Silencing of TLR2 expression attenuates the increased susceptibility to HIV.

<p>Expression of TLR2 increases at the transcription level in cells stimulated with <i>M. bovis</i> BCG when compared to cells stimulated with <i>M. smegmatis</i> (A). The expression levels of TLR4 and TLR9 do not show a significant difference in cells stimulated with different strains of mycobacteria as evidenced by RT-PCR. Comparison of HIV infectivity of CD4+ cells transfected with scrambled siRNA and subsequently stimulated with <i>M. bovis</i> BCG to CD4+ cells transfected with TLR2 siRNA and subsequently stimulated with <i>M. bovis</i> BCG (B) **p<0.005. TLR2 expression on CD4+ cells after stimulation with <i>M. bovis</i> BCG, <i>M. tuberculosis</i>, and <i>M. smegmatis</i> compared to TLR2 expression on CD4+ cells transfected with TLR2 siRNA and subsequently stimulated with <i>M. bovis</i> BCG (C). The expression showed similar trends for the four samples tested. Dot plots comparing the percentage of surface expression of TLR2 on unstimulated CD4+ cells and CD4+ cells stimulated with <i>M. bovis</i> BCG, <i>M. tuberculosis</i> (CDC1551), <i>M. smegmatis</i> (MC²155), and CD4+ cells transfected with TLR2 siRNA and subsequently stimulated with <i>M. bovis</i> BCG (D).</p

Differential susceptibility to HIV infection is not dependent on increased expression of the immune activation markers CD38 and HLA DR.

<p>Cells stimulated with different antigens are activated to a similar extent as evidenced by expression of immune activation marker CD38 (A). Dot plots from unstimulated CD4+ cells and CD4+ cells stimulated with PHA (150 g/ml), <i>M. bovis</i> BCG (Copenhagen) and <i>M. tuberculosis</i> (CDC1551) depicting the expression of immune activation marker CD38 (B). Cells stimulated with <i>M. bovis</i> BCG (Copenhagen) and <i>M. tuberculosis</i> (CDC1551) show lower expression of MHC II molecule HLA DR when compared to cells stimulated with <i>M. smegmatis</i> MC²155 (C) *p<0.05.</p

<i>M. tuberculosis</i> complex increases susceptibility to HIV infectivity.

<p>The percentage of unstimulated CD4+ cells and CD4+ cells stimulated with PHA (50 µg/ml), <i>M. bovis</i> BCG (Copehnhagen), <i>M. tuberculosis</i> (CDC1551), <i>M. smegmatis</i> (MC²155) from individual subjects infected with X4-tropic pseudovirus (A) or R5-tropic pseudovirus (B). Results from individual donors are color-coded. Statistical analysis was performed using student T test (** p<0.005). Flow cytometry analysis of the representative CD4+ cells after infection with GFP-expressing pseudovirus (C).</p

<em>Mycobacterium tuberculosis</em> Complex Enhances Susceptibility of CD4 T Cells to HIV through a TLR2-Mediated Pathway

<div><p>Among HIV-infected individuals, co-infection with <em>Mycobacterium tuberculosis</em> is associated with faster progression to AIDS. We investigated the hypothesis that <em>M. bovis</em> BCG and <em>M. tuberculosis</em> (Mtb complex) could enhance susceptibility of CD4+ cells to HIV infection. Peripheral blood mononuclear cells (PBMCs) collected from healthy donors were stimulated with <em>M. bovis</em> BCG, <em>M. tuberculosis</em> CDC1551 and <em>M. smegmatis</em> MC²155, and stimulated CD4+ cells were infected with R5-and X4-tropic single replication-competent pseudovirus. CD4+ cells stimulated with Mtb complex showed enhanced infection with R5- and X4-tropic HIV, compared to unstimulated cells or cells stimulated with <em>M. smegmatis</em> (p<0.01). Treatment with TLR2 siRNA reversed the increased susceptibility of CD4+ cells with R5- and X4-tropic virus induced by Mtb complex. These findings suggest that TB infection and/or BCG vaccination may be a risk factor for HIV acquisition.</p> </div

Classification of NIPG.

<p>(A) Distribution of NIPG within each study participant. Purple: hypermutation; Green: Gag deletion; Red: intact near-full length, replication-deficient due to nonsense mutations or INDELs; Blue: undefined non-Gag deletion; Orange: defined non-Gag deletion; empty circle: not evaluable. (B) Distribution of NIPG across study participants after Gag and near-full length sequencing. <i>NIPG</i>: <i>non-induced proviral genomes</i>.</p

Deletions in NIPG with defined junctions.

<p>Letters and numbers correspond to participant identifiers listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170548#pone.0170548.t001" target="_blank">Table 1</a>. A) Sequences are aligned to HXB2. Sequenced positions appear as a black polygon. Gray thin lines indicate deleted regions of genomes. B) Regions with relatively high coverage were extracted for phylogenetic analysis. Location shown is according to HXB2 numbering. Colors correspond to nucleotides on the left. Sequences designated with a red dot were not used in subsequent analysis due to missing sequence segments. C) Phylogenetic analysis of sequences rooted using HXB2 and aligned to positions 3350–4327 and 9105–9597.</p

HIV DNA concentrations from cultured resting CD4+ T cell QVOA wells with and without NIPG.

<p>“N” represents the number of QVOA culture wells.</p

HIV-specific antibody responses of plasma samples.

<p>HIV-specific antibody responses of plasma samples.</p

Characteristics of the study population.

<p>Characteristics of the study population.</p

Fluorescence-activated cell sorting (FACS) strategies for PBMC samples (A-G) and GI tract samples (H-M; ileum shown as example).

<p><b> </b>For PBMC, all cells were included (panel A), doublets excluded (panel B), and residual non-viable cells excluded by LIVE/DEAD violet cell staining ("Viab dump," panel C). Low-frequency CCR5+ events were then collected in one sorting tube (inside box gate, panel D). Of the remaining, CCR5- events (outside box gate, panel D), CD3+ events negative for CD14 and CD11c were included for further gating (inside polygon gate, panel E), with remaining events collected in a second sorting tube (outside polygon gate, panel E). CCR5-CD3+ events negative for CD14 and CD11c that were also CD8- (panel F) and T cell receptor-γδ-, CD20-, and CD56- ("Lin dump," panel G) were collected in a third sorting tube as presumptive CD4+ T cells. Remaining CD3+ events that were either CD8+ or Lin dump+ were combined in a fourth sorting tube. For ileum and rectum, all cells were included (panel H) and then doublets excluded (panel I). Viable CD45+ events were included for further gating (inside polygon gate, panel J), with all events outside this gate collected in one sorting tube as non-hematopoietic cells. CD3+ events negative for CD14 and CD11c were included for further gating (inside polygon gate, panel K), with remaining events collected in a second sorting tube (outside polygon gate, panel K). CD3+ events negative for CD14 and CD11c that were also CD8- (panel L) and T cell receptor-γδ-, CD20-, and CD56- ("Lin dump," panel M) were collected in a third sorting tube as presumptive CD4+ T cells. Remaining CD3+ events that were either CD8+ or Lin dump+ were combined in a fourth sorting tube. Numbers in upper-right corners of flow plots indicate the percentages of events on plots falling inside gates shown. </p