PI3K is critical for the nuclear translocation of IRF-7 and type I IFN production by human plasmacytoid predendritic cells in response to TLR activation - PubMed (original) (raw)
PI3K is critical for the nuclear translocation of IRF-7 and type I IFN production by human plasmacytoid predendritic cells in response to TLR activation
Cristiana Guiducci et al. J Exp Med. 2008.
Abstract
Plasmacytoid predendritic cells (pDCs) are the main producers of type I interferon (IFN) in response to Toll-like receptor (TLR) stimulation. Phosphatidylinositol-3 kinase (PI3K) has been shown to be activated by TLR triggering in multiple cell types; however, its role in pDC function is not known. We show that PI3K is activated by TLR stimulation in primary human pDCs and demonstrate, using specific inhibitors, that PI3K is required for type I IFN production by pDCs, both at the transcriptional and protein levels. Importantly, PI3K was not involved in other proinflammatory responses of pDCs, including tumor necrosis factor alpha and interleukin 6 production and DC differentiation. pDCs preferentially expressed the PI3K delta subunit, which was specifically involved in the control of type I IFN production. Although uptake and endosomal trafficking of TLR ligands were not affected in the presence of PI3K inhibitors, there was a dramatic defect in the nuclear translocation of IFN regulatory factor (IRF) 7, whereas nuclear factor kappaB activation was preserved. Thus, PI3K selectively controls type I IFN production by regulating IRF-7 nuclear translocation in human pDCs and could serve as a novel target to inhibit pathogenic type I IFN in autoimmune diseases.
Figures
Figure 1.
TLR triggering activates the PI3K pathway in human pDCs. Purified pDCs were cultured with (A) 1 μM CpG-C or (B) Flu (multiplicity of infection [MOI] = 2) for 20 or 90 min with or without the PI3K inhibitor LY at 1 μM. Cells were stained with anti–p-AKT, as specified in Materials and methods. Representative histograms of at least three separate experiments are shown.
Figure 2.
P13K inhibition selectively inhibits TLR7- and 9-mediated IFN-α response in human pDCs. (A) Purified pDCs were cultured with 1 μM CpG-C, HSV (MOI = 5), or Flu (MOI = 1) either alone or in combination with various concentration of the PI3K inhibitor LY (in micromolars) for 16 h. IFN-α production was evaluated by ELISA. Means of one experiment with 3 independent donors (representative of >15 donors) are shown. (B) Purified pDCs were cultured with 1 μM CpG-C alone or in the presence of 2 μM of LY inhibitor for 2 and 5 h. The expression levels of IFN-α, -ω, and -β were measured by real-time PCR. The mean of three independent donors is shown. (C) Purified pDCs were cultured with CpG-C or Flu, either alone or in the presence of LY inhibitor. The supernatants were collected after 5 h, after which the cells were washed twice and restimulated with CpG-C or Flu for another 12 h. IFN-α production was evaluated by ELISA. The mean of three independent donors is shown. Data were analyzed using a two-tailed Student's t test. *, P < 0.05.
Figure 3.
P13K inhibition does not affect inflammatory cytokines or maturation of pDCs in response to TLR7/9 triggering. Purified pDCs were cultured with (A) 1 μM CpG-C immunostimulatory sequence (ISS) or HSV (MOI = 5) or (B) Flu (MOI = 1) either alone or in combination with various concentrations of the PI3K inhibitor LY (in micromolars) for 16 h. IL-6 and TNF-α production were evaluated by ELISA. Means of one experiment with 3 independent donors (representative of >15 donors) are shown. (C) Purified pDCs were cultured with 1 μM CpG-C alone or in the presence of 2 μM of LY inhibitor for 2 and 5 h. The expression levels of IL-6 and TNF-α were measured by real-time PCR. The mean of three independent donors is shown. (D) Cells were stimulated as indicated and characterized for CD80 and CD86 expression by flow cytometry analysis. Data shown are representative of at least 10 donors (see Fig. S4).
Figure 4.
PI3K δ is essential for IFN-α production by pDCs in response to TLR stimulation. (A) Expression of class I A p110 (α, β, and δ) and class I B (γ) PI3K subunits in human pDCs. Purified pDCs were cultured for 6 h as indicated, and RNA was extracted and analyzed by quantitative PCR. Expression levels are expressed after normalization to β-actin. Data are shown as the mean with standard deviation from three independent donors. (B) Purified pDCs were cultured with 1 μM CpG-C, either alone or in combination with various concentrations of the PI3K inhibitors p110 γ AS 604850, p110 δ IC 87114, or LY for 16 h. IFN-α production was measured by ELISA. The mean of three independent donors is shown.
Figure 5.
PI3K is critical for the nuclear translocation of IRF-7 in pDCs but does not block IRF-7 up-regulation. (A) 105 purified pDCs were cultured with or without 1 μM CpG-C ISS alone or in the presence of 5 μM of LY inhibitor. IRF-7 expression was evaluated 2 and 5 h after stimulation by real-time PCR. The mean of three independent donors is shown. (B) 2 × 105 purified pDCs were left untreated or stimulated with CpG alone or in the presence of LY inhibitor for 3 h. Cells were visualized using the membrane staining of class II molecule (FITC), whereas the nucleus was identified using DAPI. IRF-7 nuclear translocation was visualized by immunofluorescence with IRF-7 antibody (Alexa Fluor 555, red). Representative cells of at least four independent donors are shown. Bar, 5 μm. (C) Between 50 and 70 cells from at least four different donors were analyzed for IRF-7 translocation in the nuclei. Cells were considered positive when at least 20% of the IRF-7 fluorescence was localized in the nucleus. (D, left) Purified pDCs were cultured with 1 μM CpG-C ISS with or without 1 or 5 μM of the PI3K inhibitor LY, or 0.5 μM of NF-κB inhibitor for 90 min (representative histograms are shown). (right) Expression intensity values as mean fluorescent intensity (MFI). The mean of three experiments is shown. *, P < 0.05. (E) Purified pDCs were cultured with 1 μM CpG-C ISS with or without 5 μM of the PI3K inhibitor LY for 4 h. Nuclear extracts were analyzed for the binding activity of NF-κB p50 and p65 family members. Data are shown as the fold of increase to unstimulated (mean ± SEM) of three separate experiments.
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