Downregulation of CCR5 on brain perivascular macrophages in simian immunodeficiency virus-infected rhesus macaques.

BACKGROUND: C-C chemokine receptor 5 (CCR5) is a major coreceptor for Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) cell entry; however, its role in brain pathogenesis is largely understudied. Thus, we sought to examine cell type-specific protein expression of CCR5 during SIV infection of the brain. METHODS: We examined occipital cortical tissue from uninfected rhesus macaques and SIV-infected animals with or without encephalitis using immunohistochemistry and immunofluorescence microscopy to determine the number and distribution of CCR5-positive cells. RESULTS: An increase in the number of CCR5+ cells in the brain of SIV-infected animals with encephalitis was accounted for by increased CD3+CD8+ cells expressing CCR5, but not by increased CCR5+ microglia or perivascular macrophages (PVMs), and a concurrent decrease in the percentage of CCR5+ PVMs was observed. Levels of CCR5 and SIV Gag p28 protein expression were examined on a per-cell basis, and a significant, negative relationship was established indicating decreased CCR5 expression in productively infected cells. While investigating the endocytosis-mediated CCR5 internalization as a mechanism for CCR5 downregulation, we found that phospho-ERK1/2, an indicator of clathrin-mediated endocytosis, was colocalized with infected PVMs and that macrophages from infected animals showed significantly increased expression of clathrin heavy chain 1. CONCLUSIONS: These findings show a shift in CCR5-positive cell types in the brain during SIV pathogenesis with an increase in the number of CCR5+ CD8 T cells, and downregulated CCR5 expression on infected PVMs, likely through ERK1/2-driven, clathrin-mediated endocytosis.

Development of Droplet Digital PCR-Based Assays to Quantify HIV Proviral and Integrated DNA in Brain Tissues from Viremic Individuals with Encephalitis and Virally Suppressed Aviremic Individuals.

Although combination antiretroviral therapy (cART) can suppress the replication of HIV, the virus persists and rebounds when treatment is stopped. To find a cure that can eradicate latent reservoir, a method should be able to quantify the lingering HIV. Unlike other digital PCR technologies, droplet digital PCR (ddPCR), provides absolute quantification of target DNA molecules using fluorescent dually labeled probes by massively partitioning the sample into droplets. ddPCR enables exquisitely sensitive detection and quantification of viral DNA from very limiting clinical samples, including brain tissues. We developed and optimized duplex ddPCR assays for the detection and quantification of HIV proviral DNA and integrated DNA in the brain of HIV-1-infected patients. We have applied these approaches to successfully analyze 77 human brain tissues obtained from 27 HIV-1-infected individuals, either fully virally suppressed or with encephalitis, and were able to quantify low levels of viral DNA. Further developments and advancement of digital PCR technology is promising to aid in accurate quantification and characterization of the persistent HIV reservoir. IMPORTANCE We developed ddPCR assays to quantitatively measure HIV DNA and used this ddPCR assays to detect and quantitatively measure HIV DNA in the archived brain tissues from HIV patients. The tissue viral loads assessed by ddPCR was highly correlative with those assessed by qPCR. HIV DNA in the brain was detected more frequently by ddPCR than by qPCR. ddPCR also showed higher sensitivity than qPCR since ddPCR detected HIV DNA signals in some tissues from virally suppressed individuals while qPCR could not.

A Zika virus primary isolate induces neuroinflammation, compromises the blood-brain barrier and upregulates CXCL12 in adult macaques.

Zika virus (ZIKV) is a flavivirus that can cause neuropathogenesis in adults and fetal neurologic malformation following the infection of pregnant women. We used a nonhuman primate model, the Indian-origin Rhesus macaque (IRM), to gain insight into virus-associated hallmarks of ZIKV-induced adult neuropathology. We find that the virus causes prevalent acute and chronic neuroinflammation and chronic disruption of the blood-brain barrier (BBB) in adult animals. ZIKV infection resulted in specific short- and long-term augmented expression of the chemokine CXCL12 in the central nervous system (CNS)of adult IRMs. Moreover, CXCL12 expression persists long after the initial viral infection is apparently cleared. CXCL12 plays a key role both in regulating lymphocyte trafficking through the BBB to the CNS and in mediating repair of damaged neural tissue including remyelination. Understanding how CXCL12 expression is controlled will likely be of central importance in the definition of ZIKV-associated neuropathology in adults.

Perivascular macrophages in the neonatal macaque brain undergo massive necroptosis after simian immunodeficiency virus infection.

We previously showed that rhesus macaques neonatally infected with simian immunodeficiency virus (SIV) do not develop SIV encephalitis (SIVE) and maintain low brain viral loads despite having similar plasma viral loads compared to SIV-infected adults. We hypothesize that differences in myeloid cell populations that are the known target of SIV and HIV in the brain contribute to the lack of neonatal susceptibility to lentivirus-induced encephalitis. Using immunohistochemistry and immunofluorescence microscopy, we examined the frontal cortices from uninfected and SIV-infected infant and adult macaques (n = 8/ea) as well as adults with SIVE (n = 4) to determine differences in myeloid cell populations. The number of CD206+ brain perivascular macrophages (PVMs) was significantly greater in uninfected infants than in uninfected adults and was markedly lower in SIV-infected infants while microglia numbers were unchanged across groups. CD206+ PVMs, which proliferate after infection in SIV-infected adults, did not undergo proliferation in infants. While virtually all CD206+ cells in adults are also CD163+, infants have a distinct CD206 single-positive population in addition to the double-positive population commonly seen in adults. Notably, we found that more than 60% of these unique CD206+CD163- PVMs in SIV-infected infants were positive for cleaved caspase-3, an indicator of apoptosis, and that nearly 100% of this subset were concomitantly positive for the necroptosis marker receptor-interacting protein kinase-3 (RIP3). These findings show that distinct subpopulations of PVMs found in infants undergo programmed cell death instead of proliferation following SIV infection, which may lead to the absence of PVM-dependent SIVE and the limited size of the virus reservoir in the infant brain.

Macrophages but not Astrocytes Harbor HIV DNA in the Brains of HIV-1-Infected Aviremic Individuals on Suppressive Antiretroviral Therapy.

The question of whether the human brain is an anatomical site of persistent HIV-1 infection during suppressive antiretroviral therapy (ART) is critical, but remains unanswered. The presence of virus in the brains of HIV patients whose viral load is effectively suppressed would demonstrate not only the potential for CNS to act as an anatomical HIV reservoir, but also the urgent need to understand the factors contributing to persistent HIV behind the blood-brain barrier. Here, we investigated for the first time the presence of cells harboring HIV DNA and RNA in the brains from subjects with undetectable plasma viral load and sustained viral suppression, as identified by the National NeuroAIDS Tissue Consortium. Using new, highly sensitive in situ hybridization techniques, RNAscope and DNAscope, in combination with immunohistochemistry, we were able to detect HIV-1 in the brains of all virally suppressed cases and found that brain macrophages and microglia, but not astrocytes, were the cells harboring HIV DNA in the brain. This study demonstrated that HIV reservoirs persist in brain macrophages/microglia during suppressive ART, which cure/treatment strategies will need to focus on targeting.

Lentiviral infection of proliferating brain macrophages in HIV and simian immunodeficiency virus encephalitis despite sterile alpha motif and histidine-aspartate domain-containing protein 1 expression.

OBJECTIVE: HIV-1 infection of the brain and related cognitive impairment remain prevalent in HIV-1-infected individuals despite combination antiretroviral therapy. Sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) is a newly identified host restriction factor that blocks the replication of HIV-1 and other retroviruses in myeloid cells. Cell cycle-regulated phosphorylation at residue Thr592 and viral protein X (Vpx)-mediated degradation of SAMHD1 have been shown to bypass SAMHD1 restriction in vitro. Herein, we investigated expression and phosphorylation of SAMHD1 in vivo in relation to macrophage infection and proliferation during the neuropathogenesis of HIV-1 and simian immunodeficiency virus (SIV) encephalitis. METHODS: Using brain and other tissues from uninfected and SIV-infected macaques with or without encephalitis, we performed immunohistochemistry, multilabel fluorescence microscopy and western blot to examine the expression, localization and phosphorylation of SAMHD1. RESULTS: The number of SAMHD1 nuclei increased in encephalitic brains despite the presence of Vpx. Many of these cells were perivascular macrophages, although subsets of SAMHD1 microglia and endothelial cells were also observed. The SAMHD1 macrophages were shown to be both infected and proliferating. Moreover, the presence of cycling SAMHD1 brain macrophages was confirmed in the tissue of HIV-1-infected patients with encephalitis. Finally, western blot analysis of brain-protein extracts from SIV-infected macaques showed that SAMHD1 protein exists in the brain mainly as an inactive Thr592-phosphorylated form. CONCLUSION: The ability of SAMHD1 to act as a restriction factor for SIV/HIV in the brain is likely bypassed in proliferating brain macrophages through the phosphorylation-mediated inactivation, not Vpx-mediated degradation of SAMHD1.