Central Nervous System Inflammation and Infection during Early, Nonaccelerated Simian-Human Immunodeficiency Virus Infection in Rhesus Macaques

Abstract

Studies utilizing highly pathogenic simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV) have largely focused on the immunopathology of the central nervous system (CNS) during end-stage neurological AIDS and SIV encephalitis. However, this may not model pathophysiology in earlier stages of infection. In this nonaccelerated SHIV model, plasma SHIV RNA levels and peripheral blood and colonic CD4⁺ T cell counts mirrored early human immunodeficiency virus (HIV) infection in humans. At 12 weeks postinfection, cerebrospinal fluid (CSF) detection of SHIV RNA and elevations in IP-10 and MCP-1 reflected a discrete neurovirologic process. Immunohistochemical staining revealed a diffuse, low-level CD3⁺ CD4^- cellular infiltrate in the brain parenchyma without a concomitant increase in CD68/CD163⁺ monocytes, macrophages, and activated microglial cells. Rare SHIV-infected cells in the brain parenchyma and meninges were identified by RNAScope in situ hybridization. In the meninges, there was also a trend toward increased CD4⁺ infiltration in SHIV-infected animals but no differences in CD68/CD163⁺ cells between SHIV-infected and uninfected control animals. These data suggest that in a model that closely recapitulates human disease, CNS inflammation and SHIV in CSF are predominantly mediated by T cell-mediated processes during early infection in both brain parenchyma and meninges. Because SHIV expresses an HIV rather than SIV envelope, this model could inform studies to understand potential HIV cure strategies targeting the HIV envelope.IMPORTANCE Animal models of the neurologic effects of HIV are needed because brain pathology is difficult to assess in humans. Many current models focus on the effects of late-stage disease utilizing SIV. In the era of antiretroviral therapy, manifestations of late-stage HIV are less common. Furthermore, new interventions, such as monoclonal antibodies and therapeutic vaccinations, target HIV envelope. We therefore describe a new model of central nervous system involvement in rhesus macaques infected with SHIV expressing HIV envelope in earlier, less aggressive stages of disease. Here, we demonstrate that SHIV mimics the early clinical course in humans and that early neurologic inflammation is characterized by predominantly T cell-mediated inflammation accompanied by SHIV infection in the brain and meninges. This model can be utilized to assess the effect of novel therapies targeted to HIV envelope on reducing brain inflammation before end-stage disease.

Keywords: HIV; SHIV; early infection; neuropathology.

FIG 1

SHIV RNA in plasma and CSF during early infection. Gray circles represent individual plasma viremia after intrarectal (n = 9) or intravaginal (n = 3) SHIV inoculation at week 0. Red box-and-whisker plots depict medians, interquartile ranges, and ranges of plasma SHIV levels at weeks 2 and 12 postinfection. Blue squares represent CSF SHIV RNA levels in the four animals with detectable SHIV RNA in the CSF at week 12 postinfection, corresponding to the four animals with the highest plasma viremia at the same time point.

FIG 2

Peripheral and colonic CD4⁺ T cell depletion occurs early in infection. (A) Longitudinal peripheral blood CD4⁺ T cell counts at preinfection baseline, acute infection (W3), and set point (W12). (B) CD4⁺ T cell percentages from colonic biopsy specimens at acute infection (W3) and set point (W12) with respect to uninfected controls. Horizontal lines represent mean values. **, P ≤ 0.005; *, P ≤ 0.05. P values were calculated with Wilcoxon matched-pairs signed rank tests (A) or Mann-Whitney tests (B).

FIG 3

Soluble markers of inflammation in CSF are distinct from those in plasma. (A to C) Longitudinal depiction of soluble IL-15 (A), MCP-1 (B), and IP-10 (C) in plasma prior to SHIV infection at week 0 (solid circles), at peak viremia 2 weeks postinfection (solid triangles), and at set point viremia 12 weeks postinfection (solid squares). Horizontal lines represent mean values. Levels in CSF 12 weeks postinfection (open squares) were compared to those in CSF from healthy uninfected control macaques. ***, P ≤ 0.0005; **, P ≤ 0.005; *, P ≤ 0.05. P values were calculated with Wilcoxon matched-pairs signed rank tests (plasma) or Mann-Whitney tests (CSF).

FIG 4

CD3⁺ T cell infiltrate into brain parenchyma in early SHIV infection. (A) Representative images of CD3⁺ T cell staining in the superficial cortex and midbrain of SHIV-infected animals and an uninfected control. CD3⁺ T cells (brown) are depicted pavementing along the endothelium of a blood vessel in the superficial cortex (top left) in an SHIV-infected animal and in the midbrain parenchyma of a different SHIV-infected animal (top middle). In contrast, CD3⁺ T cell staining in uninfected control animals was limited to intravascular spaces in all regions (top right) without evidence of endothelial pavementing. (B) Quantification of CD3⁺ T cell counts per 40 HPF in midbrain, basal ganglia, and superficial cortex in the six SHIV-infected animals with the highest viremia 12 weeks postinfection (solid squares) versus uninfected controls (open squares). Horizontal lines represent mean values. **, P < 0.01; calculated with the Mann-Whitney test.

FIG 5

No significant increase in CD68/CD163⁺ cell infiltrate in brain parenchyma in early SHIV infection. (A) Representative images of CD68/CD163⁺ staining in the superficial cortex and midbrain of SHIV-infected animals and an uninfected control. White arrows indicate cells positive for CD68/CD163 (red). DAPI staining of nuclei is depicted in aqua. (B) Quantification of CD68/CD163⁺ cell counts per 40 HPF in midbrain, basal ganglia, and superficial cortex in the six SHIV-infected animals with the highest viremia 12 weeks postinfection (solid squares) versus uninfected controls (open squares). Horizontal lines represent mean values. P > 0.05; calculated with the Mann-Whitney test.

FIG 6

CD4⁺ cell infiltrate into meninges of SHIV-infected macaques without concomitant increase of CD168/CD163⁺ cell infiltrate. (A) Representative immunohistochemical staining of meninges revealing a mild CD4⁺ cell infiltrate (dark brown) into meninges 12 weeks after SHIV infection (left) but not in uninfected controls (right). Light brown represents nonspecific staining inside meningeal vessels, whereas dark brown represents extravascular CD4⁺ cells within the meningeal space. (B) Quantification of CD4⁺ cell counts per 40 HPF in meninges in SHIV-infected macaques (solid squares) versus uninfected controls (open squares), depicting increased CD4⁺ cell infiltrate in four of six animals relative to healthy controls, three of whom had detectable SHIV RNA in CSF. Horizontal lines represent mean values. P > 0.05; calculated with the Mann-Whitney test. (C) Representative immunofluorescent staining of meninges in an SHIV-infected animal and uninfected control, with CD68/CD163⁺ cells depicted in red and DAPI-stained nuclei depicted in aqua. (D) Quantification of CD68/CD163⁺ cell counts per 40 HPF in meninges in SHIV-infected macaques (solid squares) versus uninfected controls (open squares), showing no significant differences between groups. Horizontal lines represent mean values. P > 0.05; calculated with the Mann-Whitney test.

FIG 7

SHIV RNA⁺ cells in superficial cortex and meninges 12 weeks postinfection. RNAScope staining of tissue using light (A and E) or confocal (B, C, D, and F) microscopy. (A) SHIV RNA⁺ cells (brown) in superficial cortex near a cortical sulcus (10×, left) and at higher magnification (60×, right). (B) SHIV RNA⁺ cells (red) in midbrain parenchyma with DAPI costaining of cell nuclei (aqua). (C) SHIV RNA⁺ cells (red) colocated with cells along the endothelium of a cortical blood vessel with DAPI costaining of cell nuclei (aqua). (D) SHIV RNA⁺ cells (red) within the meninges with DAPI costaining of cell nuclei (aqua). (E and F) SHIV RNA⁺ cells and virions within mesenteric lymph node follicles as positive controls (brown and red).

FIG 8

SHIV-1157ipd3N4 replication in MDMs. MDMs were exposed to SHIV-1157ipd3N4 at an MOI of 0.03 (see Materials and Methods). (A) Uninfected control MDMs; (B) syncytium formation in SHIV-1157ipd3N4-exposed MDMs. Black arrows indicate multinucleated giant cells; magnification, ×10. (C) p27 levels in supernatants collected from SHIV-1157ipd3N4-exposed MDMs. A blood pack of an anonymous human donor was used to prepare the cells used in this experiment. Data are representative of two independent experiments; error bars represent means ± standard deviations.