HIV-1 infection-induced apoptotic microparticles inhibit human DCs via CD44

Abstract

Acute HIV-1 infection results in dysregulated immunity, which contributes to poor control of viral infection. DCs are key regulators of both adaptive and innate immune responses needed for controlling HIV-1, and we surmised that factors elicited during acute HIV-1 infection might impede DC function. We derived immature DCs from healthy donor peripheral blood monocytes and treated them with plasma from uninfected control donors and donors with acute HIV-1 infections. We found that the plasma from patients with HIV specifically inhibited DC function. This suppression was mediated by elevated apoptotic microparticles derived from dying cells during acute HIV-1 infection. Apoptotic microparticles bound to and inhibited DCs through the hyaluronate receptor CD44. These data suggest that targeting this CD44-mediated inhibition by apoptotic microparticles could be a novel strategy to potentiate DC activation of HIV-specific immunity.

Figures

Figure 1. AHIV plasma inhibits TLR-stimulated DC function.

(A) DCs were treated overnight with 10% uninfected donor plasma or plasma taken from patients with AHIV during Fiebig stages 1 and 2. The next day, DCs were stimulated with the TLR3 agonist poly I:C, and cytokine production was analyzed the following day. Mean is shown by horizontal bars, with each dot representing individual plasma samples. (B) DCs were tested as described in A with AHIV plasma (Fiebig stages 1 and 2), including seroconversion panels obtained from ZeptoMetrix Corp. (black bars), University of North Carolina Center for Infectious Diseases (white bar), Duke University Medical Center (light gray bar), Queen Elizabeth Central Hospital (medium gray bars), and Lilongwe Central Hospital (dark gray bars). The level of IL-12p70 production of AHIV plasma–treated DCs is shown, expressed as a percentage of the IL-12p70 levels produced by uninfected donor plasma–treated DCs (100% represents no inhibition). This figure combines multiple experiments compared against multiple uninfected control donor plasma samples (ranging from n = 12 to n = 50). (C) DCs were treated with plasma as described in A. DCs were then stimulated with poly I:C, washed, and cocultured with allogeneic naive CD4+ T cells. T cell cytokine production was analyzed at day 6. Mean is shown by horizontal bars, with each dot representing individual plasma samples. (D) DCs were treated as described in C and then cocultured with NK cells. NK cell cytokine production was analyzed the next day. Representative experiments are shown.

Figure 2. AHIV plasma–mediated DC inhibition occurs concurrently with VR.

DCs were treated overnight with 10% AHIV, acute HBV, or acute HCV plasma collected at indicated time points before and after VR. DCs were also treated with 10% normal, uninfected donor plasma taken at various time points, generally 2–7 days apart. DCs were then poly I:C stimulated. IL-12p70 levels (as percentages of the levels from uninfected plasma–treated DCs) secreted by DCs treated with plasma from before or after viremia (red line) are graphed versus the plasma viral load (VL) (blue line). The dotted line denotes 100% of control IL-12p70 levels, indicating no inhibition. Arrows indicate start of VR.

Figure 3. AHIV plasma–mediated DC inhibition is independent of virus and occurs in primary myeloid DCs.

(A) DCs were treated overnight with 10% uninfected plasma containing live HIV-1 (ADA laboratory strain or founder strain; dose of 300 ng/ml of p24 protein) or no virus (–). DCs were subsequently stimulated and cytokine production assessed. (B) PBMCs from subjects at different stages of HIV infection (Fiebig stages [F] 1–6 or a time point in early infection, approximately 6 months after seroconversion) or uninfected subjects were stimulated with CL097 (Neg) (TLR7/8 agonist). Percentages of IL-12+ or TNF-α+ myeloid DCs assessed by intracellular cytokine staining are indicated. Mean is shown by horizontal bars, with each dot representing individual patient samples. The P values (Kruskal-Wallis test followed by Dunn’s multiple comparison post-test) for the indicated comparisons are shown.

Figure 4. Apoptotic MPs inhibit TLR-stimulated DC function.

(A) DCs were treated overnight with apoptotic MPs (Apo-MP), control MPs (Cont-MP), or no MPs. DCs were subsequently poly I:C stimulated, and cytokine production was analyzed. (B) DCs were treated with MPs as described in A. DCs were then stimulated with 100 ng/ml flagellin, and IL-6 production was measured (flagellin-stimulated DCs do not produce substantial IL-12p70). (C) DCs were treated with MPs as described in A. After being poly I:C stimulated for 4 to 6 hours, DCs were washed, cocultured with isolated allogeneic naive CD4+ T cells or (D) naive CD8+ T cells for 6 days, and T cell cytokine production was assessed. (E) DCs were treated with MPs and stimulated as described in C. DCs were then cocultured overnight with isolated NK cells, and NK cell cytokine production was assessed. Data are representative of at least 3 independent experiments. The P values (unpaired Student’s t test) for indicated comparisons are shown. (F) DCs were treated with MPs derived from AHIV plasma from indicated donors. DCs were then poly I:C stimulated and IL-12p70 levels were analyzed. IL-12p70 levels were normalized to levels from DCs treated with MPs derived from uninfected plasma (at least 2 different control donors per experiment). The dotted line denotes 100% of control levels, indicating no inhibition.

Figure 5. Inhibition of DCs by AHIV plasma is dependent on CD44.

(A) Control or apoptotic MPs were stained with FITC-labeled CD44-Fc (30 μg/ml; blue line) and analyzed by FACS. Control staining was with FITC-labeled human IgG-Fc (red line). (B) MP preparations were pretreated with 50 μg/ml CD44-Fc chimeric protein or human IgG-Fc protein (hIgG-Fc). DCs were treated overnight with control or apoptotic MPs and subsequently poly I:C stimulated, and cytokine production was analyzed the following day. Data are representative of at least 3 independent experiments. The P value (unpaired Student’s t test) for indicated comparisons is shown. (C) DCs were pretreated with 50 μg/ml anti-CD44 blocking monoclonal antibody (BD Biosciences; clone 515) or mouse IgG isotype control. An alternative method involved pretreating plasma-derived MPs with CD44-Fc or hIgG-Fc control. DCs were then treated with MPs derived from uninfected or AHIV donors and subsequently stimulated with poly I:C. IL-12p70 secretion by DCs was analyzed the next day, and levels of IL-12p70 from AHIV MP-treated DCs were normalized to levels from DCs treated with MPs derived from uninfected donor plasma. Results are representative of at least 5 separate experiments with different control and AHIV donors. The P values (paired Student’s t test) for indicated comparisons are shown. (D) DCs were treated with microbead-bound anti-CD44, mouse IgG1 control, or microbeads only. DCs were subsequently poly I:C or LPS stimulated, and cytokine levels were assessed (IL-12p70 for poly I:C; IL-6 for LPS; LPS-stimulated DCs do not produce substantial IL-12p70). The P values (unpaired Student’s t test) for indicated comparisons are shown.

Figure 6. Mechanisms of apoptotic MP-mediated DC inhibition.

(A) DCs were pretreated with 50 μM Rac1 inhibitor (EMD Chemicals) for 30 minutes and then treated with control MPs, apoptotic MPs, or no MPs. DCs were subsequently washed and poly I:C stimulated, and cytokine production was analyzed the following day. (B) DCs were pretreated with 50 μM Rac1 inhibitor for 30 minutes and then treated with of CFSE-labeled control or apoptotic MPs (green). After 3 hours, DCs were mounted, stained with LAMP-1 (red) and DAPI nuclear stain (blue), and analyzed by confocal microscopy (original magnification, ×63; inset, ×252). Arrows indicate individual cells shown in insets. (C) DCs were pretreated with 10 μM cytochalasin D (CCD; Sigma-Aldrich) for 30 minutes and then treated with control MPs, apoptotic MPs, or no MPs. DCs were subsequently washed and poly I:C stimulated, and cytokine production was analyzed the following day.