Using an antiretroviral treatment regimen considered clinically to have low CNS efficacy, instituted in SIV-infected animals following the resolution of the acute viremic stage, we confirmed and expanded upon our prior results that a reduction of plasma viral load induced by the therapy is beneficial for preventing/treating CNS dysfunction. Here, modeling an early commencement of treatment, for example after an individual is exposed, develops an acute viremic syndrome, is recognized as infected and begins therapy, we find that treatment leads to a significant reduction in the viral load in the brain. Although the magnitude of the infiltrating immune cells into the brain is unchanged, treatment induces an alteration in the memory phenotypes in the brain as well as a peripheral organ, the liver. Finally, alterations of the host transcriptional response to infection occur, with a decrease in IFNα and the IFN-responsive G1P3, and an increase in CCL5.
Our studies and those of others reveal that virus enters the brain early, and is easily detectable by the second week after inoculation [25
]. The RNA viral load then drops in the brain as it does outside of the brain, likely due to immune control. The decrease in viral load in the brain that we observed following treatment is likely either to a lower level of continued infection of the brain from peripheral virus, which itself is markedly diminished, and/or more effective immune control of the virus within the brain. The overall number of infiltrating immune cells is unchanged by antiretroviral treatment in the infected brain. However there was an overall enrichment of effector CD4 cells, and decline in CD8 central memory T cells. Such a shift in phenotype argues for increased active immune control of virus in the brain or for less killing of target cells. Although we did not quantify the percentage of virus-specific cells, the shifting to an effector phenotype, correlating with a successful decrease of viral load, may contribute to the generation of a more focused response, with less bystander activity, which may be rather beneficial both to control the virus and avoid damage to neuronal components.
Other studies have found that monkeys with chronic SIV infection and treated with antiretrovirals showed changes in the distribution of the memory phenotype within SIV-specific cytotoxic T lymphocytes in the blood, from TEM to TCM [28
]. We did not see such a shift in blood; possibly due to the earlier institution of treatment as well as our examination of bulk CD8 T cells as opposed to viral epitope-specific CTL. In any regard, in the brain we found the opposite in both CD4 and CD8 T cells, with a shift from TCM to TEM. This shift may represent changes in the brain CD8 population that favor more effective immune control of virus, resulting in the lowered brain viral load. However, the antiretroviral drugs may reduce the brain viral load by controlling virus peripherally. This can result in lowered infection and killing of active TEM cells, this increasing their frequency. Regardless the mechanism, the enrichment of effector memory caused by early antiretroviral treatment resembles the phenotype observed in live attenuated vaccination protocols [29
], suggesting the potential of development of a long lasting protective environment and priming by reducing the initial set point viral load.
Treatment, and the subsequent lowering of brain and blood virus, resulted in a significant decrease of IFNα in the infected brain. This finding is a potential key for the amelioration of CNS dysfunction, manifested in HIV infection as cognitive and motor symptoms, and in our experiments as abnormal delay in sensory evoked potentials. Although we cannot exclude a direct effect from the drugs themselves, this is correlated with the decrease of infected cells in the CNS. Increased IFNα in the basal ganglia in patients has been shown to contribute to neurological and behavioral changes [30
]. Patients with HIV dementia have high levels of IFNα in the CSF [36
]. Also, in a mouse model of neuroAIDS, in which HIV-infected macrophages are intracerebrally injected in SCID mice, cognitive dysfunction correlates with the expression of IFNα on neurons and glial cells [38
]. Interestingly IFNα treatment is used therapeutically in some viral infections and cancer, but such treatment often results in neurological deficits and behavioral alterations [40
], strongly suggesting a direct correlation between levels of this cytokine and CNS dysfunction.
IFNα can also influence the local T cell response. It supports an effector phenotype on T cells, but by transiently increasing apoptosis also contributes to the elimination of activated cells [45
]. In addition, similar to IL15, IFNα can support the division of memory cells in an antigen independent way, facilitating the enrichment of bystander cells [46
]. However whether there is a linkage between the reduction in IFNα and the changes found in T cell phenotypes in the brain is not known.
In contrast to the decrease in IFNα, CCL5 is increased in the brains of treated animals. Whether this increase found with treatment may result from, or possibly contribute to, the difference in T cell memory phenotype is not known. In addition to its chemokine properties, CCL5 is among the soluble inhibitory factors with the ability to inhibit HIV-1 infection and spread [48
] and thus may contribute to the reduction in brain viral load. Furthermore, CCL5 treatment of microglia was found to reduce their expression of the proinflammatory molecules, thus may act in protecting neurons from their deleterious effects [49
Interestingly, the inflammatory mediator profiling revealed that, irrespective of treatment, higher levels of their mRNAs could be found in the hippocampus as opposed to other brain regions. Certainly region-specific gene expression occurs in the primate brain[50
], but whether this contributes to these region-specific differences found in the infected brain is unknown. However it has been found that hippocampal microglia, compared to microglia from other regions of the brain, express higher levels of some inflammatory mediators [51
]. Our findings may reflect such increased proclivity of hippocampal microglia to express inflammatory molecules.
Overall, our results reveal the significance of early treatment, even with antiretroviral agents that do not penetrate the BBB. Early administration of antiretrovirals is effective in lowering virus in the brain as well as preventing early signs of CNS dysfunction. Whether such early treatment will be effective in preventing the development of HAND is not known. However a later introduction of antiretrovirals may not have such a beneficial effect, given the chronicity of virus-host interaction and its resulting effects in the brain. The effect of early treatment on the development of CNS disorders is worthy of study, since both early as well as chronic changes in the brain, including levels of virus and cytokines, can have a profound impact over the course of infection and overall patient prognosis.