A type I interferon autocrine-paracrine loop is involved in Toll-like receptor-induced interleukin-12p70 secretion by dendritic cells

Abstract

Dendritic cells (DC) produce interleukin-12 (IL-12) in response to Toll-like receptor (TLR) activation. Two major TLR signaling pathways participate in the response to pathogens: the nuclear factor-kappaB (NF-kappaB)-dependent pathway leading to inflammatory cytokine secretion including IL-12 and the interferon (IFN)-dependent pathway inducing type I IFN and IFN-regulated genes. Here we show that the two pathways cooperate and are likely both necessary for inducing an optimal response to pathogens. R-848/Resiquimod (TLR7 ligand in the mouse and TLR7/8 ligand in human) synergized with poly(I:C) (TLR3 ligand) or lipopolysaccharide (LPS; TLR4 ligand) in inducing high levels of bioactive IL-12p70 secretion and IFN-beta mRNA accumulation by mouse bone marrow-derived DC (BM-DC). Strikingly, IL-12p70 but not IL-12p40 secretion was strongly reduced in BM-DC from STAT1(-/-) and IFNAR(-/-) mice. STAT1 tyrosine-phosphorylation, IL-12p35, and IFN-beta mRNA accumulation were strongly inhibited in IFNAR(-/-) BM-DC activated with the TLR ligand combinations. Similar observation were obtained in human TLR8-expressing monocyte-derived DC (moDC) using neutralizing anti-IFNAR2 antibodies, although results also pointed to a possible involvement of IFN-lambda1 (also known as IL-29). This suggests that TLR engagement on DC induces endogenous IFNs that further synergize with the NF-kappaB pathway for optimal IL-12p70 secretion. Moreover, analysis of interferon regulatory factors (IRF) regulation in moDC suggests a role for IRF7/8 in mediating IRF3-independent type I IFN and possibly IL-12p35 synthesis in response to TLR7/8.

Figure 1.

R-848 synergizes with LPS or poly(I:C) in inducing IL-12p70 secretion by mouse BM-DCs without IFN-γ priming. BM-DCs from wild-type mice (129Sv) were activated with LPS (25 ng/ml), R-848 (10 μM), or poly(I:C) (25 μg/ml) alone or in combination, or with LPS plus IFN-γ (20 ng/ml) in the presence of 10 μg/ml anti-IL10 receptor mAb (anti–IL-10R) or rat IgG1 control mAb. After 24 h, supernatants were collected and (A) IL-12p40 and (B) IL-12p70 were measured using specific ELISA. Data are expressed as mean ± SD of culture triplicates and are representative of four experiments.

Figure 2.

STAT1 is required for optimal IL-12p70 production by BM-DCs. (A) Similar phenotype of BM-DCs derived from wild-type (WT), STAT1^−/−, and IFNAR^−/− mice. Expression of CD8α, CD11b, CD11c, and CD86 was analyzed by flow cytometry on BM-DCs derived from the indicated mice. (B–E) BM-DCs from wild-type or STAT1^−/− mice were activated for 24 h with different TLR ligands and their combinations in the presence of anti–IL-10R mAb as described in Fig. 1. Concentrations of (B) IL-6 , (C) CXCL10, (D) IL-12p70, and (E) IL-12p40 were quantified in supernatants by specific ELISA. Data are expressed as mean ± SD of culture triplicates and are representative of three experiments. Statistical significance is indicated (**P < 0.01).

Figure 3.

Type I IFNs are required for optimal IL-12p70 production by mouse BM-DCs. (A and C) BM-DCs from wild-type (wt) or IFNAR^−/− mice were activated with the TLR ligands indicated and their combinations in the presence of anti–IL-10R mAb as described in Fig. 1. (B) BM-DCs from wild-type, STAT1^−/−, or IFNAR^−/− mice were activated with the combination R-848 plus LPS and with or without 20 ng/ml IFN-γ. Supernatants were collected after 24 h and (A and B) IL-12p70 and (C) IL-12p40 were measured using specific ELISA. Data are expressed as mean ± SD of culture triplicates and are representative of three experiments. Statistical significance comparing results from each deficient mice to wild-type mice is indicated (*P < 0.05; **P < 0.01).

Figure 4.

Tyrosine phosphorylation of STAT1 by TLR ligands and their combinations requires a type I IFN pathway. BM-DCs from wild-type or IFNAR1^−/− mice were activated with IFN-α (1,000 U/ml), IFN-γ (20 ng/ml), or different TLR ligands at concentration described in Fig. 1. After 2 h, stimulation was stopped with cold PBS and BM-DCs were resuspended in lysis buffer. After SDS-PAGE and Western blot, membranes were incubated with anti-phosphotyrosine STAT1 (pY-STAT1) or anti-STAT1 antibodies and analyzed as described in Materials and methods. Results are representative of three experiments.

Figure 5.

Induction of IL-12p35, IL-12p40, IFN-β, and IFN-α mRNA in TLR-stimulated wild-type and IFNAR1^−/− BM-DCs. BM-DCs from wild-type or IFNAR1^−/− mice were activated with TLR ligands indicated in the presence of anti-IL10R antibodies as described in Fig. 1. After 4.5 h, stimulations were stopped with cold PBS and mRNA was extracted and reverse transcribed. Real-time PCR was performed using primers specific for mouse (A) IL-12p35, (B) IL-12p40 , (C) IFN-β, or (D) IFN-α (all genes) genes on cDNA normalized with GAPDH. Data are expressed as -fold induction of the gene of interest over GAPDH calculated with the following formula (1.8^{[CT GAPDH − CT gene]}). Percentages of inhibition are indicated. Statistical significance is indicated (*P < 0.05; **P < 0.01). Results are representative of three experiments.

Figure 6.

TLR-induced IL-12p70 secretion by human moDCs is correlated with the capacity of the TLR ligand to induce STAT1 tyrosine phosphorylation. (A and B) moDCs were activated for 24 h with or without optimal concentrations of LPS, R-848, poly(I:C), PGN, or their combinations. (A) IL-12p70 and (B) IL-12p40 were measured in supernatants using specific ELISA. Data are expressed as mean ± SD of six experiments performed with different donors. (C) moDCs were cultured with or without 30 ng/ml IFN-γ, LPS, PGN, poly(I:C), R-848, or DMSO (1/1,000 final dilution for R-848 control). (D) moDCs were activated with or without the TLR ligands as in C for 1 or 4 h (C) or 2 h (D), or a mixture of IFN-α and IFN-β (500 U/ml each) in presence or not of CHX (25 μg/ml) added 15 min before activation. Activations were stopped with cold PBS, moDCs were resuspended in lysis buffer. After SDS-PAGE and Western blot, membranes were incubated with anti-pY-STAT1, anti-STAT1, or anti-IRF1 antibodies and analyzed as described in Materials and methods. Results are representative of three independent experiments.

Figure 7.

Neutralizing type I IFN partially blocks LPS- and R-848–induced IL-12p70 secretion and STAT1 tyrosine phosphorylation in human moDCs. (A, B, and D) moDCs were activated for 24 h with or without LPS or R-848 in the presence or the absence of 30 μg/ml neutralizing anti-IFNAR2 antibodies (CD118) or IgG2a control mAb. Concentrations of (A) IL-12p70, (B) IL-12p40, and (D) CXCL10 were quantified by specific ELISA in supernatants. Statistical significance of results with anti-IFNAR2 mAb compared with control mAb is indicated (*P < 0.05; ** P < 0.01). (C) moDCs were activated with LPS or R-848 with or without exogenous IFN-α, IFN-β, or IFN-γ and IL-12p70 was quantified by ELISA after a 24-h incubation. Data are expressed as mean ± SD of culture triplicates and are representative of three experiments with different donors. Statistical significance is indicated (*P < 0.05; **P < 0.01). (E) moDCs were pretreated for 30 min with or without 30 μg/ml neutralizing anti-IFNAR2 antibodies or IgG2a control mAb and then activated for 2 h with or without LPS, R-848, or 1,000 U/ml IFN-β. Analysis of pY-STAT1 in moDCs lysates was performed by Western blot analysis as described in Fig. 6. Results are representative of three independent experiments.

Figure 8.

Induction of IFN-β, IFN-α, and IFN-λ1 (also know as IL-29) mRNA in TLR-stimulated human moDCs. moDCs were activated for 4 h with or without LPS or R-848. mRNA were extracted and reverse transcribed. Real-time PCR was performed using primers specific for human IFN-β, IFN-α all genes, or IFN-λ1 (also known as IL-29) with cDNA normalized to GAPDH expression. Expression of the gene of interest was quantified by applying the formula 1.8^{(CT GAPDH − CT gene of interest)}. Data are expressed as the -fold induction of the gene of interest in the different activation conditions compared with medium and are representative of four independent experiments.

Figure 9.

(A) LPS and poly(I:C) but not R-848, induce nuclear translocation of phosphorylated IRF3 in moDCs. moDCs were activated with or without the indicated TLR ligands. At the time indicated, stimulations were stopped with cold PBS and nuclei were isolated as described in Materials and methods. After SDS-PAGE of nuclear extracts and Western blot, membranes were incubated with anti-pY-STAT1 or anti-phospho-IRF3 (p-IRF3) antibodies and analyzed as described in Materials and methods. Results are representative of three independent experiments. (B) Expression of IRF7, IRF8, IRF4, and IRF5 mRNA in TLR-stimulated human moDCs. moDCs were activated for 1 or 4 h with or without LPS, poly(I:C) or R-848. mRNA were extracted and reverse transcribed. Real-time PCR was performed using primers specific for the human IRF indicated with cDNA normalized to GAPDH expression. Expression of the gene of interest was quantified by applying the formula 1.8^{(CT GAPDH − CT gene of interest)}. Data are expressed as the -fold induction of the gene of interest in the different activation conditions compared with medium and are representative of three independent experiments.