CCR5 antagonist reduces HIV-induced amyloidogenesis, tau pathology, neurodegeneration, and blood-brain barrier alterations in HIV-infected hu-PBL-NSG mice.

BACKGROUND: Neurocognitive impairment is present in 50% of HIV-infected individuals and is often associated with Alzheimer's Disease (AD)-like brain pathologies, including increased amyloid-beta (Aß) and Tau hyperphosphorylation. Here, we aimed to determine whether HIV-1 infection causes AD-like pathologies in an HIV/AIDS humanized mouse model, and whether the CCR5 antagonist maraviroc alters HIV-induced pathologies. METHODS: NOD/scid-IL-2Rγcnull mice engrafted with human blood leukocytes were infected with HIV-1, left untreated or treated with maraviroc (120 mg/kg twice/day). Human cells in animal's blood were quantified weekly by flow cytometry. Animals were sacrificed at week-3 post-infection; blood and tissues viral loads were quantified using p24 antigen ELISA, RNAscope, and qPCR. Human (HLA-DR+) cells, Aß-42, phospho-Tau, neuronal markers (MAP 2, NeuN, neurofilament-L), gamma-secretase activating protein (GSAP), and blood-brain barrier (BBB) tight junction (TJ) proteins expression and transcription were quantified in brain tissues by immunohistochemistry, immunofluorescence, immunoblotting, and qPCR. Plasma Aß-42, Aß-42 cellular uptake, release and transendothelial transport were quantified by ELISA. RESULTS: HIV-1 significantly decreased human (h)CD4+ T-cells and hCD4/hCD8 ratios; decreased the expression of BBB TJ proteins claudin-5, ZO-1, ZO-2; and increased HLA-DR+ cells in brain tissues. Significantly, HIV-infected animals showed increased plasma and brain Aß-42 and phospho-Tau (threonine181, threonine231, serine396, serine199), associated with transcriptional upregulation of GSAP, an enzyme that catalyzes Aß formation, and loss of MAP 2, NeuN, and neurofilament-L. Maraviroc treatment significantly reduced blood and brain viral loads, prevented HIV-induced loss of neuronal markers and TJ proteins; decreased HLA-DR+ cells infiltration in brain tissues, significantly reduced HIV-induced increase in Aß-42, GSAP, and phospho-Tau. Maraviroc also reduced Aß retention and increased Aß release in human macrophages; decreased the receptor for advanced glycation end products (RAGE) and increased low-density lipoprotein receptor-related protein-1 (LRP1) expression in human brain endothelial cells. Maraviroc induced Aß transendothelial transport, which was blocked by LRP1 antagonist but not RAGE antagonist. CONCLUSIONS: Maraviroc significantly reduced HIV-induced amyloidogenesis, GSAP, phospho-Tau, neurodegeneration, BBB alterations, and leukocytes infiltration into the CNS. Maraviroc increased cellular Aß efflux and transendothelial Aß transport via LRP1 pathways. Thus, therapeutically targeting CCR5 could reduce viremia, preserve the BBB and neurons, increased brain Aß efflux, and reduce AD-like neuropathologies.

SIV/SHIV-Zika co-infection does not alter disease pathogenesis in adult non-pregnant rhesus macaque model.

Due to the large geographical overlap of populations exposed to Zika virus (ZIKV) and human immunodeficiency virus (HIV), understanding the disease pathogenesis of co-infection is urgently needed. This warrants the development of an animal model for HIV-ZIKV co-infection. In this study, we used adult non-pregnant macaques that were chronically infected with simian immunodeficiency virus/chimeric simian human immunodeficiency virus (SIV/SHIV) and then inoculated with ZIKV. Plasma viral loads of both SIV/SHIV and ZIKV co-infected animals revealed no significant changes as compared to animals that were infected with ZIKV alone or as compared to SIV/SHIV infected animals prior to ZIKV inoculation. ZIKV tissue clearance of co-infected animals was similar to animals that were infected with ZIKV alone. Furthermore, in co-infected macaques, there was no statistically significant difference in plasma cytokines/chemokines levels as compared to prior to ZIKV inoculation. Collectively, these findings suggest that co-infection may not alter disease pathogenesis, thus warranting larger HIV-ZIKV epidemiological studies in order to validate these findings.

Preliminary Studies on Immune Response and Viral Pathogenesis of Zika Virus in Rhesus Macaques.

Zika Virus (ZIKV) is primarily transmitted through mosquito bites. It can also be transmitted during sexual intercourse and in utero from mother to fetus. To gain preliminary insight into ZIKV pathology and immune responses on route of transmission, rhesus macaques (RMs) were inoculated with ZIKV (PRVABC59) via intravaginal (IVAG) (n = 3) or subcutaneous (sub Q) (n = 2) routes. Systemic ZIKV infection was observed in all RMs, regardless of the route of inoculation. After 9 days postinfection (dpi), ZIKV was not detected in the plasma of IVAG- and sub-Q-inoculated RMs. Importantly, RMs harbored ZIKV up to 60 dpi in various anatomical locations. Of note, ZIKV was also present in several regions of the brain, including the caudate nucleus, parietal lobe, cortex, and amygdala. These observations appear to indicate that ZIKV infection may be systemic and persistent regardless of route of inoculation. In addition, we observed changes in key immune cell populations in response to ZIKV infection. Importantly, IVAG ZIKV infection of RMs is associated with increased depletion of CD11C hi myeloid cells, reduced PD-1 expression in NK cells, and elevated frequencies of Ki67⁺ CD8⁺ central memory cells as compared to sub Q ZIKV-infected RMs. These results need to interpreted with caution due to the small number of animals utilized in this study. Future studies involving large groups of animals that have been inoculated through both routes of transmission are needed to confirm our findings.

Immune Cell Dynamics in Rhesus Macaques Infected with a Brazilian Strain of Zika Virus.

Zika virus (ZIKV) is a mosquito-borne and sexually transmitted flavivirus that is associated with fetal CNS-damaging malformations during pregnancy in humans. This study documents the viral kinetics and immune responses in rhesus macaques infected with a clinical ZIKV Brazilian isolate. We evaluated the viral kinetics and immune responses induced after an i.v. infection with a Brazilian ZIKV clinical isolate (HS-2015-BA-01) in rhesus macaques for up to 142 d. ZIKV-specific Ab-secreting cells, germinal center reactions, and monocyte, dendritic cell, NK, and T cell frequencies were monitored. ZIKV loads were readily detected in plasma (until day 5 or 7), semen and urine (until days 7 and 14), and saliva (until day 42), but the viremia was rapidly controlled. No detectable clinical manifestations were observed. However, lymph node hyperplasia was clearly visible postviremia but was associated with low frequencies of ZIKV-specific Ab-secreting cells in lymph nodes and bone marrow, correlating with low Ab titers. CD14+/CD16- monocytes and myeloid CD11chi dendritic cells decreased in blood, whereas NK and T cell numbers were only marginally altered during the course of the study. ZIKV infection caused a significant lymphoid tissue activation but limited induction of ZIKV-specific B cells, suggesting that these parameters need to be considered for ZIKV vaccine design.

Data in support of NFκB and JNK pathways involvement in TLR3-mediated HIV-1 transactivation, expression of IL-6 and transcription factors associated with HIV-1 replication.

In the present article, using human monocyte-derived macrophages and cell lines containing integrated copies of the HIV-1 promoter, we show the effects of TLR3 ligands on the pro-inflammatory cytokine IL-6. We further show the effects of TLR3 ligands on HIV-1 transactivation and transcription factors involved in HIV-1 replication. This article complements the data reported by the authors, "Toll-Like receptor-3 mediates HIV-1 transactivation via NFκB and JNK pathways, and histone acetylation, but prolonged activation suppresses Tat and HIV-1 replication" (Bhargavan et al., 2015) [1], and the interpretation of these data can be found in the research article published by the authors in Cellular Signaling in 2015 (Bhargavan et al., 2015) [1].

Toll-like receptor-3 mediates HIV-1 transactivation via NFκB and JNK pathways and histone acetylation, but prolonged activation suppresses Tat and HIV-1 replication.

TLR3 has been implicated in the pathogenesis of several viral infections, including SIV- and HIV-1-induced inflammation and AIDS. However the molecular mechanisms of these TLR3-mediated effects are not known, and it is not known whether HIV interacts with cellular TLR3 to affect disease process. Here we investigate the effects of TLR3 ligands on HIV-1 transactivation using both primary human macrophages and cells containing integrated copies of the HIV-1 promoter. We demonstrate that TLR3 activation induced upregulation of transcription factors such as c-Jun, CCAAT/enhancer-binding protein alpha (CEBPA), signal transducer and activator of transcription (STAT)-1, STAT-2, RELB, and nuclear factor kappa-B1 (NFκB1), most of which are known to regulate the HIV promoter activity. We also demonstrate that TLR3 activation increased HIV-1 transactivation via the c-Jun N-terminal kinase (JNK) and NFκB pathways. This was associated with epigenetic modifications, including decreased histone deacetylase activity, increased histone acetyl transferase (HAT) activity, and increased acetylation of histones H3 and H4 at lysine residues in the nucleosome-0 and nucleosome-1 of the HIV-1 promoter. However, prolonged TLR3 activation decreased HIV-1 transactivation, decreased HAT activity and Tat transcription, and suppressed viral replication. Overall, data suggests that TLR3 can act as viral sensor to mediate viral transactivation, cellular signaling, innate immune response, and inflammation in HIV-infected humans. Our study provides novel insights into the molecular basis for these TLR3-mediated effects.

Maraviroc: a review of its use in HIV infection and beyond.

The human immunodeficiency virus-1 (HIV-1) enters target cells by binding its envelope glycoprotein gp120 to the CD4 receptor and/or coreceptors such as C-C chemokine receptor type 5 (CCR5; R5) and C-X-C chemokine receptor type 4 (CXCR4; X4), and R5-tropic viruses predominate during the early stages of infection. CCR5 antagonists bind to CCR5 to prevent viral entry. Maraviroc (MVC) is the only CCR5 antagonist currently approved by the United States Food and Drug Administration, the European Commission, Health Canada, and several other countries for the treatment of patients infected with R5-tropic HIV-1. MVC has been shown to be effective at inhibiting HIV-1 entry into cells and is well tolerated. With expanding MVC use by HIV-1-infected humans, different clinical outcomes post-approval have been observed with MVC monotherapy or combination therapy with other antiretroviral drugs, with MVC use in humans infected with dual-R5- and X4-tropic HIV-1, infected with different HIV-1 genotype or infected with HIV-2. This review discuss the role of CCR5 in HIV-1 infection, the development of the CCR5 antagonist MVC, its pharmacokinetics, pharmacodynamics, drug-drug interactions, and the implications of these interactions on treatment outcomes, including viral mutations and drug resistance, and the mechanisms associated with the development of resistance to MVC. This review also discusses available studies investigating the use of MVC in the treatment of other diseases such as cancer, graft-versus-host disease, and inflammatory diseases.

Differential effects of Tat proteins derived from HIV-1 subtypes B and recombinant CRF02_AG on human brain microvascular endothelial cells: implications for blood-brain barrier dysfunction.

HIV-1 genetic differences influence viral replication and progression to AIDS. HIV-1 circulating recombinant form (CRF)02_AG is the predominant viral subtype infecting humans in West and Central Africa, but its effects on HIV neuropathogenesis are not known. In the present study, we investigated the effects of Tat proteins from HIV-1 subtype B (Tat.B) and HIV-1 CRF02_AG (Tat.AG) on primary human brain microvascular endothelial cells (HBMEC), the major component of the blood-brain barrier (BBB). Using Affymetrix GeneChip Human Gene 1.0.ST arrays, we showed that Tat.AG had minimal effects while Tat.B induced transcriptional upregulation of 90 genes in HBMEC, including proinflammatory chemokines, complement components C3, C7, and complement factor B, matrix metalloproteinases (MMP)-3, MMP-10, and MMP-12. These results were confirmed by real-time PCR. Compared with Tat.AG, Tat.B significantly increased MMP-3, MMP-10, and MMP-12 activities in HBMEC, and the MMPs tissue inhibitor of metalloproteinase-2 blocked Tat-induced increase in MMPs activity. Western blot analyses also showed that Tat increased the expression of C3 and its cleaved fragment C3b in HBMEC. These data suggest that genetic differences between HIV-1 subtypes B and CRF02_AG influence the effects of Tat proteins from these two clades on HBMEC, including molecular and cellular functions, and canonical pathways, which would affect BBB dysfunction and viral neuropathogenesis.

HIV-1 induces cytoskeletal alterations and Rac1 activation during monocyte-blood-brain barrier interactions: modulatory role of CCR5.

BACKGROUND: Most HIV strains that enter the brain are macrophage-tropic and use the CCR5 receptor to bind and infect target cells. Because the cytoskeleton is a network of protein filaments involved in cellular movement and migration, we investigated whether CCR5 and the cytoskeleton are involved in endothelial-mononuclear phagocytes interactions, adhesion, and HIV-1 infection. RESULTS: Using a cytoskeleton phospho-antibody microarray, we showed that after co-culture with human brain microvascular endothelial cells (HBMEC), HIV-1 infected monocytes increased expression and activation of cytoskeleton-associated proteins, including Rac1/cdc42 and cortactin, compared to non-infected monocytes co-cultured with HBMEC. Analysis of brain tissues from HIV-1-infected patients validated these findings, and showed transcriptional upregulation of Rac1 and cortactin, as well as increased activation of Rac1 in brain tissues of HIV-1-infected humans, compared to seronegative individuals and subjects with HIV-1-encephalitis. Confocal imaging showed that brain cells expressing phosphorylated Rac1 were mostly macrophages and blood vessels. CCR5 antagonists TAK-799 and maraviroc prevented HIV-induced upregulation and phosphorylation of cytoskeleton-associated proteins, prevented HIV-1 infection of macrophages, and diminished viral-induced adhesion of monocytes to HBMEC. Ingenuity pathway analysis suggests that during monocyte-endothelial interactions, HIV-1 alters protein expression and phosphorylation associated with integrin signaling, cellular morphology and cell movement, cellular assembly and organization, and post-translational modifications in monocytes. CCR5 antagonists prevented these HIV-1-induced alterations. CONCLUSIONS: HIV-1 activates cytoskeletal proteins during monocyte-endothelial interactions and increase transcription and activation of Rac1 in brain tissues. In addition to preventing macrophage infection, CCR5 antagonists could diminish viral-induced alteration and phosphorylation of cytoskeletal proteins, monocyte adhesion to the brain endothelium and viral entry into the central nervous system.