Subcellular localization and function study of a secreted phospholipase C from Nocardia seriolae

Fish nocardiosis is a chronic systemic granulomatous disease, andNocardia seriolaeis the main pathogen that causes this disease. But the pathogenesis and virulence factors ofN. seriolaeare not fully understood. A phospholipase C (PLC), which was likely to be a secreted protein targeting host cell mitochondria, was found by the bioinformatics analysis on the whole genome sequence ofN. seriolae. In order to determine the subcellular localization and study the preliminary function of PLC fromN. seriolae(NsPLC), the gene cloning, secreted protein identification, subcellular localization in host cells and apoptosis detection of NsPLC were carried out in this study. The results showed that NsPLC was a secreted protein by mass spectrometry analysis of extracellular products fromN. seriolae. Subcellular localization of NsPLC-GFP fusion protein in FHM cells revealed that the green fluorescence exhibited a punctate distribution near the nucleus and did not co-localize with mitochondria. In addition, apoptosis assay suggested that apoptosis was induced in FHM cells by the overexpression of NsPLC. This study may lay the foundation for further study on the function of NsPLC and promote the understanding of the virulence factors and pathogenic mechanism ofN. seriolae

T-regulatory cells and vaccination “pay attention and do not neglect them”: Lessons from HIV and cancer vaccine trials

Efficient vaccines are characterized by the establishment of long-lived memory T cells, including T-helper (effectors and follicular) and T-regulatory cells (Tregs). While the former induces cytotoxic or antibody responses, the latter regulates immune responses by maintaining homeostasis. The role of Tregs in inflammatory conditions is ambiguous and their systematic monitoring in vaccination along with effector T-cells is not instinctive. Recent studies from the cancer field clearly showed that Tregs suppress vaccine-induced immune responses and correlate with poor clinical benefit. In HIV infection, Tregs are needed during acute infection to preserve tissue integrity from an overwhelmed activation, but are not beneficial in chronic infection as they suppress anti-HIV responses. Current assays used to evaluate vaccine-induced specific responses are limited as they do not take into account antigen-specific Tregs. However, new assays, such as the OX40 assay, which allow for the simultaneous detection of a full range of Th-responses including antigen-specific Tregs responses, can overcome these issues. In this review article we will revise the role of Tregs in vaccination and review the recent work performed in the field, including the available tools to monitor them, from novel assays to humanized mouse models

Liver immune-pathogenesis and therapy of human liver tropic virus infection in humanized mouse models

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infect and replicate primarily in human hepatocytes. Few reliable and easy accessible animal models are available for studying the immune system’s contribution to the liver disease progression during hepatitis virus infection. Humanized mouse models reconstituted with human hematopoietic stem cells (HSCs) have been developed to study human immunology, human immunodeficiency virus 1 infection, and immunopathogenesis. However, a humanized mouse model engrafted with both human immune and human liver cells is needed to study infection and immunopathogenesis of HBV/HCV infection in vivo. We have recently developed the humanized mouse model with both human immune and human liver cells (AFC8-hu HSC/Hep) to study immunopathogenesis and therapy of HCV infection in vivo. In this review, we summarize the current models of HBV/HCV infection and their limitations in immunopathogenesis. We will then present our recent findings of HCV infection and immunopathogenesis in the AFC8-hu HSC/Hep mouse, which supports HCV infection, human T-cell response and associated liver pathogenesis. Inoculation of humanized mice with primary HCV isolates resulted in long-term HCV infection. HCV infection induced elevated infiltration of human immune cells in the livers of HCV-infected humanized mice. HCV infection also induced HCV-specific T-cell immune response in lymphoid tissues of humanized mice. Additionally, HCV infection induced liver fibrosis in humanized mice. Anti-human alpha smooth muscle actin (αSMA) staining showed elevated human hepatic stellate cell activation in HCV-infected humanized mice. We discuss the limitation and future improvements of the AFC8-hu HSC/Hep mouse model and its application in evaluating novel therapeutics, as well as studying both HCV and HBV infection, human immune responses, and associated human liver fibrosis and cancer

Regulatory T Cells Contribute to HIV-1 Reservoir Persistence in CD4 + T Cells Through Cyclic Adenosine Monophosphate-Dependent Mechanisms in Humanized Mice in Vivo

Background. Regulatory T cells (Tregs) suppress T-cell immune activation and human immunodeficiency virus type 1 (HIV-1) replication, but the role of Tregs in HIV-1 reservoir persistence is poorly defined. Methods. Tregs were depleted by denileukin diftitox in humanized mice with chronic HIV-1 infection. Viral replication in lineage cells was determined by p24 expression. Levels of HIV-1 RNA and DNA in human cells, as well as replication-competent-virus- producing cells, were measured to quantified viral replication and reservoirs. Results. Treg depletion resulted in a blip of HIV-1 replication in T cells but not in myeloid cells. The major activated reservoir cells were memory CD4+ T cells in vivo. Interestingly, the transient activation of viral replication led to HIV-1 reservoir reduction after viremia resuppression, as indicated by the quantity of HIV-1 DNA and replication-competent-virus-producing cells. Furthermore, we demonstrated that Tregs use cyclic adenosine monophosphate (cAMP)-dependent protein kinase A pathway to inhibit HIV-1 activation and replication in resting conventional T cells in vitro. Conclusion. Tregs suppress HIV-1 replication in T cells and contribute to HIV-1 reservoir persistence. cAMP produced in Tregs is involved in their suppression of viral gene activation and expression. Treg depletion combined with combination antiretroviral therapy provides a novel strategy for HIV-1 cure

Type I interferons suppress viral replication but contribute to T cell depletion and dysfunction during chronic HIV-1 infection

The direct link between sustained type I interferon (IFN-I) signaling and HIV-1-induced immunopathogenesis during chronic infection remains unclear. Here we report studies using a monoclonal antibody to block IFN-α/β receptor 1 (IFNAR1) signaling during persistent HIV-1 infection in humanized mice (hu-mice). We discovered that, during chronic HIV-1 infection, IFNAR blockade increased viral replication, which was correlated with elevated T cell activation. Thus, IFN-Is suppress HIV-1 replication during the chronic phase but are not essential for HIV-1-induced aberrant immune activation. Surprisingly, IFNAR blockade rescued both total human T cell and HIV-specific T cell numbers despite elevated HIV-1 replication and immune activation. We showed that IFNAR blockade reduced HIV-1-induced apoptosis of CD4+ T cells. Importantly, IFNAR blockade also rescued the function of human T cells, including HIV-1-specific CD8+ and CD4+ T cells. We conclude that during persistent HIV-1 infection, IFN-Is suppress HIV-1 replication, but contribute to depletion and dysfunction of T cells

Minicircle HBV cccDNA with a Gaussia luciferase reporter for investigating HBV cccDNA biology and developing cccDNA-targeting drugs

Chronic Hepatitis B Virus (HBV) infection is generally not curable with current anti-viral drugs. Virus rebounds after stopping treatment from the stable HBV covalently-closed-circular DNA (cccDNA). The development of drugs that directly target cccDNA is hampered by the lack of robust HBV cccDNA models. We report here a novel HBV cccDNA technology that will meet the need. We engineered a minicircle HBV cccDNA with a Gaussia Luciferase reporter (mcHBV-GLuc cccDNA), which serves as a surrogate to measure cccDNA activity. The mcHBV-GLuc cccDNA was easily produced in bacteria, and it formed minichromosomes as HBV cccDNA episome DNA does when it was transfected into human hepatocytes. Compared to non-HBV minicircle plasmids, mcHBV-GLuc cccDNA showed persistent HBV-GLuc activity and HBx-dependent gene expression. Importantly, the mcHBV-GLuc cccDNA showed resistance to interferons (IFN) treatment, indicating its unique similarity to HBV cccDNA that is usually resistant to long-term IFN treatment in chronic HBV patients. Most importantly, GLuc illuminates cccDNA as a surrogate of cccDNA activity, providing a very sensitive and quick method to detect trace amount of cccDNA. The mcHBV-GLuc cccDNA model is independent of HBV infection, and will be valuable for investigating HBV cccDNA biology and for developing cccDNA-targeting drugs

Specific activation in vivo of HIV-1 by a bromodomain inhibitor from monocytic cells in humanized mice under antiretroviral therapy

Combination antiretroviral therapy (cART) effectively suppresses HIV-1 replication and enables HIV-infected individuals to live long, productive lives. However, the persistence of HIV-1 reservoirs of both T and myeloid cells with latent or low-replicating HIV-1 in patients under cART makes HIV-1 infection an incurable disease. Recent studies have focused on the development of strategies to activate and purge these reservoirs. Bromodomain and extraterminal domain proteins (BETs) are epigenetic readers involved in modulating gene expression. Several bromodomain inhibitors (BETi) are reported to activate viral transcription in vitro in HIV-1 latency cell lines in a P-TEFb (CDK9/cyclin T1)-dependent manner. Little is known about BETi efficacy in activating HIV-1 reservoir cells under cART in vivo. Here we report that a BETi (I-BET151) efficiently activated HIV-1 reservoirs under effective cART in humanized mice in vivo. Interestingly, I-BET151 during suppressive cART in vivo activated HIV-1 gene expression only in monocytic cells and not in CD4 T cells. We further demonstrate that BETi preferentially enhanced HIV-1 gene expression in monocytic cells rather than in T cells and that whereas CDK9 was involved in activating HIV-1 by I-BET151 in both monocytic and T cells, CDK2 enhanced HIV-1 transcription in monocytic cells but inhibited it in T cells. Our findings reveal a role for CDK2 in differential modulation of HIV-1 gene expression in myeloid cells and in T cells and provide a novel strategy to reactivate monocytic reservoirs with BETi during cART. IMPORTANCE Bromodomain inhibitors have been reported to activate HIV-1 transcription in vitro, but their effect on activation of HIV-1 reservoirs during cART in vivo is unclear. We found that BETi (I-BET151) treatment reactivated HIV-1 gene expression in humanized mice during suppressive cART. Interestingly, I-BET151 preferentially reactivated HIV-1 gene expression in monocytic cells, but not in CD4 T cells, in cART-treated mice. Furthermore, I-BET151 significantly increased HIV-1 transcription in monocytic cells, but not in HIV-1-infected CD4 T cells, via CDK2-dependent mechanisms. Our findings suggest that BETi can preferentially activate monocytic HIV-1 reservoir cells and that a combination of reservoir activation agents targeting different cell types and pathways is needed to achieve reactivation of different HIV-1 reservoir cells during cART