Reciprocal regulation of farnesoid X receptor α activity and hepatitis B virus replication in differentiated HepaRG cells and primary human hepatocytes.

Hepatitis B virus (HBV) and bile salt metabolism seem tightly connected. HBV enters hepatocytes by binding to sodium taurocholate cotransporting polypeptide (NTCP), the genome of which contains 2 active farnesoid X receptor (FXR) α response elements that participate in HBV transcriptional activity. We investigated in differentiated HepaRG cells and in primary human hepatocytes (PHHs) effects of FXR activation on HBV replication and of infection on the FXR pathway. In HepaRG cells, FXR agonists (6-ethyl chenodeoxycholic acid and GW4064), but no antagonist, and an FXR-unrelated bile salt inhibited viral mRNA, DNA, and protein production (IC50, 0.1-0.5 µM) and reduced covalently closed circular DNA pool size. These effects were independent of the NTCP inhibitor cyclosporine-A, which suggests inhibition occurred at a postentry step. Similar results were obtained in PHHs with GW4064. Infection of these cells increased expression of FXR and modified expression of FXR-regulated genes SHP, APOA1, NTCP, CYP7A1, and CYP8B1 with a more pronounced effect in PHHs than in HepaRG cells. FXR agonists reversed all but one of the HBV-induced FXR gene profile modifications. HBV replication and FXR regulation seem to be interdependent, and altered bile salt metabolism homeostasis might contribute to the persistence of HBV infection.-Radreau, P., Porcherot, M., Ramière, C., Mouzannar, K., Lotteau, V., André, P. Reciprocal regulation of farnesoid X receptor α activity and hepatitis B virus replication in differentiated HepaRG cells and primary human hepatocytes.

Hepatitis C virus/human interactome identifies SMURF2 and the viral protease as critical elements for the control of TGF-ß signaling.

TGF-ß signaling induces epithelial to mesenchymal transition (EMT) and plays an important role in hepatocellular carcinoma (HCC) development. Clinical observations indicate that hepatitis C virus (HCV) chronic infection, which is a major cause of HCC, induces TGF-ß signaling perturbations. Here, we investigate the mechanisms by which HCV nonstructural proteins interfere with TGF-ß signaling, in human hepatoma cell lines expressing HCV subgenomic replicon. A transcriptomic study showed that TGF-ß stimulation of these cells resulted in a protumoral gene expression profile and in up-regulation of EMT-related genes compared to control interferon-treated cells not expressing HCV proteins. We found that the viral protease NS3-4A interacted with SMURF2, a negative regulator of TGF-ß signaling. In cells expressing HCV subgenomic replicon or NS3-4A, TGF-ß stimulation induced an increased expression of SMAD-dependent genes compared to control cells. This enhanced signaling was suppressed by SMURF2 overexpression and mimicked by SMURF2 silencing. In addition, NS3-4A expression resulted in an increased and prolonged TGF-ß-induced phosphorylation of SMAD2/3 that was abrogated by SMURF2 overexpression. Neither NS3-4A protease activity nor SMURF2 ubiquitin-ligase activity was required to affect TGF-ß signaling. Therefore, by targeting SMURF2, NS3-4A appears to block the negative regulation of TGF-ß signaling, increasing the responsiveness of cells to TGF-ß.

Improved adenovirus type 5 vector-mediated transduction of resistant cells by piggybacking on coxsackie B-adenovirus receptor-pseudotyped baculovirus.

Taking advantage of the wide tropism of baculoviruses (BVs), we constructed a recombinant BV (BV(CAR)) pseudotyped with human coxsackie B-adenovirus receptor (CAR), the high-affinity attachment receptor for adenovirus type 5 (Ad5), and used the strategy of piggybacking Ad5-green fluorescent protein (Ad5GFP) vector on BV(CAR) to transduce various cells refractory to Ad5 infection. We found that transduction of all cells tested, including human primary cells and cancer cell lines, was significantly improved using the BV(CAR)-Ad5GFP biviral complex compared to that obtained with Ad5GFP or BV(CAR)GFP alone. We determined the optimal conditions for the formation of the complex and found that a high level of BV(CAR)-Ad5GFP-mediated transduction occurred at relatively low adenovirus vector doses, compared with transduction by Ad5GFP alone. The increase in transduction was dependent on the direct coupling of BV(CAR) to Ad5GFP via CAR-fiber knob interaction, and the cell attachment of the BV(CAR)-Ad5GFP complex was mediated by the baculoviral envelope glycoprotein gp64. Analysis of the virus-cell binding reaction indicated that the presence of BV(CAR) in the complex provided kinetic benefits to Ad5GFP compared to the effects with Ad5GFP alone. The endocytic pathway of BV(CAR)-Ad5GFP did not require Ad5 penton base RGD-integrin interaction. Biodistribution of BV(CAR)-Ad5Luc complex in vivo was studied by intravenous administration to nude BALB/c mice and compared to Ad5Luc injected alone. No significant difference in viscerotropism was found between the two inocula, and the liver remained the preferred localization. In vitro, coagulation factor X drastically increased the Ad5GFP-mediated transduction of CAR-negative cells but had no effect on the efficiency of transduction by the BV(CAR)-Ad5GFP complex. Various situations in vitro or ex vivo in which our BV(CAR)-Ad5 duo could be advantageously used as gene transfer biviral vector are discussed.

Th1 and Th17 balance in inflammatory myopathies: interaction with dendritic cells and possible link with response to high-dose immunoglobulins.

To clarify the interactions between dendritic cells (DCs) and Th1 and Th17 T cell subsets and the mode of action of IVIG in inflammatory myopathies, Expression of CD4(+) and CD8(+) T cells, immature (CD1a) and mature (DC-LAMP) DCs, interleukin-17 (IL-17) and interferon-gamma (IFN-gamma), was quantified by immunohistochemistry in muscle biopsies from 13 patients (11 with polymyositis (PM) and 2 dermatomyositis (DM)) obtained before treatment with IVIG. The Th1/Th17 cytokine and the immature/mature DC ratio were studied according to the response to IVIG. Immature DCs were rarely detected compared to mature DCs, observed in all samples except one PM. IFN-gamma-producing cell count was higher than IL-17 count. Neither the expression of IFN-gamma nor IL-17 was correlated with that of DC subsets. Seven of the 13 patients (6 PM and 1 DM) responded to IVIG. T cells and DC subsets were not differentially expressed between responders and non-responders. The frequency of IFN-gamma-producing cells was significantly higher in non-responders with an increased IFN-gamma/IL-17-producing-cell ratio. In conclusion, mature rather than immature DC and IFN-gamma-rather than IL-17-producing cells accumulate in inflamed muscle. Increased IFN-gamma-producing cell count and IFN-gamma/IL-17-ratio were found in IVIG non-responders, suggesting a role for the Th17 mediated pathway in the response to IVIG.