Infection Of Epithelial Cells Research Articles

STAT1/MUC4 activation promotes antimicrobial peptide production to reduce intestinal epithelium barrier injury caused by enteropathogenic Escherichia coli infection

IntroductionAntimicrobial peptides (AMPs) are endogenous peptides that have been identified to alleviate intestinal epithelial barrier inflammation and dysfunction caused by enteropathogenic Escherichia coli (EPEC) infection; nonetheless, the upstream molecular mechanism of the production of AMPs is poorly understood.Material and methodsThe binding of signal transducer and activator of transcription (STAT) 1 (STAT1) to mucin 4 (MUC4) was examined by Co-Immunoprecipitation assay. To detect the influence of STAT1 and MUC4 expression, C57BL/6 mice model of EPEC infection in vivo and EPEC infected intestinal epithelial cells (IEC) in vitro model was established. Expressions of STAT1, MUC4, phosphorylated (p)-STAT1, proinflammatory cytokines, zonula occludens-1 (ZO-1) and AMP-related genes in mouse ileum and/or IEC were analyzed by immunohistochemical test, immunofluorescence assay, Western blot, and/or qRT-PCR. Meanwhile, IEC viability and apoptosis were measured using CCK-8 assay and flow cytometry.Resultsp-STAT1, MUC4, ZO-1 and AMP-related gene were lowly expressed in the ileum of EPEC-infected mice. p-STAT1 and MUC4 bound to each other. The expressions of STAT1 and MUC4 were decreased in EPEC-infected IEC. Overexpression of STAT1 resisted EPEC-induced viability decrease, apoptosis promotion, ZO-1 activity inhibition, release of proinflammatory cytokines, and downregulation of MUC4 and AMP-related genes in IEC. MUC4 knockdown partly counteracted the effect of STAT1 overexpression, but did not affect the forced STAT1 overexpression in EPEC-infected IEC.ConclusionsSTAT1/MUC4 pathway activation promotes AMP production to mitigate intestinal epithelium barrier injury caused by EPEC infection.

Modelling BK Polyomavirus dissemination and cytopathology using polarized human renal tubule epithelial cells.

Most humans have a lifelong imperceptible BK Polyomavirus (BKPyV) infection in epithelial cells lining the reno-urinary tract. In kidney transplant recipients, unrestricted high-level replication of donor-derived BKPyV in the allograft underlies polyomavirus-associated nephropathy, a condition with massive epithelial cell loss and inflammation causing premature allograft failure. There is limited understanding on how BKPyV disseminates throughout the reno-urinary tract and sometimes causes kidney damage. Tubule epithelial cells are tightly connected and have unique apical and basolateral membrane domains with highly specialized functions but all in vitro BKPyV studies have been performed in non-polarized cells. We therefore generated a polarized cell model of primary renal proximal tubule epithelial cells (RPTECs) and characterized BKPyV entry and release. After 8 days on permeable inserts, RPTECs demonstrated apico-basal polarity. BKPyV entry was most efficient via the apical membrane, that in vivo faces the tubular lumen, and depended on sialic acids. Progeny release started between 48 and 58 hours post-infection (hpi), and was exclusively detected in the apical compartment. From 72 hpi, cell lysis and detachment gradually increased but cells were mainly shed by extrusion and the barrier function was therefore maintained. The decoy-like cells were BKPyV infected and could transmit BKPyV to uninfected cells. By 120 hpi, the epithelial barrier was disrupted by severe cytopathic effects, and BKPyV entered the basolateral compartment mimicking the interstitial space. Addition of BKPyV-specific neutralizing antibodies to this compartment inhibited new infections. Taken together, we propose that during in vivo low-level BKPyV replication, BKPyV disseminates inside the tubular system, thereby causing minimal damage and delaying immune detection. However, in kidney transplant recipients lacking a well-functioning immune system, replication in the allograft will progress and eventually cause denudation of the basement membrane, leading to an increased number of decoy cells, high-level BKPyV-DNAuria and DNAemia, the latter a marker of allograft damage.

169 Involvement of PTX3 and MBL in the clearance of SARS-CoV-2 infected lung airway epithelial cells

169 Involvement of PTX3 and MBL in the clearance of SARS-CoV-2 infected lung airway epithelial cells

Candida albicans stimulates formation of a multi-receptor complex that mediates epithelial cell invasion during oropharyngeal infection.

Fungal invasion of the oral epithelium is central to the pathogenesis of oropharyngeal candidiasis (OPC). Candida albicans invades the oral epithelium by receptor-induced endocytosis but this process is incompletely understood. We found that C. albicans infection of oral epithelial cells induces c-Met to form a multi-protein complex with E-cadherin and the epidermal growth factor receptor (EGFR). E-cadherin is necessary for C. albicans to activate both c-Met and EGFR and to induce the endocytosis of C. albicans. Proteomics analysis revealed that c-Met interacts with C. albicans Hyr1, Als3 and Ssa1. Both Hyr1 and Als3 are required for C. albicans to stimulate c-Met and EGFR in oral epithelial cells in vitro and for full virulence during OPC in mice. Treating mice with small molecule inhibitors of c-Met and EGFR ameliorates OPC, demonstrating the potential therapeutic efficacy of blocking these host receptors for C. albicans.

Protective role of CFTR during fungal infection of cystic fibrosis bronchial epithelial cells with Aspergillus fumigatus.

Lung infection with the fungus Aspergillus fumigatus (Af) is a common complication in cystic fibrosis (CF) and is associated with loss of pulmonary function. We established a fungal epithelial co-culture model to examine the impact of Af infection on CF bronchial epithelial barrier function using Af strains 10AF and AF293-GFP, and the CFBE41o- cell line homozygous for the F508del mutation with (CF+CFTR) and without (CF) normal CFTR expression. Following exposure of the epithelial surface to Af conidia, formation of germlings (early stages of fungal growth) was detected after 9-12 hours and hyphae (mature fungal growth) after 12-24 hours. During fungal morphogenesis, bronchial epithelial cells showed signs of damage including rounding, and partial detachment after 24 hours. Fluorescently labeled conidia were internalized after 6 hours and more internalized conidia were observed in CF compared to CF+CFTR cells. Infection of the apical surface with 10AF conidia, germlings, or hyphae was performed to determine growth stage-specific effects on tight junction protein zona occludens protein 1 (ZO-1) expression and transepithelial electrical resistance (TER). In response to infection with conidia or germlings, epithelial barrier function degraded time-dependently (based on ZO-1 immunofluorescence and TER) with a delayed onset in CF+CFTR cell monolayers and required viable fungi and apical application. Infection with hyphae caused an earlier onset and faster rate of decline in TER compared to conidia and germlings. Gliotoxin, a major Af virulence factor, caused a rapid decline in TER and induced a transient chloride secretory response in CF+CFTR but not CF cells. Our findings suggest growth and internalization of Af result in deleterious effects on bronchial epithelial barrier function that occurred more rapidly in the absence of CFTR. Bronchial epithelial barrier breakdown was time-dependent and morphotype-specific and mimicked by acute administration of gliotoxin. Our study also suggests a protective role for CFTR by turning on CFTR-dependent chloride transport in response to gliotoxin, a mechanism that will support mucociliary clearance, and could delay the loss of epithelial integrity during fungal development in vivo.

Rhinovirus infection induces secretion of endothelin-1 from airway epithelial cells in both in vitro and in vivo models

BackgroundRhinovirus (RV) infection of airway epithelial cells triggers asthma exacerbations, during which airway smooth muscle (ASM) excessively contracts. Due to ASM contraction, airway epithelial cells become mechanically compressed. We previously reported that compressed human bronchial epithelial (HBE) cells are a source of endothelin-1 (ET-1) that causes ASM contraction. Here, we hypothesized that epithelial sensing of RV by TLR3 and epithelial compression induce ET-1 secretion through a TGF-β receptor (TGFβR)-dependent mechanism.MethodsTo test this, we used primary HBE cells well-differentiated in air–liquid interface culture and two mouse models (ovalbumin and house dust mite) of allergic airway disease (AAD). HBE cells were infected with RV-A16, treated with a TLR3 agonist (poly(I:C)), or exposed to compression. Thereafter, EDN1 (ET-1 protein-encoding gene) mRNA expression and secreted ET-1 protein were measured. We examined the role of TGFβR in ET-1 secretion using either a pharmacologic inhibitor of TGFβR or recombinant TGF-β1 protein. In the AAD mouse models, allergen-sensitized and allergen-challenged mice were subsequently infected with RV. We then measured ET-1 in bronchoalveolar lavage fluid (BALF) and airway hyperresponsiveness (AHR) following methacholine challenge.ResultsOur data reveal that RV infection induced EDN1 expression and ET-1 secretion in HBE cells, potentially mediated by TLR3. TGFβR activation was partially required for ET-1 secretion, which was induced by RV, poly(I:C), or compression. TGFβR activation alone was sufficient to increase ET-1 secretion. In AAD mouse models, RV induced ET-1 secretion in BALF, which positively correlated with AHR.ConclusionsOur data provide evidence that RV infection increased epithelial-cell ET-1 secretion through a TGFβR-dependent mechanism, which contributes to bronchoconstriction during RV-induced asthma exacerbations.

Streptococcus pneumoniae disrupts the structure of the golgi apparatus and subsequent epithelial cytokine response in an H2O2-dependent manner

BackgroundLung infections caused by Streptococcus pneumonia are a global leading cause of death. The reactive oxygen species H2O2 is one of the virulence factors of Streptococcus pneumoniae. The Golgi apparatus is essential for the inflammatory response of a eukaryotic cell. Golgi fragmentation was previously shown to be induced by bacterial pathogens and in response to H2O2 treatment. This led us to investigate whether the Golgi apparatus is actively involved and targeted in host–pathogen interactions during pneumococcal infections.MethodsFollowing in vitro infection of BEAS-2B bronchial epithelial cells with Streptococcus pneumoniae for 16 h, the structure of the Golgi apparatus was assessed by fluorescence staining of the Golgi-associated protein, Golgin-97. To investigate the effect of H2O2 production on Golgi structure, BEAS-2B cells were treated with H2O2 or the H2O2 degrading enzyme Catalase, prior to Golgi staining. Artificial disruption of the Golgi apparatus was induced by treatment of cells with the GBF1 inhibitor, Golgicide A. A proinflammatory cellular response was induced by treatment of cells with the bacterial cell wall component and TLR4 ligand lipoteichoic acid.ResultsIn vitro infection of bronchial epithelial cells with wild type Streptococcus pneumoniae led to a disruption of normal Golgi structure. Golgi fragmentation was not observed after deletion of the pneumococcal H2O2-producing gene, spxB, or neutralization of H2O2 by catalase treatment, but could be induced by H2O2 treatment. Streptococcus pneumoniae infection significantly reduced host cell protein glycosylation and artificial disruption of Golgi structure significantly reduced bacterial adherence, but increased bacterial counts in the supernatant. To understand if this effect depended on cell-contact or soluble factors, pneumococci were treated with cell-supernatant of cells treated with Golgicide A and/or lipoteichoic acid. This approach revealed that lipoteichoic acid conditioned medium inhibits bacterial replication in presence of host cells. In contrast, artificial Golgi fragmentation by Golgicide A treatment prior to lipoteichoic acid treatment rescued bacterial replication. This effect was associated with an increase of IL-6 and IL-8 in the supernatant of lipoteichoic acid treated cells. The increased cytokine release was abolished if cells were treated with Golgicide A prior to lipoteichoic acid treatment.ConclusionStreptococcus pneumoniae disrupts the Golgi apparatus in an H2O2-dependent manner, thereby inhibiting paracrine anti-infective mechanisms.EnamKsCfhZcCbpPUq5E2ztVideo

Epithelial Galectin-3 Induced the Mitochondrial Complex Inhibition and Cell Cycle Arrest of CD8+ T Cells in Severe/Critical COVID-19.

Previous research suggested that the dramatical decrease in CD8+ T cells is a contributing factor in the poor prognosis and disease progression of COVID-19 patients. However, the underlying mechanisms are not fully understood. In this study, we conducted Single-cell RNA sequencing (scRNA-seq) and single-cell T cell receptor sequencing (scTCR-seq) analysis, which revealed a proliferative-exhausted MCM+FASLGlow CD8+ T cell phenotype in severe/critical COVID-19 patients. These CD8+ T cells were characterized by G2/M cell cycle arrest, downregulation of respiratory chain complex genes, and inhibition of mitochondrial biogenesis. CellChat analysis of infected lung epithelial cells and CD8+ T cells found that the galectin signaling pathway played a crucial role in CD8+ T cell reduction and dysfunction. To further elucidate the mechanisms, we established SARS-CoV-2 ORF3a-transfected A549 cells, and co-cultured them with CD8+ T cells for ex vivo experiments. Our results showed that epithelial galectin-3 inhibited the transcription of the mitochondrial respiratory chain complex III/IV genes of CD8+ T cells by suppressing the nuclear translocation of nuclear respiratory factor 1 (NRF1). Further findings showed that the suppression of NRF1 translocation was associated with ERK-related and Akt-related signaling pathways. Importantly, the galectin-3 inhibitor, TD-139, promoted nuclear translocation of NRF1, thus enhancing the expression of the mitochondrial respiratory chain complex III/IV genes and the mitochondrial biogenesis of CD8+ T cells. Our study provided new insights into the immunopathogenesis of COVID-19 and identified potential therapeutic targets for the prevention and treatment of severe/critical COVID-19 patients.

Oxylipin concentration shift in exhaled breath condensate (EBC) of SARS-CoV-2 infected patients

Infection of airway epithelial cells with severe acute respiratory coronavirus 2 (SARS-CoV-2) can lead to severe respiratory tract damage and lung injury with hypoxia. It is challenging to sample the lower airways non-invasively and the capability to identify a highly representative specimen that can be collected in a non-invasive way would provide opportunities to investigate metabolomic consequences of COVID-19 disease. In the present study, we performed a targeted metabolomic approach using liquid chromatography coupled with high resolution chromatography (LC-MS) on exhaled breath condensate (EBC) collected from hospitalized COVID-19 patients (COVID+) and negative controls, both non-hospitalized and hospitalized for other reasons (COVID−). We were able to noninvasively identify and quantify inflammatory oxylipin shifts and dysregulation that may ultimately be used to monitor COVID-19 disease progression or severity and response to therapy. We also expected EBC-based biochemical oxylipin changes associated with COVID-19 host response to infection. The results indicated ten targeted oxylipins showing significative differences between SAR-CoV-2 infected EBC samples and negative control subjects. These compounds were prostaglandins A2 and D2, LXA4, 5-HETE, 12-HETE, 15-HETE, 5-HEPE, 9-HODE, 13-oxoODE and 19(20)-EpDPA, which are associated with specific pathways (i.e. P450, COX, 15-LOX) related to inflammatory and oxidative stress processes. Moreover, all these compounds were up-regulated by COVID+, meaning their concentrations were higher in subjects with SAR-CoV-2 infection. Given that many COVID-19 symptoms are inflammatory in nature, this is interesting insight into the pathophysiology of the disease. Breath monitoring of these and other EBC metabolites presents an interesting opportunity to monitor key indicators of disease progression and severity.

Development and analytical validation of a novel nasopharynx swab-based Epstein-Barr virus C promoter methylation quantitative assay for nasopharyngeal carcinoma detection.

Epstein-Barr virus (EBV) C promoter (Cp) hypermethylation, a crucial factor for EBV latent infection of nasopharyngeal epithelial cells, has been recognized as a promising biomarker for nasopharyngeal carcinoma (NPC) detection. In this study, we develop a novel EBV Cp methylation quantification (E-CpMQ) assay and evaluate its diagnostic performance for NPC detection. A novel qPCR assay for simultaneous quantification of methylated- and unmethylated EBV Cp was developed by the combinational modification of MethyLight and QASM, with an innovative calibrator to improve the detection accuracy and consistency. The NP swab samples and synthetic standards were used for the analytical validation of the E-CpMQ. The diagnostic efficacy of the developed E-CpMQ assay was validated in 137 NPC patients and 137 non-NPC controls. The E-CpMQ assay can detect the EBV Cp methylation ratio in one reaction system under 10 copies with 100 % recognition specificity, which is highly correlated to pyrosequencing with a correlation coefficient over 0.99. The calibrated E-CpMQ assay reduces the coefficient of variation by an average of 55.5 % with a total variance of less than 0.06 units standard deviation (SD). Linear methylation ratio detection range from 4.76 to 99.01 %. The sensitivity and specificity of the E-CpMQ respectively are 96.4 % (95 % CI: 91.7-98.8 %), 89.8 % (95 % CI: 83.5-94.3 %). The developed E-CpMQ assay with a calibrator enables accurate and reproducible EBV Cp methylation ratio quantification and offers a sensitive, specific, cost-effective method for NPC early detection.

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.

The Impact of CC16 on Pulmonary Epithelial-Driven Host Responses during Mycoplasma pneumoniae Infection in Mouse Tracheal Epithelial Cells.

Club Cell Secretory Protein (CC16) plays many protective roles within the lung; however, the complete biological functions, especially regarding the pulmonary epithelium during infection, remain undefined. We have previously shown that CC16-deficient (CC16-/-) mouse tracheal epithelial cells (MTECs) have enhanced Mp burden compared to CC16-sufficient (WT) MTECs; therefore, in this study, we wanted to further define how the pulmonary epithelium responds to infection in the context of CC16 deficiency. Using mass spectrometry and quantitative proteomics to analyze proteins secreted apically from MTECs grown at an air-liquid interface, we investigated the protective effects that CC16 elicits within the pulmonary epithelium during Mycoplasma pneumoniae (Mp) infection. When challenged with Mp, WT MTECs have an overall reduction in apical protein secretion, whereas CC16-/- MTECs have increased apical protein secretion compared to their unchallenged controls. Following Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) assessment, many of the proteins upregulated from CC16-/- MTECS (unchallenged and during Mp infection) were related to airway remodeling, which were not observed by WT MTECs. These findings suggest that CC16 may be important in providing protection within the pulmonary epithelium during respiratory infection with Mp, which is the major causative agent of community-acquired pneumoniae.

The outer membrane protein of Fusobacterium necrophorum, 43K OMP, stimulates inflammatory cytokine production through nuclear factor kappa B activation

ObjectiveFusobacterium necrophorum causes bovine hepatic abscess, foot rot, mastitis, and endometritis. The 43 kDa outer membrane protein (43 K OMP) of F. necrophorum is a porin protein that plays an important role in infections by this bacterium, but the biological function and the pathogenesis of this protein are largely unknown. MethodsIn this study, we investigated the role of the 43 K OMP in bacterial infection of bovine mammary epithelial cells (MAC-T cells) by Tandem Mass Tag proteomic analysis. The RAW264.7 cells were incubated with recombinant 43 K OMP (12.5 μg/mL) for 2 h, 4 h, 6 h, and 12 h, and then the inflammatory related protein and inflammatory cytokine production were measured by Western blot analysis and ELISA, the mRNA expression levels of inflammatory cytokine were measured by Real-Time PCR. ResultsProteomic analysis results demonstrated there were 224 differentially expressed proteins in the MAC-T cells stimulated with the 43 K OMP compared with control, and 118 proteins were upregulated and 106 proteins were downregulated. These differentially expressed proteins were mainly involved in NF-kappa B signaling, bacterial invasion of epithelial cells, cell adhesion, complement and coagulation cascades. The top six differentially expressed proteins were; MMP9, PLAU, STOM, PSMD13, PLAUR, and ITGAV, which were involved in a protein-protein interaction network. Furthermore, TLR/MyD88/NF-κB pathway related proteins and inflammatory cytokines (IL-6, TNF-α, and IL-1β) were assessed by Western blot analysis and ELISA. Results showed the 43 K OMP to enhance the expression of TLR4 protein at 2 h (P < 0.01) and the MyD88 protein at 4 h (P < 0.05) post-stimulation, and to decrease IκBα expression at 4 h, 6 h and 12 h (P < 0.05) post-infection, as well as induce phosphorylation at Ser536 (P < 0.01). Levels of IL-6, IL-1β, and TNF-α in the supernatants of mouse macrophages were increased (P < 0.05), as were mRNA expression levels of IL-6, IL-1β, and TNF-α (P < 0.05), while IL-4 mRNA expression was decreased (P < 0.05). ConclusionsTaken together, these results suggested the important role for 43 K OMP in F. necrophorum infection, promoting the production of pro-inflammatory cytokines (IL-6 and TNF-α) by activation of the TLR/MyD88/NF-κB pathway. These findings provided a theoretical basis for a better understanding of the pathogenesis of F. necrophorum infection.

The Transcriptome Landscape of the In Vitro Human Airway Epithelium Response to SARS-CoV-2.

Airway-liquid interface cultures of primary epithelial cells and of induced pluripotent stem-cell-derived airway epithelial cells (ALI and iALI, respectively) are physiologically relevant models for respiratory virus infection studies because they can mimic the in vivo human bronchial epithelium. Here, we investigated gene expression profiles in human airway cultures (ALI and iALI models), infected or not with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), using our own and publicly available bulk and single-cell transcriptome datasets. SARS-CoV-2 infection significantly increased the expression of interferon-stimulated genes (IFI44, IFIT1, IFIT3, IFI35, IRF9, MX1, OAS1, OAS3 and ISG15) and inflammatory genes (NFKBIA, CSF1, FOSL1, IL32 and CXCL10) by day 4 post-infection, indicating activation of the interferon and immune responses to the virus. Extracellular matrix genes (ITGB6, ITGB1 and GJA1) were also altered in infected cells. Single-cell RNA sequencing data revealed that SARS-CoV-2 infection damaged the respiratory epithelium, particularly mature ciliated cells. The expression of genes encoding intercellular communication and adhesion proteins was also deregulated, suggesting a mechanism to promote shedding of infected epithelial cells. These data demonstrate that ALI/iALI models help to explain the airway epithelium response to SARS-CoV-2 infection and are a key tool for developing COVID-19 treatments.

Cholinergic α7 nAChR signaling suppresses SARS-CoV-2 infection and inflammation in lung epithelial cells.

Cholinergic α7 nAChR signaling suppresses SARS-CoV-2 infection and inflammation in lung epithelial cells.

Several Alphaherpesviruses Interact Similarly with the NF-κB Pathway and Suppress NF-κB-Dependent Gene Expression.

Alphaherpesvirus infection is associated with attenuation of different aspects of the host innate immune response that is elicited to confine primary infections at the mucosal epithelia. Here, we report that infection of epithelial cells with several alphaherpesviruses of different species, including herpes simplex virus 1 and 2 (HSV-1 and HSV-2), feline alphaherpesvirus 1 (FHV-1), and bovine alphaherpesvirus 1 (BoHV-1) results in the inactivation of the responses driven by the nuclear factor kappa B (NF-κB) pathway, considered a pillar of the innate immune response. The mode to interact with and circumvent NF-κB-driven responses in infected epithelial cells is seemingly conserved in human, feline, and porcine alphaherpesviruses, consisting of a persistent activation of the NF-κB cascade but a potent repression of NF-κB-dependent transcription activity, which relies on replication of viral genomes. However, BoHV-1 apparently deviates from the other investigated members of the taxon in this respect, as BoHV-1-infected epithelial cells do not display the persistent NF-κB activation observed for the other alphaherpesviruses. In conclusion, this study suggests that inhibition of NF-κB transcription activity is a strategy used by several alphaherpesviruses to prevent NF-κB-driven responses in infected epithelial cells. IMPORTANCE The current study provides a side-by-side comparison of the interaction of different alphaherpesviruses with NF-κB, a key and central player in the (proinflammatory) innate host response, in infected nontransformed epithelial cell lines. We report that all studied viruses prevent expression of the hallmark NF-κB-dependent gene IκB, often but not always via similar strategies, pointing to suppression of NF-κB-dependent host gene expression in infected epithelial cells as a common and therefore likely important aspect of alphaherpesviruses.

Alternative Splicing of RIOK3 Engages the Noncanonical NFκB Pathway during Rift Valley Fever Virus Infection.

Although the noncanonical NFκB pathway was originally identified as a cellular pathway contributing to lymphoid organogenesis, in the past 20 years, its involvement in innate immunity has become more appreciated. In particular, the noncanonical NFκB pathway has been found to be activated and even exploited by some RNA viruses during infection. Intriguingly, activation of this pathway has been shown to have a role in disrupting transcription of type 1 interferon (IFN), suggesting a rationale for why this response could be co-opted by some viruses. Rift Valley fever virus (RVFV) is a trisegmented ambisense RNA virus that poses a considerable threat to domestic livestock and human health. Previously, we showed the atypical kinase RIOK3 is important for mounting an IFN response to RVFV infection of human epithelial cells, and shortly following infection with RVFV (MP12 strain), RIOK3 mRNA is alternatively spliced to its X2 isoform that encodes a truncated RIOK3 protein. Alternative splicing of RIOK3 mRNA has an inhibitory effect on the IFN response but also stimulates an NFκB-mediated inflammatory response. Here, we demonstrate alternative splicing of RIOK3 mRNA is associated with activation of the noncanonical NFκB pathway and suggest this pathway is co-opted by RVFV (MP12) to enhance viral success during infection.

Src-Dependent NM2A Tyrosine Phosphorylation Regulates Actomyosin Remodeling.

Non-muscle myosin 2A (NM2A) is a key cytoskeletal enzyme that, along with actin, assembles into actomyosin filaments inside cells. NM2A is fundamental for cell adhesion and motility, playing important functions in different stages of development and during the progression of viral and bacterial infections. Phosphorylation events regulate the activity and the cellular localization of NM2A. We previously identified the tyrosine phosphorylation of residue 158 (pTyr158) in the motor domain of the NM2A heavy chain. This phosphorylation can be promoted by Listeria monocytogenes infection of epithelial cells and is dependent on Src kinase; however, its molecular role is unknown. Here, we show that the status of pTyr158 defines cytoskeletal organization, affects the assembly/disassembly of focal adhesions, and interferes with cell migration. Cells overexpressing a non-phosphorylatable NM2A variant or expressing reduced levels of Src kinase display increased stress fibers and larger focal adhesions, suggesting an altered contraction status consistent with the increased NM2A activity that we also observed. We propose NM2A pTyr158 as a novel layer of regulation of actomyosin cytoskeleton organization.

A novel in vitro model to study prolonged Pseudomonas aeruginosa infection in the cystic fibrosis bronchial epithelium.

Pseudomonas aeruginosa (PA) is known to chronically infect airways of people with cystic fibrosis (CF) by early adulthood. PA infections can lead to increased airway inflammation and lung tissue damage, ultimately contributing to decreased lung function and quality of life. Existing models of PA infection in vitro commonly utilize 1-6-hour time courses. However, these relatively early time points may not encompass downstream airway cell signaling in response to the chronic PA infections observed in people with cystic fibrosis. To fill this gap in knowledge, the aim of this study was to establish an in vitro model that allows for PA infection of CF bronchial epithelial cells, cultured at the air liquid interface, for 24 hours. Our model shows with an inoculum of 2 x 102 CFUs of PA for 24 hours pro-inflammatory markers such as interleukin 6 and interleukin 8 are upregulated with little decrease in CF bronchial epithelial cell survival or monolayer confluency. Additionally, immunoblotting for phosphorylated phospholipase C gamma, a well-known downstream protein of fibroblast growth factor receptor signaling, showed significantly elevated levels after 24 hours with PA infection that were not seen at earlier timepoints. Finally, inhibition of phospholipase C shows significant downregulation of interleukin 8. Our data suggest that this newly developed in vitro "prolonged PA infection model" recapitulates the elevated inflammatory markers observed in CF, without compromising cell survival. This extended period of PA growth on CF bronchial epithelial cells will have impact on further studies of cell signaling and microbiological studies that were not possible in previous models using shorter PA exposures.

EPs® 7630 Stimulates Tissue Repair Mechanisms and Modifies Tight Junction Protein Expression in Human Airway Epithelial Cells

Airway epithelium repair after infection consists of wound repair, re-synthesis of the extracellular matrix (ECM), and tight junction proteins. In humans, EPs® 7630 obtained from Pelargonium sidoides roots reduces the severity and duration of acute respiratory tract infections. The effect of EPs® 7630 on tissue repair of rhinovirus-16 (RV-16) infected and control human airway epithelial cells was assessed for: (i) epithelial cell proliferation by manual cell counts, (ii) epithelial wound repair by “scratch assay”, (iii) ECM composition by Western-blotting and cell-based ELISA, and (iv) epithelial tight junction proteins by Western-blotting. EPs® 7630 stimulated cell proliferation through cAMP, CREB, and p38 MAPK. EPs® 7630 significantly improved wound repair. Pro-inflammatory collagen type-I expression was reduced by EPs® 7630, while fibronectin was increased. Virus-binding tight junction proteins desmoglein2, desmocollin2, ZO-1, claudin1, and claudin4 were downregulated by EPs® 7630. The RV16-induced shift of the ECM towards the pro-inflammatory type was prevented by EPs® 7630. Most of the effects of EPs® 7630 on tissue repair and regeneration were sensitive to inhibition of cAMP-induced signaling. The data suggest that EPs® 7630-dependent modification of epithelial cell metabolism and function might underlie the faster recovery time from viral infections, as reported by others in clinical studies.