scholarly journals Entamoeba histolytica-Induced Mucin Exocytosis Is Mediated by VAMP8 and Is Critical in Mucosal Innate Host Defense

mBio ◽  
2017 ◽  
Vol 8 (5) ◽  
Author(s):  
Steve Cornick ◽  
France Moreau ◽  
Herbert Y. Gaisano ◽  
Kris Chadee
Keyword(s):  
Epithelial Cells ◽  
Entamoeba Histolytica ◽  
Host Defense ◽  
Critical Role ◽  
Single Layer ◽  
Goblet Cells ◽  
Mucus Secretion ◽  
Mucosal Barrier ◽  
Proinflammatory Response

ABSTRACT Intestinal mucus secretion is critical in maintaining mucosal host defense against a myriad of pathogens by preventing direct association with the epithelium. Entamoeba histolytica specifically binds colonic MUC2 mucin and also induces potent hypersecretion from goblet cells; however, characterization of the nature of the mechanisms controlling mucus release remains elusive. In this report, we identify vesicle SNARE vesicle-associated membrane protein 8 (VAMP8) present on mucin granules as orchestrating regulated exocytosis in human goblet cells in response to the presence of E. histolytica. VAMP8 was specifically activated during E. histolytica infection, and ablation of VAMP8 led to impaired mucin secretion. As a consequence, loss of VAMP8 increased E. histolytica adherence to epithelial cells associated with enhanced cell death through apoptosis characterized by caspase 3 and 9 cleavages and DNA fragmentation. With the mucosal barrier compromised in Vamp8 −/− animals, E. histolytica induced an aggressive proinflammatory response with elevated levels of interleukin-1 alpha (IL-1α), IL-1β, and tumor necrosis factor alpha (TNF-α) secretion. This report is the first to characterize regulated mucin exocytosis in intestinal goblet cells in response to a pathogen and the downstream consequences of improper mucin secretion in mucosal barrier defense. IMPORTANCE The intestinal tract is exposed to countless substances and pathogens, and yet homeostasis is maintained, in part by the mucus layer that houses the microbiota and spatially separates potential threats from the underlying single layer of epithelium. Despite the critical role of mucus in innate host defense, characterization of the mechanisms by which mucus is secreted from specialized goblet cells in the gut remains elusive. Here, we describe the machinery that regulates mucus secretion as well as the consequence during infection with the colonic pathogen Entamoeba histolytica. Abolishment of the key machinery protein VAMP8 abrogated mucus release in cultured human colonic goblet cells and during E. histolytica infection in Vamp8 −/− mice, which showed enhanced amoeba contact and killing of epithelial cells, triggering a potent proinflammatory response. This report highlights the importance of the VAMP8 secretory machinery in facilitating mucus release from intestinal goblet cells and the dire consequences that occur during disease pathogenesis if these pathways are not functional. IMPORTANCE The intestinal tract is exposed to countless substances and pathogens, and yet homeostasis is maintained, in part by the mucus layer that houses the microbiota and spatially separates potential threats from the underlying single layer of epithelium. Despite the critical role of mucus in innate host defense, characterization of the mechanisms by which mucus is secreted from specialized goblet cells in the gut remains elusive. Here, we describe the machinery that regulates mucus secretion as well as the consequence during infection with the colonic pathogen Entamoeba histolytica. Abolishment of the key machinery protein VAMP8 abrogated mucus release in cultured human colonic goblet cells and during E. histolytica infection in Vamp8 −/− mice, which showed enhanced amoeba contact and killing of epithelial cells, triggering a potent proinflammatory response. This report highlights the importance of the VAMP8 secretory machinery in facilitating mucus release from intestinal goblet cells and the dire consequences that occur during disease pathogenesis if these pathways are not functional.

scholarly journals Epithelial GPR35 protects from Citrobacter rodentium infection by preserving goblet cells and mucosal barrier integrity

2021 ◽  
Author(s):  
Hassan Melhem ◽  
Berna Kaya ◽  
Tanay Kaymak ◽  
Philipp Wuggenig ◽  
Emilio Flint ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Goblet Cell ◽  
Cell Function ◽  
Critical Role ◽  
Goblet Cells ◽  
Mucosal Barrier ◽  
Critical Element ◽  
Citrobacter Rodentium ◽  
Invasive Bacterial Infection ◽  
Genetic Deletion

Goblet cells are essential for maintaining intestinal health and for the defense against invasive bacterial infection. However, the molecular pathways that regulate goblet cell function remain largely unknown. Although GPR35 is highly expressed in colonic epithelial cells, its importance in promoting the epithelial barrier is unclear. Here we found that epithelial Gpr35 plays a critical role in goblet cell function. Genetic deletion of Gpr35 in epithelial cells but not in from macrophages results in goblet cell depletion and dysbiosis, rendering these mice more susceptible to Citrobacter rodentium infection. Mechanistically, scRNA-seq analysis indicates that signaling of epithelial Gpr35 is essential to maintain normal pyroptosis levels in goblet cells. Our work shows how the epithelial presence of Gpr35 is a critical element for the function of goblet cell-mediated symbiosis between host and microbiota.

scholarly journals A24 FCGBP MAINTAINS MUC2 MUCUS STRUCTURAL INTEGRITY BY STABILIZING THE MUCUS LAYER IN RESPONSE TO THE COLONIC PATHOGEN, ENTAMOEBA HISTOLYTICA

2021 ◽  
Vol 4 (Supplement_1) ◽  
pp. 212-213
Author(s):  
H Gorman ◽  
F Moreau ◽  
A Kim ◽  
K Chadee
Keyword(s):  
Cell Surface ◽  
Entamoeba Histolytica ◽  
Host Defense ◽  
Inflammatory Cytokine ◽  
Structural Integrity ◽  
Cysteine Proteinase ◽  
Goblet Cells ◽  
Dependent Manner ◽  
Mucus Layer ◽  
Fluorescent Beads

Abstract Background MUC2 mucin is the major component of the colonic mucus bilayer that serves as the first line of innate host defense against pathogens while supporting a healthy microbiota and regulating epithelial barrier function. Proteomic studies of colonic mucus have identified various mucus-associated proteins. One of the most abundant is FCGBP, similar to MUC2 mucin, but its interaction with MUC2 or function is not known. Here, we elucidated FCGBP functional role in stabilizing MUC2 mucus and in innate host defence against Entamoeba histolytica (Eh). Aims Hypothesis: MUC2 mucin and FCGBP are coordinately produced and play an important role in innate host defense. The specific aims are: 1. To determine if FCGBP alters the structural integrity of the mucus layer 2. To determine the role of FCGBP in Eh infection Methods FCGBP mRNA and protein expression induced by Eh, in WT and FCGBP CRISPR/Cas9 LS174T goblet cells were analysed by RT-PCR and Western blotting. To compare integrity of the mucus layer, fluorescent Eh and 1μM fluorescent beads were inoculated on WT and KO monolayers and adherent Eh and bead penetrability analyzed. To quantify MUC2 and FCGBP degradation by Eh, purified MUC2 and recombinant FCGBP were incubated with Eh proteases (SPs) and Western blotted using highly specific antibodies against various regions of the proteins. Results In response to live Eh, FCGBP and MUC2 mRNA and protein expressions were significantly increased in a time-dependent manner. Surprisingly, FCGBP KO cells elicited robust expression of pro-inflammatory cytokine mRNA and protein as compared to WT cells. More fluorescent Eh were attached to the mucus layer of FCGBP KO cells as compared to WT or MUC2 KO cells. Fluorescent beads penetrated further towards the epithelial cell surface in KO as compared to WT cells. Interestingly, while both MUC2 and FCGBP from purified polymeric mucins were degraded by Eh SPs, FCGBP cleavage occurred at a faster rate than MUC2. Degradation of FCGBP and MUC2 was mediated by EhCP-A5 cysteine proteinase using purified MUC2 and recombinant FCGBP. Conclusions In WT goblet cells, FCGBP and MUC2 were upregulated temporally in response to Eh. The increase in pro-inflammatory cytokine expression in FCGBP KO cells in response to Eh suggests that Eh directly interacted with the cell surface suggesting an impaired protective mucus layer. In support of this, fluorescent beads penetrated the mucus layer close to the cell surface and more Eh were attached to FCGBP KO mucus demonstrating that FCGBP was critical in providing structural integrity of the mucus layer. In response to Eh, FCGBP degradation was a prerequisite for MUC2 cleavage, providing direct evidence that FCGBP and MUC2 interactions conferred biophysical properties of the protective functions of the mucus gel. Funding Agencies CIHR

scholarly journals In Vitro Models of Tissue Penetration and Destruction by Porphyromonas gingivalis

2004 ◽  
Vol 72 (8) ◽  
pp. 4689-4698 ◽  
Author(s):  
Elisoa Andrian ◽  
Daniel Grenier ◽  
Mahmoud Rouabhia
Keyword(s):  
Epithelial Cells ◽  
Porphyromonas Gingivalis ◽  
Critical Role ◽  
Specific Inhibitor ◽  
In Vitro Models ◽  
Null Mutant ◽  
Tissue Destruction ◽  
Tissue Penetration ◽  
Proinflammatory Response

ABSTRACT Porphyromonas gingivalis is a gram-negative anaerobic bacterium that is considered the key etiologic agent of chronic periodontitis. Arg- and Lys-gingipain cysteine proteinases produced by P. gingivalis are key virulence factors and are believed to be essential for significant tissue component degradation, leading to host tissue invasion by periodontopathogens. Two in vitro models were used to determine the extent to which P. gingivalis can reach connective tissue. The tissue penetration potential of P. gingivalis was first investigated by using an engineered human oral mucosa model composed of normal human epithelial cells and fibroblasts. Internalized bacteria were assessed by transmission electron microscopy. Bacteria were observed within multilayered gingival epithelial cells and in the space between the stratified epithelium and the lamina propria. A gingipain-null mutant strain of P. gingivalis was found to be less potent in penetrating tissue than the wild-type strain. Proinflammatory responses to P. gingivalis infection were evaluated. P. gingivalis increased the secretion of interleukin-1β, interleukin-6, interleukin-8, and tumor necrosis factor alpha. In the second part of the study, the contribution of P. gingivalis gingipains to tissue penetration was investigated by using a reconstituted basement membrane model (Matrigel). The penetration of 14C-labeled P. gingivalis cells through Matrigel was significantly reduced when leupeptin, a specific inhibitor of Arg-gingipain activity, was added or when a gingipain-null mutant was used. The results obtained with these two relevant models support the capacities of P. gingivalis to infiltrate periodontal tissue and to modulate the proinflammatory response and suggest a critical role of gingipains in tissue destruction.

Innate mechanisms of epithelial host defense: spotlight on intestine

1999 ◽  
Vol 277 (3) ◽  
pp. C351-C358 ◽  
Author(s):  
Gail Hecht
Keyword(s):  
Epithelial Cells ◽  
Host Defense ◽  
Defense Mechanisms ◽  
Single Layer ◽  
Intestinal Lumen ◽  
Intestinal Epithelial ◽  
Antimicrobial Proteins ◽  
Commensal Flora ◽  
Secretion Expression ◽  
Water Secretion

The single layer of epithelial cells lining the intestinal tract is charged with a most difficult task: protecting the underlying biological compartments from both the normal commensal flora that reside within the intestinal lumen as well as the uninvited pathogens. To such an end, the intestinal epithelial cells are equipped with a panoply of defense mechanisms, both constitutive and inducible. This review focuses only on those defense mechanisms that are initiated and executed by the intestinal epithelial cell. Fitting these strict criteria are three major categories of epithelial host defense: enhanced salt and water secretion, expression of antimicrobial proteins and peptides, and production of intestinal mucins. Each of these areas is discussed in this review.

scholarly journals Human β-defensin-3 reduces excessive autophagy in intestinal epithelial cells and in experimental necrotizing enterocolitis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Liping Chen ◽  
Zhibao Lv ◽  
Zhimei Gao ◽  
Guijie Ge ◽  
Xueli Wang ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Necrotizing Enterocolitis ◽  
Rat Model ◽  
Intestinal Epithelial Cells ◽  
Critical Role ◽  
Neonatal Rat ◽  
Mucosal Barrier ◽  
Intestinal Epithelial ◽  
Barrier Dysfunction ◽  
Autophagy Activity

AbstractNecrotizing enterocolitis (NEC) is a leading cause of mortality in preterm newborns. Intestinal barrier dysfunction is one key event in NEC pathogenesis. Human β-defensin-3 (hBD3), one member of cationic host defence peptides, was reported to reduce the development of necrotizing enterocolitis in a neonatal rat model. And autophagy was induced in the intestine of human and animals with NEC. We hypothesized that regulation of autophagy might play a critical role in hBD3-mediated protection against NEC injury. Autophagy activity was evaluated both in intestinal epithelial cells and in NEC models. Newborn Sprague-Dawley rats were divided randomly into four groups: Control + NS, Control + rapamycin, NEC + NS, and NEC + hBD3. Body weight, histological score, survival time, enterocyte migration and mucosal barrier were recorded. Our results showed that hBD3 pretreatment could effectively inhibit autophagy activity in cultured IEC-6 and Caco2 enterocytes, and CXCR4 might be involved in hBD3-mediated autophagy suppression. Moreover, hBD3-induced inhibition of autophagy significantly promoted the intestinal epithelial cell migration by wound healing assay and transwell migration assay. In the rat model of NEC, hBD3 could noticeably reduce the expression of autophagy-activated proteins, down-regulate the expression of inflammatory mediators, and promote the mucosal integrity. Our data suggest an additional role of hBD3-mediated protection against intestinal mucosal injury: inhibition of over-activated autophagy in enterocytes.

scholarly journals Prostaglandin E2 Produced by Entamoeba histolytica Binds to EP4 Receptors and Stimulates Interleukin-8 Production in Human Colonic Cells

2008 ◽  
Vol 76 (11) ◽  
pp. 5158-5163 ◽  
Author(s):  
Indranil Dey ◽  
Kris Chadee
Keyword(s):  
Epithelial Cells ◽  
Entamoeba Histolytica ◽  
Interleukin 8 ◽  
Cyclooxygenase Inhibitors ◽  
Prostaglandin E ◽  
Proinflammatory Response ◽  
Colonic Epithelial Cells ◽  
Ep4 Receptor ◽  
Interfering Rna ◽  
Colonic Cells

ABSTRACT Entamoeba histolytica pathogenesis in the colon occurs in a stepwise fashion. It begins with colonization of the mucin layer, which is followed by stimulation of a proinflammatory response that causes nonspecific tissue damage that may facilitate parasite invasion of the underlying colonic mucosa. Unfortunately, the parasite and/or host factors that stimulate a proinflammatory response in the gut are poorly understood. In this study, we found that live E. histolytica or secretory or proteins (SP) and soluble ameba components (SAP) can markedly increase interleukin-8 (IL-8) mRNA expression and protein production in colonic epithelial cells. The IL-8-stimulating molecule produced by live amebae was identified as prostaglandin E2 (PGE2) as trophozoites treated with cyclooxygenase inhibitors inhibited the biosynthesis of PGE2 and eliminated IL-8 production induced by live parasites or ameba components. Moreover, using specific prostaglandin EP2 and EP4 receptor agonists and antagonists, we found that PGE2 binds exclusively through EP4 receptors in colonic epithelial cells to stimulate IL-8 production. Silencing of EP4 receptors with EP4 small interfering RNA completely eliminated SP- and SAP-induced IL-8 production. These studies identified bioactive PGE2 as a one of the major virulence factors produced by E. histolytica that can stimulate the potent neutrophil chemokine and activator IL-8, which can trigger an acute host inflammatory response. Thus, the induction of IL-8 production in response to E. histolytica-derived PGE2 may be a mechanism that explains the initiation and amplification of acute inflammation associated with intestinal amebiasis.

Characterization of a monolayer graphitic structure

1994 ◽  
Vol 52 ◽  
pp. 760-761
Author(s):  
X. Lin ◽  
X. K. Wang ◽  
V. P. Dravid ◽  
J. B. Ketterson ◽  
R. P. H. Chang
Keyword(s):  
Three Dimensional ◽  
Single Layer ◽  
Synthesis Process ◽  
Graphitic Structure ◽  
Positive Gaussian Curvature ◽  
Graphitic Sheet ◽  
Structural And Electronic Properties ◽  
New Material ◽  
Hexagonal Network

For small curvatures of a graphitic sheet, carbon atoms can maintain their preferred sp2 bonding while allowing the sheet to have various three-dimensional geometries, which may have exotic structural and electronic properties. In addition the fivefold rings will lead to a positive Gaussian curvature in the hexagonal network, and the sevenfold rings cause a negative one. By combining these sevenfold and fivefold rings with sixfold rings, it is possible to construct complicated carbon sp2 networks. Because it is much easier to introduce pentagons and heptagons into the single-layer hexagonal network than into the multilayer network, the complicated morphologies would be more common in the single-layer graphite structures. In this contribution, we report the observation and characterization of a new material of monolayer graphitic structure by electron diffraction, HREM, EELS.The synthesis process used in this study is reported early. We utilized a composite anode of graphite and copper for arc evaporation in helium.

scholarly journals Shiga Toxin (Stx)-Binding Glycosphingolipids of Primary Human Renal Cortical Epithelial Cells (pHRCEpiCs) and Stx-Mediated Cytotoxicity

Toxins ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 139
Author(s):  
Johanna Detzner ◽  
Elisabeth Krojnewski ◽  
Gottfried Pohlentz ◽  
Daniel Steil ◽  
Hans-Ulrich Humpf ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Epithelial Cells ◽  
Shiga Toxin ◽  
Saturated Fatty Acids ◽  
Human Kidney ◽  
Enterohemorrhagic Escherichia Coli ◽  
Highly Sensitive ◽  
Liquid Ordered ◽  
Uremic Syndrome

Human kidney epithelial cells are supposed to be directly involved in the pathogenesis of the hemolytic–uremic syndrome (HUS) caused by Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC). The characterization of the major and minor Stx-binding glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), respectively, of primary human renal cortical epithelial cells (pHRCEpiCs) revealed GSLs with Cer (d18:1, C16:0), Cer (d18:1, C22:0), and Cer (d18:1, C24:1/C24:0) as the dominant lipoforms. Using detergent-resistant membranes (DRMs) and non-DRMs, Gb3Cer and Gb4Cer prevailed in the DRM fractions, suggesting their association with microdomains in the liquid-ordered membrane phase. A preference of Gb3Cer and Gb4Cer endowed with C24:0 fatty acid accompanied by minor monounsaturated C24:1-harboring counterparts was observed in DRMs, whereas the C24:1 fatty acid increased in relation to the saturated equivalents in non-DRMs. A shift of the dominant phospholipid phosphatidylcholine with saturated fatty acids in the DRM to unsaturated species in the non-DRM fractions correlated with the GSL distribution. Cytotoxicity assays gave a moderate susceptibility of pHRCEpiCs to the Stx1a and Stx2a subtypes when compared to highly sensitive Vero-B4 cells. The results indicate that presence of Stx-binding GSLs per se and preferred occurrence in microdomains do not necessarily lead to a high cellular susceptibility towards Stx.

scholarly journals Mammary gland adipocytes in lactation cycle, obesity and breast cancer

2021 ◽  
Author(s):  
Georgia Colleluori ◽  
Jessica Perugini ◽  
Giorgio Barbatelli ◽  
Saverio Cinti
Keyword(s):  
Breast Cancer ◽  
Mammary Gland ◽  
Epithelial Cells ◽  
Close Association ◽  
Critical Role ◽  
Exocrine Gland ◽  
Plastic Properties ◽  
Lactation Cycle ◽  
Gland Development

AbstractThe mammary gland (MG) is an exocrine gland present in female mammals responsible for the production and secretion of milk during the process of lactation. It is mainly composed by epithelial cells and adipocytes. Among the features that make the MG unique there are 1) its highly plastic properties displayed during pregnancy, lactation and involution (all steps belonging to the lactation cycle) and 2) its requirement to grow in close association with adipocytes which are absolutely necessary to ensure MG’s proper development at puberty and remodeling during the lactation cycle. Although MG adipocytes play such a critical role for the gland development, most of the studies have focused on its epithelial component only, leaving the role of the neighboring adipocytes largely unexplored. In this review we aim to describe evidences regarding MG’s adipocytes role and properties in physiologic conditions (gland development and lactation cycle), obesity and breast cancer, emphasizing the existing gaps in the literature which deserve further investigation.

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