scholarly journals Requirement of the Shigella flexneri Virulence Plasmid in the Ability To Induce Trafficking of Neutrophils across Polarized Monolayers of the Intestinal Epithelium

1998 ◽  
Vol 66 (9) ◽  
pp. 4237-4243 ◽  
Author(s):  
Beth A. McCormick ◽  
Andrew M. Siber ◽  
Anthony T. Maurelli
Keyword(s):  
Intestinal Epithelium ◽  
Shigella Flexneri ◽  
Basolateral Membrane ◽  
Epithelial Cell Line ◽  
Virulence Plasmid ◽  
Intestinal Epithelial ◽  
Epithelial Monolayers ◽  
Human Pmns

ABSTRACT Attachment of an array of enteric pathogens to epithelial surfaces is accompanied by recruitment of polymorphonuclear leukocytes (PMN) across the intestinal epithelium. In this report, we examine howShigella-intestinal epithelium interactions evoke the mucosal inflammatory response. We modeled these interactions in vitro by using polarized monolayers of the human intestinal epithelial cell line, T84, isolated human PMNs, and Shigella flexneri. We show that Shigella attachment to T84-cell basolateral membranes was a necessary component in the signaling cascade for induction of basolateral-to-apical directed transepithelial PMN migration, the direction of PMN transepithelial migration in vivo. In contrast, attachment of Shigella to the T84-cell apical membrane failed to stimulate a directed PMN transepithelial migration response. Importantly, the ability of Shigella to induce PMN migration across epithelial monolayers was dependent on the presence of the 220-kb virulence plasmid. Moreover, examination ofShigella genes necessary to signal subepithelial neutrophils established the requirement of a functional type III secretion system. Our results indicate that the ability ofShigella to elicit transepithelial signaling to neutrophils from the basolateral membrane of epithelial cells represents a mechanism involved in Shigella-elicited enteritis in humans.

scholarly journals Virulent Shigella flexneri subverts the host innate immune response through manipulation of antimicrobial peptide gene expression

2008 ◽  
Vol 205 (5) ◽  
pp. 1121-1132 ◽  
Author(s):  
Brice Sperandio ◽  
Béatrice Regnault ◽  
Jianhua Guo ◽  
Zhi Zhang ◽  
Samuel L. Stanley ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Shigella Flexneri ◽  
Host Cells ◽  
Virulence Plasmid ◽  
Cationic Peptides ◽  
Immunity Genes ◽  
Genes Encoding ◽  
Peptide Gene

Antimicrobial factors are efficient defense components of the innate immunity, playing a crucial role in the intestinal homeostasis and protection against pathogens. In this study, we report that upon infection of polarized human intestinal cells in vitro, virulent Shigella flexneri suppress transcription of several genes encoding antimicrobial cationic peptides, particularly the human β-defensin hBD-3, which we show to be especially active against S. flexneri. This is an example of targeted survival strategy. We also identify the MxiE bacterial regulator, which controls a regulon encompassing a set of virulence plasmid-encoded effectors injected into host cells and regulating innate signaling, as being responsible for this dedicated regulatory process. In vivo, in a model of human intestinal xenotransplant, we confirm at the transcriptional and translational level, the presence of a dedicated MxiE-dependent system allowing S. flexneri to suppress expression of antimicrobial cationic peptides and promoting its deeper progression toward intestinal crypts. We demonstrate that this system is also able to down-regulate additional innate immunity genes, such as the chemokine CCL20 gene, leading to compromised recruitment of dendritic cells to the lamina propria of infected tissues. Thus, S. flexneri has developed a dedicated strategy to weaken the innate immunity to manage its survival and colonization ability in the intestine.

scholarly journals Protein Kinase C Signaling Mediates a Program of Cell Cycle Withdrawal in the Intestinal Epithelium

2000 ◽  
Vol 151 (4) ◽  
pp. 763-778 ◽  
Author(s):  
Mark R. Frey ◽  
Jennifer A. Clark ◽  
Olga Leontieva ◽  
Joshua M. Uronis ◽  
Adrian R. Black ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Intestinal Epithelium ◽  
Molecular Mechanisms ◽  
Model Systems ◽  
Intestinal Epithelial ◽  
Kinase C

Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G0. PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21waf1/cip1 and p27kip1, thus targeting all of the major G1/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G0 as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCα alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt–villus axis revealed that PKCα activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit–specific events in situ. Together, these data point to PKCα as a key regulator of cell cycle withdrawal in the intestinal epithelium.

scholarly journals Bifidobacterium bifidum reduces apoptosis in the intestinal epithelium in necrotizing enterocolitis

2010 ◽  
Vol 299 (5) ◽  
pp. G1118-G1127 ◽  
Author(s):  
Ludmila Khailova ◽  
Sarah K. Mount Patrick ◽  
Kelly M. Arganbright ◽  
Melissa D. Halpern ◽  
Toshi Kinouchi ◽  
...  
Keyword(s):  
Necrotizing Enterocolitis ◽  
Intestinal Epithelium ◽  
Mucosal Injury ◽  
In Vitro Models ◽  
Bifidobacterium Bifidum ◽  
Epithelial Cell Line ◽  
Prostaglandin E ◽  
Cox 2

Necrotizing enterocolitis (NEC) is a devastating intestinal disease of neonates, and clinical studies suggest the beneficial effect of probiotics in NEC prevention. Recently, we have shown that administration of Bifidobacterium bifidum protects against NEC in a rat model. Intestinal apoptosis can be suppressed by activation of cyclooxygenase-2 (COX-2) and increased production of prostaglandin E2 (PGE2). The present study investigates the effect of B. bifidum on intestinal apoptosis in the rat NEC model and in an intestinal epithelial cell line (IEC-6), as a mechanism of protection against mucosal injury. Premature rats were divided into the following three groups: dam fed, hand fed with formula (NEC), or hand fed with formula supplemented with B. bifidum (NEC + B. bifidum ). Intestinal Toll-like receptor-2 (TLR-2), COX-2, PGE2, and apoptotic regulators were measured. The effect of B. bifidum was verified in IEC-6 cells using a model of cytokine-induced apoptosis. Administration of B. bifidum increased expression of TLR-2, COX-2, and PGE2 and significantly reduced apoptosis in the intestinal epithelium of both in vivo and in vitro models. The Bax-to-Bcl-w ratio was shifted toward cell survival, and the number of cleaved caspase-3 positive cells was markedly decreased in B. bifidum -treated rats. Experiments in IEC-6 cells showed anti-apoptotic effect of B. bifidum . Inhibition of COX-2 signaling blocked the protective effect of B. bifidum treatment in both in vivo and in vitro models. In conclusion, oral administration of B. bifidum activates TLR-2 in the intestinal epithelium. B. bifidum increases expression of COX-2, which leads to higher production of PGE2 in the ileum and protects against intestinal apoptosis associated with NEC. This study indicates the ability of B. bifidum to downregulate apoptosis in the rat NEC model and in IEC-6 cells by a COX-2-dependent matter and suggests a molecular mechanism by which this probiotic reduces mucosal injury and preserves intestinal integrity.

scholarly journals Jnk2 deletion disrupts intestinal mucosal homeostasis and maturation by differentially modulating RNA-binding proteins HuR and CUGBP1

2014 ◽  
Vol 306 (12) ◽  
pp. C1167-C1175 ◽  
Author(s):  
Hee Kyoung Chung ◽  
Jaladanki N. Rao ◽  
Tongtong Zou ◽  
Lan Liu ◽  
Lan Xiao ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Intestinal Epithelium ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Intestinal Epithelial ◽  
Targeted Gene Deletion ◽  
Epithelial Homeostasis

Homeostasis and maturation of the mammalian intestinal epithelium are preserved through strict regulation of cell proliferation, apoptosis, and differentiation, but the exact mechanism underlying this process remains largely unknown. c-Jun NH2-terminal kinase 2 (JNK2) is highly expressed in the intestinal mucosa, and its activation plays an important role in proliferation and also mediates apoptosis in cultured intestinal epithelial cells (IECs). Here, we investigated the in vivo function of JNK2 in the regulation of intestinal epithelial homeostasis and maturation by using a targeted gene deletion approach. Targeted deletion of the jnk2 gene increased cell proliferation within the crypts in the small intestine and disrupted mucosal maturation as indicated by decreases in the height of villi and the villus-to-crypt ratio. JNK2 deletion also decreased susceptibility of the intestinal epithelium to apoptosis. JNK2-deficient intestinal epithelium was associated with an increase in the level of the RNA-binding protein HuR and with a decrease in the abundance of CUG-binding protein 1 (CUGBP1). In studies in vitro, JNK2 silencing protected intestinal epithelial cell-6 (IEC-6) cells against apoptosis and this protection was prevented by inhibiting HuR. Ectopic overexpression of CUGBP1 repressed IEC-6 cell proliferation, whereas CUGBP1 silencing enhanced cell growth. These results indicate that JNK2 is essential for maintenance of normal intestinal epithelial homeostasis and maturation under biological conditions by differentially modulating HuR and CUGBP1.

Superoxide released from neutrophils causes a reduction in nitric oxide gas

1998 ◽  
Vol 275 (6) ◽  
pp. L1120-L1126 ◽  
Author(s):  
Kimberly L. Jones ◽  
Ty W. Bryan ◽  
Patricia A. Jinkins ◽  
Keith L. Simpson ◽  
Matthew B. Grisham ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cystic Fibrosis ◽  
Epithelial Cell ◽  
Distress Syndrome ◽  
Polymorphonuclear Neutrophils ◽  
Epithelial Cell Line ◽  
Lung Epithelial ◽  
Human Pmns

Exhaled nitric oxide (NO) is increased in some inflammatory airway disorders but not in others such as cystic fibrosis and acute respiratory distress syndrome. NO can combine with superoxide ([Formula: see text]) to form peroxynitrite, which can decompose into nitrate. Activated polymorphonuclear neutrophils (PMNs) releasing[Formula: see text] could account for a reduction in exhaled NO in disorders such as cystic fibrosis. To test this hypothesis in vitro, we stimulated confluent cultures of LA-4 cells, a murine lung epithelial cell line, to produce NO. Subsequently, human PMNs stimulated to produce [Formula: see text] were added to the LA-4 cells. A gradual increase in NO in the headspace above the cultures was observed and was markedly reduced by the addition of PMNs. An increase in nitrate in the culture supernatant fluids was measured, but no increase in nitrite was detected. Superoxide dismutase attenuated the PMN effect, and xanthine/xanthine oxidase reproduced the effect. No changes in epithelial cell inducible NO synthase protein or mRNA were observed. These data demonstrate that [Formula: see text]released from PMNs can decrease NO by conversion to nitrate and suggest a potential mechanism for modulation of NO levels in vivo.

Dynamics of endogenous ATP7A (Menkes protein) in intestinal epithelial cells: copper-dependent redistribution between two intracellular sites

2007 ◽  
Vol 292 (4) ◽  
pp. G1181-G1194 ◽  
Author(s):  
L. Nyasae ◽  
R. Bustos ◽  
L. Braiterman ◽  
B. Eipper ◽  
A. Hubbard
Keyword(s):  
Cell Surface ◽  
Basolateral Membrane ◽  
Cell Periphery ◽  
Intestinal Epithelial ◽  
Intestinal Epithelia ◽  
Dependent Behavior ◽  
Cell Surface Biotinylation ◽  
Vitro Model

We report for the first time on the copper-dependent behavior of endogenous ATP7A in two types of polarized intestinal epithelia, rat enterocytes in vivo and filter-grown Caco-2 cells, an accepted in vitro model of human small intestine. We used high-resolution, confocal immunofluorescence combined with quantitative cell surface biotinylation and found that the vast majority of endogenous ATP7A was localized intracellularly under all copper conditions. In copper-depleted cells, virtually all of the ATP7A localized to a post-TGN compartment, with <3% of the total protein detectable at the basolateral cell surface. When copper levels were elevated, ATP7A dispersed to the cell periphery in punctae whose pattern did not overlap with the steady-state distributions of post-Golgi, endosomal, or basolateral membrane markers; only ∼8–10% of the recovered ATP7A was detected at the basolateral cell surface. These results raise several questions regarding prevailing models of ATP7A dynamics and the mechanism of copper efflux.

Three-dimensional Growth of Renal Epithelial Cells in Vitro: A Tool in Toxicity Testing

1993 ◽  
Vol 21 (2) ◽  
pp. 191-195 ◽  
Author(s):  
Knut-Jan Andersen ◽  
Erik Ilsø Christensen ◽  
Hogne Vik
Keyword(s):  
Epithelial Cells ◽  
Three Dimensional ◽  
Toxicity Testing ◽  
Renal Tissue ◽  
Epithelial Cell Line ◽  
Multicellular Spheroids ◽  
Renal Epithelial Cell ◽  
Renal Epithelial Cells

The tissue culture of multicellular spheroids from the renal epithelial cell line LLC-PK1 (proximal tubule) is described. This represents a biological system of intermediate complexity between renal tissue in vivo and simple monolayer cultures. The multicellular structures, which show many similarities to kidney tubules in vivo, including a vectorial water transport, should prove useful for studying the potential nephrotoxicity of drugs and chemicals in vitro. In addition, the propagation of renal epithelial cells as multicellular spheroids in serum-free culture may provide information on the release of specific biological parameters, which may be suppressed or masked in serum-supplemented media.

scholarly journals Villin Severing Activity Enhances Actin-based Motility In Vivo

2007 ◽  
Vol 18 (3) ◽  
pp. 827-838 ◽  
Author(s):  
Céline Revenu ◽  
Matthieu Courtois ◽  
Alphée Michelot ◽  
Cécile Sykes ◽  
Daniel Louvard ◽  
...  
Keyword(s):  
Shigella Flexneri ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Loss Of Function ◽  
Mesenchymal Transition ◽  
Capping Protein ◽  
Function Strategy ◽  
In Cells

Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition.

scholarly journals Porcine Milk-Derived Small Extracellular Vesicles Promote Intestinal Immunoglobulin Production through pIgR

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1522
Author(s):  
Bin Zeng ◽  
Hailong Wang ◽  
Junyi Luo ◽  
Meiying Xie ◽  
Zhengjiang Zhao ◽  
...  
Keyword(s):  
Small Intestine ◽  
Extracellular Vesicles ◽  
Immunoglobulin A ◽  
Luciferase Reporter ◽  
Epithelial Cell Line ◽  
Intestinal Immunity ◽  
In Vivo Experiments ◽  
Porcine Milk

Secretory immunoglobulin A (SIgA) plays an important role in gut acquired immunity and mucosal homeostasis. Breast milk is the irreplaceable nutritional source for mammals after birth. Current studies have shown the potential functional role of milk-derived small extracellular vesicles (sEVs) and their RNAs cargo in intestinal health and immune regulation. However, there is a lack of studies to demonstrate how milk-derived sEVs affect intestinal immunity in recipient. In this study, through in vivo experiments, we found that porcine milk small extracellular vesicles (PM-sEVs) promoted intestinal SIgA levels, and increased the expression levels of polymeric immunoglobulin receptor (pIgR) both in mice and piglet. We examined the mechanism of how PM-sEVs increased the expression level of pIgR in vitro by using a porcine small intestine epithelial cell line (IPEC-J2). Through bioinformatics analysis, dual-luciferase reporter assays, and overexpression or knockdown of the corresponding non-coding RNAs, we identified circ-XPO4 in PM-sEVs as a crucial circRNA, which leads to the expression of pIgR via the suppression of miR-221-5p in intestinal cells. Importantly, we also observed that oral administration of PM-sEVs increased the level of circ-XPO4 and decreased the level of miR-221-5p in small intestine of piglets, indicating that circRNAs in milk-derived sEVs act as sponge for miRNAs in recipients. This study, for the first time, reveals that PM-sEVs have a capacity to stimulate intestinal SIgA production by delivering circRNAs to receptors and sponging the recipient’s original miRNAs, and also provides valuable data for insight into the role and mechanism of animal milk sEVs in intestinal immunity.

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