Modulation of MARKs-p38 signalling pathway in the presence of the structural hepatitis C virus (HCV) proteins

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide and often leads to chronic liver disease and hepatocellular carcinoma. The virus typically establishes a chronic infection in the liver that can promote serious hepatic disorders, such as hepatocellular carcinoma, over a period of decades. Although HCV is an enveloped virus, naked nucleocapsids have been reported in the serum of infected patients, and recently novel HCV subgenomes with deletions of the envelope proteins have been identified. Initial studies from our laboratory, previously published, indicated that expression of HCV core in insect cells can direct the formation of capsid-like particles lacking the envelope glycoproteins. These protein nanospheres, morphologically similar to natural capsids, were shown to be taken up by human hepatic cells and to produce cell signalling events. To follow the intracellular fate of these particles we fused the core protein to eGFP. We demonstrate that the chimeric proteins can be efficiently expressed, self-assembled, and form fluorescent non-enveloped capsid-like particles. Using FACS analysis, we provide evidence that the fluorescent particles can enter human immune cells such as T and B lymphocytes, as well as human myeloid leukaemia cells differentiated along the monocyte/macrophage-like pathway. Despite the fact that HCV is mainly a hepatotropic virus, it has been shown to infect and actively replicate in cells of the immune system altering their function and their cytokine expression. Using the baculovirus expression system we have generated recombinant non-enveloped capsid-like particles (HCVne) in the absence of other viral proteins and investigated their interference with signalling pathways in T-cells. HCVne internalisation was verified in Jurkat and Hut 78 T-cells, as well as primary human PBMCs and intrahepatic mononuclear cells. HCVne uptake leads to activation of the MAPKs (p38 and ERKs) signalling pathway. Using specific phosphoantibodies, signalling pathways inhibitors and chemical agents, was demonstrated that p38 activation in T-cells correlated with IL-2 transcriptional activation. c-fos and egr-1, two transcription factors, essential for IL-2 promoter activity, were also found elevated. In parallel, phosphorylated p38 translocates to the nucleus and phosphorylates CREB. All these transcription factors bind to IL-2 promoter and activate IL-2 gene transcription. Furthermore, HCVne uptake was accompanied with a parallel increase of IL-2 cytokine secretion. We propose that HCVne uptake by T-lymphocytes results in increased MAPKs activity and IL-2 expression, thus altering the host immune response.Ο ιός της ηπατίτιδας C (ΗCV) αποτελεί έναν από τους κύριους αιτιολογικούς παράγοντες της χρόνιας ηπατίτιδας και αποτελεί ένα από τα σημαντικότερα προβλήματα της δημόσιας υγείας. Το αυξημένο ποσοστό μετάπτωσης της HCV λοίμωξης σε χρόνια νόσο είναι δυνατόν να οφείλεται στην ικανότητα του ιού να μολύνει και να πολλαπλασιάζεται τόσο στα ηπατοκύτταρα, που είναι ο κύριος ξενιστής του ιού, όσο και στα λεμφοκύτταρα. Ο HCV ανήκει στην οικογένεια Flaviviridae και καψίδιο μικρής διαμέτρου που περιβάλλεται από μεμβρανικό φάκελο. Παρόλα αυτά έχουν ανιχνευτεί διαφορετικοί πληθυσμοί ιικών σωματιδίων στον ορό των ασθενών, με χαρακτηριστική την ύπαρξη γυμνών νουκλεοκαψιδίων χωρίς φάκελο. Ο ρόλος των γυμνών καψιδίων στη φυσική πορεία της λοίμωξης καθώς και ο μηχανισμός απελευθέρωσης τους στην κυκλοφορία του αίματος των ασθενών δεν είναι ακόμη γνωστά. Επίσης έχει αναφερθεί η παρουσία ελλειμματικών γονιδιωμάτων του ιού, στο ήπαρ και τον ορό ορισμένων ασθενών, ικανών να περικλείονται σε καψίδια. Η παρούσα εργασία επικεντρώθηκε στην κατασκευή ανασυνδυασμένων βακουλοιών, μέσω των οποίων επιτεύχθηκε απομόνωση και χαρακτηρισμός γυμνών core καψιδίων με ή χωρίς την ιδιότητα φθορισμού. Στη συνέχεια, έγινε διερεύνηση της πρόσδεσης και της εισόδου των GFP-συζευγμένων καψιδίων σε κύτταρα του ανοσοποιητικού συστήματος, με τη χρήση κυτταρομετρίας ροής. Ειδικότερα σε ότι αφορά τα γυμνά καψίδια (HCVne), επιβεβαιώθηκε η πρόσδεση και η είσοδος τους στα Jurkat και Hut-78 Τ-λεμφοκύτταρα, καθώς και σε πρωτογενή Τ-λεμφοκύτταρα, είτε περιφερικά είτε ηπατο-διηθούμενα. Επιπρόσθετα, μελετήθηκε και βρέθηκε ότι η είσοδος των HCVne οδηγεί στην παράλληλη ενεργοποίηση των MAPKs σηματοδοτικών μονοπατιών p38 και ERK. Με τη χρήση ειδικών αντισωμάτων, αναστολέων καθώς και πλασμιδίων αναφοράς μελετήθηκε το p38 σηματοδοτικό μονοπάτι που καταλήγει στη μεταγραφική ενεργοποίηση και έκκριση της ιντερλευκίνης IL-2. Στην παρούσα εργασία δείχθηκε ότι η HCVne-επαγόμενη p38 φωσφοσφορυλίωση οδηγεί στην ενεργοποίηση των μεταγραφικών παραγόντων CREB, EGR-1 και c-Fos, οι οποίοι στη συνέχεια προσδένονται στον υποκινητή της IL-2 και ενεργοποιούν τη μεταγραφή της. Η μελέτη των μηχανισμών που εμπλέκονται στην παρεμπόδιση της λειτουργίας του ανοσοποιητικού συστήματος του ξενιστή αποτελούν αντικείμενα εκτεταμένης έρευνας καθώς θα συμβάλλουν στην κατανόηση των μηχανισμών της παθογένειας της ηπατίτιδας C καθώς και της χρόνιας εμμένουσας λοίμωξης. Βασικός στόχος της παρούσας διατριβής είναι να συμβάλλει προς αυτήν την κατεύθυνση, μελετώντας την HCVne- επαγόμενη σηματοδότηση στα Τ-λεμφοκύτταρα. Συμπερασματικά η παρούσα μελέτη δείχνει ότι η είσοδος των HCVne οδηγεί σε αυξημένη ενεργότητα των MAPKs και σε αύξηση της έκφρασης της IL-2, τροποποιώντας έτσι την ανοσολογική απόκριση του ξενιστή

Phenotypic and functional alterations of primary human PBMCs induced by HCV non-enveloped capsid-like particles uptake

Hepatitis C virus non-enveloped particles circulate in the serum of HCV-infected patients and are believed to be involved in viral persistence. It was previously demonstrated that recombinant HCVne particles can efficiently enter T cells. In this study we investigated the effect of this entry on the phenotype and function of PBMCs, focused on the CD4+ and CD8+ T-cells. We have generated recombinant HCVne in the absence of other viral proteins. PBMCs from healthy donors were sampled after incubation either with HCVne or the control at different time points. Levels of expression of CD107a, CD25, CTLA-4, and T regulatory cells were estimated and cytokine expression and secretion were also monitored. Peripheral T cells expressed elevated CD127. The intracellular expression of the inhibitory marker CTLA-4 (CD152) increased significantly on peripheral T cells at late hours post-treatment, compared to the respective non-treated group. Despite the fact that there was an initial immune response due to HCVne uptake, T cells were driven to a partial exhausted phenotype. A significant induction of CD4+CD25+(hi)CD127-regulatory T cells at late hours was observed. Consistently, Foxp3+CD4+ T cells were also increased. In parallel, a significant transcriptional activation and increased secretion of IL-2, IL-10, and IFN-gamma, was recorded. Moreover, mRNA transcription of TGF-beta was considerably elevated. HCVne particles have the potential to shape the immune response by modifying specific phenotypic and functional markers mainly on CD4+ T cells and driving them to partial exhaustion as well as to Treg expansion

Exhausted-like CD8+ T cell phenotypes linked to C-peptide preservation in alefacept-treated T1D subjects

Clinical trials of biologic therapies in type 1 diabetes (T1D) aim to mitigate autoimmune destruction of pancreatic β cells through immune perturbation and serve as resources to elucidate immunological mechanisms in health and disease. In the T1DAL trial of alefacept (LFA3-Ig) in recent-onset T1D, endogenous insulin production was preserved in 30% of subjects for 2 years after therapy. Given our previous findings linking exhausted-like CD8+ T cells to beneficial response in T1D trials, we applied unbiased analyses to sorted CD8+ T cells to evaluate their potential role in T1DAL. Using RNA sequencing, we found that greater insulin C-peptide preservation was associated with a module of activation- and exhaustion-associated genes. This signature was dissected into 2 CD8 memory phenotypes through correlation with cytometry data. These cells were hypoproliferative, shared expanded rearranged TCR junctions, and expressed exhaustion-associated markers including TIGIT and KLRG1. The 2 phenotypes could be distinguished by reciprocal expression of CD8+ T and NK cell markers (GZMB, CD57, and inhibitory killer cell immunoglobulin-like receptor [iKIR] genes), versus T cell activation and differentiation markers (PD-1 and CD28). These findings support previous evidence linking exhausted-like CD8+ T cells to successful immune interventions for T1D, while suggesting that multiple inhibitory mechanisms can promote this beneficial cell state

Early changes in interferon signaling define natural killer cell response and refractoriness to interferon-based therapy of hepatitis C patients

Natural killer (NK) cells exhibit a polarized phenotype with increased cytotoxicity and decreased interferon gamma (IFN-γ) production in chronic hepatitis C virus (HCV) infection. Here, we asked whether this is caused by type I interferon (IFN)-induced expression and phosphorylation levels of signal transducer and activator of transcription (STAT) molecules in NK cells and whether it affects the response and refractoriness of NK cells to IFN-α-based therapy of HCV. STAT1 levels in NK cells were significantly higher in patients with chronic HCV infection than in uninfected controls. STAT1 levels and induction of phosphorylated STAT1 (pSTAT1) increased further during IFN-α-based therapy with preferential STAT1 over STAT4 phosphorylation. Induction of pSTAT1 correlated with increased NK cytotoxicity (tumor necrosis factor-apoptosis-inducing ligand [TRAIL] expression and degranulation) and decreased IFN-γ production. NK cells from patients with a greater than 2 log 10 first-phase HCV RNA decline to IFN-α-based therapy (>99% IFN effectiveness) displayed strong pSTAT1 induction in vivo and were refractory to further stimulation in vitro. In contrast, NK cells from patients with a less than 2 log 10 first-phase HCV RNA decline exhibited lower pSTAT1 induction in vivo (P = 0.024), but retained greater IFN-α responsiveness in vitro (P = 0.024). NK cells of all patients became refractory to in vivo and in vitro stimulation by IFN-α during the second-phase virological response. Conclusion: These data show that IFN-α-induced modulation of STAT1/4 phosphorylation underlies the polarization of NK cells toward increased cytotoxicity and decreased IFN-γ production in HCV infection, and that NK cell responsiveness and refractoriness correlate to the antiviral effectiveness of IFN-α-based therapy

Modulation of IL-2 expression after uptake of hepatitis C virus non-enveloped capsid-like particles: the role of p38 kinase

Hepatitis C virus (HCV) has been shown to actively replicate in cells of the immune system, altering both their function and cytokine expression. Naked nucleocapsids have been reported in the serum of infected patients. We investigated interference of recombinant non-enveloped capsid-like particles with signaling pathways in T cells. HCV non-enveloped particles (HCVne) internalization was verified in Jurkat and Hut 78 T cells, as well as primary human peripheral blood and intrahepatic mononuclear cells. HCVne uptake leads to activation of the MAPKs-p38 signaling pathway. Using specific phosphoantibodies, signaling pathways inhibitors, and chemical agents, it was demonstrated that p38 activation in T cells correlated with IL-2 transcriptional activation and was accompanied by a parallel increase of IL-2 cytokine secretion. c-fos and egr-1, two transcription factors, essential for IL-2 promoter activity, were also found to be elevated. We propose that HCVne uptake by T lymphocytes results in increased MAPKs-p38 activity and IL-2 expression, thus altering the host immune response

Ribavirin improves the IFN-γ response of natural killer cells to IFN-based therapy of hepatitis C virus infection

Ribavirin (RBV) is an important component of interferon (IFN)-based and direct antiviral treatment regimens for hepatitis C virus (HCV) infection. Immunomodulation, in particular improvement of the host IFN response, has been proposed as RBV's mechanism of action. Natural killer (NK) cells are sensitive biomarkers for IFN-α/β receptor signaling, as NK cell cytotoxicity and IFN-γ production are regulated by signal transducer and activator of transcription (STAT)1- and STAT4-phosphorylation, respectively. Specifically, pSTAT1-dependent NK cell cytotoxicity increases and pSTAT4-dependent IFN-γ production decreases in response to endogenous, virus-induced IFN-α and during IFN-α-based therapy. To assess whether RBV has a direct effect on NK cells and/or improves the IFN-γ response of NK cells in the presence of IFN-α, we prospectively studied 22 HCV patients with and 32 patients without 4 weeks of RBV pretreatment, who all received subsequent pegylated (Peg)IFN/ribavirin combination therapy. During RBV pretreatment, both the frequency of CD56dim NK cells with cytotoxic effector functions and the frequency of CD56bright NK cells with the capacity to produce IFN-γ decreased (P=0.049 and P=0.001, respectively). In vitro or in vivo exposure of NK cells to RBV improved the pSTAT4 (P<0.01) but not pSTAT1 response of NK cells to subsequent stimulation with IFN-α. This was associated with an increase in IFN-γ production but not cytotoxicity of NK cells during subsequent IFN-α-based therapy. The frequency of IFN-γ-producing NK cells was greater in fast second-phase virological responders than in slow responders. Conclusion: RBV enhances the pSTAT4 and IFN-γ response of NK cells to IFN-α-stimulation

Hepatitis C virus modulates lipid regulatory factor Angiopoietin-like 3 gene expression by repressing HNF-1 alpha activity

Background &amp; Aims: HCV relies on host lipid metabolism to complete its life cycle and HCV core is crucial to this interaction. Liver secreted ANGPTL-3 is an LXR-and HNF-1 alpha-regulated protein, which plays a key role in lipid metabolism by increasing plasma lipids via inhibition of lipase enzymes. Here we aimed to investigate the modulation of ANGPTL-3 by HCV core and identify the molecular mechanisms involved. Methods: qRT-PCR and ELISA were used to assess ANGPTL-3 mRNA and protein levels in HCV patients, the JFH-1 infectious system and liver cell lines. Transfections, chromatin immunoprecipitation and immunofluorescence delineated parts of the molecular mechanisms implicated in the core-mediated regulation of ANGPTL-3 gene expression. Results: ANGPTL-3 gene expression was decreased in HCV-infected patients and the JFH-1 infectious system. mRNA and promoter activity levels were down-regulated by core. The response was lost when an HNF-1 alpha element in ANGPTL-3 promoter was mutated, while loss of HNF-1 alpha DNA binding to this site was recorded in the presence of HCV core. HNF-1 alpha mRNA and protein levels were not altered by core. However, trafficking between nucleus and cytoplasm was observed and then blocked by an inhibitor of the HNF-1 alpha-specific kinase Mirk/Dyrk1B. Transactivation of LXR/RXR signalling could not restore coremediated down-regulation of ANGPTL-3 promoter activity. Conclusions: ANGPTL-3 is negatively regulated by HCV in vivo and in vitro. HCV core represses ANGPTL-3 expression through loss of HNF-1 alpha binding activity and blockage of LXR/RXR transactivation. The putative ensuing increase in serum lipid clearance and uptake by the liver may sustain HCV virus replication and persistence. (C) 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved

CD4+CD25+CD127hi cell frequency predicts disease progression in type 1 diabetes

Transient partial remission, a period of low insulin requirement experienced by most patients soon after diagnosis, has been associated with mechanisms of immune regulation. A better understanding of such natural mechanisms of immune regulation might identify new targets for immunotherapies that reverse type 1 diabetes (T1D). In this study, using Cox model multivariate analysis, we validated our previous findings that patients with the highest frequency of CD4+CD25+CD127hi (127-hi) cells at diagnosis experience the longest partial remission, and we showed that the 127-hi cell population is a mix of Th1- and Th2-type cells, with a significant bias toward antiinflammatory Th2-type cells. In addition, we extended these findings to show that patients with the highest frequency of 127-hi cells at diagnosis were significantly more likely to maintain β cell function. Moreover, in patients treated with alefacept in the T1DAL clinical trial, the probability of responding favorably to the antiinflammatory drug was significantly higher in those with a higher frequency of 127-hi cells at diagnosis than those with a lower 127-hi cell frequency. These data are consistent with the hypothesis that 127-hi cells maintain an antiinflammatory environment that is permissive for partial remission, β cell survival, and response to antiinflammatory immunotherapy

Efficacy and safety of azithromycin versus placebo to treat lower respiratory tract infections associated with low procalcitonin: a randomised, placebo-controlled, double-blind, non-inferiority trial

BACKGROUND: Lower respiratory tract infections are frequently treated with antibiotics, despite a viral cause in many cases. It remains unknown whether low procalcitonin concentrations can identify patients with lower respiratory tract infection who are unlikely to benefit from antibiotics. We aimed to compare the efficacy and safety of azithromycin versus placebo to treat lower respiratory tract infections in patients with low procalcitonin. METHODS: We conducted a randomised, placebo-controlled, double-blind, non-inferiority trial at five health centres in the USA. Adults aged 18 years or older with clinically suspected non-pneumonia lower respiratory tract infection and symptom duration from 24 h to 28 days were eligible for enrolment. Participants with a procalcitonin concentration of 0·25 ng/mL or less were randomly assigned (1:1), in blocks of four with stratification by site, to receive over-encapsulated oral azithromycin 250 mg or matching placebo (two capsules on day 1 followed by one capsule daily for 4 days). Participants, non-study clinical providers, investigators, and study coordinators were masked to treatment allocation. The primary outcome was efficacy of azithromycin versus placebo in terms of clinical improvement at day 5 in the intention-to-treat population. The non-inferiority margin was -12·5%. Solicited adverse events (abdominal pain, vomiting, diarrhoea, allergic reaction, or yeast infections) were recorded as a secondary outcome. This trial is registered with ClinicalTrials.gov, NCT03341273. FINDINGS: Between Dec 8, 2017, and March 9, 2020, 691 patients were assessed for eligibility and 499 were enrolled and randomly assigned to receive azithromycin (n=249) or placebo (n=250). Clinical improvement at day 5 was observed in 148 (63%, 95% CI 54 to 71) of 238 participants with full data in the placebo group and 155 (69%, 61 to 77) of 227 participants with full data in the azithromycin group in the intention-to-treat analysis (between-group difference -6%, 95% CI -15 to 2). The 95% CI for the difference did not meet the non-inferiority margin. Solicited adverse events and the severity of solicited adverse events were not significantly different between groups at day 5, except for increased abdominal pain associated with azithromycin (47 [23%, 95% CI 18 to 29] of 204 participants) compared with placebo (35 [16%, 12 to 21] of 221; between-group difference -7% [95% CI -15 to 0]; p=0·066). INTERPRETATION: Placebo was not non-inferior to azithromycin in terms of clinical improvement at day 5 in adults with lower respiratory tract infection and a low procalcitonin concentration. After accounting for both the rates of clinical improvement and solicited adverse events at day 5, it is unclear whether antibiotics are indicated for patients with lower respiratory tract infection and a low procalcitonin concentration. FUNDING: National Institute of Allergy and Infectious Diseases, bioMérieux