Necrotizing enterocolitis (NEC) is a gastrointestinal disease of complex etiology resulting in devastating systemic inflammation and often death in premature newborns. We previously demonstrated that formula feeding inhibits ileal expression of heat shock protein-70 (Hsp70), a critical stress protein within the intestine. Barrier function for the premature intestine is critical. We sought to determine whether reduced Hsp70 protein expression increases neonatal intestinal permeability.
Young adult mouse colon cells (YAMC) were utilized to evaluate barrier function as well as intestine from Hsp70−/− pups (KO). Sections of intestine were analyzed by Western blot, immunohistochemistry, and real time PCR. YAMC cells were sub-lethally heated or treated with expressed milk (EM) to induce Hsp70.
Immunostaining demonstrates co-localized Hsp70 and tight junction protein zona occludens-1 (ZO-1), suggesting physical interaction to protect tight junction function. The permeability of YAMC monolayers increases following oxidant injury and is partially blocked by Hsp70 induction either by prior heat stress or EM. RT-PCR analysis demonstrated that the Hsp70 isoforms, 70.1 and 70.3, predominate in WT pup; however, Hsp70.2 predominates in the KO pups. While Hsp70 is present in WT milk, it is not present in KO EM. Hsp70 associates with ZO-1 to maintain epithelial barrier function.
Both induction of Hsp70 and exposure to EM prevent stress-induced increased permeability. Hsp70.2 is present in both WT and KO neonatal intestine, suggesting a crucial role in epithelial integrity. Induction of the Hsp70.2 isoform appears to be mediated by mother’s milk. These results suggest that mother’s milk feeding modulates Hsp70.2 expression and could attenuate injury leading to NEC.
Level of evidence
Necrotizing enterocolitis (NEC) is a gastrointestinal disease of multifactorial etiology resulting in devastating systemic inflammation and often death in premature newborns. It affects approximately 7% of premature very low birth weight (VLBW) infants with an overall mortality of 20–30% depending on the disease severity [1, 2]. NEC occurs almost exclusively in preterm newborns in the first 2–6 weeks of life, primarily affecting the ileum and cecum. Importantly, breast milk feeding has been shown to reduce the risk and severity of NEC [3, 4] and significantly decrease complications associated with prematurity including feeding intolerance and late-onset sepsis, decreasing re-hospitalizations and improving neurodevelopmental outcomes at 30 months [5,6,7,8].
The gastrointestinal tract functions as a barrier between the luminal contents of the gut and the host , changing with the neonatal developmental needs for nutrient absorption [10,11,12,13]. Differences exist between immature and mature enterocytes. Immature and mature neonatal enterocytes form a single layer of enterocytes bathed in amniotic fluid, allowing nutrients and beneficial macromolecules to cross. Ultimately, if the molecules are recognized as harmful, a more robust and exaggerated response of the inflammatory cascade is initiated [14,15,16]. This exaggerated response drives the inflammatory response which further damages enterocyte integrity [17,18,19], predisposing the neonate to sepsis and NEC [16, 20, 21].
Enterocyte functional integrity and the stability of tight junctions are altered and disrupted by stressors and NEC ensues, leading to increased bacterial translocation and inflammation . Preservation of the intestinal barrier implies reduction in translocation and inhibition of the inflammatory response. Heat shock protein-70 (Hsp70) has specifically been shown to maintain barrier function in part by stabilizing the tight junctions between intestinal epithelial cells [23,24,25]. We previously demonstrated that formula feeding inhibits ileal expression of Hsp70 .
Hsp70 is a critical stress protein within the intestine and that may serve to raise the injury threshold for subsequent stressors. It is a chaperone protein that has both inducible and constitutively expressed isoforms, protecting cells from stressful stimuli such as extremes of temperature, reactive oxygen and nitrogen species and during radiation and ischemic conditions . In NEC, the intracellular role of Hsp70 has been found to be protective against activation and upregulation of toll like receptor-4 (TLR4), the signaling pathway which leads to increased enterocyte apoptosis . We previously demonstrated that in an in vivo model, Hsp70 colocalized with the tight junction protein, zona occludens-1 (ZO-1), to stabilize the epithelial barrier and that formula feeding inhibited its expression . Finally, pretreatment with sublethal thermal injury reduced the incidence of early experimental NEC by possibly inhibiting nuclear factor-kB (NF-kB) activation through increased Hsp-70 expression .
Hsp70 has three isoforms: Hsp70.1 (HspA1a), Hsp70.2 (HspA2) and Hsp70.3 (HspA1b). Hsp70.1 and Hsp70.3 are well characterized and are important in gut protection, but Hsp70.2 has not been well characterized. In piglets, Hsp70 expression in the immature neonatal intestine is modulated through weaning . It is unknown which of the isoforms is responsible for the protective effect of Hsp70 in the immature intestine.
We hypothesized that expression of the Hsp70.2 isoform of Hsp70 is responsible for the protective effects of Hsp70 on intestinal barrier function in the protection against NEC.
Neonatal rodent animal model
Animal experiments were approved by the animal care committee at the University of Chicago. Wild-type (WT) mice were C57B16. Knockout (KO) mice were provided as a generous gift by Tao et al/Dr David Dix (National Institutes of Health) as heterozygous for Hsp70 deletion (Hsp70.1+/− and 70.3+/− mice), on a mixed 129S/C57Bl6/J background, and had been interbred . Genotyping for the adults was confirmed. WT and double-mutant Hsp70−/−littermates were used for experiments. Wild-type (WT) and knockout (KO) mouse pups were spontaneously delivered and then remained with the mother for rearing. There were a total of six neonatal rodents from two different litters that were each raised for a total of 4 days following delivery.
Mouse colonic epithelial cells or young adult mouse colon cells (YAMC) (ATCC; Manasas, Virginia, USA) were used as neonatal cells. At non-permissive temperatures, YAMC cells express minimal Hsp70, and following sublethal stress the cells express Hsp70. Cells were maintained as described at 33 °C . To stimulate ‘heat stress/heat shock’, cells were exposed to 42 °C for 40 min. Three wells of cells were utilized for each condition and performed four times.
Cellular oxidative stress
Monochloramine (NH2Cl) is an oxidant molecule. This molecule is released from white blood cells as part of the inflammatory response, producing oxidant-induced injury. In our cellular model, we utilized NH2Cl to stimulate oxidant injury when applied to cells at a 0.1 mM concentration as previously described [31, 32].
Hsp70 and ZO-1 immunofluorescence staining
The YAMC cells were grown on coverslips to confluence. Staining was performed for three slides, for each repeated three times. Fixation utilized ice-cold methanol exposure for 30 s. The specimens were then treated as described  with incubation with mouse anti-Hsp70 antibody (R&D Systems, Minneapolis, MN MAB1663) or rabbit anti-ZO-1 (Invitrogen, Carlsbad, CA 40-2200) .
Young adult mouse cells (YAMC) were grown at non-permissive temperature (33 °C) on 0.4 µm inserts (Becton Dickson, LePont De Claix, France) to confluence. Staining was performed for three slides, for each repeated three times. Cells were then treated with either freshly prepared 0.1 mM monochloramine (NH2Cl) for 60 min, exposed to 42 °C for 40 min heat shock, NH2Cl and heat shock, or were treated with wild-type (WT) collected mouse milk. A 10% solution of FITC dextran 70 (FD-70, Sigma, St Louis, MO) was added to the apical surface of the monolayer. At 60 min, 100 μL of the media from the basolateral compartment was removed for measurement of fluorescence to determine translocation across the monolayer. Translocated FITC–dextran was quantified by fluorescence and concentration determined by a standard curve of known amounts of FITC–dextran, expressed as μg/mL.
Intestinal segments were removed and processed as previously described . The specimens were incubated with mouse anti-Hsp70 antibody (R&D Systems, Minneapolis, MN MAB1663) and mouse anti-Hsp70.2/HspA2 (ABCAM, Cambridge, MA ab1428) at 4 °C. Staining was performed for three slides, for each repeated three times.
Ribonucleic acid extraction and RT-PCR
Real-time polymerase chain reaction (RT-PCR) assays were performed to quantitate the expression of Hsp70, Hsp70.1 and 70.3 and Hsp70.2 with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as an internal gene control. Total ribonucleic acid (RNA) was isolated from ileal sections with Qiagen RNeasyMini Kit (Qiagen Valenia, CA, USA) as previously described [31, 33]. Primers and probes were designed using the National Institutes of Health/National Center for Biotechnology Information (NIH/NCBI) Program Reference Sequence Accession No. GAPDH (NM_017008.3), Hsp70.2, Hsp70.1/70.3 Hsp70. RNA expression was compared using the Pfaffl ΔΔCt method. Messenger RNA (mRNA) levels are expressed as relative to control and are presented as mean fold change of standard error (SE) of the mean.
Western blot for Hsp70
Protein quantification was done using the bicinchoninic acid reagent (Pierce, Rockford, IL, USA)  from each intestinal homogenate following procurement of the pup wild-type and knockout mouse ileum, mid small bowel and colon. Two centimeters of each of the desired intestine sections were dissected from the mesentery and placed in a sucrose-based potassium buffer. As described , the buffer was used to bring the total volume to 15 µL. Primary antibodies used were mouse, anti-Hsp70 (1:7500; Abcam) and monoclonal anti-β-actin antibody to control for loading (1:7500; Santa Cruz). Secondary antibodies, goat anti-mouse IgG HRP (1:7500; Santa Cruz), were incubated for 1 h at room temperature. Bands were detected using SuperSignal West Pico according to the manufacturer’s instructions (Thermo Scientific Rockford, IL). Western blot band intensities were quantified using UN-SCAN-IT software (ver 5.3: Silk Scientific, In, Orem, UT). Values were normalized to total protein expressed as percent of control.
Quantitative measurement of Hsp70 in mouse dam milk
Collected mouse milk from five lactating female mice was centrifuged and supernatant Hsp70 was measured by ELISA (EKS-700B; Assay Designs, Boulder, CO) as previously described .
Data were analyzed using PRISM software (ver. 7; Graphpad, Inc., La Jolla, California, USA). Data were analyzed using nonparametric tests to adjust for outliers and skewed data. Comparisons were made between all groups and are reported as means and standard deviation. Medians and interquartile ranges were reported for skewed data, and groups were compared using a Mann–Whitney U test. Multiple comparisons were corrected using the Bonferroni method. Differences were considered statistically significant for p value < 0.05.
Hsp70 associates with ZO-1 in YAMC cells
In vivo Hsp70 associates with ZO-1 in the neonatal intestinal epithelium to form tight junctions. Cells grown to confluence at non-permissive temperature and stained with antibodies against ZO-1 and Hsp70 demonstrated minimal expression of Hsp70. As shown (Fig. 1a), ZO-1 (green stain) is present in YAMC cells in the monolayer. Following sublethal thermal stress, immunohistochemistry demonstrated the presence of inducible Hsp70 (red stain) in association with ZO-1. Where co-localization of Hsp70 and ZO-1 occurred is demonstrated as yellow staining on the heat shock slide (Fig. 1a).
Thermal stress improves barrier function in YAMC cells
An increase in Hsp70 protein expression is demonstrated compared to control cells following thermal stress (Fig. 1b). Integrity of the barrier of the cellular monolayer increases following sublethal thermal stress (Fig. 1c). Young adult mouse colon cell (YAMC) monolayer permeability following exposure to physiologic concentrations of monochloramine increases FITC flux by 29-fold (p < 0.007). In cells exposed to thermal stress alone, an increase in permeability was seen, but did not reach statistical significance. With exposure to thermal stress followed by monochloramine exposure, cells have an attenuated increase in permeability compared with control cells (3.5-fold increase in FITC flux) (p < 0.05). However, comparing cells treated with monochloramine alone vs. those treated first with thermal stress demonstrated a fourfold decrease in FITC flux (p < 0.05) (Fig. 1C).
Milk preserves barrier function in YAMC cells
Detected by Western blot, pretreatment with small amounts of milk from WT mice induced Hsp70 protein expression in the cells (Fig. 1b). To determine if mother’s milk alone could improve barrier function, cells were pretreated with milk collected from WT mice prior to treatment with monochloramine. As shown again (Fig. 1c), there was a 29-fold increase in FITC flux in cells exposed to monochloramine in the absence of Hsp70 (control untreated vs. monochloramine treated cells, p < 0.007). In cells exposed to milk from wild-type (WT) mice alone, no alteration in permeability was seen. However, milk exposure prior to oxidant injury resulted in a 2.4-fold reduction in FITC detected in the media (p < 0.001). This permeability was 12-fold greater in monochloramine-treated cells compared to that of milk-pretreated cells prior to oxidative stress. Thus, the permeability of YAMC monolayers increases following oxidant injury and is partially blocked by Hsp70 induction following exposure to expressed mother’s milk (Fig. 1c).
Hsp70.2 is present in the Hsp70 knockout mouse small intestine, but is not present in mother’s milk
Multiple heat shock proteins may be induced in response to thermal stress. To confirm the importance of Hsp70 in the intestine, intestinal tissue from Hsp70 knockout (KO) mice was evaluated. Hsp70 KO mice retain Hsp70.2 isoform function, while Hsp70.1 and 70.3 are not present. Ileal section genotyping of the mice was completed, confirming the absence of Hsp70.1 and 70.3, but the persistence of Hsp70.2. Staining with an antibody specific for all three isoforms of Hsp70 revealed protein expression in the neonatal ileum, i.e., positive IHC for Hsp70, despite less intense staining of the ileal villi (Fig. 2a). To confirm the retention of some Hsp70 expression, both western analyses (Fig. 2b) revealed continued expression of non-specific Hsp70 protein. To demonstrate the isoform responsible for continued Hsp70 expression, RT-PCR (Fig. 2c) demonstrated that while Hsp70.1/3 is present in the wild-type mouse, it is absent in the knockout (Fig. 2c), but that Hsp70.2 was significantly elevated.
Hsp70 is present in measurable quantities in WT mouse milk. However, Hsp70 is present in the milk expressed from wild-type animals, but absent in the knockout dams’ milk (Fig. 2d).
In this study, Hsp70 associates with ZO-1 to maintain epithelial barrier function. Both induction of Hsp70 and exposure to expressed mother’s milk attenuated stress-induced increased permeability. We previously demonstrated that Hsp70 associates with the tight junction protein, ZO-1, in the rat ileum with breast milk feeds, and maintains barrier function in a rat model of NEC . In our experiments, we found that Hsp70.2 isoform is present in both WT and KO neonatal intestine, suggesting a crucial role in neonatal epithelial integrity. Induction of the Hsp70.2 isoform appears to be mediated by mother’s milk. These results suggest that mother’s milk feeding modulates Hsp70.2 expression and could attenuate injury leading to NEC.
Hsp70 has three isoforms, Hsp70.1, Hsp70.2 and Hsp70.3, but Hsp70.2 has not been well characterized. In the available literature, Hsp70.2 is required for gametogenesis in the male [35, 36], lens and retinal development , and is expressed early in embryonic gut development . Hsp70.2 has a key role in inflammatory bowel disease polymorphisms of Hsp70.2  and celiac disease [39, 40]. Furthermore Hsp70.2 has correlatory implications for pneumonia  and survival in pancreatitis or after severe trauma, breast , colorectal , gastric , and renal cell cancer .
The mechanisms underlying the pathogenesis of NEC in the premature infant remain largely unknown. Investigation has revealed that exposure to mother’s milk is important for the induction of neonatal intestinal Hsp70.2 protein expression. Induction of expression is independent of the presence or absence of Hsp70 in the milk. The intestinal epithelial barrier is a key component in the health of the preterm infant. While this barrier is porous in utero, allowing growth factors and nutrients through, at birth it must become less permeable to protect the newborn from proteins, bacteria and bacterial products that may lead to inflammation and bacteremia. This barrier consists of multiple components which work together.
Previously, we have demonstrated that the presence of the cytoprotective protein, Hsp70, is induced by a diet of mother’s milk and colocalizes with ZO-1 in the neonatal rat ileum. We have also shown that formula feeding alone inhibits Hsp70 expression . Additionally, formula feeding results in NF-κB translocation and expression of downstream cytokines within the first 24 h of life . These studies demonstrate that expressed milk can induce Hsp70 and is able to attenuate barrier dysfunction in a cellular monolayer. Moreover, the Hsp70.2 isoform of Hsp70 is expressed predominately in the intestinal epithelium of the neonatal mouse and rat.
One of the most interesting findings was that Hsp70.2 expression was induced via exposure to expressed mother’s milk. While many studies describe the benefits of breast milk, it is still unknown which proteins are of importance or are induced by mother’s milk in the neonatal setting. This study therefore has implications for the bioactivity of proteins in mother’s breast milk and the milk-induced expression of Hsp70.2. Mother’s milk contains many bioactive proteins that are likely to be involved in the better outcomes of breast-fed infants compared with those fed infant formula. These proteins and their effect may ultimately help better understand the preterm infant and human milk feeding effects in decreasing NEC and ultimately maximizing the benefits of human milk feeding .
Hsp70 levels in breast milk, mechanisms of action, and implications for the neonatal intestine have not been previously studied. The mechanism by which Hsp70 is transferred into breast milk is not known, nor is the mechanism by which milk without Hsp70 is able to induce intestinal expression in the knockout pup. The intracellular role of Hsp70 in NEC has begun to be elucidated as being a downregulating mediator of NF-kB with pretreatment heat shock  as well as attenuating TLR4 signaling . In animal models, intestinal epithelial expression of Hsp70 is induced by mother’s milk feeds and co-localizes with intestinal barrier proteins, but is inhibited by formula feeding [25, 46]. However, there may be a paradoxical reaction whereby induction of cells primed by inflammation prior to induction of Hsp70 can have damaging effects . Exposure to extracellular Hsp70, contained in mother’s milk in WT mice, may yet place a role, but has not been evaluated.
This study provides insight into Hsp70 and its isoforms; however, limitations in our study exist. Our data are limited by complexity of mother’s milk milieu and the amount of milk tested. Additionally, we have still not fully elucidated the effect of extracellular Hsp70 and its association with NEC. Also, the precise effects of Hsp70 at varying stages of development within the enterocyte have yet to be elucidated. Further studies are underway to characterize Hsp70.2 induction as well as the interplay between Hsp70 and the NF-κB/IκB pathway.
In conclusion, both induction of Hsp70 and exposure to expressed mother’s milk prevent stress-induced increased intestinal permeability. Hsp70.2 is present in both WT and KO neonatal intestine, suggesting a crucial role in epithelial integrity. Induction of the Hsp70.2 isoform appears to be mediated by mother’s milk. Thus, these results demonstrate that expression of Hsp70 in the neonatal intestine is regulated by exposure to mother’s milk and not exposure to Hsp70. Further, mother’s milk feeding modulates Hsp70.2 expression and could attenuate injury leading to NEC. These findings also suggest that biologically active factors in milk induce specific protective isoforms of Hsp70. The importance of the protective effects of mother’s milk feeding is suggested, as well as the potential molecular targets to prevent and decrease enteric injury leading to NEC have been highlighted.
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Rentea, R.M., Guo, Y., Zhu, X. et al. Role of intestinal Hsp70 in barrier maintenance: contribution of milk to the induction of Hsp70.2. Pediatr Surg Int 34, 323–330 (2018). https://doi.org/10.1007/s00383-017-4211-3
- Necrotizing enterocolitis (NEC)
- Heat shock protein (HSP)
- Intestinal barrier
- Tight junction
- Breast milk