Basolateral Infection Research Articles

Representative measles virus infection requires appropriate airway epithelia culture conditions.

For many years, measles virus (MeV) was assumed to first enter the host via the apical surface of airway epithelial cells and subsequently spread systemically. We and others reported that MeV has an overwhelming preference for entry at the basolateral surface of airway epithelial cells, which led to a fundamental new understanding of how MeV enters a human host. This unexpected observation using well-differentiated primary cultures of airway epithelia from human donors contradicted previous studies using immortalized cultured cells. Here, we show that appropriate differentiation and cell morphology of primary human airway epithelial cells are critical to recapitulate MeV infection patterns and pathogenesis of the in vivo airways. By simply culturing primary cells in media containing serum or passaging primary cultures, erroneous results quickly emerge. These results have broad implications for data interpretation related to respiratory virus infection, spread, and release from human airway epithelial cells.

Infection of porcine enteroids and 2D differentiated intestinal epithelial cells with rotavirus A to study cell tropism and polarized immune response

ABSTRACT Intestinal epithelial cell interactions with enteric pathogens have been incompletely elucidated owing to the lack of model systems that recapitulate the cellular diversity, architecture and functionality of the intestine. To analyze rotavirus (RV) infection and the subsequent innate immune response, we established cultures of differentiated porcine intestinal epithelial cells in three different variations: basolateral-out enteroids, apical-out enteroids and two-dimensional (2D) filter-grown intestinal epithelial cells. Application of specific antibodies for fluorescent staining indicated that enteroids and enteroid-derived cell cultures contain multiple intestinal epithelial cell types. Infection studies indicated that both apical-out enteroids and 2D intestinal epithelial cells are susceptible to porcine RV infection. However, 2D intestinal epithelial cells are more useful for a detailed characterization and comparison of apical and basolateral infection than apical-out enteroids. Virus-induced apoptosis was observed in apical-out enteroids at 24 h post infection but not at earlier time points after infection. RV infected not only enterocytes but also goblet cells and Paneth cells in apical-out enteroids and 2D intestinal epithelial cells. Interestingly, despite the lack of significant differences in the efficiency of infection after apical and basolateral infection of 2D intestinal epithelial cells, stronger innate immune and inflammatory responses were observed after basolateral infection as compared to infection via the apical route. Therefore, apical-out enteroids and 2D intestinal epithelial cells provide useful primary cell culture models that can be extended to analyze invasion and replication strategies of agents implicated in enteric diseases or to study immune and inflammatory responses of the host induced by enteric pathogens.

Asymmetric profiles of infection and innate immunological responses in human iPS cell-derived small intestinal epithelial-like cell monolayers following infection with mammalian reovirus

The intestinal mucosa plays an important role as an immune barrier due to its continual exposure to invading pathogens, including viruses. It is thus highly important to evaluate virus infection profiles in the intestinal mucosa for prevention of virus infection and development of antivirus medicines; however, only a few enterocyte lines are available as in vitro intestinal models for the evaluation of virus infection. In this study, we evaluated profiles of infection and innate immune responses following infection with a mammalian orthoreovirus (hereafter reovirus), which has often been used as a tractable model for studies of viral pathogenesis, in human iPS cell-derived small intestinal epithelial-like cell (hiPS-SIEC) monolayers and cells of a human colon adenocarcinoma cell line, Caco-2. The levels of reovirus infection were similar between hiPS-SIEC and Caco-2 cell monolayers, which are often used as an intestinal model, after apical and basolateral infection. In hiPS-SIEC monolayers, more efficient replication of the virus genome was observed following basolateral infection than apical infection, while apical infection resulted in higher levels of virus protein expression and progeny virus production than basolateral infection. Reovirus significantly induced innate immune responses, including expression of type I and III interferons (IFNs), in hiPS-SIEC monolayers more efficiently than Caco-2 cells. Higher levels of type I and III interferon (IFN) expression were found in hiPS-SIEC monolayers following apical infection than basolateral infection. These results suggested that hiPS-SIECs are a promising in vitro model for the evaluation of virus infection.

Infection of polarized bovine respiratory epithelial cells by bovine viral diarrhea virus (BVDV)

ABSTRACT Bovine viral diarrhea virus (BVDV) is affecting cattle populations all over the world causing acute disease, immunosuppressive effects, respiratory diseases, gastrointestinal, and reproductive failure in cattle. The virus is taken up via the oronasal route and infection of epithelial and immune cells contributes to the dissemination of the virus throughout the body. However, it is not known how the virus gets across the barrier of epithelial cells encountered in the airways. Here, we analyzed the infection of polarized primary bovine airway epithelial cells (BAEC). Infection of BAEC by a non-cytopathogenic BVDV was possible via both the apical and the basolateral plasma membrane, but the infection was most efficient when the virus was applied to the basolateral plasma membrane. Irrespective of the site of infection, BVDV was efficiently released to the apical site, while only minor amounts of virus were detected in the basal medium. This indicates that the respiratory epithelium can release large amounts of BVDV to the environment and susceptible animals via respiratory fluids and aerosols, but BVDV cannot cross the airway epithelial cells to infect subepithelial cells and establish systemic infection. Further experiments showed that the receptor, bovine CD46, for BVDV is expressed predominantly on the apical membrane domain of the polarized epithelial cells. In a CD46 blocking experiment, the addition of an antibody directed against CD46 almost completely inhibited apical infection, whereas basolateral infection was not affected. While CD46 serves as a receptor for apical infection of BAEC by BVDV, the receptor for basolateral infection remains to be elucidated.

Endothelial cells are not productively infected by SARS-CoV-2.

ObjectivesThrombotic and microvascular complications are frequently seen in deceased COVID‐19 patients. However, whether this is caused by direct viral infection of the endothelium or inflammation‐induced endothelial activation remains highly contentious.MethodsHere, we use patient autopsy samples, primary human endothelial cells and an in vitro model of the pulmonary epithelial–endothelial cell barrier.ResultsWe show that primary human endothelial cells express very low levels of the SARS‐CoV‐2 receptor ACE2 and the protease TMPRSS2, which blocks their capacity for productive viral infection, and limits their capacity to produce infectious virus. Accordingly, endothelial cells can only be infected when they overexpress ACE2, or are exposed to very high concentrations of SARS‐CoV‐2. We also show that SARS‐CoV‐2 does not infect endothelial cells in 3D vessels under flow conditions. We further demonstrate that in a co‐culture model endothelial cells are not infected with SARS‐CoV‐2. Endothelial cells do however sense and respond to infection in the adjacent epithelial cells, increasing ICAM‐1 expression and releasing pro‐inflammatory cytokines.ConclusionsTaken together, these data suggest that in vivo, endothelial cells are unlikely to be infected with SARS‐CoV‐2 and that infection may only occur if the adjacent pulmonary epithelium is denuded (basolateral infection) or a high viral load is present in the blood (apical infection). In such a scenario, whilst SARS‐CoV‐2 infection of the endothelium can occur, it does not contribute to viral amplification. However, endothelial cells may still play a key role in SARS‐CoV‐2 pathogenesis by sensing adjacent infection and mounting a pro‐inflammatory response to SARS‐CoV‐2.

Polar Infection of Echovirus-30 Causes Differential Barrier Affection and Gene Regulation at the Blood-Cerebrospinal Fluid Barrier.

Echovirus-30 (E-30) is responsible for the extensive global outbreaks of meningitis in children. To gain access to the central nervous system, E-30 first has to cross the epithelial blood–cerebrospinal fluid barrier. Several meningitis causing bacteria preferentially infect human choroid plexus papilloma (HIBCPP) cells in a polar fashion from the basolateral cell side. Here, we investigated the polar infection of HIBCPP cells with E-30. Both apical and basolateral infections caused a significant decrease in the transepithelial electrical resistance of HIBCPP cells. However, to reach the same impact on the barrier properties, the multiplicity of infection of the apical side had to be higher than that of the basolateral infection. Furthermore, the number of infected cells at respective time-points after basolateral infection was significantly higher compared to apical infection. Cytotoxic effects of E-30 on HIBCPP cells during basolateral infection were observed following prolonged infection and appeared more drastically compared to the apical infection. Gene expression profiles determined by massive analysis of cDNA ends revealed distinct regulation of specific genes depending on the side of HIBCPP cells’ infection. Altogether, our data highlights the polar effects of E-30 infection in a human in vitro model of the blood–cerebrospinal fluid barrier leading to central nervous system inflammation.

Zinc ameliorates intestinal barrier dysfunctions in shigellosis by reinstating claudin-2 and -4 on the membranes.

Whether zinc (Zn2+) regulates barrier functions by modulating tight-junction (TJ) proteins when pathogens such as Shigella alter epithelial permeability is still unresolved. We investigated the potential benefits of Zn2+ in restoring impaired barrier function in vivo in Shigella-infected mouse tissue and in vitro in T84 cell monolayers. Basolateral Shigella infection triggered a time-dependent decrease in transepithelial resistance followed by an increase in paracellular permeability of FITC-labeled dextran and altered ion selectivity. This led to ion and water loss into the intestinal lumen. Immunofluorescence studies revealed redistribution of claudin-2 and -4 to an intracellular location and accumulation of these proteins in the cytoplasm following infection. Zn2+ ameliorated this perturbed barrier by redistribution of claudin-2 and -4 back to the plasma membrane and by modulating the phosphorylation state of TJ proteins t hough extracellular signal-regulated kinase (ERK)1/2 dependency. Zn2+ prevents elevation of IL-6 and IL-8. Mice challenged with Shigella showed that oral Zn2+supplementation diminished diverse pathophysiological symptoms of shigellosis. Claudin-2 and -4 were susceptible to Shigella infection, resulting in altered barrier function and increased levels of IL-6 and IL-8. Zn2+ supplementation ameliorated this barrier dysfunction, and the inflammatory response involving ERK-mediated change of phosphorylation status for claudin-2 and -4. Thus, Zn2+ may have potential therapeutic value in inflammatory diarrhea and shigellosis. NEW & NOTEWORTHY Our study addresses whether Zn2+ could be an alternative strategy to reduce Shigella-induced inflammatory response and epithelial barrier dysfunction. We have defined a mechanism in terms of intracellular signaling pathways and tight-junction protein expression by Zn2+. Claudin-2 and -4 are susceptible to Shigella infection, whereas in the presence of Zn2+ they are resistant to infection-related barrier dysfunction involving ERK-mediated change of phosphorylation status of claudins.

Heparan sulfate is an important mediator of Ebola virus infection in polarized epithelial cells

BackgroundCurrently, no FDA-approved vaccines or treatments are available for Ebola virus disease (EVD), and therapy remains largely supportive. Ebola virus (EBOV) has broad tissue tropism and can infect a variety of cells including epithelial cells. Epithelial cells differ from most other cell types by their polarized phenotype and barrier function. In polarized cells, the apical and basolateral membrane domains are demarcated by tight junctions, and specialized sorting machinery, which results in a difference in composition between the two membrane domains. These specialized sorting functions can have important consequences for viral infections. Differential localization of a viral receptor can restrict virus entry to a particular membrane while polarized sorting can lead to a vectorial virus release. The present study investigated the impact of cell polarity on EBOV infection.MethodsCharacteristics of EBOV infection in polarized cells were evaluated in the polarized Caco-2 model grown on semipermeable transwells. Transepithelial resistance (TEER), which is a function of tight junctions, was used to assess epithelial cell polarization. EBOV infection was assessed with immunofluorescence microscopy and qPCR. Statistical significance was calculated using one-way ANOVA and significance was set at p < 0.05.ResultsOur data indicate that EBOV preferentially infects cells from the basolateral route, and this preference may be influenced by the resistance across the Caco-2 monolayer. Infection occurs without changes in cellular permeability. Further, our data show that basolateral infection bias may be dependent on polarized distribution of heparan sulfate, a known viral attachment factor. Treatment with iota-carrageenan, or heparin lyase, which interrupts viral interaction with cellular heparan sulfate, significantly reduced cell susceptibility to basolateral infection, likely by inhibiting virus attachment.ConclusionsOur results show cell polarity has an impact on EBOV infection. EBOV preferentially infects polarized cells through the basolateral route. Access to heparan sulfate is an important factor during basolateral infection and blocking interaction of cellular heparan sulfate with virus leads to significant inhibition of basolateral infection in the polarized Caco-2 cell model.

Cellular Tight Junctions Prevent Effective Campylobacter jejuni Invasion and Inflammatory Barrier Disruption Promoting Bacterial Invasion from Lateral Membrane in Polarized Intestinal Epithelial Cells.

Campylobacter jejuni invasion is closely related to C. jejuni pathogenicity. The intestinal epithelium contains polarized epithelial cells that form tight junctions (TJs) to provide a physical barrier against bacterial invasion. Previous studies indicated that C. jejuni invasion of non-polarized cells involves several cellular features, including lipid rafts. However, the dynamics of C. jejuni invasion of polarized epithelial cells are not fully understood. Here we investigated the interaction between C. jejuni invasion and TJ formation to characterize the mechanism of C. jejuni invasion in polarized epithelial cells. In contrast to non-polarized epithelial cells, C. jejuni invasion was not affected by depletion of lipid rafts in polarized epithelial cells. However, depletion of lipid rafts significantly decreased C. jejuni invasion in TJ disrupted cells or basolateral infection and repair of cellular TJs suppressed lipid raft-mediated C. jejuni invasion in polarized epithelial cells. In addition, pro-inflammatory cytokine, TNF-α treatment that induce TJ disruption promote C. jejuni invasion and lipid rafts depletion significantly reduced C. jejuni invasion in TNF-α treated cells. These data demonstrated that TJs prevent C. jejuni invasion from the lateral side of epithelial cells, where they play a main part in bacterial invasion and suggest that C. jejuni invasion could be increased in inflammatory condition. Therefore, maintenance of TJs integrity should be considered important in the development of novel therapies for C. jejuni infection.

Interleukin-8, CXCL1, and MicroRNA miR-146a Responses to Probiotic Escherichia coli Nissle 1917 and Enteropathogenic E. coli in Human Intestinal Epithelial T84 and Monocytic THP-1 Cells after Apical or Basolateral Infection.

Bacterium-host interactions in the gut proceed via directly contacted epithelial cells, the host's immune system, and a plethora of bacterial factors. Here we characterized and compared exemplary cytokine and microRNA (miRNA) responses of human epithelial and THP-1 cells toward the prototype enteropathogenic Escherichia coli (EPEC) strain E2348/69 (O127:H6) and the probiotic strain Escherichia coli Nissle 1917 (EcN) (O6:K5:H1). Human T84 and THP-1 cells were used as cell culture-based model systems for epithelial and monocytic cells. Polarized T84 monolayers were infected apically or basolaterally. Bacterial challenges from the basolateral side resulted in more pronounced cytokine and miRNA responses than those observed for apical side infections. Interestingly, the probiotic EcN also caused a pronounced transcriptional increase of proinflammatory CXCL1 and interleukin-8 (IL-8) levels when human T84 epithelial cells were infected from the basolateral side. miR-146a, which is known to regulate adaptor molecules in Toll-like receptor (TLR)/NF-κB signaling, was found to be differentially regulated in THP-1 cells between probiotic and pathogenic bacteria. To assess the roles of flagella and flagellin, we employed several flagellin mutants of EcN. EcN flagellin mutants induced reduced IL-8 as well as CXCL1 responses in T84 cells, suggesting that flagellin is an inducer of this cytokine response. Following infection with an EPEC type 3 secretion system (T3SS) mutant, we observed increased IL-8 and CXCL1 transcription in T84 and THP-1 cells compared to that in wild-type EPEC. This study emphasizes the differential induction of miR-146a by pathogenic and probiotic E. coli strains in epithelial and immune cells as well as a loss of probiotic properties in EcN interacting with cells from the basolateral side.

680. Optimization of rAAV-Mediated Expression for Large Transgenes Using a Synthetic Promoter and Tandem Array Enhancers

rAAV is a widely used viral vector for the human gene therapy, but its inherently small 4.679kb genome is a significant challenge for delivering effective expression of a large gene. Our main goal is to develop rAAV vectors for delivering the cystic fibrosis transmembrane conductance regulator (CFTR) for cystic fibrosis lung disease gene therapy. To express the CFTR gene of 4.43kb coding sequence from a rAAV vector, a short promoter well functioned in airway epithelium is required. In the present study, we screened a set of 100-mer synthetic enhancer elements for their ability to augment CFTR expression from a short 83-bp synthetic promoter. With an empirical approach, screening for their effectiveness in the delivery of reporter gene expression were conducted in step-wise fashion – in plasmids, proviral vectors, and rAAV vectors. Our initial studies in monolayer (non-polarized) cultures of human airway cell lines and primary ferret airway cells revealed that three of these synthetic enhancers (F1, F5, and F10) significantly promoted transcription of a luciferase transgene in the context of plasmid transfection. Analysis in polarized cultures of human and ferret airway epithelia at an air-liquid interface (ALI) in the context of AAV vector infection found that the combination of F5tg83 (183bp) is the most efficient promoter in both ALI cultures, leading to 19.6-fold and 57.5-fold increases reporter expression, respectively, over the enhancer-less counterpart. We also demonstrated that the F5tg83 promoter produced the highest level of transgene expression in the ferret airway in vivo. Since rAAV-CFTR vectors greater than 5.0kb in size are dramatically impaired with respect to vector efficacy, we utilized a shortened ferret CFTR minigene with a 159 bp deletion in the R-domain to construct a rAAV vector (AV2/2. F5tg83-fCFTRDR). This vector yielded an ≈17-fold increase in vector derived CFTR mRNA transcription and significantly improved Cl- currents in human CF ALI cultures when the vectors were administrated through basolateral infection. We also tested the F5tg83 promoter activity in the context of rAAV/HBoV1 vector, which packages an oversized rAAV genome up to 5.5kb in human bocavirus virus 1 (HBoV1) caspid and efficiently transduces HAE-ALI from apical membrane. We found that the F5tg83 promoter delivered robust and persistent expression of different reporter genes (firefly luciferase or the secreted guassia luciferase) from the HAE-ALI following apical infections of the AV2/HBoV1 chimeric vectors. The expanded package capacity of rAAV2/HBoV1 vector makes it possible to further optimize the F5tg83 promoter directed CFTR gene expression with the full-length CFTR gene. In summary, our study has identified a small enhancer/promoter combination that may have broad utility for rAAV-mediated CF gene therapy to the airway.Furthermore, the enhancer screening approach may be useful for rAAV applications in other organs that seek to deliver large transgenes that approach the packing capacity of rAAV.

Directional Release of Reovirus from the Apical Surface of Polarized Endothelial Cells

ABSTRACT Bloodstream spread is a critical step in the pathogenesis of many viruses. However, mechanisms that promote viremia are not well understood. Reoviruses are neurotropic viruses that disseminate hematogenously to the central nervous system. Junctional adhesion molecule A (JAM-A) is a tight junction protein that serves as a receptor for reovirus. JAM-A is required for establishment of viremia in infected newborn mice and viral spread to sites of secondary replication. To determine how viruses gain access to the circulatory system, we examined reovirus infection of polarized human brain microvascular endothelial cells (HBMECs). Reovirus productively infects polarized HBMECs, but infection does not alter tight junction integrity. Apical infection of polarized HBMECs is more efficient than basolateral infection, which is attributable to viral engagement of sialic acid and JAM-A. Viral release occurs exclusively from the apical surface via a mechanism that is not associated with lysis or apoptosis of infected cells. These data suggest that infection of endothelial cells routes reovirus apically into the bloodstream for systemic dissemination in the host. Understanding how viruses invade the bloodstream may aid in the development of therapeutics that block this step in viral pathogenesis.IMPORTANCE Bloodstream spread of viruses within infected hosts is a critical but poorly understood step in viral disease. Reoviruses first enter the host through the oral or respiratory route and infect cells in the central nervous system. Spread of reoviruses to the brain occurs by blood or nerves, which makes reoviruses useful models for studies of systemic viral dissemination. In this study, we examined how reoviruses infect endothelial cells, which form the walls of blood vessels. We found that reovirus infection of endothelial cells allows the virus to enter blood vessels and serves as a means for the virus to reach high titers in the circulation. Understanding how reovirus is routed through endothelial cells may aid in the design of antiviral drugs that target this important step in systemic viral infections.

Distinct transduction difference between adeno-associated virus type 1 and type 6 vectors in human polarized airway epithelia

Of the many biologically isolated AAV serotypes, AAV1 and AAV6 share the highest degree of sequence homology, with only six different capsid residues. We compared the transduction efficiencies of rAAV1 and rAAV6 in primary polarized human airway epithelia (HAE) and found significant differences in their abilities to transduce epithelia from the apical and basolateral membranes. rAAV1 transduction was ~10-fold higher than rAAV6 following apical infection, while rAAV6 transduction was ~10-fold higher than rAAV1 following basolateral infection. Furthermore, rAAV6 demonstrated significant polarity of transduction (100-fold; basolateral≫apical), while rAAV1 transduced from both membranes with equal efficiency. To evaluate capsid residues responsible for the observed serotype differences, we mutated the six divergent amino acids either alone or in combination. Results from these studies demonstrated that capsid residues 418 and 513 most significantly controlled membrane polarity differences in transduction between serotypes, with the rAAV6-D418E/K513E mutant demonstrating decreased (~10-fold) basolateral transduction and the rAAV1-E418D/E513K mutant demonstrating a transduction polarity identical to rAAV6-WT. However, none of the rAAV6 mutants obtained apical transduction efficiencies of rAAV1-WT, suggesting that all six divergent capsid residues in AAV1 act in concert to improve apical transduction of HAE.

Tumor Cell Marker PVRL4 (Nectin 4) Is an Epithelial Cell Receptor for Measles Virus

Vaccine and laboratory adapted strains of measles virus can use CD46 as a receptor to infect many human cell lines. However, wild type isolates of measles virus cannot use CD46, and they infect activated lymphocytes, dendritic cells, and macrophages via the receptor CD150/SLAM. Wild type virus can also infect epithelial cells of the respiratory tract through an unidentified receptor. We demonstrate that wild type measles virus infects primary airway epithelial cells grown in fetal calf serum and many adenocarcinoma cell lines of the lung, breast, and colon. Transfection of non-infectable adenocarcinoma cell lines with an expression vector encoding CD150/SLAM rendered them susceptible to measles virus, indicating that they were virus replication competent, but lacked a receptor for virus attachment and entry. Microarray analysis of susceptible versus non-susceptible cell lines was performed, and comparison of membrane protein gene transcripts produced a list of 11 candidate receptors. Of these, only the human tumor cell marker PVRL4 (Nectin 4) rendered cells amenable to measles virus infections. Flow cytometry confirmed that PVRL4 is highly expressed on the surfaces of susceptible lung, breast, and colon adenocarcinoma cell lines. Measles virus preferentially infected adenocarcinoma cell lines from the apical surface, although basolateral infection was observed with reduced kinetics. Confocal immune fluorescence microscopy and surface biotinylation experiments revealed that PVRL4 was expressed on both the apical and basolateral surfaces of these cell lines. Antibodies and siRNA directed against PVRL4 were able to block measles virus infections in MCF7 and NCI-H358 cancer cells. A virus binding assay indicated that PVRL4 was a bona fide receptor that supported virus attachment to the host cell. Several strains of measles virus were also shown to use PVRL4 as a receptor. Measles virus infection reduced PVRL4 surface expression in MCF7 cells, a property that is characteristic of receptor-associated viral infections.

Adherent-invasive Escherichia coli, strain LF82 disrupts apical junctional complexes in polarized epithelia

BackgroundAlthough bacteria are implicated in the pathogenesis of chronic inflammatory bowel diseases (IBD), mechanisms of intestinal injury and immune activation remain unclear. Identification of adherent-invasive Escherichia coli (AIEC) strains in IBD patients offers an opportunity to characterize the pathogenesis of microbial-induced intestinal inflammation in IBD. Previous studies have focused on the invasive phenotype of AIEC and the ability to replicate and survive in phagocytes. However, the precise mechanisms by which these newly identified microbes penetrate the epithelial lining remain to be clarified. Therefore, the aim of this study was to delineate the effects of AIEC, strain LF82 (serotype O83:H1) on model polarized epithelial monolayers as a contributor to intestinal injury in IBD.ResultsInfection of T84 and Madin-Darby Canine Kidney-I polarized epithelial cell monolayers with AIEC, strain LF82 led to a reduction in transepithelial electrical resistance and increased macromolecular (10 kilodalton dextran) flux. Basolateral AIEC infection resulted in more severe disruption of the epithelial barrier. Increased permeability was accompanied by a redistribution of the tight junction adaptor protein, zonula occludens-1, demonstrated by confocal microscopy and formation of gaps between cells, as shown by transmission electron microscopy. After 4 h of infection of intestine 407 cells, bacteria replicated in the cell cytoplasm and were enclosed in membrane-bound vesicles positive for the late endosomal marker, LAMP1.ConclusionThese findings indicate that AIEC, strain LF82 disrupts the integrity of the polarized epithelial cell barrier. This disruption enables bacteria to penetrate into the epithelium and replicate in the host cell cytoplasm. These findings provide important links between microbes related to IBD, the intestinal epithelial cell barrier and disease pathogenesis.

Reovirus preferentially infects the basolateral surface and is released from the apical surface of polarized human respiratory epithelial cells.

Mammalian reoviruses infect respiratory and gastrointestinal epithelia and cause disease in neonates. Junctional adhesion molecule-A (JAM-A) is a serotype-independent receptor for reovirus. JAM-A localizes to tight junctions and contributes to paracellular permeability in polarized epithelia. To investigate the mechanisms of reovirus infection of polarized epithelial cells, we assessed reovirus replication, release, and spread after apical and basolateral adsorption to primary human airway epithelial cultures. Reovirus infection of human airway epithelia was more efficient after adsorption to the basolateral surface than after adsorption to the apical surface, and it was dependent on JAM-A. Reovirus binding to carbohydrate coreceptor sialic acid inhibited apical infection, which was partially ameliorated by treatment of the cultures with neuraminidase. Despite the preference for basolateral infection, reovirus was released from the apical surface of respiratory epithelia and did not disrupt tight junctions. These results establish the existence of an infectious circuit for reovirus in polarized human respiratory epithelial cells.

Enteropathogenic Escherichia coli (EPEC) inactivate innate immune responses prior to compromising epithelial barrier function

Enteropathogenic Escherichia coli (EPEC) infection of the human small intestine induces severe watery diarrhoea linked to a rather weak inflammatory response despite EPEC's in vivo capacity to disrupt epithelial barrier function. Here, we demonstrate that EPEC flagellin triggers the secretion of the pro-inflammatory cytokine, interleukin (IL)-8, from small (Caco-2) and large (T84) intestinal epithelia model systems. Interestingly, IL-8 secretion required basolateral infection of T84 cells implying that flagellin must penetrate the epithelial barrier. In contrast, apical infection of Caco-2 cells induced IL-8 secretion but less potently than basolateral infections. Importantly, infection of Caco-2, but not T84 cells rapidly inhibited IL-8 secretion by a mechanism dependent on the delivery of effectors through a translocation system encoded on the locus of enterocyte effacement (LEE). Moreover, EPEC prevents the phosphorylation-associated activation of multiple kinase pathways regulating IL-8 gene transcription by a mechanism apparently independent of LEE-encoded effectors and four non-LEE-encoded effectors. Crucially, our studies reveal that EPEC inhibits the capacity of the cells to secrete IL-8 in response to bacterial antigens and inflammatory cytokines prior to disrupting barrier function by a distinct mechanism. Thus, these findings also lend themselves to a plausible mechanism to explain the absence of a strong inflammatory response in EPEC-infected humans.

Disruption of Tight Junctions and Induction of Proinflammatory Cytokine Responses in Colonic Epithelial Cells by Campylobacter jejuni

Campylobacter jejuni is a leading cause of human enterocolitis and is associated with postinfectious complications, including irritable bowel syndrome and Guillain-Barré syndrome. However, the pathogenesis of C. jejuni infection remains poorly understood. Paracellular pathways in intestinal epithelial cells are gated by intercellular junctions (tight junctions and adherens junctions), providing a functional barrier between luminal microbes and host immune cells in the lamina propria. Here we describe alterations in tight junctions in intestinal epithelial monolayers following C. jejuni infection. Apical infection of polarized T84 monolayers caused a time-dependent decrease in transepithelial electrical resistance (TER). Immunofluorescence microscopy revealed a redistribution of the tight junctional transmembrane protein occludin from an intercellular to an intracellular location. Subcellular fractionation using equilibrium sucrose density gradients demonstrated decreased hyperphosphorylated occludin in lipid rafts, Triton X-100-soluble fractions, and the Triton X-100-insoluble pellet following apical infection. Apical infection with C. jejuni also caused rapid activation of NF-kappaB and AP-1, phosphorylation of extracellular signal-regulated kinase, Jun N-terminal protein kinase, and p38 mitogen-activated protein kinases, and basolateral secretion of the CXC chemokine interleukin-8 (IL-8). Basolateral infection with C. jejuni caused a more rapid decrease in TER, comparable redistribution of tight-junction proteins, and secretion of more IL-8 than that seen with apical infection. These results suggest that compromised barrier function and increased chemokine expression contribute to the pathogenesis of C. jejuni-induced enterocolitis.

Access to Nectin Favors Herpes Simplex Virus Infection at the Apical Surface of Polarized Human Epithelial Cells

Viral entry may preferentially occur at the apical or the basolateral surfaces of polarized cells, and differences may impact pathogenesis, preventative strategies, and successful implementation of viral vectors for gene therapy. The objective of these studies was to examine the polarity of herpes simplex virus (HSV) entry using several different human epithelial cell lines. Human uterine (ECC-1), colonic (CaCo-2), and retinal pigment (ARPE-19) epithelial cells were grown on collagen-coated inserts, and the polarity was monitored by measuring the transepithelial cell resistance. Controls were CaSki cells, a human cervical cell line that does not polarize in vitro. The polarized cells, but not CaSki cells, were 16- to 50-fold more susceptible to HSV infection at the apical surface than at the basolateral surface. Disruption of the tight junctions by treatment with EGTA overcame the restriction on basolateral infection but had no impact on apical infection. No differences in binding at the two surfaces were observed. Confocal microscopy demonstrated that nectin-1, the major coreceptor for HSV entry, sorted preferentially to the apical surface, overlapping with adherens and tight junction proteins. Transfection with small interfering RNA specific for nectin-1 resulted in a significant reduction in susceptibility to HSV at the apical surface but had little impact on basolateral infection. Infection from the apical but not the basolateral surface triggered focal adhesion kinase phosphorylation and led to nuclear transport of viral capsids and viral gene expression. These studies indicate that access to nectin-1 contributes to preferential apical infection of these human epithelial cells by HSV.

499. Species-Specific Differences in the Polarity of rAAV5 and rAAV2 Transduction of Mouse and Human Airway Epithelia

Top of pageAbstract Species-specific differences in the tropism of viral vectors between mice and humans have made it difficult to anticipate the most appropriate adeno-associated virus (AAV) serotypes for clinical trials. In the context of developing gene therapies for cystic fibrosis lung disease, human air-liquid interface (ALI) models of airway epithelium have been useful as an intermediate surrogate for evaluating the human tropisms of various rAAV serotypes. In this model, we have previously shown that apical infection with rAAV2 and rAAV5 vectors have very similar transduction efficiencies. Additionally, in the presence of proteasome inhibitors, transduction is enhanced |[sim]|1000-fold with both serotypes following apical infection. In contrast to these findings in the human ALI model, in vivo rAAV5 transduction is 50-100-fold greater than that of rAAV2 in mouse lung. These differences could be the result of model system dependencies or species-specific differences in the tropism of AAV2 and AAV5 for airway epithelium in mice and humans. To resolve whether species-specific differences (human vs. mouse) or model system-specific differences (in vitro ALI culture vs. in vivo lung) are the cause of the discrepancies in rAAV5 and rAAV2 transduction in these various studies, we have compared transduction of these rAAV serotypes in mouse and human airway epithelia in ALI culture using pseudotyped luciferase rAAV vectors. Differentiated mouse airway epithelia in ALI culture gave electrophysiologic characteristics similar to those previously reported for murine tracheal epithelium, but distinct from that of human ALI cultures. Following apical infection of mouse airway epithelia in ALI culture, rAAV2/5 gave |[sim]|100-fold greater levels of transduction than infection with rAAV2/2. These results were similar to the differences in transduction between these two serotypes seen in vivo in mouse lung. These findings demonstrate that ALI cultures of mouse airway epithelium retain species-specific differences in comparison to human airway epithelium, which is reflected in a greater apical tropism for rAAV5 as compared to rAAV2 capsid serotypes. Interestingly, basolateral infection of mouse ALI cultures also retained a greater transduction efficiency (10-fold) with rAAV2/5, as compared to rAAV2/2. These differences in basolateral tropisms were reversed for human ALI cultures. Viral transduction for both rAAV2/2 and rAAV2/5 from the apical membrane of both mouse and human ALI cultures was enhanced 2-3 logs by the addition of proteasome modulating agents Doxorubicin and LLnL at the time of infection. Basolateral infection in the absence of proteasome modulating was always more effective than apical infection regardless of the species. However, the addition of proteasome modulating agents appeared to normalize transduction from the apical membrane to maximal levels regardless of the serotype or species. These findings suggest that rAAV2 and rAAV5 vectors have different transduction efficiencies for mouse and human airway epithelium due to serotype-specific differences in post-entry blocks modulated by the proteasome pathway.