Howard S. Fox

A Central Role for CD4+ T Cells and RANTES in Virus-Induced Central Nervous System Inflammation and Demyelination

ABSTRACT Infection of C57BL/6 mice with mouse hepatitis virus (MHV) results in a demyelinating encephalomyelitis characterized by mononuclear cell infiltration and white matter destruction similar to the pathology of the human demyelinating disease multiple sclerosis. The contributions of CD4+ and CD8+ T cells in the pathogenesis of the disease were investigated. Significantly less severe inflammation and demyelination were observed in CD4−/− mice than in CD8−/− and C57BL/6 mice (P ≤ 0.002 andP ≤ 0.001, respectively). Immunophenotyping of central nervous system (CNS) infiltrates revealed that CD4−/− mice had a significant reduction in numbers of activated macrophages/microglial cells in the brain compared to the numbers in CD8−/− and C57BL/6 mice, indicating a role for these cells in myelin destruction. Furthermore, CD4−/−mice displayed lower levels of RANTES (a C-C chemokine) mRNA transcripts and protein, suggesting a role for this molecule in the pathogenesis of MHV-induced neurologic disease. Administration of RANTES antisera to MHV-infected C57BL/6 mice resulted in a significant reduction in macrophage infiltration and demyelination (P ≤ 0.001) compared to those in control mice. These data indicate that CD4+ T cells have a pivotal role in accelerating CNS inflammation and demyelination within infected mice, possibly by regulating RANTES expression, which in turn coordinates the trafficking of macrophages into the CNS, leading to myelin destruction.

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.

Neurological Abnormalities Associated with Feline Immunodeficiency Virus Infection

Specific pathogen-free cats were infected with the Maryland strain of FIV (FIV-MD) for the purpose of assessing the effects of FIV infection on the central nervous system (CNS). Two separate studies were performed, involving a total of 13 infected cats and six age-matched, sham-inoculated controls. All animals infected with FIV-MD seroconverted by 8 weeks post-infection and virus was recovered from peripheral blood mononuclear cells of all infected cats. All of the infected animals had lower absolute CD4+ cells counts and decreased CD4+/CD8+ ratios. Virus was recovered from the cerebrospinal fluid (CSF) of certain infected individuals, and antiviral antibody and pleocytosis were evident in the CSF of the majority of infected cats. Additionally, virus was recovered from tissue explants from the cerebellum, midbrain and brainstem of one sacrificed FIV+ cat. Specific neurological changes included anisocoria, delayed righting reflex and delayed pupillary reflex, as well as delayed visual and auditory evoked potentials, and marked alterations in sleep patterns similar to those reported for human immunodeficiency virus (HIV)-positive individuals. Histological evaluation revealed the presence of perivascular cuffing and glial nodules in FIV-infected cats. These results indicate that FIV causes an acute neurological disease that closely resembles the early neurological effects of HIV infection in humans and should serve well as an animal model for lentivirus-induced CNS disease.

Interferon-Independent, Human Immunodeficiency Virus Type 1 gp120-Mediated Induction of CXCL10/IP-10 Gene Expression by Astrocytes In Vivo and In Vitro

ABSTRACT The CXC chemokine gamma interferon (IFN-γ)-inducible protein CXCL10/IP-10 is markedly elevated in cerebrospinal fluid and brain of individuals infected with human immunodeficiency virus type 1 (HIV-1) and is implicated in the pathogenesis of HIV-associated dementia (HAD). To explore the possible role of CXCL10/IP-10 in HAD, we examined the expression of this and other chemokines in the central nervous system (CNS) of transgenic mice with astrocyte-targeted expression of HIV gp120 under the control of the glial fibrillary acidic protein (GFAP) promoter, a murine model for HIV-1 encephalopathy. Compared with wild-type controls, CNS expression of the CC chemokine gene CCL2/MCP-1 and the CXC chemokine genes CXCL10/IP-10 and CXCL9/Mig was induced in the GFAP-HIV gp120 mice. CXCL10/IP-10 RNA expression was increased most and overlapped the expression of the transgene-encoded HIV gp120 gene. Astrocytes and to a lesser extent microglia were identified as the major cellular sites for CXCL10/IP-10 gene expression. There was no detectable expression of any class of IFN or their responsive genes. In astrocyte cultures, soluble recombinant HIV gp120 protein was capable of directly inducing CXCL10/IP-10 gene expression a process that was independent of STAT1. These findings highlight a novel IFN- and STAT1-independent mechanism for the regulation of CXCL10/IP-10 expression and directly link expression of HIV gp120 to the induction of CXCL10/IP-10 that is found in HIV infection of the CNS. Finally, one function of IP-10 expression may be the recruitment of leukocytes to the CNS, since the brain of GFAP-HIV gp120 mice had increased numbers of CD3+ T cells that were found in close proximity to sites of CXCL10/IP-10 RNA expression.

Disruption of neuronal autophagy by infected microglia results in neurodegeneration.

There is compelling evidence to support the idea that autophagy has a protective function in neurons and its disruption results in neurodegenerative disorders. Neuronal damage is well-documented in the brains of HIV-infected individuals, and evidence of inflammation, oxidative stress, damage to synaptic and dendritic structures, and neuronal loss are present in the brains of those with HIV-associated dementia. We investigated the role of autophagy in microglia-induced neurotoxicity in primary rodent neurons, primate and human models. We demonstrate here that products of simian immunodeficiency virus (SIV)-infected microglia inhibit neuronal autophagy, resulting in decreased neuronal survival. Quantitative analysis of autophagy vacuole numbers in rat primary neurons revealed a striking loss from the processes. Assessment of multiple biochemical markers of autophagic activity confirmed the inhibition of autophagy in neurons. Importantly, autophagy could be induced in neurons through rapamycin treatment, and such treatment conferred significant protection to neurons. Two major mediators of HIV-induced neurotoxicity, tumor necrosis factor-α and glutamate, had similar effects on reducing autophagy in neurons. The mRNA level of p62 was increased in the brain in SIV encephalitis and as well as in brains from individuals with HIV dementia, and abnormal neuronal p62 dot structures immunoreactivity was present and had a similar pattern with abnormal ubiquitinylated proteins. Taken together, these results identify that induction of deficits in autophagy is a significant mechanism for neurodegenerative processes that arise from glial, as opposed to neuronal, sources, and that the maintenance of autophagy may have a pivotal role in neuroprotection in the setting of HIV infection.

Metabolomic analysis of the cerebrospinal fluid reveals changes in phospholipase expression in the CNS of SIV-infected macaques

HIV infiltrates the CNS soon after an individual has become infected with the virus, and can cause dementia and encephalitis in late-stage disease. Here, a global metabolomics approach was used to find and identify metabolites differentially regulated in the cerebrospinal fluid (CSF) of rhesus macaques with SIV-induced CNS disease, as we hypothesized that this might provide biomarkers of virus-induced CNS damage. The screening platform used a non-targeted, mass-based metabolomics approach beginning with capillary reverse phase chromatography and electrospray ionization with accurate mass determination, followed by novel, nonlinear data alignment and online database screening to identify metabolites. CSF was compared before and after viral infection. Significant changes in the metabolome specific to SIV-induced encephalitis were observed. Metabolites that were increased during infection-induced encephalitis included carnitine, acyl-carnitines, fatty acids, and phospholipid molecules. The elevation in free fatty acids and lysophospholipids correlated with increased expression of specific phospholipases in the brains of animals with encephalitis. One of these, a phospholipase A2 isoenzyme, is capable of releasing a number of the fatty acids identified. It was expressed in different areas of the brain in conjunction with glial activation, rather than linked to regions of SIV infection and inflammation, indicating widespread alterations in infected brains. The identification of specific metabolites as well as mechanisms of their increase illustrates the potential of mass-based metabolomics to address problems in CNS biochemistry and neurovirology, as well as neurodegenerative diseases.

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.

Neurologic dysfunctions caused by a molecular clone of feline immunodeficiency virus, FIV-PPR.

FIV is a lentivirus of domestic cats that causes a spectrum of diseases that is remarkably similar to the clinical syndrome produced by HIV infection in people. Both HIV and FIV has been shown to cause neurologic dysfunction. Specific Pathogen-Free (SPF) cats were placed into one of three groups: FIV-PPR infected; DU-FIV-PPR (a dUTPase mutant of the FIV-PPR clone) infected; or an age-matched control group. In both infected groups, the general clinical signs of infection included lymphadenopathy, oral ulcerations, rough hair coat, and conjuntivitis. Specific neurological changes in the FIV-PPR infected cats included hind limb paresis; delayed righting and pupillary reflexes; behavioral changes; delayed visual and auditory evoked potentials; decreased spinal and peripheral nerve conduction velocities; and marked alterations in sleep patterns. Most of these changes were also observed in the DU-FIV-PPR infected cats. However, these cats tended to have a slightly less severe disease. In this study, we have demonstrated that an infectious molecular clone of FIV closely parallels the disease course of wild type FIV-infected cats. By using a knockout gene mutant of this clone, we were able to demonstrate that the dUTPase gene is not essential for neuropathogenesis. Further use of the FIV-PPR clone should prove useful in determining the essential viral elements that are important in the neuropathogenesis of lentiviral infections.

Neurovirological correlation with HIV-associated neurocognitive disorders and encephalitis in a HAART-era cohort.

Objective:Replicating HIV-1 in the brain is present in HIV encephalitis (HIVE) and microglial nodule encephalitis (MGNE) and is putatively linked with HIV-associated neurocognitive disorders (HAND). A cliniconeurovirological correlation was conducted to elucidate the relationship between brain viral load and clinical phenotype. Subjects and assays:HIV gag/pol RNA and DNA copies were quantified with reverse transcriptase-polymerase chain reaction or polymerase chain reaction in 148 HAART-era brain specimens. Comparison with HAND, HIVE, and MGNE and correlation with neuropsychological (NP) test scores were done using one-way ANOVA with Tukey-Kramer and Spearman tests, respectively. Results:Brain HIV RNA was higher in subjects with HAND plus HIVE versus without HAND (delta = 2.48 log10 units, n = 27 versus 36, P < 0.001). In HAND without HIVE or MGNE, brain HIV RNA was not significantly different versus without HAND (P = 0.314). Worse NP scores correlated significantly with higher HIV RNA and interferon responses in brain specimens (P < 0.001) but not with HIV RNA levels in premortem blood plasma (n = 114) or cerebrospinal fluid (n = 104). In subjects with MGNE, brain HIV RNA was slightly higher versus without MGNE (P < 0.01) and much lower versus with HIVE (P < 0.001). Conclusions:Brain HIV RNA and to a lesser extent HIV DNA are correlated with worse NP performance in the 6 months before death. Linkage occurs primarily in patients with HIVE and MGNE, and these patients could obtain added NP improvement by further reducing brain HIV while on HAART. Patients not in those groups are less certain to obtain added NP benefit.