Identification of Differentiated Intestinal Epithelial Cells Using Immunostaining and Fluorescence Microscopy.

Immunofluorescence imaging enables visualization of a wide range of molecules in diverse cells and tissues. Determining the localization and endogenous protein levels in cells using immunostaining can be highly informative for researchers studying cell structure and function. The small intestinal epithelium is composed of numerous cell types including absorptive enterocytes, mucus-producing goblet cells, lysozyme positive Paneth cells, proliferative stem cells, chemosensing tuft cells, and hormone-producing enteroendocrine cells. Each cell type in the small intestine has unique functions and structures that are critical for maintaining intestinal homeostasis and identifiable by immunofluorescence labeling. In this chapter we provide a detailed protocol and representative images of immunostaining of paraffin-embedded mouse small intestinal tissue. The method highlights antibodies and micrographs that identify differentiated cell types. These details are important because quality immunofluorescence imaging can provide novel insights and a greater understanding of healthy and disease states.

Bioinformatics reveal elevated levels of Myosin Vb in uterine corpus endometrial carcinoma patients which correlates to increased cell metabolism and poor prognosis.

Carcinoma of the endometrium of the uterus is the most common female pelvic malignancy. Although uterine corpus endometrial cancer (UCEC) has a favorable prognosis if removed early, patients with advanced tumor stages have a low survival rate. These facts highlight the importance of understanding UCEC biology. Computational analysis of RNA-sequencing data from UCEC patients revealed that the molecular motor Myosin Vb (MYO5B) was elevated in the beginning stages of UCEC and occurred in all patients regardless of tumor stage, tumor type, age, menopause status or ethnicity. Although several mutations were identified in the MYO5B gene in UCEC patients, these mutations did not correlate with mRNA expression. Examination of MYO5B methylation revealed that UCEC patients had undermethylated MYO5B and undermethylation was positively correlated with increased mRNA and protein levels. Immunostaining confirmed elevated levels of apical MYO5B in UCEC patients compared to adjacent tissue. UCEC patients with high expressing MYO5B tumors had far worse prognosis than UCEC patients with low expressing MYO5B tumors, as reflected by survival curves. Metabolic pathway analysis revealed significant alterations in metabolism pathways in UCE patients and key metabolism genes were positively correlated with MYO5B mRNA. These data provide the first evidence that MYO5B may participate in UCEC tumor development.

Serum 3-phenyllactic acid level is reduced in benign multiple sclerosis and is associated with effector B cell ratios.

BACKGROUND: 3-phenyllactic acid (PLA) is produced by both intestinal bacteria and the human host. PLA exists in its D- and L- chiral forms. It modulates human immune functions, thereby acting as a mediator of bacterial-host interactions. We aim to determine the amount and potential influence of PLA on clinical and immunological features of MS. METHODS: We measured D- and L-PLA levels in bacterial supernatants and in sera of 60 MS patients and 25 healthy controls. We investigated potential associations between PLA levels, clinical features of MS, serum cytokine levels and ratios of peripheral blood lymphocyte subsets. RESULTS: Multiple gut commensal bacteria possessed the capacity to generate D- and L-PLA. MS patients with benign phenotype showed markedly lower PLA levels than healthy controls or other MS patients. Fingolimod resistant patients had higher PLA levels at baseline. Furthermore, MS patients with higher PLA levels tended to display increased memory B and plasma cell ratios, elevated IL-4 levels and increased ratios of IL-4 and IL-10 producing T cell subsets. CONCLUSION: Collectively, our work indicates that reduced serum levels of PLA could be associated with a favorable clinical course in MS and possibly be used as a biomarker.

Myosins and membrane trafficking in intestinal brush border assembly.

Myosins are a class of motors that participate in a wide variety of cellular functions including organelle transport, cell adhesion, endocytosis and exocytosis, movement of RNA, and cell motility. Among the emerging roles for myosins is regulation of the assembly, morphology, and function of actin protrusions such as microvilli. The intestine harbors an elaborate apical membrane composed of highly organized microvilli. Microvilli assembly and function are intricately tied to several myosins including Myosin 1a, non-muscle Myosin 2c, Myosin 5b, Myosin 6, and Myosin 7b. Here, we review the research progress made in our understanding of myosin mediated apical assembly.

Loss of H2R Signaling Disrupts Neutrophil Homeostasis and Promotes Inflammation-Associated Colonic Tumorigenesis in Mice.

BACKGROUND & AIMS: We previously showed that histamine suppressed inflammation-associated colonic tumorigenesis through histamine type 2 receptor (H2R) signaling in mice. This study aimed to precisely elucidate the downstream effects of H2R activation in innate immune cells. METHODS: Analyses using online databases of single-cell RNA sequencing of intestinal epithelial cells in mice and RNA sequencing of mouse immune cells were performed to determine the relative abundances of 4 histamine receptors among different cell types. Mouse neutrophils, which expressed greater amounts of H2R, were collected from the peritoneum of wild-type and H2R-deficient mice, of which low-density and high-density neutrophils were extracted by centrifugation and were subjected to RNA sequencing. The effects of H2R activation on neutrophil differentiation and its functions in colitis and inflammation-associated colon tumors were investigated in a mouse model of dextran sulfate sodium-induced colitis. RESULTS: Data analysis of RNA sequencing and quantitative reverse-transcription polymerase chain reaction showed that Hrh2 is highly expressed in neutrophils, but barely detectable in intestinal epithelial cells. In mice, the absence of H2R activation promoted infiltration of neutrophils into both sites of inflammation and colonic tumors. H2R-deficient high-density neutrophils yielded proinflammatory features via nuclear factor-κB and mitogen-activated protein kinase signaling pathways, and suppressed T-cell proliferation. On the other hand, low-density neutrophils, which totally lack H2R activation, showed an immature phenotype compared with wild-type low-density neutrophils, with enhanced MYC pathway signaling and reduced expression of the maturation marker Toll-like receptor 4. CONCLUSIONS: Blocking H2R signaling enhanced proinflammatory responses of mature neutrophils and suppressed neutrophil maturation, leading to accelerated progression of inflammation-associated colonic tumorigenesis.

Fusobacterium nucleatum Secretes Outer Membrane Vesicles and Promotes Intestinal Inflammation.

Multiple studies have implicated microbes in the development of inflammation, but the mechanisms remain unknown. Bacteria in the genus Fusobacterium have been identified in the intestinal mucosa of patients with digestive diseases; thus, we hypothesized that Fusobacterium nucleatum promotes intestinal inflammation. The addition of >50 kDa F. nucleatum conditioned media, which contain outer membrane vesicles (OMVs), to colonic epithelial cells stimulated secretion of the proinflammatory cytokines interleukin-8 (IL-8) and tumor necrosis factor (TNF). In addition, purified F. nucleatum OMVs, but not compounds <50 kDa, stimulated IL-8 and TNF production; which was decreased by pharmacological inhibition of Toll-like receptor 4 (TLR4). These effects were linked to downstream effectors p-ERK, p-CREB, and NF-κB. F. nucleatum >50-kDa compounds also stimulated TNF secretion, p-ERK, p-CREB, and NF-κB activation in human colonoid monolayers. In mice harboring a human microbiota, pretreatment with antibiotics and a single oral gavage of F. nucleatum resulted in inflammation. Compared to mice receiving vehicle control, mice treated with F. nucleatum showed disruption of the colonic architecture, with increased immune cell infiltration and depleted mucus layers. Analysis of mucosal gene expression revealed increased levels of proinflammatory cytokines (KC, TNF, IL-6, IFN-γ, and MCP-1) at day 3 and day 5 in F. nucleatum-treated mice compared to controls. These proinflammatory effects were absent in mice who received F. nucleatum without pretreatment with antibiotics, suggesting that an intact microbiome is protective against F. nucleatum-mediated immune responses. These data provide evidence that F. nucleatum promotes proinflammatory signaling cascades in the context of a depleted intestinal microbiome.IMPORTANCE Several studies have identified an increased abundance of Fusobacterium in the intestinal tracts of patients with colon cancer, liver cirrhosis, primary sclerosing cholangitis, gastroesophageal reflux disease, HIV infection, and alcoholism. However, the direct mechanism(s) of action of Fusobacterium on pathophysiological within the gastrointestinal tract is unclear. These studies have identified that F. nucleatum subsp. polymorphum releases outer membrane vesicles which activate TLR4 and NF-κB to stimulate proinflammatory signals in vitro Using mice harboring a human microbiome, we demonstrate that F. nucleatum can promote inflammation, an effect which required antibiotic-mediated alterations in the gut microbiome. Collectively, these results suggest a mechanism by which F. nucleatum may contribute to intestinal inflammation.

Exploring the impact of intestinal ion transport on the gut microbiota.

The gut microbiota and the host are intimately connected. The host physiology dictates the intestinal environment through regulation of pH, ion concentration, mucus production, etc., all of which exerts a selective pressure on the gut microbiota. Since different regions of the gastrointestinal tract are characterized by their own physicochemical conditions, distinct microbial communities are present in these locations. While it is widely accepted that the intestinal microbiome influences the host (tight junctions, cytokine/immune responses, diarrhea, etc.), the reciprocal interaction of the host on the microbiome is under-explored. This review aims to address these gaps in knowledge by focusing on how the host intestinal ion transport influences the luminal environment and thereby modulates the gut microbiota composition.

Therapeutic Opportunities for Intestinal Angioectasia- Targeting PPARγ and Oxidative Stress.

Recurrent and acute bleeding from intestinal tract angioectasia (AEC) presents a major challenge for clinical intervention. Current treatments are empiric, with frequent poor clinical outcomes. Improvements in understanding the pathophysiology of these lesions will help guide treatment. Using data from the US Food and Drug Administration (FDA)'s Adverse Event Reporting System (FAERS), we analyzed 12 million patient reports to identify drugs inversely correlated with gastrointestinal bleeding and potentially limiting AEC severity. FAERS analysis revealed that drugs used in patients with diabetes and those targeting PPARγ-related mechanisms were associated with decreased AEC phenotypes (P < 0.0001). Electronic health records (EHRs) at University of Cincinnati Hospital were analyzed to validate FAERS analysis. EHR data showed a 5.6% decrease in risk of AEC and associated phenotypes in patients on PPARγ agonists. Murine knockout models of AEC phenotypes were used to construct a gene-regulatory network of candidate drug targets and pathways, which revealed that wound healing, vasculature development and regulation of oxidative stress were impacted in AEC pathophysiology. Human colonic tissue was examined for expression differences across key pathway proteins, PPARγ, HIF1α, VEGF, and TGFß1. In vitro analysis of human AEC tissues showed lower expression of PPARγ and TGFß1 compared with controls (0.55 ± 0.07 and 0.49 ± 0.05). National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) RNA-Seq data was analyzed to substantiate human tissue findings. This integrative discovery approach showing altered expression of key genes involved in oxidative stress and injury repair mechanisms presents novel insight into AEC etiology, which will improve targeted mechanistic studies and more optimal medical therapy for AEC.

Rotavirus induces intercellular calcium waves through ADP signaling.

Rotavirus causes severe diarrheal disease in children by broadly dysregulating intestinal homeostasis. However, the underlying mechanism(s) of rotavirus-induced dysregulation remains unclear. We found that rotavirus-infected cells produce paracrine signals that manifested as intercellular calcium waves (ICWs), observed in cell lines and human intestinal enteroids. Rotavirus ICWs were caused by the release of extracellular adenosine 5'-diphosphate (ADP) that activated P2Y1 purinergic receptors on neighboring cells. ICWs were blocked by P2Y1 antagonists or CRISPR-Cas9 knockout of the P2Y1 receptor. Blocking the ADP signal reduced rotavirus replication, inhibited rotavirus-induced serotonin release and fluid secretion, and reduced diarrhea severity in neonatal mice. Thus, rotavirus exploited paracrine purinergic signaling to generate ICWs that amplified the dysregulation of host cells and altered gastrointestinal physiology to cause diarrhea.

Reuterin disrupts Clostridioides difficile metabolism and pathogenicity through reactive oxygen species generation.

Antibiotic resistance is one of the world's greatest public health challenges and adjunct probiotic therapies are strategies that could lessen this burden. Clostridioides difficile infection (CDI) is a prime example where adjunct probiotic therapies could decrease disease incidence through prevention. Human-derived Lactobacillus reuteri is a probiotic that produces the antimicrobial compound reuterin known to prevent C. difficile colonization of antibiotic-treated fecal microbial communities. However, the mechanism of inhibition is unclear. We show that reuterin inhibits C. difficile outgrowth from spores and vegetative cell growth, however, no effect on C. difficile germination or sporulation was observed. Consistent with published studies, we found that exposure to reuterin stimulated reactive oxygen species (ROS) in C. difficile, resulting in a concentration-dependent reduction in cell viability that was rescued by the antioxidant glutathione. Sublethal concentrations of reuterin enhanced the susceptibility of vegetative C. difficile to vancomycin and metronidazole treatment and reduced toxin synthesis by C. difficile. We also demonstrate that reuterin is protective against C. difficile toxin-mediated cellular damage in the human intestinal enteroid model. Overall, our results indicate that ROS are essential mediators of reuterin activity and show that reuterin production by L. reuteri is compatible as a therapeutic in a clinically relevant model.

Enhancing responsiveness of human jejunal enteroids to host and microbial stimuli.

KEY POINTS: Enteroids are a physiologically relevant model to examine the human intestine and its functions. Previously, the measurable cytokine response of human intestinal enteroids has been limited following exposure to host or microbial pro-inflammatory stimuli. Modifications to enteroid culture conditions facilitated robust human cytokine responses to pro-inflammatory stimuli. This new human enteroid culture methodology refines the ability to study microbiome:human intestinal epithelium interactions in the laboratory. ABSTRACT: The intestinal epithelium is the primary interface between the host, the gut microbiome and its external environment. Since the intestinal epithelium contributes to innate immunity as a first line of defence, understanding how the epithelium responds to microbial and host stimuli is an important consideration in promoting homeostasis. Human intestinal enteroids (HIEs) are primary epithelial cell cultures that can provide insights into the biology of the intestinal epithelium and innate immune responses. One potential limitation of using HIEs for innate immune studies is the relative lack of responsiveness to factors that stimulate epithelial cytokine production. We report technical refinements, including removal of extracellular antioxidants, to facilitate enhanced cytokine responses in HIEs. Using this new method, we demonstrate that HIEs have distinct cytokine profiles in response to pro-inflammatory stimuli derived from host and microbial sources. Overall, we found that host-derived cytokines tumour necrosis factor and interleukin-1α stimulated reactive oxygen species and a large repertoire of cytokines. In contrast, microbial lipopolysaccharide, lipoteichoic acid and flagellin stimulated a limited number of cytokines and histamine did not stimulate the release of any cytokines. Importantly, HIE-secreted cytokines were functionally active, as denoted by the ability of human blood-derived neutrophil to migrate towards HIE supernatant containing interleukin-8. These findings establish that the immune responsiveness of HIEs depends on medium composition and stimuli. By refining the experimental culture medium and creating an environment conducive to epithelial cytokine responses by human enteroids, HIEs can facilitate exploration of many experimental questions pertaining to the role of the intestinal epithelium in innate immunity.

Human intestinal enteroids as a model of Clostridioides difficile-induced enteritis.

Clostridioides difficile is an important nosocomial pathogen that produces toxins to cause life-threatening diarrhea and colitis. Toxins bind to epithelial receptors and promote the collapse of the actin cytoskeleton. C. difficile toxin activity is commonly studied in cancer-derived and immortalized cell lines. However, the biological relevance of these models is limited. Moreover, no model is available for examining C. difficile-induced enteritis, an understudied health problem. We hypothesized that human intestinal enteroids (HIEs) express toxin receptors and provide a new model to dissect C. difficile cytotoxicity in the small intestine. We generated biopsy-derived jejunal HIE and Vero cells, which stably express LifeAct-Ruby, a fluorescent label of F-actin, to monitor actin cytoskeleton rearrangement by live-cell microscopy. Imaging analysis revealed that toxins from pathogenic C. difficile strains elicited cell rounding in a strain-dependent manner, and HIEs were tenfold more sensitive to toxin A (TcdA) than toxin B (TcdB). By quantitative PCR, we paradoxically found that HIEs expressed greater quantities of toxin receptor mRNA and yet exhibited decreased sensitivity to toxins when compared with traditionally used cell lines. We reasoned that these differences may be explained by components, such as mucins, that are present in HIEs cultures, that are absent in immortalized cell lines. Addition of human-derived mucin 2 (MUC2) to Vero cells delayed cell rounding, indicating that mucus serves as a barrier to toxin-receptor binding. This work highlights that investigation of C. difficile infection in that HIEs can provide important insights into the intricate interactions between toxins and the human intestinal epithelium.NEW & NOTEWORTHY In this article, we developed a novel model of Clostridioides difficile-induced enteritis using jejunal-derived human intestinal enteroids (HIEs) transduced with fluorescently tagged F-actin. Using live-imaging, we identified that jejunal HIEs express high levels of TcdA and CDT receptors, are more sensitive to TcdA than TcdB, and secrete mucus, which delays toxin-epithelial interactions. This work also optimizes optically clear C. difficile-conditioned media suitable for live-cell imaging.

Editing Myosin VB Gene to Create Porcine Model of Microvillus Inclusion Disease, With Microvillus-Lined Inclusions and Alterations in Sodium Transporters.

BACKGROUND & AIMS: Microvillus inclusion disease (MVID) is caused by inactivating mutations in the myosin VB gene (MYO5B). MVID is a complex disorder characterized by chronic, watery, life-threatening diarrhea that usually begins in the first hours to days of life. We developed a large animal model of MVID to better understand its pathophysiology. METHODS: Pigs were cloned by transfer of chromatin from swine primary fetal fibroblasts, which were edited with TALENs and single-strand oligonucleotide to introduce a P663-L663 substitution in the endogenous swine MYO5B (corresponding to the P660L mutation in human MYO5B, associated with MVID) to fertilized oocytes. We analyzed duodenal tissues from patients with MVID (with the MYO5B P660L mutation) and without (controls), and from pigs using immunohistochemistry. Enteroids were generated from pigs with MYO5B(P663L) and without the substitution (control pigs). RESULTS: Duodenal tissues from patients with MVID lacked MYO5B at the base of the apical membrane of intestinal cells; instead MYO5B was intracellular. Intestinal tissues and derived enteroids from MYO5B(P663L) piglets had reduced apical levels and diffuse subapical levels of sodium hydrogen exchanger 3 and SGLT1, which regulate transport of sodium, glucose, and water, compared with tissues from control piglets. However, intestinal tissues and derived enteroids from MYO5B(P663L) piglets maintained CFTR on apical membranes, like tissues from control pigs. Liver tissues from MYO5B(P663L) piglets had alterations in bile salt export pump, a transporter that facilitates bile flow, which is normally expressed in the bile canaliculi in the liver. CONCLUSIONS: We developed a large animal model of MVID that has many features of the human disease. Studies of this model could provide information about the functions of MYO5B and MVID pathogenesis, and might lead to new treatments.

Healthy Human Gastrointestinal Microbiome: Composition and Function After a Decade of Exploration.

The human gastrointestinal (GI) tract contains communities of microbes (bacteria, fungi, viruses) that vary by anatomic location and impact human health. Microbial communities differ in composition based on age, diet, and location in the gastrointestinal tract. Differences in microbial composition have been associated with chronic disease states. In terms of function, microbial metabolites provide key signals that help maintain healthy human physiology. Alterations of the healthy gastrointestinal microbiome have been linked to the development of various disease states including inflammatory bowel disease, diabetes, and colorectal cancer. While the definition of a healthy GI microbiome cannot be precisely identified, features of a healthy gut microbiome include relatively greater biodiversity and relative abundances of specific phyla and genera. Microbes with desirable functional profiles for the human host have been identified, in addition to specific metabolic features of the microbiome. This article reviews the composition and function of the healthy human GI microbiome, including the relative abundances of different bacterial taxa and the specific metabolic pathways and classes of microbial metabolites contributing to human health and disease prevention.

ClC transporter activity modulates histidine catabolism in Lactobacillus reuteri by altering intracellular pH and membrane potential.

BACKGROUND: Histamine is a key mediator of the anti-inflammatory activity conferred by the probiotic organism Lactobacillus reuteri ATCC PTA 6475 in animal models of colitis and colorectal cancer. In L. reuteri, histamine synthesis and secretion requires L-histidine decarboxylase and a L-histidine/histamine exchanger. Chloride channel (ClC)-family proton/chloride antiporters have been proposed to act as electrochemical shunts in conjunction with amino acid decarboxylase systems, correcting ion imbalances generated by decarboxylation through fixed ratio exchange of two chloride ions for one proton. This family is unique among transporters by facilitating ion flux in either direction. Here we examine the histidine decarboxylase system in relation to ClC antiporters in the probiotic organism Lactobacillus reuteri. RESULTS: In silico analyses reveal that L. reuteri possesses two ClC transporters, EriC and EriC2, as well as a complete histidine decarboxylase gene cluster (HDC) for the synthesis and export of histamine. When the transport activity of either proton/chloride antiporter is disrupted by genetic manipulation, bacterial histamine output is reduced. Using fluorescent reporter assays, we further show that ClC transporters affect histamine output by altering intracellular pH and membrane potential. ClC transport also alters the expression and activity of two key HDC genes: the histidine decarboxylase (hdcA) and the histidine/histamine exchanger (hdcP). CONCLUSIONS: Histamine production is a potentially beneficial feature for intestinal microbes by promoting long-term colonization and suppression of inflammation and host immune responses. ClC transporters may serve as tunable modulators for histamine production by L. reuteri and other gut microbes.

Phagocytosis by macrophages depends on histamine H2 receptor signaling and scavenger receptor 1.

The histamine H2 receptor (H2R) is a G protein-coupled receptor that mediates cyclic AMP production, protein kinase A activation, and MAP kinase signaling. In order to explore the multifaceted effects of histamine signaling on immune cells, phagocytosis was evaluated using primary mouse-derived macrophages. Phagocytosis is initiated by signaling via surface-bound scavenger receptors and can be regulated by autophagy. Absence of H2R signaling resulted in diminished phagocytosis of live bacteria and synthetic microspheres by primary macrophages from histamine H2 receptor gene (Hrh2)-deficient mice. Flow cytometry and immunofluorescence microscopy were used to quantify phagocytosis of phylogenetically diverse bacteria as well as microspheres of defined chemical composition. Autophagy and scavenger receptor gene expression were quantified in macrophages after exposure to Escherichia coli. Expression of the autophagy genes, Becn1 and Atg12, was increased in Hrh2-/- macrophages, indicating upregulation of autophagy pathways. Expression of the Macrophage Scavenger Receptor 1 gene (Msr1) was diminished in Hrh2-deficient macrophages, supporting the possible importance of histamine signaling in scavenger receptor abundance and macrophage function. Flow cytometry confirmed diminished MSR1 surface abundance in Hrh2-/- macrophages. These data suggest that H2R signaling is required for effective phagocytosis by regulating the process of autophagy and scavenger receptor MSR1 abundance in macrophages.

Distinct roles of histamine H1- and H2-receptor signaling pathways in inflammation-associated colonic tumorigenesis.

Inflammatory bowel disease (IBD) is a well-known risk factor for the development of colorectal cancer. Prior studies have demonstrated that microbial histamine can ameliorate intestinal inflammation in mice. We tested the hypothesis whether microbe-derived luminal histamine suppresses inflammation-associated colon cancer in Apcmin/+ mice. Mice were colonized with the human-derived Lactobacillus reuteri. Chronic inflammation was induced by repeated cycles of low-dose dextran sulfate sodium (DSS). Mice that were given histamine-producing L. reuteri via oral gavage developed fewer colonic tumors, despite the presence of a complex mouse gut microbiome. We further demonstrated that administration of a histamine H1-receptor (H1R) antagonist suppressed tumorigenesis, while administration of histamine H2-receptor (H2R) antagonist significantly increased both tumor number and size. The bimodal functions of histamine include protumorigenic effects through H1R and antitumorigenic effects via H2R, and these results were supported by gene expression profiling studies on tumor specimens of patients with colorectal cancer. Greater ratios of gene expression of H2R ( HRH2) vs. H1R ( HRH1) were correlated with improved overall survival outcomes in patients with colorectal cancer. Additionally, activation of H2R suppressed phosphorylation of mitogen-activated protein kinases (MAPKs) and inhibited chemokine gene expression induced by H1R activation in colorectal cancer cells. Moreover, the combination of a H1R antagonist and a H2R agonist yielded potent suppression of lipopolysaccharide-induced MAPK signaling in macrophages. Given the impact on intestinal epithelial and immune cells, simultaneous modulation of H1R and H2R signaling pathways may be a promising therapeutic target for the prevention and treatment of inflammation-associated colorectal cancer. NEW & NOTEWORTHY Histamine-producing Lactobacillus reuteri can suppress development of inflammation-associated colon cancer in an established mouse model. The net effects of histamine may depend on the relative activity of H1R and H2R signaling pathways in the intestinal mucosa. Our findings suggest that treatment with H1R or H2R antagonists could yield opposite effects. However, by harnessing the ability to block H1R signaling while stimulating H2R signaling, novel strategies for suppression of intestinal inflammation and colorectal neoplasia could be developed.

Loss of MYO5B Leads to Reductions in Na+ Absorption With Maintenance of CFTR-Dependent Cl- Secretion in Enterocytes.

BACKGROUND & AIMS: Inactivating mutations in MYO5B cause microvillus inclusion disease (MVID), but the physiological cause of the diarrhea associated with this disease is unclear. We investigated whether loss of MYO5B results in aberrant expression of apical enterocyte transporters. METHODS: We studied alterations in apical membrane transporters in MYO5B-knockout mice, as well as mice with tamoxifen-inducible, intestine-specific disruption of Myo5b (VilCreERT2;Myo5bflox/flox mice) or those not given tamoxifen (controls). Intestinal tissues were collected from mice and analyzed by immunostaining, immunoelectron microscopy, or cultured enteroids were derived. Functions of brush border transporters in intestinal mucosa were measured in Ussing chambers. We obtained duodenal biopsy specimens from individuals with MVID and individuals without MVID (controls) and compared transporter distribution by immunocytochemistry. RESULTS: Compared to intestinal tissues from littermate controls, intestinal tissues from MYO5B-knockout mice had decreased apical localization of SLC9A3 (also called NHE3), SLC5A1 (also called SGLT1), aquaporin (AQP) 7, and sucrase isomaltase, and subapical localization of intestinal alkaline phosphatase and CDC42. However, CFTR was present on apical membranes of enterocytes from MYO5B knockout and control mice. Intestinal biopsies from patients with MVID had subapical localization of NHE3, SGLT1, and AQP7, but maintained apical CFTR. After tamoxifen administration, VilCreERT2;Myo5bflox/flox mice lost apical NHE3, SGLT1, DRA, and AQP7, similar to germline MYO5B knockout mice. Intestinal tissues from VilCreERT2;Myo5bflox/flox mice had increased CFTR in crypts and CFTR localized to the apical membranes of enterocytes. Intestinal mucosa from VilCreERT2;Myo5bflox/flox mice given tamoxifen did not have an intestinal barrier defect, based on Ussing chamber analysis, but did have decreased SGLT1 activity and increased CFTR activity. CONCLUSIONS: Although trafficking of many apical transporters is regulated by MYO5B, trafficking of CFTR is largely independent of MYO5B. Decreased apical localization of NHE3, SGLT1, DRA, and AQP7 might be responsible for dysfunctional water absorption in enterocytes of patients with MVID. Maintenance of apical CFTR might exacerbate water loss by active secretion of chloride into the intestinal lumen.