Understanding early toll-like receptor signalling through Myddosome examination

De Nardo D1,2, Balka KR1,2, Cardona Gloria Y3, Rao VR4, Latz E3,5,6 and Masters SL1,2

  1. Inflammation Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria, 3052, Australia.
  2. Department of Medical Biology, The University of Melbourne, Parkville 3010, Australia.
  3. Institute of Innate Immunity, University Hospital, University of Bonn, Sigmund Freud Str. 25, 53127, Bonn, Germany.
  4. Inflammation and Immunology, Pfizer Inc., Cambridge, Massachusetts 02139, USA.
  5. Department of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
  6. German Center for Neurodegenerative Diseases, Bonn 53175, Germany.

Toll-like receptors (TLRs) form part of the host innate immune system, where they act as sensors of microbial and endogenous danger signals. Upon activation, the intracellular Toll/Interleukin-1 receptor (TIR) domains of TLR dimers form a platform for oligomerisation of a multiprotein signalling platform comprising MyD88 and members of the IRAK family, termed the Myddosome. Formation of the Myddosome complex initiates signal transduction pathways leading to the activation of transcription factors and ultimately, the production of inflammatory cytokines. Despite the critical role Myddosome formation plays in initiating TLR-induced signalling, the molecular mechanisms controlling Myddosome function remain poorly defined. Using immunoprecipitation approaches we successfully isolated Myddosome complexes from whole cell lysates of TLR activated primary mouse bone marrow-derived macrophages (BMDMs) and from IRAK-deficient immortalised BMDMs reconstituted with WT and mutant forms of IRAKs via retroviral transduction. Through the use of a selective IRAK4 inhibitor we were able to examine the role of IRAK4 kinase activity within the Myddosome. Immunoblot and immunofluorescence techniques were used to assess TLR signalling and NF-κB translocation respectively, while ELISAs were employed to examine the secretion of pro-inflammatory cytokines into the supernatant. Here we demonstrate the kinetics of the Myddosome upon TLR activation, revealing rapid assembly and slow disassembly. Furthermore, we show that inhibition of IRAK4 kinase activity leads to increased stability of the Myddosome complex as demonstrated by greater associations between MyD88 and IRAK4. Importantly, we found that the kinase activity of IRAK4 is dispensable for TLR-mediated NF-κB and MAPK signalling but essential for production of inflammatory cytokines. To our knowledge this is the first full examination of the kinetics of the Myddosome from macrophages. We further demonstrated that a loss of IRAK4 activity by either chemical inhibition or genetic manipulation resulted in a significantly more stable Myddosome structure. This increase in stability is suggestive of a prominent protein scaffold role of IRAK4, independent of its kinase activity, in which IRAK4 interacts with MyD88 and IRAK1, tethering them together into the Myddosome complex. Our findings may help explain why, to date, therapeutic targeting IRAK4 kinase activity has not been as successful as hoped, and highlights that targeting the scaffold function of IRAK4 may be an attractive alternative.