Increase In Intestinal Permeability Research Articles

Polystyrene micro- and nanoparticles exposure induced anxiety-like behaviors, gut microbiota dysbiosis and metabolism disorder in adult mice

Plastics have been proven to be a potential threat to the ecosystem, and their toxicity mechanism is still uncertain. In the ecological environment, plastics can be degraded into microplastics (MPs) and nanoplastics (NPs), which can be contaminated and ingested through the food chain. MPs and NPs are associated with severe intestinal injury, intestinal microbiota disorder, and neurotoxicity, but it is still unclear whether MPs- and NPs-induced intestinal microbiota dysbiosis will affect the brain through the gut-brain axis. In the current study, we determined the effects of exposure to polystyrene (PS)-MPs and PS-NPs on anxiety-like behaviors and explored the underlying mechanisms. This study explored the behavioral effects of 30-day and 60-day exposure to PS-NPs and PS-MPs using the open field test (OFT) and elevated plus maze (EPM) test. Behavioral tests showed PS-NPs and PS-MPs treatment remarkedly induced anxiety-like behaviors compared with the control group. Using 16 S rRNA gene sequencing and untargeted metabolomics analyses, we observed that PS-MPs and PS-NPs exposure reduced the beneficial gut microbiota expression level, such as Lachnoclostridium and Lactobacillus, and increased the conditionally pathogenic bacteria expressions level, such as Proteobacteria, Actinobacteria, and Desulfovibrio. In addition, PS-NPs and PS-MPs reduce intestinal mucus secretion and increase intestinal permeability. The results of serum metabonomics suggested that the metabolic pathways, such as ABC transporter pathways, aminoacyl-tRNA biosynthesis, biosynthesis of amino acids, and bile secretion were enriched after PS-NPs and PS-MPs treatment. Besides, neurotransmitter metabolites were also altered by PS-NPs and PS-MPs. It is noteworthy that the correlation analysis showed that the disorder of intestinal microbiota was related to anxiety-like behaviors and neurotransmitter metabolites disorder. The regulation of intestinal microbiota may be a promising treatment strategy for PS-MPs- and PS-NPs-induced anxiety disorder.

Faecal calprotectin: Marker of intestinal inflammatory process in anorexia nervosa? A preliminary study.

Anorexia Nervosa (AN) is a serious and potentially lethal mental disorder characterised by a deliberate quest to reduce one's weight. It can have multiple physical and psychological consequences. The clinical presentation of AN can include gastrointestinal symptoms, however, the pathophysiology of these symptoms in the context of AN remains uncertain. It is hypothesised that patients with AN may have an increase in intestinal permeability, which could lead to an increase in faecal calprotectin (fCP), a marker of intestinal inflammation. No relation between AN and elevation of fCP has been previously described in literature. Eight patients hospitalised for AN have a dosage of fCP. Calprotectine was found to be elevated in 50% of cases, with or without any underlying comorbid gastrointestinal disease. Only the duration of illness tended to be associated with the increase in fCP suggesting a greater alteration as a function related to the time of denutrition. Although these findings provide insights in the potential pathophysiology of gastrointestinal symptoms in AN, further studies that evaluate the factors associated with elevated fCP in patients with AN are needed.

KALP BAĞIRSAK EKSENİ

Many interactions play a role in the gut-heart axis. These include intestinal epithelial dysfunction, dysbiosis, butyrate-producing bacteria, bile acids, and intestinal microbe-derived metabolites. In patients with heart failure (HF), mucosal malabsorption, intestinal wall edema and barrier dysfunction develop as a result of microcirculation disorders in the gut due to decreased perfusion, increased congestion and sympathetically mediated vasoconstriction. Toxic, pathogenic, immunogenic and inflammatory factors, through the increase in intestinal permeability as a result of damaged tight junctions in the intestine, pass through the mucosa and reach the systemic circulation, causing local-systemic inflammation. Many factors that cause dysbiosis by changing the intestinal flora, which are frequently seen in HF, lead to bacterial overgrowth, bacterial translocation and formation of many toxic substances, including lipopolysaccharide (LPS), trimethylamine N-oxide (TMAO), p-cresylsulfate (PCS) and indoxyl sulfate (IS). Depending on the increase in intestinal permeability, these toxic substances reach the systemic circulation; it increases the risk of atherosclerosis by playing a role in thrombosis, platelet invasion, foam cell formation and inflammation processes. Decreased levels of butyrate, one of the short-chain fatty acids that have many effects on the gastrointestinal tract, including maintaining intestinal barrier integrity; It promotes foam cell formation, exacerbates dysbiosis, and plays a role in the disruption of intestinal barrier function, causing endotoxins to reach the general circulation. With this review, it is aimed to inform about the physiopathological processes in the gut-heart axis, in the light of the current literature.

Adherent-invasive Escherichia coli LF82 aggravated intestinal inflammation in colitis mice by affecting the gut microbiota and Th17/Treg cell differentiation balance.

The imbalance of Th17 and Treg cell differentiation, intestinal flora imbalance, and intestinal mucosal barrier damage may be important links in the occurrence and development of inflammatory bowel disease (IBD) since Th17 and Treg differentiation are affected by the intestinal flora. This study aimed to explore the effect of Escherichia coli (E. coli) LF82 on the differentiation of Th17 and Treg cells and the role of the intestinal flora in mouse colitis. The effects of E. coli LF82 infection on intestinal inflammation were evaluated by analyzing the disease activity index, histology, myeloperoxidase activity, FITC-D fluorescence value, and claudin-1 and ZO-1 expression. The effects of E. coli LF82 on the Th17/Treg balance and intestinal flora were analyzed by flow cytometry and 16S rDNA sequencing. Inflammatory markers, changes in the intestinal flora, and Th17/Treg cells were then detected after transplanting fecal bacteria from normal mice into colitis mice infected by E. coli LF82. We found that E. coli LF82 infection can aggravate the intestinal inflammation of mice colitis, destroy their intestinal mucosal barrier, increase intestinal mucosal permeability, and aggravate the imbalance of Th17/Treg differentiation and the disorder of intestinal flora. After improving the intestinal flora imbalance by fecal bacteria transplantation, intestinal inflammation and intestinal mucosal barrier damage were reduced, and the differentiation balance of Th17 and Treg cells was restored. This study showed that E. coli LF82 infection aggravates intestinal inflammation and intestinal mucosal barrier damage in colitis by affecting the intestinal flora composition and indirectly regulating the Th17 and Treg cell differentiation balance.

Bifidobacterium bifidum prevents the IL-1B induced increase in intestinal permeability by a novel mechanism: TLR-2 dependent activation of PPAR-gamma and inhibition of NF-kB signaling pathway

Background: Bifidobacterium bifidum (BB) is one of the most common probiotic bacteria strains that have been shown to have protective effects against intestinal inflammation, primarily by enhancing the intestinal epithelial tight junction (TJ) barrier function. IL-1β contributes to the development of intestinal inflammation in inflammatory bowel disease (IBD) in part by causing an increase in intestinal permeability. The IL-1β induced increase in intestinal epithelial TJ permeability is mediated by an NF-κB-dependent activation of myosin light chain kinase (MLCK) gene and disruption of the intestinal TJ barrier. The peroxisome proliferator-activated receptor gamma (PPAR-γ) is an important nuclear receptor in enterocytes which is known to have anti-inflammatory activity by interfering with NF-κB activation. However, the role of BB/PPAR-γ (and the possible mechanism involved) in protecting against IL-1β increase in intestinal permeability remain unclear. Aims: The major purpose of this study was to delineate the protective effect of BB against the IL-1β induced increase in intestinal TJ permeability and the intracellular mechanism involved. Methods: Filter-grown Caco-2 monolayers ( in vitro) and recycling intestinal perfusion of live mice ( in vivo) were used to assess intestinal permeability by using a paracellular marker (dextran-10kDa). Results: IL-1β caused a rapid activation of NF-κB and NF-κB and MLCK-dependent increase in intestinal epithelial TJ permeability in vitro and in vivo. A specific strain of BB (1x108 CFU/ml) referred to as BB1 strain inhibited the IL-1β increase in intestinal TJ permeability while strain BB4 had no effect. Other BB strains examined (5 total strains including BB4) did not inhibit the IL-1β increase in intestinal TJ permeability, suggesting that the BB enhancement of Caco-2 TJ barrier function was strain-specific. BB1, not BB4 enhancement of the TJ barrier was associated with an increase in TLR-2 expression, and PPAR-γ activation; BB1 also inhibited NF-kB activation and MLCK activity in Caco-2 monolayers and in mouse enterocyte. The inhibitory effect of BB on 1L-1β induced increase intestinal permeability, NF-κB activation, MLCK expression and activity was blocked by siRNA-induced knockdown of TLR-2 or PPAR-γ in Caco-2 monolayers. We also generated Villin-cre intestinal epithelial specific PPAR-γ knockout mice to study the involvement of enterocytes TLR-2/PPAR-γ on BB1 effect. BB1 did not inhibit the IL-1β-induced activation of NF-κB, increase in MLCK expression and increase in mouse intestinal permeability in TLR-2 or PPAR-γ intestinal deficient mice. Conclusion: These studies provide a novel insight into the BB the protective mechanism against the IL-1β-induced increase in intestinal TJ permeability in -vitro and in-vivo. Our data show that BB protects against the IL-1β induced increase in intestinal TJ permeability by a novel mechanism involving TLR-2/PPAR-g mediated inhibition of NF-kB and MLCK gene activatiation. NIH funding This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

High sodium diet and intestinal permeability in young, healthy adults

High sodium diets (HSD) have been shown to both increase blood pressure (BP) and lead to gut dysbiosis in adults. Gut dysbiosis can increase intestinal permeability (IP), allowing for translocation of bacteria and their products into the systemic circulation. There, bacteria and their products are recognized by the immune system and can activate pro-inflammatory pathways such as TH17 in a dose dependent manner. HSD has been shown to activate those pathways and increase BP in men. However, the effect of a HSD on IP has not been explored as a potential mechanism of HSD induced BP changes. The objective of this research was to investigate if a HSD can increase IP and inflammatory markers. We hypothesized that plasma concentrations of two common biomarkers of IP, zonulin and LPS binding protein (LBP), and proinflammatory TH17 lymphocytes would increase, while anti-inflammatory Treg lymphocytes would decrease on a HSD compared to a recommended sodium diet (RSD). Twenty-seven healthy participants (15W/12M, age 24±3 yrs, BP 112±9/65±6 mmHg) underwent a 10-day HSD (6,900mg sodium/d, supplemented by salt pills) and a 10-day RSD (2,300 mg sodium/d) intervention in randomized order. On day 9 of each intervention, participants collected urine and wore an ambulatory BP monitor for 24hr. Blood was collected to measure plasma zonulin and LBP by ELISA. TH17 and Treg populations were characterized from PBMCs by flow cytometry. Paired t-tests were used to compare variables on day 9 between diets. Pearson correlations were used to assess the relation between markers of IP and BP. Twenty-four-hour sodium excretion was increased on the HSD (RSD: 135±57 mmol/24h, HSD: 290±68 mmol/24h, p<0.0001). Mean 24-hr SBP (RSD: 116±10, HSD: 117±11 p=0.70) and DBP (RSD: 66±6 mmHg, HSD: 66±5 mmHg, p=0.84) were not different between diets. Plasma zonulin concentrations (RSD: 2.6±1.6 ng/ml, HSD: 2.6±1.5 ng/ml p=0.88) and LBP concentrations (RSD: 3.2±1.7 μg/ml, HSD: 3.2±1.8 μg/ml, p=0.72) were not different between diets. The TH17/Treg ratio was not different between diets (RSD: 0.54±0.51, HSD: 0.41±0.38; p=0.21) however the ratio was inversely associated with zonulin (r=-0.57 p=0.04), and LBP (r=-0.61; p=0.012) on HSD when controlling for age and sex but not on RSD (all p>0.05). These data suggest that 10 days of a HSD did not change IP, BP or T lymphocyte populations in young, healthy individuals. However, the TH17/Treg ratio was associated with zonulin and LBP suggesting that the immune system may respond to changes in IP. Future research is needed to determine whether a HSD can affect IP and inflammation in salt sensitive populations. NIH Grants R01 HL145055 and P20GM113125 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Intestinal Epithelial Cell PTPN2 Restricts Intestinal Permeability and Tight-Junction Remodelling Caused by Adherent-invasive E. coli in Mice

Background: Alterations in epithelial tight junction proteins can compromise the ability of the intestinal mucosa to act as a barrier between luminal contents and underlying tissues. The resulting increase in intestinal permeability can give rise to conditions such as inflammatory bowel disease (IBD). Additionally, expansion of pathobionts, such as adherent-invasive Escherichia coli (AIEC), are associated with IBD. Mice deficient in the IBD gene candidate, Ptpn2, exhibit increased intestinal permeability and a pronounced expansion of a novel murine AIEC ( mAIEC). This study aimed to investigate how PTPN2 expression in intestinal epithelial cells protects intestinal barrier properties after mAIEC infection. Methods: Tamoxifen-inducible, intestinal epithelial cell (IEC)-specific knockout mice ( Ptpn2 ΔIEC ) and control littermates ( Ptpn2 fl/fl ) were infected with either PBS, non-invasive E. coli K12, or mcherry-tagged mAIEC ( mAIEC red ). FITC-Dextran (FD4) and Rhodamine-Dextran (RD70) permeability probes were administered by oral-gavage and serum collected after 5hrs. Results: Ptpn2 ΔIEC mice exhibited intestinal region-specific higher mAIEC red - but not K12 - bacterial load compared to Ptpn2 fl/fl mice ( P=0.038). E. coli K12 increased FD4 but not RD70 permeability in Ptpn2 ΔIEC mice compared with Ptpn2 fl/fl mice ( P=0.0025). mAIEC red also had no effect on RD70, but caused an even greater increase in FD4 permeability in Ptpn2 ΔIEC vs. Ptpn2 fl/fl mice ( P< 0.0001). Ptpn2 fl/fl mice infected with mAIEC red also exhibited increased FD4 permeability compared to PBS-treated littermates ( P =0.0210). To identify if increased FD4 permeability was due to changes in expression of relevant tight-junction proteins, we performed western blot analysis on isolated IECs. Ptpn2 ΔIEC + mAIEC red mice had decreased expression of occludin ( P=0.0030) and the adherens junction protein, E-cadherin ( P=0.0142). Expression of the cation pore, claudin-2, was increased in PBS and K12-infected Ptpn2 ΔIEC vs. Ptpn2 fl/fl mice ( P=0.015). Interestingly, mAIEC red increased claudin-2 expression in Ptpn2 fl/fl mice compared to PBS controls ( P=0.0105) however, Ptpn2 ΔIEC + mAIEC red mice exhibited decreased claudin-2 levels compared to Ptpn2 fl/fl + mAIEC red mice ( P=0.0436). Immunofluorescence staining of claudin-2 confirmed reduced claudin-2 in Ptpn2 ΔIEC + mAIEC red mice. Furthermore, we observed gaps in ZO-1 membrane localization in Ptpn2 ΔIEC mice treated with PBS and K12 but not in their Ptpn2 fl/fl controls. ZO-1 membrane localization was dramatically depleted in Ptpn2 ΔIEC + mAIEC red mice compared to the Ptpn2 fl/fl + mAIEC red controls. Conclusion: Intestinal epithelial PTPN2 exhibits a major role in maintaining intestinal homeostasis by preserving intestinal barrier function and the integrity of tight-junction protein localization. Given that loss of PTPN2 in vivo also provokes expansion of adherent-invasive E. coli, these studies indicate that PTPN2 is an important mediator of host-microbiota crosstalk. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Maternal Vitamin A deficiency during pregnancy and lactation induced damaged intestinal structure and intestinal flora homeostasis in offspring mice.

The small intestine serves as the first channel of dietary Vitamin A (VA) and the unique organ of VA absorption and metabolism. However, there have not been extensive investigations on the exact mechanisms within VA-related changes in intestinal metabolic disorders. This research is designed to analyze whether and how VA affects intestinal metabolic phenotypes. Male C57BL/6 mice after weaning were randomly fed a VA control diet (VAC) or a VA-deficient diet (VAD) during the entire pregnancy and lactation process. After a total of 11 weeks, cohorts of VA deprived were next fed to a VA control diet (VAD-C) for another 8 weeks. The concentration of retinol was measured by a high-performance liquid chromatography system. The 16S gene sequencing was used to evaluate the intestinal microbiota changes. Through the use of histological staining, western blots, quantitative PCR, and enzyme-linked immunosorbent assays, the intestinal morphology, inflammatory factors, and intestinal permeability were all evaluated. Following the decrease of the tissue VA levels, VAD mice show a decrease in tissue VA levels, community differences, and the richness and diversity of intestinal microbiota. VAD diet-driven changes occur in intestinal microbiota, accompanied by a higher mRNA expression of intestinal inflammatory cytokines and an increase in intestinal permeability. As dietary VA is reintroduced into VAD diet-fed mice, the tissue VA levels, inflammatory response, and intestinal homeostasis profiles are all restored, which are similar to those found after the occurrence of VA-controlled changes within intestinal microbiota. VA deficiency caused the imbalance of intestinal metabolic phenotypes through a mechanism involving changes in intestinal microbiota. It is thought that intestinal microbiota metabolic influences represent a new salient and additional mechanism, which can be used as a new method to achieve the onset and treatment of the effect of VAD on intestinal homeostasis impairment.

Bile Acid Derivatives Effectively Prevented High-Fat Diet-Induced Colonic Barrier Dysfunction.

Bile acids (BAs) have recently emerged as important regulators of many physiological and pathological processes. However, the change of colonic BAs induced by high-fat diet (HFD) and their effects on colonic barrier function remain to be further elucidated. C57BL/6 mice are divided into two groups and feed 12weeks with diets differing for fat content. Higher levels of serum diamine oxidase (DAO) activity, endotoxin (ET), and d-lactate (d-LA) are observed in HFD-fed mice, indicating an increase in intestinal permeability. Real-time quantitative PCR and western blot analyses demonstrate that HFD downregulates tight junction proteins (TJs, including zonula-occludens 1 [ZO-1], Occludin, and Claudin1) and Muc2 expression in the colon. The colonic BA profiles are analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). HFD induces an increase in primary BAs but a decrease in secondary BAs. In human colonic cell line Caco-2, secondary BAs (deoxycholic acid [DCA], lithocholic acid [LCA], their 3-oxo- and iso- derivates) upregulate the expression of TJs and counteract DSS-induced increase in intestinal permeability at physiological concentrations. IsoDCA and isoLCA are the most effective ones. Moreover, supplementation of isoDCA or isoLCA also effectively prevents HFD-induced colonic barrier dysfunction in mice. These results demonstrate that secondary BAs (especially isomerized derivatives) may be important protectors for the colonic barrier function.

An Association Between the Intestinal Permeability Biomarker Zonulin and the Development of Diabetic Retinopathy in Type II Diabetes Mellitus.

Increased intestinal permeability (IP) and gut microbiota dysbiosis have been implicated in low-grade chronic inflammation, which is an important factor in the pathogenesis of diabetic retinopathy (DR). This study aims to demonstrate the relationship between the IP biomarker zonulin and DR in patients with type 2 diabetes mellitus (T2DM). This study was conducted with a total of 89 T2DM patients, including 33 non-DR, 28 with non-proliferative DR (NPDR), and 28 with proliferative DR (PDR), and 32 healthy controls. Zonulin levels were determined from blood samples using an enzyme-linked immunosorbent assay kit. There was no difference between the four groups in terms of age (p=0.236), gender (p=0.952), and body mass index (p=0.134) of the participants. Zonulin levels were significantly higher in the PDR group compared to the other three groups, as well as in the non-DR and NPDR groups compared to the control group. In multivariate logistic regression analysis, zonulin was found to be an independent predictor of DR (odds ratio: 1,781, 95% confidence interval: 1,122-2,829, p=0.014). Our study showed that elevated zonulin levels may play a significant role in the development of DR, particularly during the transition to the proliferative stage. This suggests that regulation of IP could be one of the targets of DR treatment. More studies are needed to determine whether a eubiotic gut microbiota and IP have a direct relationship with DR.

75 Effect of probiotic supplementation on intestinal permeability in subjects with overweight and obesity: A systematic review of randomized controlled trials

OBJECTIVES/GOALS: Obesity is associated with gut dysbiosis, inflammation, and increased intestinal permeability. Probiotic consumption may reverse these outcomes. The goal of this study is to evaluate the evidence linking probiotic consumption to changes in intestinal permeability in subjects with overweight or obesity. METHODS/STUDY POPULATION: Articles were searched in Pubmed, Web of Science, and CAB Direct through February 2022 using search terms: intestinal permeability, overweight or obesity, and probiotic supplementation. 694 articles were exported, and 289 duplicates were identified. Titles and abstract were screened in the 405 remaining references by two investigators to determine eligibility. Eligible studies had data extracted on study participant characteristics, probiotic strain used, probiotic dosage, length of intervention, and intestinal barrier outcomes. Results were summarized in tabular form based on intestinal permeability response to probiotics. Quality of the studies was assessed based on Cochrane–Risk of Bias’ tool (RoB2). RESULTS/ANTICIPATED RESULTS: Thirteen eligible studies were identified. Probiotic genera included Akkermansia, Bifidobacterium, Lactobacillus, Streptococcus, Lactococcus, and Bacillus. Single strain probiotics were used in 3 studies, while the other 10 used multi-strain formulas. Dosage and length of probiotic supplementation ranged from 2.4 x 10^7 to 5 x 10^10 CFU/person/day and 3 to 26 weeks, respectively. The most widely used gut permeability outcomes were serum lipopolysaccharide (LPS) (n=10) and mixed sugar solution consumption with urine analysis (n=6). Five of the 10 studies reported decreases in serum LPS following probiotic consumption, while the other 5 showed no effect. Urinary lactulose/mannitol ratio decreased in 1 of the 4 studies, and urinary lactulose percent decreased in 2 studies. DISCUSSION/SIGNIFICANCE: Probiotic supplementation may be remediating an obesity-induced increase in intestinal permeability as evidenced from the effect on serum LPS and mixed sugar solution assays. However, additional studies are needed to further clarify which strain of probiotic bacteria is most effective and the optimal intervention length in subjects with obesity.

Cis-Nerolidol Inhibits MAP Kinase and NF-κB Signaling Pathways and Prevents Epithelial Tight Junction Dysfunction in Colon Inflammation: In Vivo and In Vitro Studies

Inflammation of the GI tract leads to compromised epithelial barrier integrity, which increases intestine permeability. A compromised intestinal barrier is a critical event that leads to microbe entry and promotes inflammatory responses. Inflammatory bowel diseases that comprise Crohn's disease (CD) and ulcerative colitis (UC) show an increase in intestinal permeability. Nerolidol (NED), a naturally occurring sesquiterpene alcohol, has potent anti-inflammatory properties in preclinical models of colon inflammation. In this study, we investigated the effect of NED on MAPKs, NF-κB signaling pathways, and intestine epithelial tight junction physiology using in vivo and in vitro models. The effect of NED on proinflammatory cytokine release and MAPK and NF-κB signaling pathways were evaluated using lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophages. Subsequently, the role of NED on MAPKs, NF-κB signaling, and the intestine tight junction integrity were assessed using DSS-induced colitis and LPS-stimulated Caco-2 cell culture models. Our result indicates that NED pre-treatment significantly inhibited proinflammatory cytokine release, expression of proteins involved in MAP kinase, and NF-κB signaling pathways in LPS-stimulated RAW macrophages and DSS-induced colitis. Furthermore, NED treatment significantly decreased FITC-dextran permeability in DSS-induced colitis. NED treatment enhanced tight junction protein expression (claudin-1, 3, 7, and occludin). Time-dependent increases in transepithelial electrical resistance (TEER) measurements reflect the formation of healthy tight junctions in the Caco-2 monolayer. LPS-stimulated Caco-2 showed a significant decrease in TEER. However, NED pre-treatment significantly prevented the fall in TEER measurements, indicating its protective role. In conclusion, NED significantly decreased MAPK and NF-κB signaling pathways and decreased tight junction permeability by enhancing epithelial tight junction protein expression.

Protective Effect of Limosilactobacillus fermentum ME-3 against the Increase in Paracellular Permeability Induced by Chemotherapy or Inflammatory Conditions in Caco-2 Cell Models.

Chemotherapy- or inflammation-induced increase in intestinal permeability represents a severe element in disease evolution in patients suffering from colorectal cancer and gut inflammatory conditions. Emerging data strongly support the gut microbiota's role in preserving intestinal barrier integrity, whilst both chemotherapy and gut inflammation alter microbiota composition. Some probiotics might have a strong re-balancing effect on the gut microbiota, also positively affecting intestinal barrier integrity. In this study, we asked whether Limosilactobacillus fermentum ME-3 can prevent the intestinal paracellular permeability increase caused by the chemotherapeutic drug Irinotecan or by inflammatory stimuli, such as lipopolysaccharide (LPS). As an intestinal barrier model, we used a confluent and polarized Caco-2 cell monolayer and assessed the ME-3-induced effect on paracellular permeability by transepithelial electrical resistance (TEER) and fluorescent-dextran flux assays. The integrity of tight and adherens junctions was examined by confocal microscopy analysis. Transwell co-cultures of Caco-2 cells and U937-derived macrophages were used as models of LPS-induced intestinal inflammation to test the effect of ME-3 on release of the pro-inflammatory cytokines Tumor Necrosis Factor α, Interleukin-6, and Interleukin-8, was measured by ELISA. The results demonstrate that ME-3 prevents the IRI-induced increment in paracellular permeability, possibly by modulating the expression and localization of cell junction components. In addition, ME-3 inhibited both the increase in paracellular permeability and the release of pro-inflammatory cytokines in the co-culture model of LPS-induced inflammation. Our findings sustain the validity of L. fermentum ME-3 as a valuable therapeutic tool for preventing leaky gut syndrome, still currently without an available specific treatment.

Faecal Microbiota Transplantation, Paving the Way to Treat Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is currently the most prevalent cause of chronic liver disease (CLD). Currently, the only therapeutic recommendation available is a lifestyle change. However, adherence to this approach is often difficult to guarantee. Alteration of the microbiota and an increase in intestinal permeability seem to be key in the development and progression of NAFLD. Therefore, the manipulation of microbiota seems to provide a promising therapeutic strategy. One way to do so is through faecal microbiota transplantation (FMT). Here, we summarize the key aspects of FMT, detail its current indications and highlight the most recent advances in NAFLD.

Microbiota-gut-adipose axis: butyrate-mediated the improvement effect on inflammatory response and fatty acid oxidation dysregulation attenuates obesity in sleep-restricted mice

BackgroundInsufficient sleep was considered as a substantial cause of obesity. The present study further explored the mechanism whereby sleep restriction (SR)-mediated intestinal dysbiosis induced metabolic disorder and ultimately lead to obesity in mice and the improvement effect of butyrate exerting on it. MethodsA continuous 3 months SR mouse model with or without butyrate supplementation and fecal microbiota transplantation to explore the key role of intestinal microbiota in butyrate improving inflammatory response in inguinal white adipose tissue (iWAT) and fatty acid oxidation dysfunction in brown adipose tissue (BAT), further ameliorating SR-induced obesity. ResultsSR-mediated gut microbiota dysbiosis (down-regulation in butyrate level and up-regulation in LPS level) induced intestinal permeability increase and inflammatory response in iWAT and fatty acid oxidation dysfunction in BAT, ultimately resulting in obesity. Further, we demonstrated butyrate ameliorated gut microbiota homeostasis, suppressed inflammatory response via GPR43/LPS/TLR4/MyD88/GSK-3β/β-catenin loop in iWAT and restored fatty acid oxidation function via HDAC3/PPARα/PGC-1α/UCP1/Calpain1 pathway in BAT, ultimately reversing SR-induced obesity. ConclusionsWe revealed that gut dysbiosis is a key factor for SR-induced obesity and provided a better understanding of the effects of butyrate. We further expected that reversing SR-induced obesity by improving microbiota–gut–adipose axis disorder could be a possible treatment for metabolic diseases.

Immunomodulatory effects of different strains of Lactococcus lactis in DSS-induced colitis.

Inflammatory bowel diseases (IBD) are gastrointestinal disorders characterized by a breakdown in intestinal homeostasis by inflammatory immune responses to luminal antigens. Novel strategies for ameliorating IBD have been proposed in many studies using animal models. Our group has demonstrated that administration of Lactococcus lactis NCDO 2118 can improve clinical parameters of colitis induced by oral administration of dextran sulphate sodium (DSS). However, it is not clear whether other strains of L. lactis can yield the same effect. The objective of present study was to analyze the effects of three different L. lactis strains (NCDO2118, IL1403 and MG1363) in the development of DSS-induced colitis in C57BL/6 mice. Acute colitis was induced in C57/BL6 mice by the administration of 2% DSS during 7 consecutive days. Body weight loss and shortening of colon length were observed in DSS-treated mice, and none of L. lactis strains had an impact in these clinical signs of colitis. On the other hand, all strains improved the global macroscopical disease index and prevented goblet cells depletion as well as the increase of intestinal permeability. TNF-α production was reduced in gut mucosa of L. lactis DSS-treated mice indicating a modulation of a critical pro-inflammatory response by all strains tested. However, only L. lactis NCDO2118 and MG1363 induced a higher frequency of CD11c+CD11b-CD103+ tolerogenic dendritic cells in lymphoid organs of mice at steady state. We conclude that all tested strains of L. lactis improved the clinical scores and parameters of colitis, which confirm their anti-inflammatory properties in this model of colitis.

Intestinal barrier dysfunction in murine sickle cell disease is associated with small intestine neutrophilic inflammation, oxidative stress, and dysbiosis.

The intestinal microbiome has emerged as a potential contributor to the severity of sickle cell disease (SCD). We sought to determine whether SCD mice exhibit intestinal barrier dysfunction, inflammation, and dysbiosis. Using the Townes humanized sickle cell mouse model, we found a 3-fold increase in intestinal permeability as assessed via FITC-dextran (4 kDa) assay in SS (SCD) mice compared to AA (wild type) mice (n=4, p < 0.05). This was associated with 25 to 50% decreases in claudin-1, 3, and 15 and zonula occludens-1 gene expression (n=8-10, p < 0.05) in the small intestine. Increased Ly6G staining demonstrated more neutrophils in the SS small intestine (3-fold, n=5, p < 0.05) associated with increased expression of TNFα, IL-17A, CXCL1, and CD68 (2.5 to 5-fold, n=7-10, p < 0.05). In addition, we observed 30 to 55% decreases in superoxide dismutase-1, glutathione peroxidase-1, and catalase antioxidant enzyme expression (n=7-8, p < 0.05) concomitant to an increase in superoxide (2-fold, n=4, p < 0.05). Importantly, all significant observations of a leaky gut phenotype and inflammation were limited to the small intestine and not observed in the colon. Finally, characterization of the composition of the microbiome within the small intestine revealed dysbiosis in SS mice compared to their AA littermates with 47 phyla to species-level significant alterations in amplicon sequence variants. We conclude that the intestinal barrier is compromised in SCD, associated with decreased gene expression of tight junction proteins, enhanced inflammation, oxidative stress, and gut microbiome dysbiosis, all specific to the small intestine.

Abstract WMP117: Exploring The Contribution Of Matrix Metalloproteinase 7 In Post-stroke Gut Permeability

Background: Stroke is associated with profound immunodepression and aberrations of autonomic signaling which render the body more susceptible to infection from without and from within. Dramatic alterations of sensitive signaling networks within the gut after stroke along with systemic inflammation may promote pathways that evoke a leaky gut. The enzyme matrix metalloproteinase 7 (MMP-7) has been shown to play a prominent role in evoking gut permeability in models of systemic inflammation. We found that stoke elicits a profound increase in MMP-7 across the small intestine rapidly after injury. Hypothesis: We hypothesize that this increase in MMP-7 in the gut after stroke contributes to post-stroke gut permeability and that mice deficient in MMP-7 are protected from this effect. Methods: Young (2-5-month-old) Mmp7 knockout ( Mmp7 -/- ) and wild type (WT) control littermates were subjected to 45min-tMCAO. At 3h post-tMCAO, mice were injected intraocularly with 200mg/kg FITC-inulin at 3h post-tMCAO and euthanized 1h later. Aged (24-month-old) Mmp7 -/- mice and WT control littermates were subjected to pMCAO, injected intraocularly with 200mg/kg FITC-inulin at 47h, and euthanized 1h later. Intestinal tissue was collected, homogenized, and used to detect FITC-inulin as well as for Western blotting and RT-qPCR. Results: We demonstrate that MMP-7 levels rapidly increase throughout the small intestine after stroke at 6h and 24h post-stroke. This increase in MMP-7 is accompanied by a concurrent decrease in tight junction proteins and increase in intestinal permeability at 3h post-transient middle cerebral artery occlusion (tMCAO) and 48h post-permanent (p)MCAO. Interestingly, MMP-7 deficient mice are protected from gut permeability after stroke. Conclusion: Our data identify a novel causative agent in the phenomenon of gut permeability observed after stroke and prompt further study of the role of MMP’s in promoting gut permeability after stroke.

Faeces-derived extracellular vesicles participate in the onset of barrier dysfunction leading to liver diseases.

The role of extracellular vesicles (EVs) from faeces (fEVs) and small circulating EVs (cEVs) in liver diseases such as non-alcoholic fatty diseases (NAFLD) and non-alcoholic steatohepatitis (NASH) has not been demonstrated. fEVs and cEVs of healthy donors, NAFLD and NASH patients were isolated and characterized. The effects of EVs were evaluated in intestinal, endothelial, Kupffer and stellate cells. Non-muscular myosin light chain kinase (nmMLCK) deficient mice were used in vivo. Bacterial origins of fEVs were analysed by 16s rDNA gene sequencing. fEVs and small cEVs were composed of prokaryotic and eukaryotic origins. Only NASH-fEVs exerted deleterious effects. NASH-fEVs increased intestinal permeability and reduced expression of tight junction proteins that were prevented by nmMLCK inhibition, increased endothelial cell permeability and inflammatory cytokines and chemokines requiring TLR4/lipopolysaccharide pathway. NASH-fEVs and NASH-cEVs activated profibrotic and proinflammatory proteins of hepatic stellate cells. Treatment with NASH-fEVs evoked an increase in intestinal permeability in wild type but not in nmMLCK deficient mice. Bacterial origins of fEVs were different between NAFLD and NASH patients and 16 amplicon sequence variants were differentially abundant. We demonstrate that fEVs actively participate in barrier dysfunctions leading to liver injuries underscoring the role of nmMLCK and lipopolysaccharide carried by fEVs.

Features of assessing the intestinal barrier permeability in chronic kidney disease

Numerous studies have shown that changes in the intestinal microbial-tissue complex are a risk factor for the progression of chronic kidney disease (CKD) to end-stage renal disease and, therefore, a potential target for new therapeutic interventions. Thus, reliable and sensitive diagnostic tools for measuring intestinal permeability in the clinical setting are necessary. Modern genome sequencing and multi-omics technologies have established that patients with CKD are characterized by a specific imbalance between the saccharolytic and proteolytic microbiota, contributing to the accumulation of numerous toxic microbial products, such as indoxyl sulphate, p-cresyl sulphate, trimethylamine-N-oxide. Progressive kidney function decline leads to compensatory urea accumulation in the gastrointestinal tract. In the intestinal lumen, urea is hydrolyzed by microbial urease, forming a large amount of ammonium hydroxide, which may be accompanied by disruption of the epithelial barrier integrity with an increase in intestinal permeability for microbial molecules that initiate systemic inflammation. Experimental approaches to studying the intestinal barrier in CKD include the assessment of electrophysiological parameters of the intestinal epithelium and the transport of fluorescently labelled tracers in the Ussing chamber. Actively improving various cell-based in vitro methods, which may be useful for studying the effect of microbiota on the barrier functions of the intestinal epithelium. Gene expression and protein content of tight junctions are estimated using polymerase chain reaction, immunohistochemical methods and Western blotting. Using various biomolecular methods, it was found that renal failure is characterized by the presence of inflammatory and atrophic changes throughout the gastrointestinal tract, destruction of the mucin layer, damage to tight junctions with a decrease in the amount of claudine-1, occludin and ZO-1 as well as a decrease in transepithelial electrical resistance. Clinical examination of intestinal permeability by methods based on the urine excretion of orally administered sugars, polyethylene glycol polymers and labelled tracers indicate a distortion of the results in patients with CKD due to altered renal clearance. Alternatively, quantitative determination of bacterial DNA and D-lactate levels in the blood is considered. Identification of serum non-coding microRNAs, confocal laser endomicroscopy and impedance spectroscopy have the potential to be used as methods for assessing intestinal barrier function.