Laminin alpha 5 influences the architecture of the mouse small intestine mucosa.
ABSTRACT: The mammalian intestine displays two distinct patterns of mucosal organization. The small intestine contains mucosal epithelial invaginations (the crypts of Lieberkühn) that are continuous with evaginations (villi) into the lumen. The colon also contains crypts of Lieberkühn, but its epithelial surface is lined by flat surface cuffs. The epithelial cells of both organs communicate with the underlying mesenchyme through a basement membrane that is composed of a variety of extracellular matrix proteins, including members of the laminin family. The basement membranes of the small intestine and colon contain distinct laminin subtypes; notably, the villus basement membrane is rich in laminin alpha 5. Here, we show that the diminution of laminin alpha 5 in a mouse model led to a compensatory deposition of colonic laminins, which resulted in a transformation from a small intestinal to a colonic mucosal architecture. The alteration in mucosal architecture was associated with reduced levels of nuclear p27Kip1 - a cell-cycle regulator - and altered intestinal epithelial cell proliferation, migration and differentiation. Our results suggest that laminin alpha 5 has a crucial role in establishing and maintaining the specific mucosal pattern of the mouse small intestine.
Project description:The colonic epithelial lining undergoes constant replacement, driven by epithelial stem cells in crypts of Lieberkühn. Stem cells lost because of damage or disease can be replaced by adjacent crypts that undergo fission. The close proximity of an extraordinary number of luminal microbes creates a challenge for this repair process; infection must be prevented while immune system activation and epithelial stem cell genetic damage must be minimized. To understand the factors that modulate crypt/stem cell replacement in the mouse colon, we developed an in vivo acute injury system analogous to punch biopsy of the skin. In contrast to epidermal stem cells, colonic epithelial progenitors did not migrate over the wound bed. Instead, their proliferative expansion was confined to crypts adjacent to wound beds and was delayed to the latter phase of healing. This increased epithelial proliferation was coincident with the infiltration of Trem2 expressing macrophages and increased expression of IL-4 and IL-13 in the wound bed. Interestingly, Trem2(-/-) mice displayed slow and incomplete wound healing of colonic mucosal injuries. We found the latter phase of healing in Trem2(-/-) mice showed a diminished burst of epithelial proliferation, increased expression of IFN-gamma and TNF-alpha, diminished expression of IL-4 and IL-13, and increased markers of classical macrophage activation. Ablation of these cytokines in injured WT and Trem2(-/-) mice demonstrated that their expression ultimately determined the rate and nature of wound healing. These studies show that Trem2 signaling is an important pathway to promote healing of wounds in the colon where stem cell replacement is necessary.
Project description:BACKGROUND: The functions of urokinase in intestinal epithelia are unknown. AIMS: To determine the relation of urokinase expressed by intestinal epithelial cells to their position in the crypt-villus/surface axis and of mucosal urokinase activity to epithelial proliferative kinetics in the distal colon. METHODS: Urokinase expression was examined immunohistochemically in human intestinal mucosa. Urokinase activity was measured colorimetrically in epithelial cells isolated sequentially from the crypt-villus axis of the rat small intestine. In separate experiments, urokinase activity and epithelial kinetics (measured stathmokinetically) were measured in homogenates of distal colonic mucosa of 14 groups of eight rats fed diets known to alter epithelial turnover. RESULTS: From the crypt base, an ascending gradient of expression and activity of urokinase was associated with the epithelial cells. Median mucosal urokinase activities in each of the dietary groups of rats correlated positively with autologous median number of metaphase arrests per crypt (r=0.68; p<0.005) and per 100 crypt cells (r=0.75; p<0.001), but not with crypt column height. CONCLUSIONS: Localisation of an enzyme capable of leading to digestion of cell substratum in the region where cells are loosely attached to their basement membrane, and the association of its activity with indexes of cell turnover, suggest a role for urokinase in facilitating epithelial cell loss in the intestine.
Project description:Inhibition of growth of the intestinal epithelium, a rapidly self-renewing tissue, is commonly found in various critical disorders. The RNA-binding protein HuR is highly expressed in the gut mucosa and modulates the stability and translation of target mRNAs, but its exact biological function in the intestinal epithelium remains unclear. Here, we investigated the role of HuR in intestinal homeostasis using a genetic model and further defined its target mRNAs. Targeted deletion of HuR in intestinal epithelial cells caused significant mucosal atrophy in the small intestine, as indicated by decreased cell proliferation within the crypts and subsequent shrinkages of crypts and villi. In addition, the HuR-deficient intestinal epithelium also displayed decreased regenerative potential of crypt progenitors after exposure to irradiation. HuR deficiency decreased expression of the Wnt coreceptor LDL receptor-related protein 6 (LRP6) in the mucosal tissues. At the molecular level, HuR was found to bind the Lrp6 mRNA via its 3'-untranslated region and enhanced LRP6 expression by stabilizing Lrp6 mRNA and stimulating its translation. These results indicate that HuR is essential for normal mucosal growth in the small intestine by altering Wnt signals through up-regulation of LRP6 expression and highlight a novel role of HuR deficiency in the pathogenesis of intestinal mucosal atrophy under pathological conditions.
Project description:Paneth cells (PCs) are terminally differentiated, highly specialized secretory cells located at the base of the crypts of Lieberkühn in the small intestine. Besides their antimicrobial function, PCs serve as a component of the intestinal stem cell niche. By secreting granules containing bactericidal proteins like defensins/cryptdins and lysozyme, PCs regulate the microbiome of the gut. Here we study the control of PC degranulation in primary epithelial organoids in culture. We show that PC degranulation does not directly occur upon stimulation with microbial antigens or bacteria. In contrast, the pro-inflammatory cytokine Interferon gamma (IFN-?) induces rapid and complete loss of granules. Using live cell imaging, we show that degranulation is coupled to luminal extrusion and death of PCs. Transfer of supernatants from in vitro stimulated iNKT cells recapitulates degranulation in an IFN-?-dependent manner. Furthermore, endogenous IFN-? secretion induced by anti-CD3 antibody injection causes Paneth loss and release of goblet cell mucus. The identification of IFN-? as a trigger for degranulation and extrusion of PCs establishes a novel effector mechanism by which immune responses may regulate epithelial status and the gut microbiome.
Project description:<h4>Background</h4>Dietary yeast inclusions in a pig diet may drive changes both in gut bacterial composition and bacterial functional profile. This study investigated the effect of Cyberlindnera jadinii as a protein to replace 40% of the conventional proteins in a diet for weanling pigs on the microbiota in the small and large intestine, colonic short-chain fatty acid concentration, and colonic histopathology parameters. Seventy-two pigs weaned at 28?days of age were randomly assigned to either a control or a C. jadinii-based diet and followed for 2?weeks.<h4>Results</h4>Compared with the controls, higher numbers of cultivable lactic acid-producing bacteria in the small and large intestine were registered in the yeast group. Alpha and beta bacterial diversity were different between the diet groups with lower alpha-diversity and distinct bacterial composition in the large intestine in the yeast group compared with those of the controls. The large intestine microbiota in the yeast group had higher numbers of Prevotella, Mitsuokella and Selenomonas compared with those of the controls. The concentrations of colonic acetate and butyrate were higher in the controls compared with that of the yeast group. The colonic crypt depth was deeper in the control group. The gut histopathology of colonic tissues revealed no differences between the diets. The colonic crypt depth tended to be deeper with higher relative abundance of an unclassified Spirochetes, higher colonic butyrate concentration, and higher bacterial richness. The concentration of colonic butyrate was positively associated with the relative abundance of the Faecalibacterium prausnitzii, Dialister, and an unclassified amplicon of the Spirochaetaceae family in the colon.<h4>Conclusions</h4>The replacement of the conventional proteins by proteins from Cyberlindnera jadinii in a weanling pig diet reshaped the large intestine microbiota structure. The novel yeast diet appeared to be selective for Lactobacillus spp., which may represent an added value resulting from using the sustainably produced yeast protein ingredient as an alternative to conventional protein ingredients in animal diets. The large intestine bacterial composition and their metabolites may be involved in an adaptive alteration of the colonic crypts without pathological consequences.
Project description:The CDC25 protein phosphatases (CDC25A, B, and C) drive cell cycle transitions by activating key components of the cell cycle engine. CDC25A and CDC25B are frequently overproduced in human cancers. Disruption of Cdc25B or Cdc25C individually or in combination has no effect on mouse viability. Here we report that CDC25A is the only family member to provide an essential function during early embryonic development, and that other family members compensate for its loss in adult mice. In contrast, conditional disruption of the entire family is lethal in adults due to a loss of small intestinal epithelial cell proliferation in crypts of Lieberkühn. Cdc25 loss induced Wnt signaling, and overall crypt structures were preserved. In the face of continuous Wnt signaling, nearly all crypt epithelial progenitors differentiated into multiple cell lineages, including crypt base columnar cells, a proposed stem cell. A small population of Musashi/Dcamkl-1/nuclear beta-catenin-positive epithelial cells was retained in these crypts. These findings have implications for the development of novel, less cytotoxic cancer chemotherapeutic drugs that specifically target the cell cycle.
Project description:Laminins are major constituents of basement membranes and are essential for tissue homeostasis. Laminin-511 is highly expressed in the intestine and its absence causes severe malformation of the intestine and embryonic lethality. To understand the mechanistic role of laminin-511 in tissue homeostasis, we used RNA profiling of embryonic intestinal tissue of lama5 knockout mice and identified a lama5 specific gene expression signature. By combining cell culture experiments with mediated knockdown approaches, we provide a mechanistic link between laminin ?5 gene deficiency and the physiological phenotype. We show that laminin ?5 plays a crucial role in both epithelial and mesenchymal cell behavior by inhibiting Wnt and activating PI3K signaling. We conclude that conflicting signals are elicited in the absence of lama5, which alter cell adhesion, migration as well as epithelial and muscle differentiation. Conversely, adhesion to laminin-511 may serve as a potent regulator of known interconnected PI3K/Akt and Wnt signaling pathways. Thus deregulated adhesion to laminin-511 may be instrumental in diseases such as human pathologies of the gut where laminin-511 is abnormally expressed as it is shown here.
Project description:Paneth cells were first described in the late 19th century by Gustav Schwalbe and Josef Paneth as columnar epithelial cells possessing prominent eosinophilic granules in their cytoplasm. Decades later there is continued interest in Paneth cells as they play an integral role in maintaining intestinal homeostasis and modulating the physiology of the small intestine and its associated microbial flora. Paneth cells are highly specialized secretory epithelial cells located in the small intestinal crypts of Lieberkühn. The dense granules produced by Paneth cells contain an abundance of antimicrobial peptides and immunomodulating proteins that function to regulate the composition of the intestinal flora. This in turn plays a significant role in secondary regulation of the host microvasculature, the normal injury and repair mechanisms of the intestinal epithelial layer, and the levels of intestinal inflammation. These critical functions may have even more importance in the immature intestine of premature infants. While Paneth cells begin to develop in the middle of human gestation, they do not become immune competent or reach their adult density until closer to term gestation. This leaves preterm infants deficient in normal Paneth cell biology during the greatest window of susceptibility to develop intestinal pathology such as necrotizing enterocolitis (NEC). As 10% of infants worldwide are currently born prematurely, there is a significant population of infants contending with an inadequate cohort of Paneth cells. Infants who have developed NEC have decreased Paneth cell numbers compared to age-matched controls, and ablation of murine Paneth cells results in a NEC-like phenotype suggesting again that Paneth cell function is critical to homeostasis to the immature intestine. This review will provide an up to date and comprehensive look at Paneth cell ontogeny, the impact Paneth cells have on the host-microbial axis in the immature intestine, and the repercussions of Paneth cell dysfunction or loss on injury and repair mechanisms in the immature gut.
Project description:Interleukin 33 (IL-33) is a cytokine preferentially elevated in acute ulcerative colitis (UC), inferring a role in its pathogenesis. The role of IL-33 in intestinal inflammation is incompletely understood, with both pro-inflammatory and regulatory properties described. There are also conflicting reports on cellular sources and subcellular location of IL-33 in the colonic mucosa, justifying a closer look at IL-33 expression in well-defined clinical stages of UC. A total of 50 study participants (29?UC patients and 21 healthy controls) were included from a prospective cohort of inflammatory bowel disease patients treated to disease remission with infliximab, a tumour necrosis factor alpha (TNF) inhibitor. To our knowledge this is the first study examining mucosal IL-33 expression before and after anti-TNF therapy. In colonic mucosal biopsies we found a 3-fold increase in IL-33 gene expression comparing acute UC to healthy controls (p?<?0.01). A significant reduction of IL33 between acute UC and disease remission was observed when TNF normalised in the mucosa (p?=?0.02). Immunostaining revealed IL-33 in the nuclei of epithelial cells of scattered colonic crypts in acute disease, while at disease remission, IL-33 was undetectable, a novel finding suggesting that enterocyte-derived IL-33 is induced and maintained by inflammatory mediators.
Project description:Commensal bacteria and their products provide beneficial effects to the mammalian gut by stimulating epithelial cell turnover and enhancing wound healing, without activating overt inflammation. We hypothesized that N-formylpeptide receptors, which bind bacterial N-formylpeptides and are expressed by intestinal epithelial cells, may contribute to these processes. Here we report that formylpeptide receptor-2 (FPR2), which we show is expressed on the apical and lateral membranes of colonic crypt epithelial cells, mediates N-formylpeptide-dependent epithelial cell proliferation and renewal. Colonic epithelial cells in FPR2-deficient mice displayed defects in commensal bacterium-dependent homeostasis as shown by the absence of responses to N-formylpeptide stimulation, shortened colonic crypts, reduced acute inflammatory responses to dextran sulfate sodium (DSS) challenge, delayed mucosal restoration after injury, and increased azoxymethane-induced tumorigenesis. These results indicate that FPR2 is critical in mediating homeostasis, inflammation, and epithelial repair processes in the colon.