although the HS HX treatment had the highest TEER, it also had increased TJ protein fluorescence and higher remodeling. This could be unrelated to endosomal recycling or may be due to effects on other junctional complexes that were not measured within the scope of the current study.

The changes in the amount of tight junction proteins of heat shock proteins. The levels of heat shock proteins, such as HSP70, are increased during HS, and they act as molecular chaperones and protect protein function (Alfieri et al., 2004; Chen et al., 2009). In pig intestinal tissues, we have reported that HS increases levels of SP70, in addition to levels of HIF1α (Pearce et al., 2013a). However, hypoxia alone may not increase the amount of heat shock proteins. Heat shock proteins act as molecular chaperones that assist in protein folding and are regulated by heat shock factors (HSFs). Heat shock protein 70 is one of the most inducible HSPs and is a chaperone for nascent polypeptide chains (Wang et al., 2018). It is also thought that HSPs act as intestinal gatekeepers (Liu et al., 2014). Following HS, intestinal epithelial cells produce heat shock proteins as a protective mechanism (Malago et al., 2002). HSP90 has been shown to be involved in several survival signaling pathways, including those involved in antioxidant response and apoptosis (Zhang et al., 2020). As expected, levels of HSP70 and HSP90 were increased in both HS-treated groups, indicating that both groups experienced HS. There was no difference between the normoxia- and hypoxia-treated groups; therefore hypoxia did not alter the HSP response. The total fluorescence of TJ proteins was increased in treatment groups where HSP activation was also observed.

Interleukin 8 (IL-8/CXCL-8) is one of the most abundant inflammatory cytokines/chemokines produced by the intestinal epithelium and epithelial cells. Its main functions are to induce chemotaxis in neutrophils, induce phagocytosis, and protect cells against damage (Maheshwari et al., 2002). Interestingly, our results show a decrease in IL-8 protein secretion in HS-treated cells, which would mimic systemic IL-8 concentrations in the blood. This is counter-intuitive to a potential inflammatory response to environmental hyperthermia; however, this phenomenon has also been observed in vivo (Pearce et al., 2013a; Varasteh et al., 2015) and may be a species-specific or timing-specific response. In our in vitro model, there is no immune cell component, thus the IL-8 response must be due solely to intestinal epithelial cell production. Previous research has shown that the induction of HSPs may inhibit IL-8 production or secretion. Interestingly, heat shock has been reported to co-activate interleukin-8 gene transcription (Singh et al., 2008; Chung et al., 2009; Zhong et al., 2012).

We believe hypoxia is a driving factor in barrier dysfunction, as HS alone in our model was not enough to disrupt the intestinal barrier at a functional level but was able to cause changes at a molecular level. This may be partially due to an increase in HSP70, which is a molecular chaperone and protectant during HS that is not observed in hypoxia. It may also be due to a slightly higher starting temperature to mimic normal porcine body temperatures. There are likely other factors or proteins involved in this process that were not measured in the current study. In conclusion, this short research communication provides new insights into how HS and hypoxia modulate intestinal barrier integrity. These data show for the first time in IPEC-J2 cells that hypoxia appears to cause a large majority of the epithelial damage and dysfunction during a bout of high ambient heat, including increased intestinal permeability, tight junction remodeling, and early endosome disruption. Interestingly, these changes are in the absence of an IL-8 response. Future studies should elucidate mechanisms and provide potential nutritional mitigation strategies to prevent HS and hypoxia-induced intestinal dysfunction.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

All authors contributed to the design of the study, editing the manuscript, and the interpretation of results. SP conducted the experiments, collected the samples and wrote the manuscript. All authors contributed to the article and approved the submitted version.