Background
Human immunodeficiency virus type 1 (HIV-1) is associated with the development of neurological complications in many infected individuals, most especially a broad spectrum of motor impairments and cognitive deficits. Approximately 80-90% of autopsied cases of HIV-1-infected people demonstrated neuropathological changes [
1‐
4]. The histopathology of HIV-associated dementia (HAD) is characterized by brain infiltration of mononuclear cells, formation of multinucleated giant cells, astrogliosis, and neuronal damage sometimes with neuronal loss [
5,
6]. The underlying mechanisms of HAD leading to neurological disorders and its complete understanding is still lacking. In addition, after the introduction of highly active antiretroviral therapy (HAART), the prevalence of HAD has risen due to prolonged life expectancy of HIV-infected patients [
7‐
9].
HIV-1 penetration of the central nervous system is a vital event in the neuropathogenesis of HAD. The presence of HIV in the cerebrospinal fluid (CSF) is one of the factors implicated in HAD [
10‐
12], although high plasma viral load do not necessarily correlate with dementia. The principal cell types infected by HIV in the CNS and implicated in HIV related neuronal dysfunction are macrophages and microglia, which are known to secrete cytokines and factors toxic to neurons [
13]. It is also widely believed that monocytes or monocyte-derived macrophages may be required for neurologic manifestation of HIV disease [
14,
15]. Blood-borne macrophages can transmit the virus into the CNS and then infect or stimulate other perivascular macrophages and microglia [
12,
16]. However, HAD usually occurs at an advanced stage of HIV disease, while HIV entry into the CNS has been reported to occur early after primary infection [
17,
18]. The most popular explanation for this discrepancy is the collapse of immune functions mediated by T cells because cytotoxic T lymphocytes, which are believed to be the principal regulatory elements that control viral production in the periphery and CNS [
19‐
23]. Both CD4+ and CD8+ T lymphocytes have been shown to accumulate in AIDS patients with HIV encephalitis along with the demonstration that brain CD8-CTL are HIV-specific and are associated with HIV encephalitis [
24‐
27].
Although some studies have shown evidence in favor of frequency and topographical distribution of HIV core protein P24 [
28,
29], detailed investigations with focus on quantity, quality, topographical distribution and infiltration of macrophages, CD8+ T cells, especially in relation to HIV, in diverse regions of the brain from patients with and without dementia, which might elucidate entry mechanism of HIV into the CNS and explain regional involvement in the development of HAD, are seriously lacking. Therefore, we have carried out a detailed and simultaneous tracking of activation and infiltration patterns of macrophages, CD8+ T cells in relation to HIV P24 antigen in diverse areas of the brain of HIV+ patients with and without dementia. We analyzed 53 different brain regions from 4 HIV+ non-dementia patients and 46 regions from patients with 2 HIV+ severely demented, rapidly progressing patients. Our study is novel in revealing the predilection of HIV movements together with cellular infiltrates of macrophages and CD8+ T cells to the deeper mid-line and mesial structures uniquely in patients with HAD.
Discussion
In this study, we have investigated cellular infiltration of macrophages and CD8+ T cells and their activation states in diverse areas of the brain, especially in concomitance with HIV in order to define regional predilection of these cell types and their possible association with neurologic manifestation of HIV disease or HAD. Here, we provide the first evidence showing 1. That macrophage and CD8+ T cell infiltration and their activation alone was not enough for the development of dementia. 2. That the topographic patterns of macrophage/CD8+T cell distribution and levels of activation in diverse areas the CNS were comparable between patients with and without dementia. 3. That the major distinction between HAD and HIV non-dementia patients was the predilection of HIV-infected macrophages and CD8+ T cells to the deeper midline and mesial temporal structures (thalamus, medulla, hippocampus, cerebellum, basal ganglia and pons) uniquely in HAD patients. This further correlated with HIV-related pathology in these parts of the CNS and also with the neurological manifestation of HIV disease in both HAD patients. Further, this distribution pattern was also consistent with the activation patterns, confirmed by S-100A8 immunohistochemical staining, which was also pronounced in the deeper midline and mesial temporal structures. Thus the preponderance of activated/infected CD8+ T cells and CD68+ macrophages in the midline and mesial temporal structures of the brain is the feature unique to HAD patients. In contrast, even though considerable macrophage and CD8 infiltration was observed in diverse areas of the brain of HIV non-dementia patients, there was complete lack of P24 antigen staining and low level of cellular activation, which indicates either the absence or lower than detectable level of HIV in the CNS of HIV non-dementia patients.
It is known that HIV in the periphery and the CSF may impair the integrity of the blood-brain barrier (BBB), which makes the BBB permeable to cellular infiltrates or the recruitment of immune cells into the CNS [
37‐
39]. However, the strength of the immune system and the quality of immune cells migrating to the CNS determine the onset of neurological manifestation of HIV disease. Thus, the immuno-suppression of the host by HIV may have strong bearing on the appearance of productively infected macrophages, which was the critical feature observed in both HAD patients examined in our study. The actual relationship, between HIV-1 proviral load and clinical diagnosis of HIV-associated dementia (HAD), remains to be established. A clear determination of neuroanatomic distribution of HIV-1 proviral loads in diverse areas of the brain (including deeper midline and mesial temporal structures) of individuals with and without HAD may facilitate elucidation of the relationship between tissue proviral load and HAD, and may also explain whether regional predilection of provirus also correlates with macrophage and CD8+ T cell infiltration and active replication of HIV in HAD patients, as we have observed. Taken together, our findings lead to the supposition that the predilection of HIV-infected macrophages and CD8+ T cells to the deeper midline and mesial temporal structures (which includes the brainstem), along with the unique architecture of proviral sequences appear to play a vital role in the development of dementia in HIV patients.
These and our recently published data [
30] are consistent with previous findings showing that the infection was most frequent in the deeper midline and mesial temporal structures, whereas the choroids plexus (CPx) showed no productive infection [
28,
40]. Further, another semi-quantitative study by Neuen-Jacob [
29], which analyzed topographical distribution of HIV core antigen in relation to macrophages showed that deep grey matter, in particular putamen and thalamus, were involved in every case, irrespective of disease stage. It is also important to reiterate that HIV infection may incur indirect modalities and our data confirm the active HIV infection of the CNS being the vital feature for neurologic manifestation. Without productive infection of macrophages in the deeper midline and mesial temporal structures by HIV, the trigger for HAD does not occur. Thus, our supposition for the localization of HIV-infected/activated macrophages and CD8+ T cells in the context of the development of dementia in HIV patients is highly relevant.
Previously, CD8+ cytotoxic T lymphocytes specific for viral antigens have been shown to accumulate and correlate with the central nervous system dysfunction in SIV or HIV infection [
25,
27]. However, studies on macaques by Sopper [
20], have shown that increased infiltration of CD8+ cytotoxic T ymphocytes specific for viral antigens were detected only in the CSF of slow progressors. Consistently, we observed no differences at the level of CD8+ T cell infiltration and infection in two HAD patients analyzed in our study, but much higher CTL in the patient who died within 6 years, and very little in the patient who died within 40 weeks. Thus, our data suggest that slow progressors may display strong intrathecal immune response suggesting a possible protective role or delay of the virus-specific immunity in HIV-induced central nervous system disease, as opposed to rapidly progressing HAD patients.
Overall, why deeper parts appear to be so much more involved rather than the cortex proximal to the meningeal or ventricle area? And why the whole midline and mesial temporal parts of the brain are so severely affected histopathologically? Specific tissue vulnerability may explain part of the puzzle, but not the underlying reasons regarding the specific topographic distribution of HIV and cellular infiltrates in HAD patients. It is known that the brainstem disturbance can be associated with dementia through interference with arousal systems. Pathologically, gross examination of the brain in progressive supranuclear palsy (PSP) shows midbrain atrophy. Further, in PSP neuronal loss and neurofibrillary tangles in the basal ganglia, diencephalon and brainstem have been observed. The substantia nigra, subthalamic nucleus and pontine base are typically involved, as well as the ventral anterior and lateral thalamic nuclei. The cerebellar dentate nucleus may show degeneration [
41]. In addition, in the context of HIV basal ganglia hyperatrophy has been shown to often accompany mild cognitive impairment [
42]. Thus, taken together, it leads us to postulate that the productive infection of HIV in the midline and mesial temporal structures is crucial to the occurrence of dementia in HIV patients. We wish to reiterate that we do not imply that dementia can exclusively be caused by brainstem disease -more that it can exacerbate probably through several mechanisms - diminished arousal and therefore attention.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
LZ fully performed the work, analyzed data and wrote the paper. RR, KH and CG performed the work on HIV activation using S-100, VV contributed to immunohistochemical analysis, TN extensively contributed to neurologic and immunohistochemical results interpretation and analysis, YSH contributed to phylogenetic analysis, NKS provided ideas, designed the research project, planned and supervised this work and contributed to the writing.