Diversity of tight junctions (TJs) between gastrointestinal epithelial cells and their function in maintaining the mucosal barrier

The gastrointestinal epithelium, which is covered by a single layer of epithelial cells, including enterocytes, intraepithelial lymphocytes, goblet cells, microfold cells, and dendritic cells, serves as a protective barrier separating luminal contents from the underlying tissue compartments. The epithelium plays an important role in the first line of host defense against a variety of pathogens, as well as maintaining the homeostasis in gastrointestinal tract. All these epithelial cells express junction complex proteins and form cell junctions such as adherens and TJs, although the TJs have small differences among different epithelial cells. The TJs, located most apically on the lateral membrane, are required for the proper formation of epithelial cell polarity as well as sustaining of the mucosal barrier. Furthermore, TJs are the key cell junctions modulating the paracellular pathway. Understanding the diversity of the TJs between intestinal epithelial cells and their different roles in defending pathogens' invasion and modifying the paracellular pathway are attractive to exploration.     

Cryptosporidium parvum disrupts intestinal epithelial barrier function via altering expression of key tight junction and adherens junction proteins

Infection with the protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a widespread diarrhoeal disease. Impaired intestinal epithelial barrier function and increased permeability are most commonly associated with diarrhoeal diseases caused by enteric infections. However, studies on barrier disruption and underlying mechanisms in cryptosporidiosis are extremely limited. Epithelial tight junctions (TJs) and adherens junctions (AJs) are important in maintaining barrier integrity. Therefore, we examined the effects of CP infection on paracellular permeability and on the expression of the major TJ and AJ proteins utilising in vitro, ex vivo, and in vivo models. CP infection (0.5 × 10⁶ oocysts/well in Transwell inserts, 24 hr) increased paracellular permeability (FITC‐dextran flux) in Caco‐2 cell monolayers and substantially decreased the protein levels of occludin, claudin 4, and E‐cadherin. Claudin 3, zonula occludens‐1 (ZO1) and α‐catenin were also significantly decreased, whereas claudins 1 and 2 and β‐catenin were not altered. Substantial downregulation of occludin, claudin 4, and E‐cadherin was also observed in response to CP infection ex vivo in mouse enteroid‐derived monolayers and in vivo in the ileal and jejunal mocosa of C57BL/6 mice. The mRNA levels of these proteins were also significantly decreased in CP‐infected mouse ileum and jejunum but were unaltered in Caco‐2 cells. Further, bafilomycin‐A, an inhibitor of lysosomal proton pump, partially abrogated CP effects on occludin expression in Caco‐2 cells, suggesting a potential role of posttranslational mechanisms, such as induction of protein degradation pathways, in mediating the effects of the parasite. Our studies suggest that disruption of barrier function via downregulation of specific key components of TJ and AJ could be a major mechanism underlying CP infection‐induced diarrhoea.     

Autophagy Controls an Intrinsic Host Defense to Bacteria by Promoting Epithelial Cell Survival: A Murine Model

Cell death is a critical host response to regulate the fate of bacterial infections, innate immune responses, and ultimately, disease outcome. Shigella spp. invade and colonize gut epithelium in human and nonhuman primates but adult mice are naturally resistant to intra-gastric Shigella infection. In this study, however, we found Shigella could invade the terminal ileum of the mouse small intestine by 1 hour after infection and be rapidly cleared within 24 h. These early phase events occurred shortly after oral infection resulting in epithelial shedding, degranulation of Paneth cells, and cell death in the intestine. During this process, autophagy proceeded without any signs of inflammation. In contrast, blocking autophagy in epithelial cells enhanced host cell death, leading to tissue destruction and to inflammation, suggesting that autophagic flow relieves cellular stress associated with host cell death and inflammation. Herein we propose a new concept of “epithelial barrier turnover” as a general intrinsic host defense mechanism that increases survival of host cells and inhibits inflammation against enteric bacterial infections, which is regulated by autophagy.     

In vitro adhesion and invasion inhibition of <Emphasis Type="Italic">Shigella dysenteriae</Emphasis>, <Emphasis Type="Italic">Shigella flexneri</Emphasis> and <Emphasis Type="Italic">Shigella sonnei</Emphasis> clinical strains by human milk proteins

Background  

Shigella is the etiological agent of shigellosis, a disease responsible for more than 500,000 deaths of children per year, in developing countries. These pathogens colonize the intestinal colon, invade, spreading to the other enterocytes. Breastfeeding plays a very important role in protecting infants from intestinal infections. Amongst milk compounds, glycosylated proteins prevent the adhesion of many enteropathogens in vitro. The aim of this work was to determine the effect of human milk proteins on the colonization potential of Shigella dysenteriae, S. flexneri and S. sonnei. To fulfill this purpose, pooled milk samples from five donors, were fractionated by gel filtration and affinity chromatography. Using tissue culture, the milk fractions obtained were tested in Shigella adhesion and invasion assays.     

Acetate,a metabolic product of Heligmosomoides polygyrus,facilitates intestinal epithelial barrier breakdown in a FFAR2-dependent manner

Approximately 2 billion people worldwide and a significant part of the domestic livestock are infected with soil-transmitted helminths, of which many establish chronic infections causing substantial economic and welfare burdens. Beside intensive research on helminth-triggered mucosal and systemic immune responses, the local mechanism that enables infective larvae to cross the intestinal epithelial barrier and invade mucosal tissue remains poorly addressed. Here, we show that Heligmosomoides polygyrus infective L3s secrete acetate and that acetate potentially facilitates paracellular epithelial tissue invasion by changed epithelial tight junction claudin expression. In vitro, impedance-based real-time epithelial cell line barrier measurements together with ex vivo functional permeability assays in intestinal organoid cultures revealed that acetate decreased intercellular barrier function via the G-protein coupled free fatty acid receptor 2 (FFAR2, GPR43). In vivo validation experiments in FFAR2^?/? mice showed lower H. polygyrus burdens, whereas oral acetate-treated C57BL/6 wild type mice showed higher burdens. These data suggest that locally secreted acetate – as a metabolic product of the energy metabolism of H. polygyrus L3s – provides a significant advantage to the parasite in crossing the intestinal epithelial barrier and invading mucosal tissues. This is the first and a rate-limiting step for helminths to establish chronic infections in their hosts and if modulated could have profound consequences for their life cycle.     

Shigella host: Pathogen interactions: Keeping bacteria in the loop

Shigella spp. are Gram‐negative enteric pathogens and the leading cause of bacterial dysentery worldwide. Since the discovery more than three decades ago that the large virulence plasmid of Shigella is essential for pathogenesis, our understanding of how the bacterium orchestrates inflammation and tissue destruction at the mucosal surface has been informed by studies employing the rabbit ileal loop model. Here, we outline how Phillippe Sansonetti, together with his co‐workers and collaborators, exploited this model to provide a holistic view of how Shigella survives in the intestinal tract, traverses the intestinal epithelial barrier, and manipulates the host immune system to cause disease.     

Differential Infection of Polarized Epithelial Cell Lines by Sialic Acid-Dependent and Sialic Acid-Independent Rotavirus Strains

Infection of epithelial cells by some animal rotaviruses, but not human or most animal rotaviruses, requires the presence of N-acetylneuraminic (sialic) acid (SA) on the cell surface for efficient infectivity. To further understand how rotaviruses enter susceptible cells, six different polarized epithelial cell lines, grown on permeable filter membrane supports containing 0.4-μm pores, were infected apically or basolaterally with SA-independent or SA-dependent rotaviruses. SA-independent rotaviruses applied apically or basolaterally were capable of efficiently infecting both sides of the epithelium of all six polarized cell lines tested, while SA-dependent rotaviruses only infected efficiently through the apical surface of five of the polarized cell lines tested. Regardless of the route of virus entry, SA-dependent and SA-independent rotaviruses were released almost exclusively from the apical domain of the plasma membrane of polarized cells before monolayer disruption or cell lysis. The transepithelial electrical resistance (TER) of cells decreased at the same time, irrespective of whether infection with SA-independent rotaviruses occurred apically or basolaterally. The TER of cells infected apically with SA-dependent rotaviruses decreased earlier than that of cells infected basolaterally. Rotavirus infection decreased TER before the appearance of cytopathic effect and cell death and resulted in an increase in the paracellular permeability to [³H]inulin as a function of loss of TER. The presence of SA residues on either the apical or basolateral side was determined using a Texas Red-conjugated lectin, wheat germ agglutinin (WGA), which binds SA residues. WGA bound exclusively to SA residues on the apical surface of the cells, confirming the requirement for SA residues on the apical cell membrane for efficient infectivity of SA-dependent rotaviruses. These results indicate that the rotavirus SA-independent cellular receptor is present on both sides of the epithelium, but SA-dependent and SA-independent rotavirus strains infect polarized epithelial cells by different mechanisms, which may be relevant for pathogenesis and selection of vaccine strains. Finally, rotavirus-induced alterations of the epithelial barrier and paracellular permeability suggest that common mechanisms of pathogenesis may exist between viral and bacterial pathogens of the intestinal tract.     

Gadd45�� activity is the principal effector of Shigella mitochondria-dependent epithelial cell death in vitro and ex vivo

Modulation of death is a pathogen strategy to establish residence and promote survival in host cells and tissues. Shigella spp. are human pathogens that invade colonic mucosa, where they provoke lesions caused by their ability to manipulate the host cell responses. Shigella spp. induce various types of cell death in different cell populations. However, they are equally able to protect host cells from death. Here, we have investigated on the molecular mechanisms and cell effectors governing the balance between survival and death in epithelial cells infected with Shigella. To explore these aspects, we have exploited both, the HeLa cell invasion assay and a novel ex vivo human colon organ culture model of infection that mimics natural conditions of shigellosis. Our results definitely show that Shigella induces a rapid intrinsic apoptosis of infected cells, via mitochondrial depolarization and the ensuing caspase-9 activation. Moreover, for the first time we identify the eukaryotic stress-response factor growth arrest and DNA damage 45α as a key player in the induction of the apoptotic process elicited by Shigella in epithelial cells, revealing an unexplored role of this molecule in the course of infections sustained by invasive pathogens.     

Cell wall fraction of Enterococcus hirae ameliorates TNF-alpha-induced barrier impairment in the human epithelial tight junction

Aims: The evaluation of the effects of Enterococcus hirae, an intestinal bacterium in the adjacent mucosa (mucosal bacterium), on tumour necrosis factor‐alpha (TNF‐α)‐induced barrier impairment in human epithelial Caco‐2 cells. Methods and Results: The filter‐grown Caco‐2 monolayers were used as an intestinal epithelial model system. In Caco‐2 cells, heat‐killed E. hirae ATCC 9790^T suppressed the TNF‐α‐induced barrier impairment and increase in interleukin‐8 (IL‐8) secretion, but lipase‐ and mutanolysin‐treated E. hirae ATCC 9790^T did not have these effects. It was demonstrated that lipoteichoic acid (LTA) from E. hirae ATCC 9790^T is responsible for Caco‐2 cells’ recovery from TNF‐α‐induced impairments. In addition, Caco‐2 cells had the same response to Toll‐like receptor 2 (TLR2) ligand, Pam₃Cys‐Ser‐(Lys)₄ as they did to LTA. Increased expression of zonula occludens‐1 was observed by the addition of E. hirae ATCC 9790^T to TNF‐α‐treated Caco‐2 cells, and decreased expression of myosin light chain kinase was observed by the addition of LTA and Pam₃Cys‐Ser‐(Lys)₄; this, in turn, led to barrier enforcement. Conclusions: Enterococcus hirae ATCC 9790^T cell wall fractions, such as LTA, protect against intestinal impairment by regulation of epithelial tight junction via TLR2 signalling. Significance and Impact of the Study: Enterococcus hirae could be useful in the treatment of inflammatory bowel disease, as well as other intestinal disorders.     

Phosphodiesterase inhibition attenuates alterations to the tight junction proteins occludin and ZO-1 in immunostimulated Caco-2 intestinal monolayers

AimsUnder normal conditions, the intestinal mucosa acts as a local barrier to prevent the influx of luminal contents. The intestinal epithelial tight junction is comprised of several membrane associated proteins, including zonula occludens-1 (ZO-1) and occludin. Disruption of this barrier can lead to the production of pro-inflammatory mediators and ultimately multiple organ failure. We have previously shown that Pentoxifylline (PTX) decreases histologic gut injury and pro-inflammatory mediator synthesis. We hypothesize that PTX prevents the breakdown of ZO-1 and occludin in an in vitro model of immunostimulated intestinal cell monolayers.Main methodsCaco-2 human enterocytes were grown as confluent monolayers and incubated under control conditions, or with PTX (2 mM), Cytomix (TNF-α, IFN-γ, IL-1), or Cytomix + PTX for 24 h. Occludin and ZO-1 protein levels were analyzed by Western blot. Confocal microscopy was used to assess the cytoplasmic localization of ZO-1 and occludin.Key findingsCytomix stimulation of Caco-2 cells resulted in a 50% decrease in both occludin and ZO-1 protein. Treatment with Cytomix + PTX restored both occludin and ZO-1 protein to control levels. Confocal microscopy images show that Cytomix caused an irregular, undulating appearance of ZO-1 and occludin at the cell junctions. Treatment with PTX prevented the Cytomix-induced changes in ZO-1 and occludin localization.SignificanceTreatment with PTX decreases the pro-inflammatory cytokine induced changes in the intestinal tight junction proteins occludin and ZO-1. Pentoxifylline may be a useful adjunct in the treatment of sepsis and shock by attenuating intestinal barrier breakdown.     

Characterization of enteroinvasive Escherichia coli and Shigella strains by RAPD analysis

Genetic variation of 33 enteroinvasive Escherichia coli (EIEC), 12 non-EIEC and 39 Shigella strains (representing the 4 species of this genus) was analyzed using the random amplified polymorphic DNA (RAPD) technique. Reproducible polymorphisms were generated and the combined data allowed us to construct a dendrogram using Jaccard's distance. Two main groups were obtained: one for Shigella and the other for EIEC and non-EIEC strains. The first group contained four clusters, one for each Shigella species. The second group contained one cluster for EIEC and another for non-EIEC strains. The main clusters encompassed many small clusters corresponding to different serotypes. It was possible to characterize each one of the 84 strains under study as well as the boundaries among Shigella species and between this genus and EIEC strains.     

Differential effects of TNF (TNFSF2) and IFN-γ on intestinal epithelial cell morphogenesis and barrier function in three-dimensional culture

Background

The cytokines TNF (TNFSF2) and IFNγ are important mediators of inflammatory bowel diseases and contribute to enhanced intestinal epithelial permeability by stimulating apoptosis and/or disrupting tight junctions. Apoptosis and tight junctions are also important for epithelial tissue morphogenesis, but the effect of TNF and IFNγ on the process of intestinal epithelial morphogenesis is unknown.

Methods/Principal Findings

We have employed a three-dimensional cell culture system, reproducing in vivo-like multicellular organization of intestinal epithelial cells, to study the effect of TNF on intestinal epithelial morphogenesis and permeability. We show that human intestinal epithelial cells in three-dimensional culture assembled into luminal spheres consisting of a single layer of cells with structural, internal, and planar cell polarity. Exposure of preformed luminal spheres to TNF or IFNγ enhanced paracellular permeability, but via distinctive mechanisms. Thus, while both TNF and IFNγ, albeit in a distinguishable manner, induced the displacement of selected tight junction proteins, only TNF increased paracellular permeability via caspase-driven apoptosis and cell shedding. Infliximab and adalumimab inhibited these effects of TNF. Moreover, we demonstrate that TNF via its stimulatory effect on apoptosis fundamentally alters the process of intestinal epithelial morphogenesis, which contributes to the de novo generation of intestinal epithelial monolayers with increased permeability. Also IFNγ contributes to the de novo formation of monolayers with increased permeability, but in a manner that does not involve apoptosis.

Conclusions

Our study provides an optimized 3D model system for the integrated analysis of (real-time) intestinal epithelial paracellular permeability and morphogenesis, and reveals apoptosis as a pivotal mechanism underlying the enhanced permeability and altered morphogenesis in response to TNF, but not IFNγ.     

Staphylococcus aureus protein A mediates invasion across airway epithelial cells through activation of RhoA GTPase signaling and proteolytic activity

Staphyococcus aureus and especially the epidemic methicillin-resistant S. aureus strains cause severe necrotizing pneumonia. The mechanisms whereby these organisms invade across the mucosal epithelial barrier to initiate invasive infection are not well understood. Protein A (SpA), a highly conserved and abundant surface protein of S. aureus, activates TNF receptor 1 and EGF receptor (EGFR) signaling cascades that can perturb the cytoskeleton. We demonstrate that wild-type S. aureus, but not spa mutants, invade across polarized airway epithelial cell monolayers via the paracellular junctions. SpA stimulated a RhoA/ROCK/MLC cascade, resulting in the contraction of the cytoskeleton. SpA(+) but not SpA(-) mutants stimulated activation of EGFR and along with subsequent calpain activity cleaved the membrane-spanning junctional proteins occludin and E-cadherin, facilitating staphylococcal transmigration through the cell-cell junctions. Treatment of polarized human airway epithelial monolayers with inhibitors of ROCK, EGFR, MAPKs, or calpain prevented staphylococcal penetration through the monolayers. In vivo, blocking calpain activity impeded bacterial invasion into the lung parenchyma. Thus, S. aureus exploits multiple receptors available on the airway mucosal surface to facilitate invasion across epithelial barriers.     

Defensins play a crucial role in protecting mice against oral Shigella flexneri infection

An earlier study revealed that 4-day-old mice, but not older mice, were infected with invasive Shigella strains. Here we attempted to determine the underlying mechanism that induces inflammation in the intestines of neonate mice after oral Shigella infection. Wild-type BALB/c mice of different ages (7, 14, and 35 days old) were orally administered GFP-expressing Shigella flexneri 5a M90T strain (5 × 10⁹ CFU) and analyzed for colonization 6 h following infection. We found that Shigella localized in the epithelium, lamina propria, and crypt regions of the small intestines of 7-day-old BALB/c mice. Microarray analysis revealed that expression levels of cryptdin and various types of cryptdin-related mRNA (e.g., cryptrs-2, -5, -7, -12 and lysozyme) in the small intestines were significantly lower in 7-day-old than in older mice regardless of Shigella infection status. Interestingly, matrix metalloprotease-7 (matrilysin)-deficient (MAT^−/−) mice of B6 background had more colonies and more severe symptoms of inflammation in the intestines than did wild-type B6 mice after oral Shigella challenge. This suggests that cryptdin-related antimicrobial molecules are indispensable for efficient protection against oral Shigella infection.     

Tight junction proteins claudin-2 and -12 are critical for vitamin D-dependent Ca2+ absorption between enterocytes

Ca²⁺ is absorbed across intestinal epithelial monolayers via transcellular and paracellular pathways, and an active form of vitamin D₃, 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃], is known to promote intestinal Ca²⁺ absorption. However, the molecules driving the paracellular Ca²⁺ absorption and its vitamin D dependency remain obscure. Because the tight junction proteins claudins are suggested to form paracellular channels for selective ions between neighboring cells, we hypothesized that specific intestinal claudins might facilitate paracellular Ca²⁺ transport and that expression of these claudins could be induced by 1α,25(OH)₂D₃. Herein, we show, by using RNA interference and overexpression strategies, that claudin-2 and claudin-12 contribute to Ca²⁺ absorption in intestinal epithelial cells. We also provide evidence showing that expression of claudins-2 and -12 is up-regulated in enterocytes in vitro and in vivo by 1α,25(OH)₂D₃ through the vitamin D receptor. These findings strongly suggest that claudin-2- and/or claudin-12-based tight junctions form paracellular Ca²⁺ channels in intestinal epithelia, and they highlight a novel mechanism behind vitamin D-dependent calcium homeostasis.     

Cloning and characterization of a new intestinal inflammation-associated colonic epithelial Ste20-related protein kinase isoform

Intestinal epithelial cells respond to inflammatory extracellular stimuli by activating mitogen activated protein kinase (MAPK) signaling, which mediates numerous pathophysiological effects, including intestinal inflammation. Here, we show that a novel isoform of SPS1-related proline alanine-rich kinase (SPAK/STE20) is involved in this inflammatory signaling cascade. We cloned and characterized a SPAK isoform from inflamed colon tissue, and found that this SPAK isoform lacked the characteristic PAPA box and alphaF loop found in SPAK. Based on genomic sequence analysis the lack of PAPA box and alphaF loop in colonic SPAK isoform was the result of specific splicing that affect exon 1 and exon 7 of the SPAK gene. The SPAK isoform was found in inflamed and non-inflamed colon tissues as well as Caco2-BBE cells, but not in other tissues, such as liver, spleen, brain, prostate and kidney. In vitro analyses demonstrated that the SPAK isoform possessed serine/threonine kinase activity, which could be abolished by a substitution of isoleucine for the lysine at position 34 in the ATP-binding site of the catalytic domain. Treatment of Caco2-BBE cells with the pro-inflammatory cytokine, interferon γ, induced expression of the SPAK isoform. Over-expression of the SPAK isoform in Caco2-BBE cells led to nuclear translocation of an N-terminal fragment of the SPAK isoform, as well as activation of p38 MAP kinase signaling cascades and increased intestinal barrier permeability. These findings collectively suggest that pro-inflammatory cytokine signaling may induce expression of this novel SPAK isoform in intestinal epithelia, triggering the signaling cascades that govern intestinal inflammation.     

MicroRNA‐21 regulates intestinal epithelial tight junction permeability

Increased tight junction (TJ) barrier permeability, induced by tumour necrosis factor (TNF)‐α, may lead to the defects in TJ barrier and subsequent development of inflammation. Recent evidence suggests that miR‐21 is implicated in inflammatory diseases. However, the physiological role of miR‐21 in intestinal permeability remains elusive. This study aimed to explore the role of miR‐21 in intestinal epithelial tight junction permeability. The filter‐grown Caco‐2 monolayers model system was established to mimic intestinal barrier defect. The tight junction proteins were detected by immunofluorescence and western blot analysis. The expression of miR‐21 was assessed by real‐time polymerase chain reaction (PCR). We found that the expression of miR‐21 was increased significantly in TNF‐α induced intestinal TJ barrier defect model. miR‐21 overexpression significantly enhanced while miR‐21 knockdown significantly decreased intestinal permeability. In addition, miR‐21 overexpression significantly increased while miR‐21 knockdown significantly decreased the levels of interleukin‐6, interleukin‐8 and prostaglandin E2 in cell culture medium. Furthermore, miR‐21 positively regulated Akt phosphorylation and negatively regulated Phosphatase and tensin homolog (PTEN) expression in Caco‐2 cells. Our results suggest that miR‐21 may regulate intestinal epithelial tight junction permeability through PTEN/PI3K/Akt signalling pathway. This promotes the feasibility of targeting miR‐21 in the clinical to preserve the intestinal barrier. Copyright © 2015 John Wiley & Sons, Ltd.