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Differential Mechanisms of Inflammation and Endothelial Dysfunction by HIV-1 Subtype-B and Recombinant CRF02_AG Tat Proteins on Human Brain Microvascular Endothelial Cells: Implications for Viral Neuropathogenesis.

ABSTRACT: The recombinant HIV-1 CRF02_AG is prevalent in West-Central Africa but its effects on the blood-brain barrier (BBB) and HIV-associated neurocognitive disorders (HAND) are not known. We analyzed the effects of Tat from HIV-1 subtype-B (Tat.B) and CRF02_AG (Tat.AG) on primary human brain microvascular endothelial cells (HBMEC), the major BBB component. Exposure of HBMEC to Tat.B increased IL-6 expression and transcription by 9- (P < 0.001) and 113-fold (P < 0.001), respectively, whereas Tat.AG increased IL-6 expression and transcription by 2.7-3.8-fold and 35.7-fold (P < 0.001), respectively. Tat.B induced IL-6 through the interleukin-1 receptor-associated kinase (IRAK)-1/4/mitogen-activated protein kinase kinase(MKK)/C-jun N-terminal kinase(JNK) pathways, in an activator protein-1(AP1)- and nuclear factor-kappaB (NF?B)-independent manner, whereas Tat.AG effects occurred via MKK/JNK/AP1/NF?B pathways. Tat-induced effects were associated with activation of c-jun (serine-63) and SAPK/JNK (Thr183/Tyr185). We demonstrated increased expression of transcription factors associated with these pathways (Jun, RELB, CEBPA), with higher levels in Tat.B-treated cells compared to Tat.AG. Functional studies showed that Tat.B and Tat.AG decreased the expression of tight junction proteins claudin-5 and ZO-1 and decreased the trans-endothelial electric resistance (TEER); Tat.B induced greater reduction in TEER, claudin-5, and ZO-1, compared to Tat.AG. Overall, our data showed increased inflammation and BBB dysfunction with Tat.B, compared to Tat.AG. This suggests these two HIV-1 subtypes differentially affect the BBB and central nervous system; our data provides novel insights into the molecular basis of these differential Tat-mediated effects.

SUBMITTER: Bhargavan B

PROVIDER: S-EPMC6507400 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Differential Mechanisms of Inflammation and Endothelial Dysfunction by HIV-1 Subtype-B and Recombinant CRF02_AG Tat Proteins on Human Brain Microvascular Endothelial Cells: Implications for Viral Neuropathogenesis.

Bhargavan Biju B Kanmogne Georgette D GD

Molecular neurobiology 20170127 2

The recombinant HIV-1 CRF02_AG is prevalent in West-Central Africa but its effects on the blood-brain barrier (BBB) and HIV-associated neurocognitive disorders (HAND) are not known. We analyzed the effects of Tat from HIV-1 subtype-B (Tat.B) and CRF02_AG (Tat.AG) on primary human brain microvascular endothelial cells (HBMEC), the major BBB component. Exposure of HBMEC to Tat.B increased IL-6 expression and transcription by 9- (P < 0.001) and 113-fold (P < 0.001), respectively, whereas Tat.AG inc ...[more]

PMID: 28127697

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Project description:Sepsis is characterized by injury of pulmonary microvascular endothelial cells (PMVEC) leading to barrier dysfunction. Multiple mechanisms promote septic PMVEC barrier dysfunction, including interaction with circulating leukocytes and PMVEC apoptotic death. Our previous work demonstrated a strong correlation between septic neutrophil (PMN)-dependent PMVEC apoptosis and pulmonary microvascular albumin leak in septic mice in vivo; however, this remains uncertain in human PMVEC. Thus, we hypothesize that human PMVEC apoptosis is required for loss of PMVEC barrier function under septic conditions in vitro. To assess this hypothesis, human PMVECs cultured alone or in coculture with PMN were stimulated with PBS or cytomix (equimolar interferon γ, tumor necrosis factor α, and interleukin 1β) in the absence or presence of a pan-caspase inhibitor, Q-VD, or specific caspase inhibitors. PMVEC barrier function was assessed by transendothelial electrical resistance (TEER), as well as fluoroisothiocyanate-labeled dextran and Evans blue-labeled albumin flux across PMVEC monolayers. PMVEC apoptosis was identified by (1) loss of cell membrane polarity (Annexin V), (2) caspase activation (FLICA), and (3) DNA fragmentation [terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)]. Septic stimulation of human PMVECs cultured alone resulted in loss of barrier function (decreased TEER and increased macromolecular flux) associated with increased apoptosis (increased Annexin V, FLICA, and TUNEL staining). In addition, treatment of septic PMVEC cultured alone with Q-VD decreased PMVEC apoptosis and prevented septic PMVEC barrier dysfunction. In septic PMN-PMVEC cocultures, there was greater trans-PMVEC macromolecular flux (both dextran and albumin) vs. PMVEC cultured alone. PMN presence also augmented septic PMVEC caspase activation (FLICA staining) vs. PMVEC cultured alone but did not affect septic PMVEC apoptosis. Importantly, pan-caspase inhibition (Q-VD treatment) completely attenuated septic PMN-dependent PMVEC barrier dysfunction. Moreover, inhibition of caspase 3, 8, or 9 in PMN-PMVEC cocultures also reduced septic PMVEC barrier dysfunction whereas inhibition of caspase 1 had no effect. Our data demonstrate that human PMVEC barrier dysfunction under septic conditions in vitro (cytomix stimulation) is clearly caspase-dependent, but the mechanism differs depending on the presence of PMN. In isolated PMVEC, apoptosis contributes to septic barrier dysfunction, whereas PMN presence enhances caspase-dependent septic PMVEC barrier dysfunction independently of PMVEC apoptosis.

Project description:BackgroundThe Integrase (IN) strand transfer inhibitor (INSTI), Dolutegravir (DTG), has been given the green light to form part of first-line combination antiretroviral therapy (cART) by the World Health Organization (WHO). DTG containing regimens have shown a high genetic barrier against HIV-1 isolates carrying specific resistance mutations when compared with other class of regimens.MethodsWe evaluated the HIV-1 CRF02_AG IN gene sequences from Cameroon for the presence of resistance-associated mutations (RAMs) against INSTIs and naturally occurring polymorphisms (NOPs), using study sequences (n = 20) and (n = 287) sequences data derived from HIV Los Alamos National Laboratory database. The possible impact of NOPs on protein structure caused by HIV-1 CRF02_AG variations was addressed within the context of a 3D model of the HIV-1 IN complex and interaction analysis was performed using PyMol to validate DTG binding to the Wild type and seven mutant structures.ResultsWe observed 12.8% (37/287) sequences to contain RAMs, with only 1.0% (3/287) of the sequences having major INSTI RAMs: T66A, Q148H, R263K and N155H. Of these,11.8% (34/287) of the sequences contained five different IN accessory mutations; namely Q95K, T97A, G149A, E157Q and D232N. NOPs occurred at a frequency of 66% on the central core domain (CCD) position, 44% on the C-terminal domain (CTD) position and 35% of the N-terminal domain (NTD) position. The interaction analysis revealed that DTG bound to DNA, 2MG ions and DDE motif residues for T66A, T97A, Q148H, N155H and R263K comparable to the WT structure. Except for accessory mutant structure E157Q, only one MG contact was made with DTG, while DTG had no MG ion contacts and no DDE motif residue contacts for structure D232N.ConclusionsOur analysis indicated that all RAM's that resulted in a change in the number of interactions with encompassing residues does not affect DTG binding, while accessory mutations E157Q and D232N could affect DTG binding leading to possible DTG resistance. However, further experimental validation is required to validate the in silico findings of our study.

Dataset Information

Differential Mechanisms of Inflammation and Endothelial Dysfunction by HIV-1 Subtype-B and Recombinant CRF02_AG Tat Proteins on Human Brain Microvascular Endothelial Cells: Implications for Viral Neuropathogenesis.

Publications

Differential Mechanisms of Inflammation and Endothelial Dysfunction by HIV-1 Subtype-B and Recombinant CRF02_AG Tat Proteins on Human Brain Microvascular Endothelial Cells: Implications for Viral Neuropathogenesis.

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