Human Gut–Brain Interaction Chip for Dissecting the Gut-Derived LPS and Butyrate Regulation of the Blood–Brain Barrier

Probiotic Bacteria Induce Maturation of Intestinal Claudin 3 Expression and Barrier Function

Potential conflict of interest: Nothing to report. See Article on Page 883 An intact intestinal epithelial barrier plays a major role in preventing intestinal invasion of luminal pathogens and antigenic molecules and their subsequent migration to the liver and, potentially, the systemic immune system. The clinical relevance of intestinal barrier dysfunction has been recognized by its pathogenic association with several disease states, ranging from inflammatory bowel disease to rheumatoid arthritis to Alzheimer's disease, and, of direct relevance to this editorial, to alcoholic liver disease (ALD).1 Integrity of intestinal barrier function is regulated, in part, by the epithelial tight junction (TJ) complex composed of proteins such as zonula occludens, occludin, and claudins.3 The TJ complex establishes intestinal barrier integrity by connecting the interepithelial cell spaces and inhibiting the paracellular passage of microbes and microbial products and other luminal contents.4 However, intestinal epithelial barrier dysfunction is frequently observed in several acute and chronic enteropathic disorders, such as inflammatory bowel disease, irritable bowel syndrome, and infectious diarrhea.1 Intestinal production of proinflammatory cytokines, including interferon (IFN)‐γ, tumor necrosis factor (TNF)‐α, interleukin (IL)−1β, IL‐6, IL‐13, and the TNF superfamily member, LIGHT, have been documented to promote intestinal epithelial barrier dysfunction.5 Particularly, cytokine‐initiated inflammatory signaling plays a major role in breaching TJ integrity.9 Although the role of cytokines and inflammatory signaling in intestinal epithelial barrier dysfunction has been well documented, the underlying mechanisms are still being defined, and potential therapeutic strategies for reestablishing intestinal barrier function are still under investigation. Work by Chen et al. examined the role of TNF‐α‐induced receptor 1 (TNFR1) signaling as a critical factor in alcohol‐associated loss of intestinal barrier function and the development of liver disease.10 TNF‐α plays a major pathogenic role in many diseases associated with intestinal epithelial barrier dysfunction.11 An interesting aspect of the ALD model of intestinal dysbiosis and barrier dysfunction, in comparison to other enteropathic diseases, such as inflammatory bowel disease, intestinal ischemia, and graft versus host disease, is that the damage to the epithelium is much less extensive, thereby allowing investigation of the pathogenic interaction between proinflammatory signaling and TJ integrity. The data presented in the context of the ALD model showed that chronic alcohol feeding induces intestinal inflammation in the jejunum, as indicated by an increase in TNF‐α production by monocytes and macrophages and an increase in the intestinal permeability. The clinical relevance of these findings was supported by similar findings in duodenal biopsies from patients with chronic alcohol abuse. This is especially important because there are multiple experimental models of ALD (usually early stages of ALD), and it is valuable to connect animal findings to human disease. Chen et al.10 were able to specifically address and establish the causal role of TNF/TNFR1 interaction and signaling in the development of alcohol‐induced barrier dysfunction and intestinal permeability in this model of ALD. Specifically, the investigators were able to demonstrate that TNFR1 mutant mice are protected from alcohol‐induced intestinal barrier dysfunction and liver disease. Importantly, selective reinstatement of TNFR1 expression on intestinal epithelial cells in a TNFR1 mutant mouse carrying a conditional gain‐of‐function allele for this receptor caused a resumption of intestinal pathology and liver disease similar to wild‐type mice. These data certainly support the notion that intestinal TNF/TNFR1 signaling plays an essential pathogenic role in alcohol‐induced intestinal barrier dysfunction and subsequent development of liver disease (Fig. 1). These data also suggest that hepatic TNFR1 is not required for development of ALD in this experimental model system.Figure 1: TNFR1 signaling in the intestine is necessary for subsequent development of alcohol‐induced liver injury. This demonstrates the critical link between alcohol‐induced intestinal inflammation and the subsequent development of alcohol‐induced liver injury (gut:liver axis).In comparison to the research presented by Chen et al., work done by Wang et al.6 investigating intestinal epithelial barrier dysfunction using an IFN‐γ‐primed human intestinal epithelial cell line, Caco‐2 cells, showed that TNFR2, and not TNFR1, is required for TNF‐dependent barrier dysfunction. Findings from these two studies indicate that the type of TNFR relevant for mediating TNF‐dependent TJ disruption could be contextual and dictated by species and/or disease‐type specificity. Importantly, these studies show the need for care in drawing mechanistic conclusions with regard to the development of therapeutic approaches to target inflammatory signaling for reestablishing intestinal epithelial barrier function. Significantly, TNF‐mediated intestinal epithelial TNFR1 signaling in the mouse model of alcohol feeding as well as TNFR2 signaling in a human intestinal epithelial cell line both lead to myosin light chain (MLC) phosphorylation by activation of MLC kinase (MLCK) that plays a major role in the proinflammatory cytokine‐induced intestinal barrier disruption.9 These data indicate that, regardless of which type of receptor is activated, TNF‐dependent disruption of TJ and consequent loss of intestinal barrier function involve MLCK phosphorylation. Work done by Chen et al.10raises some important questions regarding the mechanisms underlying the pathogenesis of ALD. Particularly, an intriguing aspect of the findings is that in TNFR1 knockout animals, restoration of TNFR1 expression only in the intestinal epithelial cells was sufficient to cause liver pathology, even in the absence of TNFR1‐induced inflammatory and cytotoxic signaling in the liver. These data suggest that hepatic TNFR1 activation is not required for the initiation and perpetuation of ALD in this animal model system. Overall, the findings are significant in that they provide an improved understanding of intestinal proinflammatory signaling and malfunction of TJ integrity in the context of chronic alcohol consumption that leads to loss of intestinal epithelial barrier function and development of ALD. These findings raise other important questions, including those related to the best approach to prevent/treat ALD. A quarter of a century ago, we reported increased peripheral blood monocyte production of TNF in human alcoholic hepatitis.12 Chen et al. showed that intestinal monocyte TNF production is increased in experimental ALD and that TNF is increased in intestinal biopsies from human alcoholics. Is it only the intestinal TNF that is important, or is the TNF produced by the liver and systemic cells, which recycles back to activate intestinal TNFR1, also important? Chen et al. also showed that intestinal TNFR1 (but not hepatic TNFR1) was critical for the development of experimental ALD. Multiple approaches, such as antibiotics, probiotics, prebiotics, and several dietary factors, have been shown to stabilize gut‐barrier function, decrease inflammation, and protect against experimental ALD. Perhaps we should be placing greater emphasis on intestinal inflammation and intestinal barrier disruption as a therapeutic target in human ALD.

Reply to: “Uncovering the molecular events associated with increased intestinal permeability in liver cirrhosis: The pivotal role of enterocyte tight junctions and future perspectives”

2021 Workshop: Neurodegenerative Diseases in the Gut-Brain Axis—Parkinson's Disease

Erratum: Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips.

Intestinal epithelial barrier dysfunction is intricately linked to the pathogenesis of ulcerative colitis (UC). Dietary interventions that bolster intestinal epithelial barrier function can effectively thwart UC onset. Our prior research revealed that p-Hydroxy benzaldehyde (HD), a phenolic compound from Nostoc commune (an edible cyanobacterium), markedly upregulated the expression of E-cadherin, a pivotal protein in intestinal mucosa, thereby mitigating mucosal damage in mice afflicted with dextran sulfate sodium (DSS)-induced colitis. Nevertheless, the precise molecular mechanisms underpinning HD's ameliorative effects on intestinal epithelial barrier dysfunction remain elusive. Dextran sodium sulfate (DSS)-induced colitis mouse model was established, and the successful establishment of the model was determined by evaluating the changes in body weight, disease activity index (DAI), colonic histopathology, and white blood cell count. Transmission electron microscopy (TEM) observed the ultrastructural changes of intestinal villi. The levels of inflammatory factors ( IFN-γ IL-13 ) and intestinal permeability indicators (FITC-Dextran, DAO, ET, and D-LA ) were detected by Enzyme-linked immunosorbent assay (ELISA). Western blotting (WB) and immunohistochemistry (IHC) were used to detect the expression of intestinal barrier integrity-related factors such as tight junction protein TJs (ZO-1, occludin) and adhesion junction protein AJs (E-cadherin). Furthermore, WB, Pull-down assay, drug affinity reaction target stability (DARTS) assay, molecular docking and molecular dynamics (MD) simulation were used to determine the potential target and molecular mechanism of HD. HD intervention significantly alleviated the symptoms of colitis mice, inhibited the weight loss and colon shortening, reduced DAI score and colon pathological score, maintained the ultrastructure of intestinal villi in colon tissue, and significantly reduced the inflammatory factors IFN-γ, IL-13 and the number of white blood cells in colon tissue of colitis mice. HD could also reduce the levels of FITC-Dextran, DAO, ET, and D-LA and increase the expression of ZO-1, occludin, and E-cadherin in the colonic tissues of colitis mice, thereby maintaining the impaired intestinal barrier function caused by colitis. Mechanically, HD augmented the expression of hepatocyte nuclear factor 1β (HNF-1β) and DRA. Adeno-associated virus (AAV)-HNF-1β shRNA or Lentivirus-mediated HNF-1β knockdown effectively abolished HD-induced intestinal barrier protection, as well as the promotion of solute carrier family 26 member 3 (SLC26A3) expression levels. SLC26A3 siRNA effectively reversed the inhibition of intestinal permeability by HD. Pull-down assay, DARTS analysis, molecular docking, and MD results showed high binding strength, interaction efficiency and remarkable stability between HNF-1β and HD. This study elucidates HD's role in forestalling intestinal epithelial barrier disruption under colitis conditions. Mechanistic investigations revealed that HD fortifies TJs and AJs expression via the HNF-1β/SLC26A3 pathway, thus preserving the lower intestinal epithelial barrier's integrity in UC.

TRPV6 channel mediates alcohol-induced gut barrier dysfunction and systemic response.

LIGHT Signals Directly to Intestinal Epithelia to Cause Barrier Dysfunction via Cytoskeletal and Endocytic Mechanisms

Significance of gastrointestinal tract in the therapeutic mechanisms of exercise in depression: Synchronism between brain and intestine through GBA

Testing of a hypothesis for osmotic opening of the blood-brain barrier.

The impermeant nature of the intestinal barrier is maintained by tight junctions (TJs) formed between adjacent intestinal epithelial cells. Disruption of TJs and loss of barrier function are associated with a number of gastrointestinal diseases, including neonatal necrotizing enterocolitis (NEC), the leading cause of death from gastrointestinal diseases in preterm infants. Human milk is protective against NEC, and the human milk factor erythropoietin (Epo) has been shown to protect endothelial cell-cell and blood-brain barriers. We hypothesized that Epo may also protect intestinal epithelial barriers, thereby lowering the incidence of NEC. Our data demonstrate that Epo protects enterocyte barrier function by supporting expression of the TJ protein ZO-1. As immaturity is a key factor in NEC, Epo regulation of ZO-1 in the human fetal immature H4 intestinal epithelial cell line was examined and demonstrated Epo-stimulated ZO-1 expression in a dose-dependent manner through the PI3K/Akt pathway. In a rat NEC model, oral administration of Epo lowered the incidence of NEC from 45 to 23% with statistical significance. In addition, Epo treatment protected intestinal barrier function and prevented loss of ZO-1 at the TJs in vivo. These effects were associated with elevated Akt phosphorylation in the intestine. This study reveals a novel role of Epo in the regulation of intestinal epithelial TJs and barrier function and suggests the possible use of enteral Epo as a therapeutic agent for gut diseases.

Magnolol preserves the integrity of the intestinal epithelial barrier and mitigates intestinal injury through activation of PPAR γ in COPD rat

Background and Objectives: Irritable Bowel Syndrome (IBS) is a common gastrointestinal disorder often exacerbated by stress, influencing the brain-gut axis (BGA). BGA dysregulation, disrupted intestinal barrier function, altered visceral sensitivity and immune imbalance defects underlying IBS pathogenesis have been emphasized in recent investigations. Phosphoproteomics reveals unique phosphorylation details resulting from environmental stress. Here, we employ phosphoproteomics to explore the molecular mechanisms underlying IBS-like symptoms, mainly focusing on the role of ZO-1 and IL-1RAP phosphorylation. Materials and Methods: Morris water maze (MWM) was used to evaluate memory function for single prolonged stress (SPS). To assess visceral hypersensitivity of IBS-like symptoms, use the Abdominal withdrawal reflex (AWR). Colonic bead expulsion and defecation were used to determine fecal characteristics of the IBS-like symptoms. Then, we applied a phosphoproteomic approach to BGA research to discover the molecular mechanisms underlying the process of visceral hypersensitivity in IBS-like mice following SPS. ZO-1, p-S179-ZO1, IL-1RAP, p-S566-IL-1RAP and GFAP levels in BGA were measured by western blotting, immunofluorescence staining, and enzyme-linked immunosorbent assay to validate phosphorylation quantification. Fluorescein isothiocyanate-dextran 4000 and electron-microscopy were performed to observe the structure and function of the intestinal epithelial barrier. Results: The SPS group showed changes in learning and memory ability. SPS exposure affects visceral hypersensitivity, increased fecal water content, and significant diarrheal symptoms. Phosphoproteomic analysis displayed that p-S179-ZO1 and p-S566-IL-1RAP were significantly differentially expressed following SPS. In addition, p-S179-ZO1 was reduced in mice's DRG, colon, small intestine, spinal and hippocampus and intestinal epithelial permeability was increased. GFAP, IL-1β and p-S566-IL-1RAP were also increased at the same levels in the BGA. And IL-1β showed no significant difference was observed in serum. Our findings reveal substantial alterations in ZO-1 and IL-1RAP phosphorylation, correlating with increased epithelial permeability and immune imbalance. Conclusions: Overall, decreased p-S179-ZO1 and increased p-S566-IL-1RAP on the BGA result in changes to tight junction structure, compromising the structure and function of the intestinal epithelial barrier and exacerbating immune imbalance in IBS-like stressed mice.

Loss of intestinal epithelial barrier function is a hallmark in digestive tract inflammation. The detailed mechanisms remain unclear due to the lack of suitable cell-based models in barrier research. Here we performed a detailed functional characterization of human intestinal organoid cultures under different conditions with the aim to suggest an optimized ex-vivo model to further analyse inflammation-induced intestinal epithelial barrier dysfunction. Differentiated Caco2 cells as a traditional model for intestinal epithelial barrier research displayed mature barrier functions which were reduced after challenge with cytomix (TNFα, IFN-γ, IL-1ß) to mimic inflammatory conditions. Human intestinal organoids grown in culture medium were highly proliferative, displayed high levels of LGR5 with overall low rates of intercellular adhesion and immature barrier function resembling conditions usually found in intestinal crypts. WNT-depletion resulted in the differentiation of intestinal organoids with reduced LGR5 levels and upregulation of markers representing the presence of all cell types present along the crypt-villus axis. This was paralleled by barrier maturation with junctional proteins regularly distributed at the cell borders. Application of cytomix in immature human intestinal organoid cultures resulted in reduced barrier function that was accompanied with cell fragmentation, cell death and overall loss of junctional proteins, demonstrating a high susceptibility of the organoid culture to inflammatory stimuli. In differentiated organoid cultures, cytomix induced a hierarchical sequence of changes beginning with loss of cell adhesion, redistribution of junctional proteins from the cell border, protein degradation which was accompanied by loss of epithelial barrier function. Cell viability was observed to decrease with time but was preserved when initial barrier changes were evident. In summary, differentiated intestinal organoid cultures represent an optimized human ex-vivo model which allows a comprehensive reflection to the situation observed in patients with intestinal inflammation. Our data suggest a hierarchical sequence of inflammation-induced intestinal barrier dysfunction starting with loss of intercellular adhesion, followed by redistribution and loss of junctional proteins resulting in reduced barrier function with consecutive epithelial death.

The integrity of intercellular junctions that form the "terminal bar" in intestinal epithelium is crucial for sealing the intestinal barrier. Whereas specific roles of tight and adherens junctions are well known, the contribution of desmosomal adhesion for maintaining the intestinal epithelial barrier has not been specifically addressed. For the present study, we generated a desmoglein 2 antibody directed against the extracellular domain (Dsg2 ED) to test whether impaired Dsg2-mediated adhesion affects intestinal epithelial barrier functions in vitro. This antibody was able to specifically block Dsg2 interaction in cell-free atomic-force microscopy experiments. For in vitro studies of the intestinal barrier we used Caco2 cells following differentiation into tight enterocyte-like epithelial monolayers. Application of Dsg2 ED to Caco2 monolayers resulted in increased cell dissociation compared with controls in a dispase-based enterocyte dissociation assay. Under similar conditions, Dsg2 antibody significantly decreased transepithelial electrical resistance and increased FITC-dextran flux, indicating that Dsg2 interaction is critically involved in the maintenance of epithelial intestinal barrier functions. As revealed by immunostaining, this was due to Dsg2 ED antibody-induced rupture of tight junctions because tight junction proteins claudins 1, 4, and 5, occludin, and tight junction-associated protein zonula occludens-1 were partially removed from cell borders by Dsg2 ED treatment. Similar results were obtained by application of a commercial monoclonal antibody directed against the ED of Dsg2. Antibody-induced effects were blocked by absorption experiments using Dsg2-Fc-coated beads. Our data indicate that Dsg2-mediated adhesion affects tight junction integrity and is required to maintain intestinal epithelial barrier properties.