Debbie D. Watry

Induction of Pathogenic Sets of Genes in Macrophages and Neurons in NeuroAIDS

The etiology of the central nervous system (CNS) alterations after human immunodeficiency virus (HIV) infection, such as dementia and encephalitis, remains unknown. We have used microarray analysis in a monkey model of neuroAIDS to identify 98 genes, many previously unrecognized in lentiviral CNS pathogenesis, whose expression is significantly up-regulated in the frontal lobe of simian immunodeficiency virus-infected brains. Further, through immunohistochemical illumination, distinct classes of genes were found whose protein products localized to infiltrating macrophages, endothelial cells and resident glia, such as CD163, Glut5, and ISG15. In addition we found proteins induced in cortical neurons (ie, cyclin D3, tissue transglutaminase, alpha1-antichymotrypsin, and STAT1), which have not previously been described as participating in simian immunodeficiency virus or HIV-related CNS pathology. This molecular phenotyping in the infected brains revealed pathways promoting entry of macrophages into the brain and their subsequent detrimental effects on neurons. These data support the hypothesis that in HIV-induced CNS disease products of activated macrophages and astrocytes lead to CNS dysfunction by directly damaging neurons, as well as by induction of altered gene and protein expression profiles in neurons themselves which are deleterious to their function.

Selective decrease in paracellular conductance of tight junctions: Role of the first extracellular domain of claudin-5

ABSTRACT Claudin-5 is a protein component of many endothelial tight junctions, including those at the blood-brain barrier, a barrier that limits molecular exchanges between the central nervous system and the circulatory system. To test the contribution of claudin-5 to this barrier function of tight junctions, we expressed murine claudin-5 in Madin-Darby canine kidney II cells. The result was a fivefold increase in transepithelial resistance in claudin-5 transductants and a reduction in conductance of monovalent cations. However, the paracellular flux of neither neutral nor charged monosaccharides was significantly changed in claudin-5 transductants compared to controls. Therefore, expression of claudin-5 selectively decreased the permeability to ions. Additionally, site-directed mutations of particular amino acid residues in the first extracellular domain of claudin-5 altered the properties of the tight junctions formed in response to claudin-5 expression. In particular, the conserved cysteines were crucial: mutation of either cysteine abolishted the ability of claudin-5 to increase transepithelial resistance, and mutation of Cys64 strikingly increased the paracellular flux of monosaccharides. These new insights into the functions of claudin-5 at the molecular level in tight junctions may account for some aspects of the blood-brain barriers selective permeability.

CD4 independence of simian immunodeficiency virus Envs is associated with macrophage tropism, neutralization sensitivity, and attenuated pathogenicity

ABSTRACT To investigate the basis for envelope (Env) determinants influencing simian immunodeficiency virus (SIV) tropism, we studied a number of Envs that are closely related to that of SIVmac239, a pathogenic, T-tropic virus that is neutralization resistant. The Envs from macrophage-tropic (M-tropic) virus strains SIVmac316, 1A11, 17E-Fr, and 1100 facilitated infection of CCR5-positive, CD4-negative cells. In contrast, the SIVmac239 Env was strictly dependent upon the presence of CD4 for membrane fusion. We also found that the Envs from M-tropic virus strains, which are less pathogenic in vivo, were very sensitive to antibody-mediated neutralization. Antibodies to the V3-loop, as well as antibodies that block SIV gp120 binding to CCR5, efficiently neutralized CD4-independent, M-tropic Envs but not the 239 Env. However, triggering the 239 Env with soluble CD4, presumably resulting in exposure of the CCR5 binding site, made it as neutralization sensitive as the M-tropic Envs. In addition, mutations of N-linked glycosylation sites in the V1/V2 region, previously shown to enhance antigenicity and immunogenicity, made the 239 Env partially CD4 independent. These findings indicate that Env-based determinants of M tropism of these strains are generally associated with decreased dependence on CD4 for entry into cells. Furthermore, CD4 independence and M tropism are also associated with neutralization sensitivity and reduced pathogenicity, suggesting that the humoral immune response may exert strong selective pressure against CD4-independent M-tropic SIVmac strains. Finally, genetic modification of viral Envs to enhance CD4 independence may also result in improved humoral immune responses.

Highly Activated CD8+ T Cells in the Brain Correlate with Early Central Nervous System Dysfunction in Simian Immunodeficiency Virus Infection

One of the consequences of HIV infection is damage to the CNS. To characterize the virologic, immunologic, and functional factors involved in HIV-induced CNS disease, we analyzed the viral loads and T cell infiltrates in the brains of SIV-infected rhesus monkeys whose CNS function (sensory evoked potential) was impaired. Following infection, CNS evoked potentials were abnormal, indicating early CNS disease. Upon autopsy at 11 wk post-SIV inoculation, the brains of infected animals contained over 5-fold more CD8+ T cells than did uninfected controls. In both infected and uninfected groups, these CD8+ T cells presented distinct levels of activation markers (CD11a and CD95) at different sites: brain > CSF > spleen = blood > lymph nodes. The CD8+ cells obtained from the brains of infected monkeys expressed mRNA for cytolytic and proinflammatory molecules, such as granzymes A and B, perforin, and IFN-γ. Therefore, the neurological dysfunctions correlated with increased numbers of CD8+ T cells of an activated phenotype in the brain, suggesting that virus-host interactions contributed to the related CNS functional defects.

Human Immunodeficiency Virus-1/Surface Glycoprotein 120 Induces Apoptosis through RNA-Activated Protein Kinase Signaling in Neurons

Previous work has demonstrated that the surface glycoprotein (gp120) of human immunodeficiency virus-1 (HIV-1) can induce damage and apoptosis of neurons both in vitro and in vivo. In this report, we provide evidence that double-stranded RNA-activated protein kinase (PKR), a stress kinase, is involved in HIV/gp120-associated neurodegeneration. In cultures of mixed cortical cells, HIV/gp120 increased the protein level of PKR. Additionally, PKR was phosphorylated in neurons but not glia after exposure to gp120. The use of two independent pharmacological inhibitors of PKR activity abrogated neuronal cell death induced by gp120. Cortical neurons from PKR knock-out mice were significantly protected from neurotoxicity induced by gp120, further validating the pivotal proapoptotic function of PKR. gp120-induced phosphorylated PKR localized prominently to neuronal nuclei; PKR inhibition or the NMDA receptor antagonist MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate] abrogated this effect. PKR inactivation also inhibited gp120-induced caspase-3 activation, consistent with its neuroprotective effect. Finally, brain tissue from individuals diagnosed with HIV-associated dementia (HAD), but not HIV infection alone, contained the activated form of PKR, which localized predominantly to neuronal nuclei. Together, these results identify PKR as a critical mediator of gp120 neurotoxicity, suggesting that activation of PKR contributes to the neuronal injury and cell death observed in HAD.

Methamphetamine Increases Brain Viral Load and Activates Natural Killer Cells in Simian Immunodeficiency Virus-Infected Monkeys

Methamphetamine (Meth) abuse increases risky behaviors that contribute to the spread of HIV infection. In addition, because HIV and Meth independently affect physiological systems including the central nervous system, HIV-induced disease may be more severe in drug users. We investigated changes in blood and brain viral load as well as differences in immune cells in chronically simian immunodeficiency virus-infected rhesus macaques that were either administered Meth or used as controls. Although Meth administration did not alter levels of virus in the plasma, viral load in the brain was significantly increased in Meth-treated animals compared with control animals. Meth treatment also resulted in an activation of natural killer cells. Given the prevalence of Meth use in HIV-infected and HIV at-risk populations, these findings reveal the likely untoward effects of Meth abuse in such individuals.

Regulation of Indoleamine 2,3-Dioxygenase Expression in Simian Immunodeficiency Virus-Infected Monkey Brains

ABSTRACT The human immunodeficiency virus type 1-associated cognitive-motor disorder, including the AIDS dementia complex, is characterized by brain functional abnormalities that are associated with injury initiated by viral infection of the brain. Indoleamine 2,3-dioxygenase (IDO), the first and rate-limiting enzyme in tryptophan catabolism in extrahepatic tissues, can lead to neurotoxicity through the generation of quinolinic acid and immunosuppression and can alter brain chemistry via depletion of tryptophan. Using the simian immunodeficiency virus (SIV)-infected rhesus macaque model of AIDS, we demonstrate that cells of the macrophage lineage are the main source for expression of IDO in the SIV-infected monkey brain. Animals with SIV encephalitis have the highest levels of IDO mRNA, and the level of IDO correlates with gamma interferon (IFN-γ) and viral load levels. In vitro studies on mouse microglia reveal that IFN-γ is the primary inducer of IDO expression. These findings demonstrate the link between IDO expression, IFN-γ levels, and brain pathology signs observed in neuro-AIDS.

Enrichment and persistence of virus-specific CTL in the brain of simian immunodeficiency virus-infected monkeys is associated with a unique cytokine environment

The host reaction to infection of the brain contributes to a number of CNS pathologies including neuro-AIDS. In this study, we have identified the accumulation of SIV-specific CTL in the brains of SIV-infected animals who have neurophysiological abnormalities but are otherwise asymptomatic. SIV-specific CTL enter the brain early after viral infection and are maintained in the brain even when those reactive with an immunodominant epitope in Tat are lost from the rest of the body. The specialized CNS environment contributes to this unique outcome. Following SIV infection, brain levels of IL-15 were significantly elevated whereas IL-2 was absent, creating an environment that favors CTL persistence. Furthermore, in response to IL-15, brain-derived CD8+ T cells could expand in greater numbers than those from spleen. The accumulation, persistence, and maintenance of CTL in the brain are closely linked to the increased levels of IL-15 in the absence of IL-2 in the CNS following SIV infection.

Efficient infection of brain microvascular endothelial cells by an in vivo-selected neuroinvasive SIVmac variant

A neuroinvasive/neuropathogenic SIV variant termed SIVmac182 was previously isolated and characterized (Watry et al, 1994). This neuroinvasive strain was derived from the uncloned strain SIVmac251 through serial animal passage of infected microglia, unlike previously reported neurovirulent strains. Importantly, the virus described here was isolated from a strain which already demonstrates limited neuroinvasiveness in vivo, through a route of inoculation which exerts selective pressure for variants in the periphery that can naturally cross the blood-brain barrier and gain access to the brain. Examination of animal tissues indicated that the neuroinvasive strain was capable of replicating in brain microvascular endothelial cells (BMEC). Therefore, we developed an in vitro model of BMEC infection in which to examine mechanisms of virus neuroinvasiveness and neuropathogenicity as well as to address mechanisms of HIV-induced dementia. Results obtained with this in vitro system indicate that growth in BMEC may predict neuroinvasiveness in vivo, and furthermore, that brain passage of virus results in the generation of neuroinvasive strains which demonstrate an increased efficiency of BMEC infection in vitro.

IFN-γ induced IDO and WRS expression in microglia is differentially regulated by IL-4

Indoleamine 2,3‐dioxygenase (IDO), a tryptophan catabolizing enzyme, has been implicated in the pathogenesis of various neurological disorders. IDO expression is induced by IFN‐γ and leads to neurotoxicity by generating quinolinic acid. Additionally, it inhibits the immune response through both tryptophan depletion and generating other tryptophan catabolites. IL‐4 and IL‐13 have been shown to control IDO expression by antagonizing the effects of IFN‐γ in different cell types. Here, we investigated the effects of these cytokines on IDO expression in microglia. Interestingly, we observed that both IL‐4 and IL‐13 greatly enhanced IFN‐γ‐induced IDO expression. However, tryptophanyl‐tRNA synthetase (WRS), which is coinduced with IDO by IFN‐γ, is downregulated by IL‐4 and IL‐13. The effect of IL‐4 and IL‐13 was independent of STAT‐6. Modulation of IDO but not WRS was eliminated by inhibition of protein phosphatase 2A (PP2A) activity. The phosphatidylinositol 3‐kinase (PI3K) pathway further differentiated the regulation of these two enzymes, as inhibiting the PI3K pathway eliminated IFN‐γ induction of IDO, whereas such inhibition greatly enhanced WRS expression. These findings show discordance between modulations of expression of two distinct enzymes utilizing tryptophan as a common substrate, and raise the possibility of their involvement in regulating immune responses in various neurological disorders.