Morphine potentiates neuropathogenesis of SIV infection in rhesus macaques

Abstract

Despite the advent of antiretroviral therapy, complications of HIV-1 infection with concurrent drug abuse are an emerging problem. Opiates are well known to modulate immune responses by preventing the development of cell-mediated immune responses. Their effect on the pathogenesis of HIV-1 infection however remains controversial. Using the simian immunodeficiency virus/macaque model of HIV pathogenesis, we sought to explore the impact of morphine on disease progression and pathogenesis. Sixteen rhesus macaques were divided into two groups; four were administered saline and 12 others morphine routinely. Both groups of animals were then inoculated with SIVmacR71/17E and followed longitudinally for disease pathogenesis. The morphine group (M+V) exhibited a trend towards higher mortality rates and retardation in weight gain compared to the virus-alone group. Interestingly, a subset of M+V animals succumbed to disease within weeks post-infection. These rapid progressors also exhibited a higher incidence of other end-organ pathologies. Despite the higher numbers of circulating CD4+ and CD8+ T cells in the M+V group, CD4/CD8 ratios between the groups remained unchanged. Plasma and CSF viral load in the M+V group was at least a log higher than the control group. Similarly, there was a trend toward increased virus build-up in the brains of M+V animals compared with controls. A novel finding of this study was the increased influx of infected monocyte/macrophages in the brains of M+V animals.

Figure 1. Morphine-treated animals exhibit rapid disease progression, retardation in weight gain and higher mortality rates

(A) Average weight gain over time, in the M and M+V groups, expressed as percent of body weight at week 36 prior to viral inoculation. *p<0.05 vs V group using the Wilcox signed rank test. (B) Survival (expressed as percentage of total animals) of morphine-treated and untreated animals infected with SIV.

Figure 2. Pathological findings in the brains of the M+V and V rapid progressors were comparable

Focal histiocytic nodules in the basal ganglia of M+V group [Macaques A1 (A) & B4 (B)]; note a hint of golden pigment (associated with oxidative stress) in several cells (arrows). Histiocytic infiltrates around a blood vessel in the frontal cortex of A1 (D); note the syncytial cell formation (black arrow) and 2 single mononuclear cells containing golden pigment (white arrows). Perivascular cuff in the Parietal cortex of A1 (E). No Histiocytic (C) nodule and histiocytic infiltrates (F) in the basal ganglia from V group (macaque C3). Magnification: 40×.

Figure 3. Viral load in plasma in SIV-infected monkeys treated with morphine compared to those without morphine

(A) Scattergram expressing viral loads in plasma of M + V animals at various time points PI. (B) Scattergram expressing viral loads in plasma of V animals at various time points PI. (C) Average virus load in the plasma from MV RP, V RP, MV LP and V LP groups over time.

Figure 4. Viral load in CSF of SIV-infected monkeys treated with morphine compared to those without morphine

(A) Scattergram expressing viral loads in CSF of M + V animals at various time points PI. (B) Scattergram expressing viral loads in CSF of V animals at various time points PI. (C) Average of virus load in the CSF from MV RP, V RP, MV LP and V LP groups over time.

Figure 5. CD4+T cell counts in PBMC of SIV-infected macaques treated with or without morphine

(A) Scattergram expressing CD4+T cells in PBMC of M + V animals at various time points PI. (B) Scattergram expressing CD4+T cells in PBMC of M + V animals at various time points PI. (C) Average of CD4+T cell counts in PBMC from V and MV animals at various time points PI. ***p<0.001 vs V group.

Figure 6

CD8+T cell counts in PBMC of SIV-infected macaques treated with or without morphine. (A) Scattergram expressing CD8+T cells in PBMC of M + V animals at various time points PI. (B) Scattergram expressing CD8+T cells in PBMC of M + V animals at various time points PI. (C) Average of CD8+T cell counts in PBMC from V and MV animals at various time points PI. (D) CD4/CD8 ratios of averaged cell counts from V and MV animals at various time points PI. ***p<0.001 vs V group.

Figure 7. Virus RNA titer in different brain regions of SIV-infected macaque with or without morphine

(A) Scattergram expressing viral loads in different brain region of RP animals at various time points PI. Black dots: A1, B4, B5, C3; Red dots: B6. (B) Scattergram expressing viral loads in different brain regions of LP animals at various time points PI. Black dots: A3, A4, C1, C2, C4; Red dots: A2. (C) SIV mRNA in the basal ganglia of macaque brain. In situ hybridization was used to identify SIV RNA in macaque brain sections from M+V and V group. The sections were hybridized to ³⁵S-labelled SIV antisense probes. After washing, digestion with RNases, the sections were coated with nuclear track emulsion, exposed, and developed. When the radioautographs are illuminated with epipolarized and transmitted light, the light reflected from the silver grain imparts a greenish yellow color to cells that have relatively high levels of SIV-1 RNA. (Scale bar=500 μm for upper two panels; Scale bar=100 μm for lower two panels).

Figure 8. Localization of viral gp120 in the brain macrophages

(A) Morphine treated macaques (M+V) showed more viral antigen and CD68+ macrophages in the basal ganglia compared to the SIV-treated (V) animal. The CD68 positive cells corresponded to regions that are also positive for the viral antigen gp120 indicating that macrophages constitute the majority of infected cells. (B) Representative image for CD68 staining in the basal ganglia section from V and M+V groups.