Joannie M. Allaire

Epithelial BMP signaling is required for proper specification of epithelial cell lineages and gastric endocrine cells

Bone morphogenetic protein (BMP) signaling within the gastrointestinal tract is complex. BMP ligands and their receptors are expressed in both epithelial and mesenchymal compartments, suggesting bidirectional signaling between these two entities. Despite an increasing interest in BMP signaling in gut physiology and pathologies, the distinct contribution of BMP signaling in the epithelium vs. the mesenchyme in gastrointestinal homeostasis remains to be established. We aimed to investigate the role of epithelial BMP signaling in gastric organogenesis, gland morphogenesis, and maintenance of epithelial cell functions. Using the Cre/loxP system, we generated a mouse model with an early deletion during development of BMP receptor 1A (Bmpr1a) exclusively in the foregut endoderm. Bmpr1a(ΔGEC) mice showed no severe abnormalities in gastric organogenesis, gland epithelial proliferation, or morphogenesis, suggesting only a minor role for epithelial BMP signaling in these processes. However, early loss of BMP signaling in foregut endoderm did impact on gastric patterning, leading to an anteriorization of the stomach. In addition, numbers of parietal cells were reduced in Bmpr1a(ΔGEC) mice. Epithelial BMP deletion significantly increased the numbers of chromogranin A-, ghrelin-, somatostatin-, gastrin-, and serotonin-expressing gastric endocrine cells. Cancer never developed in young adult (<100 days) Bmpr1a-inactivated mice although a marker of spasmolytic polypeptide-expressing metaplasia was upregulated. Using this model, we have uncovered that BMP signaling negatively regulates the proliferation and commitment of endocrine precursor cells. Our data also indicate that loss of BMP signaling in epithelial gastric cells alone is not sufficient to induce gastric neoplasia.

Loss of Smad5 leads to the disassembly of the apical junctional complex and increased susceptibility to experimental colitis

The regulation of intestinal epithelial cell adhesion and migratory properties is often compromised in inflammatory bowel disease (IBD). Despite an increasing interest in bone morphogenetic protein (Bmp) signaling in gut pathologies, little is known of the specific roles played by individual Smads in intestinal epithelial functions. In the present study, we generated a mouse model with deletion of Smad5 transcriptional effector of the Bmp signaling pathway exclusively in the intestinal epithelium. Proliferation, migration, and apical junctional complex (AJC) protein expression were analyzed by immunofluorescence and Western blot. Human intestinal biopsies from control and IBD patients were analyzed for SMAD5 gene transcript expression by quantitative PCR (qPCR). Smad5(ΔIEC) and control mice were subjected to dextran sulfate sodium (DSS)-induced experimental colitis, and their clinical and histological symptoms were assessed. Loss of Smad5 led to intestinal epithelial hypermigration and deregulation of the expression of claudin-1 and claudin-2. E-cadherin was found to be equally expressed but displaced from the AJC to the cytoplasm in Smad5(ΔIEC) mice. Analysis of SMAD5 gene expression in human IBD patient samples revealed a significant downregulation of the gene transcript in Crohns disease and ulcerative colitis samples. Smad5(ΔIEC) mice exposed to experimental DSS colitis were significantly more susceptible to the disease and had impaired wound healing during the recovery phase. Our results support that Smad5 is partly responsible for mediating Bmp signals in intestinal epithelial cells. In addition, deficiency in epithelial Smad5 leads to the deregulation of cell migration by disassembling the AJC with increasing susceptibility to experimental colitis and impairment in wound healing.

Milk Fat Globule Membrane Supplementation in Formula Modulates the Neonatal Gut Microbiome and Normalizes Intestinal Development

Breast milk has many beneficial properties and unusual characteristics including a unique fat component, termed milk fat globule membrane (MFGM). While breast milk yields important developmental benefits, there are situations where it is unavailable resulting in a need for formula feeding. Most formulas do not contain MFGM, but derive their lipids from vegetable sources, which differ greatly in size and composition. Here we tested the effects of MFGM supplementation on intestinal development and the microbiome as well as its potential to protect against Clostridium difficile induced colitis. The pup-in-a-cup model was used to deliver either control or MFGM supplemented formula to rats from 5 to 15 days of age; with mother’s milk (MM) reared animals used as controls. While CTL formula yielded significant deficits in intestinal development as compared to MM littermates, addition of MFGM to formula restored intestinal growth, Paneth and goblet cell numbers, and tight junction protein patterns to that of MM pups. Moreover, the gut microbiota of MFGM and MM pups displayed greater similarities than CTL, and proved protective against C. difficile toxin induced inflammation. Our study thus demonstrates that addition of MFGM to formula promotes development of the intestinal epithelium and microbiome and protects against inflammation.

Bmp signaling in colonic mesenchyme regulates stromal microenvironment and protects from polyposis initiation

In the colon, myofibroblasts are primary contributors in the establishment of the microenvironment involved in tissue homeostasis. Alterations in myofibroblast functions lead to changes resulting in a toxic microenvironment nurturing tumorigenesis. Bone morphogenetic proteins (Bmps) are morphogens known to play key roles in adult gut homeostasis. Studies in genetically‐modified mice have shown that Bmp disruption in all cell layers leads to the development of gut polyposis. In contrast, our studies showed that loss of Bmp exclusively in the gastrointestinal epithelium resulted in increased epithelial proliferation without polyposis initiation, thus suggesting a key role for mesenchymal Bmp signaling in polyposis initiation. In order to identify the role of mesenchymal Bmp signaling on the microenvironment and its impact on colonic mucosa, a mouse model was generated with suppression of Bmp signaling exclusively in myofibroblasts (Bmpr1aΔMES). Bmpr1aΔMES mice exhibited increased subepithelial proliferation with changes in cellular composition leading to the development of a primed stroma with modulation of extracellular matrix proteins, immune cells and cytokines as early as 90 days of age. This microenvironmental deregulation was associated with increased polyposis initiation at one year of age. These results are the first to demonstrate that mesenchymal Bmpr1a inactivation alone is sufficient to prompt an expansion of myofibroblasts leading to the development of a reactive mesenchyme that contributes to polyposis initiation in the colon. These findings support the novel concept that inhibition of Bmp signaling in mesenchymal cells surrounding the normal epithelium leads to important changes instructing a toxic microenvironment sufficient to induce colonic polyposis.

Loss of Sonic Hedgehog Leads to Alterations in Intestinal Secretory Cell Maturation and Autophagy

Background Intestinal epithelial cells express the Sonic and Indian hedgehog ligands. Despite the strong interest in gut hedgehog signaling in GI diseases, no studies have specifically addressed the singular role of intestinal epithelial cell Sonic hedgehog signaling. The aim of this study was to investigate the specific role of Sonic hedgehog in adult ileal epithelial homeostasis. Methodology/Principal Findings A Sonic hedgehog intestinal epithelial conditional knockout mouse model was generated. Assessment of ileal histological abnormalities, crypt epithelial cell proliferation, epithelial cell fate, junctional proteins, signaling pathways, as well as ultrastructural analysis of intracellular organelles were performed in control and mutant mice. Mice lacking intestinal epithelial Sonic Hedgehog displayed decreased ileal crypt/villus length, decreased crypt proliferation as well as a decrease in the number of ileal mucin-secreting goblet cells and antimicrobial peptide-secreting Paneth cells during adult life. These secretory cells also exhibited disruption of their secretory products in mutant mice. Ultrastructural microscopy analysis revealed a dilated ER lumen in secretory cells. This phenotype was also associated with a decrease in autophagy. Conclusions/Significance Altogether, these findings indicate that the loss of Sonic hedgehog can lead to ileal secretory cell modifications indicative of endoplasmic reticulum stress, accompanied by a significant reduction in autophagy.

Frontline defenders: goblet cell mediators dictate host-microbe interactions in the intestinal tract during health and disease

Goblet cells (GCs) are the predominant secretory epithelial cells lining the luminal surface of the mammalian gastrointestinal (GI) tract. Best known for their apical release of mucin 2 (Muc2), which is critical for the formation of the intestinal mucus barrier, GCs have often been overlooked for their active contributions to intestinal protection and host defense. In part, this oversight reflects the limited tools available to study their function but also because GCs have long been viewed as relatively passive players in promoting intestinal homeostasis and host defense. In light of recent studies, this perspective has shifted, as current evidence suggests that Muc2 as well as other GC mediators are actively released into the lumen to defend the host when the GI tract is challenged by noxious stimuli. The ability of GCs to sense and respond to danger signals, such as bacterial pathogens, has recently been linked to inflammasome signaling, potentially intrinsic to the GCs themselves. Moreover, further work suggests that GCs release Muc2, as well as other mediators, to modulate the composition of the gut microbiome, leading to both the expansion as well as the depletion of specific gut microbes. This review will focus on the mechanisms by which GCs actively defend the host from noxious stimuli, as well as describe advanced technologies and new approaches by which their responses can be addressed. Taken together, we will highlight current insights into this understudied, yet critical, aspect of intestinal mucosal protection and its role in promoting gut defense and homeostasis.

Gata4 is critical to maintain gut barrier function and mucosal integrity following epithelial injury

The intestinal epithelial barrier is critical to limit potential harmful consequences from exposure to deleterious luminal contents on the organism. Although this barrier is functionally important along the entire gut, specific regional regulatory mechanisms involved in the maintenance of this barrier are poorly defined. Herein, we identified Gata4 as a crucial regulator of barrier integrity in the mouse proximal intestinal epithelium. Conditional deletion of Gata4 in the intestine led to a drastic increase in claudin-2 expression that was associated with an important increase of gut barrier permeability without causing overt spontaneous inflammation. Administration of indomethacin, a non-steroidal anti-inflammatory drug (NSAID) that causes enteritis, led to rapid and restricted proximal small intestinal injuries in Gata4 mutant mice as opposed to control mice. Comparative analysis of gene transcript profiles from indomethacin-challenged control and Gata4 mutant mice identified defects in epithelial cell survival, inflammatory cell recruitment and tissue repair mechanisms. Altogether, these observations identify Gata4 as a novel crucial regulator of the intestinal epithelial barrier and as a critical epithelial transcription factor implicated in the maintenance of proximal intestinal mucosal integrity after injury.

Loss of mesenchymal bone morphogenetic protein signaling leads to development of reactive stroma and initiation of the gastric neoplastic cascade

Bmps are morphogens involved in various gastric cellular functions. Studies in genetically-modified mice have shown that Bmp disruption in gastric epithelial and stromal cell compartments leads to the development of tumorigenesis. Our studies have demonstrated that abrogation of gastric epithelial Bmp signaling alone was not sufficient to recapitulate the neoplastic features associated with total gastric loss of Bmp signaling. Thus, epithelial Bmp signaling does not appear to be a key player in gastric tumorigenesis initiation. These observations suggest a greater role for stromal Bmp signaling in gastric polyposis initiation. In order to identify the specific roles played by mesenchymal Bmp signaling in gastric homeostasis, we generated a mouse model with abrogation of Bmp signaling exclusively in the gastro-intestinal mesenchyme (Bmpr1aΔMES). We were able to expose an unsuspected role for Bmp loss of signaling in leading normal gastric mesenchyme to adapt into reactive mesenchyme. An increase in the population of activated-fibroblasts, suggesting mesenchymal transdifferentiation, was observed in mutant stomach. Bmpr1aΔMES stomachs exhibited spontaneous benign polyps with presence of both intestinal metaplasia and spasmolytic-polypeptide-expressing metaplasia as early as 90 days postnatal. These results support the novel concept that loss of mesenchymal Bmp signaling cascade acts as a trigger in gastric polyposis initiation.