In Vivo Multivesicular Body and Exosome Secretion in the Intestinal Epithelial Cells of Turtles During Hibernation

2019 ◽  
Vol 25 (6) ◽  
pp. 1341-1351
Author(s):  
Waseem Ali Vistro ◽  
Yufei Huang ◽  
Xuebing Bai ◽  
Ping Yang ◽  
Abdul Haseeb ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Susceptibility Gene ◽  
Multivesicular Body ◽  
Intestinal Epithelial ◽  
Transmission Electron ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Cluster Of Differentiation

AbstractThe present study was designed to investigate the in vivo biological processes of multivesicular bodies (MVBs) and exosomes in mitochondria-rich cells (MRCs), goblet cells (GCs), and absorptive cells (ACs) in turtle intestines during hibernation. The exosome markers, cluster of differentiation 63 (CD63) and tumor susceptibility gene 101 (TSG101), were positively expressed in intestinal villi during turtle hibernation. The distribution and formation processes of MVBs and exosomes in turtle MRCs, GCs, and ACs were further confirmed by transmission electron microscopy. During hibernation, abundantly secreted early endosomes (ees) were localized in the luminal and basal cytoplasm of the MRCs and ACs, and late endosomes (les) were dispersed with the supranuclear parts of the MRCs and ACs. Many “heterogeneous” MVBs were identified throughout the cytoplasm of the MRCs and ACs. Interestingly, the ees, les, and MVBs were detected in the cytoplasm of the GCs during hibernation; however, they were absent during nonhibernation. Furthermore, the exocytosis pathways of exosomes and autophagic vacuoles were observed in the MRCs, GCs, and ACs during hibernation. In addition, the number of different MVBs with intraluminal vesicles (ILVs) and heterogeneous endosome–MVB–exosome complexes was significantly increased in the MRCs, GCs, and ACs during hibernation. All these findings indicate that intestinal epithelial cells potentially perform a role in the secretion of MVBs and exosomes, which are essential for mucosal immunity, during hibernation.

Luminal antigens access late endosomes of intestinal epithelial cells enriched in MHC I and MHC II molecules: in vivo study in Crohn's ileitis

2007 ◽  
Vol 293 (4) ◽  
pp. G798-G808 ◽  
Author(s):  
Gheorghe Hundorfean ◽  
Klaus-Peter Zimmer ◽  
Stephan Strobel ◽  
Andreas Gebert ◽  
Diether Ludwig ◽  
...  
Keyword(s):  
T Cells ◽  
Epithelial Cells ◽  
Antigen Processing ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Mhc I ◽  
Mhc Ii ◽  
Late Endosomes ◽  
Early Endosomes

In contrast to healthy conditions, intestinal epithelial cells (IECs) stimulate proinflammatory CD4+and CD8+T cells during Crohn's disease (CD). The underlying regulatory mechanisms remain unknown. Here we investigated the epithelial expression of major histocompatibility complex (MHC) I and MHC II and its interference with endocytic pathways, in vivo. During ileoscopy, ovalbumin (OVA) was sprayed onto ileal mucosa of CD patients (ileitis and remission) and controls. The epithelial traffic of OVA and MHC I/II pathways were studied in biopsies using fluorescence and electron microscopy. We found MHC I and MHC II to accumulate within multivesicular late endosomes (MVLE) of IECs. Faint labeling for these molecules was seen in early endosomes and lysosomes. MVLE were entered by OVA 10 min after exposure. Exosomes carrying MHC I, MHC II, and OVA were detected in intercellular spaces of the epithelium. OVA trafficking and labeling patterns for MHC I and MHC II in IECs showed no differences between CD patients and controls. Independent of inflammatory stimuli, MHC I and MHC II pathways intersect MVLE in IECs, which were efficiently targeted by luminal antigens. Similar to MHC II-enriched compartments in professional antigen presenting cells, these MVLE might be critically involved in MHC I- and MHC II-related antigen processing in IECs and the source of epithelial-released exosomes. The access of luminal antigens to MHC I in MVLE might indicate that the presentation of exogenous antigens by IECs must not be restricted to MHC II but might also occur as “cross-presentation” via MHC I to CD8+T cells.

scholarly journals Human Cytomegalovirus Infects Caco-2 Intestinal Epithelial Cells Basolaterally Regardless of the Differentiation State

2000 ◽  
Vol 74 (1) ◽  
pp. 513-517 ◽  
Author(s):  
Audrey Esclatine ◽  
Michel Lemullois ◽  
Alain L. Servin ◽  
Anne-Marie Quero ◽  
Monique Geniteau-Legendre
Keyword(s):  
Epithelial Cells ◽  
Human Cytomegalovirus ◽  
Intestinal Epithelial Cells ◽  
Severe Disease ◽  
Epithelial Cell Line ◽  
Intestinal Epithelial ◽  
Cmv Infection ◽  
Transmission Electron ◽  
Immunosuppressed Patients

ABSTRACT Human cytomegalovirus (CMV) causes severe disease in immunosuppressed patients and notably infects the gastrointestinal tract. To understand the interaction of CMV with intestinal epithelial cells, which are highly susceptible to CMV infection in vivo, we used the intestinal epithelial cell line Caco-2 and demonstrated that CMV enters predominantly through the basolateral surface of polarized Caco-2 cells. As shown by expression of all three classes of CMV proteins and by visualization of nucleocapsids by transmission electron microscopy, both poorly and fully differentiated Caco-2 cells were permissive to CMV replication. However, infection failed to produce infectious particles in Caco-2 cells, irrespective of the state of differentiation.

scholarly journals Expression of lysophosphatidic acid receptor 5 is necessary for the regulation of intestinal Na+/H+ exchanger 3 by lysophosphatidic acid in vivo

2018 ◽  
Vol 315 (4) ◽  
pp. G433-G442 ◽  
Author(s):  
Kayte A. Jenkin ◽  
Peijian He ◽  
C. Chris Yun
Keyword(s):  
Epithelial Cells ◽  
Lysophosphatidic Acid ◽  
Direct Evidence ◽  
Intestinal Epithelial Cells ◽  
Regulatory Role ◽  
Intestinal Epithelial ◽  
Fluid Absorption ◽  
Wild Type

Lysophosphatidic acid (LPA) is a bioactive lipid molecule, which regulates a broad range of pathophysiological processes. Recent studies have demonstrated that LPA modulates electrolyte flux in the intestine, and its potential as an antidiarrheal agent has been suggested. Of six LPA receptors, LPA5 is highly expressed in the intestine. Recent studies by our group have demonstrated activation of Na+/H+ exchanger 3 (NHE3) by LPA5. However, much of what has been elucidated was achieved using colonic cell lines that were transfected to express LPA5. In the current study, we engineered a mouse that lacks LPA5 in intestinal epithelial cells, Lpar5ΔIEC, and investigated the role of LPA5 in NHE3 regulation and fluid absorption in vivo. The intestine of Lpar5ΔIEC mice appeared morphologically normal, and the stool frequency and fecal water content were unchanged compared with wild-type mice. Basal rates of NHE3 activity and fluid absorption and total NHE3 expression were not changed in Lpar5ΔIEC mice. However, LPA did not activate NHE3 activity or fluid absorption in Lpar5ΔIEC mice, providing direct evidence for the regulatory role of LPA5. NHE3 activation involves trafficking of NHE3 from the terminal web to microvilli, and this mobilization of NHE3 by LPA was abolished in Lpar5ΔIEC mice. Dysregulation of NHE3 was specific to LPA, and insulin and cholera toxin were able to stimulate and inhibit NHE3, respectively, in both wild-type and Lpar5ΔIEC mice. The current study for the first time demonstrates the necessity of LPA5 in LPA-mediated stimulation of NHE3 in vivo. NEW & NOTEWORTHY This study is the first to assess the role of LPA5 in NHE3 regulation and fluid absorption in vivo using a mouse that lacks LPA5 in intestinal epithelial cells, Lpar5ΔIEC. Basal rates of NHE3 activity and fluid absorption, and total NHE3 expression were not changed in Lpar5ΔIEC mice. However, LPA did not activate NHE3 activity or fluid absorption in Lpar5ΔIEC mice, providing direct evidence for the regulatory role of LPA5.

Progastrin1–80stimulates growth of intestinal epithelial cells in vitro via high-affinity binding sites

2003 ◽  
Vol 284 (2) ◽  
pp. G328-G339 ◽  
Author(s):  
P. Singh ◽  
X. Lu ◽  
S. Cobb ◽  
B. T. Miller ◽  
N. Tarasova ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Binding Sites ◽  
Intestinal Epithelial Cells ◽  
Full Length ◽  
Intestinal Epithelial ◽  
Growth Effects ◽  
High Affinity ◽  
Significant Difference

Proliferation and carcinogenesis of the large intestinal epithelial cells (IEC) cells is significantly increased in transgenic mice that overexpress the precursor progastrin (PG) peptide. It is not known if the in vivo growth effects of PG on IEC cells are mediated directly or indirectly. Full-length recombinant human PG (rhPG1–80) was generated to examine possible direct effects of PG on IEC cells. Surprisingly, rhPG (0.1–1.0 nM) was more effective than the completely processed gastrin 17 (G17) peptide as a growth factor. Even though IEC cells did not express CCK1and CCK2receptors (-R), fluorescently labeled G17 and Gly-extended G17 (G-Gly) were specifically bound to the cells, suggesting the presence of binding proteins other than CCK1-R and CCK2-R on IEC cells. High-affinity ( Kd= 0.5–1.0 nM) binding sites for125I-rhPG were discovered on IEC cells that demonstrated relative binding affinity for gastrin-like peptides in the order PG ≥ COOH-terminally extended G17 ≥ G-Gly > G17 > *CCK-8 (* significant difference; P< 0.05). In conclusion, our studies demonstrate for the first time direct growth effects of the full-length precursor peptide on IEC cells in vitro that are apparently mediated by the high-affinity PG binding sites that were discovered on these cells.

Comparison of gene expression and activation of transcription factors in organoid-derived monolayer intestinal epithelial cells and organoids

2021 ◽  
Author(s):  
Yu Takahashi ◽  
Yu Inoue ◽  
Keitaro Kuze ◽  
Shintaro Sato ◽  
Makoto Shimizu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Gene Expression Regulation ◽  
Intestinal Epithelial Cells ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Intestinal Epithelial

Abstract Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

7 LACTOBACILLUS REUTERI SUPPRESSES PRO-INFLAMMATORY DRIVEN REACTIVE OXYGEN SPECIES IN VITRO IN HUMAN INTESTINAL EPITHELIAL CELLS AND IN VIVO IN A TNBS COLITIS MOUSE MODEL

2020 ◽  
Vol 26 (Supplement_1) ◽  
pp. S41-S41 ◽  
Author(s):  
Wenly Ruan ◽  
Melinda Engevik ◽  
Alexandra Chang-Graham ◽  
Joseph Hyser ◽  
James Versalovic
Keyword(s):  
Reactive Oxygen Species ◽  
Epithelial Cells ◽  
Lactobacillus Reuteri ◽  
Intestinal Inflammation ◽  
Intestinal Epithelial Cells ◽  
Oxygen Species ◽  
Intestinal Epithelial ◽  
Colitis Model

Abstract Background Reactive oxygen species (ROS) play a role in maintaining intestinal epithelial homeostasis and are normally kept at low levels via antioxidant compounds. Dysregulation of ROS can lead to intestinal inflammation and contribute to inflammatory bowel disease (IBD). Select gut microbes possess the enzymatic machinery to produce antioxidants whereas others can dysregulate levels of ROS. Our model microbe, Lactobacillus reuteri (ATCC PTA 6475), has been demonstrated to reduce intestinal inflammation in mice models. It contains the genes encoding two distinct GshA-like glutamylcysteine ligases. We hypothesize that L. reuteri can secrete γ-glutamylcysteine to suppress ROS, minimize NFκB activation and regulate secretion of e pithelial cytokines. Methods & Results Conditioned media from L. reuteri was analyzed via mass spectrometry to confirm the presence of γ-glutamylcysteine. All cysteine containing products including γ-glutamylcysteine were fluorescently tagged in the conditioned media and then incubated with HT29 cell monolayers as well as human jejunal enteroid (HJE) monolayers. γ-glutamylcysteine was demonstrated to enter intestinal epithelial cells based on microscopy. Next, a Thioltracker assay was used to show increased intracellular glutathione levels by L. reuteri secreted γ-glutamylcysteine. HT29 cells and HJEs were then treated with IL-1β or hydrogen peroxide, and L. reuteri metabolites as well as γ-glutamylcysteine significantly suppressed pro-inflammatory cytokine driven ROS and IL-8 production. L. reuteri secreted products also reduced activity of NFκB as determined by a luciferase reporter assay. γ-glutamylcysteine deficient mutants were generated by targeted mutagenesis of GshA genes, and these mutant L. reuteri strains had a diminished ability to suppress IL-8 production and ROS. To further test the role of L. reuteri secreted γ-glutamylcysteine in vivo, a 2,4,6-Trinitrobenzenesulfonic acid (TNBS)- induced mouse colitis model was used. Adolescent mice were orogavaged with PBS, L. reuteri, L. reuteri GshA2 mutant, or γ-glutamylcysteine for a week after which TNBS was rectally administered to induce colitis. We demonstrate that L. reuteri and γ-glutamylcysteine can suppress histologic inflammation compared to PBS control and L. reuteri GshA2 mutant groups. Conclusions Together these data indicate that L. reuteri secretes γ-glutamylcysteine which can enter the intestinal epithelial cells and modulate epithelial cytokine production. It acts via suppression of ROS and NFκB which then decreases IL-8 production. We are able to demonstrate this in vitro in both HT 29 cells and HJEs. We now also demonstrate this in vivo in a mouse colitis model. These experiments highlight a prominent role for ROS intermediates in microbiome-mammalian cell signaling processes involved in immune responses and intestinal inflammation.

scholarly journals Decreased SLC26A3 expression and function in intestinal epithelial cells in response to Cryptosporidium parvum infection

2019 ◽  
Vol 317 (6) ◽  
pp. C1205-C1212 ◽  
Author(s):  
Anoop Kumar ◽  
Dulari Jayawardena ◽  
Arivarasu N. Anbazhagan ◽  
Ishita Chatterjee ◽  
Shubha Priyamvada ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cryptosporidium Parvum ◽  
Diarrheal Disease ◽  
Intestinal Epithelial Cells ◽  
Ex Vivo ◽  
Jejunal Mucosa ◽  
Intestinal Epithelial ◽  
Chloride Absorption ◽  
And Function

The protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a diarrheal disease worldwide. Infection in immunocompetent hosts typically results in acute, self-limiting, or recurrent diarrhea. However, in immunocompromised individuals infection can cause fulminant diarrhea, extraintestinal manifestations, and death. To date, the mechanisms underlying CP-induced diarrheal pathogenesis are poorly understood. Diarrheal diseases most commonly involve increased secretion and/or decreased absorption of fluid and electrolytes. We and others have previously shown impaired chloride absorption in infectious diarrhea due to dysregulation of SLC26A3 [downregulated in adenoma (DRA)], the human intestinal apical membrane Cl−/[Formula: see text] exchanger protein. However, there are no studies on the effects of CP infection on DRA activity. Therefore, we examined the expression and function of DRA in intestinal epithelial cells in response to CP infection in vitro and in vivo. CP infection (0.5 × 106 oocysts/well in 24-well plates, 24 h) of Caco-2 cell monolayers significantly decreased Cl−/[Formula: see text] exchange activity (measured as DIDS-sensitive 125I uptake) as well as DRA mRNA and protein levels. Substantial downregulation of DRA mRNA and protein was also observed following CP infection ex vivo in mouse enteroid-derived monolayers and in vivo in the ileal and jejunal mucosa of C57BL/6 mice for 24 h. However, at 48 h after infection in vivo, the effects on DRA mRNA and protein were attenuated and at 5 days after infection DRA returned to normal levels. Our results suggest that impaired chloride absorption due to downregulation of DRA could be one of the contributing factors to CP-induced acute, self-limiting diarrhea in immunocompetent hosts.

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