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* School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland;
Centre for Veterinary Science, Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom; and
Department of Infectious Diseases and Immunology, University of Massachusetts; Medical School, Worcester, MA 01605
Although a clear role for the adaptor protein myeloid differentiation factor-88 (MyD88) adaptor-like (Mal, or TIRAP) in TLR4 signaling has been demonstrated, there is limited information on its role in TLR2 signaling. Here we have systematically analyzed the role of Mal in signaling by TLR2, TLR4, and as a control TLR3 in murine macrophages and dendritic cells. Mal was not required for the induction of IL-6 or NF
B activation at high concentrations of the TLR1/2 ligand Pam3Cys-Ser-(Lys)4 or the TLR2/6 ligand macrophage-activating lipopeptide-2 and was required for these responses only at low ligand concentrations. Similarly, induction of IL-6 by Salmonella typhimurium, which is sensed by TLR2, required Mal only at low levels of bacteria. Mal was required for IL-6 induction at all concentrations of the TLR4 ligand LPS. Mal deficiency boosted IL-6 induction by the TLR3 ligand polyinosinic-polycytidylic acid. Activation of JNK, but not p38 or IB degradation, was similarly potentiated in response to polyinosinic-polycytidylic acid in Mal-deficient macrophages. MyD88 was vital for all TLR2 and TLR4 responses and, similar to Mal, was also inhibitory for TLR3-dependent IL-6 and JNK induction. MyD88 interacted with the Toll/IL-1R domains of TLR1, TLR2, TLR4, and TLR6. Mal interacted with the Toll/Il-1R domains of TLR1, TLR2, and TLR4 but not with TLR6. Our study, therefore, reveals that Mal is dispensable in TLR2 signaling at high ligand concentrations in macrophages and dendritic cells, with MyD88 probably coupling to the TLR2 receptor complex at sufficient levels to allow activation. An inhibitory role for Mal in TLR3 signaling to JNK was also demonstrated.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Irish Research Council for Science, Engineering and Technology, Science Foundation Ireland and by grants (to C.E.B.) from the Biotechnology and Biological Sciences Research Council and the Royal Society.
2 Address correspondence and reprint requests to Prof. Luke A. J. O'Neill, School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland. E-mail address: laoneill{at}tcd.ie
3 Abbreviations used in this paper: TIR, Toll/IL-1R; BMDC, bone marrow-derived dendritic cell; BMDM, bone marrow-derived macrophage; HEK-293T, human embryonic kidney T cell; IRAK, IL-1R-associated kinase; Mal, MyD88 adaptor-like; MyD88, myeloid differentiation factor-88; TRIF, Toll/Il-1R domain-containing adaptor-inducing IFN-β; TRAM, TRIF-related adaptor molecule; IRF3, IFN-regulatory factor 3; Pam3CSK4, Pam3Cys-Ser-(Lys)4; Malp-2, macrophage-activating lipopeptide-2; MOI, multiplicity of infection; HA, hemagglutinin; poly(I:C), polyinosinic-polycytidylic acid.
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