HIV-1 and HIV-2 differentially mature plasmacytoid dendritic cells into IFN-producing cells or APCs

Abstract

HIV-1 causes a progressive impairment of immune function. HIV-2 is a naturally attenuated form of HIV, and HIV-2 patients display a slow-progressing disease. The leading hypothesis for the difference in disease phenotype between HIV-1 and HIV-2 is that more efficient T cell-mediated immunity allows for immune-mediated control of HIV-2 infection, similar to that observed in the minority of HIV-1-infected long-term nonprogressors. Understanding how HIV-1 and HIV-2 differentially influence the immune function may highlight critical mechanisms determining disease outcome. We investigated the effects of exposing primary human peripheral blood cells to HIV-1 or HIV-2 in vitro. HIV-2 induced a gene expression profile distinct from HIV-1, characterized by reduced type I IFN, despite similar upregulation of IFN-stimulated genes and viral restriction factors. HIV-2 favored plasmacytoid dendritic cell (pDC) differentiation into cells with an APC phenotype rather than IFN-α-producing cells. HIV-2, but not HIV-1, inhibited IFN-α production in response to CpG-A. The balance between pDC maturation into IFN-α-producing cells or development of an APC phenotype differentiates the early response against HIV-1 and HIV-2. We propose that divergent paths of pDC differentiation driven by HIV-1 and HIV-2 cause the observed differences in pathogenicity between the two viruses.

Figure 1. Gene expression profile of PBMC stimulated with HIV-1, HIV-2 or CpG-A

(A) Heat map of genes showing significant differences in expression (at least 2-fold change and P<0.05 as per ANOVA) and belonging to enriched gene ontology categories in PBMC from three independent donors stimulated with HIV-1 (blue), HIV-2 (green), CpG-A (yellow) or media alone (orange) for 6 (left heat map) and 12 hours (right heat map). Each column represents the response from a single donor in each condition; colour and grayscale bars on top of each heat map indicate culture conditions and donor code (A, B or C), according to the legend on the right; columns were ordered by unbiased hierarchical clustering. (B) Heat maps of IFN-I, IFN-II, IFN signalling genes in PBMC stimulated with HIV-1 (blue), HIV-2 (green), CpG-A (yellow) or media alone (orange) for 6 (top panels) and 12 hours (bottom panels). Each heat map represents responses in one individual donor. In both (A) and (B), gene expression levels are indicated by colour transitions from blue (lowest expression) to red (highest expression) according to the legend displayed below each graph.

Figure 2. IFN-α secretion in response to HIV-1 and HIV-2 stimulation

(A), (B), (C) and (D) show the concentration of IFN-α detected by ELISA in PBMC culture supernatants after stimulation for 6 to 48 hours with varying concentrations of virus, as indicated in the top left of each graph. Responses to HIV-1 are denoted by open boxes, HIV-2 by diagonal pattern filled boxes, and responses to media alone are shown by solid boxes. Horizontal lines within boxes represent median values, boxes show interquartile ranges (IQR) and lines extent to 90^th and 10^th percentiles. (E) Shows IFN-α detected by ELISA in PBMC culture supernatants after stimulation for 24 hours with HIV-1_MN/H9, HIV-1_MN/CEMx174, HIV-1_IIIB/H9, aldrithiol (At)-2 treated HIV-1_MN/H9, At-2 HIV-1_MN/CEMx174, At-2 HIV-1_{Ada/SupT1-CCR5}, HIV-2_NIH-Z/Hut78 or HIV-2_ST/CEMx174. Solid circles indicate responses against replication-competent HIV-1 isolates, solid squares represent At-2-treated isolates and open circles HIV-2 isolates. Horizontal lines represent median values. (F) shows Kyn/Trp ratios in PBMC culture supernatants after stimulation for 24 hours with HIV-1 (solid circles) or HIV-2 (open circles), at 13 × 10⁹ RNA copies/ml (left panel) and 1.3 × 10⁹ RNA copies/ml (right panel), or media alone (triangles). Horizontal lines indicate median values and vertical lines show the IQR. In all graphs, responses to HIV-1, HIV-2 and media within individual time points and virus concentrations were compared using a Friedman test with a Dunn’s post test for multiple analyses. *p≤0.05, **p≤0.01, ***p≤0.001

Figure 3. Expression of co-stimulatory molecules and CD83 on pDC after HIV-1 and HIV-2 stimulation

Expression of CD80 (A), CD86 (B) and CD83 (C) on pDC was analysed by flow cytometry after 9 and 24 hours stimulation with 13 ×10⁹ or 1.3 ×10⁹ viral RNA copies/ml of either HIV-1 (solid circles) or HIV-2 (open circles), or with cell culture media alone (triangles). Left panels show the frequencies of pDC expressing the marker of interest, and right panels show the MFI for each marker. Each dot represents one individual donor. Horizontal bars represent median values and vertical lines extend to the IQR. Responses to HIV-1, HIV-2 and media alone within individual time points and virus concentrations were compared using a Friedman test with a Dunn’s post test for multiple analyses. *p≤0.05, **p≤0.01, ***p≤0.001

Figure 4. Differentiation of pDC into IFN-I-producing or phenotypic antigen-presenting cells following HIV-1 or HIV-2 stimulation

(A) and (B) show the correlation between the frequency of IFN-α secreting pDC detected by flow cytometry and the concentration of IFN-α secreted in cell culture supernatants measured by ELISA after 9 (A) and 24 hours (B). Solid circles show responses to HIV-1 and open circles denote responses to HIV-2. Correlation was performed using a non-parametric spearman rank test. (C) and (D) show SPICE analysis of IFN-α secretion, CD83 and CD86 expression at 9 (C) and 24 hours (D). Differences in the frequencies of pDC expressing different combinations of CD83, CD86 and secreted IFN-α in response to media alone (orange), HIV-1 (blue) and HIV-2 (green) were analysed using a Friedman test with a Dunn’s post test for multiple analyses. *p≤0.05.

Figure 5. HIV-2 suppresses IFN-α secretion in response to CpG-A

(A) Shows IFN-α production measured by ELISA in supernatants from PBMC cultured for 24 hours with CpG-A in the presence or absence of 13 ×10⁹ viral RNA copies/ml HIV-1 or HIV-2. CpG-A alone and the addition of either HIV-1 or HIV-2 were compared using a Friedman test with a Dunn’s post test for multiple analyses; *p≤0.05, **p≤0.01, ***p≤0.001. (B) Shows the relative change in IFN-α secretion after the addition of HIV-1 or HIV-2 compared to CpG-A alone, calculated as ((HIV+CpG)-CpG)/CpG). In both (A) and (B), each dot represents one individual donor. Horizontal bars represent median values and vertical lines extend to the IQR. (C) Shows IFN-α production measured by ELISA in supernatants from PBMC pre-incubated with 13 ×10⁹ viral RNA copies/ml HIV-1 or HIV-2 for three hours prior to the addition of CpG-A, and then cultured for 24 hours. Each symbol represents an independent donor. (D) Shows IFN-α production measured by ELISA in supernatants from PBMC cultured for 24 hours with CpG-A in presence or absence of 13 ×10⁹ viral RNA copies/ml HIV-1 or HIV-2 added at the same time of CpG-A stimulation (0h) or one, two or three hours after stimulation (1h, 2h and 3h, respectively). Each symbol represents an independent donor.