HIV Delays IFN-α Production from Human Plasmacytoid Dendritic Cells and Is Associated with SYK Phosphorylation

Plasmacytoid dendritic cells (pDC) are the major producers of type I interferons (IFNs) in humans and rapidly produce IFN-α in response to virus exposure. Although HIV infection is associated with pDC activation, it is unclear why the innate immune response is unable to effectively control viral replication. We systematically compared the effect of HIV, Influenza, Sendai, and HSV-2 at similar target cell multiplicity of infection (M.O.I.) on human pDC function. We found that Influenza, Sendai, HSV-2 and imiquimod are able to rapidly induce IFN-α production within 4 hours to maximal levels, whereas HIV had a delayed induction that was maximal only after 24 hours. In addition, maximal IFN-α induction by HIV was at least 10 fold less than that of the other viruses in the panel. HIV also induced less TNF-α and MIP-1β but similar levels of IP-10 compared to other viruses, which was also mirrored by delayed upregulation of pDC activation markers CD83 and CD86. BDCA-2 has been identified as an inhibitory receptor on pDC, signaling through a pathway that involves SYK phosphorylation. We find that compared to Influenza, HIV induces the activation of the SYK pathway. Thus, HIV delays pDC IFN-α production and pDC activation via SYK phosphorylation, allowing establishment of viral populations

IFN-αproduction from pDCs following stimulation with HIV or Influenza.

<p>(A) Gating strategy used to identify CD14 negative BDCA-2 positive CD123 positive pDCs. (B) PBMCs from HIV negative individuals were treated with either Influenza (M.O.I = 0.1) or HIV_BAL (M.O.I = 0.1) in the presence of GolgiPlug™ for 8 hours and stained for intracellular IFN-α expression. Shown is a representative flow cytometry plot. (C) Summary data of pDCs from 3 individuals measuring intracellular IFN-α expression following stimulation with Influenza (M.O.I = 0.1) or HIV_BAL (M.O.I = 0.1) (D) Isolated pDCs from 3 HIV negative patients were stimulated with Influenza (M.O.I = 0.5) and HIV_BAL (M.O.I = 0.5) for 8 hours, and levels of IFN-α in supernatant were quantified by ELISA. Mean ± SEM, * = p<0.05.</p

Kinetics of CD83 and CD86 upregulation.

<p>Isolated pDCs from HIV negative donors were treated with Influenza (M.O.I = 0.5), imiquimod (20 µg/ml), or HIV (M.O.I = 0.5) for various time points and harvested for analysis by flow cytometry for surface expression of CD83 and CD86. (A) Representative contour plots of CD83 and CD86 upregulation at various time points post stimulation by Influenza, showing distinct kinetics of CD83 and CD86 upregulation. (B) Percentage of pDCs that are CD83 single positive (SP, Blue bars), CD83/CD86 double positive (DP, Purple bars), CD86SP (Red bars), and double negative for both markers (DN, gray bars) at time points post stimulation with Influenza, imiquimod or HIV_BAL. (C) Percentages of pDCs that are CD83⁺ or CD86⁺ at time points post stimulation. Data shown are representative of at least 3 different donors stimulated and stained in duplicate.</p

HIV induces upregulation of pSYK.

<p>(<b>A</b>) Purified pDCs were treated with Influenza and HIV_BAL for various time points. Positive control with anti-BDCA-2 antibody (1∶100) treated for 30 minutes is shown. Cells were harvested, permeabalized and stained for pSYK (Y352) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037052#s4" target="_blank">materials and methods</a>. Shown are representative histograms from 3 independent experiments. (<b>B</b>) PDCs were stimulated with HIV_BAL, HIV_NL43, Influenza, or medium alone for 30 minutes and percentages of pDCs staining positive for pSYK (Y352) and pSYK (Y525/526) were evaluated by flow cytometry (n = 3). In (<b>C</b>) gp120 induces pSyk signaling in pDCs. Purified pDCs were stimulated for 90 minutes in the presence of 1 mg/ml monomeric gp120, AN1, (blue); 20 µg/ml imiquimod, (red); or the absence of stimulation (black). Cells were harvested and stained for pSyk (pY348) and analyzed by flow cytometry. Shown is a representative flow cytometric plot of two experiments. (<b>D</b>) pDCs were stimulated with 0.5 M.O.I. of HIV_BAL, HIV_NL43, Influenza, and imiquimod, or medium alone for 24 hours in the presence or absence of anti-BDCA2 antibody or an IgG1 isotype control and IFN-α was measured by ELISA (n = 2, performed with two independent replicates, *<i>p</i><0.001, **<i>p</i><0.00005). BDCA2 treated pDC completely abrogated IFN-α production in the presence of HIV (bars not seen).</p

Kinetics of IFN-αproduction.

<p>Isolated pDCs from five HIV negative donors were rested overnight, and treated with a panel of stimuli as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037052#s4" target="_blank">materials and methods</a>. Levels of IFN-α in supernatants were quantified by CBA as part of a panel of other cytokines. (<b>A</b>) Quantified IFN-α (pg/ml) present in culture supernatant of 3–5 donors (N1-N5) at various time points (represented by blue shaded bars) following stimulation by HIV_BAL, HIV_NL43 or Influenza (<b>B</b>) Mean ± SEM concentrations of IFN-α present in supernatants at various time points following stimulation by HIV_BAL, (red), HIV_NL43 (orange), Influenza (yellow), HSV-1 (green), Imiquimod (light blue), Sendai virus (dark blue), and medium control (white) from data from 5 donors.</p

Cytokine expression profiles following stimulation.

<p>Isolated pDCs from HIV negative donors were rested overnight, and treated with a panel of stimuli as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037052#s4" target="_blank">materials and methods</a>. Cytokines were quantified in culture supernatants by cytometric bead array. Mean ± SEM concentrations of (<b>A</b>) MIP-1β, (<b>B</b>) IP-10, and (<b>C</b>) TNF-α from 5 donors are shown at various time points following stimulation by HIV_BAL, (red), HIV_NL43 (orange), Influenza (yellow), HSV (green), Imiquimod (light blue), Sendai virus (dark blue), and medium control (white). Neither IL-10 nor IL-12p70 was detected in any of the supernatant samples.</p