The SWI/SNF-Related, Matrix Associated, Actin-Dependent Regulator of Chromatin A4 Core Complex Represses Respiratory Syncytial Virus-Induced Syncytia Formation and Subepithelial Myofibroblast Transition.

Epigenetics plays an important role in the priming the dynamic response of airway epithelial cells to infectious and environmental stressors. Here, we examine the epigenetic role of the SWI/SNF Related, Matrix Associated, Actin Dependent Regulator of Chromatin A4 (SMARCA4) in the epithelial response to RSV infection. Depletion of SMARCA4 destabilized the abundance of the SMARCE1/ARID1A SWI/SNF subunits, disrupting the innate response and triggering a hybrid epithelial/mesenchymal (E/M) state. Assaying SMARCA4 complex-regulated open chromatin domains by transposase cleavage -next generation sequencing (ATAC-Seq), we observed that the majority of cleavage sites in uninfected cells have reduced chromatin accessibility. Paradoxically, SMARCA4 complex-depleted cells showed enhanced RSV-inducible chromatin opening and gene expression in the EMT pathway genes, MMP9, SNAI1/2, VIM, and CDH2. Focusing on the key MMP9, we observed that SMARCA4 complex depletion reduced basal BRD4 and RNA Polymerase II binding, but enhanced BRD4/Pol II binding in response to RSV infection. In addition, we observed that MMP9 secretion in SMARCA4 complex deficient cells contributes to mesenchymal transition, cellular fusion (syncytia) and subepithelial myofibroblast transition. We conclude the SMARCA4 complex is a transcriptional repressor of epithelial plasticity, whose depletion triggers a hybrid E/M state that affects the dynamic response of the small airway epithelial cell in mucosal remodeling via paracrine MMP9 activity.

Microparticle-mediated VZV propagation and endothelial activation: Mechanism of VZV vasculopathy.

OBJECTIVE: Varicella zoster virus (VZV) can spread anterogradely and infect cerebral arteries causing VZV vasculopathy and arterial ischemic stroke. In this study, we tested the hypothesis that virus-infected cerebrovascular fibroblasts undergo phenotypic changes that promote vascular remodeling and facilitate virus transmission in an in vitro model of VZV vasculopathy. The aims of this project were therefore to examine the changes that virus-infected human brain adventitial vascular fibroblasts (HBVAFs) undergo in an in vitro model of VZV vasculopathy and to identify disease biomarkers relating to VZV-related vasculopathy. METHODS: HBVAFs were infected with VZV, and their ability to migrate, proliferate, transdifferentiate, and interact with endothelial cells was studied with flow cytometry. Microparticles (MPs) released from these cells were isolated and imaged with transmission electron microscopy, and their protein content was analyzed with mass spectrometry. Circulating MP profiles were also studied in children with VZV and non-VZV vasculopathy and compared with controls. RESULTS: VZV-infected HBVAFs transdifferentiated into myofibroblasts with enhanced proliferative and migratory capacity. Interaction of VZV-infected HBVAFs with endothelial cells resulted in endothelial dysfunction. These effects were, in part, mediated by the release of MPs from VZV-infected HBVAFs. These MPs contained VZV virions that could transmit VZV to neighboring cells, highlighting a novel model of VZV cell-to-cell viral dissemination. MPs positive for VZV were significantly higher in children with VZV-related vasculopathy compared to children with non-VZV vasculopathy (p = 0.01) and controls (p = 0.007). CONCLUSIONS: VZV-infected HBVAFs promote vascular remodeling and facilitate virus transmission. These effects were mediated by the release of apoptotic MPs that could transmit VZV infection to neighboring cells through a Trojan horse means of productive viral infection. VZV+ MPs may represent a disease biomarker worthy of further study.

Varicella Zoster Virus Alters Expression of Cell Adhesion Proteins in Human Perineurial Cells via Interleukin 6.

BACKGROUND: In temporal arteries (TAs) from patients with giant cell arteritis, varicella zoster virus (VZV) is seen in perineurial cells that surround adventitial nerve bundles and form the peripheral nerve-extrafascicular tissue barrier (perineurium). We hypothesized that during VZV reactivation from ganglia, virus travels transaxonally and disrupts the perineurium to infect surrounding cells. METHODS: Mock- and VZV-infected primary human perineurial cells (HPNCs) were examined for alterations in claudin-1, E-cadherin, and N-cadherin. Conditioned supernatant was analyzed for a soluble factor(s) mediating these alterations and for the ability to increase cell migration. To corroborate in vitro findings, a VZV-infected TA was examined. RESULTS: In VZV-infected HPNCs, claudin-1 redistributed to the nucleus; E-cadherin was lost and N-cadherin gained, with similar changes seen in VZV-infected perineurial cells in a TA. VZV-conditioned supernatant contained increased interleukin 6 (IL-6) that induced E-cadherin loss and N-cadherin gain and increased cell migration when added to uninfected HPNCs; anti-IL-6 receptor antibody prevented these changes. CONCLUSIONS: IL-6 secreted from VZV-infected HPNCs facilitated changes in E- and N-cadherin expression and cell migration, reminiscent of an epithelial-to-mesenchymal cell transition, potentially contributing to loss of perineurial cell barrier integrity and viral spread. Importantly, an anti-IL-6 receptor antibody prevented virus-induced perineurial cell disruption.

Establishment of porcine enterocyte/myofibroblast co-cultures for the growth of porcine rota- and coronaviruses.

A stable culture of primary porcine enterocytes is necessary to study porcine enteric virus replication characteristics. Because the direct cultivation of primary porcine enterocytes is difficult, alternatives have to be considered. As subepithelial myofibroblasts secrete extracellular matrix and growth factors contributing to the attachment, proliferation and differentiation of epithelial cells, co-cultures of primary porcine enterocytes (ileocytes and colonocytes) with myofibroblasts were developed and evaluated for their susceptibility to enteric viruses. First, it was demonstrated that the co-cultured ileocytes and colonocytes were susceptible to an archival rotavirus strain RVA/pig-tc/BEL/RV277/1977/G1P[7] and different other rotavirus genotypes (fecal samples containing G5P[7], G5P[13], G9P[23], G4P[6]). Next, the TGEV Purdue strain infected both ileocytes and colonocytes whereas the Miller strain only infected ileocytes. Last, the PEDV CV777 Vero adapted and non-adapted (fecal suspension) strains could infect co-cultured ileocytes but not colonocytes. The infectivity of the CV777 Vero adapted strain was higher when the cells were cultured without fetal bovine serum and the CV777 fecal suspension only infected the ileocytes cultured without fetal bovine serum. In conclusion, a novel co-culture of porcine enterocytes with myofibroblasts was established, which can be used for the investigation of the replication of enteric viruses.

Reelin expression in human liver of patients with chronic hepatitis C infection.

Reelin is a secreted extracellular glycoprotein that plays a critical role during brain development. Several studies have described Reelin expression in hepatic stellate cells of the human liver. In order to investigate the possible role of Reelin in the process of hepatic fibrogenesis, in this study we investigated Reelin expression in the liver tissue of patients infected with the Hepatitis C Virus (HCV). On this basis, Reelin expression was analysed by immunohistochemistry during liver biopsies of 81 patients with HCV-related chronic hepatitis. A Knodell score was used to stage liver fibrosis. Hepatic stellate cells/myofibroblast immunohistochemical markers (CRBP-1, alpha-SMA) were also evaluated. As further confirmed by co-localization experiments (Reelin +CRBP-1), Reelin protein was expressed by hepatic stellate cells/myofibroblasts, and a significant positive correlation was found between Reelin expression and the stage of liver fibrosis (P=0.002). Moreover, Reelin correlated with CRBP-1 positive cells (P=0.002), but not with alpha-SMA, suggesting that Reelin should not be regarded as a marker of hepatic stellate cells/myofibroblasts differentiation but rather as a functional protein expressed during some phases of liver fibrosis. Furthermore, Disabled-1 (Dab1), a Reelin adaptor protein, was expressed in cells of ductular reaction suggesting a paracrine role for Reelin with regards these elements. In conclusion, Reelin was expressed by human hepatic stellate cells/myofibroblasts and the number of these cells increased significantly in the lobule as the liver fibrosis progressed, suggesting a role for Reelin in the activation of hepatic stellate cells/myofibroblasts during liver injury. Reelin may potentially be incorporated into liver injury evaluations in combination with other histological data.

Posttranscriptional Regulation in Adenovirus Infected Cells.

A deeper understanding of how viruses reprogram their hosts for production of progeny is needed to combat infections. Most knowledge on the regulation of cellular gene expression during adenovirus infection is derived from mRNA studies. Here, we investigated the changes in protein expression during the late phase of adenovirus type 2 (Ad2) infection of the IMR-90 cell line by stable isotope labeling in cell culture with subsequent liquid chromatography-high resolution tandem mass spectrometric analysis. Two biological replicates of samples collected at 24 and 36 h post-infection (hpi) were investigated using swapped labeling. In total, 2648 and 2394 proteins were quantified at 24 and 36 hpi, respectively. Among them, 659 and 645 were deregulated >1.6-fold at the two time points. The protein expression was compared with RNA expression using cDNA sequencing data. The correlation was surprisingly low (r = 0.3), and several examples of posttranscriptional regulation were observed; e.g., proteins related to carbohydrate metabolism were up-regulated at the protein level but unchanged at the RNA level, whereas histone proteins were down-regulated at the protein level but up-regulated at the RNA level. The deregulation of cellular gene expression by adenovirus is mediated at multiple levels and more complex than hitherto believed.

Activation of AMP-activated protein kinase reduces collagen production via p38 MAPK in cardiac fibroblasts induced by coxsackievirus B3.

Collagen deposition is the major cause of myocardial fibrosis, contributing to impaired cardiac contractile function in coxsackie virus B3 (CVB3)-infected hearts. Adenosine monophosphate-activated protein kinase (AMPK) has been considered as a cellular fuel gauge and super metabolic regulator, however, whether AMPK has an effect on collagen production in CVB3­infected heart remains to be elucidated. In the present study, the association between AMPK activation and CVB3­infected neonatal rat cardiac fibroblasts (NRCFs) was investigated. Collagen production was determined by the hydroxyproline content of the supernatant and by the expression of type I/IV collagen in the cell lysate. Rat hydroxyproline ELISA was used to detect hydroxyproline content in the supernatant. The expression of type I/IV collagen, and the phosphorylation of AMPKα­Thr172 and p38 in the cell lysate were evaluated using western blotting. As expected, it was found that the hydroxyproline content in the supernatant, and the production of collagen I/IV in the cell lysate were significantly promoted at 48 h post­CVB3­infection. However, this effect was inhibited in a dose­dependent manner when pretreated with 5­aminoimidazole­4­carboxamide­1­4­ribofuranoside (AICAR) for 2 h prior to CVB3­infection. However, if the cells were preincubated with compound C or SB203580 for 30 min prior the treatment with AICAR, the inhibitive effects of AICAR were reversed. The results of the western blotting indicated that the phosphorylation of AMPKα­Thr172 and p38 were significantly increased by AICAR in the NRCFs. However, only the phosphorylation of p38 mitogen­activated protein kinase (MAPK) was inhibited by SB203580. In conclusion, AMPK activation reduced collagen production via the p38 MAPK­dependent pathway in the cardiac fibroblasts induced by CVB3. The results of the present study may contribute to identifying an effective therapy for CVB3­induced myocarditis and CVB3-associated dilated cardiomyopathy.

Infection of Human Liver Myofibroblasts by Hepatitis C Virus: A Direct Mechanism of Liver Fibrosis in Hepatitis C.

BACKGROUND: Chronic hepatitis C is a major cause of liver fibrosis and cirrhosis. It is generally accepted that inflammation that occurs in response to hepatocyte infection by the hepatitis C virus (HCV) is the main mechanism that triggers myofibroblast differentiation and stimulation in chronic hepatitis C. The aim of this study was to determine if HCV might infect human liver myofibroblasts (HLMF) and directly stimulate their fibrogenic activities. METHODS: We evaluated the expression of the viral entry receptors, levels of HCV-RNA and HCV-protein and the expression of fibrosis markers in HLMF by using quantitative PCR, western blot and immunofluorescence analyses. Pseudoparticles (HCVpp) and cell culture-derived HCV (HCVcc) were used to study the ability of HLMF to support viral entry, replication and fibrosis induction. RESULTS: We showed that HLMF expressed all known molecules of the HCV receptor complex, i.e. CD81, LDL-R, scavenger receptor-BI, claudin-1 and occludin. These cells were also permissive to HCVpp entry. Inoculation with HCVcc caused short-term infection of these cells, as shown by their content in positive- and negative-strand HCV RNA, in core and NS3 viral proteins, and by their release of core protein levels in the culture supernatants. HCV infection stimulated myofibroblastic differentiation, proliferation and collagen production in these cells. In addition, evidence of in vivo infection was provided by the detection of positive- and negative-strand HCV RNA in preparations of HLMF obtained from HCV-infected patients. CONCLUSION: These findings indicate that HCV infection of HLMF can occur and trigger extracellular matrix overproduction, thereby contributing to the development of HCV-related liver fibrosis.

Epstein-Barr virus infection induces aberrant TLR activation pathway and fibroblast-myofibroblast conversion in scleroderma.

Scleroderma (SSc) is a complex and heterogeneous connective tissue disease mainly characterized by autoimmunity, vascular damage, and fibrosis that mostly involve the skin and lungs. Epstein-Barr virus (EBV) is a lymphotropic γ-herpesvirus that has co-evolved with human species, infecting >95% of the adult population worldwide, and has been a leading candidate in triggering several autoimmune diseases. Here we show that EBV establishes infection in the majority of fibroblasts and endothelial cells in the skin of SSc patients, characterized by the expression of the EBV noncoding small RNAs (EBERs) and the increased expression of immediate-early lytic and latency mRNAs and proteins. We report that EBV is able to persistently infect human SSc fibroblasts in vitro, inducing an aberrant innate immune response in infected cells. EBV-Toll-like receptor (TLR) aberrant activation induces the expression of selected IFN-regulatory factors (IRFs), IFN-stimulated genes (ISGs), transforming growth factor-ß1 (TGFß1), and several markers of fibroblast activation, such as smooth muscle actin and Endothelin-1, and all of these genes play a key role in determining the profibrotic phenotype in SSc fibroblasts. These findings imply that EBV infection occurring in mesenchymal, endothelial, and immune cells of SSc patients may underlie the main pathological features of SSc including autoimmunity, vasculopathy, and fibrosis, and provide a unified disease mechanism represented by EBV reactivation.

Role of TGF-ß1/p38 MAPK pathway in hepatitis B virus-induced tubular epithelial-myofibroblast transdifferentiation.

OBJECTIVE: This study is to investigate the hepatitis B virus (HBV)-induced tubular epithelial-myofibroblast transdifferentiation (TEMT) in human renal tubular epithelial HK-2 cells. METHODS: Human proximal tubular epithelial HK-2 cells were cultured. These HK-2 cells were divided into 4 groups: the blank control group, the vector control group, the HBV-transfected group, and the inhibitor-treated group. Transfection was performed with lipofectamine. Measurements of hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) in culture supernatant were determined by electrochemiluminescence immunoassay. Immunocytochemical staining, reverse transcription PCR (RT-PCR), and Western blot analysis were performed to detect the mRNA and protein expression levels, respectively. RESULTS: The immunocytochemical staining showed that, the expression level of E-cadherin was dramatically decreased, while the α-SMA expression level was significantly elevated, in HBV-transfected HK-2 cells. The mRNA level of TGF-ß1 and the protein level of p-p38 mitogen-activated protein kinase (MAPK) were elevated in HK-2 cells transfected with HBV. When treated with the p38 MAPK-specific inhibitor, the activation of p38 MAPK was eliminated in HBV-transfected HK-2 cells. In addition, the altered expression levels of E-cadherin and α-SMA, the increased contents of HBeAg and HBsAg in the culture supernatant, as well as the morphological changes of TEMT in HBV-transfected HK-2 cells, were all reversed by the inhibiter treatment. CONCLUSION: HBV transfection could induce TEMT in HK-2 cells, which was mediated by the TGF-ß1/p38 MAPK pathway. These findings provide new insights into the prevention and treatment of HBV-associated glomerulonephritis.

NADPH oxidase NOX4 mediates stellate cell activation and hepatocyte cell death during liver fibrosis development.

A role for the NADPH oxidases NOX1 and NOX2 in liver fibrosis has been proposed, but the implication of NOX4 is poorly understood yet. The aim of this work was to study the functional role of NOX4 in different cell populations implicated in liver fibrosis: hepatic stellate cells (HSC), myofibroblats (MFBs) and hepatocytes. Two different mice models that develop spontaneous fibrosis (Mdr2(-/-)/p19(ARF-/-), Stat3(Δhc)/Mdr2(-/-)) and a model of experimental induced fibrosis (CCl(4)) were used. In addition, gene expression in biopsies from chronic hepatitis C virus (HCV) patients or non-fibrotic liver samples was analyzed. Results have indicated that NOX4 expression was increased in the livers of all animal models, concomitantly with fibrosis development and TGF-ß pathway activation. In vitro TGF-ß-treated HSC increased NOX4 expression correlating with transdifferentiation to MFBs. Knockdown experiments revealed that NOX4 downstream TGF-ß is necessary for HSC activation as well as for the maintenance of the MFB phenotype. NOX4 was not necessary for TGF-ß-induced epithelial-mesenchymal transition (EMT), but was required for TGF-ß-induced apoptosis in hepatocytes. Finally, NOX4 expression was elevated in patients with hepatitis C virus (HCV)-derived fibrosis, increasing along the fibrosis degree. In summary, fibrosis progression both in vitro and in vivo (animal models and patients) is accompanied by increased NOX4 expression, which mediates acquisition and maintenance of the MFB phenotype, as well as TGF-ß-induced death of hepatocytes.

Toll-like receptor-4 expression by hepatic progenitor cells and biliary epithelial cells in HCV-related chronic liver disease.

Notwithstanding numerous evidences implicating toll-like receptor-4 (TLR4) in the pathogenesis of chronic hepatitis C virus (HCV) infection, the localization and level of TLR4 expression in the liver of patients with hepatitis C have never been investigated. We aimed to evaluate, by means of immunohistochemistry and real-time PCR (rt-PCR), hepatic TLR4 expression in patients with chronic HCV infection. Fifty patients who had undergone liver biopsy and 11 patients transplanted because of chronic HCV infection, and 12 controls free of liver disease, were included in the study. Each case was analyzed by immunohistochemistry for TLR4, α-smooth muscle actin and cytokeratin-7 (CK-7), and a subgroup of patients and all controls by rt-PCR for TLR4. Immunohistochemistry for α-smooth muscle actin was used to derive a score of activation of hepatic stellate cells and portal/septal myofibroblasts, while immunohistochemistry for CK-7 was used to evaluate and count hepatic progenitor cells, interlobular bile ducts and intermediate hepatocytes. In patients, the parenchymal elements responsible for the highest TLR4 level of expression were hepatic progenitor cells and biliary epithelial cells of interlobular bile ducts. Double-labeling experiments between anti-TLR4 and anti-CK7, anti-CD133, anti-CD44, anti-neural cell adhesion molecule, anti-epithelial cell adhesion molecule and anti-sex determining region Y-box 9, confirmed these findings. TLR4-positive hepatic progenitor cells and interlobular bile ducts were significantly correlated with the stage of liver disease (P<0.001), the grade of inflammation (P<0.001), and the activity of portal/septal myofibroblasts (P<0.001). rt-PCR study confirmed an increased TLR4 expression in the 26 patients analyzed with respect to controls (P<0.001). TLR4 expression positively correlated with fibrosis (P<0.05) and inflammation (P<0.05). The present results suggest that TLR4 expression by hepatic progenitor cells and biliary epithelial cells contributes to the progression of liver damage in the course of chronic HCV-related infection.