Depletion of CD4 T lymphocytes in human lymphoid tissue infected ex vivo with doxycycline-dependent HIV-1

AbstractWe investigated whether CD4+ T cells that do not produce HIV-1 are killed in HIV-infected human lymphoid tissue. Tissue blocks were inoculated with high amount of doxycycline-dependent HIV-rtTA. Doxycycline triggered productive infection and loss of CD4+ T cells in these tissues, whereas without doxycycline, neither productive infection nor CD4+ T cell depletion was detected in spite of the massive presence of virions in the tissue and of viral DNA in the cells. Thus, HIV-1 alone is sufficient to deplete productively infected CD4+ T cells but is not sufficient to cause the death of uninfected or latently infected CD4+ T cells

Contrasting Roles for TLR Ligands in HIV-1 Pathogenesis

The first line of a host's response to various pathogens is triggered by their engagement of cellular pattern recognition receptors (PRRs). Binding of microbial ligands to these receptors leads to the induction of a variety of cellular factors that alter intracellular and extracellular environment and interfere directly or indirectly with the life cycle of the triggering pathogen. Such changes may also affect any coinfecting microbe. Using ligands to Toll-like receptors (TLRs) 5 and 9, we examined their effect on human immunodeficiency virus (HIV)-1 replication in lymphoid tissue ex vivo. We found marked differences in the outcomes of such treatment. While flagellin (TLR5 agonist) treatment enhanced replication of CC chemokine receptor 5 (CCR 5)-tropic and CXC chemokine receptor 4 (CXCR4)-tropic HIV-1, treatment with oligodeoxynucleotide (ODN) M362 (TLR9 agonist) suppressed both viral variants. The differential effects of these TLR ligands on HIV-1 replication correlated with changes in production of CC chemokines CCL3, CCL4, CCL5, and of CXC chemokines CXCL10, and CXCL12 in the ligand-treated HIV-1-infected tissues. The nature and/or magnitude of these changes were dependent on the ligand as well as on the HIV-1 viral strain. Moreover, the tested ligands differed in their ability to induce cellular activation as evaluated by the expression of the cluster of differentiation markers (CD) 25, CD38, CD39, CD69, CD154, and human leukocyte antigen D related (HLA)-DR as well as of a cell proliferation marker, Ki67, and of CCR5. No significant effect of the ligand treatment was observed on apoptosis and cell death/loss in the treated lymphoid tissue ex vivo. Our results suggest that binding of microbial ligands to TLRs is one of the mechanisms that mediate interactions between coinfected microbes and HIV-1 in human tissues. Thus, the engagement of appropriate TLRs by microbial molecules or their mimetic might become a new strategy for HIV therapy or prevention

The Nonnucleoside Reverse Transcriptase Inhibitor UC-781 Inhibits Human Immunodeficiency Virus Type 1 Infection of Human Cervical Tissue and Dissemination by Migratory Cells

Heterosexual transmission of human immunodeficiency virus remains the major route of transmission worldwide; thus, there is an urgent need for additional prevention strategies, particularly those that could be controlled by women. Using cellular and tissue explant models, we have evaluated the potential activity of thiocarboxanilide nonnucleoside analogue reverse transcriptase inhibitor UC-781 as a vaginal microbicide. We were able to demonstrate a potent dose-dependent effect against R5 and X4 infections of T cells. In human cervical explant cultures, UC-781 was not only able to inhibit direct infection of mucosal tissue but was able to prevent dissemination of virus by migratory cells. UC-781 formulated into a carbopol gel (0.1%) retained significant activity against both direct tissue infection and transinfection mediated by migratory cells. Furthermore, UC-781 demonstrated prolonged inhibitory effects able to prevent both localized and disseminated infections up to 6 days post compound treatment. Additional studies were carried out to determine the concentration of compound that might be required to block a primary infection within draining lymph nodes. While a greater dose of compound was required to inhibit both X4 and R5 infections of lymphoid versus cervical explants, this was equivalent to a 1:3,000 dilution of the 0.1% formulation. Furthermore, a 2-h exposure to the compound prevented infection of lymphoid tissue when challenged up to 2 days later. The prolonged protection observed following pretreatment of both genital and lymphoid tissues with UC-781 suggests that this class of inhibitors may have unique advantages over other classes of potential microbicide candidates

Stimulation of the Liver X Receptor Pathway Inhibits HIV-1 Replication via Induction of ATP-Binding Cassette Transporter A1

Cholesterol plays an important role in the HIV life cycle, and infectivity of cholesterol-depleted HIV virions is significantly impaired. Recently, we demonstrated that HIV-1, via its protein Nef, inhibits the activity of the major cellular cholesterol transporter ATP binding cassette transporter A1 (ABCA1), suggesting that the virus may use this mechanism to get access to cellular cholesterol. In this study, we investigated the effect on HIV infection of a synthetic liver X receptor (LXR) ligand, N-(2,2,2-trifluoro-ethyl)-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-benzenesulfonamide (TO-901317), which is a potent stimulator of ABCA1 expression. We demonstrate that TO-901317 restores cholesterol efflux from HIV-infected T lymphocytes and macrophages. TO-901317 potently suppressed HIV-1 replication in both cell types and inhibited HIV-1 replication in ex vivo cultured lymphoid tissue and in RAG-hu mice infected in vivo. This anti-HIV activity was dependent on ABCA1, because the effect of the drug was significantly reduced in ABCA1-defective T cells from a patient with Tangier disease, and RNA interference-mediated inhibition of ABCA1 expression eliminated the effect of TO-901317 on HIV-1 replication. TO-901317-mediated inhibition of HIV replication was due to reduced virus production and reduced infectivity of produced virions. The infectivity defect was in part due to reduced fusion activity of the virions, which was directly linked to reduced viral cholesterol. These results describe a novel approach to inhibiting HIV infection by stimulating ABCA1 expression

Effects of flagellin on chemokines production in HIV-1-infected lymphoid tissue <i>ex vivo</i>.

<p>Blocks of tonsillar tissue were treated with flagellin and subsequently infected with HIV-1 X4_LAI.04 or R5_SF162. Release of CCL3, CCL4, CCL5, CXCL10, and CXCL12 was monitored in the culture medium with the multiplexed fluorescent microsphere immunoassay. Concentrations of released chemokines at individual time points were normalized to their total cumulative release in matched untreated cultures. Presented data are means of these normalized values (± SEM) from 4 to 7 experiments. Statistical analysis (see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012831#s2" target="_blank">Results</a>) revealed significant difference between (i) uninfected and X4_LAI.04 –infected (but not R5-infected) tissues in the release of CCL3, CCL4, CCL5, and CXCL10 (<i>p</i><0.046, <i>n</i> = 5 to 7); (ii) R5_SF162-infected tissues and these tissues treated with flagellin in the release of CCL3, CCL4, CCL5, CXCL12, and CXCL10 (<i>p</i><0.016, <i>n</i> = 6); (ii) X4_LAI.04 -infected tissues and these tissues treated with flagellin in the release of CCL3, CCL4, and CXCL10 (<i>p</i><0.046, <i>n</i> = 6).</p

Effect of ODN M362 and flagellin on CD4⁺ T-cell activation.

<p>Blocks of tonsillar tissue were treated with either flagellin or ODN M362 for 7 days. Expression of activation markers CD25, HLA-DR, CD38, CD69, CD39, CD154 on CD4⁺CD8⁻ CD3⁺ lymphocytes (Panels A & C) and of cell-proliferation marker Ki67 in these cells (panels B & D) were monitored with flow cytometry in tissue blocks (Panels A & B) and in the fraction of cells emigrated from the tonsillar tissues (Panels C & D). Panels A & B show representative staining of CD3⁺CD4⁺CD8⁻ lymphocytes for CD38 and Ki67. Data were compensated and analyzed with FlowJo version 9 (Treestar, Ashland, OR, USA). The plotted data represent means (± SEM) of fractions of CD4⁺CD8⁻ CD3⁺ lymphocytes positive for particular activation marker from four experiments. Data are normalized to the corresponding fraction of CD4⁺CD8⁻ CD3⁺ lymphocytes from matched untreated control tissue. <b>⁺</b> marks statistically significant (p<0.04) difference between flagellin- and ODN M362-treated tissue for a particular activation marker.</p

ODN M362 and flagellin modulate CXCL12 release HIV-1 X4_LAI.04 -infected lymphoid tissue <i>ex vivo</i>.

<p>Blocks of tonsillar tissue were treated either with flagellin (▴) or with ODN M362 (▪) or mock-treated (•), and subsequently infected with HIV-1 X4_LAI.04. Concentrations of CXCL12 and p24 were determined in the culture supernatants every 3^rd day for 12 days following the infection. Data were normalized to the maximums released in matched X4_LAI.04-infected ligand-untreated cultures. Each datum point represents a ratio between released CXCL12 and HIV-1 p24 in each of 47 HIV-1 positive supernatants. On average, in ODN M362 treated cultures, the relative release of CXCL12 per unit of HIV-1 p24 was 5 times higher (<i>p</i><0.001) whereas in flagellin-pretreated cultures, it was twice lower (<i>p</i> = 0.01) than in ligand-untreated culture.</p

Effects of ODN M362 on chemokines production in HIV-1-infected lymphoid tissue <i>ex vivo</i>.

<p>Blocks of tonsillar tissue were treated with ODN M362 and subsequently infected with HIV-1 X4_LAI.04 or R5_SF162. Release of CCL3, CCL4, CCL5, CXCL10, and CXCL12 was monitored in the culture medium with the multiplexed fluorescent microsphere immunoassay. Concentrations of released chemokines at individual time points were normalized to their total cumulative release in matched untreated cultures. Presented data are means of these normalized values (± SEM) from 4 to 7 experiments. Statistical analysis revealed significant difference between: (i) uninfected and X4_LAI.04 –infected (but not R5-infected) tissues in the release of CCL3, CCL4, CCL5, CXCL10, and CXCL12 (<i>p</i><0.046, <i>n</i> = 5 to 7); (ii) uninfected mock- and ODN M362-treated tissues in production of CCL3, CCL4, CCL5, and CXCL12 (<i>p</i><0.031, <i>n</i> = 5); (iii) R5_SF162-infected tissues and these tissues treated with ODN M362 in the production of CCL3, CCL4, CCL5, and CXCL12 (<i>p</i><0.04; <i>n</i> = 6); (iv) X4_LAI.04- infected tissues and these tissues treated with ODN M362, in production of CCL4 and CXCL12 (<i>p</i><0.046, <i>n</i> = 6).</p