TLR7 stimulation in human plasmacytoid dendritic cells leads to the induction of early IFN-inducible genes in the absence of type I IFN

Abstract

On recognition of influenza virus (Flu) by TLR7, plasmacytoid dendritic cells (pDCs) produce type I IFN in significant amounts. Synthetic TLR7 ligands induce the maturation of pDCs, as evidenced by the expression of costimulatory molecules and the production of proinflammatory cytokines; however, they induce only low-level production of IFN-alpha. To dissect the TLR7 signaling in pDCs and how these different profiles are induced, we studied the effects of 2 TLR7 ligands (Flu and CL097) on the activation of blood-isolated pDCs and the human GEN2.2 pDC cell line. Type I IFN production by pDCs correlates with differential interferon regulatory factor 7 (IRF7) translocation into the nucleus induced by the 2 TLR7 ligands. Surprisingly, with both activators we nevertheless observed the rapid expression of the IFN-inducible genes mxa, cxcl10, and trail within 4 hours of stimulation. This expression, controlled by STAT1 phosphorylation, was independent of type I IFN. STAT1 activation was found to be strictly dependent on the PI3K-p38MAPK pathway, showing a new signaling pathway leading to rapid expression of IFN-inducible genes after TLR7 triggering. Thus, pDCs, through this unusual TLR7 signaling, have the capacity to promptly respond to viral infection during the early phases of the innate immune response.

Figure 1. Influenza virus and CL097 induce human pDC activation by triggering TLR7

(A) Left panel: Flow cytometry evaluation of purity of unstimulated pDCs enriched from healthy donor blood labeled with anti-BDCA2 and anti-CD123 antibodies. Cells were untreated (medium) or stimulated with UV-formol-inactivated Influenza virus (Flu) or synthetic TLR7 ligand (CL097). Expression of CD40 was measured by flow cytometry on CD123⁺ cells. Dot plots are shown, the percentage of CD40 positive cells is indicated on each plot. Results shown are representative of four independent experiments. (B) GEN2.2 cells were untreated or stimulated with Flu or CL097 for 24 h. CD40 expression was evaluated on forward scatter (FSC)/side scatter (SSC) gated live cells by flow cytometry. Percentages indicated on dot correspond to the proportion of CD40 positive cells. Results shown are representative of at least five independent experiments. (C) Expression levels of TLR7 and TLR9 in the GEN2.2 cell line and in lentiviral shRNA TLR7 transfected GEN2.2 cells (GENshTLR7) measured by real-time PCR. Expression levels are normalized to G6PDH. Data are shown as the mean and standard deviation from duplicate values of two independent experiments. (D) GEN2.2 and GENshTLR7 cells were untreated or stimulated with Flu, CL097 or two different synthetic TLR9 ligands (CpG A and CpG B) for 24 h. Expression of CD40 was measured by flow cytometry. The mean percentages and standard deviation from duplicate values of three independent experiments are shown.

Figure 2. Differential pDC maturation is triggered by different TLR7 ligands

GEN2.2 cells were untreated or stimulated with Flu or CL097 for 24 h. (A) Production of pro-inflammatory cytokines was measured in culture supernatants by ELISA and CBA. The mean and standard deviation from duplicate values of three independent experiments are shown. (B) After 24 h of culture, RNA was extracted and RNA expression levels of type I IFNs: IFN-α1, -α2, -β1 and -ω1 were measured by quantitative PCR. Data shown are normalized to G6PDH and are representative of two independent experiments. (C) PBMCs from healthy donors were cultured in the absence or presence of Flu or CL097 for 3 h. Secretion was blocked by adding brefeldin A for a further 4 h. Intracellular IFN-α production was measured in HLA-DR⁺ BDCA4⁺ pDCs by flow cytometry. Dot plots show the percentage of IFN-α-producing cells among pDCs. Representative results from three different donors are shown. (D) GEN2.2 cells were cultured in the absence or presence of Flu or CL097 for 3 h. Cells were lyzed and proteins extracted. The different NFκB subunits were quantified in nuclear fractions using the TransAM kit. The mean OD and standard deviation from duplicate values of three independent experiments are shown. (E) GEN2.2 cells were cultured in the absence or presence of Flu or CL097 for 3 h. Cells were immunostained for IRF7 and evans blue colored. Immunofluorescence was assessed by microscopy. Representative images from three independent experiments are shown.

Figure 3. TLR7 triggers expression of IFN-inducible genes in a type I IFN-independent manner

GEN2.2 cells were untreated or stimulated with Flu or CL097 for different times. Supernatants were collected for evaluation of (A) IFN-α and (B) CXCL10 production. (C) RNA was extracted for measurement of MxA transcription levels by real-time PCR. (D) TRAIL expression was evaluated by flow cytometry. GEN2.2 cells were untreated or stimulated with Flu or CL097 in the absence or presence of anti-IFN-α/β and anti-IFN-α/βR neutralizing antibodies for 4 h. (E) TRAIL expression was evaluated by flow cytometry and (F) CXCL10 production was measured in culture supernatants by CBA. Data shown are the means and standard deviation from duplicate values from two independent experiments except data in (C) which are representative of two independent experiments.

Figure 4. Expression of IFN-inducible genes is independent of the NFκB pathway and of extracellular factors

GEN2.2 cells were untreated or stimulated with Flu or CL097 in the absence or presence of the NFκB inhibitors BAY and BMS. TRAIL (A) and CD40 (B) expression were evaluated by flow cytometry after 4 h and 24 h of culture, respectively. The mean percentages and standard deviation from duplicate values of four independent experiments are shown. (C) GEN2.2 cells were untreated or stimulated for 2 h. After washing, cells were placed in 24-well plates; untreated cells were added in transwells. After 4 h of culture, TRAIL expression was evaluated by flow cytometry on cells in wells and transwells. Data shown are the means and standard deviation from duplicate values of three independent experiments. (D) GEN2.2 cells were untreated or stimulated for 2 h in the absence or presence of anti-IFN-α/β and anti-IFN-α/βR neutralizing antibodies. After washing, cells were placed in 24-well plates; untreated cells were added in transwells. After 4 h of culture, TRAIL expression was evaluated by flow cytometry on cells in wells and transwells. Data from one representative experiment are shown.

Figure 5. STAT1 is phosphorylated independently of type I IFN after TLR7 triggering with either ligand

GEN2.2 cells were untreated or stimulated with Flu, CL097 or IFN-α for 30 min, 2 h and 3 h. Whole cell protein extracts were prepared. (A) Phospho-STAT1 (pY701) was quantified in the protein extracts by CBA. Data shown are representative of two independent experiments. (B) Western-blot analysis of phospho-STAT1 (pY701) and phospho-STAT2 (pY690) following activation of GEN2.2 cells. Data shown are representative of two independent experiments (C) After 2 h of stimulation, cells were fixed and permeabilised for phospho-STAT1 (pY701) analysis by flow cytometry. Representative dot plots of four independent experiments are shown. (D) Activation of GEN2.2 cells in the absence or presence of anti-IFN-α/β and anti-IFN-α/βR neutralizing antibodies for 2 h. phospho-STAT1 (pY701) was analysed by flow cytometry. Data shown are the means and standard deviation from duplicate values of two independent experiments.

Figure 6. STAT1 phosphorylation and expression of IFN-inducible genes following TLR7 triggering depends on the PI3K-p38MAPK pathway

GEN2.2 cells were untreated or stimulated with Flu, CL097 or IFN-α for 30 min or 3 h. (A) phospho-p38MAPK (pT180/pY182) was quantified on whole-cell lysates by CBA. Data shown are representative of two independent experiments. (B) GEN2.2 cells were untreated or stimulated with Flu, CL097 or IFN-α for 30 min in the absence or presence of the PI3K inhibitor LY. Cells were fixed and permeabilised for phosphop38MAPK (pT180/pY182) analysis by flow cytometry. The mean percentages and standard deviation from duplicate values of two independent experiments are shown. (C) phospho-STAT1 (pY701) and (D) TRAIL expression were analysed by flow cytometry after 2 h or 4 h of stimulation of GEN2.2 cells with Flu, CL097 or IFN-α in the absence or presence of LY and the specific p38MAPK inhibitor SB203580 (SB). The mean percentages and standard deviation from duplicate values of three independent experiments are shown. Blood-isolated pDCs were unstimulated or stimulated with Flu or CL097 for 4 h in the absence or presence of LY and SB. (E) Expression of TRAIL was evaluated by flow cytometry. Dot plots representative of two experiments are shown. The percentage of TRAIL positive cells is indicated on each plot. (F) CXCL10 production was measured in cell culture supernatants by CBA. The mean and standard deviation from duplicate values of two experiments performed with two different donors are shown.