Intestinal Epithelial Barrier Permeability Research Articles

Effect of intestinal microbiota on myocardial resistance to ischemia-reperfusion injury

The last 10–15 years have been marked by a rapid increase in the volume of knowledge about the role of the intestinal microbiota in the regulation of various physiological processes. In particular, the interrelations between disturbances in the composition of the intestinal microflora and an increase in the risk of developing cardiovascular diseases were noted. There are also emerging works that demonstrate the possibility of reducing the severity of myocardial ischemia-reperfusion injury as a result of various effects on the composition of intestinal microbiota. This review analyzes the recent literature on the influence of intestinal microbiota on myocardial resistance to ischemia-reperfusion injury. It has been shown that modulation of the intestinal microbiome by changing the nature of nutrition, probiotics, antibiotics, and intestinal microflora transplantation leads to a reduction in the size of myocardial infarction. Normalization of increased permeability of intestinal epithelial barrier with reduction of manifestations of systemic inflammation, changes in the level of secretion of some hormones, increased production of short-chain fatty acids and modulation of bile acid metabolism under the action of intestinal bacterial enzymes are considered as possible mechanisms of cardioprotection mediated by changes in the functional state of microbiota. The concept of increasing myocardial resistance to ischemia-reperfusion injury by modulating the composition of the intestinal microbiota is a completely new scientific direction that requires detailed study in experiment with the possibility of rapid implementation of the results obtained in clinical practice.

Diet high in linoleic acid dysregulates the intestinal endocannabinoid system and increases susceptibility to colitis in Mice

ABSTRACT Inflammatory bowel disease (IBD) is a multifactorial disease with increasing incidence in the U.S. suggesting that environmental factors, including diet, are involved. It has been suggested that excessive consumption of linoleic acid (LA, C18:2 omega-6), which must be obtained from the diet, may promote the development of IBD in humans. To demonstrate a causal link between LA and IBD, we show that a high fat diet (HFD) based on soybean oil (SO), which is comprised of ~55% LA, increases susceptibility to colitis in several models, including IBD-susceptible IL10 knockout mice. This effect was not observed with low-LA HFDs derived from genetically modified soybean oil or olive oil. The conventional SO HFD causes classical IBD symptoms including immune dysfunction, increased intestinal epithelial barrier permeability, and disruption of the balance of isoforms from the IBD susceptibility gene Hepatocyte Nuclear Factor 4α (HNF4α). The SO HFD causes gut dysbiosis, including increased abundance of an endogenous adherent invasive Escherichia coli (AIEC), which can use LA as a carbon source. Metabolomic analysis shows that in the mouse gut, even in the absence of bacteria, the presence of soybean oil increases levels of LA, oxylipins and prostaglandins. Many compounds in the endocannabinoid system, which are protective against IBD, are decreased by SO both in vivo and in vitro. These results indicate that a high LA diet increases susceptibility to colitis via microbial and host-initiated pathways involving alterations in the balance of bioactive metabolites of omega-6 and omega-3 polyunsaturated fatty acids, as well as HNF4α isoforms.

Disordered gut microbiota promotes atrial fibrillation by aggravated conduction disturbance and unbalanced linoleic acid/SIRT1 signaling

Emerging evidence suggests an association of dysbiotic gut microbiota (GM) with atrial fibrillation (AF). The current study aimed to determine whether aberrant GM promotes AF development. A fecal microbiota transplantation (FMT) mouse model demonstrated that dysbiotic GM is sufficient to enhance AF susceptibility assessed by transesophageal burst pacing. Compared with recipients transplanted with GM obtained from healthy subjects (FMT-CH), the prolonged P wave duration and an enlarging tendency for the left atrium were detected in recipients transplanted with AF GM (FMT-AF). Meanwhile, the disrupted localizations of connexin 43 and N-cadherin and increased expression levels of phospho-CaMKII and phospho-RyR2, were observed in the atrium of FMT-AF, which indicated aggravated electrical remodeling caused by the altered gut flora. Specifically, exacerbated fibrosis disarray, collagen deposition, α-SMA expression, and inflammation in the atrium were also confirmed to be transmissible by the GM. Furthermore, deteriorated intestinal epithelial barrier and intestinal permeability, accompanied by disturbing metabolomic features in both feces and plasma, especially decreased linoleic acid (LA), were identified in FMT-AF mice. Subsequently, the anti-inflammatory role of LA among the imbalanced SIRT1 signaling discovered in the atrium of FMT-AF was confirmed in mouse HL-1 cells treated with LPS/nigericin, LA, and SIRT1 knockdown. This study provides preliminary insights into the causal role of aberrant GM in the pathophysiology of AF, suggesting the GM-intestinal barrier-atrium axis might participate in the vulnerable substrates for AF development, and the GM could be utilized as an environmental target in AF management.

Akkermansia muciniphila improves heat stress-impaired intestinal barrier function by modulating HSP27 in Caco-2 cells

ObjectiveHeat stress causes an elevation of intestinal epithelial barrier permeability and leads to multiple organ dysfunction in heatstroke. Akkermansia muciniphila (A. muciniphila) plays a role in maintaining intestinal integrity and improving the inflammatory state. This study aimed to investigate whether A. muciniphila could alleviate heat stress-induced dysfunction of intestinal permeability in Caco-2 monolayers and have the preventive effects on heatstroke. MethodsHuman intestinal epithelial Caco-2 cells were preincubated with live or pasteurized A. muciniphila then exposed to heat stress at 43 °C. Transepithelial electrical resistance (TEER) and the flux of horseradish peroxidase (HRP) across cell monolayers were measured to determine intestinal permeability. The levels of the tight junction proteins Occludin, ZO-1 and HSP27 were analyzed by Western blotting. These proteins were immunostained and localized by fluorescence microscopy. TJ morphology was observed using transmission electron microscopy (TEM). ResultsBoth live and pasteurized A. muciniphila effectively attenuated the decrease in TEER and impairment of intestinal permeability in HRP flux induced by heat exposure. A. muciniphila significantly elevated the expression of Occludin and ZO-1 by promoting HSP27 phosphorylation. The distortion and redistribution of tight junction proteins and disruption of morphology were also effectively prevented by pretreatment with A. muciniphila. ConclusionThis study indicates for the first time that both live and pasteurized A. muciniphila play an important protective role against heat-induced permeability dysfunction and epithelial barrier damage.

Probiotic Therapy with Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis Results in Infarct Size Limitation in Rats with Obesity and Chemically Induced Colitis

In this study, we investigated the effect of three different probiotics, namely, a combination of Lactobacillus acidophilus (LA-5) and Bifidobacterium animalis subsp. lactis (BB-12), Saccharomyces boulardii, and Enterococcus faecium L3 on myocardial infarct size in rats with diet-induced obesity (DIO) and chemically-induced colitis (CIC). Potential associations between the effects of probiotics on myocardial ischemia-reperfusion injury and gut microbiome patterns as well as the serum levels of pro- and anti-inflammatory cytokines, lipopolysaccharide, and short chain fatty acids were also studied. Intragastric administration of lyophilized Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis at a dose of 1.2 × 108 CFU/mL for 15 days resulted in myocardial infarct size reduction in rats with DIO, CIC, and antibiotic-induced dysbiosis. This cardioprotective effect was associated with specific changes in cytokine concentrations, namely reduced levels of IL-1β, TNF-α, IL-2, and IL-8. At the same time, the use of Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis was accompanied by a significant reduction in lipopolysaccharide level, suggesting normalization of intestinal epithelial barrier permeability. However, the cardioprotective effect of Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis is not secondary to improved healing of the intestinal mucosa in CIC, as evidenced by the lack of difference in histopathological scores.

Role of Nutrition in Pediatric Patients with Respiratory Failure

Respiratory failure is a consequence of malfunction of the respiratory system including neuronal and cellular aspects. The most common causes in children are pneumonitis, vasculitis, pulmonary edema, cystic fibrosis, tuberculosis, asthma, foreign-body aspiration, and respiratory infections of the upper and lower airways. Reduced immunological response due to critical illness, atrophy, and increased intestinal epithelial barrier permeability results in increased susceptibility to infections and the spread of pathogens. The search strategy included a PubMed search for articles from 2000 to 2022. Malnutrition is acquired in 50% of patients admitted to the pediatric intensive care unit, with the added burden of nutritional deficits worsening preexisting malnutrition. Nutrition forms an essential component in managing respiratory conditions, with the potential to change outcomes. Enteral nutrition (EN) can reduce inflammatory cytokine activation and release, as well as maintain gastrointestinal (GI) mucosal integrity, which lowers systemic bacterial invasion and sepsis. Therefore, EN should be the preferred mode of nutrition (when clinically indicated) to parenteral nutrition. ASPEN guidelines recommend the administration of a minimum of 1.5 g/kg/ day of protein in critically ill children. Reduction in the respiratory quotient may be achieved by lowering the carbohydrate intake in infants suffering from prolonged lung disease; however, a balance of carbohydrate and fat ratios is recommended. Immunonutrition helps in reducing inflammation and pro-inflammatory cytokine levels. During pediatric acute respiratory distress syndrome, an essential target to improve lung inflammation is the GI tract. However, no disease-specific recommendation for probiotics and immunonutrients has been established in children yet.

Intestinal histomorphological and molecular alterations in patients with Parkinson's disease.

Changes in gut microbiota composition, enteric inflammation, impairments of the intestinal epithelial barrier and neuroplastic changes in the enteric nervous systemhave been reported in Parkinson's disease (PD) patients and could contribute to the onset of both neurological and gastrointestinal symptoms. However, their mutual interplay has rarely been investigated. This study evaluated, in an integrated manner, changes in faecal microbiota composition, morphofunctional alterations of colonic mucosal barrier and changes of inflammatory markers in blood and stools of PD patients. Nineteen PD patients and nineteen asymptomatic subjects were enrolled. Blood lipopolysaccharide binding protein (LBP, marker of altered intestinal permeability) and interleukin-1β (IL-1β) levels, as well as stool IL-1β and tumour necrosis factor (TNF) levels, were evaluated. Gut microbiota analysis was performed. Epithelial mucins, collagen fibres, claudin-1 and S100-positive glial cells as markers of an impairment of the intestinal barrier, mucosal remodelling and enteric glial activationwere evaluated on colonic mucosal specimens collected during colonoscopy. Faecal microbiota analysis revealed a significant difference in the α-diversity in PD patients compared to controls, while no differences were found in the β-diversity. Compared to controls, PD patients showed significant chenags in plasma LBP levels, as well as faecal TNF and IL-1β levels. The histological analysis showed a decrease in epithelial neutral mucins and claudin-1 expression and an increased expression of acidic mucins, collagen fibres and S100-positive glial cells. Parkinson's disease patients are characterized by enteric inflammation and increased intestinal epithelial barrier permeability, as well as colonic mucosal barrier remodelling, associated with changes in gut microbiota composition.

P101 Isosteviol Attenuates Dextran Sulfate Sodium-Induced Colitis by Maintaining the Intestinal Barrier Through PDK1/AKT/NF-κB Signaling

Abstract Background Inflammatory bowel disease (IBD) is a chronic debilitating inflammatory disorder of the gastrointestinal tract that is characterized by intestinal epithelial barrier dysfunction and excessive activation of the mucosal immune system. Isosteviol (IS) has been reported to possess anti-inflammatory properties. Thus, in this study, we aimed to investigate the therapeutic effects and detailed mechanisms of IS use against intestinal inflammation using both in vivo and in vitro models. Methods C57BL/6 mice were randomly divided into sham, dextran sodium sulfate (DSS), and DSS+IS groups. Colitis was established using 3.0% DSS through oral gavage for five consecutive days. In vitro, tumor necrosis factor-α (TNF-α)-treated Caco-2 cells were used as an inflammatory model. Clinical characteristics, histological performance, proinflammatory cytokine expression, and intestinal barrier function were measured. In addition, activation of the pyruvate dehydrogenase kinase 1/protein kinase B/nuclear factor-κB (PDK1/AKT/NF-κB) signaling pathway was determined by western blotting, quantitative polymerase chain reaction, and immunofluorescence assays. Results IS mitigated DSS-induced colitis by significantly reducing body weight loss, colonic shortening, and the disease activity index score via inhibition of colonic proinflammatory cytokine production of IL-1β, IL-6, and TNF-α. IS restored impaired barrier function by regulating tight junctions and intestinal epithelial barrier permeability. Furthermore, we found that IS ameliorated intestinal barrier injury by regulating the PDK1-dependent AKT/NF-κB signaling pathway. Conclusion Our results demonstrate that IS attenuates experimental colitis by preserving intestinal barrier function, probably mediated by the PDK1/AKT/NF-κB signaling pathway. These findings highlight the potential of IS as a novel therapeutic agent for IBD.

Impact of fermentation supernatants of rich in β-glucans mushrooms on the intestinal barrier: an in vitro study

Rationale: Intestinal epithelial barrier permeability can be modulated by gut microbiota, composition and metabolism. The aim of the study was to examine the in vitro effect of fermentation supernatants (FS) from Pleurotus eryngii lyophilized mushrooms (PEWS) cultivated in wheat straw and of their enriched in β-glucan extracts (PEWSE) by fecal microbiota, on the expression levels of tight junctions (TJs) genes in Caco-2 cells monolayer stimulated by bacterial lipopolysaccharides (LPS).

Deletion of intestinal epithelial AMP-activated protein kinase alters distal colon permeability but not glucose homeostasis

ObjectiveThe intestinal epithelial barrier (IEB) restricts the passage of microbes and potentially harmful substances from the lumen through the paracellular space, and rupture of its integrity is associated with a variety of gastrointestinal disorders and extra-digestive diseases. Increased IEB permeability has been linked to disruption of metabolic homeostasis leading to obesity and type 2 diabetes. Interestingly, recent studies have uncovered compelling evidence that the AMP-activated protein kinase (AMPK) signaling pathway plays an important role in maintaining epithelial cell barrier function. However, our understanding of the function of intestinal AMPK in regulating IEB and glucose homeostasis remains sparse. MethodsWe generated mice lacking the two α1 and α2 AMPK catalytic subunits specifically in intestinal epithelial cells (IEC AMPK KO) and determined the physiological consequences of intestinal-specific deletion of AMPK in response to high-fat diet (HFD)-induced obesity. We combined histological, functional, and integrative analyses to ascertain the effects of gut AMPK loss on intestinal permeability in vivo and ex vivo and on the development of obesity and metabolic dysfunction. We also determined the impact of intestinal AMPK deletion in an inducible mouse model (i-IEC AMPK KO) by measuring IEB function, glucose homeostasis, and the composition of gut microbiota via fecal 16S rRNA sequencing. ResultsWhile there were no differences in in vivo intestinal permeability in WT and IEC AMPK KO mice, ex vivo transcellular and paracellular permeability measured in Ussing chambers was significantly increased in the distal colon of IEC AMPK KO mice. This was associated with a reduction in pSer425 GIV phosphorylation, a marker of leaky gut barrier. However, the expression of tight junction proteins in intestinal epithelial cells and pro-inflammatory cytokines in the lamina propria were not different between genotypes. Although the HFD-fed AMPK KO mice displayed suppression of the stress polarity signaling pathway and a concomitant increase in colon permeability, loss of intestinal AMPK did not exacerbate body weight gain or adiposity. Deletion of AMPK was also not sufficient to alter glucose homeostasis or the acute glucose-lowering action of metformin in control diet (CD)- or HFD-fed mice. CD-fed i-IEC AMPK KO mice also presented higher permeability in the distal colon under homeostatic conditions but, surprisingly, this was not detected upon HFD feeding. Alteration in epithelial barrier function in the i-IEC AMPK KO mice was associated with a shift in the gut microbiota composition with higher levels of Clostridiales and Desulfovibrionales. ConclusionsAltogether, our results revealed a significant role of intestinal AMPK in maintaining IEB integrity in the distal colon but not in regulating glucose homeostasis. Our data also highlight the complex interaction between gut microbiota and host AMPK.

Analysis of enteric nervous system and intestinal epithelial barrier to predict complications in Hirschsprung\u2019s disease

In Hirschsprung’s disease (HSCR), postoperative course remains unpredictable. Our aim was to define predictive factors of the main postoperative complications: obstructive symptoms (OS) and Hirschsprung-associated enterocolitis (HAEC). In this prospective multicentre cohort study, samples of resected bowel were collected at time of surgery in 18 neonates with short-segment HSCR in tertiary care hospitals. OS and HAEC were noted during postoperative follow-up. We assessed the enteric nervous system and the intestinal epithelial barrier (IEB) in ganglionic segments by combining immunohistochemical, proteomic and transcriptomic approaches, with functional ex vivo analysis of motility and para/transcellular permeability. Ten HSCR patients presented postoperative complications (median follow-up 23.5 months): 6 OS, 4 HAEC (2 with OS), 2 diarrhoea (without OS/HAEC). Immunohistochemical analysis showed a significant 41% and 60% decrease in median number of nNOS-IR myenteric neurons per ganglion in HSCR with OS as compared to HSCR with HAEC/diarrhoea (without OS) and HSCR without complications (p = 0.0095; p = 0.002, respectively). Paracellular and transcellular permeability was significantly increased in HSCR with HAEC as compared to HSCR with OS/diarrhoea without HAEC (p = 0.016; p = 0.009) and HSCR without complications (p = 0.029; p = 0.017). This pilot study supports the hypothesis that modulating neuronal phenotype and enhancing IEB permeability may treat or prevent postoperative complications in HSCR.

Contributions of HO-1-Dependent MAPK to Regulating Intestinal Barrier Disruption.

The mitogen-activated protein kinase (MAPK) pathway controls intestinal epithelial barrier permeability by regulating tight junctions (TJs) and epithelial cells damage. Heme oxygenase-1 (HO-1) and carbon monoxide (CO) protect the intestinal epithelial barrier function, but the molecular mechanism is not yet clarified. MAPK activation and barrier permeability were studied using monolayers of Caco-2 cells treated with tissue necrosis factor α (TNF-α) transfected with FUGW-HO-1 or pLKO.1-sh-HO-1 plasmid. Intestinal mucosal barrier permeability and MAPK activation were also investigated using carbon tetrachloride (CCl4) administration with CoPP (a HO-1 inducer), ZnPP (a HO-1 inhibitor), CO releasing molecule 2 (CORM-2), or inactived-CORM-2-treated wild-type mice and mice with HO-1 deficiency in intestinal epithelial cells. TNF-α increased epithelial TJ disruption and cleaved caspase-3 expression, induced ERK, p38, and JNK phosphorylation. In addition, HO-1 blocked TNF-α-induced increase in epithelial TJs disruption, cleaved caspase-3 expression, as well as ERK, p38, and JNK phosphorylation in an HO-1-dependent manner. CoPP and CORM-2 directly ameliorated intestinal mucosal injury, attenuated TJ disruption and cleaved caspase-3 expression, and inhibited epithelial ERK, p38, and JNK phosphorylation after chronic CCl4 injection. Conversely, ZnPP completely reversed these effects. Furthermore, mice with intestinal epithelial HO-1 deficient exhibited a robust increase in mucosal TJs disruption, cleaved caspase-3 expression, and MAPKs activation as compared to the control group mice. These data demonstrated that HO-1-dependent MAPK signaling inhibition preserves the intestinal mucosal barrier integrity by abrogating TJ dysregulation and epithelial cell damage. The differential targeting of gut HO-1-MAPK axis leads to improved intestinal disease therapy.

Cold stress promotes IL-33 expression in intestinal epithelial cells to facilitate food allergy development

BackgroundThe causative factors and pathogenesis of food allergy (FA) is not fully understood yet. Cold stress (CS) occurs frequently in human life that influences physiological activities in the body. In this study, we aimed to investigate the chronic CS (CS) effects on promoting the expression of IL-33 in intestinal epithelial cells. MethodsCS was carried out by placing mice at 4 °C for 1 h daily for 7 consecutive days. We developed a mouse model used to test the effects of CS on the FA development. ResultsWe found that, similar to conventional FA mouse model, CS induced the core body temperature to drop markedly in mice, increased intestinal epithelial barrier permeability and facilitated FA development. CS promoted interleukin (IL)-33 expression in intestinal epithelial cells through the adrenocorticotropic hormone (ACTH)/cortisol axis and via inducing the Il33 promoter methylation. CS facilitated the FA development in mice, that could be blocked by depletion of IL-33 expression in intestinal epithelial cells. ConclusionsCS induces IL-33 expression in intestinal epithelial cells to promote Th2 polarization in the intestinal tissues and facilitates FA development.

Palmitic Acid Affects Intestinal Epithelial Barrier Integrity and Permeability In Vitro.

Palmitic acid (PA), a long-chain saturated fatty acid, might activate innate immune cells. PA plays a role in chronic liver disease, diabetes and Crohn’s disease, all of which are associated with impaired intestinal permeability. We investigated the effect of PA, at physiological postprandial intestinal concentrations, on gut epithelium as compared to lipopolysaccharide (LPS) and ethanol, using an in vitro gut model, the human intestinal epithelial cell line Caco-2 grown on transwell inserts. Cytotoxicity and oxidative stress were evaluated; epithelial barrier integrity was investigated by measuring the paracellular flux of fluorescein, and through RT-qPCR and immunofluorescence of tight junction (TJ) and adherens junction (AJ) mRNAs and proteins, respectively. In PA-exposed Caco-2 monolayers, cytotoxicity and oxidative stress were not detected. A significant increase in fluorescein flux was observed in PA-treated monolayers, after 90 min and up to 360 min, whereas with LPS and ethanol, this was only observed at later time-points. Gene expression and immunofluorescence analysis showed TJ and AJ alterations only in PA-exposed monolayers. In conclusion, PA affected intestinal permeability without inducing cytotoxicity or oxidative stress. This effect seemed to be faster and stronger than those with LPS and ethanol. Thus, we hypothesized that PA, besides having an immunomodulatory effect, might play a role in inflammatory and functional intestinal disorders in which the intestinal permeability is altered.

Microbial hydrogen economy alleviates colitis by reprogramming colonocyte metabolism and reinforcing intestinal epithelial barrier

Abstract Several studies have found exogenous H2 treatment can alleviate inflammatory damage in inflammatory bowel disease (IBD) animal models, but the mechanisms behind it are less clearly understood. The present study aimed to investigate whether hydrogen-rich saline (HS) attenuates IBD in mice model and the underlying mechanisms. The results showed that HS administration significantly inhibit weight loss and reduce inflammatory damage in dextran sulfate sodium (DSS)-induced acute ulcerative colitis (UC) mice model. We observed that H2 regulates the composition of microbiota by up-regulating the abundance of intestinal specific short chain fatty acids (SCFAs)-producing bacteria. Increased butyrate-producing microbes activated the intracellular butyrate sensor peroxisome proliferator–activated receptor γ (PPAR-γ) signaling pathway, meanwhile decreased the epithelial expression of Nos2, the gene encoding inducible nitric oxide synthase, and thus promoted the recovery of colonic anaerobic environment. Our results also indicated that HS administration reduce intestinal epithelial barrier permeability by decreasing the dextran concentrations in serum, inhibit dysbiotic Enterobacteriaceae expansion, increasing mucus secretion in the colonic lumen, and promoting the expression of intestinal interepithelial tight junction protein. Taken together, we identified the mechanism by which exogenous H2-improved “microbial hydrogen economy” regulates metabolic reprogramming of colonocyte through microbiota-activated PPAR-γ signaling pathway to provide colonic anaerobic environment, and reinforces intestinal epithelial barrier function, thereby promoting the recovery of intestinal homeostasis.

MCL1 Is Required for Maintenance of Intestinal Homeostasis and Prevention of Carcinogenesis in Mice

Background & AimsIntestinal epithelial homeostasis depends on a tightly regulated balance between intestinal epithelial cell (IEC) death and proliferation. While the disruption of several IEC death regulating factors result in intestinal inflammation, the loss of the anti-apoptotic BCL2 family members BCL2 and BCL2L1 has no effect on intestinal homeostasis in mice. We investigated the functions of the antiapoptotic protein MCL1, another member of the BCL2 family, in intestinal homeostasis in mice.MethodsWe generated mice with IEC-specific disruption of Mcl1 (Mcl1ΔIEC mice) or tamoxifen-inducible IEC-specific disruption of Mcl1 (i-Mcl1ΔIEC mice); these mice and mice with full-length Mcl1 (controls) were raised under normal or germ-free conditions. Mice were analyzed by endoscopy and for intestinal epithelial barrier permeability. Intestinal tissues were analyzed by histology, in situ hybridization, proliferation assays, and immunoblots. Levels of calprotectin, a marker of intestinal inflammation, were measured in intestinal tissues and feces.ResultsMcl1ΔIEC mice spontaneously developed apoptotic enterocolopathy, characterized by increased IEC apoptosis, hyperproliferative crypts, epithelial barrier dysfunction, and chronic inflammation. Loss of MCL1 retained intestinal crypts in a hyperproliferated state and prevented the differentiation of intestinal stem cells. Proliferation of intestinal stem cells in MCL1-deficient mice required WNT signaling and was associated with DNA damage accumulation. By 1 year of age, Mcl1ΔIEC mice developed intestinal tumors with morphologic and genetic features of human adenomas and carcinomas. Germ-free housing of Mcl1ΔIEC mice reduced markers of microbiota-induced intestinal inflammation but not tumor development.ConclusionThe antiapoptotic protein MCL1, a member of the BCL2 family, is required for maintenance of intestinal homeostasis and prevention of carcinogenesis in mice. Loss of MCL1 results in development of intestinal carcinomas, even under germ-free conditions, and therefore does not involve microbe-induced chronic inflammation. Mcl1ΔIEC mice might be used to study apoptotic enterocolopathy and inflammatory bowel diseases.

Orexin-A Prevents Lipopolysaccharide-Induced Neuroinflammation at the Level of the Intestinal Barrier.

In states of intestinal dysbiosis, a perturbation of the normal microbiome composition, the intestinal epithelial barrier (IEB) permeability is increased as a result of the disruption of the epithelial tight junction protein network, in which occludin is mostly affected. The loss of IEB integrity promotes endotoxemia, that is, bacterial lipopolysaccharide (LPS) translocation from the intestinal lumen to the circulatory system. This condition induces an enhancement of pro-inflammatory cytokines, which leads to neuroinflammation through the gut-brain axis. Orexin-A (OX-A), a neuropeptide implicated in many physiological functions and produced mainly in the brain lateral hypothalamic area, is expressed also in several peripheral tissues. Orexin-producing neurons have been found in the myenteric plexus to project to orexin receptor 1 (OX-1R)-expressing enterocytes of the intestinal villi. In the present study we investigated the protective role of OX-A against LPS-induced increase of IEB permeability and microglia activation in both an in vivo and in vitro model of the gut-brain axis. By exploiting biochemical, immunocytochemical, immunohistochemical, and functional approaches, we demonstrate that OX-A preserves the IEB and occludin expression, thus preventing endotoxemia and subsequent neuroinflammation.

Herbal cake-partitioned moxibustion improves intestinal epithelial barrier dysfunction by suppressing TNF-α-mediated apoptosis pathway of intestinal epithelium in rats with Crohn's disease

To observe the effect of herbal cake-partitioned moxibustion (Moxi) on tumor necrosis factor (TNF)-α/ TNF receptor 1 (TNFR1)-associated death domain (TRADD) / Fas-associated death domain (FADD) pathway-mediated apoptosis of intestinal epithelial cells in Crohn's disease (CD) rats, so as to explore its underlying mechanisms in the treatment of CD. Forty-eight SD male rats were randomly divided into normal, model, Moxi and medication groups (n＝12 rats in each). The CD model was established by intra-annual perfusion of 2，4，6-trinitrobenzene sulfonic acid (TNBS) solution (TNBS∶50% alcohol＝2∶1, 3 mL/kg), once every 7 days, 4 times altogether. For rats of the Moxi group, moxibustion was given to "Tianshu" (ST25) and "Qihai" (CV6), two moxa-cones every time, once daily for 10 days. For rats of the medication group, intragastric perfusion of mesalazine solution was given twice daily for 10 days. After the treatment, the colonic epithelium tissue was sampled. The epithelial cells were purified and cultured to establish an in vitro intestinal epithelial barrier, and added with TNF-α (a pro-inflammatory factor, 100 ng/mL) in the culture medium for 24 h for making an increased epithelial permeability model. The permeability of intestinal epithelial cell barrier was evaluated by detecting the fluorescence yellow transmittance of the TNF-α-incubated cell medium. Western blot was used to detect the expression levels of TNFR1, TRADD, receptor-interacting protein 1 (RIP1), FADD and zinc finger protein A20 (A20, a ubiquitination enzyme for inhibiting activation of TRADD and RIP1) of the cultured intestinal epithelium cells. The apoptosis of the TNF-α-incubated intestinal epithelial cells was detected by flow cytometry. After modeling and compared with the normal group, the fluorescence yellow transmittance of intestinal epithelia cells, apoptosis rate, and expression levels of TNFR1, TRADD, and RIP1 proteins were significantly increased (P<0.001, P<0.01), and the expression of A20 was significantly decreased (P<0.01) in the model group. In comparison with the model group, the fluorescence yellow transmittance of intestinal epithelial cells, the apoptosis rate and expression levels of TRADD, RIP1 and FADD were remarkably down-regulated (P<0.001, P<0.01), and the expression of A20 was significantly up-regulated (P<0.01) in both the Moxi and medication groups. Herbal cake-partitioned moxibustion may down-regulate the permeability of intestinal epithelial barrier and the apoptosis of intestinal epithelial cells by way of suppressing TNF-α-mediated cellular apoptosis pathway of intestinal epithelium in CD rats.

IFN-γ, IL-17A, or zonulin rapidly increase the permeability of the blood-brain and small intestinal epithelial barriers: Relevance for neuro-inflammatory diseases.

Breakdown of the blood-brain barrier (BBB) precedes lesion formation in the brains of multiple sclerosis (MS) patients. Since recent data implicate disruption of the small intestinal epithelial barrier (IEB) in the pathogenesis of MS, we hypothesized that the increased permeability of the BBB and IEB are mechanistically linked. Zonulin, a protein produced by small intestine epithelium, can rapidly increase small intestinal permeability. Zonulin blood levels are elevated in MS, but it is unknown whether zonulin can also disrupt the BBB. Increased production of IL-17A and IFN-γ has been implicated in the pathogenesis of MS, epilepsy, and stroke, and these cytokines impact BBB integrity after 24 h. We here report that primary human brain microvascular endothelial cells expressed the EGFR and PAR2 receptors necessary to respond to zonulin, and that zonulin increased BBB permeability to a 40 kDa dextran tracer within 1 h. Moreover, both IL-17A and IFN-γ also rapidly increased BBB and IEB permeability. By using confocal microscopy, we found that exposure of the IEB to zonulin, IFN-γ, or IL-17A invitro rapidly modified the localization of the TJ proteins, ZO-1, claudin-5, and occludin. TJ disassembly was accompanied by marked depolymerization of the peri-junctional F-actin cytoskeleton. Our data indicate that IFN-γ, IL-17A, or zonulin can increase the permeability of the IEB and BBB rapidly invitro, by modifying TJs and the underlying actin cytoskeleton. These observations may help clarify how the gut-brain axis mediates the pathogenesis of neuro-inflammatory diseases.

Cytokine Tuning of Intestinal Epithelial Function.

The intestine serves as both our largest single barrier to the external environment and the host of more immune cells than any other location in our bodies. Separating these potential combatants is a single layer of dynamic epithelium composed of heterogeneous epithelial subtypes, each uniquely adapted to carry out a subset of the intestine’s diverse functions. In addition to its obvious role in digestion, the intestinal epithelium is responsible for a wide array of critical tasks, including maintaining barrier integrity, preventing invasion by microbial commensals and pathogens, and modulating the intestinal immune system. Communication between these epithelial cells and resident immune cells is crucial for maintaining homeostasis and coordinating appropriate responses to disease and can occur through cell-to-cell contact or by the release or recognition of soluble mediators. The objective of this review is to highlight recent literature illuminating how cytokines and chemokines, both those made by and acting on the intestinal epithelium, orchestrate many of the diverse functions of the intestinal epithelium and its interactions with immune cells in health and disease. Areas of focus include cytokine control of intestinal epithelial proliferation, cell death, and barrier permeability. In addition, the modulation of epithelial-derived cytokines and chemokines by factors such as interactions with stromal and immune cells, pathogen and commensal exposure, and diet will be discussed.