Differential expression of IFN-alpha and TRAIL/DR5 in lymphoid tissue of progressor versus nonprogressor HIV-1-infected patients

Abstract

Loss of CD4+ T cells, the hallmark of HIV pathogenesis, was suggested to be partly due to apoptosis. We recently reported that IFN-alpha produced by HIV-1-activated plasmacytoid dendritic cells (pDCs) contributes to CD4+ T cell apoptosis by the TNF-related apoptosis-inducing ligand (TRAIL)/death receptor (DR)5 pathway. Here, we show that HIV-1-induced intracellular expression of IFN-alpha in pDCs is coupled to increased expression of IFN regulatory factor 7 and MyD88 by pDCs in vivo and in vitro. Expression of IFN-alpha was increased in lymphoid tonsillar tissue (LT) of patients with progressive (HIV(prog)) compared with nonprogressive (HIV(NP)) HIV-1 disease and to uninfected controls. LT from HIV(prog) exhibited higher TRAIL and DR5 mRNA levels than LT from HIV(NP) or controls. TRAIL mRNA levels in LT correlated with plasma viral load. We show that HIV-1 induces IFN-alpha and the TRAIL/DR5 apoptotic pathway in LT, suggesting a role for these cytokines in HIV-1 immunopathogenesis.

Fig. 1.

Characterization of circulating pDCs from HIV⁺ patients (HIV+) compared with HIV⁻ donors (HIV−). (a) Percentage of pDCs in HIV⁺ and HIV⁻ individuals. CD123⁺ BDCA-2⁺ CD11c⁻ pDC percentages were determined in PBMCs from 22 HIV⁻ and 28 HIV⁺ individuals. (b Upper) IFN-α in serum from HIV⁻ and HIV⁺ individuals. Red line indicates limit of detection. Compare intracellular levels of IFN-α in pDCs from isotype to HIV⁻ (Lower Left) and from HIV⁻ to HIV⁺ (Lower Right). Samples representative of 11 patients (HIV⁺) and 10 controls (HIV⁻) are shown. (c) Intracellular expression of MyD88 and IRF7 in pDCs from HIV⁻ and HIV⁺ individuals. (Left and Center) Comparison is shown between irrelevant isotype-matched antibody to MyD88 and IRF7 expression in uninfected donors. (Right) Compare IRF7 and MyD88 levels in pDCs from HIV⁻ to HIV⁺ individuals. Mean fluorescence intensity (MFI) of controls and patients for MyD88 and IRF7. (d) Activation of pDC determined by extracellular expression of CCR7 and HLA-DR by pDCs from HIV⁻ and HIV⁺ patients. P values were determined by using two-tailed Student’s t test.

Fig. 2.

HIV-1 activation of human pDCs from HIV⁻ donors. (a) Confocal immunofluorescence images of BDCA-2 (green) and IFN-α (red) staining in pDCs cultured with AT-2 HIV-1 (Lower) and pDCs cultured with media alone (Upper). Combined BDCA-2/IFN-α images overlaid onto corresponding images are shown (Right). (Scale bar, 10 μm.) (b Left) FACS analysis of pDCs using BDCA-2 and CD123. (b Center) FACS profiles of IFN-α staining of CD123⁺ BDCA-2⁺ CD4⁺-gated cells cultured with microvesicles [Mock versus isotype (Iso)]. (b Right) pDCs cultured overnight with AT-2 HIV-1 compared with pDCs cultured with microvesicles (Mock). (c) Intracellular expression of IRF7 and MyD88 in pDCs cultured with infectious or AT-2 HIV-1. (Left) Compare isotype controls (Iso, light gray line) with MyD88 and IRF7 expression in pDCs cultured with microvesicles (Mock, heavy black line). (Center) Compare MyD88 and IRF7 expression in pDCs cultured with microvesicles (Mock, light black line) and infectious HIV-1 (HIV, heavy red line). (Right) Compare MyD88 and IRF-7 expression in pDCs cultured with microvesicles (Mock, light black line) and noninfectious HIV-1 (AT-2 HIV, heavy red line). (d) Extracellular expression of HLADR and CCR7. (c and d) Mean fluorescence intensity (MFI) for pDCs cultured with microvesicles (Mock), HIV-1, or AT-2 HIV-1 (combined data).

Fig. 3.

IFN-α production and apoptotic pathway analyses in human LT. (a) IFN-α expression (brown) in LT sections from HIV⁻ (Upper Left), HIV_prog (Upper Right), HIV_NP (Lower Left), and an HIV_ai individual (Lower Right). Cell nuclei were counterstained with hematoxylin (blue). Magnification, ×380. (b) An LT section from HIV_prog was stained with IFN-α (green) and CD3 (red) (Left) or IFN-α (red) and CD4 (green) (Right). Images show IFN-α expression in parafollicular T cell-rich areas. (Scale bar, 10 μm.) IFN-α-expressing cells were proximal to CD3⁺ and CD4⁺ cells in LT. (c Left) TRAIL mRNA expression in LT of HIV⁻, HIV_prog, and HIV_NP normalized on GAPDH. (c Right) Correlation between TRAIL mRNA expression in LT and HIV-1 plasma viral load (n = 11). (d Left) FasL mRNA expression in LT of HIV_prog, HIV_NP, and HIV⁻ normalized on GAPDH. (d Right). Correlation between FasL mRNA expression in LT and plasma HIV-1 viral load (n = 11). (e) DR5 (Left) and Fas (Right) mRNA expression in LT of HIV_prog, HIV_NP, and HIV⁻ controls normalized on CD4 mRNA. LT biopsy data of b and c were compared by the Mann–Whitney test. Horizontal bars indicate median values; upper and lower limits of boxes, 75th and 25th percentiles; vertical lines indicate 90th and 10th percentiles. ∗, P < 0.05 compared with uninfected control.