Woong-Ki Kim

Magnetic resonance spectroscopy reveals that activated monocytes contribute to neuronal injury in SIV neuroAIDS

Difficulties in understanding the mechanisms of HIV neuropathogenesis include the inability to study dynamic processes of infection, cumulative effects of the virus, and contributing host immune responses. We used H magnetic resonance spectroscopy and studied monocyte activation and progression of CNS neuronal injury in a CD8 lymphocyte depletion model of neuroAIDS in SIV-infected rhesus macaque monkeys. We found early, consistent neuronal injury coincident with viremia and SIV infection/activation of monocyte subsets and sought to define the role of plasma virus and monocytes in contributing to CNS disease. Antiretroviral therapy with essentially non-CNS-penetrating agents resulted in slightly decreased levels of plasma virus, a significant reduction in the number of activated and infected monocytes, and rapid, near-complete reversal of neuronal injury. Robust macrophage accumulation and productive virus replication were found in brains of infected and CD8 lymphocyte-depleted animals, but no detectable virus and few scattered infiltrating macrophages were observed in CD8 lymphocyte-depleted animals compared with animals not receiving antiretroviruses that were sacrificed at the same time after infection. These results underscore the role of activated monocytes and monocyte infection outside of the brain in driving CNS disease.

The level of monocyte turnover predicts disease progression in the macaque model of AIDS

It is widely accepted that destruction of CD4(+) T cells and viral load are the primary markers for immunodeficiency in HIV-1-infected humans and in simian immunodeficiency virus (SIV)-infected macaques. However, monocyte/macrophages are also important targets of HIV/SIV infection and a critical link between innate and adaptive immunity. We therefore examined whether changes in cells of the monocyte/macrophage lineage could be linked to the pathogenesis of AIDS in the rhesus macaque model. Here, we show that massive turnover of peripheral monocytes associated with death of tissue macrophages correlates with AIDS progression in macaques. More importantly, the level of monocyte turnover was not linked to the CD4(+) T-cell count and was a better predictive marker for AIDS progression than was viral load or lymphocyte activation. Our results show the importance of monocyte/macrophages in the pathogenesis of AIDS and suggest the dynamic changes of the monocyte/macrophages as a new marker for AIDS progression.

Monocyte/macrophage traffic in HIV and SIV encephalitis.

This short review focuses on the role of central nervous system (CNS) perivascular macrophages as targets of productive infection of the CNS. Data discussed include the importance of these cells as early targets of infection and their productive infection with AIDS. Many of the immune molecules on perivascular macrophages are also found on subsets of blood monocyte/macrophages, some of which are expanded during human immunodeficiency virus (HIV) infection. These observations paired with the known bone marrow (BM) origin of perivascular macrophages and the BM as a site of HIV infection underscore the importance of the study of monocyte populations in the BM and blood, which are activated and infected as a source of virus that enters the CNS. Data presented and discussed herein suggest a role of HIV‐infected BM‐derived monocytes as “Trojan horse” cells that traffic to the CNS to become perivascular macrophages. The study of such cells including their timing of infection, activation, and traffic and the role of HIV‐specific immune responses controlling their accumulation in the CNS warrant study with regard to CNS neuropathogenesis.

Monocyte heterogeneity underlying phenotypic changes in monocytes according to SIV disease stage

Infection by HIV is associated with the expansion of monocytes expressing CD16 antigens, but the significance of this in HIV pathogenesis is largely unknown. In rhesus macaques, at least three subpopulations of blood monocytes were identified based on their expression of CD14 and CD16: CD14highCD16−, CD14highCD16low, and CD14lowCD16high. The phenotypes and functions of these subpopulations, including CD16+ monocytes, were investigated in normal, uninfected rhesus macaques and macaques that were infected with SIV or chimeric SHIV. To assess whether these different monocyte subpopulations expand or contract in AIDS pathogenesis, we conducted a cross‐sectional study of 54 SIV‐ or SHIV‐infected macaques and 48 uninfected controls. The absolute numbers of monocyte populations were examined in acutely infected animals, chronically infected animals with no detectable plasma virus RNA, chronically infected animals with detectable plasma virus RNA, and animals that died with AIDS. The absolute numbers of CD14highCD16low and CD14lowCD16high monocytes were elevated significantly in acutely infected animals and chronically infected animals with detectable plasma virus RNA compared with uninfected controls. Moreover, a significant, positive correlation was evident between the number of CD14highCD16low or CD14lowCD16high monocytes and plasma viral load in the infected cohort. These data show the dynamic changes of blood monocytes, most notably, CD14highCD16low monocytes during lentiviral infection, which are specific to disease stage.

Changes in MRS neuronal markers and T cell phenotypes observed during early HIV infection.

Objective: To determine if changes in brain metabolites are observed during early HIV infection and correlate these changes with immunologic alterations. Methods: Eight subjects with early HIV infection, 9 HIV-seronegative controls, and 10 chronically HIV-infected subjects without neurologic impairment underwent 1H magnetic resonance spectroscopy. Subjects with early stage infection were identified near the time of HIV seroconversion and imaged within 60 days of an evolving Western blot, while still having detectable plasma virus. Subjects had blood drawn for viral RNA and T cell quantification. Results: Both N-acetylaspartate (NAA) and Glx (glutamate + glutamine) were decreased in the frontal cortical gray matter of seropositive subjects. NAA levels were found to be decreased in the centrum semiovale white matter of chronically HIV-infected subjects, but not in those with early infection. Both HIV-infected cohorts demonstrated a lower number of CD4+ T lymphocytes and a higher number of CD8+ T lymphocytes in their blood. Lower NAA levels in the frontal cortex of subjects with early infection were associated with an expansion of CD8+ T cells, especially effector CD8+ T cells. Conclusions: These results verify metabolism changes occurring in the brain early during HIV infection. Lower NAA and Glx levels in the cortical gray matter suggests that HIV causes neuronal dysfunction soon after infection, which correlates to the expansion of CD8+ T cells, specifically to an activated phenotype. Utilizing magnetic resonance spectroscopy to track NAA levels may provide important information on brain metabolic health while allowing better understanding of the virus–host interactions involved in CNS functional deficits.

Recently Infiltrating MAC387+ Monocytes/ Macrophages: A Third Macrophage Population Involved in SIV and HIV Encephalitic Lesion Formation

Monocytes/macrophages are critical components of HIV and SIV encephalitic lesions. We used in vivo BrdU labeling and markers specific to stages of macrophage differentiation or inflammation to define macrophage heterogeneity and to better define the role of macrophage populations in lesion formation and productive infection. Lesions were heterogeneously composed of resident macrophages (CD68(+)HAM56(+)), perivascular macrophages (CD163(+) CD68(+)MAC387(-)), and recently infiltrated MAC387(+) CD68(-)CD163(-) monocytes/macrophages. At 24 and 48 hours after BrdU inoculation, 30% of MAC387(+) monocytes/macrophages were BrdU(+), consistent with their being recently infiltrated. In perivascular cuffs with low-level SIV replication, MAC387(+) monocytes/macrophages outnumbered CD68(+) macrophages. Conversely, lesions with numerous SIV-p28(+) macrophages and multinucleated giant cells had fewer MAC387(+) monocytes/macrophages. The MAC387(+) cells were not productively infected nor did they express detectable CCR2, unlike perivascular macrophages. Overall, we found that the proportion of MAC387(+) cells tends to be higher than the proportion of CD68(+) macrophages in the brain of animals with mild encephalitis; the ratio was reversed with more severe encephalitis. These results suggest that development of SIV and HIV encephalitis is an active and ongoing process that involves the recruitment and accumulation of: i) nonproductively infected MAC387(+) monocytes/macrophages that are present with inflammation (potentially M1-like macrophages), ii) CD163(+) perivascular macrophages (consistent with M2-like macrophages), and iii) CD68(+) or HAM56(+) resident macrophages. The latter two populations are cellular reservoirs for productive infection.

Characterization of lymphocytic infiltrates in progressive multifocal leukoencephalopathy: Co-localization of CD8+ T cells with JCV-infected glial cells

We characterized inflammatory infiltrates in archival brain biopsy and autopsy samples from 26 HIV+ and 20 HIV− patients with progressive multifocal leukoencephalopathy (PML). The predominant inflammatory cells were CD8+ T lymphocytes. We defined CD8+ T cell distribution with regard to JCV-infected glial cells, PML lesions and the extent of demyelination. In most samples from either HIV+ and HIV− patients, we found positive correlations between the parenchymal CD8+ T cells and JCV-infected glial cells and conversely, negative correlations between the perivascular CD8+ T cells and JCV-infected glial cells in the surrounding brain. Most of these correlations remained significant after accounting for the degree of demyelination and location of the cells relative to lesions. Moreover, high numbers of CD8+ T cells were found within and at the border of active PML lesions. These results suggest that CD8+ T cells are primarily associated with JCV-infected glial cells in most PML cases and that an active ongoing recruitment of CD8+ T cells and possibly viral antigen-specific retention could occur. These observations are discussed in the context of the recent evidence of PML in multiple sclerosis and Crohn’s patients treated with natalizumab, underscoring the role of CD8+ T lymphocytes in continued immunosurveillance of the CNS.

Identification of T lymphocytes in simian immunodeficiency virus encephalitis: Distribution of CD8+ T cells in association with central nervous system vessels and virus

T lymphocytes are found within brains infected with human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) where they are a minor, but consistently identified, population. However, little analysis of their phenotypes has been done, and questions concerning whether or not they are viral antigen specific has not been thoroughly examined. We investigated the central nervous system (CNS) of SIV-infected rhesus macaques to identify T-lymphocyte subsets in relation to virus-infected cells and brain microvessels. We have found that a sensitive antigen-retrieval technique greatly enhanced immunohistochemical detection of CD4+ and CD8+ T lymphocytes in control studies. In encephalitic brains of SIV-infected monkeys with acquired immunodeficiency syndrome (AIDS), we found a significant accumulation of CD8+ T lymphocytes but little-to-no accumulation of CD4+ T lymphocytes. CD4+ cells, when detected, were mostly monocyte/macrophages closely associated with CNS vessels. Using a combination of in situ hybridization for SIV RNA, and immunohistochemistry for CD8+ T lymphocytes and/or Glut-1 for endothelial cells on brain microvessels, we found CD8+ T lymphocytes with an angiocentric distribution often adjacent to virus-infected cells. In the CNS of animals with SIV encephalitis, there was a trend of CD8+ T lymphocytes that were not directly juxtaposed with CNS vessels. These data suggest that in brains of SIV-infected monkeys and HIV-infected humans, CD8+ T lymphocytes traffic to and are retained in the CNS in an angiocentric and possibly antigen-specific manner.

SMAD proteins of oligodendroglial cells regulate transcription of JC virus early and late genes coordinately with the Tat protein of human immunodeficiency virus type 1

JC virus (JCV) is the aetiological agent of progressive multifocal leukoencephalopathy (PML), a fatal, demyelinating disease of the brain affecting people with AIDS. Although immunosuppression is involved in infection of the brain by JCV, a direct influence of human immunodeficiency virus type 1 (HIV-1) has also been established. The Tat protein of HIV-1 has been implicated in activation of the cytokine transforming growth factor (TGF)-beta in HIV-1-infected cells and in stimulating JCV gene transcription and DNA replication in oligodendroglia, the primary central nervous system cell type infected by JCV in PML. This study demonstrated that Tat can cooperate with SMAD proteins, the intracellular effectors of TGF-beta, at the JCV DNA control region (CR) to stimulate JCV gene transcription. Tat stimulated JCV early gene transcription in KG-1 oligodendroglial cells when expressed via transfection or added exogenously. Using chromatin immunoprecipitation, it was shown that exogenous Tat enhanced binding of SMAD2, -3 and -4 and their binding partner Fast1 to the JCV CR in living cells. When SMAD2, -3 and -4 were expressed together, Tat, expressed from plasmid pTat, stimulated transcription from both early and late gene promoters, with the early promoter exhibiting stimulation of >100-fold. Tat, SMAD4 and JCV large T-antigen were all visualized in oligodendroglial cells at the border of an active PML lesion in the cerebral frontal lobe. These results revealed a positive reinforcement system in which the SMAD mediators of the TGF-beta system act cooperatively with Tat to stimulate JCV gene transcription.

The role of monocytes and perivascular macrophages in HIV and SIV neuropathogenesis: Information from non-human primate models

Perivascular macrophages are located in the perivascular space of cerebral microvessels and thus uniquely situated at the intersection between the brain parenchyma and blood. Connections between the nervous and immune systems are mediated in part through these cells that are ideally located to sense perturbations in the periphery and turnover by cells entering the central nervous system (CNS) from the circulation. It has become clear that unique subsets of brain macrophages exist in normal and SIV- or HIV-infected brains, and perivascular macrophages and similar cells in the meninges and choroid plexus play a central role in lentiviral neuropathogenesis. Common to all these cell populations is their likely replacement within the CNS by monocytes. Studies of SIV-infected non-human primates and HIV-infected humans underscore the importance of virus-infected and activated monocytes, which traffic to the CNS from blood to become perivascular macrophages, potentially drive blood-brain barrier damage and cause neuronal injury. This review summarizes what we know about SIV- and HIV-induced neuropathogenesis focusing on brain perivascular macrophages and their precursors in blood that may mediate HIV infection and injury in the CNS.