Kristen A. Engevik

Human Clostridium difficile infection: inhibition of NHE3 and microbiota profile.

Clostridium difficile infection (CDI) is principally responsible for hospital acquired, antibiotic-induced diarrhea and colitis and represents a significant financial burden on our healthcare system. Little is known about C. difficile proliferation requirements, and a better understanding of these parameters is critical for development of new therapeutic targets. In cell lines, C. difficile toxin B has been shown to inhibit Na(+)/H(+) exchanger 3 (NHE3) and loss of NHE3 in mice results in an altered intestinal environment coupled with a transformed gut microbiota composition. However, this has yet to be established in vivo in humans. We hypothesize that C. difficile toxin inhibits NHE3, resulting in alteration of the intestinal environment and gut microbiota. Our results demonstrate that CDI patient biopsy specimens have decreased NHE3 expression and CDI stool has elevated Na(+) and is more alkaline compared with stool from healthy individuals. CDI stool microbiota have increased Bacteroidetes and Proteobacteria and decreased Firmicutes phyla compared with healthy subjects. In vitro, C. difficile grows optimally in the presence of elevated Na(+) and alkaline pH, conditions that correlate to changes observed in CDI patients. To confirm that inhibition of NHE3 was specific to C. difficile, human intestinal organoids (HIOs) were injected with C. difficile or healthy and CDI stool supernatant. Injection of C. difficile and CDI stool decreased NHE3 mRNA and protein expression compared with healthy stool and control HIOs. Together these data demonstrate that C. difficile inhibits NHE3 in vivo, which creates an altered environment favored by C. difficile.

Human Clostridium difficile infection: altered mucus production and composition.

The majority of antibiotic-induced diarrhea is caused by Clostridium difficile (C. difficile). Hospitalizations for C. difficile infection (CDI) have tripled in the last decade, emphasizing the need to better understand how the organism colonizes the intestine and maintain infection. The mucus provides an interface for bacterial-host interactions and changes in intestinal mucus have been linked host health. To assess mucus production and composition in healthy and CDI patients, the main mucins MUC1 and MUC2 and mucus oligosaccharides were examined. Compared with healthy subjects, CDI patients demonstrated decreased MUC2 with no changes in surface MUC1. Although MUC1 did not change at the level of the epithelia, MUC1 was the primary constituent of secreted mucus in CDI patients. CDI mucus also exhibited decreased N-acetylgalactosamine (GalNAc), increased N-acetylglucosamine (GlcNAc), and increased terminal galactose residues. Increased galactose in CDI specimens is of particular interest since terminal galactose sugars are known as C. difficile toxin A receptor in animals. In vitro, C. difficile is capable of metabolizing fucose, mannose, galactose, GlcNAc, and GalNAc for growth under healthy stool conditions (low Na(+) concentration, pH 6.0). Injection of C. difficile into human intestinal organoids (HIOs) demonstrated that C. difficile alone is sufficient to reduce MUC2 production but is not capable of altering host mucus oligosaccharide composition. We also demonstrate that C. difficile binds preferentially to mucus extracted from CDI patients compared with healthy subjects. Our results provide insight into a mechanism of C. difficile colonization and may provide novel target(s) for the development of alternative therapeutic agents.

The Development of Spasmolytic Polypeptide/TFF2-Expressing Metaplasia (SPEM) During Gastric Repair Is Absent in the Aged Stomach

Background & Aims During aging, physiological changes in the stomach result in more tenuous gastric tissue that is less capable of repairing injury, leading to increased susceptibility to chronic ulceration. Spasmolytic polypeptide/trefoil factor 2–expressing metaplasia (SPEM) is known to emerge after parietal cell loss and during Helicobacter pylori infection, however, its role in gastric ulcer repair is unknown. Therefore, we sought to investigate if SPEM plays a role in epithelial regeneration. Methods Acetic acid ulcers were induced in young (2–3 mo) and aged (18–24 mo) C57BL/6 mice to determine the quality of ulcer repair with advancing age. Yellow chameleon 3.0 mice were used to generate yellow fluorescent protein–expressing organoids for transplantation. Yellow fluorescent protein–positive gastric organoids were transplanted into the submucosa and lumen of the stomach immediately after ulcer induction. Gastric tissue was collected and analyzed to determine the engraftment of organoid-derived cells within the regenerating epithelium. Results Wound healing in young mice coincided with the emergence of SPEM within the ulcerated region, a response that was absent in the aged stomach. Although aged mice showed less metaplasia surrounding the ulcerated tissue, organoid-transplanted aged mice showed regenerated gastric glands containing organoid-derived cells. Organoid transplantation in the aged mice led to the emergence of SPEM and gastric regeneration. Conclusions These data show the development of SPEM during gastric repair in response to injury that is absent in the aged stomach. In addition, gastric organoids in an injury/transplantation mouse model promoted gastric regeneration.

Epithelial Regeneration After Gastric Ulceration Causes Prolonged Cell-Type Alterations

Background & Aims The peptic ulcer heals through a complex process, although the ulcer relapse often occurs several years later after healing. Our hypothesis is that even after visual evidence of healing of gastric ulceration, the regenerated epithelium is aberrant for an extended interval, increasing susceptibility of the regenerated epithelium to damage and further diseases. Methods Gastric ulcers were induced in mice by serosal topical application of acetic acid. Results Gastric ulcers induced by acetic acid visually healed within 30 days. However, regenerated epithelial architecture was poor. The gene profile of regenerated tissue was abnormal, indicating increased stem/progenitor cells, deficient differentiated gastric cell types, and deranged cell homeostasis. Despite up-regulation of PDX1 in the regenerated epithelium, no mature antral cell type was observed. Four months after healing, the regenerated epithelium lacks parietal cells, trefoil factor 2 (TFF2) and (sex-determining region Y)-box 9 (SOX9) remain up-regulated deep in the gastric gland, and the Na/H exchanger 2 (a TFF2 effector in gastric healing) remains down-regulated. Gastric ulcer healing was strongly delayed in TFF2 knockout mice, and re-epithelialization was accompanied with mucous metaplasia. After Helicobacter pylori inoculum 30 days after ulceration, we observed that the gastric ulcer selectively relapses at the same site where it originally was induced. Follow-up evaluation at 8 months showed that the relapsed ulcer was not healed in H pylori–infected tissues. Conclusions These findings show that this macroscopically regenerated epithelium has prolonged abnormal cell distribution and is differentially susceptible to subsequent damage by H pylori.

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.

Organoids as a Model to Study Infectious Disease

The advent of the gastric organoid culture system has provided a new model to emulate native epithelial tissue in vitro. Gastric organoids grow from isolated epithelial stem cells and develop into three dimensional structures that can be used to study host physiology. Here we describe current laboratory protocols for growing gastric organoids and the microinjection of pathogens such as Helicobacter pylori into the lumen of gastric organoids in order to study the cellular response following infection.

Cell injury triggers actin polymerization to initiate epithelial restitution

ABSTRACT The role of the actin cytoskeleton in the sequence of physiological epithelial repair in the intact epithelium has yet to be elucidated. Here, we explore the role of actin in gastric repair in vivo and in vitro gastric organoids (gastroids). In response to two-photon-induced cellular damage of either an in vivo gastric or in vitro gastroid epithelium, actin redistribution specifically occurred in the lateral membranes of cells neighboring the damaged cell. This was followed by their migration inward to close the gap at the basal pole of the dead cell, in parallel with exfoliation of the dead cell into the lumen. The repair and focal increase of actin was significantly blocked by treatment with EDTA or the inhibition of actin polymerization. Treatment with inhibitors of myosin light chain kinase, myosin II, trefoil factor 2 signaling or phospholipase C slowed both the initial actin redistribution and the repair. While Rac1 inhibition facilitated repair, inhibition of RhoA/Rho-associated protein kinase inhibited it. Inhibitors of focal adhesion kinase and Cdc42 had negligible effects. Hence, initial actin polymerization occurs in the lateral membrane, and is primarily important to initiate dead cell exfoliation and cell migration to close the gap. Summary: The role of actin in epithelial repair is explored in experiments using gastric organoids, demonstrating that initial actin polymerization in the lateral membrane is of primary importance for initiation of repair.