Hui Qin Xing

In vivo expression of proinflammatory cytokines in HIV encephalitis: an analysis of 11 autopsy cases

As the pathogenesis of AIDS dementia complex (ADC), cytokines such as TNF‐α and IL‐1β have been thought to have toxic effects on CNS cells and induce neuronal cell death. However, many of the discussions have been based on the studies done by in vitro experiments. There are only a few reports which demonstrate proinflammatory cytokines directly in vivo in HIV encephalitis (HIVE) brains, and roles of these cytokines with relation to HIV‐1 infection are not yet clarified. In the present study, we examined 11 autopsy cases of HIVE using immunohistochemistry, and explored which cell types expressed these cytokines and whether expression of cytokines was related to viral infection. IL‐1β was detected in the frontal white matter of all 11 cases where microglial nodules were observed to varying degrees, whereas TNF‐α was detected in seven cases. IL‐1β− or TNF‐α‐positive cells were almost restricted to CD68‐positive macrophages/microglia and mild expression of these cytokines by astrocytes was observed in two cases with severe HIVE. IL‐1β was detected in some HIVp24‐positive multinucleated giant cells. However, we could not detect TNF‐α expression in the HIVp24‐positive cells, which indicates that IL‐1β is induced by HIV‐1 infection. In conclusion, a macrophage/microglia lineage is the main cell type to release cytokines in HIVE, and IL‐1β expression by HIV‐1‐infected cells may be one of the important factors for induction of HIVE. In addition, many non‐infected macrophages/microglia as well as some astrocytes express IL‐1β and TNF‐α, which might contribute to pathogenesis of ADC.

Reduced expression of excitatory amino acid transporter 2 and diffuse microglial activation in the cerebral cortex in AIDS cases with or without HIV encephalitis.

To determine the relationship between the human immunodeficiency virus type 1 (HIV-1) encephalitis (HIVE) and diffuse poliodystrophy in the acquired immunodeficiency syndrome dementia complex, we examined the neuropathologic features in brain autopsy tissue specimens of HIV-1-infected patients with (n = 11) or without HIVE (n = 9). The brains were free of opportunistic diseases and major cerebrovascular lesions. In both groups, there was diffuse microglial activation, astrocytic gliosis, and decreased excitatory amino acid transporter 2 (EAAT-2) immunoreactivity. These changes did not correlate either with the severity of encephalitis or local HIV-1 infection as detected by p24 immunostaining. Some activated microglia expressed EAAT-2; interleukin-1&bgr; and tumor necrosis factor were detected only in microglial nodules of HIVE cases but not in areas with diffusely activated microglia. There was a significant negative correlation between the areas of EAAT-2 expression and numbers of activated microglia (p < 0.01) in cases with decreased EAAT-2. These data indicate that diffuse cortical changes may occur independently of HIVE in acquired immunodeficiency syndrome patients. The expression of EAAT-2 by activated microglia suggests that they might exert a compensatory effect that protects neurons from glutamate neurotoxicity.

Expression of proinflammatory cytokines and its relationship with virus infection in the brain of macaques inoculated with macrophage-tropic simian immunodeficiency virus

The pathogenesis of acquired immunodeficiency syndrome dementia complex (ADC) is still poorly understood. Many studies suggest that proinflammatory cytokines such as IL‐1β and TNF‐α released by microglia/macrophages or astrocytes play a role in CNS injury. A microscopic finding of a microglial nodule with multinucleated giant cells (MNGCs) is a histopathologic hallmark of ADC and named HIV encephalitis. However, in vivo expression of these cytokines in this microenvironment of HIV encephalitis is not yet clarified. One of the main reasons is complexities of brain pathology in patients who have died from terminal AIDS. In this study, we infected two macaques with macrophage‐tropic Simian immunodeficiency virus SIV239env/MERT and examined expression of TNF‐α and IL‐1β in inflammatory lesions with MNGCs and its relation to virus‐infected cells using immunohistochemistry. One macaque showed typical inflammatory lesions with MNGCs in the frontal white matter. Small microglial nodules were also detected in the basal ganglia and the spinal cord. SIVenv positive cells were detected mainly in inflammatory lesions, and seemed to be microglia/macrophages and MNGCs based on their morphology. Expression of IL‐1β and TNF‐α were detected in the inflammatory lesions with MNGCs, and these positive cells were found to be negative for SIVenv by double‐labeling immunohistochemistry or immunohistochemistry of serial sections. There were a few TNF‐α positive cells and almost no IL‐1β positive cells in the area other than inflammatory lesions. Another macaque showed scattered CD3+ cells and CD68+ cells in the perivascular regions of the white matter. SIVenv and TNF‐α was demonstrated in a few perivascular macrophages. These findings indicate that virus‐infected microglia/macrophages do not always express IL‐1β and TNF‐α, which suggests an indirect role of HIV‐1‐infected cells in cytokine‐mediated pathogenesis of ADC. Our macaque model for human ADC may be useful for better understanding of its pathogenesis.

Simian Immunodeficiency Virus Encephalitis in the White Matter and Degeneration of the Cerebral Cortex Occur Independently in Simian Immunodeficiency Virus-Infected Monkey

Highly active antiretroviral therapy (HAART) has been successful to reduce progression of acquired immunodeficiency syndrome (AIDS). Nevertheless, recent autopsy analysis of the brain from patients with human immunodeficiency virus (HIV)-1 infection reported same or even increasing numbers of AIDS encephalopathy. This insufficient effect of HAART for central nervous system (CNS) complication might be explained by independent pathogenetic processes in lymph node and CNS. We inoculated macaques with three Simian immunodeficiency virus (SIV) strains and investigated relationship between degree of the lymph node pathology and that of AIDS-related brain pathology. Animals infected with T-cell-tropic viruses SIVmac239 and SHIV-RT developed typical AIDS pathology in the lymph node 46 to 156 weeks after infection. The cerebral cortex of these animals showed focal or diffuse gliosis, and electron microscopic analysis demonstrated degenerative changes, such as accumulation of dense lamellar bodies in the dendrites and swelling of astrocytic processes. However, there was no evidence of microglial nodules or multinucleated giant cells in the white mater. The animals infected with macrophage-tropic SIV239env/MERT did not develop lymph node pathology of AIDS in the same or longer period of infection. The white mater of the animal, however, showed microglial nodules with multinucleated giant cells, a pathological hallmark of AIDS encephalopathy. SIV immunoreactivity was demonstrated in these giant cells as well as macrophage/microglia cells. On the other hand, there was no abnormality in the cerebral cortex. These findings suggest that there are two independent pathogenetic processes in AIDS encephalopathy: immune response against virus infected macrophage/microglial cells in the white mater without immunodeficiency and cortical degeneration caused in the late stage of AIDS.

CD8 positive T-cell infiltration in the dentate nucleus of paraneoplastic cerebellar degeneration

Recent reports have discussed the presence of cytotoxic T cells in paraneoplastic cerebellar degeneration (PCD). We report an autopsy case of PCD associated with anti-Hu antibody, in which we revealed infiltration of CD8+ T cells in and around the dentate nucleus but not in the cerebellar cortex, in addition to severe Purkinje cell loss. Some infiltrated mononuclear cells expressed cytotoxic cell marker, Granzyme B. Decrease of neurons and reduced presynapses were demonstrated in the dentate nucleus. This is the first report that suggests the possibility of the dentate nucleus being primarily attacked followed by Purkinje cell loss in PCD.

Impaired Astrocytes and Diffuse Activation of Microglia in the Cerebral Cortex in Simian Immunodeficiency Virus-Infected Macaques Without Simian Immunodeficiency Virus Encephalitis

Various types of neuronal damage have been reported in acquired immunodeficiency syndrome (AIDS) dementia. We previously demonstrated that inflammation and cortical damage occur independently according to viral tropism in a simian immunodeficiency virus (SIV)-infected macaque model of AIDS dementia. To elucidate the pathogenesis of cortical degeneration, we examined the frontal cortex of SIV-infected macaques and found apoptosis and decreased expression of the excitatory amino acid transporter 2 in astrocytes and diffuse activation of microglia in association with limited neuronal damage. Some activated microglia also expressed excitatory amino acid transporter 2 but not proinflammatory cytokines. No inflammatory changes were seen in the cortex or the white matter, and SIV-infected cells were not detected in or around cortical lesions either by immunohistochemistry or by the polymerase chain reaction detection of SIV genomes of extracted DNA from microdissected tissue samples. These results indicate that an astrocytic abnormality and a compensatory activation of microglia might provide a protective effect against neuronal degeneration in the frontal cortex of SIV-infected macaques without SIV encephalitis.

Decrease of aquaporin-4 and excitatory amino acid transporter-2 indicate astrocyte dysfunction for pathogenesis of cortical degeneration in HIV-associated neurocognitive disorders.

Human immunodeficiency virus (HIV) encephalitis and degeneration of cerebral cortex are established histopathologies of HIV‐associated neurocognitive disorders (HAND). We previously reported decreased excitatory amino acid transporter‐2 (EAAT‐2) and astrocytic apoptosis in cortical degeneration using SIVmac239 and simian‐human immunodeficiency virus (SHIV)‐infected macaques and human AIDS autopsy cases. In the present study, we added highly pathogenic SIVsm543‐3‐infected macaques. These animals showed similar degenerative changes in the frontal cortex. Using 11 SIV‐infected macaques, three SIVsm543‐3, five SIVmac239 and three SHIV, we compared brain pathology caused by three different viruses and further analyzed the pathogenic process of HAND. We noticed vacuolar changes in perivascular processes of astrocytes by electron microscopy, and examined expression of astrocyte‐specific protein aquaporin‐4 (AQP4) by immunohistochemistry. APQ4 was diffusely positive in the neuropil and perivascular area in control brains. There was patchy or diffuse decrease of AQP4 staining in the neuropil of SIV‐infected macaques, which was associated with EAAT‐2 staining by double immunostaining. A quantitative analysis demonstrated significant positive correlation between areas of AQP4 and EAAT‐2. Some astrocytes express EAAT‐2 but not AQP4, and decrease of EAAT‐2 expression tended to be less than the decrease of AQP4. Active‐caspase‐3 immunostaining demonstrated apoptosis of neurons and astrocytes in the area of AQP4/EAAT‐2 reduction. These results suggest that AQP4 is damaged first and decrease of EAAT‐2 may follow in pathogenesis of cortical degeneration. This is the first demonstration of decrease of AQP4 and its association with EAAT‐2 decrease in AIDS brain, suggesting a role in the pathogenesis of HAND.

Menin mediates Tat-induced neuronal apoptosis in brain frontal cortex of SIV-infected macaques and in Tat-treated cells

The molecular mechanisms involved in human immunodeficiency virus (HIV)-associated neurocognitive disorder (HAND) remain poorly understood. It has been recently reported that HIV-1 Tat transactivation requires menin, suggesting that menin may be involved in HAND pathogenesis. But the role of menin is not clear. Here, we found that protein level of menin was increased in simian-human immunodeficiency chimeric virus (SHIV)-SF162.P4 and simian immunodeficiency virus (SIV) sm543-3-infected rhesus macaques compared with the controls by immunohistochemistry (IHC) and western blot. Menin mainly expressed in the frontal cortex neurons of the brain, more importantly, the number of menin-staining cells was positively correlated with cleaved-caspase-3-positive cells while it was negatively correlated with a neuron-specific nuclear protein NeuN-positive cells, suggesting that expression of menin may induce neuronal apoptosis. Further studies showed that menin level was significantly increased during Tat-induced apoptosis, while downregulation of menin by pll3.7-MEN1-shRNA attenuated the Tat-induced cleavage of caspase-3 and caspase-8 in SY5Y cells and primary neuron cultures. Together, our findings reveal a pro-apoptotic role of menin in the brains of the SIV-infected macaques and the cultured neurons, indicating that targeting menin may be potential to block the HIV-1 Tat induced neuronal damage in HAND.

HIV-1 Tat inhibits EAAT-2 through AEG-1 upregulation in models of HIV-associated neurocognitive disorder

During HIV-associated neurocognitive disorder (HAND), decreasing in excitatory amino acid transporter 2 (EAAT-2) in astrocyte plasma membranes leads to elevated levels of extracellular glutamate and, in turn, neuronal apoptosis. We used immunohistochemistry, western blot, qRT-PCR, and RNA interference to elucidate the molecular mechanisms underlying the decreased EAAT-2 expression during HAND at the tissue and cellular levels. We used simian immunodeficiency virus-human immunodeficiency virus chimeric virus (SHIV)-infected macaques as an in vivo model of HAND. Our results show that EAAT-2 expression was decreased in the cerebral cortex, while AEG-1 expression was increased, and the expression levels of these proteins were negatively correlated. In vitro analyses showed that HIV-1 Tat inhibited EAAT-2 expression by inducing overexpression of AEG-1. More specifically, HIV-1 Tat increased AEG-1 expression via the PI3-K signaling pathway, while increasing EAAT-2 inhibition by YinYan-1 (YY-1) via the NF-κB signaling pathway. These results warrant testing AEG-1 as a potential therapeutic target for treating HAND.

Multiple spotty lesions of the spinal cord in a Chinese patient with human T-lymphotropic virus type 1-associated myelopathy/tropical spastic paraparesis

The case of a Chinese patient with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), who showed typical neurological symptoms of the disease, is reported here. Since the presence of anti-HTLV-1 antibody was not investigated, this patients diagnosis of HAM/TSP was delayed for 4 years. Magnetic resonance imaging revealed multiple spotty lesions in the cervical spinal cord, probably reflecting pathological changes known as perivascular lymphocytic infiltrations of the spinal cord. As this is the first case report of a HAM/TSP patient in China, it is suggested that serological testing for HTLV-1 should be considered in patients with spastic paraparesis even in areas that are non-endemic for HTLV-1.