Umbilical cord mesenchymal stem cell exosomes alleviate necrotizing enterocolitis in neonatal mice by regulating intestinal epithelial cells autophagy.

BACKGROUND: Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease that affects premature infants. Although mounting evidence supports the therapeutic effect of exosomes on NEC, the underlying mechanisms remain unclear. AIM: To investigate the mechanisms underlying the regulation of inflammatory response and intestinal barrier function by umbilical cord mesenchymal stem cell (UCMSCs) exosomes, as well as their potential in alleviating NEC in neonatal mice. METHODS: NEC was induced in 5-d-old C57BL/6 pups through hypoxia and gavage feeding of formula containing lipopolysaccharide (LPS), after which the mice received human UCMSC exosomes (hUCMSC-exos). The control mice were allowed to breastfeed with their dams. Ileal tissues were collected from the mice and analyzed by histopathology and immunoblotting. Colon tissues were collected from NEC neonates and analyzed by immunofluorescence. Molecular biology and cell culture approaches were employed to study the related mechanisms in intestinal epithelial cells. RESULTS: We found that autophagy is overactivated in intestinal epithelial cells during NEC, resulting in reduced expression of tight junction proteins and an increased inflammatory response. The ability of hUCMSC-exos to ameliorate NEC in a mouse model was dependent on decreased intestinal autophagy. We also showed that hUCMSC-exos alleviate the inflammatory response and increase migration ability in intestinal epithelial cells induced by LPS. CONCLUSION: These results contribute to a better understanding of the protective mechanisms of hUCMSC-exos against NEC and provide a new theoretical and experimental foundation for NEC treatment. These findings also enhance our understanding of the role of the autophagy mechanism in NEC, offering potential avenues for identifying new therapeutic targets.

Identification of human host factors required for beta-defensin-2 expression in intestinal epithelial cells upon a bacterial challenge.

The human intestinal tract is colonized with microorganisms, which present a diverse array of immunological challenges. A number of antimicrobial mechanisms have evolved to cope with these challenges. A key defense mechanism is the expression of inducible antimicrobial peptides (AMPs), such as beta-defensins, which rapidly inactivate microorganisms. We currently have a limited knowledge of mechanisms regulating the inducible expression of AMP genes, especially factors from the host required in these regulatory mechanisms. To identify the host factors required for expression of the beta-defensin-2 gene (HBD2) in intestinal epithelial cells upon a bacterial challenge, we performed a RNAi screen using a siRNA library spanning the whole human genome. The screening was performed in duplicate to select the strongest 79 and 110 hit genes whose silencing promoted or inhibited HBD2 expression, respectively. A set of 57 hits selected among the two groups of genes was subjected to a counter-screening and a subset was subsequently validated for its impact onto HBD2 expression. Among the 57 confirmed hits, we brought out the TLR5-MYD88 signaling pathway, but above all new signaling proteins, epigenetic regulators and transcription factors so far unrevealed in the HBD2 regulatory circuits, like the GATA6 transcription factor involved in inflammatory bowel diseases. This study represents a significant step toward unveiling the key molecular requirements to promote AMP expression in human intestinal epithelial cells, and revealing new potential targets for the development of an innovative therapeutic strategy aiming at stimulating the host AMP expression, at the era of antimicrobial resistance.

Alleviating Pyroptosis of Intestinal Epithelial Cells to Restore Mucosal Integrity in Ulcerative Colitis by Targeting Delivery of 4-Octyl-Itaconate.

Current therapies primarily targeting inflammation often fail to address the root relationship between intestinal mucosal integrity and the resulting dysregulated cell death and ensuing inflammation in ulcerative colitis (UC). First, UC tissues from human and mice models in this article both emphasize the crucial role of Gasdermin E (GSDME)-mediated pyroptosis in intestinal epithelial cells (IECs) as it contributes to colitis by releasing proinflammatory cytokines, thereby compromising the intestinal barrier. Then, 4-octyl-itaconate (4-OI), exhibiting potential for anti-inflammatory activity in inhibiting pyroptosis, was encapsulated by butyrate-modified liposome (4-OI/BLipo) to target delivery for IECs. In brief, 4-OI/BLipo exhibited preferential accumulation in inflamed colonic epithelium, attributed to over 95% of butyrate being produced and absorbed in the colon. As expected, epithelium barriers were restored significantly by alleviating GSDME-mediated pyroptosis in colitis. Accordingly, the permeability of IECs was restored, and the resulting inflammation, mucosal epithelium, and balance of gut flora were reprogrammed, which offers a hopeful approach to the effective management of UC.

Nicotinamide Supplementation Mitigates Oxidative Injury of Bovine Intestinal Epithelial Cells through Autophagy Modulation.

The small intestine is important to the digestion and absorption of rumen undegradable nutrients, as well as the barrier functionality and immunological responses in ruminants. Oxidative stress induces a spectrum of pathophysiological symptoms and nutritional deficits, causing various gastrointestinal ailments. Previous studies have shown that nicotinamide (NAM) has antioxidant properties, but the potential mechanism has not been elucidated. The aim of this study was to explore the effects of NAM on hydrogen peroxide (H2O2)-induced oxidative injury in bovine intestinal epithelial cells (BIECs) and its potential mechanism. The results showed that NAM increased the cell viability and total antioxidant capacity (T-AOC) and decreased the release of lactate dehydrogenase (LDH) in BIECs challenged by H2O2. The NAM exhibited increased expression of catalase, superoxide dismutase 2, and tight junction proteins. The expression of autophagy-related proteins was increased in BIECs challenged by H2O2, and NAM significantly decreased the expression of autophagy-related proteins. When an autophagy-specific inhibitor was used, the oxidative injury in BIECs was not alleviated by NAM, and the T-AOC and the release of LDH were not affected. Collectively, these results indicated that NAM could alleviate oxidative injury in BIECs by enhancing antioxidant capacity and increasing the expression of tight junction proteins, and autophagy played a crucial role in the alleviation.

Traditional Chinese medicine improved diabetic kidney disease through targeting gut microbiota.

CONTEXT: Diabetic kidney disease (DKD) affects nearly 40% of diabetic patients, often leading to end-stage renal disease that requires renal replacement therapies, such as dialysis and transplantation. The gut microbiota, an integral aspect of human evolution, plays a crucial role in this condition. Traditional Chinese medicine (TCM) has shown promising outcomes in ameliorating DKD by addressing the gut microbiota. OBJECTIVE: This review elucidates the modifications in gut microbiota observed in DKD and explores the impact of TCM interventions on correcting microbial dysregulation. METHODS: We searched relevant articles from databases including Web of Science, PubMed, ScienceDirect, Wiley, and Springer Nature. The following keywords were used: diabetic kidney disease, diabetic nephropathy, gut microbiota, natural product, TCM, Chinese herbal medicine, and Chinese medicinal herbs. Rigorous criteria were applied to identify high-quality studies on TCM interventions against DKD. RESULTS: Dysregulation of the gut microbiota, including Lactobacillus, Streptococcus, and Clostridium, has been observed in individuals with DKD. Key indicators of microbial dysregulation include increased uremic solutes and decreased short-chain fatty acids. Various TCM therapies, such as formulas, tablets, granules, capsules, and decoctions, exhibit unique advantages in regulating the disordered microbiota to treat DKD. CONCLUSION: This review highlights the importance of targeting the gut-kidney axis to regulate microbial disorders, their metabolites, and associated signaling pathways in DKD. The Qing-Re-Xiao-Zheng formula, the Shenyan Kangfu tablet, the Huangkui capsule, and the Bekhogainsam decoction are potential candidates to address the gut-kidney axis. TCM interventions offer a significant therapeutic approach by targeting microbial dysregulation in patients with DKD.

Wuliangye strong aroma baijiu promotes intestinal homeostasis by improving gut microbiota and regulating intestinal stem cell proliferation and differentiation.

BACKGROUND: Wuliangye strong aroma baijiu (hereafter, Wuliangye baijiu) is a traditional Chinese grain liquor containing short-chain fatty acids, ethyl caproate, ethyl lactate, other trace components, and a large proportion of ethanol. The effects of Wuliangye baijiu on intestinal stem cells and intestinal epithelial development have not been elucidated. Here, the role of Wuliangye baijiu in intestinal epithelial regeneration and gut microbiota modulation was investigated by administering a Lieber-DeCarli chronic ethanol liquid diet in a mouse model to mimic long-term (8 weeks') light/moderate alcohol consumption (1.6 g kg-1 day-1) in healthy human adults. RESULTS: Wuliangye baijiu promoted colonic crypt proliferation in mice. According to immunofluorescence and reverse transcription-quantitative polymerase chain reaction analyses, compared with the ethanol-only treatment, Wuliangye baijiu increased the number of intestinal stem cells and goblet cells and the expression of enteroendocrine cell differentiation markers in the mouse colon. Furthermore, gut microbiota analysis showed an increase in the relative abundance of microbiota related to intestinal homeostasis following Wuliangye baijiu administration. Notably, increased abundance of Bacteroidota, Faecalibaculum, Lachnospiraceae, and Blautia may play an essential role in promoting stem-cell-mediated intestinal epithelial development and maintaining intestinal homeostasis. CONCLUSIONS: In summary, these findings suggest that Wuliangye baijiu can be used to regulate intestinal stem cell proliferation and differentiation in mice and to alter gut microbiota distributions, thereby promoting intestinal homeostasis. This research elucidates the mechanism by which Wuliangye baijiu promotes intestinal health. © 2024 Society of Chemical Industry.

The Effects of Resveratrol and Apigenin on Jejunal Oxidative Injury in Ducks and on Immortalized Duck Intestinal Epithelial Cells Exposed to H2O2.

Oxidative stress increases the apoptosis of intestinal epithelial cells and impairs intestinal epithelial cell renewal, which further promotes intestinal barrier dysfunction and even death. Extensive evidence supports that resveratrol and apigenin have antioxidant, anti-inflammatory, and antiproliferative properties. Here, we investigated the ability of these two compounds to alleviate diquat-induced jejunal oxidative stress and morphological injury, using the duck as a model, as well as the effects of apigenin on oxidative stress induced by H2O2 in immortalized duck intestinal epithelial cells (IDECs). Ducks were randomly assigned to the following four groups, with five replicates: a control (CON) group, a diquat-challenged (DIQ) group, a resveratrol (500 mg/kg) + diquat (RES) group, and an apigenin (500 mg/kg) + diquat (API) group. We found that serum catalase (CAT) activity and total antioxidant capacity (T-AOC) markedly reduced in the RES and API groups as compared to the DIQ group (p < 0.05); moreover, serum S superoxide dismutase (SOD) levels increased significantly in the API group as compared to the DIQ group (p < 0.05). In jejunal mucosa, the malondialdehyde (MDA) content in the RES and API groups decreased more than that in the DIQ group (p < 0.05). In addition, the jejunal expression levels of the NRF2 and GCLM genes in the RES and API groups increased notably compared with those in the DIQ group (p < 0.05); meanwhile, CAT activity in the RES and API groups was markedly elevated compared with that in the CON group (p < 0.05). In IDECs, apigenin significantly restrained the H2O2-mediated increase in MDA content and decrease in CAT levels (p < 0.05). Furthermore, apigenin increased the protein expression of p-NRF2, NRF2, p-AKT, and p-P38; downregulated that of cleaved caspase-3 and cleaved caspase-9; and reduced the ratio of Bax/Bcl-2 in H2O2-treated IDECs (p < 0.05). In conclusion, resveratrol and apigenin can be used as natural feed additives to protect against jejunal oxidative stress in ducks.

REV-ERBα negatively regulates NLRP6 transcription and reduces the severity of Salmonella infection in mice.

Non-typhoidal Salmonella infection is among the most frequent foodborne diseases threatening human health worldwide. The host circadian clock orchestrates daily rhythms to adapt to environmental changes, including coordinating immune function in response to potential infections. However, the molecular mechanisms underlying the interplay between the circadian clock and the immune system in modulating infection processes are incompletely understood. Here, we demonstrate that NLRP6, a novel nucleotide-oligomerization domain (NOD)-like receptor (NLR) family member highly expressed in the intestine, is closely associated with the differential day-night response to Salmonella infection. The core clock component REV-ERBα negatively regulates NLRP6 transcription, leading to the rhythmic expression of NLRP6 and the secretion of IL-18 in intestinal epithelial cells, playing a crucial role in mediating the differential day-night response to Salmonella infection. Activating REV-ERBα with agonist SR9009 in wild-type mice attenuated the severity of infection by decreasing the NLRP6 level in intestinal epithelial cells. Our findings provide new insights into the association between the host circadian clock and the immune response to enteric infections by revealing the regulation of Salmonella infection via the inhibitory effect of REV-ERBα on NLRP6 transcription. Targeting REV-ERBα to modulate NLRP6 activation may be a potential therapeutic strategy for bacterial infections.

Zinc alleviates thermal stress-induced damage to the integrity and barrier function of cultured chicken embryonic primary jejunal epithelial cells via the MAPK and PI3K/AKT/mTOR signaling pathways.

Zinc (Zn) could alleviate the adverse effect of high temperature (HT) on intestinal integrity and barrier function of broilers, but the underlying mechanisms remain unclear. We aimed to investigate the possible protective mechanisms of Zn on primary cultured broiler jejunal epithelial cells exposed to thermal stress (TS). In Exp.1, jejunal epithelial cells were exposed to 40℃ (normal temperature, NT) and 44℃ (HT) for 1, 2, 4, 6, or 8 h. Cells incubated for 8 h had the lowest transepithelial resistance (TEER) and the highest phenol red permeability under HT. In Exp.2, the cells were preincubated with different Zn sources (Zn sulfate as iZn and Zn proteinate with the moderate chelation strength as oZn) and Zn supplemental levels (50 and 100 µmol/L) under NT for 24 h, and then continuously incubated under HT for another 8 h. TS increased phenol red permeability, lactate dehydrogenase (LDH) activity and p-PKC/PKC level, and decreased TEER, cell proliferation, mRNA levels of claudin-1, occludin, zona occludens-1 (ZO-1), PI3K, AKT and mTOR, protein levels of claudin-1, ZO-1 and junctional adhesion molecule-A (JAM-A), and the levels of p-ERK/ERK, p-PI3K/PI3K and p-AKT/AKT. Under HT, oZn was more effective than iZn in increasing TEER, occludin, ZO-1, PI3K, and AKT mRNA levels, ZO-1 protein level, and p-AKT/AKT level; supplementation with 50 µmol Zn/L was more effective than 100 µmol Zn/L in increasing cell proliferation, JAM-A, PI3K, AKT, and PKC mRNA levels, JAM-A protein level, and the levels of p-ERK/ERK and p-PI3K/PI3K; furthermore, supplementation with 50 µmol Zn/L as oZn had the lowest LDH activity, and the highest ERK, JNK-1, and mTOR mRNA levels. Therefore, supplemental Zn, especially 50 µmol Zn/L as oZn, could alleviate the TS-induced integrity and barrier function damage of broiler jejunal epithelial cells possibly by promoting cell proliferation and tight junction protein expression via the MAPK and PI3K/AKT/mTOR signaling pathways.

Mechanisms of intestinal epithelial cell damage by Clostridiumperfringens.

Clostridium perfringens, a Gram-positive bacterium, causes intestinal diseases in humans and livestock through its toxins, related to alpha toxin (CPA), beta toxin (CPB), C. perfringens enterotoxin (CPE), epsilon toxin (ETX), Iota toxin (ITX), and necrotic enteritis B-like toxin (NetB). These toxins disrupt intestinal barrier, leading to various cell death mechanisms such as necrosis, apoptosis, and necroptosis. Additionally, non-toxin factors like adhesins and degradative enzymes contribute to virulence by enhancing colonization and survival of C. perfringens. A vicious cycle of intestinal barrier breach, misregulated cell death, and subsequent inflammation is at the heart of chronic inflammatory and infectious gastrointestinal diseases. Understanding these mechanisms is essential for developing targeted therapies against C. perfringens-associated intestinal diseases.

Sophoricoside improved Crohn's disease-like colitis by inhibiting intestinal epithelial cell apoptosis through PI3K/AKT signaling.

BACKGROUND AND AIMS: Increased apoptosis of intestinal epithelial cells (IECs) is a significant cause of intestinal barrier dysfunction in Crohn's disease (CD). Sophoricoside (SOP) is an isoflavone glycoside known for its anti-apoptotic properties. The aim of this study was to investigate the effects of SOP on mice with CD-like colitis and to understand the underlying mechanisms. METHODS: Mice treated with 2,4,6-trinitrobenzene sulfonic acid (TNBS) were used to examine the therapeutic effect of SOP on CD-like colitis and intestinal barrier damage. To further explore SOP's impact on IECs apoptosis and intestinal barrier protection, an in vitro colonic organoid apoptosis model induced by TNF-α was utilized. Network pharmacology was employed to predict the relevant pathways and molecular processes associated with SOP in the treatment of CD. RESULTS: Treatment with SOP significantly improved colitis symptoms in TNBS mice, as demonstrated by reductions in the Disease Activity Index (DAI), weight loss, colon shortening, macroscopic scores, colonic tissue inflammatory scores, and the expression of pro-inflammatory factors. Our experiments confirmed that SOP protects the intestinal barrier by counteracting IECs apoptosis. Additionally, this study established that SOP reduced IECs apoptosis by inhibiting the PI3K/AKT signaling pathway. CONCLUSIONS: SOP can reduce IECs apoptosis through the inhibition of the PI3K/AKT signaling pathway, thereby protecting the intestinal barrier. This study is the first to illustrate how SOP ameliorates colitis and protects the intestinal barrier, suggesting SOP has potential clinical application in treating CD.

The intestinal epithelial-macrophage-crypt stem cell axis plays a crucial role in regulating and maintaining intestinal homeostasis.

The intestinal tract plays a vital role in both digestion and immunity, making its equilibrium crucial for overall health. This equilibrium relies on the dynamic interplay among intestinal epithelial cells, macrophages, and crypt stem cells. Intestinal epithelial cells play a pivotal role in protecting and regulating the gut. They form vital barriers, modulate immune responses, and engage in pathogen defense and cytokine secretion. Moreover, they supervise the regulation of intestinal stem cells. Macrophages, serving as immune cells, actively influence the immune response through the phagocytosis of pathogens and the release of cytokines. They also contribute to regulating intestinal stem cells. Stem cells, known for their self-renewal and differentiation abilities, play a vital role in repairing damaged intestinal epithelium and maintaining homeostasis. Although research has primarily concentrated on the connections between epithelial and stem cells, interactions with macrophages have been less explored. This review aims to fill this gap by exploring the roles of the intestinal epithelial-macrophage-crypt stem cell axis in maintaining intestinal balance. It seeks to unravel the intricate dynamics and regulatory mechanisms among these essential players. A comprehensive understanding of these cell types' functions and interactions promises insights into intestinal homeostasis regulation. Moreover, it holds potential for innovative approaches to manage conditions like radiation-induced intestinal injury, inflammatory bowel disease, and related diseases.

[Corrigendum] FKBP11 protects intestinal epithelial cells against inflammation­induced apoptosis via the JNK­caspase pathway in Crohn's disease.

Subsequently to the publication of the above article, the authors realized that Fig. 4 in their paper had been assembled containing two erroneously placed gel slices; essentially, the GAPDH bands featured in Fig. 4A had also been included in Fig. 5, and the data for the FKBP11 bands in Fig. 4A had also been included to show the GRP78 bands in Fig. 4. The authors were able to revisit their original data and to correct the data that had been featured incorrectly in Fig. 4. The corrected version of Fig. 4, now showing the true data for the GRP78 protein bands in Fig. 4C and the correct GAPDH protein bands for Fig. 4A, is shown on the next page. Note that these errors did not significantly affect the results or the conclusions reported in this paper. All the authors agree to the publication of this Corrigendum, and are grateful to the Editor of Molecular Medicine Reports for allowing them the opportunity to correct this error. Moreover, the authors apologize to the readership for any inconvenience caused. [Molecular Medicine Reports 18: 4428­4438, 2018; DOI: 10.3892/mmr.2018.9485].

Transcription factor EB modulates the homeostasis of reactive oxygen species in intestinal epithelial cells to alleviate inflammatory bowel disease.

Transcription factor EB (TFEB), a master lysosomal biogenesis and autophagy regulator, is crucial for cellular homeostasis, and its abnormality is related to diverse inflammatory diseases. Genetic variations in autophagic genes are associated with susceptibility to inflammatory bowel disease (IBD); however, little is known about the role and mechanism of TFEB in disease pathogenesis. In this study, we found that the genetic deletion of TFEB in mouse intestinal epithelial cells (IEC) caused intestinal barrier dysfunction, leading to increased susceptibility to experimental colitis. Mechanistically, TFEB functionally protected IEC in part through peroxisome proliferator-activated receptor gamma coactivator 1alpha (TFEB-PGC1α axis) induction, which consequently suppressed reactive oxygen species. TFEB can directly regulate PGC-1α transcription to control antioxidation level. Notably, TFEB expression is impaired and downregulated in the colon tissues of IBD patients. Collectively, our results indicate that intestinal TFEB participates in oxidative stress regulation and attenuates IBD progression.

Bacillus subtilis Induces Human Beta Defensin-2 Through its Lipoproteins in Human Intestinal Epithelial Cells.

Human intestinal epithelial cells (IECs) play an important role in maintaining gut homeostasis by producing antimicrobial peptides (AMPs). Bacillus subtilis, a commensal bacterium, is considered a probiotic. Although its protective effects on intestinal health are widely reported, the key component of B. subtilis responsible for its beneficial effects remains elusive. In this study, we tried to identify the key molecules responsible for B. subtilis-induced AMPs and their molecular mechanisms in a human IEC line, Caco-2. B. subtilis increased human beta defensin (HBD)-2 mRNA expression in a dose- and time-dependent manner. Among the B. subtilis microbe-associated molecular patterns, lipoprotein (LPP) substantially increased the mRNA expression and protein production of HBD-2, whereas lipoteichoic acid and peptidoglycan did not show such effects. Those results were confirmed in primary human IECs. In addition, both LPP recognition and HBD-2 secretion mainly took place on the apical side of fully differentiated and polarized Caco-2 cells through Toll-like receptor 2-mediated JNK/p38 MAP kinase/AP-1 and NF-κB pathways. HBD-2 efficiently inhibited the growth of the intestinal pathogens Staphylococcus aureus and Bacillus cereus. Furthermore, LPPs pre-incubated with lipase or proteinase K decreased LPP-induced HBD-2 expression, suggesting that the lipid and protein moieties of LPP are crucial for HBD-2 expression. Q Exactive Plus mass spectrometry identified 35 B. subtilis LPP candidates within the LPP preparation, and most of them were ABC transporters. Taken together, these results suggest that B. subtilis promotes HBD-2 secretion in human IECs mainly with its LPPs, which might enhance the protection from intestinal pathogens.

A novel avian intestinal epithelial cell line: its characterization and exploration as an in vitro infection culture model for Eimeria species.

BACKGROUND: The gastrointestinal epithelium plays an important role in directing recognition by the immune system, and epithelial cells provide the host's front line of defense against microorganisms. However, it is difficult to cultivate avian intestinal epithelial cells in vitro for lengthy periods, and the lack of available cell lines limits the research on avian intestinal diseases and nutritional regulation. Chicken coccidiosis is a serious intestinal disease that causes significant economic losses in the poultry industry. In vitro, some cell line models are beneficial for the development of Eimeria species; however, only partial reproduction can be achieved. Therefore, we sought to develop a new model with both the natural host and epithelial cell phenotypes. METHODS: In this study, we use the SV40 large T antigen (SV40T) gene to generate an immortalized cell line. Single-cell screening technology was used to sort positive cell clusters with epithelial characteristics for passage. Polymerase chain reaction (PCR) identification, immunofluorescence detection, and bulk RNA sequencing analysis and validation were used to check the expression of epithelial cell markers and characterize the avian intestinal epithelial cell line (AIEC). AIECs were infected with sporozoites, and their ability to support the in vitro endogenous development of Eimeria tenella was assessed. RESULTS: This novel AIEC consistently expressed intestinal epithelial markers. Transcriptome assays revealed the upregulation of genes associated with proliferation and downregulation of genes associated with apoptosis. We sought to compare E. tenella infection between an existing fibroblast cell line (DF-1) and several passages of AIEC and found that the invasion efficiency was significantly increased relative to that of chicken fibroblast cell lines. CONCLUSIONS: An AIEC will serve as a better in vitro research model, especially in the study of Eimeria species development and the mechanisms of parasite-host interactions. Using AIEC helps us understand the involvement of intestinal epithelial cells in the digestive tract and the immune defense of the chickens, which will contribute to the epithelial innate defense against microbial infection in the gastrointestinal tract.

Anthrax lethal toxin and tumor necrosis factor-α synergize on intestinal epithelia to induce mouse death.

Bacillus anthracis lethal toxin (LT) is a determinant of lethal anthrax. Its function in myeloid cells is required for bacterial dissemination, and LT itself can directly trigger dysfunction of the cardiovascular system. The interplay between LT and the host responses is important in the pathogenesis, but our knowledge on this interplay remains limited. Tumor necrosis factor-α (TNF-α) is a pleiotropic pro-inflammatory cytokine induced by bacterial infections. Since LT accumulates and cytokines, predominantly TNF, amass during B. anthracis infection, co-treatment of TNF + LT in mice was used to mimic in vivo conditions for LT to function in inflamed hosts. Bone marrow transplantation and genetically engineered mice showed unexpectedly that the death of intestinal epithelial cells (IECs) rather than that of hematopoietic cells led to LT + TNF-induced lethality. Inhibition of p38α mitogen-activated protein kinase (MAPK) signaling by LT in IECs promoted TNF-induced apoptosis and necroptosis of IECs, leading to intestinal damage and mouse death. Consistently, p38α inhibition by LT enhanced TNF-mediated cell death in human colon epithelial HT-29 cells. As intestinal damage is one of the leading causes of lethality in anthrax patients, the IEC damage caused by LT + TNF would most likely be a mechanism underneath this clinical manifestation and could be a target for interventions.

Effects of functional nutrients on chicken intestinal epithelial cells induced with oxidative stress.

The objective of this study was to investigate the protective effects of functional nutrients including various functional amino acids, vitamins, and minerals on chicken intestinal epithelial cells (cIECs) treated with oxidative stress. The cIECs were isolated from specific pathogen free eggs. Cells were exposed to 0 mM supplement (control), 20 mM threonine (Thr), 0.4 mM tryptophan (Trp), 1 mM glycine (Gly), 10 µM vitamin C (VC), 40 µM vitamin E (VE), 5 µM vitamin A (VA), 34 µM chromium (Cr), 0.42 µM selenium (Se), and 50 µM zinc (Zn) for 24 h with 6 replicates for each treatment. After 24 h, cells were further incubated with fresh culture medium (positive control, PC) or 1 mM H2O2 with different supplements (negative control, NC and each treatment). Oxidative stress was measured by cell proliferation, whereas tight junction barrier function was analyzed by fluorescein isothiocyanate (FITC)-dextran permeability and transepithelial electrical resistance (TEER). Results indicated that cell viability and TEER values were less (p < 0.05) in NC treatments with oxidative stress than in PC treatments. In addition, FITC-dextran values were greater (p < 0.05) in NC treatments with oxidative stress than in PC treatments. The supplementations of Thr, Trp, Gly, VC, and VE in cells treated with H2O2 showed greater (p < 0.05) cell viability than the supplementation of VA, Cr, Se, and Zn. The supplementations of Trp, Gly, VC, and Se in cells treated with H2O2 showed the least (p < 0.05) cellular permeability. In addition, the supplementation of Thr, VE, VA, Cr, and Zn in cells treated with H2O2 decreased (p < 0.05) cellular permeability. At 48 h, the supplementations of Thr, Trp, and Gly in cells treated with H2O2 showed the greatest (p < 0.05) TEER values among all treatments, and the supplementations of VC and VE in cells treated with H2O2 showed greater (p < 0.05) TEER values than the supplementations of VA, Cr, Se, and Zn in cells treated with H2O2. In conclusion, Thr, Trp, Gly, and VC supplements were effective in improving cell viability and intestinal barrier function of cIECs exposed to oxidative stress.

Identification of a functional peptide of a probiotic bacterium-derived protein for the sustained effect on preventing colitis.

Several probiotic-derived factors have been identified as effectors of probiotics for exerting beneficial effects on the host. However, there is a paucity of studies to elucidate mechanisms of their functions. p40, a secretory protein, is originally isolated from a probiotic bacterium, Lactobacillus rhamnosus GG. Thus, this study aimed to apply structure-functional analysis to define the functional peptide of p40 that modulates the epigenetic program in intestinal epithelial cells for sustained prevention of colitis. In silico analysis revealed that p40 is composed of a signal peptide (1-28 residues) followed by a coiled-coil domain with uncharacterized function on the N-terminus, a linker region, and a ß-sheet domain with high homology to CHAP on the C-terminus. Based on the p40 three-dimensional structure model, two recombinant p40 peptides were generated, p40N120 (28-120 residues) and p40N180 (28-180 residues) that contain first two and first three coiled coils, respectively. Compared to full-length p40 (p40F) and p40N180, p40N120 showed similar or higher effects on up-regulating expression of Setd1b (encoding a methyltransferase), promoting mono- and trimethylation of histone 3 on lysine 4 (H3K4me1/3), and enhancing Tgfb gene expression and protein production that leads to SMAD2 phosphorylation in human colonoids and a mouse colonic epithelial cell line. Furthermore, supplementation with p40F and p40N120 in early life increased H3K4me1, Tgfb expression and differentiation of regulatory T cells (Tregs) in the colon, and mitigated disruption of epithelial barrier and inflammation induced by DSS in adult mice. This study reveals the structural feature of p40 and identifies a functional peptide of p40 that could maintain intestinal homeostasis.