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Integral role of IRF-5 in the gene induction programme activated by Toll-like receptors

Abstract

The activation of Toll-like receptors (TLRs) is central to innate and adaptive immunity1,2,3. All TLRs use the adaptor MyD88 for signalling4, but the mechanisms underlying the MyD88-mediated gene induction programme are as yet not fully understood. Here, we demonstrate that the transcription factor IRF-5 is generally involved downstream of the TLR–MyD88 signalling pathway for gene induction of proinflammatory cytokines, such as interleukin-6 (IL-6), IL-12 and tumour-necrosis factor-α. In haematopoietic cells from mice deficient in the Irf5 gene (Irf5-/- mice), the induction of these cytokines by various TLR ligands is severely impaired, whereas interferon-α induction is normal. We also provide evidence that IRF-5 interacts with and is activated by MyD88 and TRAF6, and that TLR activation results in the nuclear translocation of IRF-5 to activate cytokine gene transcription. Consistently, Irf5-/- mice show resistance to lethal shock induced by either unmethylated DNA or lipopolysaccharide, which correlates with a marked decrease in the serum levels of proinflammatory cytokines. Thus, our study identifies IRF-5 as a new, principal downstream regulator of the TLR–MyD88 signalling pathway and a potential target of therapeutic intervention to control harmful immune responses.

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Figure 1: Generation of Irf5-/- mice.
Figure 2: Impaired induction of proinflammatory cytokines and their mRNAs in haematopoietic cells from Irf5-/- mice in response to TLR ligands.
Figure 3: Interaction of IRF-5 with the MyD88 and TRAF6 adaptors for TLR signalling.
Figure 4: IRF-5 activation and binding to IL-12p40 promoter upon TLR stimulation.
Figure 5: Resistance of Irf5-/- mice to CpG-B ODN- or LPS-induced lethal shock.

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Acknowledgements

We thank J. Vilcek, R. Flavell, H. Rosen and N. Fujiwara for advice, and M. Shishido for technical assistance. This work was supported in part by a grant for Advanced Research on Cancer and a Grant-In-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Uehara Memorial Foundation, Mitsukoshi Foundation, the Princess Takamatsu Cancer Research Fund and Canadian Institute of Health Research. H.Y. is a research fellow of the Japan Society for the Promotion of Science. H.N. and S.K. were supported by an Ishidu Shun Memorial Scholarship.

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Correspondence to Tak W. Mak or Tadatsugu Taniguchi.

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Supplementary information

Supplementary Figures 1a,b,c

a, Tissue distribution of IRF-5 mRNA expression. b, Upregulation of IRF-5 mRNA expression by stimulation of the TLR9 ligand CpG-B oligodeoxynucleotide (ODN) in conventional dendritic cells (cDCs) (left) or by LPS and poly(I:C) in splenic macrophages (right). c, Hematopoietic cell population in IRF-5-/- mice. (PDF 592 kb)

Supplementary Figure 2 and Figure 3a and b

Supplementary Figure 2: Induction of IFN-α and IFN-β mRNAs by poly(I:C) in splenic cells. Supplementary Figure 3a and b: a, Induction of IL-6 in splenic B cells by CpG ODN stimulation. b, Induction of IL-6 mRNA in splenic B cells by CpG ODNs. (PDF 660 kb)

Supplementary Figures 4a, b and c

a, Expression of TLR mRNAs in splenic macrophages. b, Splenic B cells from WT or IRF-5-/- mice were treated with the indicated amounts of CpG-B in RPMI, 10%FCS. (left) Splenic B cells from WT or IRF-5-/- mice were treated with 1.0 M of CpG-B in RPMI, 10%FCS. (right). c, WT and IRF-5-/- splenic B cells were treated for 12 h with 1.0 μM CpG-A or CpG-B, or left untreated (Medium). (left). Splenocytes from WT and IRF-5-/- mice were cultured for 12 h with 1.0 μM CpG-B or left untreated (Medium) (right). (PDF 718 kb)

Supplementary Figures 4d and Figure 5

Supplementary Figure 4d: d, Macrophage cell viability after TLR simulation or X-ray irradiation. Supplementary Figure 5: Potential ISREs within the murine IL-6, IL-12p40, TNF-α and IκBζ genes. (PDF 1033 kb)

Supplementary Figures 6a and b

Nuclear translocation of IRF-5 by LPS or CpG-B ODN stimulation. (PDF 447 kb)

Supplementary Legends

Legends to Supplementary Figures 1-6 (DOC 67 kb)

Supplementary Methods and references (DOC 73 kb)

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Takaoka, A., Yanai, H., Kondo, S. et al. Integral role of IRF-5 in the gene induction programme activated by Toll-like receptors. Nature 434, 243–249 (2005). https://doi.org/10.1038/nature03308

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