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

HIV-1 expressing the envelopes of transmitted/founder or control/reference viruses have similar infection patterns of CD4 T-cells in human cervical tissue ex vivo.

Recently, it was found that 80% of sexual HIV-1 transmissions are established by a single virion/viral genome. To investigate whether the transmitted/founder (T/F) viruses have specific biological properties favoring sexual transmission, we inoculated human cervical tissue explants with isogenic HIV-1 viruses encoding Env sequences from T/F and control reference (C/R) HIV-1 variants as well as with full length T/F HIV-1 and compared their replication efficiencies, T cell depletion, and the activation status of infected cells. We found that all the HIV-1 variants were capable of transmitting infection to cervical tissue ex vivo and in this system preferentially replicate in activated CD4 T cells and deplete these cells. There was no difference in the biological properties of T/F and C/R HIV-1 variants as evaluated in ex vivo cervical tissue

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

Enhancement of HIV-1 replication in human lymphoid tissue <i>ex vivo</i> treated with the TLR5 ligand flagellin.

<p>Release of HIV-1 was monitored in the culture medium of flagellin-treated and -untreated cultures. Each datum point reflects cumulative p24 release. Data were normalized to the total release of p24 in untreated cultures over the period of 15 days. Presented data are means (± SEM) of four experiments. Differences between flagellin-treated and -untreated cultures are statistically significant (p<0.05) at days 9 to 15 for X4_LAI.04 infection and at days 12 to 15 for R5_SF162 infection.</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

Suppression of HIV-1 replication in human lymphoid tissue <i>ex vivo</i> treated with the TLR9 ligand ODN M362.

<p>Release of HIV-1 was monitored in the culture medium of ODN M362-treated and -untreated cultures. Each datum point reflects cumulative p24 release. Data were normalized to the total release of p24 in untreated cultures. Presented data are means (± SEM) of four experiments. Differences between ODN M362-treated and -untreated cultures are statistically significant (p<0.05) at days 6 to 15 for X4_LAI.04 infection and at days 9 to 15 for R5_SF162 infection.</p

Replication of various C/R and T/F HIV-1 variants in human cervical tissue <i>ex vivo</i>.

<p>Donor-matched human cervical tissue blocks were infected <i>ex-vivo</i> with C/R and T/F viruses in presence or absence of 3TC. Culture media were collected every 3 days up to day 12 and the amount of p24 was measured. In addition, 12 days post infection, the tissues were enzymatically digested and their cells were analyzed by polychromatic flow cytometry. T cells were identified by side-scatter and staining for CD3. The CD4 T cell subset was defined as CD8^– T cells since the down-regulation of CD4 in HIV-1 infected cells precludes use of staining for CD4. CD4 T cell, which were infected with HIV-1 were revealed by staining for p24.(a) For each virus, cumulative values for the net p24 production ([p24] in untreated − [p24] in 3TC-treated donor-matched tissues) were computed and plotted as box plots (median, 25^th and 75^th percentiles, and range) on a log scale (left Y-axis; empty boxes; C/R virus: n = 23; T/F viruses: n = 30). The fractions of p24⁺ positive cells among CD8⁻CD3⁺ cells were plotted on a linear axis (right Y-axis; filled boxes; n = 19, and n = 14, respectively) (b). Bivariate plots showing p24 and CD8 staining in T cells; the values represent the fraction of CD8⁻CD3⁺ cells expressing p24 as defined by the plotted gate. Plots illustrate a representative experiment.</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