Increased IL-6 expression precedes reliable viral detection in the rhesus macaque brain during acute SIV infection.

Knowledge of immune activation in the brain during acute HIV infection is crucial for the prevention and treatment of HIV-associated neurological disorders. We determined regional brain (basal ganglia, thalamus, and frontal cortex) immune and virological profiles at 7 and 14 days post infection (dpi) with SIVmac239 in rhesus macaques. The basal ganglia and thalamus had detectable viruses earlier (7 dpi) than the frontal cortex (14 dpi) and contained higher quantities of viruses than the latter. Increased immune activation of astrocytes and significant infiltration of macrophages in the thalamus at 14 dpi coincided with elevated plasma viral load, and SIV colocalized only within macrophages. RNA signatures of proinflammatory responses, including IL-6, were detected at 7 dpi in microglia and interestingly, preceded reliable detection of virus in tissues and were maintained in the chronically infected macaques. Countering the proinflammatory response, the antiinflammatory response was not detected until increased TGF-ß expression was found in perivascular macrophages at 14 dpi. But this response was not detected in chronic infection. Our data provide evidence that the interplay of acute proinflammatory and antiinflammatory responses in the brain likely contributed to the overt neuroinflammation, where the immune activation preceded reliable viral detection.

HIV infection of astrocytes compromises inter-organelle interactions and inositol phosphate metabolism: A potential mechanism of bystander damage and viral reservoir survival.

HIV-associated neurological dysfunction is observed in more than half of the HIV-infected population, even in the current antiretroviral era. The mechanisms by which HIV mediates CNS dysfunction are not well understood but have been associated with the presence of long-lasting HIV reservoirs. In the CNS, macrophage/microglia and a small population of astrocytes harbor the virus. However, the low number of HIV-infected cells does not correlate with the high degree of damage, suggesting that mechanisms of damage amplification may be involved. Here, we demonstrate that the survival mechanism of HIV-infected cells and the apoptosis of surrounding uninfected cells is regulated by inter-organelle interactions among the mitochondria/Golgi/endoplasmic reticulum system and the associated signaling mediated by IP3 and calcium. We identified that latently HIV-infected astrocytes had elevated intracellular levels of IP3, a master regulator second messenger, which diffuses via gap junctions into neighboring uninfected astrocytes resulting in their apoptosis. In addition, using laser capture microdissection, we confirmed that bystander apoptosis of uninfected astrocytes and the survival of HIV-infected astrocytes were dependent on mitochondrial function, intracellular calcium, and IP3 signaling. Blocking gap junction channels did not prevent an increase in IP3 or inter-organelle dysfunction in HIV-infected cells but reduced the amplification of apoptosis into uninfected neighboring cells. Our data provide a mechanistic explanation for bystander damage induced by surviving infected cells that serve as viral reservoirs and provide potential targets for interventions to reduce the devastating consequences of HIV within the brain.

Tunneling nanotubes, TNT, communicate glioblastoma with surrounding non-tumor astrocytes to adapt them to hypoxic and metabolic tumor conditions.

Cell-to-cell communication is essential for the development and proper function of multicellular systems. We and others demonstrated that tunneling nanotubes (TNT) proliferate in several pathological conditions such as HIV, cancer, and neurodegenerative diseases. However, the nature, function, and contribution of TNT to cancer pathogenesis are poorly understood. Our analyses demonstrate that TNT structures are induced between glioblastoma (GBM) cells and surrounding non-tumor astrocytes to transfer tumor-derived mitochondria. The mitochondrial transfer mediated by TNT resulted in the adaptation of non-tumor astrocytes to tumor-like metabolism and hypoxia conditions. In conclusion, TNT are an efficient cell-to-cell communication system used by cancer cells to adapt the microenvironment to the invasive nature of the tumor.

NK Cells Contribute to the Immune Risk Profile in Kidney Transplant Candidates.

Background: A previously proposed immune risk profile (IRP), based on T cell phenotype and CMV serotype, is associated with mortality in the elderly and increased infections post-kidney transplant. To evaluate if NK cells contribute to the IRP and if the IRP can be predicted by a clinical T cell functional assays, we conducted a cross sectional study in renal transplant candidates to determine the incidence of IRP and its association with specific NK cell characteristics and ImmuKnow® value. Material and Methods: Sixty five subjects were enrolled in 5 cohorts designated by age and dialysis status. We determined T and NK cell phenotypes by flow cytometry and analyzed multiple factors contributing to IRP. Results: We identified 14 IRP+ [CMV seropositivity and CD4/CD8 ratio < 1 or being in the highest quintile of CD8+ senescent (28CD-/CD57+) T cells] individuals equally divided amongst the cohorts. Multivariable linear regression revealed a distinct IRP+ group. Age and dialysis status did not predict immune senescence in kidney transplant candidates. NK cell features alone could discriminate IRP- and IRP+ patients, suggesting that NK cells significantly contribute to the overall immune status in kidney transplant candidates and that a combined T and NK cell phenotyping can provide a more detailed IRP definition. ImmuKnow® value was negatively correlated to age and significantly lower in IRP+ patients and predicts IRP when used alone or in combination with NK cell features. Conclusion: NK cells contribute to overall immune senescence in kidney transplant candidates.

Role of Connexin and Pannexin containing channels in HIV infection and NeuroAIDS.

Neuron-Glia crosstalk is essential for efficient synaptic communication, cell growth and differentiation, neuronal activity, neurotransmitter recycling, and brain immune response. The master regulators of this neuron-glia communication are connexin containing Gap Junctions (GJs) and Hemichannels (HCs) as well as pannexin HCs. However, the role of these channels under pathological conditions, especially in infectious diseases is still in exploratory stages. Human Immunodeficiency Virus-1 (HIV) is one such infectious agent that takes advantage of the host intercellular communication systems, GJs and HCs, to exacerbate viral pathogenesis in the brain in spite of the antiretroviral therapy effectively controlling viral replication in the periphery. Although most infectious agents lead to total "shutdown" of gap junctional communication in parenchymal cells, HIV infection maintains and "hijacks" GJs and HCs to enable few infected cells to spread toxic intracellular agents to neighboring uninfected cells aggravating viral neuropathology even in the absence of viral replication. In this mini-review, we present a comprehensive overview of the role of GJs and HCs in augmenting HIV neuropathogenesis.

Identification, Localization, and Quantification of HIV Reservoirs Using Microscopy.

The major barrier to eradicating human immunodeficiency virus-1 (HIV) infection is the generation and extended survival of HIV reservoirs. In order to eradicate HIV infection, it is essential to detect, quantify, and characterize circulating and tissue-associated viral reservoirs in infected individuals. Currently, PCR-based technologies and Quantitative Viral Outgrowth Assays (Q-VOA) are the gold standards to detect viral reservoirs. However, these methods are limited to detecting circulating viral reservoirs, and it has been shown that they misrepresent the size of the reservoirs, largely because they detect only one component of the HIV life cycle and are unable to detect viral reservoirs in tissues. Here, we described the use of multiple detection systems to identify integrated HIV DNA or viral mRNA and several HIV proteins in circulating and tissue reservoirs using improved staining and microscopy techniques. We believe that this imaging-based approach for detecting HIV reservoirs will lead to breakthroughs necessary to eradicate these reservoirs. © 2018 by John Wiley & Sons, Inc.

Connexin43 Containing Gap Junction Channels Facilitate HIV Bystander Toxicity: Implications in NeuroHIV.

Human immunodeficiency virus-1 (HIV-1) infection compromises the central nervous system (CNS) in a significant number of infected individuals, resulting in neurological dysfunction that ranges from minor cognitive deficits to frank dementia. While macrophages/microglia are the predominant CNS cells infected by HIV, our laboratory and others have shown that HIV-infected astrocytes, although present in relatively low numbers with minimal to undetectable viral replication, play key role in NeuroAIDS pathogenesis. Our laboratory has identified that HIV "hijacks" connexin (Cx) containing channels, such as gap junctions (GJs) and hemichannels (HCs), to spread toxicity and apoptosis to uninfected cells even in the absence of active viral replication. In this study, using a murine model with an astrocyte-directed deletion of Cx43 gene (hGFAP-cre Cx43fl/fl) and control Cx43fl/fl mice, we examined whether few HIV-infected human astrocytoma cells (U87-CD4-CCR5), microinjected into the mouse cortex, can spread toxicity and apoptosis through GJ-mediated mechanisms, into the mouse cells, which are resistant to HIV infection. In the control Cx43fl/fl mice, microinjection of HIV-infected U87-CD4-CCR5 cells led to apoptosis in 84.28 ± 6.38% of mouse brain cells around the site of microinjection, whereas hGFAP-cre Cx43fl/fl mice exhibited minimal apoptosis (2.78 ± 1.55%). However, simultaneous injection of GJ blocker, 18α-glycyrrhetinic acid, and Cx43 blocking peptide along with microinjection of HIV-infected cells prevented apoptosis in Cx43fl/fl mice, demonstrating the Cx43 is essential for HIV-induced bystander toxicity. In conclusion, our findings demonstrate that Cx43 expression, and formation of GJs is essential for bystander apoptosis during HIV infection. These findings reveal novel potential therapeutic targets to reduce astrocyte-mediated bystander toxicity in HIV-infected individuals because despite low to undetectable viral replication in the CNS, Cx channels hijacked by HIV amplify viral neuropathogenesis.

Pannexin1 Channels Are Required for Chemokine-Mediated Migration of CD4+ T Lymphocytes: Role in Inflammation and Experimental Autoimmune Encephalomyelitis.

Pannexin1 (Panx1) channels are large high conductance channels found in all vertebrates that can be activated under several physiological and pathological conditions. Our published data indicate that HIV infection results in the extended opening of Panx1 channels (5-60 min), allowing for the secretion of ATP through the channel pore with subsequent activation of purinergic receptors, which facilitates HIV entry and replication. In this article, we demonstrate that chemokines, which bind CCR5 and CXCR4, especially SDF-1α/CXCL12, result in a transient opening (peak at 5 min) of Panx1 channels found on CD4(+) T lymphocytes, which induces ATP secretion, focal adhesion kinase phosphorylation, cell polarization, and subsequent migration. Increased migration of immune cells is key for the pathogenesis of several inflammatory diseases including multiple sclerosis (MS). In this study, we show that genetic deletion of Panx1 reduces the number of the CD4(+) T lymphocytes migrating into the spinal cord of mice subjected to experimental autoimmune encephalomyelitis, an animal model of MS. Our results indicate that opening of Panx1 channels in response to chemokines is required for CD4(+) T lymphocyte migration, and we propose that targeting Panx1 channels could provide new potential therapeutic approaches to decrease the devastating effects of MS and other inflammatory diseases.

Mechanisms of HIV Neuropathogenesis: Role of Cellular Communication Systems.

BACKGROUND: One of the major complications of Human Immunodeficiency Virus (HIV) infection is the development of HIV-Associated Neurocognitive Disorders (HANDs) in approximately 50-60% of HIV infected individuals. Despite undetectable viral loads in the periphery owing to anti-retroviral therapy, neuroinflammation and neurocognitive impairment are still prevalent in HIV infected individuals. Several studies indicate that the central nervous system (CNS) abnormalities observed in HIV infected individuals are not a direct effect of viral replication in the CNS, rather these neurological abnormalities are associated with amplification of HIV specific signals by unknown mechanisms. We propose that some of these mechanisms of damage amplification are mediated by gap junction channels, pannexin and connexin hemichannels, tunneling nanotubes and microvesicles/exosomes. OBJECTIVE: Our laboratory and others have demonstrated that HIV infection targets cell to cell communication by altering all these communication systems resulting in enhanced bystander apoptosis of uninfected cells, inflammation and viral infection. Here we discuss the role of these communication systems in HIV neuropathogenesis. CONCLUSION: In the current manuscript, we have described the mechanisms by which HIV "hijacks" these host cellular communication systems, leading to exacerbation of HIV neuropathogenesis, and to simultaneously promote the survival of HIV infected cells, resulting in the establishment of viral reservoirs.

Involvement of extracellular signal-regulated kinase (ERK1/2)-p53-p21 axis in mediating neural stem/progenitor cell cycle arrest in co-morbid HIV-drug abuse exposure.

Neurological complications in opioid abusing Human Immunodeficiency Virus-1 (HIV-1) patients suggest enhanced neurodegeneration as compared to non-drug abusing HIV-1 infected population. Neural precursor cells (NPCs), the multipotent cells of the mammalian brain, are susceptible to HIV-1 infection and as opiates also perturb their growth kinetics, detailed mechanistic studies for their co-morbid exposure are highly warranted. Using a well characterized in vitro model of human fetal brain-derived neural precursor cells, we investigated alterations in NPC properties at both acute and chronic durations. Chronic morphine and Tat treatment attenuated proliferation in NPCs, with cells stalled at G1-phase of the cell cycle. Furthermore HIV-Tat and morphine exposure increased activation of extracellular signal-regulated kinase-1/2 (ERK1/2), enhanced levels of p53 and p21, and decreased cyclin D1 and Akt levels in NPCs. Regulated by ERK1/2 and p53, p21 was found to be indispensible for Tat and morphine mediated cell cycle arrest. Our study elaborates on the cellular and molecular machinery in NPCs and provides significant mechanistic details into HIV-drug abuse co-morbidity that may have far reaching clinical consequences both in pediatric as well as adult neuroAIDS.

A growth factor attenuates HIV-1 Tat and morphine induced damage to human neurons: implication in HIV/AIDS-drug abuse cases.

The neuropathological abnormalities of human immunodeficiency virus (HIV)-1 patients abusing illicit drugs suggest extensive interactions between the two agents, thereby leading to increased rate of progression to neurodegeneration. The role of HIV-1 transactivating protein, Tat has been elucidated in mediating neuronal damage via apoptosis, a hallmark of HIV-associated dementia (HAD), however the underlying mechanisms involved in enhanced neurodegeneration by illicit drugs remain elusive. In this study, we demonstrated that morphine enhances HIV-Tat induced toxicity in human neurons and neuroblastoma cells. Enhanced toxicity by Tat and morphine was accompanied by increased numbers of TUNEL positive apoptotic neurons, elevated caspase-3 levels and decreased ratio of anti- and pro-apoptotic proteins, Bcl2/Bax. Tat and morphine together elicited high levels of reactive oxygen species that were NADPH dependent. Significant alterations in mitochondrial membrane homeostasis were also observed with co-exposure of these agents. Extensive studies of mitogen activated protein kinase (MAPK) signaling pathways revealed the involvement of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase-1/2 (ERK1/2) pathways in enhanced toxicity of Tat and morphine. In addition to this, we found that pre-treatment of cells with platelet derived growth factor (PDGF-BB) protected neurons from HIV-Tat and morphine induced damage. PDGF-BB alleviated ROS production, maintained mitochondrial membrane potential, decreased caspase-3 activation and hence protected the cells from undergoing apoptosis. PDGF-BB mediated protection against Tat and morphine involved the phosphatidylinositol-3 kinase (PI3K) pathway, as specific inhibitor of PI3K abrogated the protection conferred by PDGF-BB. This study demonstrates the mechanism of enhanced toxicity in human neurons subjected to co-exposure of HIV protein Tat and morphine, thus implying its importance in HIV positive drug abusers, where damage to the brain is reported to be more severe than non-drug abusers. We have also showed for the first time that PDGF-BB can protect against simultaneous exposure of Tat and morphine, strengthening its role as a neuroprotective agent that could be considered for therapeutic intervention.

Human immunodeficiency virus type 1 Tat modulates proliferation and differentiation of human neural precursor cells: implication in NeuroAIDS.

Human immunodeficiency virus type 1 (HIV-1) and viral proteins affect neuronal survival and neuron-glial cell interactions, which culminate in neurological disorders. HIV-1 infects regions of neurogenesis in human adult and pediatric brain. However, little is known about the effect of HIV-1 or viral proteins on the properties of human neural precursor cells (hNPCs), particularly neurogenesis, hence a detailed investigation on these lines is warranted. Human neural precursor cells were cultured in presence and absence of HIV-1B transactivating protein Tat to investigate if HIV-1 viral protein alters the properties of human neural precursor cells. Cellular and molecular approaches were adopted to study the effect of HIV-1B transactivating protein Tat on proliferation and differentiation potential of human fetal brain-derived NPCs. Cell proliferation assays such as BrdU and Ki67 staining and pathway-specific cDNA and protein arrays were used in the study. Data reveal that HIV-1B Tat protein severely affects proliferation of hNPCs, as evident by lower incorporation of BrdU and Ki67 staining as well as neurosphere assay. HIV-1 Tat substantially attenuated neurogenesis, as evident by the smaller numbers of Tuj-1- and doublecortin-positive cells differentiated from hNPCs, without affecting their viability. These data suggest that HIV-1 Tat alters the properties of human neural precursor cells via attenuation of the cell cycle regulatory unit cyclin D1 and the mitogen-activated protein kinase (MAPK) pathway, particularly extracellular signal-related kinase 1/2 (ERK1/2). The study provides new insights into cellular and molecular mechanisms that may modulate human neural precursor cell properties in HIV/AIDS (acquired immunodeficiency syndrome) individuals. Validation with autopsy brain samples is necessary to further substantiate these important observations.