Coordinated regulation of SIV replication and immune responses in the CNS

Abstract

Central nervous system (CNS) invasion during acute-stage HIV-infection has been demonstrated in a small number of individuals, but there is no evidence of neurological impairment at this stage and virus infection in brain appears to be controlled until late-stage disease. Using our reproducible SIV macaque model to examine the earliest stages of infection in the CNS, we identified immune responses that differentially regulate inflammation and virus replication in the brain compared to the peripheral blood and lymphoid tissues. SIV replication in brain macrophages and in brain of SIV-infected macaques was detected at 4 days post-inoculation (p.i.). This was accompanied by upregulation of innate immune responses, including IFNbeta, IFNbeta-induced gene MxA mRNA, and TNFalpha. Additionally, IL-10, the chemokine CCL2, and activation markers in macrophages, endothelial cells, and astrocytes were all increased in the brain at four days p.i. We observed synchronous control of virus replication, cytokine mRNA levels and inflammatory markers (MHC Class II, CD68 and GFAP) by 14 days p.i.; however, control failure was followed by development of CNS lesions in the brain. SIV infection was accompanied by induction of the dominant-negative isoform of C/EBPbeta, which regulates SIV, CCL2, and IL6 transcription, as well as inflammatory responses in macrophages and astrocytes. This synchronous response in the CNS is in part due to the effect of the C/EBPbeta on virus replication and cytokine expression in macrophage-lineage cells in contrast to CD4+ lymphocytes in peripheral blood and lymphoid tissues. Thus, we have identified a crucial period in the brain when virus replication and inflammation are controlled. As in HIV-infected individuals, though, this control is not sustained in the brain. Our results suggest that intervention with antiretroviral drugs or anti-inflammatory therapeutics with CNS penetration would sustain early control. These studies further suggest that interventions should target HIV-infected individuals with increased CCL2 levels or HIV RNA in the CNS.

Figure 1. Quantitation of SIV virion RNA in plasma and CSF of SIV-infected macaques.

SIV RNA was isolated from 140 µl of plasma and CSF collected at terminal time points from uninfected and SIV-infected macaques sacrificed at 4, 7, 10, 14, 21, or 42 days p.i. SIV RNA copy equivalents were determined by quantitative RT-PCR, and the means (diamonds for plasma and squares for CSF) and standard deviation for each experimental group are indicated.

Figure 2. SIV RNA expression in brain of SIV-infected macaques.

SIV RNA copies were quantitated by quantitative RT-PCR in RNA isolated from brain collected at each terminal time point from uninfected and SIV-infected macaques (4, 7, 10, 14, 21, or 42 days after inoculation). Medians (black bars) and the range (vertical bars) for each experimental group are indicated. The 21 day group (12 macaques) were split into two groups that had viral RNA levels above or below the median of the 14 day macaques; the median for these two groups are shown. For the 42 day group, each diamond represents one animal, color-coded according to CNS disease severity (red - severe; blue - moderate; turquoise - mild; green–none), and the black bars represent the medians for severe/moderate (red dotted line), and mild/none animals (green dotted line).

Figure 3. Quantitation of mRNA in brain of SIV-infected macaques.

A) IFNβ; B) MxA; and C) TNFα mRNA was isolated from brain of uninfected and SIV-infected macaques at terminal time points (4, 7, 10, 14, 21, or 42 days after inoculation) and mRNA levels quantitated by quantitative RT-PCR. mRNA levels in the SIV-infected brain are represented as fold change over the average of three uninfected brain RNAs, calculated by ΔΔCt. Medians (black bars) for each experimental group are indicated. For the 42-day group, each diamond represents one animal, color-coded according to CNS disease severity (red-severe; blue-moderate; turquoise-mild; green-none), and the black bars represent the medians for severe/moderate (red dotted line), and mild/none animals (green dotted line). Outliers whose values were higher than 5 times the standard deviation for each group were excluded.

Figure 4. IFNβ protein levels in brain of SIV-infected macaques.

Brain homogenates were made from brain from uninfected and SIV-infected macaques at terminal time points (4, 7, 10, 14, 21, or 42 days after inoculation) and IFNβ protein was quantitated by quantitative western analyses as described in Methods and Materials. Medians (black bars) for each experimental group are indicated. For the 42 day group, each diamond represents one animal, color-coded according to CNS disease severity (red-severe; blue-moderate; turquoise-mild; green-none), and the black bars represent the medians for severe/moderate (red dotted line), and mild/none animals (green dotted line). Protein band intensities were normalized to GAPDH.

Figure 5. CCL2, IL6, IL10, and IL12 mRNA in brain of SIV-infected macaques.

mRNA was isolated form brain of uninfected and SIV-infected macaques at terminal time points (4, 7, 10, 14, 21, or 42 days after inoculation). mRNA levels for A) CCL2; B) IL6; C) IL10; and D) IL12 was measured by quantitative RT-PCR and mRNA levels in the SIV-infected are represented as fold change over the average of the uninfected values, calculated by ΔΔCt. Medians (black bars) for each experimental group are indicated. For the 42-day group, each diamond represents one animal, color-coded according to CNS disease severity (red-severe; blue-moderate; turquoise-mild; green-none). The black bars represent the medians for severe/moderate (red dotted line), and mild/none animals (green dotted line).

Figure 6. Expression of MHC Class II, CD68 and GFAP in brain of SIV-infected macaques.

Quantitative immunohistochemistry was used to measure A) MHC class II; B) CD68; and C) GFAP in brain from SIV-infected macaques at terminal time points (4, 7, 10, 14, 21, or 42 days p.i.). The quantification of each protein is based on the mean of 20 measures on brain from each macaque. D) MHC class II expression in brain at 4 days p.i. E) CD68 expression in brain at 4 days p.i.

Figure 7. Coordinated expression of innate immune genes and cytokines in brain of SIV-infected macaques during acute infection in brain.

A schematic of median values of mRNA levels for all cytokines measured by RT-PCR in brain tissue from uninfected and SIV-infected macaques euthanized at different time points (4, 7, 10, 14 and 21 days p.i.).