Identification of autophagy receptors for the Crohn's disease-associated adherent-invasive Escherichia coli.

Introduction: Crohn's disease (CD) is a chronic inflammatory bowel disease, of which the etiology involves genetic, environmental and microbial factors. Adherent-invasive Escherichia coli (AIEC) and polymorphisms in autophagy-related genes have been implicated in CD etiology. Autophagy is a key process for the maintenance of cellular homeostasis, which allows the degradation of damaged cytoplasmic components and pathogens via lysosome. We have shown that a functional autophagy is necessary for AIEC clearance. Here, we aimed at identifying the autophagy receptor(s) responsible to target AIEC to autophagy for degradation. Methods: The levels of autophagy receptors p62, NDP52, NBR1, TAX1BP1 and Optineurin were knocked down in human intestinal epithelial cells T84 using siRNAs. The NDP52 knock-out (KO) and p62 KO HeLa cells, as well as NDP52 KO HeLa cells expressing the wild-type NDP52 or the mutated NDP52Val248Ala protein were used. Results and discussion: We showed that, among the tested autophagy receptors (p62, NDP52, NBR1, TAX1BP1 and Optineurin), diminished expression of p62 or NDP52 increased the number of the clinical AIEC LF82 strain inside epithelial cells. This was associated with increased pro-inflammatory cytokine production. Moreover, p62 or NDP52 directly colocalized with AIEC LF82 and LC3, an autophagy marker. As the NDP52Val248Ala polymorphism has been associated with increased CD susceptibility, we investigated its impact on AIEC control. However, in HeLa cell and under our experimental condition, no effect of this polymorphism neither on AIEC LF82 intracellular number nor on pro-inflammatory cytokine production was observed. Together, our results suggest that p62 and NDP52 act as autophagy receptors for AIEC recognition, controlling AIEC intracellular replication and inflammation.

Effects of cancer-induced cachexia and administration of L-glutathione on the intestinal mucosa in rat.

Walker-256 tumor is an experimental model known to promote cachexia syndrome, oxidative stress, and systemic inflammation. This study evaluated the duodenal mucosa of rats with Walker-256 tumor administered with 1% L-glutathione, intending to evaluate the damage caused by cancer-associated cachexia in the gastrointestinal tract and the effects of antioxidant administration on mucosal protection. Twenty-four 55-day-old male Wistar rats were distributed into four groups: control (C); control administered with 1% L-glutathione (C-GSH); Walker-256 tumor (W) and Walker-256 tumor administered with 1% L-glutathione (W-GSH). After 14 days of treatment, the duodenum was harvested for morphometric analysis of the mucosa, proliferation, apoptosis, immunostaining of varicosities immunoreactive (IR) to vasoactive intestinal peptide (VIP) and 5-HT-IR cells, and quantification of mast cells and goblet cells. Walker-256 tumor-bearing rats showed cachexia syndrome, mucosal atrophy, reduced cell proliferation, reduced 5-HT-IR cells, and increased goblet cells and VIPergic varicosities, which were not reversed by L-glutathione. On the other hand, L-glutathione caused a reduction of cells in apoptosis and mast cell recruitment, demonstrating a partial recovery of the damage detected in the intestinal mucosa.

Gut microbiota as a key regulator of intestinal mucosal immunity.

Gut microbiota is a complex microbial community with the ability of maintaining intestinal health. Intestinal homeostasis largely depends on the mucosal immune system to defense external pathogens and promote tissue repair. In recent years, growing evidence revealed the importance of gut microbiota in shaping intestinal mucosal immunity. Therefore, according to the existing findings, this review first provided an overview of intestinal mucosal immune system before summarizing the regulatory roles of gut microbiota in intestinal innate and adaptive immunity. Specifically, this review delved into the gut microbial interactions with the cells such as intestinal epithelial cells (IECs), macrophages, dendritic cells (DCs), neutrophils, and innate lymphoid cells (ILCs) in innate immunity, and T and B lymphocytes in adaptive immunity. Furthermore, this review discussed the main effects of gut microbiota dysbiosis in intestinal diseases and offered future research prospects. The review highlighted the key regulatory roles of gut microbiota in intestinal mucosal immunity via various host-microbe interactions, providing valuable references for the development of microbial therapy in intestinal diseases.

Crosstalk between hypoxia-inducible factor-1α and short-chain fatty acids in inflammatory bowel disease: key clues toward unraveling the mystery.

The human intestinal tract constitutes a complex ecosystem, made up of countless gut microbiota, metabolites, and immune cells, with hypoxia being a fundamental environmental characteristic of this ecology. Under normal physiological conditions, a delicate balance exists among these complex "residents", with disruptions potentially leading to inflammatory bowel disease (IBD). The core pathology of IBD features a disrupted intestinal epithelial barrier, alongside evident immune and microecological disturbances. Central to these interconnected networks is hypoxia-inducible factor-1α (HIF-1α), which is a key regulator in gut cells for adapting to hypoxic conditions and maintaining gut homeostasis. Short-chain fatty acids (SCFAs), as pivotal gut metabolites, serve as vital mediators between the host and microbiota, and significantly influence intestinal ecosystem. Recent years have seen a surge in research on the roles and therapeutic potential of HIF-1α and SCFAs in IBD independently, yet reviews on HIF-1α-mediated SCFAs regulation of IBD under hypoxic conditions are scarce. This article summarizes evidence of the interplay and regulatory relationship between SCFAs and HIF-1α in IBD, pivotal for elucidating the disease's pathogenesis and offering promising therapeutic strategies.

Octreotide attenuates intestinal barrier damage by maintaining basal autophagy in Caco2 cells.

The intestinal mucosal barrier is of great importance for maintaining the stability of the internal environment, which is closely related to the occurrence and development of intestinal inflammation. Octreotide (OCT) has potential applicable clinical value for treating intestinal injury according to previous studies, but the underlying molecular mechanisms have remained elusive. This article is based on a cell model of inflammation induced by lipopolysaccharide (LPS), aiming to explore the effects of OCT in protecting intestinal mucosal barrier function. A Cell Counting Kit­8 assay was used to determine cell viability and evaluate the effectiveness of OCT. Gene silencing technology was used to reveal the mediated effect of somatostatin receptor 2 (SSTR2). The changes in intestinal permeability were detected through trans­epithelial electrical resistance and fluorescein isothiocyanate­dextran 4 experiments, and the alterations in tight junction proteins were detected using immunoblotting and reverse transcription fluorescence­quantitative PCR technology. Autophagosomes were observed by electron microscopy and the dynamic changes of the autophagy process were characterized by light chain (LC)3­II/LC3­I conversion and autophagic flow. The results indicated that SSTR2­dependent OCT can prevent the decrease in cell activity. After LPS treatment, the permeability of monolayer cells decreased and intercellular tight junctions were disrupted, resulting in a decrease in tight junction protein zona occludens 1 in cells. The level of autophagy­related protein LC3 was altered to varying degrees at different times. These abnormal changes gradually returned to normal levels after the combined application of LPS and SSTR2­dependent OCT, confirming the role of OCT in protecting intestinal barrier function. These experimental results suggest that OCT maintains basal autophagy and cell activity mediated by SSTR2 in intestinal epithelial cells, thereby preventing the intestinal barrier dysfunction in inflammation injury.

Neonatal intestinal mucus barrier changes in response to maturity, inflammation, and sodium decanoate supplementation.

The integrity of the intestinal mucus barrier is crucial for human health, as it serves as the body's first line of defense against pathogens. However, postnatal development of the mucus barrier and interactions between maturity and its ability to adapt to external challenges in neonatal infants remain unclear. In this study, we unveil a distinct developmental trajectory of the mucus barrier in preterm piglets, leading to enhanced mucus microstructure and reduced mucus diffusivity compared to term piglets. Notably, we found that necrotizing enterocolitis (NEC) is associated with increased mucus diffusivity of our large pathogen model compound, establishing a direct link between the NEC condition and the mucus barrier. Furthermore, we observed that addition of sodium decanoate had varying effects on mucus diffusivity depending on maturity and health state of the piglets. These findings demonstrate that regulatory mechanisms governing the neonatal mucosal barrier are highly complex and are influenced by age, maturity, and health conditions. Therefore, our results highlight the need for specific therapeutic strategies tailored to each neonatal period to ensure optimal gut health.

[How psychological stress aggravates intestinal inflammation]. / Hoe psychologische stress tot darmontstekingen leidt.

OBJECTIVE: To investigate the biological mechanisms underlying the associations of psychological stress and intestinal inflammation in inflammatory bowel disease (IBD). DESIGN: Experimental mouse models and large human cohorts have been used. METHOD: Consecutive mouse models with chemically induced colitis were used to investigate biological pathways though which psychological stress leads to gut inflammation. These results were validated in three human cohorts with patients with IBD. RESULTS: Stress induced elevated levels of glucocorticoids drive the generation of an inflammatory subset of enteric glia cells. These enteric glia cells produce the protein CSF1, that promotes monocyte accumulation in the intestinal mucosa and TNF-mediated intestinal inflammation. CONCLUSION: A pivotal role for the enteric nervous system (ENS) has been discovered in mediating the aggravating effect of psychological stress on intestinal inflammation.

Impact of Micro- and Nano-Plastics on Human Intestinal Organoid-Derived Epithelium.

The development of patient-derived intestinal organoids represents an invaluable model for simulating the native human intestinal epithelium. These stem cell-rich cultures outperform commonly used cell lines like Caco-2 and HT29-MTX in reflecting the cellular diversity of the native intestinal epithelium after differentiation. In our recent study examining the effects of polystyrene (PS), microplastics (MPs), and nanoplastics (NPs), widespread pollutants in our environment and food chain, on the human intestinal epithelium, these organoids have been instrumental in elucidating the absorption mechanisms and potential biological impacts of plastic particles. Building on previously established protocols in human intestinal organoid culture, we herein detail a streamlined protocol for the cultivation, differentiation, and generation of organoid-derived monolayers. This protocol is tailored to generate monolayers incorporating microfold cells (M cells), key for intestinal particle uptake but often absent in current in vitro models. We provide validated protocols for the characterization of MPs/NPs via scanning electron microscopy (SEM) for detailed imaging and their introduction to intestinal epithelial monolayer cells via confocal immunostaining. Additionally, protocols to test the impacts of MP/NP exposure on the functions of the intestinal barrier using transendothelial electrical resistance (TEER) measurements and assessing inflammatory responses using cytokine profiling are detailed. Overall, our protocols enable the generation of human intestinal organoid monolayers, complete with the option of including or excluding M cells, offering crucial techniques for observing particle uptake and identifying inflammatory responses in intestinal epithelial cells to advance our knowledge of the potential effects of plastic pollution on human gut health. These approaches are also amendable to the study of other gut-related chemical and biological exposures and physiological responses due to the robust nature of the systems. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Human intestinal organoid culture and generation of monolayers with and without M cells Support Protocol 1: Culture of L-WRN and production of WRN-conditioned medium Support Protocol 2: Neuronal cell culture and integration into intestinal epithelium Support Protocol 3: Immune cell culture and integration into intestinal epithelium Basic Protocol 2: Scanning electron microscopy: sample preparation and imaging Basic Protocol 3: Immunostaining and confocal imaging of MP/NP uptake in organoid-derived monolayers Basic Protocol 4: Assessment of intestinal barrier function via TEER measurements Basic Protocol 5: Cytokine profiling using ELISA post-MP/NP exposure.

Dermal injury drives a skin to gut axis that disrupts the intestinal microbiome and intestinal immune homeostasis in mice.

The composition of the microbial community in the intestine may influence the functions of distant organs such as the brain, lung, and skin. These microbes can promote disease or have beneficial functions, leading to the hypothesis that microbes in the gut explain the co-occurrence of intestinal and skin diseases. Here, we show that the reverse can occur, and that skin directly alters the gut microbiome. Disruption of the dermis by skin wounding or the digestion of dermal hyaluronan results in increased expression in the colon of the host defense genes Reg3 and Muc2, and skin wounding changes the composition and behavior of intestinal bacteria. Enhanced expression Reg3 and Muc2 is induced in vitro by exposure to hyaluronan released by these skin interventions. The change in the colon microbiome after skin wounding is functionally important as these bacteria penetrate the intestinal epithelium and enhance colitis from dextran sodium sulfate (DSS) as seen by the ability to rescue skin associated DSS colitis with oral antibiotics, in germ-free mice, and fecal microbiome transplantation to unwounded mice from mice with skin wounds. These observations provide direct evidence of a skin-gut axis by demonstrating that damage to the skin disrupts homeostasis in intestinal host defense and alters the gut microbiome.

Rab7-dependent regulation of goblet cell protein CLCA1 modulates gastrointestinal homeostasis.

Inflammation in ulcerative colitis is typically restricted to the mucosal layer of distal gut. Disrupted mucus barrier, coupled with microbial dysbiosis, has been reported to occur prior to the onset of inflammation. Here, we show the involvement of vesicular trafficking protein Rab7 in regulating the colonic mucus system. We identified a lowered Rab7 expression in goblet cells of colon during human and murine colitis. In vivo Rab7 knocked down mice (Rab7KD) displayed a compromised mucus layer, increased microbial permeability, and depleted gut microbiota with enhanced susceptibility to dextran sodium-sulfate induced colitis. These abnormalities emerged owing to altered mucus composition, as revealed by mucus proteomics, with increased expression of mucin protease chloride channel accessory 1 (CLCA1). Mechanistically, Rab7 maintained optimal CLCA1 levels by controlling its lysosomal degradation, a process that was dysregulated during colitis. Overall, our work establishes a role for Rab7-dependent control of CLCA1 secretion required for maintaining mucosal homeostasis.

Alkaline sphingomyelinase deficiency impairs intestinal mucosal barrier integrity and reduces antioxidant capacity in dextran sulfate sodium-induced colitis.

BACKGROUND: Ulcerative colitis is a chronic inflammatory disease of the colon with an unknown etiology. Alkaline sphingomyelinase (alk-SMase) is specifically expressed by intestinal epithelial cells, and has been reported to play an anti-inflammatory role. However, the underlying mechanism is still unclear. AIM: To explore the mechanism of alk-SMase anti-inflammatory effects on intestinal barrier function and oxidative stress in dextran sulfate sodium (DSS)-induced colitis. METHODS: Mice were administered 3% DSS drinking water, and disease activity index was determined to evaluate the status of colitis. Intestinal permeability was evaluated by gavage administration of fluorescein isothiocyanate dextran, and bacterial translocation was evaluated by measuring serum lipopolysaccharide. Intestinal epithelial cell ultrastructure was observed by electron microscopy. Western blotting and quantitative real-time reverse transcription-polymerase chain reaction were used to detect the expression of intestinal barrier proteins and mRNA, respectively. Serum oxidant and antioxidant marker levels were analyzed using commercial kits to assess oxidative stress levels. RESULTS: Compared to wild-type (WT) mice, inflammation and intestinal permeability in alk-SMase knockout (KO) mice were more severe beginning 4 d after DSS induction. The mRNA and protein levels of intestinal barrier proteins, including zonula occludens-1, occludin, claudin-3, claudin-5, claudin-8, mucin 2, and secretory immunoglobulin A, were significantly reduced on 4 d after DSS treatment. Ultrastructural observations revealed progressive damage to the tight junctions of intestinal epithelial cells. Furthermore, by day 4, mitochondria appeared swollen and degenerated. Additionally, compared to WT mice, serum malondialdehyde levels in KO mice were higher, and the antioxidant capacity was significantly lower. The expression of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) in the colonic mucosal tissue of KO mice was significantly decreased after DSS treatment. mRNA levels of Nrf2-regulated downstream antioxidant enzymes were also decreased. Finally, colitis in KO mice could be effectively relieved by the injection of tertiary butylhydroquinone, which is an Nrf2 activator. CONCLUSION: Alk-SMase regulates the stability of the intestinal mucosal barrier and enhances antioxidant activity through the Nrf2 signaling pathway.

Are we ready to use new endoscopic scores for ulcerative colitis?

For ulcerative colitis (UC), the variability in inflammatory activity along the colon poses a challenge in management. The focus on achieving endoscopic healing in UC is evident, where the UC Endoscopic Index of Severity and Mayo Endoscopic Subscore are commonly used for evaluation. However, these indices primarily consider the most severely affected region. Liu et al recent study validates the Toronto Inflammatory Bowel Disease Global Endoscopic Reporting (TIGER) score offering a comprehensive assessment of inflammatory activity across diverse segments of the colon and rectum and a reliable index correlating strongly with UC Endoscopic Index of Severity and moderately with Mayo Endoscopic Subscore (MES). Despite recommendation, certain aspects warrant further investigation. Fecal calprotectin, an intermediate target, correlates with TIGER and should be explored. Determining TIGER scores defining endoscopic remission and response, evaluating agreement with histological activity, and assessing inter-endoscopist agreement for TIGER require scrutiny. Exploring the correlation between TIGER and intestinal ultrasound, akin to MES, adds value.

Correlation between neuregulin receptor degradation protein-1 and Crohn's disease.

Neuregulin receptor degradation protein-1 (Nrdp1) is a newly discovered E3 ligase that plays a role in the apoptosis process of multiple diseases. Previous studies has shown that Nrdp1 exerted a proapoptotic effect in cardiac diseases. The purpose of this study is to investigate the potential involvement of Nrdp1 in the pathological processes of inflammatory bowel disease (IBD). To create a mouse model of experimental colitis, trinitrobenzenesulfonic acid (TNBS) was administered and the severity of colitis was assessed based on changes in weight and histological scores. Using Western blot and immunohistochemistry, significant increase in Nrdp1 expression was observed in intestinal epithelial cells (IECs). This was accompanied with the up-regulation of cleaved PARP and active caspase-3 in IECs, indicating a potential function in IECs. To study this further, we built an in vitro model of tumor necrosis factor-alpha (TNF-α)-induced apoptosis using human IEC line HT-29 cells. When Nrdp1 was knocked down, a decrease in apoptosis was observed, suggesting that Nrdp1 may play a proapoptotic role in IEC apoptosis. The mechanism behind this phenomenon is associated with the suppression of downstream targets of Nrdp1, such as protein kinase B (AKT). Furthermore, immunohistochemistry analysis in patients with Crohn's disease (CD) and normal controls supported the same results as observed in experimental colitis. We conclude that Nrdp1 may be a promising new therapeutic target for ameliorating IBD in humans.

DHX9 maintains epithelial homeostasis by restraining R-loop-mediated genomic instability in intestinal stem cells.

Epithelial barrier dysfunction and crypt destruction are hallmarks of inflammatory bowel disease (IBD). Intestinal stem cells (ISCs) residing in the crypts play a crucial role in the continuous self-renewal and rapid recovery of intestinal epithelial cells (IECs). However, how ISCs are dysregulated in IBD remains poorly understood. Here, we observe reduced DHX9 protein levels in IBD patients, and mice with conditional DHX9 depletion in the intestinal epithelium (Dhx9ΔIEC) exhibit an increased susceptibility to experimental colitis. Notably, Dhx9ΔIEC mice display a significant reduction in the numbers of ISCs and Paneth cells. Further investigation using ISC-specific or Paneth cell-specific Dhx9-deficient mice demonstrates the involvement of ISC-expressed DHX9 in maintaining epithelial homeostasis. Mechanistically, DHX9 deficiency leads to abnormal R-loop accumulation, resulting in genomic instability and the cGAS-STING-mediated inflammatory response, which together impair ISC function and contribute to the pathogenesis of IBD. Collectively, our findings highlight R-loop-mediated genomic instability in ISCs as a risk factor in IBD.

Gut-associated lymphoid tissue attrition associates with response to anti-α4ß7 therapy in ulcerative colitis.

Vedolizumab (VDZ) is a first-line treatment in ulcerative colitis (UC) that targets the α4ß7- mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) axis. To determine the mechanisms of action of VDZ, we examined five distinct cohorts of patients with UC. A decrease in naïve B and T cells in the intestines and gut-homing (ß7+) plasmablasts in circulation of VDZ-treated patients suggested that VDZ targets gut-associated lymphoid tissue (GALT). Anti-α4ß7 blockade in wild-type and photoconvertible (KikGR) mice confirmed a loss of GALT size and cellularity because of impaired cellular entry. In VDZ-treated patients with UC, treatment responders demonstrated reduced intestinal lymphoid aggregate size and follicle organization and a reduction of ß7+IgG+ plasmablasts in circulation, as well as IgG+ plasma cells and FcγR-dependent signaling in the intestine. GALT targeting represents a previously unappreciated mechanism of action of α4ß7-targeted therapies, with major implications for this therapeutic paradigm in UC.

Integrated analysis of gut metabolome, microbiome, and exfoliome data in an equine model of intestinal injury.

BACKGROUND: The equine gastrointestinal (GI) microbiome has been described in the context of various diseases. The observed changes, however, have not been linked to host function and therefore it remains unclear how specific changes in the microbiome alter cellular and molecular pathways within the GI tract. Further, non-invasive techniques to examine the host gene expression profile of the GI mucosa have been described in horses but not evaluated in response to interventions. Therefore, the objectives of our study were to (1) profile gene expression and metabolomic changes in an equine model of non-steroidal anti-inflammatory drug (NSAID)-induced intestinal inflammation and (2) apply computational data integration methods to examine host-microbiota interactions. METHODS: Twenty horses were randomly assigned to 1 of 2 groups (n = 10): control (placebo paste) or NSAID (phenylbutazone 4.4 mg/kg orally once daily for 9 days). Fecal samples were collected on days 0 and 10 and analyzed with respect to microbiota (16S rDNA gene sequencing), metabolomic (untargeted metabolites), and host exfoliated cell transcriptomic (exfoliome) changes. Data were analyzed and integrated using a variety of computational techniques, and underlying regulatory mechanisms were inferred from features that were commonly identified by all computational approaches. RESULTS: Phenylbutazone induced alterations in the microbiota, metabolome, and host transcriptome. Data integration identified correlation of specific bacterial genera with expression of several genes and metabolites that were linked to oxidative stress. Concomitant microbiota and metabolite changes resulted in the initiation of endoplasmic reticulum stress and unfolded protein response within the intestinal mucosa. CONCLUSIONS: Results of integrative analysis identified an important role for oxidative stress, and subsequent cell signaling responses, in a large animal model of GI inflammation. The computational approaches for combining non-invasive platforms for unbiased assessment of host GI responses (e.g., exfoliomics) with metabolomic and microbiota changes have broad application for the field of gastroenterology. Video Abstract.

Positive modulation of a new reconstructed human gut microbiota by Maitake extract helpfully boosts the intestinal environment in vitro.

The human gut is a complex environment where the microbiota and its metabolites play a crucial role in the maintenance of a healthy state. The aim of the present work is the reconstruction of a new in vitro minimal human gut microbiota resembling the microbe-microbe networking comprising the principal phyla (Bacillota, Bacteroidota, Pseudomonadota, and Actinomycetota), to comprehend the intestinal ecosystem complexity. In the reductionist model, we mimicked the administration of Maitake extract as prebiotic and a probiotic formulation (three strains belonging to Lactobacillus and Bifidobacterium genera), evaluating the modulation of strain levels, the release of beneficial metabolites, and their health-promoting effects on human cell lines of the intestinal environment. The administration of Maitake and the selected probiotic strains generated a positive modulation of the in vitro bacterial community by qPCR analyses, evidencing the prominence of beneficial strains (Lactiplantibacillus plantarum and Bifidobacterium animalis subsp. lactis) after 48 hours. The bacterial community growths were associated with the production of metabolites over time through GC-MSD analyses such as lactate, butyrate, and propionate. Their effects on the host were evaluated on cell lines of the intestinal epithelium and the immune system, evidencing positive antioxidant (upregulation of SOD1 and NQO1 genes in HT-29 cell line) and anti-inflammatory effects (production of IL-10 from all the PBMCs). Therefore, the results highlighted a positive modulation induced by the synergic activities of probiotics and Maitake, inducing a tolerogenic microenvironment.

Intestinal Piezo1 aggravates intestinal barrier dysfunction during sepsis by mediating Ca2+ influx.

INTRODUCTION: Intestinal barrier dysfunction is a pivotal factor in sepsis progression. The mechanosensitive ion channel Piezo1 is associated with barrier function; however, its role in sepsis-induced intestinal barrier dysfunction remains poorly understood. METHODS: The application of cecal ligation and puncture (CLP) modeling was performed on both mice of the wild-type (WT) variety and those with Villin-Piezo1flox/flox genetic makeup to assess the barrier function using in vivo FITC-dextran permeability measurements and immunofluorescence microscopy analysis of tight junctions (TJs) and apoptosis levels. In vitro, Caco-2 monolayers were subjected to TNF-α incubation. Moreover, to modulate Piezo1 activation, GsMTx4 was applied to inhibit Piezo1 activation. The barrier function, intracellular calcium levels, and mitochondrial function were monitored using calcium imaging and immunofluorescence techniques. RESULTS: In the intestinal tissues of CLP-induced septic mice, Piezo1 protein levels were notably elevated compared with those in normal mice. Piezo1 has been implicated in the sepsis-mediated disruption of TJs, apoptosis of intestinal epithelial cells, elevated intestinal mucosal permeability, and systemic inflammation in WT mice, whereas these effects were absent in Villin-Piezo1flox/flox CLP mice. In Caco-2 cells, TNF-α prompted calcium influx, an effect reversed by GsMTx4 treatment. Elevated calcium concentrations are correlated with increased accumulation of reactive oxygen species, diminished mitochondrial membrane potential, and TJ disruption. CONCLUSIONS: Thus, Piezo1 is a potential contributor to sepsis-induced intestinal barrier dysfunction, influencing apoptosis and TJ modification through calcium influx-mediated mitochondrial dysfunction.

Melanocortin 3,5 receptors immunohistochemical expression in colonic mucosa of inflammatory bowel disease patients: A matter of disease activity?

BACKGROUND: Melanocortin 3 and 5 receptors (i.e., MC3R and MC5R) belong to the melanocortin family. However, data regarding their role in inflammatory bowel diseases (IBD) are currently unavailable. AIM: This study aims to ascertain their expression profiles in the colonic mucosa of Crohn's disease (CD) and ulcerative colitis (UC), aligning them with IBD disease endoscopic and histologic activity. METHODS: Colonic mucosal biopsies from CD/UC patients were sampled, and immunohistochemical analyses were conducted to evaluate the expression of MC3R and MC5R. Colonic sampling was performed on both traits with endoscopic scores (Mayo endoscopic score and CD endoscopic index of severity) consistent with inflamed mucosa and not consistent with disease activity (i.e., normal appearing mucosa). RESULTS: In both CD and UC inflamed mucosa, MC3R (CD: + 7.7 fold vs normal mucosa, P < 0.01; UC: + 12 fold vs normal mucosa, P < 0.01) and MC5R (CD: + 5.5 fold vs normal mucosa, P < 0.01; UC: + 8.1 fold vs normal mucosa, P < 0.01) were significantly more expressed compared to normal mucosa. CONCLUSION: MC3R and MC5R are expressed in the colon of IBD patients. Furthermore, expression may differ according to disease endoscopic activity, with a higher degree of expression in the traits affected by disease activity in both CD and UC, suggesting a potential use of these receptors in IBD pharmacology.

Lactate Protects Intestinal Epithelial Barrier Function from Dextran Sulfate Sodium-Induced Damage by GPR81 Signaling.

The dysregulation of the intestinal epithelial barrier significantly contributes to the inflammatory progression of ulcerative colitis. Recent studies have indicated that lactate, produced by gut bacteria or derived from fermented foods, plays a key role in modulating inflammation via G-protein-coupled receptor 81 (GPR81). In this study, we aimed to investigate the potential role of GPR81 in the progression of colitis and to assess the impact of lactate/GPR81 signaling on intestinal epithelial barrier function. Our findings demonstrated a downregulation of GPR81 protein expression in patients with colitis. Functional verification experiments showed that Gpr81-deficient mice exhibited more severe damage to the intestinal epithelial barrier and increased susceptibility to DSS-induced colitis, characterized by exacerbated oxidative stress, elevated inflammatory cytokine secretion, and impaired expression of tight-junction proteins. Mechanistically, we found that lactate could suppress TNF-α-induced MMP-9 expression and prevent the disruption of tight-junction proteins by inhibiting NF-κB activation through GPR81 in vitro. Furthermore, our study showed that dietary lactate could preserve intestinal epithelial barrier function against DSS-induced damage in a GPR81-dependent manner in vivo. Collectively, these results underscore the crucial involvement of the lactate/GPR81 signaling pathway in maintaining intestinal epithelial barrier function, providing a potential therapeutic strategy for ulcerative colitis.