Interaction between microRNA-195 and HuR regulates Paneth cell function in the intestinal epithelium by altering SOX9 translation.

Paneth cells at the bottom of small intestinal crypts secrete antimicrobial peptides, enzymes, and growth factors and contribute to pathogen clearance and maintenance of the stem cell niche. Loss of Paneth cells and their dysfunction occur commonly in various pathologies, but the mechanism underlying the control of Paneth cell function remains largely unknown. Here, we identified microRNA-195 (miR-195) as a repressor of Paneth cell development and activity by altering SOX9 translation via interaction with RNA-binding protein HuR. Tissue-specific transgenic expression of miR-195 (miR195-Tg) in the intestinal epithelium decreased the levels of mucosal SOX9 and reduced the numbers of lysozyme-positive (Paneth) cells in mice. Ectopically expressed SOX9 in the intestinal organoids derived from miR-195-Tg mice restored Paneth cell development ex vivo. miR-195 did not bind to Sox9 mRNA but it directly interacted with HuR and prevented HuR binding to Sox9 mRNA, thus inhibiting SOX9 translation. Intestinal mucosa from mice that harbored both Sox9 transgene and ablation of the HuR locus exhibited lower levels of SOX9 protein and Paneth cell numbers than those observed in miR-195-Tg mice. Inhibition of miR-195 activity by its specific antagomir improved Paneth cell function in HuR-deficient intestinal organoids. These results indicate that interaction of miR-195 with HuR regulates Paneth cell function by altering SOX9 translation in the small intestinal epithelium.NEW & NOTEWORTHY Our results indicate that intestinal epithelial tissue-specific transgenic miR-195 expression decreases the levels of SOX9 expression, along with reduced numbers of Paneth cells. Ectopically expressed SOX9 in the intestinal organoids derived from miR-195-Tg mice restores Paneth cell development ex vivo. miR-195 inhibits SOX9 translation by preventing binding of HuR to Sox9 mRNA. These findings suggest that interaction between miR-195 and HuR controls Paneth cell function via SOX9 in the intestinal epithelium.

Organoid-Based Human Stomach Micro-Physiological System to Recapitulate the Dynamic Mucosal Defense Mechanism.

Several stomach diseases are attributed to the dysregulation of physiological function of gastric mucosal barrier by pathogens. Gastric organoids are a promising tool to develop treatment strategies for gastric infections. However, their functional features of in vivo gastric mucosal barrier and host-microbe interactions are limited due to the lack of physiological stimuli. Herein, a human stomach micro-physiological system (hsMPS) with physiologically relevant gastric mucosal defense system is described based on the combination of organoid and MPS technology. A fluid flow enhanced epithelial-mesenchymal interaction in the hsMPS enables functional maturation of gastric epithelial cells, which allows for the recreation of mesh-like mucus layer containing high level of mucus protective peptides and well-developed epithelial junctional complexes. Furthermore, gastroprotection mechanisms against Helicobacter pylori (H. pylori) are successfully demonstrated in this system. Therefore, hsMPS represents a new in vitro tool for research where gastric mucosal defense mechanism is pivotal for developing therapeutic strategies.

CagA-specific Gastric CD8+ Tissue-Resident T Cells Control Helicobacter pylori During the Early Infection Phase.

BACKGROUND & AIMS: Infection with Helicobacter pylori strongly affects global health by causing chronic gastritis, ulcer disease, and gastric cancer. Although extensive research into the strong immune response against this persistently colonizing bacterium exists, the specific role of CD8+ T cells remains elusive. METHODS: We comprehensively characterize gastric H pylori-specific CD8+ T-cell responses in mice and humans by flow cytometry, RNA-sequencing, immunohistochemistry, and ChipCytometry, applying functional analyses including T-cell depletion, H pylori eradication, and ex vivo restimulation. RESULTS: We define CD8+ T-cell populations bearing a tissue-resident memory (TRM) phenotype, which infiltrate the gastric mucosa shortly after infection and mediate pathogen control by executing antigen-specific effector properties. These induced CD8+ tissue-resident memory T cells (TRM cells) show a skewed T-cell receptor beta chain usage and are mostly specific for cytotoxin-associated gene A, the distinctive oncoprotein injected by H pylori into host cells. As the infection progresses, we observe a loss of the TRM phenotype and replacement of CD8+ by CD4+ T cells, indicating a shift in the immune response during the chronic infection phase. CONCLUSIONS: Our results point toward a hitherto unknown role of CD8+ T-cell response in this bacterial infection, which may have important clinical implications for treatment and vaccination strategies against H pylori.

Gastric Mucosal Protective Effects of Cinnamomum cassia in a Rat Model of Ethanol-Induced Gastric Injury.

Cinnamomum cassia (cassia) is a tropical aromatic evergreen tree of the Lauraceae family well known for its fragrance and spicy flavor and widely used in Asian traditional medicine. It has recently garnered attention for its diverse potential health benefits, including anti-inflammatory, anti-cancer, and anti-diabetic properties. However, the gastroprotective effect of C. cassia, particularly against ethanol-induced gastric damage, remains unclear. We investigated the potential gastroprotective property of C. cassia and the underlying mechanisms of action in a rat model of ethanol-induced gastric injury. To assess its effectiveness, rats were fed C. cassia for a 14-day period prior to inducing gastric damage by oral administration of ethanol. Our results indicated that pre-treatment with C. cassia mitigated ethanol-induced gastric mucosal lesions and bleeding. Reduced gastric acid secretion and expression of acid secretion-linked receptors were also observed. Additionally, pretreatment with C. cassia led to decreased levels of inflammatory factors, including TNF-α, p-p65, and IκBα. Notably, C. cassia upregulated the expressions of HO1 and HSP90, with particular emphasis on the enhanced expression of PAS and MUC, the crucial gastric mucosa defense molecules. These findings suggest that C. cassia has protective effects on the gastric mucosa and can effectively reduce oxidative stress and inflammation.

Type I interferon remodels lysosome function and modifies intestinal epithelial defense.

Organelle remodeling is critical for cellular homeostasis, but host factors that control organelle function during microbial infection remain largely uncharacterized. Here, a genome-scale CRISPR/Cas9 screen in intestinal epithelial cells with the prototypical intracellular bacterial pathogen Salmonella led us to discover that type I IFN (IFN-I) remodels lysosomes. Even in the absence of infection, IFN-I signaling modified the localization, acidification, protease activity, and proteomic profile of lysosomes. Proteomic and genetic analyses revealed that multiple IFN-I-stimulated genes including IFITM3, SLC15A3, and CNP contribute to lysosome acidification. IFN-I-dependent lysosome acidification was associated with elevated intracellular Salmonella virulence gene expression, rupture of the Salmonella-containing vacuole, and host cell death. Moreover, IFN-I signaling promoted in vivo Salmonella pathogenesis in the intestinal epithelium where Salmonella initiates infection, indicating that IFN-I signaling can modify innate defense in the epithelial compartment. We propose that IFN-I control of lysosome function broadly impacts host defense against diverse viral and microbial pathogens.

Mucins in Intestinal Mucosal Defense and Inflammation: Learning From Clinical and Experimental Studies.

Throughout the gastrointestinal (GI) tract, a distinct mucus layer composed of highly glycosylated proteins called mucins plays an essential role in providing lubrication for the passage of food, participating in cell signaling pathways and protecting the host epithelium from commensal microorganisms and invading pathogens, as well as toxins and other environmental irritants. These mucins can be broadly classified into either secreted gel-forming mucins, those that provide the structural backbone for the mucus barrier, or transmembrane mucins, those that form the glycocalyx layer covering the underlying epithelial cells. Goblet cells dispersed among the intestinal epithelial cells are chiefly responsible for the synthesis and secretion of mucins within the gut and are heavily influenced by interactions with the immune system. Evidence from both clinical and animal studies have indicated that several GI conditions, including inflammatory bowel disease (IBD), colorectal cancer, and numerous enteric infections are accompanied by considerable changes in mucin quality and quantity. These changes include, but are not limited to, impaired goblet cell function, synthesis dysregulation, and altered post-translational modifications. The current review aims to highlight the structural and functional features as well as the production and immunological regulation of mucins and the impact these key elements have within the context of barrier function and host defense in intestinal inflammation.

Interaction between HuR and circPABPN1 Modulates Autophagy in the Intestinal Epithelium by Altering ATG16L1 Translation.

Intestinal epithelial autophagy is crucial for host defense against invasive pathogens, and defects in this process occur frequently in patients with inflammatory bowel disease (IBD) and other mucosal disorders, but the exact mechanism that activates autophagy is poorly defined. Here, we investigated the role of RNA-binding protein HuR (human antigen R) in the posttranscriptional control of autophagy-related genes (ATGs) in the intestinal epithelium. We found that targeted deletion of HuR in intestinal epithelial cells (IECs) specifically decreased the levels of ATG16L1 in the intestinal mucosa. Intestinal mucosa from patients with IBD exhibited reduced levels of both HuR and ATG16L1. HuR directly interacted with Atg16l1 mRNA via its 3' untranslated region and enhanced ATG16L1 translation, without affecting Atg16l1 mRNA stability. Circular RNA circPABPN1 blocked HuR binding to Atg16l1 mRNA and lowered ATG16L1 production. HuR silencing in cultured IECs also prevented rapamycin-induced autophagy, which was abolished by overexpressing ATG16L1. These findings indicate that HuR regulates autophagy by modulating ATG16L1 translation via interaction with circPABPN1 in the intestinal epithelium.

RNA-Binding Protein HuR Regulates Paneth Cell Function by Altering Membrane Localization of TLR2 via Post-transcriptional Control of CNPY3.

BACKGROUND & AIMS: Paneth cells secrete antimicrobial proteins including lysozyme via secretory autophagy as part of the mucosal protective response. The ELAV like RNA-binding protein 1 (ELAVL1, also called HuR) regulates stability and translation of messenger RNAs (mRNAs) and many aspects of mucosal physiology. We studied the posttranscriptional mechanisms by which HuR regulates Paneth cell function. METHODS: Intestinal mucosal tissues were collected from mice with intestinal epithelium (IE)-specific disruption of HuR (IE-HuR-/-), HuRfl/fl-Cre- mice (controls), and patients with inflammatory bowel diseases and analyzed by histology and immunohistochemistry. Paneth cell functions were determined by lysozyme-immunostaining assays. We isolated primary enterocytes from IE-HuR-/- and control mice and derived intestinal organoids. HuR and the chaperone CNPY3 were overexpressed from transgenes in intestinal epithelial cells (IECs) or knocked down with small interfering RNAs. We performed RNA pulldown assays to investigate interactions between HuR and its target mRNAs. RESULTS: Intestinal tissues from IE-HuR-/- mice had reduced numbers of Paneth cells, and Paneth cells had fewer lysozyme granules per cell, compared with tissues from control mice, but there were no effects on Goblet cells or enterocytes. Intestinal mucosa from patients with inflammatory bowel diseases had reduced levels of HuR and fewer Paneth cells. IE-HuR-/- mice did not have the apical distribution of TLR2 in the intestinal mucosa as observed in control mice. IECs from IE-HuR-/- mice expressed lower levels of CNPY3. Intestinal organoids from IE-HuR-/- mice were smaller and contained fewer buds compared with those generated from controls, and had fewer lysozyme-positive cells. In IECs, knockdown of HuR decreased levels of the autophagy proteins LC3-I and LC3-II, compared with control cells, and prevented rapamycin-induced autophagy. We found HuR to interact directly with the Cnpy3 mRNA coding region and increase levels of CNPY3 by increasing the stability and translation of Cnpy3 mRNA. CNPY3 bound TLR2, and cells with knockdown of CNPY3 or HuR lost membrane localization of TLR2, but increased cytoplasmic levels of TLR2. CONCLUSIONS: In studies of mice, IECs, and human tissues, we found HuR to increase expression of CNPY3 at the posttranscriptional level. CNPY3 is required for membrane localization of TLR2 and Paneth cell function.

Expression of α-Defensins, CD20+ B-lymphocytes, and Intraepithelial CD3+ T-lymphocytes in the Intestinal Mucosa of Patients with Liver Cirrhosis: Emerging Mediators of Intestinal Barrier Function.

AIM: The present study investigates the role of innate and adaptive immune system of intestinal mucosal barrier function in cirrhosis. METHODS: Forty patients with decompensated (n = 40, group A), 27 with compensated cirrhosis (n = 27, group B), and 27 controls (n = 27, group C) were subjected to duodenal biopsy. Expression of α-defensins 5 and 6 at the intestinal crypts was evaluated by immunohistochemistry and immunofluorescence. Serum endotoxin, intestinal T-intraepithelial, and lamina propria B-lymphocytes were quantified. RESULTS: Cirrhotic patients presented higher endotoxin concentrations (p < 0.0001) and diminished HD5 and HD6 expression compared to healthy controls (p = 0.000287, p = 0.000314, respectively). The diminished HD5 and HD6 expressions were also apparent among the decompensated patients compared to compensated group (p = 0.025, p = 0.041, respectively). HD5 and HD6 expressions were correlated with endotoxin levels (r = -0.790, p < 0.0001, r = - 0.777, p < 0.0001, respectively). Although intraepithelial T-lymphocytes were decreased in group A compared to group C (p = 0.002), no notable alterations between groups B and C were observed. The B-lymphocytic infiltrate did not differ among the investigated groups. CONCLUSIONS: These data demonstrate that decreased expression of antimicrobial peptides may be considered as a potential pathophysiological mechanism of intestinal barrier dysfunction in liver cirrhosis, while remodeling of gut-associated lymphoid tissue as an acquired immune response to bio-pathogens remains an open field to illuminate.

Linking Gut Microbiota to Colorectal Cancer.

Pre-clinical and clinical data produce mounting evidence that the microbiota is strongly associated with colorectal carcinogenesis. Dysbiosis may change the course of carcinogenesis as microbial actions seem to impact genetic and epigenetic alterations leading to dysplasia, clonal expansion and malignant transformation. Initiation and promotion of colorectal cancer may result from direct bacterial actions, bacterial metabolites and inflammatory pathways. Newer aspects of microbiota and colorectal cancer include quorum sensing, biofilm formation, sidedness and effects/countereffects of microbiota and probiotics on chemotherapy. In the future, targeting the microbiota will probably be a powerful weapon in the battle against CRC as gut microbiology, genomics and metabolomics promise to uncover important linkages between microbiota and intestinal health.

Trefoil Factor Peptides and Gastrointestinal Function.

Trefoil factor (TFF) peptides, with a 40-amino acid motif and including six conserved cysteine residues that form intramolecular disulfide bonds, are a family of mucin-associated secretory molecules mediating many physiological roles that maintain and restore gastrointestinal (GI) mucosal homeostasis. TFF peptides play important roles in response to GI mucosal injury and inflammation. In response to acute GI mucosal injury, TFF peptides accelerate cell migration to seal the damaged area from luminal contents, whereas chronic inflammation leads to increased TFF expression to prevent further progression of disease. Although much evidence supports the physiological significance of TFF peptides in mucosal defenses, the molecular and cellular mechanisms of TFF peptides in the GI epithelium remain largely unknown. In this review, we summarize the functional roles of TFF1, 2, and 3 and illustrate their action mechanisms, focusing on defense mechanisms in the GI tract.

Hydrogen sulfide-based therapeutics and gastrointestinal diseases: translating physiology to treatments.

Hydrogen sulfide (H2S) is a gaseous meditator that has various physiological and pathophysiological roles in the body. It has been shown to be an important mediator of gastrointestinal (GI) mucosal defense and contributes significantly to repair of damage and resolution of inflammation. Synthesis of H2S increases markedly after mucosal injury, and inhibition of H2S in such circumstances leads to delayed healing and exacerbated inflammation. The beneficial effects of H2S may be attributable to its ability to elevate mucosal blood flow, prevent leukocyte-endothelial adhesion, reduce oxidative stress, and stimulate angiogenesis. The use of H2S-donating agents and inhibitors of the key enzymes contributing to H2S synthesis have provided strong evidence for the importance of H2S in enhancing mucosal resistance to damage, as well as modulating inflammation and repair. In recent years, significant evidence has been generated to support the notion that these positive aspects of H2S can be exploited in drug design, particularly for arthritis, inflammatory bowel disease, and colon cancer chemoprevention. Thus novel H2S-based therapies have been shown to be effective anti-inflammatories that can promote the resolution of inflammation and accelerate the healing of GI ulcers. Encouraging results have already been seen experimentally with a mesalamine derivative and with H2S-releasing derivatives of nonsteroidal anti-inflammatory drugs.