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TLR4, but not TLR2, mediates IFN-β–induced STAT1α/β-dependent gene expression in macrophages

Nature Immunology volume 3, pages 392–398 (2002)Cite this article

Abstract

Toll-like receptor 2 (TLR2) agonists induce a subset of TLR4-inducible proinflammatory genes, which suggests the use of differential signaling pathways. Murine macrophages stimulated with the TLR4 agonist Escherichia coli lipopolysaccharide (LPS), but not with TLR2 agonists, induced phosphorylation of signal transducer and activator of transcription 1α (STAT1α) and STAT1β, which was blocked by antibodies to interferon β (IFN-β) but not IFN-α. All TLR2 agonists poorly induced IFN-β, which is encoded by an immediate early LPS-inducible gene. Thus, the failure of TLR2 agonists to induce STAT1-dependent genes resulted, in part, from their inability to express IFN-β. TLR4-induced IFN-β mRNA was MyD88- and PKR (double-stranded RNA–dependent protein kinase)-independent, but TIRAP (Toll–interleukin 1 receptor domain–containing adapter protein)-dependent. Together, these findings provide the first mechanistic basis for differential patterns of gene expression activated by TLR4 and TLR2 agonists.

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Figure 1: Differential gene induction by TLR4 versus TLR2 agonists.
Figure 2: Induction of MAPK phosphorylation by E. coli LPS versus Pam3Cys.
Figure 3: TLR4, but not TLR2, agonists induce phosphorylation of STAT1α/β.
Figure 4: Monoclonal anti–IFN-β, but not monoclonal anti–IFN-α, inhibits LPS-induced STATα/β phosphorylation.
Figure 5: Differential expression of IFN-β mRNA induced by TLR2 versus TLR4 agonists in murine macrophages.
Figure 6: Exogenous murine rIFN-β restores the inducibility of expression of MCP-5, IP-10 and iNOS mRNA in Pam3Cys-treated macrophages.
Figure 7: LPS-induced STAT1α/β phosphorylation and IFN-β mRNA expression are MyD88-independent.
Figure 8: TLR4-mediated signaling for Ifnb expression is TIRAP-dependent, but PKR-independent.

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Acknowledgements

Supported by NIH grants AI-18797 (to S. V.), AI-34039 (to B. W.), CA-62220 (to T. H.) and AI-47233 (to M. F.).

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Author notes

  1. Vladimir Toshchakov and Bryan W. Jones: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, 20814, MD, USA

    Vladimir Toshchakov, Pin-Yu Perera, Karen Thomas, M. Joshua Cody, Shuling Zhang & Stefanie N. Vogel

  2. The Pulmonary Center, Boston University School of Medicine, Boston, 02118, MA, USA

    Bryan W. Jones & Matthew J. Fenton

  3. Department of Immunology, The Cleveland Clinic Research Foundation, Cleveland, 44195, OH, USA

    Bryan R. G. Williams, Jennifer Major & Thomas A. Hamilton

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Supplementary information

Web Figure 1. Kinetics of differential gene induction by TLR4 versus TLR2 agonists.

Using concentrations of E. coli K235 LPS, Pam3Cys and P. gingivalis LPS found to be optimal for induction of TNFα or IL-1β gene expression (see Figs. 1 & 2), a time course analysis of gene expression was performed. Total RNA was isolated and analyzed for the expression of individual genes by RT-PCR and Southern blotting (see Methods). Data are representative of seven separate experiments. (GIF 134 kb)

Web Figure 2. LPS-induced STAT1α/β phosphorylation is TLR4-dependent and requires de novo protein synthesis.

(a) Primary C3H/OuJ and C3H/HeJ macrophages were treated with E. coli LPS and the phosphorylation of STAT1α/β measured by immunoblotting analysis 2 h after stimulation. Total Erk1 and Erk2 proteins were also detected as a control for protein loading. (b) Primary C3H/OuJ macrophages were stimulated with E. coli LPS in the absence or presence of the protein synthesis inhibitor, CHX and phosphorylation of STAT1α/β measured by immunoblotting analysis 2 h after stimulation. CHX was added 5 min before the addition of LPS. (GIF 111 kb)

Web Figure 3. IFN-β promoter luciferase reporter activation by E. coli LPS versus araLAM in transfected RAW 264.7 macrophages.

RAW 264.7 cells were transiently transfected with the human IFN-β promoter luciferase reporter construct and stimulated with the TLR4 agonist, E. coli LPS, or the TLR2 agonist, araLAM. Luciferase activity was expressed as "fold-increase" over the background exhibited by lysates prepared from untransfected cells. Data are representative of three separate experiments. (GIF 5 kb)

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Toshchakov, V., Jones, B., Perera, PY. et al. TLR4, but not TLR2, mediates IFN-β–induced STAT1α/β-dependent gene expression in macrophages. Nat Immunol 3, 392–398 (2002). https://doi.org/10.1038/ni774

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