at the post-transcriptional levels, exerting an overall inhibitory effect on inflammatory response. On the other hand, through interaction with IRAK-2, IRAK-M inhibited TLR7-mediated production of cytokines and chemokines at translational levels. Taken together, IRAK-M mediates TLR7-induced MEKK3-dependent second wave NFjB activation to produce inhibitory molecules as a negative feedback for the pathway, while exerting inhibitory effect on translational control of cytokines and chemokines. The EMBO Journal 32, 583596. doi:10.1038/ emboj.2013.2; Published online 1 February 2013 Subject Categories: signal transduction; immunology Keywords: IRAK; myddosome; signalling; Toll-like receptor Corresponding author. Department PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19827996 of Immunology, Cleveland Clinic Foundation, Cleveland, OH 44195, USA. Tel.: 1 216 445 8706; Fax: 1 216 444 9329; E-mail: [email protected] 8 These authors contributed equally to this work. Received: 2 July 2012; accepted: 18 December 2012; published online: 1 February 2013 & 2013 European Molecular Biology Organization IRAK-M mediates TLR/IL-1R-induced NFjB activation and cytokine production H Zhou et al . The ratios of LPS-induced cytokine and chemokine mRNAs in translationactive versus translation-inactive pools were lower in IRAK-2-deficient macrophages compared with wild-type macrophages, indicating the requirement of IRAK-2 for sustained translation of these mRNAs. In this study, we investigated the functional relationships among IRAK family 
members and their roles in TLR signalling by analysing mice deficient in IRAK-1, IRAK-2 and/or IRAK-M. We found that IRAK-1 and IRAK-2, substrates of IRAK-4, are required for TAK1-dependent NFkB activation and mRNA Nigericin (sodium salt) site stabilization of cytokines and chemokines, but not for MEKK3-dependent NFkB activation. In contrast to the direct inhibitory role of IRAK-M in TLR signalling, IRAK-M was able to interact with MyD88IRAK-4 to form IRAK-M Myddosome to mediate TAK1-independent MEKK3-dependent NFkB activation in the absence of IRAK-1 and IRAK-2. This IRAK-M-dependent pathway is required for the second wave of TLR7-induced NFkB activation in the presence of IRAK-1/IRAK-2, which is uncoupled from post-transcriptional regulation. Thus, the IRAK-M-dependent pathway only induced expression of genes that are not regulated at the post-transcriptional levels, exerting an overall inhibitory effect on inflammatory response. Taken together, this study for the first time demonstrates unique positive engagement of IRAK-M in transmitting signalling upon TLR activation, providing important insight into the mechanistic roles of IRAK-M in modulating TLR signalling. Results IRAK-M mediates TLR-induced MEKK3-dependent NFjB activation in the absence of IRAK-1 and IRAK-2 Although IRAK-M has been implicated as a negative regulator in TLR signalling, the precise molecular mechanism for how IRAK-M participates and modulates TLR signalling remains unclear. We recently found that substantial TLR7-induced NFkB activation was retained in IRAK-1/2-double deficient bone marrow-derived macrophages, whereas completely abolished in MyD88- or IRAK-4-deficient BMDMs. These results suggest that MyD88IRAK-4 either themselves or in conjunction with IRAK-M can mediate NFkB activation in IRAK-1/ & 2013 European Molecular Biology Organization IRAK-M mediates TLR/IL-1R-induced NFjB activation and cytokine production H Zhou et al 2-DKO-BMDMs. To determine the role of IRAK-M in this process, we compared TLR7-mediated NFkB activation in IRAK-