Links between progressive HIV-1 infection of humanized mice and viral neuropathogenesis

Abstract

Few rodent models of human immunodeficiency virus type one (HIV-1) infection can reflect the course of viral infection in humans. To this end, we investigated the relationships between progressive HIV-1 infection, immune compromise, and neuroinflammatory responses in NOD/scid-IL-2Rγ(c)(null) mice reconstituted with human hematopoietic CD34(+) stem cells. Human blood-borne macrophages repopulated the meninges and perivascular spaces of chimeric animals. Viral infection in lymphoid tissue led to the accelerated entry of human cells into the brain, marked neuroinflammation, and HIV-1 replication in human mononuclear phagocytes. A meningitis and less commonly an encephalitis followed cM-T807 antibody-mediated CD8(+) cell depletion. We conclude that HIV-1-infected NOD/scid-IL-2Rγ(c)(null) humanized mice can, at least in part, recapitulate lentiviral neuropathobiology. This model of neuroAIDS reflects the virological, immunological, and early disease-associated neuropathological components of human disease.

Figure 1

Human cells in meninges and brain perivascular spaces. Representative paraffin-embedded 5-μm brain sections from uninfected control animal stained for human CD163, HLA-DR, and CD14 markers show cells with macrophage, microglia, and dendritic cell morphology (brown). Sections were counterstained with hematoxylin. Original magnification ×200; Insets magnification ×1000.

Figure 2

Human cells in mouse brain. The majority of human cells were in the meninges. HIV-1 infection and CD8⁺ cell depletion increased human cell infiltration into the meninges as assessed by human HLA-DR staining of macrophages and lymphocytes. Representative tissue sections (2–4 sagittal with average total area of 78.8 mm²) were examined for human HLA-DR and CD163 using a ×40 objective. Average numbers of cells in meninges, perivascular spaces, and parenchyma/section were calculated for each mouse. Mean ± SEM cells per section are shown. P values were determined by analysis of variance and animal groups were compared by non-parametric Mann–Whitney U tests: *P < 0.05, **P < 0.01 compared to control. Numbers of histologically analyzed animals per groups are placed in parentheses.

Figure 3

Influx of CD8⁺ cells in the brain of HIV-1–infected mice. Sagittal brain section of 36-week-old mouse #596 infected for 5 weeks is shown (olfactory bulb). Sections were stained for (A and D) human CD163, (B and E) human CD8, and (C and F) HIV-1p24 antigen. A–C: Distribution of human macrophages in meninges and along the vessels, scattering through parenchyma are CD8⁺ cells and rare HIV-1p24-positive cells (arrows, C). Inset in F shows magnified view of a group of infected cells in meninges. Sections were counterstained with hematoxylin. Magnifications: A–C ×100, D–F ×400 and inset ×1000.

Figure 4

Meningitis in HIV-1–infected and CD8⁺ cell–depleted mice. Horizontal brain sections of a 25-week-old mouse #209 infected for 4 weeks and CD8⁺ cell depleted for 2 weeks were stained for human HLA-DR (left column), HIV-1p24 antigen, and human CD163 (right column). Panels show the accumulation of human cells in cerebellar and olfactory bulb meninges. Insets represent magnified views of selected area (Asterisk) on adjacent section stained for human macrophage marker CD163 (MGC, middle row) and viral protein (top and bottom rows). Sections were counterstained with hematoxylin. Magnification ×10, inset magnification is ×100.

Figure 5

Meningoencephalitis in HIV-1–infected and CD8⁺ cell–depleted mice. Horizontal brain sections of a 29-week-old mouse #305 infected for 8 weeks and CD8⁺ cell–depleted for 3 weeks are shown. Sections were stained for (A) human HLA-DR, (B) HIV-1p24 antigen, and (C and D) mouse astrocytes (GFAP, Permanent Red) and microglia (Iba-1, DAB), (E and F) human CD8⁺ cells, and the inset is the adjacent section stained for HIV-1p24, and (G and H) neuronal nuclear protein (NeuN, DAB) and MAP-2 (Permanent Red). A: Accumulation of human cells in meninges and periaqueductal structures. B: Magnified views of selected area on the adjacent section stained for viral protein showing viral cytopathic effects, perivascular-infected cells, and infected cells with microglial morphology. C: Diffused activation of astrocytes and microglia, perivascular cuffs. D: Magnified view of selected region showing activated phagocytizing microglial cells (arrows). E and F: Scattering CD8⁺ cells in cerebellar white matter tracts (E) and perivascular infiltration of CD8⁺ cells near HIV-1 p24 infected cell (F and inset). G and H: Neuronal density in periaqueductal gray matter in the same mouse and in control reconstituted animal of the same age, respectively. Sections were counterstained with hematoxylin. Magnifications: A, ×1; B, ×20; inset, × 400; C, ×4; D, ×400; E and F and inset, × 400; G and H, ×20.

Figure 6

Glial reactions in HIV-1–infected and CD8⁺ cell–depleted mice. HIV-1 infection induced a diffuse astrogliosis that was increased by CD8⁺ cell depletion as seen by GFAP staining. This paralleled microglial activation responses measured by Iba-1 staining. Histopathological examination was performed on a minimum of four brain regions derived from paraffin-embedded brain tissue and recorded by two independent investigators. Findings were compared to animals that were not manipulated in any manner (score of 0). A score of 1 represents few activated astrocytes or activated microglial cells. A score of 2 is coincident with moderate activation of astrocytes and microglia. A score of 3 shows hypertrophic astrocytes and concomitant processes and microglial nodules. P values were determined by analysis of variance and animal groups were compared by nonparametric Mann–Whitney U tests. Mean ± SEM score per region are illustrated. *P < 0.05, **P < 0.01, ***P < P < 0.001 statistically different compared to uninfected controls, ^†P < 0.05 compared to HIV-1–infected.

Figure 7

HIV-1 infection and CD8⁺ cell depletion affect immune and viral brain biomarkers. RNA was extracted from cortex and cerebellar tissues, and RT-PCR was performed to assess the expression of HLA-DQ (to estimate human cells), HIV-1gag, TNF-α, and iNOS. Animals were injected with α-CD8 antibody after (post) infection with HIV-1. Uninfected animals were injected with the antibody at similar age. Expression of all genes was normalized to GAPDH used as an endogenous control. Median lines, the range and interquartile range, and number of analyzed animals (in parenthesis) are shown. Statistical analyses were determined by analysis of variance and comparison by nonparametric Mann–Whitney U test between groups. *P < 0.05, **P < 0.01 compared to uninfected controls, ***P < 0.01 to HIV-1–infected.