Diminished HIV Infection of Target CD4+ T Cells in a Toll-Like Receptor 4 Stimulated in vitro Model

Abstract

Genital inflammation is associated with increased HIV acquisition risk. Induction of an inflammatory response can occur through the recognition of pathogenic or commensal microbes by Toll-like receptors (TLRs) on various immune cells. We used a in vitro peripheral blood mononuclear cell (PBMC) system to understand the contribution of TLR stimulation in inducing inflammation and the activation of target T cells, and its effect on HIV susceptibility. PBMCs were stimulated with TLR agonists LPS (TLR4), R848 (TLR7/8), and Pam3CSK4 (TLR1/2), and then infected with HIV NL4-3 AD8. Multiplexed ELISA was used to measure 28 cytokines in cell culture supernatants. Flow cytometry was used to measure the activation state (CD38 and HLA-DR), and CCR5 expression on CD4+ and CD8+ T cells. Although TLR agonists induced higher cytokine and chemokine secretion, they did not significantly activate CD4+ and CD8+ T cells and showed decreased CCR5 expression relative to the unstimulated control. Despite several classes of inflammatory cytokines and chemokines being upregulated by TLR agonists, CD4+ T cells were significantly less infectable by HIV after TLR4-stimulation than the unstimulated control. These data demonstrate that the inflammatory effects that occur in the presence TLR agonist stimulations do not necessarily translate to the activation of T cells. Most importantly, the finding that TLR4-stimulation reduces rather than increases susceptibility of CD4+ T cells to HIV infection in this in vitro system strongly suggests that the increased chemokine and possible antiviral factor expression induced by these TLR agonists play a powerful although complex role in determining HIV infection risk.

Keywords: HIV; Toll-like receptors; cytokines; immune activation; inflammation; innate antiviral immunity.

Figure 1

Activation profiles (A,B) and CCR5 expression (C,D) of CD4+ T cells on day 3 prior to HIV infection (A,C) and day 5 post HIV infection (B,D). PHA was used at a final concentration of 10 μg/ml. TLR agonists were used at a final concentration of 2 μg/ml. A repeated measures two-way ANOVA with Dunnett's multiple comparisons test was used for immune activation, and an ordinary one-way ANOVA with a Dunnett's multiple comparisons test for CCR5 expression. Significance is displayed as ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p ≤ 0.0001 compared to the unstimulated/unstimulated infected control, unless otherwise shown. Sample size, n = 5, 4 donors run in quadruplicate, 1 donor run in duplicate.

Figure 2

Activation profiles (A,B) and CCR5 expression (C,D) of CD8+ T cells on day 3 prior to HIV infection (A,C) and day 5 post HIV infection (B,D). PHA was used at a final concentration of 10 μg/ml. TLR agonists were used at a final concentration of 2 μg/ml. A repeated measures two-way ANOVA with Dunnett's multiple comparisons test was used for immune activation, and an ordinary one-way ANOVA with a Dunnett's multiple comparisons test for CCR5 expression. Significance is displayed as ^*p < 0.05, ^**p < 0.01, ^****p ≤ 0.0001 compared to the unstimulated/unstimulated infected control, unless otherwise shown. Sample size, n = 5, 4 donors run in quadruplicate, 1 donor run in duplicate.

Figure 3

Unsupervised hierarchical cluster heat map analysis of 28 cytokines measured in cell culture supernatants on day 3 (yellow) and day 5 (brown) from the unstimulated (red), PHA (blue), LPS (green), Pam3CSK4 (purple), or R848 (orange) conditions. PHA was used at a final concentration of 10 μg/ml. TLR agonists were used at a final concentration of 2 μg/ml. Sample size, n = 5, 4 donors run in quadruplicate, 1 donor run in duplicate.

Figure 4

Box and Whisker plots showing mean ± SD Log10 concentrations of pro-inflammatory cytokines IL-1α (A,G), IL-1β (B,H), IL-6 (C,I), IL-12p70 (D,J), IFN-γ (E,K), and TNF-α (F,L) from unstimulated (red), LPS (green), R848 (orange), Pam3CSK4 (purple), and PHA (blue) conditions on day 3 prior to HIV infection (top box: A–F) and day 5 post HIV infection (bottom box: G–L). TLR agonists were used at a final concentration of 2 μg/ml. PHA was used at a final concentration of 10 μg/ml. All TLR stimulation conditions were infected. An ordinary one-way ANOVA with a Dunnett's multiple comparisons test was performed. Significance is displayed as ^*p < 0.05, ^***p < 0.001, ^****p ≤ 0.0001 compared to the unstimulated control. Sample size, n = 5, 4 donors run in quadruplicate, 1 donor run in duplicate.

Figure 5

Box and Whisker plots showing mean ± SD Log10 concentrations of chemotactic cytokines IL-8 (A,G), MIP-1α (B,H), MIP-1β (C,I), IP-10 (D,J), MCP-1 (E,K), and RANTES (F,L) from unstimulated (red), LPS (green), R848 (orange), Pam3CSK4 (purple), and PHA (blue) conditions on day 3 prior to HIV infection (top box: A–F) and day 5 post HIV infection (bottom box: G–L). TLR agonists were used at a final concentration of 2 μg/ml. PHA was used at a final concentration of 10 μg/ml. All TLR stimulation conditions were infected. An ordinary one-way ANOVA with a Dunnett's multiple comparisons test was performed. Significance displayed as ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p ≤ 0.0001 compared to the unstimulated control. Sample size, n = 5, 4 donors run in quadruplicate, 1 donor run in duplicate.

Figure 6

Box and Whisker plots showing mean ± SD Log10 concentrations of haematopoietic cytokines IL-7 (A,E) and IL-17 (B,F), the growth factor GM-CSF (C,G), and the anti-inflammatory cytokine IL-10 (D,H) from unstimulated (red), LPS (green), R848 (orange), Pam3CSK4 (purple), and PHA (blue) conditions on day 3 prior to HIV infection (top box: A–D) and day 5 post HIV infection (bottom box: E–H). TLR agonists were used at a final concentration of 2 μg/ml. PHA was used at a final concentration of 10 μg/ml. All TLR stimulation conditions were infected. An ordinary one-way ANOVA with a Dunnett's multiple comparisons test was performed. Significance is displayed as ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p ≤ 0.0001 compared to the unstimulated control. Sample size, n = 5, 4 donors run in quadruplicate, 1 donor run in duplicate.

Figure 7

Infection rates (measured by p24 expression) of CD4+ T cells either unstimulated or stimulated with PHA or TLR agonists; LPS (TLR4), Pam3CSK4 (TLR1/2) or R848 (TLR7/8). Each symbol represents a donor, while different shades of each symbol represent repeats for that donor. PHA was used at a final concentration of 10 μg/ml. TLR agonists were used at a final concentration of 2 μg/ml. Significance was assessed by two-way ANOVA with Dunnett's multiple comparisons test. Significance is displayed as ^**p < 0.01, ^***p < 0.0001 compared to the unstimulated infected control. Sample size, n = 5, 4 donors run in quadruplicate, 1 donor run in duplicate.