Epithelial Tight Junctions Research Articles

A systematic review of preclinical studies targeted toward the management of co-existing functional gastrointestinal disorders, stress, and gut dysbiosis.

Modern dietary habits and stressed lifestyle have escalated the tendency to develop functional gastrointestinal disorders (FGIDs) through alteration in the gut-brain-microbiome axis. Clinical practices use symptomatic treatments, neglect root causes, and prolong distress in patients. The past decade has seen the evolution of various interventions to attenuate FGIDs. But clinical translation of such studies is very rare mostly due to lack of awareness. The aim of this review is to meticulously integrate different studies and bridge this knowledge gap. Literature between 2013 and 2023 was retrieved from PubMed, ProQuest, and Web of Science. The data was extracted based on the PRISMA guidelines and using the SYRCLE's risk of bias and the Cochrane Risk of Bias tools, quality assessment was performed. The review has highlighted molecular insights into the coexistence of FGIDs, stress, and gut dysbiosis. Furthermore, novel interventions focusing on diet, probiotics, herbal formulations, and phytoconstituents were explored which mostly had a multitargeted approach for the management of the diseases. Scientific literature implied positive interactions between the interventions and the gut microbiome by increasing the relative abundance of beneficial bacteria and reducing stress-related hormones. Moreover, the interventions reduced intestinal inflammation and regulated the expression of epithelial tight junction proteins in different in vivo models. This systematic review delves deep into the preclinical interventions to manage coexisting FGIDs, stress, and gut dysbiosis. However, in most of the discussed studies, long-term risks and toxicity profile of the interventions are lacking. So, it is necessary to highlight them for improved clinical outcomes.

High ionic strength vector formulations enhance gene transfer to airway epithelia.

A fundamental challenge for cystic fibrosis (CF) gene therapy is ensuring sufficient transduction of airway epithelia to achieve therapeutic correction. Hypertonic saline (HTS) is frequently administered to people with CF to enhance mucus clearance. HTS transiently disrupts epithelial cell tight junctions, but its ability to improve gene transfer has not been investigated. Here, we asked if increasing the concentration of NaCl enhances the transduction efficiency of three gene therapy vectors: adenovirus, AAV, and lentiviral vectors. Vectors formulated with 3-7% NaCl exhibited markedly increased transduction for all three platforms, leading to anion channel correction in primary cultures of human CF epithelial cells and enhanced gene transfer in mouse and pig airways in vivo. The mechanism of transduction enhancement involved tonicity but not osmolarity or pH. Formulating vectors with a high ionic strength solution is a simple strategy to greatly enhance efficacy and immediately improve preclinical or clinical applications.

Lactobacillus acidophilus inhibits the TNF-α-induced increase in intestinal epithelial tight junction permeability via a TLR-2 and PI3K-dependent inhibition of NF-κB activation.

Defective intestinal epithelial tight junction (TJ), characterized by an increase in intestinal TJ permeability, has been shown to play a critical role in thepathogenesis of inflammatory bowel disease (IBD). Tumor necrosis factor-α (TNF-α) is a key pro-inflammatory cytokine involved in the immunopathology of IBD and has been shown to cause an increase in intestinal epithelial TJ permeability. Although TNF-α antibodies and other biologics have been advanced for use in IBD treatment, these therapies are associated with severe side effects and have limited efficacy, and there is an urgent need for therapies with benign profiles and high therapeutic efficacy. Probiotic bacteria have beneficial effects and are generally safe and represent an important class of potential therapeutic agents in IBD. Lactobacillus acidophilus (LA) is one of the most used probiotics for wide-ranging health benefits, including in gastrointestinal, metabolic, and inflammatory disorders. A specific strain of LA, LA1, was recently demonstrated to have protective and therapeutic effects on the intestinal epithelial TJ barrier. However, the mechanisms of actions of LA1 remain largely unknown. The primary aim of this study was to investigate microbial-epithelial interactions and novel signaling pathways that regulate the effect of LA1 on TNF-α-induced increase in intestinal epithelial TJ permeability, using cell culture and animal model systems. Pre-treatment of filter-grown Caco-2 monolayers with LA1 prevented the TNF-α-induced increase in intestinal epithelial TJ permeability by inhibiting TNF-α-induced activation of NF-κB p50/p65 and myosin light chain kinase (MLCK) gene and kinase activity in a TLR-2-dependent manner. LA1 produced a TLR-2- and MyD88-dependent activation of NF-κB p50/p65 in immune cells; however, LA1, in intestinal cells, inhibited the NF-κB p50/p65 activation in a TLR-2-dependent but MyD88-independent manner. In addition, LA1 inhibition of NF-κB p50/p65 and MLCK gene was mediated by TLR-2 pathway activation of phosphatidylinositol 3-kinase (PI3K) and IKK-α phosphorylation. Our results demonstrated novel intracellular signaling pathways by which LA1/TLR-2 suppresses the TNF-α pathway activation of NF-κB p50/p65 in intestinal epithelial cells and protects against the TNF-α-induced increase in intestinal epithelial TJ permeability.

Modified Zhenwu Decoction improved intestinal barrier function of experimental colitis through activation of sGC-mediated cGMP/PKG signaling

BackgroundThe invasion of luminal antigens and an aberrant immune response resulting from a disrupted physical epithelial barrier are the key characteristics of ulcerative colitis (UC). The restoration of damaged epithelial function is crucial for maintaining mucosal homeostasis and disease quiescence. Current therapies for UC primarily focus on suppressing inflammation. However, most patients fail to respond to therapy or develop secondary resistance over time, emphasizing the need to develop novel therapeutic targets for UC. Our study aimed to identify the potential targets of a novel modified herbal formula from the Zhen Wu Decoction, namely CDD-2103, which has demonstrated promising efficacy in treating chronic colitis. MethodsThe effect of CDD-2103 on epithelial barrier function was examined using in vitro and ex vivo models of tissue injury, as well as a chronic colitis C57BL/6 mouse model. Transcriptomic analysis was employed to profile gene expression changes in colonic tissues following treatment with CDD-2103. ResultsOur in vivo experiments demonstrated that CDD-2103 dose-dependently reduced disease severity in mice with chronic colitis. The efficacy of CDD-2103 was mediated by a reduction in goblet cell loss and the enhancement of tight junction protein integrity. Mechanistically, CDD-2103 suppressed epithelial cell apoptosis and tight junction protein breakdown by activating the soluble guanynyl cyclase (sGC)-mediated cyclic guanosine monophosphate (cGMP)/PKG signaling cascade. Molecular docking analysis revealed strong sGC ligand recognition by the CDD-2103-derived molecules, warranting further investigation. ConclusionOur study revealed a novel formulation CDD-2103 that restores intestinal barrier function through the activation of sGC-regulated cGMP/PKG signaling. Furthermore, our findings suggest that targeting sGC can be an effective approach for promoting mucosal healing in the management of UC.

Paeoniae Decoction restores intestinal barrier dysfunction by promoting the interaction between ILC3 and gut flora

BackgroundIntestinal barrier dysfunction is a significant contributor to the recurrence and refractory of ulcerative colitis (UC). Promoting the interaction between group 3 innate lymphoid cells (ILC3s) and gut flora is a valuable strategy for mucosal repair. Paeoniae decoction (PD) is a compound commonly used in clinical treatment of UC, but its exact mechanism remains unclear. PurposeWe aimed to investigate the protective effect of PD on intestinal mucosal injury induced by dextran sulfate sodium (DSS) in chronic colitis, as well as to elucidate its potential mechanism. MethodsC57BL/6 mice were induced with chronic colitis by 2 % DSS and divided into four groups: control group, model group, PD low dose (4 g/kg), and high dose (8 g/kg) group. The effectiveness of PD in treating chronic colitis mice was evaluated based on changes in body weight, colon length, colon pathological tissue scores, and the mRNA levels of inflammatory factors IL-6 and IL-1β. The expressions of intestinal epithelial tight junction proteins (ZO-1 and Occludin), IL-22, and MUC2 were observed using immunofluorescence and RT-PCR. Additionally, the proportion of ILC3 and natural cytotoxicity receptor (NCR)+ ILC3 in the colon were detected using flow cytometry. Furthermore, UHPLC-QE-MS was utilized to identify chemical components of PD and network pharmacology was employed to predict potential pathways for PD intervention in UC. Subsequently, MNK-3 cells (ILC3 in vitro cell line) and NCM460 cells were used to verify the network pharmacology results. Finally, the effects of PD on UC gut flora have been explored using in vitro fermentation and 16S rDNA techniques. ResultsThe results showed that PD significantly restored body weight and colon length in mice with chronic colitis, while also reducing colon inflammatory cell infiltration and the expression of IL-6 and IL-1β. Additionally, PD notably promoted the expression of MUC2, ZO-1, Occludin, and IL-22, as well as increasing the ratio of ILC3 and NCR+ILC3. UHPLC-QE-MS analysis identified 443 components of PD, and network pharmacology suggested that PD could target the aryl hydrocarbon receptor (AHR) signaling pathway, which was confirmed by MNK-3 cells and in vitro fermentation experiments. Furthermore, MNK-3-conditioned medium (CM) increased the expression of ZO-1 and Occludin in NCM460 cells. In addition, 16S rDNA results indicated that PD promoted the abundance of Lactobacillales, thus contributing to mucosal damage repair by activating the AHR signal in ILC3s. ConclusionIn summary, our study demonstrates that PD repairs intestinal mucosal damage in chronic colitis by regulating the interaction of gut flora with ILC3, and the specific mechanism is related to the activation of AHR signaling pathway.

Regulation of vascular endothelial integrity by mesenchymal stem cell extracellular vesicles after hemorrhagic shock and trauma

BackgroundPatients with hemorrhagic shock and trauma (HS/T) are vulnerable to the endotheliopathy of trauma (EOT), characterized by vascular barrier dysfunction, inflammation, and coagulopathy. Cellular therapies such as mesenchymal stem cells (MSCs) and MSC extracellular vesicles (EVs) have been proposed as potential therapies targeting the EOT. In this study we investigated the effects of MSCs and MSC EVs on endothelial and epithelial barrier integrity in vitro and in vivo in a mouse model of HS/T. This study addresses the systemic effects of HS/T on multiorgan EOT.MethodsIn vitro, pulmonary endothelial cell (PEC) and Caco-2 intestinal epithelial cell monolayers were treated with control media, MSC conditioned media (CM), or MSC EVs in varying doses and subjected to a thrombin or hydrogen peroxide (H2O2) challenge, respectively. Monolayer permeability was evaluated with a cell impedance assay, and intercellular junction integrity was evaluated with immunofluorescent staining. In vivo, a mouse model of HS/T was used to evaluate the effects of lactated Ringer’s (LR), MSCs, and MSC EVs on endothelial and epithelial intercellular junctions in the lung and small intestine as well as on plasma inflammatory biomarkers.ResultsMSC EVs and MSC CM attenuated permeability and preserved intercellular junctions of the PEC monolayer in vitro, whereas only MSC CM was protective of the Caco-2 epithelial monolayer. In vivo, both MSC EVs and MSCs mitigated the loss of endothelial adherens junctions in the lung and small intestine, though only MSCs had a protective effect on epithelial tight junctions in the lung. Several plasma biomarkers including MMP8 and VEGF were elevated in LR- and EV-treated but not MSC-treated mice.ConclusionsIn conclusion, MSC EVs could be a potential cell-free therapy targeting endotheliopathy after HS/T via preservation of the vascular endothelial barrier in multiple organs early after injury. Further research is needed to better understand the immunomodulatory effects of these products following HS/T and to move toward translating these therapies into clinical studies.

Ferulic Acid Inhibits Arsenic-Induced Colon Injury by Improving Intestinal Barrier Function.

The prolonged exposure to arsenic results in intestinal barrier dysfunction, which is strongly concerned with detrimental processes such as oxidative stress and the inflammatory response. Ferulic acid (FA), as a phenolic acid, possesses the capability to mitigate arsenic-induced liver damage and cardiotoxic effects dependent on inhibition of oxidative stress and inflammatory responses. FA can mitigate testicular tissue damage and alveolar epithelial dysfunction, the mechanism of which may rely on nuclear factor erythroid 2-related factor 2/heme oxygenase 1 (Nrf2/HO-1) activation and nuclear factor-kappa B (NF-κB) pathway blocking. Based on the antioxidant and anti-inflammatory properties of FA, we speculated that FA might have the potential to inhibit arsenic-induced intestinal damage. To confirm this scientific hypothesis, mice exposed to sodium arsenite were treated with FA to observe colonic histopathology and TJ protein levels, and oxidative stress and TJ protein levels in Caco-2 cells exposed to sodium arsenite were assessed after FA intervention. In addition, molecular levels of NF-κB and Nrf2/HO-1 pathway in colon and Caco-2 cells were also detected. As shown in our data, FA inhibited arsenic-induced colon injury, which was reflected in the improvement of mucosal integrity, the decrease of down-regulated expression of tight junction (TJ) proteins (Claudin-1, Occludin, and ZO-1) and the inhibition of oxidative stress. Similarly, treatment with FA attenuated the inhibitory effect of arsenic on TJ protein expression in Caco-2 cells. In addition to suppressing the activation of NF-κB pathway, FA retrieved the activation of Nrf2/HO-1 pathway in colon and intestinal epithelial cells induced by arsenic. In summary, our findings propose that FA has the potential to mitigate arsenic-induced intestinal damage by preserving the integrity of intestinal epithelial TJs and suppressing oxidative stress. These results lay the groundwork for the potential use of FA in treating colon injuries caused by arsenic.

Gut microbiota-derived acetate attenuates lung injury induced by influenza infection via protecting airway tight junctions

BackgroundGut microbiota (GM) have been implicated as important regulators of gastrointestinal symptom which is commonly occurred along with respiratory influenza A virus (IAV) infection, suggesting the involvement of the gut-to-lung axis in a host’s response to IAV. IAV primarily destroys airway epithelium tight junctions (TJs) and consequently causes acute respiratory disease syndrome. It is known that GM and their metabolism produce an anti-influenza effect, but their role in IAV-induced airway epithelial integrity remains unknown.MethodsA mouse model of IAV infection was established. GM were analyzed using 16S rRNA gene sequencing, and short-chain fatty acids (SCFAs) levels were measured. GM depletion and fecal microbiota transplantation (FMT) were conducted to validate the role of GM in IAV infection. A pair-feeding experiment was conducted to reveal whether IAV-induced GM dysbiosis is attributed to impaired food intake. Furthermore, human bronchial epithelial (HBE) cells were cocultured with IAV in the presence or absence of acetate. TJs function was analyzed by paracellular permeability and transepithelial electronic resistance (TEER). The mechanism of how acetate affects TJs integrity was evaluated in HBE cells transfected with G protein-coupled receptor 43 (GPR43) short hairpin RNA (shRNA).ResultsIAV-infected mice exhibited lower relative abundance of acetate-producing bacteria (Bacteroides, Bifidobacterium, and Akkermansia) and decreased acetate levels in gut and serum. These changes were partly caused by a decrease in food consumption (due to anorexia). GM depletion exacerbated and FMT restored IAV-induced lung inflammatory injury. IAV infection suppressed expressions of TJs (occludin, ZO-1) leading to disrupted airway epithelial barrier function as evidenced by decreased TEER and increased permeability. Acetate pretreatment activated GPR43, partially restored IAV-induced airway epithelial barrier function, and reduced inflammatory cytokines levels (TNF-α, IL-6, and IL-1β). Such protective effects of acetate were absent in HBE cells transfected with GPR43 shRNA. Acetate and GPR43 improved TJs in an AMP-activated protein kinase (AMPK)-dependent manner.ConclusionCollectively, our results demonstrated that GM protected airway TJs by modulating GPR43-AMPK signaling in IAV-induced lung injury. Therefore, improving GM dysbiosis may be a potential therapeutic target for patients with IAV infection.

Chlorination of epithelial tight junction proteins by neutrophil myeloperoxidase promotes barrier dysfunction and mucosal inflammation.

Neutrophils (polymorphonuclear leukocytes, PMNs) comprise a major component of the immune cell infiltrate during acute mucosal inflammation and have an important role in molding the inflammatory tissue environment. While PMNs are essential to clearance of invading microbes, the major PMN antimicrobial enzyme myeloperoxidase (MPO) can also promote bystander tissue damage. We hypothesized that blocking MPO would attenuate acute colitis and prevent the development of chronic colitis by limiting bystander tissue damage. Using the acute and chronic dextran sodium sulfate model of murine colitis, we demonstrated that MPO-deficient mice experienced less inflammation and more rapidly resolved colitis relative to wild-type controls. Mechanistic studies demonstrated that activated MPO disrupted intestinal epithelial barrier function through the dysregulation of the epithelial tight junction proteins. Our findings revealed that activated MPO chlorinates tyrosine within several tight junction proteins, thereby promoting tight junction mislocalization and dysfunction. These observations in cell models and in murine colitis were validated in human intestinal biopsies from individuals with ulcerative colitis and revealed a strong correlation between disease severity (Mayo score) and tissue chlorinated tyrosine levels. In summary, these findings implicate MPO as a viable therapeutic target to limit bystander tissue damage and preserve mucosal barrier function during inflammation.

Rapamycin promotes the intestinal barrier repair in ulcerative colitis via the mTOR/PBLD/AMOT signaling pathway

Intestinal barrier dysfunction characterized by the functional loss of the intestinal epithelium's tight junction (TJ) barrier is a key factor in the pathogenesis of ulcerative colitis (UC). Although rapamycin, an mTOR (mechanistic target of rapamycin) inhibitor, has shown promise in inducing clinical remission and mucosal healing in inflammatory bowel disease, its underlying mechanism remains elusive. Thus, this study investigated the role of the mTOR pathway in regulating the intestinal barrier. To investigate the molecular mechanism regulating the intestinal barrier, specific intestinal epithelial phenazine biosynthesis-like domain-containing protein (PBLD)-deficient (PBLDIEC−/−) mice and control wild-type (WT) mice were intraperitoneally injected with rapamycin or MHY1485. To determine the relevance of the findings for UC, we analyzed transcriptome data and single-cell expression profiles from public databases and intestinal mucosal tissues obtained from patients with active UC or colon cancer. We observed that mTOR activation in the intestinal epithelium of patients with active UC. Moreover, in vivo, rapamycin markedly increased the expressions of PBLD and TJ proteins and reduced intestinal inflammation in mice with dextran sulfate sodium-induced enteritis. However, the therapeutic efficacy of rapamycin was notably reduced in PBLDIEC−/− mice. In vitro, rapamycin influenced PBLD expression by modulating the nuclear transcription of transcription factor EB (TFEB). Angiomotin (AMOT) could directly bind to PBLD, and rapamycin could not effectively increase the expression of TJ proteins after the knockdown of PBLD or AMOT. In summary, the administration of rapamycin is a potential treatment for UC, and targeting the mTOR/PBLD/AMOT axis is a potential novel approach for UC treatment.

Bifidobacterium bifidum Strain BB1 Inhibits Tumor Necrosis Factor-α–Induced Increase in Intestinal Epithelial Tight Junction Permeability via Toll-Like Receptor-2/Toll-Like Receptor-6 Receptor Complex–Dependent Stimulation of Peroxisome Proliferator-Activated Receptor γ and Suppression of NF-κB p65

Bifidobacterium bifidum (BB) strain BB1 causes a strain-specific enhancement in intestinal epithelial tight junction (TJ) barrier. TNF-α induces an increase in intestinal epithelial TJ permeability and promotes intestinal inflammation. The major purpose of this study was to delineate the protective effect of BB1 against the TNF-α induced increase in intestinal TJ permeability and to unravel the intracellular mechanisms involved. Previously reported, TNF-α produces an increase in intestinal epithelial TJ permeability in Caco-2 monolayers and in mice. The addition of BB1 inhibited the TNF-α increase in Caco-2 intestinal TJ permeability and mouse intestinal permeability in a strain-specific manner. BB1 inhibited the TNF-α induced increase in intestinal TJ permeability by interfering the with TNF-α induced enterocyte NF-κB p50/p65 and MLCK gene activation. The BB1 protective effect against the TNF-α induced increase in intestinal permeability was mediated by TLR-2/TLR-6 heterodimer complex activation of PPAR-γ and PPAR-γ pathway inhibition of TNF-α induced IKK-α activation, which in turn resulted in a step-wise inhibition of NF-κB p50/p65, MLCK gene, MLCK kinase activity, MLCK-induced opening of the TJ barrier. In conclusion, these studies unravel novel intracellular mechanisms of BB1 protection against the TNF-α induced increase in intestinal TJ permeability. Our data show that BB1 protects against the TNF-α induced increase in intestinal epithelial TJ permeability via a PPAR-γ dependent inhibition of NF-κB p50/p65 and MLCK gene activation.

#2837 Novel therapeutic for crescentic glomerulonephritis through targeting CLDN1 in parietal epithelial cells

Abstract Background and Aims Crescentic glomerulonephritis (CrGN) is the final mode of kidney injury common to several immune-mediated kidney diseases. CrGN is characterized by extensive glomerular parietal epithelial cells (PECs) proliferation forming crescents. The destructive crescent is then progressively replaced by fibrosis. Currents therapies for CrGN do not directly target the deleterious response of resident kidney cells. In a pathological context, claudin-1 (CLDN1), a transmembrane protein involved in epithelial tight junctions, can be exposed outside the tight junctions and mediate pro-fibrotic pathways and extracellular matrix (ECM) remodelling. Lixudebart (formerly ALE.F02) is a first-in-class monoclonal antibody that specifically targets and blocks exposed CLDN1 in injured epithelial cells. A phase II clinical trial is under way for patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV) with rapidly progressive glomerulonephritis which induces kidney fibrosis (RENAL-F02, NCT06047171). CLDN1 is highly expressed by glomerular PECs. This study investigated the functional role of CLDN1 in CrGN and the potential benefit to target CLDN1 in CrGN. Method CLDN1 expression in renal tissues of CrGN patients was analyzed using kidney multicolor immunofluorescence staining and spatial transcriptomics. Correlation between CLDN1 expression and clinical endpoints (eGFR, proteinuria), disease biomarkers and crescent evolution were studied. A spatially resolved molecular roadmap from CLDN1+ crescentic glomeruli was conducted. Proof-of-concept studies using anti-CLDN1 mAb were performed in preclinical models of CrGN. Results In tissues (n = 150) of patients with AAV and IgAN, multicolor immunofluorescence revealed up-regulated CLDN1 expression by cellular and fibrocellular crescents. At the time of diagnostic kidney biopsy, glomerular CLDN-1 expression was correlated with podocyte loss (as measured with P57 staining) and fibronectin area. The extent of dual expression of CLDN1 and CD44 at the surface of active PECs was associated with poor renal outcome (eGFR < 30 ml/min) in AAV and IgAN patients with a median follow-up of 2.5 and 3.7 years respectively. Spatial transcriptomics analysis highlighted the association between CLDN1+ crescentic glomeruli and ECM genes. Treatment with anti-CLDN1 mAb reduced albuminuria, improved kidney function and decreased fibrosis biomarkers in nephrotoxic serum-induced CrGN in mice. Conclusion Our results suggest a functional role of CLDN1 in the pathogenesis of CrGN, providing preclinical proof-of-concept for anti-CLDN1 antibodies as a novel therapeutic approach in patients with CrGN.

#2696 The effect of laxatives on reducing serum phosphate levels of probiotics in hemodialysis patients: a subanalysis of RCT

Abstract Background and Aims Dysbiosis of the intestinal microbiota in chronic kidney disease increases uremic toxin, which damage the epithelial tight junctions and increase the permeability of the intestinal wall via endotoxemia and systemic inflammation. Some articles reported that probiotics reduced the uremic toxin and decreased inflammation biomarkers in CKD patients. Meanwhile, we have already reported that oral administration of Bifidobacterium longum in a gastro-resistant seamless capsule decreases serum phosphate levels in patients receiving hemodialysis (Clin Kidney J, 5:373-374, 2012). In addition, in this congress, we reported that three-combination probiotics decreased serum phosphate levels. Meanwhile, some articles reported the relationship hypophosphatemia and laxative abuse. The aim of this subanalysis study is to evaluate the efficacy of laxatives on reducing serum phosphate levels of three-combination probiotic in patients receiving hemodialysis. Method A 6-month prospective, randomized, double-blind, placebo-controlled study was conducted at three hemodialysis centers. As probiotic therapy, of three-combination probiotics (Bio-Three) tablets, each containing 2 mg of lactomin (Streptococcus faecalis T-110), 10 mg of Clostridium butyricum TO-A, and 10 mg of Bacillus mesentericus TO-A, were used. The patients receiving hemodialysis were randomly assigned to the Bio-Three group (n = 37) and the placebo group (n = 36). In both the Bio-Three group and the placebo group, patients orally received 6 tablets 3 times daily. We evaluated the effect of probiotic on serum phosphate level and gut microbiome before and 6 months after the start of treatment. The gut microbiome composition was analyzed by a 16s rRNA gene-based sequencing protocol. In addition, in patients with laxative (n = 24), we evaluate the effect of probiotic on serum phosphate levels and gut microbiome. Results The serum phosphate levels in the Bio-Three group significantly decreased after 6-month treatment, while did not change in the placebo group. Furthermore, the serum phosphate level in patients with Bio-Three and laxative additionally decreased. In a 16s rRNA gene-based sequencing protocol analysis, serum phosphate levels were positively correlated with the occupancy ratio of Erysipelotrichaceae, Lachnospiraceae, Oscillospiraceae, Ruminococcaceae, and Sutterellaceae and were negatively correlated with the occupancy ratio of Leuconostocaceae and Lachnospiraceae in patients with Bio-Three and laxatives. Conclusion In this subanalysis study, the effect of reducing serum phosphate levels on three-combination probiotic might be augmented by laxatives and the specific bacteria in the gut microbiome might contribute to the decrease of serum phosphate levels.

Systematic-Narrative Hybrid Literature Review: Crosstalk between Gastrointestinal Renin-Angiotensin and Dopaminergic Systems in the Regulation of Intestinal Permeability by Tight Junctions.

In the first part of this article, the role of intestinal epithelial tight junctions (TJs), together with gastrointestinal dopaminergic and renin-angiotensin systems, are narratively reviewed to provide sufficient background. In the second part, the current experimental data on the interplay between gastrointestinal (GI) dopaminergic and renin-angiotensin systems in the regulation of intestinal epithelial permeability are reviewed in a systematic manner using the PRISMA methodology. Experimental data confirmed the copresence of DOPA decarboxylase (DDC) and angiotensin converting enzyme 2 (ACE2) in human and rodent enterocytes. The intestinal barrier structure and integrity can be altered by angiotensin (1-7) and dopamine (DA). Both renin-angiotensin and dopaminergic systems influence intestinal Na+/K+-ATPase activity, thus maintaining electrolyte and nutritional homeostasis. The colocalization of B0AT1 and ACE2 indicates the direct role of the renin-angiotensin system in amino acid absorption. Yet, more studies are needed to thoroughly define the structural and functional interaction between TJ-associated proteins and GI renin-angiotensin and dopaminergic systems.

HO-1-induced autophagy establishes a HO-1-p62-Nrf2 positive feedback loop to reduce gut permeability in cholestatic liver disease

Objectives The gut-liver axis disruption is a unified pathogenetic principle of cholestatic liver disease (CSLD). Increased gut permeability is the leading cause of gut-liver axis disruption. HO-1 is capable of protecting against gut-liver axis injury. However, it has rarely been reported whether autophagy is involved in HO-1 protecting gut-liver barrier integrity and the underlying mechanism. Materials and Methods Mice underwent bile duct ligation (BDL) was established as CSLD model in vivo. Caco-2 cells with LPS treatment was established as in vitro cell model. Immunofluorescence, western blot and transepithelial electrical resistance (TER) assay were used to observe epithelial tight junction (TJ) and autophagy. Liver injury and fibrosis were evaluated as well through H&E staining, masson staining, sirius red staining and ELISA. Results and Conclusions Our study demonstrated that the epithelial TJ and TER were notably reduced both in BDL mice and in LPS treated intestinal epithelial cells. Increased HO-1 expression could significantly induce intestinal epithelial cell autophagy. Additionally, this increased autophagy level reversed the reduction effects of BDL or LPS on epithelial TJ and TER in vivo and in vitro, therefore decreased transaminase level in serum and relieved liver fibrosis in BDL mice. Besides, increased autophagy level in turn upregulated the expression of HO-1 by p62 degradation of Keap1 and subsequent activation of Nrf2 pathway. Collectively, these results indicate that HO-1 reduces gut permeability by enhancing autophagy level in CSLD, the increased autophagy establishes a HO-1-p62-Nrf2 positive feedback loop to further improve gut-liver axis disruption. Therefore, our study confirms the critical role of autophagy in HO-1 ameliorating gut-liver axis injury during CSLD, highlighting HO-1 as a promising therapeutic target.

Helicobacter pylori disrupts gastric mucosal homeostasis by stimulating macrophages to secrete CCL3

BackgroundHelicobacter pylori (H. pylori) is the predominant etiological agent of gastritis and disrupts the integrity of the gastric mucosal barrier through various pathogenic mechanisms. After H. pylori invades the gastric mucosa, it interacts with immune cells in the lamina propria. Macrophages are central players in the inflammatory response, and H. pylori stimulates them to secrete a variety of inflammatory factors, leading to the chronic damage of the gastric mucosa. Therefore, the study aims to explore the mechanism of gastric mucosal injury caused by inflammatory factors secreted by macrophages, which may provide a new mechanism for the development of H. pylori-related gastritis.MethodsThe expression and secretion of CCL3 from H. pylori infected macrophages were detected by RT-qPCR, Western blot and ELISA. The effect of H. pylori-infected macrophage culture medium and CCL3 on gastric epithelial cells tight junctions were analyzed by Western blot, immunofluorescence and transepithelial electrical resistance. EdU and apoptotic flow cytometry assays were used to detect cell proliferation and apoptosis levels. Dual-luciferase reporter assays and chromatin immunoprecipitation assays were used to study CCL3 transcription factors. Finally, gastric mucosal tissue inflammation and CCL3 expression were analyzed by hematoxylin and eosin staining and immunohistochemistry.ResultsAfter H. pylori infection, CCL3 expressed and secreted from macrophages were increased. H. pylori-infected macrophage culture medium and CCL3 disrupted gastric epithelial cells tight junctions, while CCL3 neutralizing antibody and receptor inhibitor of CCL3 improved the disruption of tight junctions between cells. In addition, H. pylori-infected macrophage culture medium and CCL3 recombinant proteins stimulated P38 phosphorylation, and P38 phosphorylation inhibitor improved the disruption of tight junctions between cells. Besides, it was identified that STAT1 was a transcription factor of CCL3 and H. pylori stimulated macrophage to secret CCL3 through the JAK1-STAT1 pathway. Finally, after mice were injected with murine CCL3 recombinant protein, the gastric mucosal injury and inflammation were aggravated, and the phosphorylation level of P38 was increased.ConclusionsIn summary, our findings demonstrate that H. pylori infection stimulates macrophages to secrete CCL3 via the JAK1-STAT1 pathway. Subsequently, CCL3 damages gastric epithelial tight junctions through the phosphorylation of P38. This may be a novel mechanism of gastric mucosal injury in H. pylori-associated gastritis.

A Biosynthetic Alternative to Human Amniotic Membrane for Use in Ocular Surface Surgery.

The biosynthetic Symatix membrane (SM) was developed to replace fresh human amniotic membrane (hAM) in ocular surgical applications. The purpose of this study was to test the biocompatibility of the SM with human limbus-derived epithelial cells with regard to their physical and biological properties. Different physical properties of SM were tested ex vivo by simulation on human corneas. In vitro, primary limbal epithelial cells from limbal explants were used to test biological properties such as cell migration, proliferation, metabolic activity, and limbal epithelial cell markers on the SM, hAM, and freeze-dried amniotic membrane (FDAM). The surgical handleability of the SM was equivalent to that of the hAM. Ultrastructural and histological studies demonstrated that epithelial cells on the SM had the typical tightly apposed, polygonal, corneal epithelial cell morphology. The epithelial cells were well stratified on the SM, unlike on the hAM and FDAM. Rapid wound healing occurred on the SM within 3days. Immunofluorescence studies showed positive expression of CK-19, Col-1, laminin, ZO-1, FN, and p-63 on the SM, plastic, and FDAM compared to positive expression of ZO-1, Col-1, laminin, FN, and p63 and negative expression of CK-19 in the hAM. These results indicate that the SM is a better substrate for limbal epithelial cell migration, proliferation, and tight junction formation. Altogether, the SM can provide a suitable alternative to the hAM for surgical application in sight-restoring operations. The hAM, currently widely used in ocular surface surgery, has numerous variations and limitations. The biocompatibility of corneal epithelial cells with the SM demonstrated in this study suggests that it can be a viable substitute for the hAM.

Bifidobacterium bifidum inhibits the TNF-α induced increase in intestinal epithelial tight junction permeability via a TLR-2/TLR-6 and PPAR-γ dependent inhibition of NF-κB p50/p65

Background: Bifidobacterium bifidum (BB) is one of the most widely used probiotic bacterial species and BB has been shown to have a protective effect against intestinal inflammation by an enhancement of the intestinal epithelial tight junction (TJ) barrier. Previous studies from our laboratory showed that a specific BB strain, referred to as BB1, caused a marked enhancement of intestinal epithelial TJ barrier, while strain BB4 had no effect. TNF-α contributes to the development of intestinal inflammation in inflammatory conditions of the gut, in part by increasing intestinal permeability and antigenic penetration. TNF-α induced increase in intestinal epithelial TJ permeability is mediated by an NF-κB p50/p65 activation of myosin light chain kinase (MLCK) gene and MLCK-induced opening of the intestinal TJ barrier. Peroxisome proliferator-activated receptor gamma (PPAR-γ) is an important nuclear receptor known to have anti-inflammatory activity by interfering with NF-κB activation. However, the protective effect of BB1 against TNF-α induced increase in intestinal TJ permeability remain unclear. Aims: The major purpose of this study was to delineate the protective effect of BB1 against the TNF-α induced increase in intestinal TJ permeability and to elucidate the intracellular mechanism involved. Methods: Filter-grown Caco-2 monolayers ( in vitro) and recycling intestinal perfusion of live mice ( in vivo) were the model systems used to assess intestinal TJ permeability. Results: 1) TNF-α caused an increase Caco-2 TJ permeability, as assed by Caco-2 trans-epithelial resistance (TER) and trans-epithelial flux of paracellular marker, FITC-Dextran 10kD. 2) Addition of BB1 (1x108 CFU/ml) to the apical membrane compartment inhibited the TNF-α increase in Caco-2 epithelial TJ permeability, while strain BB4 had no effect. 3) BB1 but not BB4, caused an increase in Caco-2 TLR-2 expression on the apical membrane surface, and siRNA induced knockdown of TLR-2 prevent the BB1 inhibition of TNF-α induced increase in Caco-2 TJ permeability. 4) TNF-α caused a rapid phosphorylation and degradation of Caco-2 IKK-α, as well as and phosphorylation and nuclear translocation of NF-κB p50/p65, indicating NF-κB activation. BB1, but not BB4, inhibited the NF-κB activation. 5) TNF-α activation of NF-κB was associated with an increase in MLCK gene (increase in MLCK promoter activity and mRNA transcription) and kinase activity. BB1, but not BB4, inhibited the TNF-α induced increase in MLCK gene and kinase activity. 6) BB1 but not BB4, caused a rapid PPAR-γ activation (PPAR-γ phosphorylation and nuclear translocation), and siRNA induced knockdown of PPAR-γ inhibited the TNF-α induced activation of NF-κB and increase in Caco-2 TJ permeability. 7) Intraperitoneal injection of TNF-α (5 mg) also caused time-dependent increase in mouse small intestinal permeability (IP) to FITC-Dextran 10kD. TNF-α caused a degradation of IkB-α in intestinal tissue and nuclear translocation of NF-kB in mouse enterocyte. 8) BB1, but not BB4, selectively activated PPAR-γ in mouse enterocytes and inhibited the TNF-α-induced activation of NF-κB, resulting in a decrease in mouse IP. 9) BB1 inhibition of TNF-α activation of mouse enterocyte NF-κB and increase in IP was prevented in mice with intestinal epithelial cell-specific knockout of PPAR-γ Villin-cre and TLR-2 Villin-cre mice. Conclusion: BB1 protects against the TNF-α induced increase in intestinal TJ permeability in a strain-specific manner. BB1 protects against the TNF-α induced increase in intestinal TJ permeability via a novel intracellular mechanism involving BB1/TLR-2 pathway activation of PPAR-γ, and PPAR-γ mediated inhibition of NF-κB p50/p65 and MLCK gene in enterocyte. This research project was supported by the National Institute of Diabetes and Digestive and Kidney Diseases R01-DK-121073-01. This is the full abstract presented at the American Physiology Summit 2024 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.

Paneth Cell-Specific TRPV6 Channel Plays a Crucial Role in Alcohol-Associated Dysbiosis of Gut Microbiota and Tissue Injury at the Gut-Liver Axis

Introduction: Endotoxemia plays an essential role in alcohol-associated tissue injury. Two factors contributing to endotoxemia are gut microbiota dysbiosis and epithelial barrier dysfunction. Previous studies indicated that TRPV6 channel-mediated Ca2+ influx plays a crucial role in alcohol-induced epithelial tight junction (TJ) disruption, mucosal barrier dysfunction, and endotoxemia. Trpv6 knockout mice were resistant to alcohol-induced intestinal barrier dysfunction and modulated microbiota dysbiosis, raising the question of how TRPV6 regulates microbiota composition. In this study, we investigated the role of Paneth cell-specific TRPV6 in alcohol-induced microbiota dysbiosis and barrier dysfunction. Methods: Adult Trpv6fl/fl (WT) and Trpv6fl/fl-Defa6Cre (KO; Paneth cell-specific TRPV6 knockout) mice were fed a Lieber-DiCarli liquid diet with EtOH (0% 2d, 1% 2d, 2% 2d, 4% 1wk, 5% 1wk, & 6% 1wk). Control groups were pair-fed an isocaloric EtOH-free diet. Intestinal permeability was measured by vascular-to-luminal flux of FITC-inulin in vivo, and mucosal inflammation was assessed by RT-PCR for cytokines/chemokines. Plasma lipopolysaccharide (LPS) was measured to evaluate endotoxemia. Gut microbiota was analyzed by 16S rRNA sequencing of colonic flushing. Lipidome in colonic flushing was analyzed using nontargeted liquid chromatography-mass spectrometry. Microbiome and lipidome data were processed by bioinformatic analyses. Results and conclusion: Data show that colon length was reduced, and colonic mucosal permeability in vivo was elevated by EtOH in WT mice. The increase in mucosal permeability was associated with increased plasma LPS levels. EtOH feeding elevated the expression of IL-6, IL-1β, and TNFα in the colon. EtOH-induced changes in colon length, mucosal permeability, cytokine expression, and plasma LPS were absent in KO mice. 16S rRNA sequencing data indicated that the microbiota composition in KO mice differed from that of WT mice. The relative abundance of Prevotellaceae, Xanthomonadaceae, and Streptococaceae were higher, and Psuedomonadaceae and Verrucomicrobiaceae abundance were lower in KO mice compared to WT mice. EtOH-induced increase in the abundance of Norcardiaceae and Psuedomonadaceae was low or absent in KO mice. EtOH increased Rhodococcus, Odoribacter, and Pseudomonas abundance at the genus level in WT but not KO mice. On the other hand, Clostridium, Turicibacter, and Helicobacter abundance were increased by EtOH in KO but not WT mice. Lipidomic analysis of colonic flushing showed that phospholipid contents were higher in KO mice than WT mice, irrespective of sex and treatments. A cluster of sphingolipids was dramatically high in KO female mice, which was reduced by EtOH feeding. Short-chain fatty acid esters of hydroxy fatty acids (AAHFA 3:0/24:0;O) were elevated by EtOH in male WT mice but not in female WT and male or female KO mice. In conclusion, these data indicate that Paneth cell-specific deletion of TRPV6 attenuates alcohol-induced changes in gut microbiota, colonic luminal lipids, mucosal barrier function, and endotoxemia in mice. NIH/NIAAA, RO1-AA029270; Veterans Administration IO1-BX003014. This is the full abstract presented at the American Physiology Summit 2024 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.

Creating new in vitro models to examine the role of Pkd1 in cell and tubular physiology

Autosomal dominant polycystic kidney disease (ADPKD) results from loss of function mutations in polycystin proteins PC1 and PC2 encoded by the PKD1 and PKD2 genes. ADPKD affects 1 in 400 to 1 in 1000 people worldwide, with approximately 50% of patients progressing to end stage renal disease. The physiological function of the polycystins and the mechanisms of cystogenesis remain largely unknown. In vitro models may provide the clearest possibility to understand the immediate cell biological effects of polycystin loss, without the confounding second order injury that occurs as cysts develop. Some of the hurdles to in vitro models include clonal variation, renal segment heterogeneity, and inconsistent growth in 3D culture conditions. To address this need, we isolated renal epithelial cells from either the cortex or the medulla of Pkd1flox/flox; Pax8-rtTA; tetO-7-Cre mice crossed with the immorto-mouse expressing the temperature sensitive SV40 large T antigen. We then cloned single cells into isogenic cell lines, one cortical and one medullary. We verified the induction of Pkd1 KO by treating the cells with either 10 ug/ml doxycycline to knock out (KO) Pkd1 or vehicle control. Treatment continued for 7 days, after which Pkd1 KO was confirmed by western blot. Next, we sought to functionally characterize the two new cell lines. When the cells were grown on transwell supports, cells formed epithelial tight junctions with a trans-epithelial resistance of 2467 ohms/cm2. Resistance increased to 3130 ohms/cm2 following Pkd1 KO in the medullary clone. We next measured the rate of media acidification with or without Pkd1 KO as a proxy for glucose utilization. Pkd1 KO cells acidified faster than controls in both clones. Next, we addressed their ability to grow into tubuloids or spheroids / cysts in 3D culture using multiple approaches. Cells were either 1) suspended directly in Matrigel and plated in a 96 well plate, 2) suspended in Matrigel droplets, with multiple droplets adhering to an individual well of a 6 well plate, 3) plated within a Matrigel “sandwich”, or 4) plated within a sandwich and pulsed with glial derived neurotropic factor (GDNF) for 48 hours. All clones suspended directly in Matrigel formed spheroids, with more and larger spheroids in the Matrigel droplet cultures. Similar results were observed in the cortical clone grown in the sandwich, however the Pkd1 KO medullary clone in the same culture conditions had several giant spheroid structures at least 3 times larger than any other organoid observed in control medullary cultures. Finally, cortical cultures grown in the sandwich and pulsed with GDNF revealed larger spheroid structures in the Pkd1 KO cells than the control structures. Medullary cultures grown with GDNF revealed complicated structures with lumens and tubuloid projections in the controls, and large hollow spheroid / cystic structures in the Pkd1 KO cultures. Taken together, these data support the Matrigel sandwich with GDNF pulse may be the most useful to interrogate physiological changes in the absence of Pkd1 expression. In summary, we have created two new clonal cell lines with inducible Pkd1 KO, with isogenic controls, and the ability to differentiate into complicated tubuloids or spheroids in a genotypical manner, that may help elucidate the most proximate events occuring in renal epithelial cells after the inactivation of Pkd1. U54 DK126114. This is the full abstract presented at the American Physiology Summit 2024 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.