Negative regulation of Toll-like-receptor signaling by IRF-4

Abstract

The recognition of microbial components by Toll-like receptors (TLRs) is an event central to the activation of innate and adaptive immune systems. TLR activation triggers the induction of downstream target genes, wherein the TLR-interacting adaptor molecule MyD88 recruits various signaling molecules and transcription factors. Two members of the IFN regulatory factor (IRF) family of transcription factors, IRF-5 and IRF-7, interact with MyD88 and induce proinflammatory cytokines and type I IFNs, respectively. Here, we show that IRF-4 also interacts with MyD88 and acts as a negative regulator of TLR signaling. IRF-4 mRNA is induced by TLR activation, and IRF-4 competes with IRF-5, but not with IRF-7, for MyD88 interaction. The TLR-dependent induction of proinflammatory cytokines is markedly enhanced in peritoneal macrophages from mice deficient in the Irf4 gene, whereas the induction is inhibited by the ectopic expression of IRF-4 in a macrophage cell line. The critical function of IRF-4 in TLR signaling in vivo is underscored by the observation that Irf4-deficient mice show hypersensitivity to DNA-induced shock, with elevated serum proinflammatory cytokine levels. This study may provide an insight into the complex regulatory mechanisms of MyD88 signaling by IRFs.

Fig. 1.

Interaction of IRF-4 with MyD88. (a) Confocal images of HEK293T cells transiently expressing YFP-IRF-4 or YFP-IRF-8 with CFP-MyD88. Arrows indicate the colocalization of IRF-4 with MyD88. (b and c) The analysis of inter-molecular FRET between CFP-MyD88 and YFP-IRFs was performed by using HEK293T cells. FRET^c was calculated and demonstrated by using pseudocolor images (b) or FRET^c/CFP values (c). (d) Cell lysates prepared from HEK293T cells transiently transfected with a combination of FLAG-MyD88 and HA-IRFs were immunoprecipitated (IP) with the anti-FLAG antibody and subjected to immunoblot (IB) analysis by using the anti-HA or anti-FLAG antibody, as indicated. WCL, whole-cell extracts.

Fig. 2.

Competition of IRF-4 with IRF-5 for MyD88 interaction. (a) Schematic diagram of MyD88 truncated mutants. +, positive interaction with IRFs; -,no interaction, assessed by immunoprecipitation assay. (b) Each FLAG-MyD88 mutant was coexpressed with HA-IRF-4 (Left) or HA-IRF-5 (Right) in HEK293T cells and subjected to coimmunoprecipitation analysis. (c) HEK293T cells were transfected with fixed amounts of the HA-IRF-5 (1.0 μg) or HA-IRF-7 (2.0 μg) expression vector and FLAG-MyD88 expression vector (1 μg) and increasing amounts of the HA-IRF-4 or HA-IRF-3 expression vector (0, 0.1, 0.2, 0.5, or 1.0 μg). Cell lysates were immunoprecipitated (IP) with the anti-FLAG antibody and subjected to immunoblot (IB) analysis by using the anti-HA or anti-FLAG antibody, as indicated.

Fig. 3.

Negative regulation of the MyD88-dependent IRF-5 activation by IRF-4. (a) Schematic diagram of IRF-4-truncated mutants. DBD, DNA-binding domain; AD, activation domain; RD, regulatory domain (17, 20). (b) Confocal images of HEK293T cells transiently expressing YFP-IRF-4, YFP-IRF-4ΔDBD, or YFP-IRF-4ΔRD. (c) HEK293T cells were transfected transiently with the indicated combination of FLAG-tagged MyD88 and HA-tagged full-length IRF-4 or deletion mutants of IRF-4 and subjected to an immunoprecipitation assay. (d) The effect of IRF-4 expression on MyD88-TRAF6-dependent IRF-5 activation. HEK293T cells were transiently cotransfected with p55C1B-Luc and the expression vectors for the indicated combinations of MyD88 (25 ng), TRAF6 (25 ng), IRF-5 (25 ng), and full-length IRF-4 or mutants of IRF-4 (0, 1, 5, or 15 ng). Luciferase activity was measured 24 h after transfection. (e) The effect of IRF-4 expression on MyD88-dependent IRF-7 activation. HEK293T cells were cotransfected transiently with p125-Luc and the expression vectors for the indicated combinations of MyD88 (25 ng), IRF-7 (25 ng), and full-length IRF-4 (0, 5, or 15 ng). Luciferase activity was measured 24 h after transfection. (f) The effect of IRF-4 on the nuclear translocation of IRF-5 induced by ODN1668 stimulation. RAW264.7 cells expressing YFP-IRF-5 alone or both YFP-IRF-5 and RFP-IRF-4 were placed on a time-lapse microscope, and images were obtained at 1-min intervals. Cells were stimulated or left unstimulated with 1 μM ODN1668 and incubated for up to 60 min in the presence of 10 ng/ml leptomycin B. The normalized YFP intensities of nuclear regions were plotted with time.

Fig. 4.

Hyperresponsivenass to TLR stimuli in Irf-4^-/- peritoneal macrophages. (a) Resident peritoneal macrophages from wild-type mice were stimulated with ODN1668, LPS, or poly(U) for indicated periods. Total RNA was prepared and analyzed for IRF-4 mRNA expression by quantitative real-time RT-PCR. (b) Resident peritoneal macrophages derived from wild-type or Irf4^-/- mice were stimulated with the indicated TLR ligands in the presence of IFN-γ for 24 h. The concentrations of IL-12p40, IL-6, and TNF-α in culture supernatants were measured by ELISA. Results shown are the means (±SD) of triplicate determinations. (c) Splenic pDCs prepared from wild-type and Irf4^-/- mice were stimulated with ODN-D19 or poly(U) for 24 h. IFN-α concentration was measured by ELISA. (d) Induction of mRNAs of proinflammatory cytokines, chemokines, and NF-κB-inducible genes. Resident peritoneal macrophages from wild-type, Irf4^-/-, or Irf5^-/- mice were stimulated with ODN1668 for indicated periods. Total RNA was prepared and subjected to quantitative real-time RT-PCR analysis of indicated genes.

Fig. 5.

Cell-type-specific contribution of IRF-4 and IRF-5 system. (a) BMMs from wild-type, Irf5^-/-,or Irf4^-/- mice were stimulated with indicated stimuli in the presence of IFN-γ. The concentrations of IL-12p40, IL-6, and TNF-α in culture supernatants were measured by ELISA. (b) RAW264.7 cells were transiently transfected with the control, full-length IRF-4, or IRF-4ΔDBD expression vector by electroporation. After 12 h, cells were stimulated with ODN1668 for indicated periods. Total RNA was prepared and subjected to quantitative real-time RT-PCR analysis.

Fig. 6.

Role of IRF-4 in vivo. Age-matched wild-type (n = 7) and Irf4^-/- (n = 4) mice were i.p. injected with ODN1668 (10 nmol) and d-GalN (20 mg). (a) The concentrations of serum IL-6, IL-12p40, and TNF-α were measured by ELISA. Results shown are the means (±SD) of serum samples. (b) The survival of these mice was monitored for 15 h.