Rodent models for HIV-associated neurocognitive disorders

Abstract

Human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) reflect the spectrum of neural impairments seen during chronic viral infection. Current research efforts focus on improving antiretroviral and adjunctive therapies, defining disease onset and progression, facilitating drug delivery, and halting neurodegeneration and viral resistance. Because HIV is species-specific, generating disease in small-animal models has proved challenging. After two decades of research, rodent HAND models now include those containing a human immune system. Antiviral responses, neuroinflammation and immunocyte blood-brain barrier (BBB) trafficking follow HIV infection in these rodent models. We review these and other rodent models of HAND and discuss their unmet potential in reflecting human pathobiology and in facilitating disease monitoring and therapeutic discoveries.

Figure 1. Schematic overview of HIVE mouse models for neuroAIDS

(i) Human monocytes differentiated into macrophages (MDM) in presence of macrophage colony stimulating factor (MCSF) are infected with HIV-1_ADA, followed by the stereotactic injection of these cells into the basal ganglia of immune deficient mice. Histopathological features of human HIVE were induced that consisted of multinucleated giant cells, astrogliosis and microglial activation and neuronal loss around the injection site (bottom panel) [107]. Standard HIVE models have been widely used to test anti-retroviral, immune modulatory and adjunctive therapies for neuroAIDS [–79]. Nonetheless, these models show limitations in reflecting aspects of HIV-1 neuropathogenesis. Although perivascular macrophages and microglia are the major drivers of brain pathology during HIV-1 disease, correlations have been noted between HIV-associated neurocognitive disorders and immune suppression [88] To this end, an alternative mouse model (called huPBL/HIVE) (ii) was developed [89] that includes an adaptive immune cell component to assess its role beyond the innate response (ie. virus-infected macrophage-associated pathologies). Herein, replicate human lymphocytes were isolated from the same donor leukopak, then injected intraperitoneally (i.p.) to reconstitute the peripheral immune system of NOD/scid mice. A week after reconstitution, HIV-1 infected MDMs were injected into the basal ganglia of these mice. In brains of huPBL-HIVE mice, lymphocytes (arrows, bottom panel) are in close contact with the infected MDMs (arrow heads). Viruses spread to lymphocytes, and the infection disseminates throughout the blood and peripheral immune organs. HIV-1 specific cytotoxic T cell responses were also detected in these mice [89]. Hence, the clearance of viral-infected MDMs was observed. huPBL-HIVE mice permits the investigation of immune modulators for disease. However, HuPBL engraftment induces graft versus host disease due to the over-activation of human lymphocytes towards the host cells. To avoid this result, a third model was developed. (iii) HIV-1/VSV infected bone marrow-derived macrophages were injected into the brains of immunocompetent C57/B16 mice. HIV-1/VSV pseudotyped chimeric virus is used to bypass the restriction of infection of murine cells. Similar to the huPBL/HIVE model, infiltration of lymphocytes (arrows) into the injected area was seen with a faster clearance of infected macrophages [59], due to the immune response from the donor mouse lymphocytes. Due to the lack of viral spread to mouse cells, there is no viral dissemination into lymphocytes and the periphery, and multinucleated giant cells are also not formed. Abbreviations: i.c., intracranial; MNGC, multinucleated giant cell; NOD/SCID, non-obese diabetic severe combined immunodeficiency; PBL, peripheral blood lymphocytes. Immunohistochemical stainings adapted, with permission, from [60] (i), [89] (ii) and [59] (iii).

Figure 2. “Humanized” NOD/NSG mouse models for chronic HIV-1 infection

A human CD34+ stem cell reconstituted mouse model was developed to permit long-term studies assessing the relationship between peripheral infection and brain pathology. Human CD34+ stem cells (HSC) isolated from umbilical cord blood were injected intrahepatically into 1 day-old irradiated pups. A complete human immune system is developed in NOD/LtSz-scid g_c^null NSG mice [91, 100, 102, 108]. The injected HSC reach mouse lymphoid organs including bone marrow, spleen, lymph nodes, gut and develop into a broad range of immune cell lineages. The presence of human CD34+ stem cells, myeloblasts, B cell precursors, erythroblasts, promyelocytes, granulocytes and monocytes in mouse bone marrow were detected. Human T cell development occurred in the mouse thymus, as evidenced by the presence of CD4/CD8 T cells. In lymphoid tissue, distinct follicles filled with human T, B lymphocytes and macrophages were observed. Lymph nodes were also reconstituted with human lymphocytes, macrophages and dendritic cells. A mature human immune system develops in the mouse by 20–22 weeks [108].

Figure 3. HIV-1 neuropathology in “humanized” NSG mice

Chronic HIV-1 infection for 8 weeks leads to reductions in CD4+ T lymphocytes in lymphoid tissue and accelerated entry of human cells into the brain. Increased ingress of HIV-1 infected blood borne macrophages into the meninges and perivascular spaces of the brain were observed. These pathobiological events induce micro- and astro- glial inflammation and neuronal dysfunction, as evidencedby protein abnormalities. A, Microglial activation and nodule formation in white matter tracts within the brain stem as evidenced by immunocytochemical staining (indicated in brown color) for the ionized calcium binding adaptor molecule-1 (anti-Iba-1, arrowheads). B, Perivascular accumulation of mouse macrophages and microglia (Iba-1 staining) in the cerebellum (arrowhead). C, Astrocyte activation in white matter tracts within the cerebellum around blood vessels (and other brain regions, not shown) is evident by the immunopositive staining for glial fibrillary acidic protein (arrows). D, Pattern of distribution of immune competent human cells visualized by staining for human HLA-DR (a MHC class II cell surface receptor, brown color). Human cells are along the cerebellar fissures, in granular cell layers and in perivascular space (arrows). Insert to D, is an adjusted section of the vessel shown with a cell stained for HIV-1p24. E, HLA-DR staining shows human leukocytes in the meninges with an adjusted section stained for HIV-1p24 shows a high proportion of infected cells (insert to E). F, Microglia-like cells stained for HIV-1p24 were rarely observed in parenchyma. The blue hematoxylin counterstaining was used for nuclei [Images adapted from [100] (A–C) and [102] (D–F)]. (G–H) Immunofluorescence labeling of dendrites (red staining for Microtubule-Associated Protein 2, MAP2) and synapses (green staining for synaptophysin, SYN) in the cortex of control uninfected (G) and HIV-1 infected (H) mice (15 weeks of age) show dendritic and synaptic protein integrity losses in the infected case. Cell nuclei indicated by 4′,6-diamidino-2-phenylindole (DAPI) staining (blue) [102]. At the figure bottom is a schematic representation of the fields of view A – F taken from the saggital sections (blue squares – bright field images), and G and H taken from coronal section (red – immunofluorescent) are shown. Images reproduced, with permission, from [100] and [102].

Figure 4

Schematic model illustrating the use of HIV-1 infected “humanized” NOD/NSG mice for testing nanoformulated antiretroviral therapies (nanoART). This schematic reflects the potential of long-acting antiretrovirals to reduce viral load and protect both CD4+ T cells and the CNS against HIV-1 associated injuries [106]. The colors reflect normal immune tissue homeostasis (green and yellow) and tissues damaged by chronic viral replication (red). CD4+ T lymphocyte decline is observed following HIV-1 infection [108, 109]. In addition, infected and immune activated lymphocytes and macrophages have been observed in lymphoid organs and peripheral blood [108, 109]. Infected animals have been treated with nanoART [106]. which can gain entry into monocyte-macrophages (center of picture) and serve as a long-term drug depot leading to the suppression of viral replication and protection of CD4+ T cell numbers (green and yellow). The nanoformulation may also facilitate ART delivery to the nervous system, although this has yet to be experimentally demonstrated.