Interleukin-1 receptor-associated kinase-1 plays an essential role for Toll-like receptor (TLR)7- and TLR9-mediated interferon-{alpha} induction

Abstract

Toll-like receptors (TLRs) recognize microbial pathogens and trigger innate immune responses. Among TLR family members, TLR7, TLR8, and TLR9 induce interferon (IFN)-alpha in plasmacytoid dendritic cells (pDCs). This induction requires the formation of a complex consisting of the adaptor MyD88, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and IFN regulatory factor (IRF) 7. Here we show an essential role of IL-1 receptor-associated kinase (IRAK)-1 in TLR7- and TLR9-mediated IRF7 signaling pathway. IRAK-1 directly bound and phosphorylated IRF7 in vitro. The kinase activity of IRAK-1 was necessary for transcriptional activation of IRF7. TLR7- and TLR9-mediated IFN-alpha production was abolished in Irak-1-deficient mice, whereas inflammatory cytokine production was not impaired. Despite normal activation of NF-kappaB and mitogen-activated protein kinases, IRF7 was not activated by a TLR9 ligand in Irak-1-deficient pDCs. These results indicated that IRAK-1 is a specific regulator for TLR7- and TLR9-mediated IFN-alpha induction in pDCs.

Figure 1.

IRAK-1 but not IRAK-4 associates with IRF7. (A) HEK293 cells were transiently transfected with FLAG–IRF7 together with Myc–IRAK–1 or Myc-IRAK-4. Cell lysates were immunoprecipitated (IP) with an anti-Myc or anti-FLAG, followed by immunoblot (IB) analysis using anti-FLAG or anti-Myc, as indicated. Slowly migrated forms of FLAG–IRF7 were shown by an asterisk. (B, top) HEK293 cells were transfected with IRF7-YFP (yellow) and IRAK-1–CFP or IRAK-4–CFP (blue) and physical interaction of these two molecules determined by FRET (pseudocolor) was visualized. (B, bottom) HEK293 cells were transfected with IRF7-YFP, IRAK-1-CFP, or IRAK-4-CFP. (Left) Fluorescence intensity of CFP emission by CFP excitation of single cells (horizontal axis) and YFP emission by CFP excitation of single cells (vertical axis). Cells that are positive for both YFP and CFP by CFP excitation are shown in gated area as FRET. (Right) Calculated FRET of IRAK-1–CFP or IRAK-4–CFP and IRF7–YFP. (C) Cell lysates prepared from HEK293 cells transiently transfected with a combination of FLAG–IRF7 deletion mutants and Myc–IRAK-1 were immunoprecipitated with an anti-Myc or anti-FLAG, followed by immunoblot analysis using anti-FLAG or anti-Myc, as indicated.

Figure 2.

IRF7 activation by IRAK-1. (A) Cell lysates prepared from HEK293 cells transiently transfected with FLAG–IRF7 together with Myc–IRAK1 or Myc–IRAK1 KN were immunoprecipitated with anti-Myc or anti-FLAG, followed by immunoblot analysis using anti-FLAG or anti-Myc, as indicated. (B) HEK293 cells were transiently transfected with FLAG–IRAK-1, FLAG–IRAK-1 KN, FLAG–IRAK-4, or FLAG–IRAK-4 KN. Cell lysates were immunoprecipitated with anti-FLAG and subjected to in vitro kinase reaction in the presence of GST–IRF7. Proteins were separated on SDS-PAGE, followed by visualized by autoradiography. (C) HEK293 cells were transiently transfected with a combination of IRF7, MyD88, and 1, 10, or 50 ng of a KN mutant of IRAK-1 (IRAK-1 KN) along with a reporter plasmid carrying an IFN-α4 promoter (left). HEK293 cells were also transfected with a combination MyD88 and 1, 10, or 50 ng of IRAK-1 KN along with a reporter plasmid carrying an ELAM promoter (right). 36 h after transfection, cells were analyzed for IFN-α4– or ELAM-dependent promoter activities by a reporter gene assay.

Figure 3.

Impaired IFN-α induction by D35 in Irak-1 ^-/Y mice. (A) Flt3L–BMDCs from Irak-1 ^{+ /Y} and Irak-1 ^{− /Y} mice were stimulated with the indicated concentration of D35 for 24 h. Concentration of IFN-α, TNF-α, IL-6, and IL-12p40 in the culture supernatants were measured by ELISA. Data are shown as the mean ± SD. (B) Flt3L–BMDCs from Irak-1^+/Y and Irak-1^{− /Y} mice were stimulated with 1 μM D35 for the indicated periods. Total RNA was extracted and subjected to Northern blot analysis. (C) Intracellular IFN-α and IL-12 staining of Flt3L–BMDCs derived from Irak-1^+/Y and Irak-1^{− /Y} mice stimulated with 3 μM CpG DNA (D35). CD11c⁺B220⁺ population of Flt3L–BMDCs was analyzed as pDC, respectively.

Figure 4.

IFN-α induction by other TLR ligands in Irak-1 ^−/Y mice. (A) Flt3L–BMDCs from Irak-1 ^{+ /Y} and Irak-1 ^{− /Y} mice were stimulated with the indicated concentration of ODN1668 for 24 h. Concentration of IFN-α, TNF-α, IL-6, and IL-12p40 in the culture supernatants was measured by ELISA. Data are shown as the mean ± SD. (B) Irak-1 ^{+ /Y} and Irak-1 ^{− /Y} mice (n = 3) were intravenously injected with 50 nmol of R-848. Samples of sera were taken and the concentrations of IFN-α and IL-12p40 were determined by ELISA. (C) Flt3L–BMDCs from Irak-1 ^{+ /Y} and Irak-1 ^{− /Y} mice were transfected with 10 μg/ml poly(I/C) for 24 h. Concentration of IFN-α in the culture supernatants was measured by ELISA. Data are shown as the mean ± SD. N.D., not detected.

Figure 5.

Impaired nuclear translocation of IRF7 in response to D35 in Irak-1 ^−/Y cells. (A) Flt3L–BMDCs from Irak-1 ^{+ /Y} and Irak-1 ^{− /Y} mice were stimulated with D35 for 1, 2, or 6 h. Nuclear proteins were prepared and subject to immunoblot analysis using anti-IRF7 and anti-RelA. An asterisk shows IRF7 protein. (B) Flt3L–BMDCs from Irak-1 ^{+ /Y} and Irak-1 ^{− /Y} mice were stimulated with D35 for 10, 30, or 60 min. Whole cell lysates were subject to immunoblot analysis using antiphospho-specific ERK1 and anti-ERK1. (C) Schematic illustration of TLR7- and TLR9-mediated signaling pathway. IRF7 interacts with MyD88 to form a complex, which provides the foundation for the induction of the IFN-α. IRAK-1 binds to IRF7 directly and is included in this complex. IRAK-1 is dispensable for the NF-κB activation but specifically regulates the IFN-α induction through the activation of IRF7. IRAK-4 may locate upstream of IRAK-1 and be involved in the activation of IRAK-1.