James R. Lokensgard - US grants

DESCRIPTION (Adapted from the Applicant's Abstract): Although the precise mechanisms whereby HIV-1 infection induces neurodegeneration have yet to be determined, a large body of evidence has incriminated glial cells and the production of proinflammatory mediators. For this reason, ideal therapeutic agents for the treatment of AIDS dementia would possess anti-inflammatory as well as anti-viral properties. Benzodiazepines, such as diazepam (Valium), are extensively prescribed drugs for anxiety disorders which readily cross the blood-brain barrier and have demonstrated immunomodulatory properties as well as antiviral activity in HIV-1-infected cell lines. In this application, the central hypothesis to be tested is that anxiolytic drugs attenuate HIV-1 neuropathogenesis through both inhibition of viral expression and suppression of brain cell-produced immune mediators. To characterize their inhibitory effects on HIV-1 expression in brain cells, human glial and mixed glial/neuronal cell cultures, as well as chronically infected promonocytes (U1 cells), will be infected with HIV-1 and maintained in the presence or absence of anxiolytic drugs. Expression of HIV-1 p24 Ag in culture supernatants will be quantified by ELISA. To test the hypothesis that the antiviral properties of anxiolytic drugs are mediated through an inhibition of cellular transcription factor activation, nuclear extracts from HIV-1-infected human glial cells as well as U1 cells, incubated in the presence or absence of anxiolytic drugs, will be probed for nuclear factor kappa B (NF-kB) activation. To link the effects of anxiolytic drug-induced inhibition of NK-kB with direct inhibition of HIV-1, transient transfection assays using HIV-1 promoter-reporter gene constructs, which contain either normal or mutated NF-kB consensus sequences, will be performed. To test the hypothesis that anxiolytic drugs attenuate HIV-1 neuropathogenesis by inhibiting the production of immune mediators, glial and mixed glial/neuronal cell cultures will be infected with HIV-1 and examined for the production of proinflammatory cytokines and beta-chemokines. The results of the proposed studies aims to contribute to a further understanding of HIV-1 neuropathogenesis and will hopefully have therapeutic implications regarding suppressing viral replication and neurodegeneration in HIV-1-infected patients.

Description (adapted from applicant's abstract): This is a submission of a revised application in which the investigators propose to study the relevance of CD8+ T cell mediated, non-cytotoxic and non-MHC-mediated suppression of human cytomegalovirus infection in human brain. As a result of the acquired immunodeficiency syndrome (AIDS) epidemic, there has been a dramatic increase in encephalitis produced by human cytomegalovirus (CMV), the most common opportunistic viral infection in AIDS patients. Intriguing preliminary studies performed in our laboratory have demonstrated that T lymphocytes, microglial cells, and selected cytokines potently inhibit CMV expression in productively infected primary human astrocytes, the most prevalent cell type in the brain. In this proposal, the central hypothesis to be tested is that CMV-specific T lymphocytes and microglial cells inhibit viral expression in brain cells through non-cytotoxic, non-MHC-restricted mechanisms mediated by the production of soluble factors. To test this hypothesis, the influence of T lymphocytes on CMV expression will be evaluated by determining if activated T cells from CMV-seropositive donors, as well as seronegative subjects, have the ability to suppress CMV gene expression in astrocytes. Through the use of transwell inserts, it will then be determined if the observed suppression of CMV expression is mediated through soluble factors. The addition of antibodies against specific cytokines will identify which soluble, inhibitory factors are involved. To determine if the suppressive ability of T lymphocytes can be mimicked by selected recombinant cytokines, the effect of exogenous cytokine treatment on CMV expression and replication in astrocytes will be examined. A human brain cell/athymic rat xenograft model will be used to investigate the antiviral effects of cytokine treatment in an in vivo model of human CMV infection in an enclosed, "immune privileged" site. To investigate the molecular mechanisms responsible for non-cytotoxic T cell and cytokine-induced viral suppression, their effect on the activation of transcription factors in astrocytes will be examined. Through the use of recombinant adenovirus vectors and reporter gene assays, it will be determined if these immune mediators lead to decreased CMV immediate-early gene promoter activity, thereby inhibiting the replication cascade. Finally, the role of microglial cells in controlling CMV infection of astrocytes will be addressed by determining if microglial cells suppress viral expression when co-cultured with CMV-infected human astrocytes and by measuring the production of antiviral cytokines from these cells in response to viral infection. Information gained from these studies will increase our understanding of the pathogenesis and host defense against this devastating central nervous system disease and may lead to innovative interventions for the management of AIDS-related CMV encephalitis based on immunotherapy.

DESCRIPTION (provided by applicant): Microglia and other brain macrophages are the principal regulators of neuroinflammation within the CNS. Herpes simplex virus (HSV)-1 is an important opportunistic pathogen in HIV-1-infected patients as well as the cause of a devastating CNS infection in normal hosts. It is well established that the brain damage seen during neuroAIDS is not caused by direct HIV infection of neurons, but rather is induced by macrophage-produced neuroinflammatory mediators. Microglial cells produce these same neuroinflammatory mediators in response to HSV, and this proposal utilizes HSV infection of mice as a small animal model to investigate the role of activated microglia during viral brain infection. The central hypothesis to be tested is that oxidative stress occurring during viral encephalitis can be modulated to prevent immune-mediated mechanisms of tissue damage. To test this hypothesis, we will (1) determine whether TLR2 mediates oxidative stress occurring during herpes encephalitis. This will be achieved by comparing virus-induced production of pro-oxidative enzymes and reactive species using cultured microglia isolated from wild-type versus TLR2 knockout mice. Additional experiments will compare oxidative stress-induced brain damage occurring during viral encephalitis following infection of these knockout animals. We will then (2) determine whether antioxidant enzymes regulate oxidative stress during viral encephalitis. These studies will examine the effect of select antioxidant enzymes on the production of reactive species and microglial cell apoptosis in response to HSV. We will go on to examine the role of these antioxidant enzymes in controlling oxidative stress-induced brain damage during viral encephalitis in vivo. Finally, we will (3) determine if virus-induced oxidative brain damage can be controlled. This will be achieved through overexpressing anti-inflammatory cytokines in the brains of iNOS-luciferase transgenic mice and assessing expression using in vivo real-time bioluminescence imaging. In the final studies, we will modulate the oxidative stress response occurring during herpes encephalitis. This will be achieved by constructing recombinant herpesviruses overexpressing vaccinia virus proteins that disrupt TLR signaling, as well as antioxidant enzymes, and assessing the resulting tissue damage in infected animals. Through studies described in this proposal, we hope to identify new approaches for treatment of herpes encephalitis and neuroAIDS, as well as other forms of viral encephalitis, which reduce the extent of immune-mediated brain damage. PUBLIC HEALTH RELEVANCE It is well established that brain damage seen during neuroAIDS is not caused by direct HIV infection of neurons, but rather is induced by microglial cell- produced neuroinflammatory mediators, such as reactive oxygen and reactive nitrogen species. Through the use of inhibitors of microglial cell activation and antioxidant enzymes, it may be possible to modulate the oxidative stress response occurring during viral encephalitis. In these experiments, we hope to identify new approaches to the treatment of herpes encephalitis and neuroAIDS, as well as other forms of viral encephalitis, which reduce the extent of immune- mediated mechanisms of brain damage.

DESCRIPTION (provided by applicant): Human cytomegalovirus is an opportunistic pathogen that produces a devastating central nervous system disease in AIDS patients and is the most common infectious cause of congenital brain disorders. Though the importance of lymphocytes in clearing viral brain infection is well appreciated, recent data have suggested that infiltrating T lymphocytes may also play an integral role in repair of brain tissue damaged during viral infection. In this proposal, the central hypothesis to be tested is that cytomegalovirus infection of neural stem cells (NSCs) compromises their neuroreparative response to T lymphocyte-mediated clearance of viral brain infection. The proposed experiments are designed to study the effect of murine cytomegalovirus (MCMV) infection on the interplay between T-cells and NSCs in vivo. To test this hypothesis, we will (1) determine if NSCs respond to T lymphocyte-mediated viral clearance. This will be achieved by depleting CD8+, CD4+, and Treg (CD4+CD25+) lymphocyte subpopulations and examining the subsequent effect on NSC migration to sites of MCMV brain infection using real-time bio-luminescence imaging. We will also investigate how T-cell-mediated immune responses affect the production of select neurotrophins in the brains of infected animals. We will then (2) determine if MCMV infects endogenous NSCs in vivo and compromises their response to T lymphocytes. These studies will examine the effect of MCMV infection on the migration of luciferase-labeled NSCs towards IFNg3-induced chemokine production and determine if MCMV infected NSCs are themselves targets for CD8+ T-cell-mediated clearance. Finally, we will (3) determine if viral infection of NSCs alters their subsequent neurogenesis. These studies will investigate persistence of transplanted luciferase-labeled NSCs and evaluate how MCMV infection alters which neural cell markers (specific for neurons, oligodendrocytes, or astrocytes) are expressed following subsequent differentiation in vivo. In the final studies, we will determine if an anti-inflammatory environment fosters regenerative processes through overexpression of interleukin (IL)-10 and transforming growth factor (TGF)b2, followed by an assessment of neurogenesis. Taken together, the studies proposed in this competitive renewal application will provide new insights into the interactions between T lymphocytes and NSCs in the maintenance of healthy brain tissue following viral infection. PUBLIC HEALTH RELEVANCE Human cytomegalovirus is an opportunistic pathogen that produces a devastating central nervous system disease in AIDS patients and the murine models described in this grant application will help us to understand interactions between antiviral immune responses and brain repair. The prognosis for full recovery in patients suffering from chronic disability following viral encephalitis is poor and the experiments outlined in this proposal are timely because the use of neural stem cells to repair brain damage is currently being explored in numerous models of neurodegenerative diseases. Because it is believed that one function of neural stem cells is to repair inflamed and damaged brain tissue, these cells may offer an innovative approach to treat the neuropathological sequelae subsequent to neuroAIDS, as well as other viral encephalitis.

DESCRIPTION (provided by applicant): Effective highly active antiretroviral therapy (HAART) reduces HIV RNA levels in cerebrospinal fluid (CSF), as well as in plasma, and has produced profound improvements in health and longevity. However, neurological complications continue to increase in prevalence as patients with the disease live longer and cognitive impairment remains one of the most feared complications; even in patients with systemically well-controlled virus. In patients receiving HAART, neuroimmune activation has been shown to persist long after reduction of viral loads. Hence, it is believed that viral antigen alone may not be responsible for driving the response. In a number of viral brain infections, it is clear that residnt microglia are activated by brain-infiltrating cells of the peripheral immune system, as well as by their mediators, rather than simply viral antigens themselves. Using a murine cytomegalovirus (MCMV) model of viral brain infection, we were surprised to detect numerous CD19(-)CD38(+)CD138(+) plasma cells and CD19(+)CD1d(high)CD5(+) regulatory B-cells (Breg) persisting in the CNS during chronic infection. Based on these preliminary findings, it is likely that antibodies present within the CNS, operating through Fc receptors, and persisting Breg cells influence neuroimmune activation. In this proposal, the central hypothesis to be tested is that B-lineage cells persisting within the CNS following viral infection modulate chronic microglial cell activation. In the proposed studies, we will first determine whether an environment capable of supporting entry and survival of B-lineage cells (i.e., B-cells, plasma blasts, plasma cells, and Breg cells) is produced following viral brain infection. We will then fin out whether antibody-secreting cell (ASC)-produced antibodies modulate microglial cell activation within the infected brain. This will be achieved by determining how the ratios of activating (i.e., Fc RI, Fc RIII) to inhibitory (i.e., Fc RIIb) Fc receptors on microglia change in response to viral brain infection and by comparing infection-induced microglial cell activation in wild-type versus Fc RI/RIII double-knockout, as well as Fc RIIb knockout animals. We will go on to investigate how regulatory B-cells modulate microglial cell activation through both anti-inflammatory cytokine- and contact-dependent mechanisms. The final set of experiments will use Foxp3 promoter-GFP- and Foxp3-DTR (diphtheria toxin receptor) expressing transgenic animals to investigate how Breg cells promote CD4(+) lymphocyte transition into a T regulatory (Treg) cell phenotype within the chronically infected brain. The interactions between brain-infiltrating cells of the B- lineage, the cytokines and antibodies they produce, and their role in regulating chronic microglial cell activation, which is the focus of this application, have largely been ignored in experimental models of NeuroAIDS. Because B-lineage cells are present in the brain during chronic viral infection, the roles they play in modulating microglial cell activation nd its associated neurodegeneration need to be explored.

DESCRIPTION (provided by applicant): Immune reconstitution inflammatory syndrome (IRIS) has emerged as a major clinical complication in the management of HIV infection following the initiation of combination antiretroviral therapy (cART). IRIS is most commonly seen in patients with severe T cell lymphopenia at the initiation of treatment, often in the presence of an opportunistic infection. cART-induced reversal of lymphopenia restores host defense, but the T- cell repertoire that comes back is often of limited diversity and hyper-responsive to particular antigens due to differential expansion of specific memory T-cells. A wide variety of opportunistic pathogens have been associated with IRIS and heterogeneous clinical manifestations are observed. However, independent of the underlying pathogen or even in the absence of identifiable opportunistic infection, IRIS is characterized by excessive immune activation with elevated frequencies of reconstituting activated T-cells. Immune recovery which specifically attacks the brain is termed central nervous system (CNS)-IRIS and it is particularly challenging due to its clinical severity. Still, the neuroimmunopathogenic mechanisms resulting in CNS-IRIS are poorly understood. Results obtained in our laboratory over the previous funding period of this grant have shown that brain-infiltrating, virus-specific T lymphocytes from the peripheral immune system activate resident microglial cells, including those in widespread areas distal to focal viral infection. Building upon these findings, the central hypothesis to be tested in this competitive renewal application is that replenished yet dysregulated T-lymphocytes drive CNS-immune reconstitution disease (IRD) by providing signals which promote hyper-activation of resident microglia and the overproduction of neurotoxic mediators. In the proposed studies, we will first determine whether T-cell reconstitution of lymphopenic mice harboring herpesvirus brain infection hyper-activates resident microglial cells. This will be achieved through adoptive transfer of CD3(+) T-cells into lymphopenic animals followed by assessment of microglial activation. We will then determine how Foxp3(+) regulatory T-cell (Treg) dysregulation contributes to CNS-IRD. These studies will employ Foxp3-DTR (diphtheria toxin receptor) expressing transgenic mice to determine the effect of depleting Tregs from CD3(+) T-cells prior to adoptive transfer into infected, lymphopenic animals. The final set of experiments will determine mechanisms by which T-cell reconstitution potentiates neurodegeneration. Identification of the precise interactions between T lymphocytes and microglia which drive hyperactive neuroimmune responses is vitally important to the field of HIV medicine. Novel therapeutic approaches (e.g., Treg immunotherapy) which target distinct neuropathogenic pathways are urgently needed. However, the mechanisms to target are still poorly understood because they are difficult to address through clinical studies. In this application, we propose to fill this gap in knowledge by studying experimental CNS-IRD using T-cell repopulation of lymphopenic murine hosts harboring opportunistic viral brain infection.

? DESCRIPTION (provided by applicant): Peripheral neuropathic pain is now the most common neurological complication in HIV-infected individuals undergoing cART. However, it is still under-studied because appropriate experimental animal models have not yet been developed. Recent data from us and others show that distal symmetric polyneuropathy (DSP) develops along with murine acquired immunodeficiency syndrome (MAIDS) following infection with the LP- BM5 retrovirus mixture. For centuries, derivatives of Cannabis sativa have been used as analgesics, and a link between cannabinoid receptor 2 (CB2R) and peripheral neuropathy has been established in animal models using nerve transection, chemical-induced pain, and various other stimuli. Diverse types of peripheral neuropathic pain respond differently to standard drug intervention and nothing is currently known regarding the effects of inflammatory modulation through CB2Rs in the context of peripheral neuropathy due to chronic retroviral infection. The experiments proposed in this application will fill this gap in knowledge. The central hypothesis to be tested is that modulation using CB2R agonists can control chronic immune activation-induced neuropathic pain seen during retroviral infection. The studies will first examine the extent of immune activation within the lumbar spinal cord (LSC) and dorsal root ganglia (DRG) in MAIDS animals with peripheral neuropathy. This will be achieved by assessing CD4+ T-cell and macrophage infiltration into the LSC and DRG, as well as activation of resident glia. We will also determine how CD4+ regulatory T-cell (Treg) dysregulation contributes to DSP. Studies will go on to determine the role of endogenous CB2Rs in controlling chronic inflammation-induced neuropathic pain. This will be achieved by giving MAIDS to CB2R knockout mice and assessing infection-induced peripheral neuropathy via mechanical sensitivity using MouseMet electronic von Frey and intra-epidermal nerve fiber loss in hind paw tissue biopsies. We will also assess its associated glial activation and inflammation-induced neurotoxicity. In the final set of experiments, we will determine whether exogenous synthetic CB2R agonists inhibit chronic immune activation, neuropathic pain, and subsequent neuronal damage. This will be achieved by treating wild-type MAIDS animals with exogenous synthetic CB2R agonists to determine if they inhibit damage within the LSC and DRG. A number of studies investigating the mechanisms leading to peripheral neuropathy have been carried out in vitro, but ultimately these in vitro findings require further validation using in vivo models. There are no FDA-approved pharmacologic agents available which are specifically designed for treatment of chronic HIV-associated neuropathy. Analgesics currently used target neurons, but are only modestly effective for chronic pain. Five decades of research focused on neurons has not brought about a real solution to neuropathic pain. New approaches which lead to development of more specific and effective analgesics are desperately needed; and immunomodulation through the CB2R system is clearly a promising strategy.

PROJECT SUMMARY/ABSTRACT Although patients on successful combination antiretroviral therapy (cART) show sustained viral suppression in the cerebrospinal fluid (CSF) as well as plasma, ?blips? indicative of transient HIV replication are often found with more frequent CSF testing. Recent studies link persistent immune activation, neuroinflammation, and CSF viral escape in cART-treated individuals to increased disease progression, as well as an increased risk for HAND. In this application, it is proposed that in patients on optimal cART, HAND is driven by recall immune responses to small amounts of antigen produced during this transient CSF viral escape. Over the previous funding period of this grant, we have described a population of CD8(+)CD103(+)CD127(+) memory T-cells which remain as permanent residents within the brain following viral infection. Building on these findings, we used a heterologous prime-boost strategy in which mice are immunized with recombinant adenovirus vectors expressing the HIV-1 p24 capsid protein (rAd5-p24), followed by a CNS boost using Pr55Gag/Env virus-like particles (HIVLPs). This approach allowed us to develop an innovative experimental model in which the murine brain becomes populated by resident CD8(+) memory T-cells specific for immunodominant HIV-1 Gag epitopes. In addition, subsequent anti-HIV-1 recall responses can then be induced in response to stimulation using defined peptide epitopes. This novel approach allows us to investigate the neuroimmune pathogenesis of recall immune responses in a powerful, yet convenient and inexpensive small animal model. Despite its importance to understanding and treating HAND, nothing is currently know about how anti-HIV-1 recall responses from brain-resident memory CD8(+) T-cells (bTRM) trigger tissue-wide innate immune responses which drive reactive gliosis-mediated neurotoxicity. Experiments proposed in this application will fill this gap in knowledge. The central hypothesis to be tested is that adaptive, anti-HIV-1 recall responses from bTRM trigger tissue-wide innate immune responses from reactive glia that promote inflammation-induced synaptic damage, neurotoxicity, and long-term neurocognitive impairment. The studies will first determine whether anti-HIV-1 recall responses from CD8(+)CD103(+)CD127(+) bTRM induce reactive gliosis and trigger broad innate neuroinflammatory responses. Experiments proposed in Aim #2 will go on to determine whether these anti- HIV-1 recall responses trigger synaptodendritic damage, neurotoxicity, and long-term neurological sequelae. Finally in Specific Aim #3, which is more translational, we will determine whether anti-HIV-1 recall response- driven neuroinflammation can be ameliorated through immunomodulatory approaches. Although patients on successful cART show sustained viral suppression, antiviral treatment alone does not fully protect against neurocognitive impairment. Still, partial protection by cART does indicate a direct connection to HIV replication. For these reasons, immunomodulation of neurotoxic, recall response-driven neuroinflammation in response to CSF viral escape is clearly a promising adjunctive approach to mitigating neural damage.

PROJECT SUMMARY/ABSTRACT Immunotherapeutic approaches which target the programmed cell death protein (PD)-1 co-inhibitory immune checkpoint have revolutionized treatment of several types of advanced cancer. However, the susceptibility of particular cell types remains highly variable and difficult to predict. We have found that both microglial cells and astrocytes govern the activity of brain-infiltrating antiviral T-cells through upregulation of PD-L1 expression in an effort to limit damaging encephalitic responses. Likewise, we have found that CD4(+) regulatory T-cells (Tregs) limit viral encephalitis through restraining expansion and activity of CD8(+) cytotoxic T-lymphocytes. While these anti-inflammatory responses within the brain are undoubtedly beneficial to the host, preventing immune-mediated damage to this vital organ, establishment of a prolonged anti-inflammatory milieu may also lead to deficiencies in viral clearance. Patients undergoing successful cART are viremically suppressed in both plasma and CSF, but persisting HIV-1 reservoirs are believed to contribute to the inability to completely cure infection. HIV-specific CD8(+) T-cell responses are critical in suppressing acute viral infection within the brain, but ultimately fail in their ability to fully eradicate virus. It is currently unknown whether glia are viable cellular targets for immune checkpoint blockade or Treg modulation. Studies proposed here are intended to fill this critical void in our understanding of how the immunosuppressive, neuroprotective brain microenvironment complicates complete clearance of viral infection. Building on our previous studies, we have developed an innovative experimental murine model in which strong CD8(+) T-cell responses specific for immunodominant HIV-1 Gag epitopes are generated within the brains of mice via heterologous prime-boost immunization with recombinant adenovirus vectors expressing the p24 capsid protein (rAd5-p24), followed by a CNS boost using Pr55Gag/Env virus-like particles (HIVLPs). This novel approach allows us to investigate the effects of immunotherapy on viral clearance from the brain in a powerful, yet convenient and inexpensive small animal model. The proposed studies will first determine whether glial cells restrain T-cell-mediated clearance of viral infection through the PD-1: PD-L1 negative immune checkpoint. Experiments proposed in Aim #2 will go on to determine whether loss or blockade of the PD-1: PD-L1 axis will facilitate anti-HIV-1 T-cell-mediated viral clearance from the brain. Finally, in Specific Aim #3 we will determine whether modulation of Treg cell activity in combination with immune checkpoint blockade will further stimulate anti-HIV-1 T-cells to promote viral clearance. The potentially synergistic combination of immune checkpoint blockade and Treg modulation is currently an area of intense investigation for treatment of a variety of immunosuppressive cancers. One of the highest priorities in contemporary HIV-1 research is to identify strategies to effect a functional cure, in which viral load is fully suppressed for extended periods in the absence of antiretroviral therapy. These studies will determine whether immunotherapy can be used to reverse immune exhaustion against the viral brain reservoir.