Imbalance Between Soluble and Membrane-Bound CD100 Regulates Monocytes Activity in Hepatitis B Virus-Associated Acute-on-Chronic Liver Failure.

CD100 is an important immune semaphorin that is a secreted and membrane bound protein involved in infectious diseases. However, CD100 expression profile and the regulation to innate immune system in hepatitis B virus (HBV)-associated acute-on-chronic liver failure (ACLF) was not previously reported. The aim of this study was to investigate CD100 level and modulatory function of CD100 to CD14+ monocytes in HBV-ACLF patients. Plasma-soluble CD100 (sCD100) level and membrane-bound CD100 (mCD100) expression on peripheral CD14+ monocytes was analyzed in HBV-ACLF patients. CD14+ monocytes-induced cytotoxicity and CD14+ monocytes-mediated T cell activation in response to CD100 stimulation was also assessed in direct and indirect contact coculture culture systems. HBV-ACLF patients had lower plasma sCD100 and higher mCD100 level on CD14+ monocytes compared with asymptomatic HBV carriers, chronic hepatitis B patients, and controls. CD14+ monocytes from HBV-ACLF patients induced limited target Huh7.5 cell death and secreted less interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and granzyme B in both direct and indirect contact coculture systems compared with controls. Recombinant sCD100 not only enhanced CD14+ monocytes-mediated Huh7.5 cell death and granzyme B secretion, but it also elevated CD14+ monocytes-induced IFN-γ/interleukin-17 production by CD4+ T cells as well as IFN-γ/TNF-α secretion by CD8+ T cells in HBV-ACLF patients. The current data indicated that severe inflammation induced sCD100/mCD100 imbalance to inactivate CD14+ monocytes response, which might be beneficial for the survival of HBV-ACLF patients.

GII.P16-GII.2 Recombinant Norovirus VLPs Polarize Macrophages Into the M1 Phenotype for Th1 Immune Responses.

Norovirus (NoV) is a zoonotic virus that causes diarrhea in humans and animals. Outbreaks in nosocomial settings occur annually worldwide, endangering public health and causing serious social and economic burdens. The latter quarter of 2016 witnessed the emergence of the GII.P16-GII.2 recombinant norovirus throughout Asia. This genotype exhibits strong infectivity and replication characteristics, proposing its potential to initiate a pandemic. There is no vaccine against GII.P16-GII.2 recombinant norovirus, so it is necessary to design a preventive vaccine. In this study, GII.P16-GII.2 type norovirus virus-like particles (VLPs) were constructed using the baculovirus expression system and used to conduct immunizations in mice. After immunization of mice, mice were induced to produce memory T cells and specific antibodies, indicating that the VLPs induced specific cellular and humoral immune responses. Further experiments were then initiated to understand the underlying mechanisms involved in antigen presentation. Towards this, we established co-cultures between dendritic cells (DCs) or macrophages (Mø) and naïve CD4+T cells and simulated the antigen presentation process by incubation with VLPs. Thereafter, we detected changes in cell surface molecules, cytokines and related proteins. The results indicated that VLPs effectively promoted the phenotypic maturation of Mø but not DCs, as indicated by significant changes in the expression of MHC-II, costimulatory factors and related cytokines in Mø. Moreover, we found VLPs caused Mø to polarize to the M1 type and release inflammatory cytokines, thereby inducing naïve CD4+ T cells to perform Th1 immune responses. Therefore, this study reveals the mechanism of antigen presentation involving GII.P16-GII.2 recombinant norovirus VLPs, providing a theoretical basis for both understanding responses to norovirus infection as well as opportunities for vaccine development.

Transcription Factor MafB Suppresses Type I Interferon Production by CD14+ Monocytes in Patients With Chronic Hepatitis C.

Transcription factor MafB regulates differentiation and activity of monocytes/macrophage and is associated with the development of atherosclerosis and cancers. However, the role of MafB in modulation of CD14+ monocytes in chronic viral hepatitis was not fully elucidated. Thus, the aim of current study was to investigate the immunoregulatory function of MafB to type I interferon (IFN) secretion by CD14+ monocytes and its contribution to pathogenesis of chronic hepatitis C virus (HCV) infection. A total of 29 chronic hepatitis C patients and 21 healthy individuals were enrolled. Serum IFN-α1 and IFN-ß was measured by ELISA, while MafB mRNA and protein expression were assessed by real-time PCR and Western blot. MafB siRNA or MafB expression plasmid was transfected into purified CD14+ monocytes to suppress or increase MafB expression. The function of MafB siRNA transfected CD14+ monocytes to HCV in cell culture (HCVcc)-infected Huh7.5 cells or CD4+ T cells was also investigated in direct and indirect contact co-culture system. Serum IFN-α1 and IFN-ß was robustly reduced in chronic hepatitis C patients. By contrast, MafB was notably elevated in chronic hepatitis C patients and negatively correlated with serum IFN-α1. Overexpression of MafB reduced the IFN-α1 production by CD14+ monocytes from healthy individuals. However, MafB inhibition elevated IFN-α1 secretion by CD14+ monocytes and interferon regulatory factor 3 phosphorylation in chronic hepatitis C. MafB inhibition also promoted CD14+ monocytes-induced viral clearance in HCVcc-infected Huh7.5 cells by up-regulation of IFN-α1 and IFN-ß without increasingly destroying hepatocytes, however, did not affect CD14+ monocytes-induced CD4+ T cells differentiation in chronic hepatitis C patients. The current data revealed that overexpression of MafB in chronic hepatitis C patients might suppress type I IFN production by CD14+ monocytes, leading to the viral persistence. MafB might be a potential therapeutic target for treatment of chronic hepatitis C.

Notch signaling suppresses CD14+ monocytes cells activity in patients with chronic hepatitis C.

Hepatitis C virus (HCV) infection always leads to chronic hepatitis via dysregulation of host immunity. Notch signaling also modulates the response of monocytes/macrophages. Thus, we aimed to investigate the regulatory role of Notch signaling to CD14+ monocytes. Forty patients with chronic hepatitis C and twenty normal controls (NC) were enrolled. CD14+ monocytes and CD4+ T cells were purified from peripheral bloods. Notch receptors' mRNA expression in CD14+ monocytes was semi-quantified by real-time PCR. Cytokine production by CD14+ monocytes in response to γ-secretase inhibitor (GSI) was investigated by ELISA. GSI-induced CD14+ monocytes activity to HCV clearance in Huh7.5 cells and to CD4+ T cell differentiation was also assessed in direct and indirect contact co-culture system. Notch1 mRNA relative level was approximately 10-fold elevated in CD14+ monocytes from chronic hepatitis C patients when compared with NC. GSI stimulation resulted in enhanced cytokines production by CD14+ monocytes from chronic hepatitis C patients. GSI-stimulated CD14+ monocytes from chronic hepatitis C patients induced suppression of HCV RNA replication in both direct and indirect contact co-culture system of CD14+ monocytes and HCVcc-infected Huh7.5 cells, and this process was accompanied by elevation of interferon-γ production but not increased target cell death. Moreover, GSI stimulation also enhanced CD14+ monocytes-induced Th1 and Th17 cells activation, and this process required direct cell-to-cell contact. Effective antiviral therapy down-regulated Notch1 mRNA expression and promoted cytokine production by CD14+ monocytes from chronic hepatitis C. Current data revealed an important immunoregulatory property of Notch signaling to CD14+ monocytes in chronic HCV infection.

Interleukin-7 Regulates T Follicular Helper Cell Function in Patients with Chronic Hepatitis C.

Signaling through interleukin (IL)-7 is essential and required for development, differentiation, proliferation, and homeostasis of T cells. However, the role of IL-7 in regulation of CD4+ T cells in chronic viral infections was not fully elucidated. Thus, the aim of the current study was to investigate the immunomodulatory activity of IL-7 to T follicular helper (Tfh) cells and its contribution to pathogenesis of chronic HCV, hepatitis C virus (HCV) infection. A total of 47 patients with chronic hepatitis C and 19 normal controls were enrolled. Serum IL-7 and proportion of Tfh cells was measured. The regulatory function of IL-7 to Tfh cells was also investigated in CD4+ T cells and CD4+ T/HCVcc-infected Huh7.5 cell cocultured system. Serum IL-7 concentration was significantly downregulated in patients with chronic hepatitis C, and was negatively correlated with HCV RNA level. Tfh frequency and Tfh-associated cytokines (IL-21 and IL-6) were also reduced in chronic HCV-infected patients. Moreover, recombinant IL-7 stimulation elevated proportion of Tfh cells and IL-21/IL-6 secretion in both HCV-specific and nonspecific manners. Furthermore, IL-7-treated CD4+ T cells exhibited elevated antiviral activities without killing infected hepatocytes, which presented as inhibition of HCV RNA, induction of antiviral proteins, and promotion of cytokine production (especially IL-21) in cocultured system. This process might be dependent on IL-6 secretion. The current data revealed that IL-7 regulated HCV-specific and nonspecific activated Tfh cells, which might contribute to viral clearance. IL-7 could be a potential therapeutic agent for the treatment of chronic hepatitis C.

Decreased CD4+CD25+CD127dim/- Regulatory T Cells and T Helper 17 Cell Responsiveness to Toll-Like Receptor 2 in Chronic Hepatitis C Patients with Daclatasvir Plus Asunaprevir Therapy.

Direct-acting antivirals (DAAs) not only rapidly inhibited hepatitis C virus (HCV) replication but also modulated innate and adaptive immune response in chronic hepatitis C patients. However, the regulatory activity of DAAs to Toll-like receptor 2 (TLR2) stimulation on CD4+CD25+CD127dim/- regulatory T cells (Tregs) and T helper (Th) 17 cells was not completely understood. In the present study, a total of 23 patients with chronic HCV genotype 1b infection were enrolled, and blood samples were collected at baseline (treatment naive), end of therapy (EOT), and 12 weeks after EOT (SVR12) with daclatasvir plus asunaprevir therapy. TLR2 expression on Tregs and Th17 cells was measured by flow cytometry. Cellular proliferation, cytokine production, and suppressive activity were also tested in purified CD4+CD25+CD127dim/- Tregs in response to the stimulation of Pam3Csk4, an agonist of TLR2. Inhibition of HCV RNA by daclatasvir and asunaprevir did not affect either percentage of Tregs/Th17 cells or TLR2 expression on Tregs/Th17 cells. Pam3Csk4 stimulation also did not influence either cellular proliferation or Tregs/Th17 proportion at each time point. Stimulation with Pam3Csk4 only enhanced the suppressive function and interleukin (IL)-35 production by Tregs purified from baseline, but not those from EOT or SVR12. Similarly, Pam3Csk4 stimulation only elevated Th17 cell frequency of CD4+ T cells from baseline, but not those from EOT or SVR12. Moreover, daclatasvir and asunaprevir therapy did not promote TLR2-induced shift of Tregs toward Th17-like phenotype and function. These data suggested that daclatasvir plus asunaprevir therapy resulted in the decreased responsiveness of Tregs/Th17 cells to TLR2 stimulation in chronic hepatitis C patients, which might provide a novel mechanism underlying DAA-induced immunoregulation.

Notch Signaling Regulates Circulating T Helper 22 Cells in Patients with Chronic Hepatitis C.

Notch signaling enhanced the response of interleukin (IL)-22-producing CD4+ T cells that were defined as T helper 22 (Th22) cells, and Notch-aryl hydrocarbon receptor (AhR)-IL-22 axis fine-tuned inflammatory response. Previous studies have demonstrated that both Notch signaling and Th22 cells took part in the pathogenesis of chronic hepatitis C virus (HCV) infection. Thus, in this study, we aimed at examining the regulatory role of Notch signaling in Th22 cells in HCV infection. A total of 59 patients with chronic hepatitis C and 22 normal controls (NCs) were enrolled in this study. The percentage of Th22 cells and mRNA expression of related transcriptional factors and cytokines were analyzed in response to γ-secretase inhibitor. Th22 cell frequency was significantly elevated in chronic hepatitis C in comparison with that in NCs. Inhibition of Notch signaling downregulated HCV-specific Th22 cells and IL-22 production, which was accompanied by the reduction of AhR and modulatory cytokines (IL-6 and tumor necrosis factor-α). Moreover, the suppression of Notch signaling also decreased the IL-22-mediated antimicrobial response in both normal and HCV-infected HepG2 cells/Huh7.5 cells. This process was also accompanied by the depression of signal transducers and activators of transcription 3 signaling. In conclusion, the current results suggested that Notch signaling acted as a critical pathway in determining the response to IL-22 in chronic hepatitis C. Thus, Notch-Th22 axis might be considered a new therapeutic target for HCV-infected patients.

Toll-Like Receptor 2 Modulates the Balance of Regulatory T Cells and T Helper 17 Cells in Chronic Hepatitis C.

Regulatory T cells (Tregs) and interleukin-17-producing T helper (Th17) cells were mutually antagonistic in the pathogenesis of chronic hepatitis C virus (HCV) infection. However, the regulation of imbalance between Tregs and Th17 cells was poorly understood in HCV infection. A recent report revealed the immunomodulatory role of Toll-like receptor (TLR) 2 in regulating the balance of Tregs/Th17 functions in multiple sclerosis. Thus, the aim of the current study was to assess the effect of TLR2 stimulation on the suppressive function of Tregs and Th17 differentiation in chronic hepatitis C. A total of 65 patients with chronic hepatitis C receiving pegylated interferon-a2a and ribavirin therapy for 48 weeks, as well as 20 of normal controls (NCs) were enrolled. Cellular proliferation and cytokine production was tested in purified CD4(+)CD25(+)CD127(dim/-) Tregs in response to the stimulation of Pam3Csk4, an agonist of TLR2. In treatment-naive patients, Tregs, but not Th17 cells, from chronic hepatitis C patients expressed higher levels of TLR2 compared with NCs. Stimulation with Pam3Csk4 enhanced the suppressive function of Tregs and production of IL-10 in chronic hepatitis C more than in NCs. However, TLR2 stimulation did not promote Th17 differentiation of Tregs in chronic hepatitis C patients. Moreover, effective anti-HCV therapy resulted in the induction of IL-17-secreting phenotypic shift of Tregs without loss of inhibitive function upon TLR2 stimulation. These data provided a novel mechanism underlying modulating the balance of Tregs/Th17 cells in chronic hepatitis C. HCV infection shifted Tregs/Th17 cells through TLR2 stimulation by inducing Tregs to produce IL-10 and enhancing inhibitive function of effector T cells, resulting in viral persistence.

Expression profile and kinetics of cytokines and chemokines in patients with chronic hepatitis C.

Cytokines and chemokines play an important role in defense against viral infection and modulating immune response. However, expression prolife of serum cytokines and chemokines, which were associated with the outcome of patients in response to anti-HCV treatment have not been fully elucidated. The current study aimed to determine the expression pattern of cytokines and chemokines in chronic HCV infection and their association with outcome in response to therapy. Seventy-two patients with HCV infection were enrolled, and fifty-one received peg-interferon α-2a and ribavirin therapy for 48 weeks. Thirty-nine cytokines and chemokines were analyzed by Luminex 200 and ELISA. In comparison to healthy individuals, production of IL-8 and IL-10 were increased in chronic hepatitis C patients. In contrast, IFN-γ, IL-7, and IL-15 were remarkably decreased, especially in HCV genotype 1b infection. HCV RNA load is closely associated with IL-10 and IL-15 expressions, and inhibition of HCV replication was accompanied by reduction in IL-10 and elevation in IL-7 and IL-15. Skewed cytokines and chemokines expression existed in chronic HCV infection, and might play an important role in persistent HCV infection. Exploiting the expression pattern of cytokines and chemokines may help to develop a better understanding of the pathogenesis of chronic HCV infection.

[Establishment of a fluorescent real-time quantitative RT-PCR assay for detection of genotype 4 hepatitis E virus in swine stools].

The primers and probes for the Real-time RT-PCR were designed based on the multiple sequence (swine and humans HEV strains) alignments of the ORF3 region of genotype 4 HEV. A rapid, sensitive and stable TaqMan Real-time RT-PCR assay was established, and its specificity and sensitivity were assessed, and comparison of the Real-time RT-PCR with conventional and nested RT-PCR was performed. The results found that the crossing points showed linearly proportional to the logarithm of the input copy number. The correlation coefficient (R2) and the slope value of the standard curves with plasmid DNA were 0.994 and -3.312, respectively. The efficiency (E) of the PCR was 100%. Coefficients of variation values of the different diluted plasmid DNA were low in the same or different repeated experimental group. In addition, the assay was able to correctly detect genotype 4 HEV RNA from swine fecal samples. The sensitivity of established assay was 100-fold higher than that of conventional RT-PCR and 10-fold higher than nested RT-PCR.