ABSTRACTS BY
INVITED SPEAKERS
Potential Role of miRNA 34a in HIV-1 biology: novel role of NQO1 in regulating Tat/Rev levels
Akhil C Banerjea
National Institute of Immunology, New Delhi, India.
HIV-1 infection + Tat & Vpr, independently up-regulate expression of miRNA34a which has earlier been shown to be involved with neuronal dysfunction/neurocognitive disorder. We show that HIV-1 infection enhances miRNA34a in a time-dependent manner in T-cells. Our overexpression and knock-down based experiments suggest a positive, feed-forward loop between miRNA34a and HIV-1 replication. Mechanistically, HIV-1 exploits the levels of a phosphatase called PNUTS which is a direct target of miRNA34a. Overexpression of PNUTS potently inhibits HIV-1 replication in T-cells. PNUTS negatively regulates HIV-1 transcription by inhibiting the assembly of transcription elongation factor p-TEFp involving cyclin T1 and CDK9. In our related studies, we show that Rev caused the decrease in the levels of cytoplasmic Tat. NQO1 causes potent stabilization of HIV-1 Tat protein and its inhibitor, Dicoumarol, results in potent degradation of Tat. Interestingly, the Rev protein caused reduction in NQO1 levels. Thus, Rev is able to induce degradation of Tat protein indirectly by down-regulating NQO1 levels.
Role of microRNA in modulating Japanese Encephalitis Virus induced neuroinflammation in microglia
Arup Banerjee
Vaccine and Infectious Disease Research Center, Translational Health Science and Technology Institute, Faridabad, India
Microglia are the CNS-resident macrophages that play an important role in both innate and adaptive immune responses in the CNS. Microglia function in normal and diseased CNS can be regulated through small (~22 nucleotide) non-coding RNAs, called microRNAs. Japanese Encephalitis Virus (JEV), a leading cause of viral encephalitis in India and other South East Asian countries, can use microglia as a long-term reservoir and may cause changes in microRNA (miRNA) and mRNA profiles. These changes in the global microRNAome may play an important role in determining the pathology of encephalitis caused by JEV infection. Here, we will discuss our recent microRNA and mRNA array data obtained from JEV infected human microglia (CHME3) cells and their involvement in modulating JEV induced neuroinflammation in the brain. Furthermore, the importance of identifying circulating microRNAs in CSF and their significance in understanding JEV induced pathogenicity will be discussed.
Insights into the neural stem/progenitor cell response to Japanese encephalitis virus infection
Sriparna Mukherjee3,1, Noopur Singh1, Nabonita Sengupta1, Mahar Fatima1, Pankaj Seth1, Anita Mahadevan2, Susarla Krishna Shankar2, Arindam Bhattacharyya3
and Anirban Basu1
1National Brain Research Centre,Manesar,India
2National Institute of Mental Health & Neurosciences, Bangalore, India
3University of Calcutta, Kolkata, India
Japanese Encephalitis Virus (JEV), one of the major neurotropic viruses in Asia, causes acute encephalopathy in both adult and infant individuals. In fact, children are more susceptible to viral infection. JEV survivors have a higher incidence of severe mental retardation, learning disabilities, behavioural abnormalities, and speech and movement disorders.
JEV has been shown to infect the subventricular zone (SVZ), one of the potent neurogenic niches of the brain that harbours neural stem/progenitor cells (NSPC). In vitro neurosphere cultures from the SVZ isolated from P-7 BALB/c pups when infected with JEV, shows reduced diameter and less BrdU incorporation with progressive viral infection. JEV induces increased expression of cell cycle inhibitory proteins and eventually proliferation arrest in infected NSPCs. Minocycline, a tetracycline, when administered in BALB/c mouse restores BrdU incorporation in NSPCs. Minocycline treated microglial culture supernatant enlarges neurosphere size along with increased basal level expression of cell cycle regulatory proteins. Furthermore, proteomic analysis of hNS1 cells (human neural stem cell) reveals endoplasmic reticulum stress. ER chaperone GRP78, mitochondrial protein Prohibitin, and heterogeneous nuclear ribonucleoprotein hnRNPC (C1/C2) were found to interact with viral RNA leading to ROS enhancement and apoptosis. Upon siRNA mediated silencing of these proteins, NSPCs inhibited viral replication and consequently caspase3 cleavage. Alterations of these proteins were also seen in multipotent neural precursor cells isolated from human foetus and autopsy samples of clinically diagnosed JE patients. Thus, to date, the diverse effects of JEV in neural stem/progenitor cells include cell cycle arrest, hampered neuronal differentiation, and proteomic alteration leading to ER stress mediated apoptosis.
The impact of RNA virus evolution on Host responses in the CNS
J. David Beckham
University of Colorado School of Medicine, Colorado, USA
Viral infections have co-evolved with eukaryotic cells and multicellular organisms. Thus, viruses have developed unique niches within the ecosystems that humans are current disrupting resulting in viral adaptation and novel infections of the central nervous system (CNS). The recent emergence of Zika virus is classical example of a novel adaptation in an emerging RNA virus infection that now has infected millions of people worldwide. Complex nervous systems have developed specialized mechanisms to prevent viral infections of the CNS. We now show that the neuronal protein, alpha-synuclein, inhibits viral infections in the CNS by preventing trafficking from the peripheral nervous system. Also, we have recently discovered that Zika virus, a novel neuroinvasive flavivirus, has conserved interactions with host XRN1 expression that determines the outcome of disease. By defining the conserved virus-host interactions in the CNS, we can develop novel vaccines and therapeutic approaches to prevent the high morbidity and mortality associated with viral infections in the CNS.
Drug abuse mediated potentiation of HAND: Blaming the messenger
Shilpa Buch, Guoku Hu, Ke Liao, Lu Yang, YeonHee Kook and Yu Cai.
University of Nebraska Medical Center, Omaha, USA
Opiate abuse and HIV-1 have been described as two linked global health crises, and despite the advent of anti-retroviral therapy, abuse of opiates has been shown to result in increased neurologic and cognitive deficits. Using the morphine-dependent rhesus macaques (RMs) infected with CCR5-utilizing SIVR71/17E we recapitulated the human syndrome demonstrating augmentation of neuropathology & neuroinflammation and rapid disease progression compared with SIV-infected RMs without opiate dependence. Mortality in these rapid progressors was associated with robust microglial activation and neuronal injury. MicroRNA (miR)-mediated regulation of disease pathogenesis represents an evolving area of research that has ramifications for identification of potential therapeutic targets for various neurodegenerative disorders for which currently there exists no cure. Herein we demonstrated that morphine & HIV Tat modulate increased neuropathology via two complementary mechanisms: a) Morphine exposed astrocytes upregulate expression & release of miR-138 in the extracellular vesicles (EVs), which, following uptake by the microglia, results in their activation via the TLR7-dependent pathway and, b) HIV Tat exposure resulted in increased induction/release of miR-9 and -29b in the EVs isolated from astrocytes. MiR-9-enriched EVs, were in turn, taken up by the neurons, resulting in neuronal injury. We further demonstrated that mutation of the GUUGUGU motif in miR-138 that is homologous to a TLR7 binding domain ablated this activation. Corroboration of these cell culture findings was further validated in vivo wherein morphine administration in wild-type mice resulted in activation of microglia. Furthermore, we also demonstrated that upregulation of miR-9 and -29b in EVs released from HIV Tat or morphine exposed astrocytes could target the tropic factor platelet-derived growth factor (PDGF) in neurons, resulting in neuronal damage. Taken together, our findings implicate that morphine and HIV protein-Tat co-operatively dysregulate EV miRNAs, thereby resulting in neuroinflammation and neuronal degeneration in HIV-infected opiates abusers.
Targeting Gut and Brain Axis to eliminate CNS reservoirs
Siddappa N. Byrareddy
University of Nebraska Medical Center, Omaha, USA
The gut and brain is connected via the gut-brain axis (GBA), in which neural and immunological signals aretransmitted between the central nervous system (CNS) and the gut. During acute infection, HIV/SIV targets gut CD4+ T cells and macrophages because these sites express high numbers of CCR5-expressing activated CD4+ T-cells. Targeting of HIV/SIV infection to the gut results in severe depletion of gut CD4+ T-cells and mucosal tissue dysfunction, resulting in leaky gut, microbial translocation, and chronic immune activation. The integrin 47 is found at high levels on the surface of some CD4+ T-cells and is involved in gut-cell trafficking. Some strains of HIV/SIV are able to bind to 47 on α4β7hi CD4+ T cells, making the cells more susceptible to infection. Increased frequencies of α4β7hi-expressing CD4+ T cells within GALT at the time of infection appear to correlate with increased viral loads and the rate of disease progression in post SIV infection. Intravenous (i.v.) administration of an anti-47 monoclonal antibody to rhesus macaques protected the gastroinestinal associated lymphoid tissues (GALT) from infection when RMs were challenged either i.v, i.r and IVAG routes, increased levels of peripheral blood naive and central memory CD4+ T cells, and inhibit the mobilization of natural killers cells and plasmacytoid dendritic cells. Remarkably, administration of anti-α4β7 mAb to ART-treated SIV-infected RMs resulted in a highly significant, unprecedented suppression of plasma/GALT viral loads even after ART treatment Interruption. Since, gut and brain is connected via the GBA, we hypothesize that by controlling viral loads in GALT resulting in reducing reservoirs in gastrointestinal tissues, which indirectly reduces the CNS reservoir. During the presentation will discuss new data on macaque models and hypothesis to target gut-brain axis.
Viral-Neural Cell Interphase and Neuroimmune Modulation in Demyelination
Jayasri Das Sarma
Indian Institute of Science Education and Research Kolkata, India.
Neurotropic mouse hepatitis virus (MHV) infection in mice provides a useful tool for studying mechanisms of demyelination in a virus-induced experimental model of Multiple Sclerosis (MS). It has been known for a long time that, in the absence of conventional ??T cells, microglia play a major role in neurotropic MHV-induced demyelination. However, the mechanisms of Central Nervous System (CNS) infection remainunknown. Our current microglial tropism studies revealed that RSA59, an isogenic demyelinating strain of MHV, can infect and activate CNS resident microglia, and microglia can help to mediate demyelination by engulfing myelin debris. Affymetrix microarray analysis was performed to compare differential spinal cord mRNA levels between mice infected with demyelinating and non-demyelinating strains of MHV to identify host immune genes expressed in this demyelinating disease model. The study reveals that during the acute stage of infection, both strains induce inflammatory innate immune response genes, whereas upregulation of several immunoglobulin genes during chronic stage infection is unique to infection with the demyelinatingstrain. Results suggest that the demyelinating strain induced an innate-immune response during acute infection that may promote switching of Ig isotype genes during chronic infection, potentially playing a role in antibody-mediated progressive demyelination even after viral clearance. Understanding the role of direct CNS resident microglial infection and activation will shed light on the pathogenesis of CNS inflammatory disease and chronic CNS disorders.
In addition, RSA59/MHV-A59 infection of astrocytes reduces intercellular communication. The attenuation of astrocytic communication appears to be a bystander effect and that infection of astrocytes by MHV causes deterioration in neural communication. As neuro-glial signaling has been shown to play an important role in synaptogenesis, microglial activation, and neurogenesis, the attenuation of neuro-glial signaling reported here may constitute an additional underlying substrate for CNS dysfunction associated with MHV induced demyelination.
Infiltrating Myeloid Cells Initiate Acute Seizures Following Viral Encephalitis
Ana Beatriz DePaula-Silva, Tyler J. Hanak, Daniel J. Doty, Jordan T. Sim, Jane E. Libbey, and Robert S. Fujinami
University of Utah School of Medicine, Salt Lake City, USA
Approximately 20% of individuals who survive viral encephalitis go on to develop epilepsy. A third of patientswith epilepsy are treatment resistant. We have developed a model for virus induced temporal lobe epilepsy(TLE) which mirrors clinical and pathologic features of individuals with TLE. Theiler’s murine encephalomyelitisvirus (TMEV) is a neurotropic picornavirus that has tropism for the hippocampus. TMEV infection of SJL/J micehas been used as a model for inflammatory demyelinating diseases such as multiple sclerosis. SJL/J mice areunable to clear TMEV and virus is able to persist in central nervous system (CNS) glia and macrophages. Incontrast C57BL/6 mice are able to clear TMEV. However, these TMEV infected C57BL/6 mice develop acuteencephalitis which is accompanied by seizures. TMEV is cleared from the CNS of these mice and after a latentperiod mice develop spontaneous recurrent seizures – epilepsy. We have found that infiltrating myeloid cellssuch as macrophages are important for the development of acute seizures during the viral encephalitis phase ofdisease. The occurrence of acute seizures is not dependent on the adaptive immune system such as anti-viralCD8+ T cells. The infiltrating macrophages produce interleukin-6 while resident microglial cells produce tumornecrosis factor. The production of these two cytokines is important for seizure development. We also havefound that the expression of the metabotropic glutamate receptor mGluR5 is altered in the hippocampus of TMEVinfected mice and this likely also contributes to the development of acute seizures. Compounds such asminocycline or wogonin can reduce the numbers of infiltrating macrophages into the CNS following TMEVinfection which results in fewer mice experiencing seizures. The TMEV infection seizure model is an importantplatform for understanding the mechanism of seizure manifestation and for testing drugs that could be used tolimit seizures.
T cells and CNS injury during viral encephalomyelitis
Diane E. Griffin, Victoria Baxter, Kirsten Kulcsar and Nina Martin
Johns Hopkins Bloomberg School of Public Health, Baltimore, USA
Alphaviruses are important mosquito-borne causes of acute encephalomyelitis. The prototypic alphavirus Sindbis virus infects neurons and causes acute encephalomyelitis in mice. The outcome of infection is dependent on virus strain, age and host genetic background. Clearance of infectious virus during nonfatal infection is dependent on antibody to the E2 glycoprotein produced by infiltrating B cells and on interferon-γ produced by infiltrating NK cells and T cells. Viral RNA decreases gradually and then persists for the life of the mouse accompanied by long-term residence of B cells producing SINV-specific antibody and SINV-specific CD4+ and CD8+ T cells producing IFN-γ. At late times after virus clearance brain T cells were primarily characterized as effector memory CD4+ and CD8+ T cells or resident memory CD8+ cells. TRM were less abundant in mice deficient in IFN-γ signaling. Fatal disease in C57BL/6 mice is due to an immunopathologic process mediated by CD4+ T cells producing IL-17 and GM-CSF modulated by regulatory T cells producing IL-10 and by local production of TGF-β. Treatments that decrease CNS inflammation decrease paralysis and improve survival.
Disruption of epithelial cell junctions in a model of chronic Human Immunodeficiency Virus exposure
Michael Koval, K. Sabrina Lynn, Barbara Schlingmann and David M. Guidot
Emory University School of Medicine, Atlanta, USA
The introduction of Highly Active Anti-Retroviral Therapy (HAART) has helped patients manage HIV-1 infection as a chronic disease, significantly extending life expectancy. Despite this, unexpected complications of chronic HIV-1 are now becoming evident. Using a transgenic rat model that simulates non-immunocompromised HIV-1, we have found that the pulmonary epithelial barrier is impaired, that may parallel the effects of HIV-1 on vascular endothelium to weaken blood brain barrier function. Primary alveolar epithelial cells (AECs) isolated from HIV-1 transgenic rats were used as an in vitro system to study how HIV-1 proteins affect the lung barrier. Compared with control cells, AECs from HIV-1 transgenic rats were leakier, based on transepithelial resistance and paracellular flux measurements. We then investigated HIV-1 induced changes to AEC tight junctions by immunoblot and immunofluorescence microscopy and found that tight junction composition in HIV tg AECs was dysregulated, with elevated protein expression of claudin-4 and decreased ZO-1 and claudin-18 expression. Immunofluorescence staining of claudin-18 showed that tight junction morphology was also disrupted. These data suggest a direct effect of HIV-1 proteins on the processing and assembly of AEC tight junctions. Current studies are underway to explore how this occurs at a molecular level, with particular emphasis on understanding how HIV-1 proteins affect the quality control pathways necessary for the correct synthesis and processing of transmembrane junction proteins. A major goal is to determine whether other classes of viruses have a comparable effect on quality control of junction proteins resulting in a negative impact on cell and tissue homeostasis.
Alteration of epigenetic state by small molecule modulators of histone acetyltransferases: Implications in therapeutics
Tapas K Kundu
Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India
Epigenetic modifications affect chromatin state and hence, altered levels of epigenetic modifications also affect physiological homeostasis along with playing a role in different pathophysiological conditions such as cancer, neurodegenerative disorders, diabetes, asthma, and COPD. Our laboratory has discovered several small molecule modulators of these enzymes, which may serve as lead scaffolds to design new generation therapeutics. Apart from showing the effect of lysine acetyltransferases in repressing oral, liver, and prostate cancer progression in xenografted animal model systems, we have also studied their effect on neurodegenerative diseases and viral infection, which will be the focus of my talk. We have shown that a p300 specific inhibitor discovered in our laboratory could potently inhibit the multiplication of HIV in a cellular system. Using a novel histone acetyltransferase activator molecule, we find that p300/CBP mediated acetylation of histones is an important inducing factor for robust neurogenesis; which contributes to long-term spatial memory to an extent where it can even rescue memory loss in a Tau mice model back to the normal level. Hence, these activators and inhibitors of lysine acetyltransferases have a wide range of therapeutic applications.