DOI: 10.1002/JLB.

MR0318-096R

REVIEW

Understanding early TLR signaling through the Myddosome

Katherine R. Balka1 Dominic De Nardo1,2

1 Inflammation Division, The Walter and Eliza

Hall Institute of Medical Research, Parkville, Abstract

Victoria, Australia
2 Department of Medical Biology, The University
of Melbourne, Parkville, Victoria, Australia
Correspondence
Dr. Dominic De Nardo, Inflammation Division,
The Walter and Eliza Hall Institute of Medical
Research, 1G Royal Parade, Parkville, Victoria,
3052, Australia.
E-mail: denardo.d@wehi.edu.au
TLRs are expressed on the plasma and endosomal membranes of innate immune cells acting as
sensors of foreign and inherent danger signals that threaten the host. Upon activation, TLRs facil-
itate the assembly of large intracellular oligomeric signaling complexes, termed Myddosomes,
which initiate key signal transduction pathways to elicit critical inflammatory immune responses.
The formation of the Myddosome is integral for TLR signaling; however, the molecular mecha-
nisms controlling its formation, disassembly, and the subsequent proximal signaling events remain
to be clearly defined. In this review, we present a brief overview of TLR signal transduction path-
ways, summarize the current understanding of the Myddosome and the proteins that comprise
its structure, including MyD88 and members of the IL-1 receptor-associated kinase (IRAK) family.
Finally, we will discuss recent advances and open questions regarding early TLR signaling in the
context of the Myddosome complex.

KEYWORDS
innate immunity, IRAK, MyD88, Myddosome, TLRs

1 AN OVERVIEW OF TLR SIGNALING
The TLRs are a family of pattern recognition receptors (PRRs) critical
for host defense against invading pathogens. The TLRs encompasses a
broad class of PRRs with 10 TLRs expressed in humans (TLR1-10), and
12 in mice (TLR1-9, 11-13) that recognize a diverse range of microbial
and host-derived ligands. Mammalian TLRs were named due to their
similarity to the Toll protein from Drosophila, which was originally iden-
tified in fly embryonic development.1 However, Toll was subsequently
shown to be critical for Drosophila immune defense against fungi and
bacteria via induction of pathways homologous with those activating
the mammalian transcription factor NF-𝜅B.2–4 Both Toll and the verte-
brate TLRs share common structural domains: an amino (N)-terminal
extracellular domain consisting of multiple leucine-rich repeat (LRR)
regions, which facilitate ligand recognition; a single transmembrane
(TM) domain; and a carboxyl (C)-terminal intracellular Toll/IL-1 recep-
tor (TIR) domain, which acts as a platform for the recruitment of down-
stream signaling molecules (Fig. 1).5 Interestingly, as TLRs and the IL-
1 receptor (IL-1R) both express TIR domains, the signaling cascades
downstream of these receptors are thought to be comparable. Indeed,
much of the fields understanding of TLR signaling originated from stud-
ies focusing on signaling downstream of the IL-1R.
TLRs can be roughly classified based on their subcellular local-
ization to either the plasma membrane or membranes within acidi-
fied endolysosomal compartments.6 In general, TLRs localized to the
plasma membrane (TLR1, 2, 4-6, and 10) recognize components of bac-
teria and fungi, whereas those located on endolysosomal membranes
(TLR3, 4, 7-9, and 11-13) detect viral nucleic acids (Fig. 2).6 A com-
prehensive list of known TLR ligands can be found elsewhere.7,8 Sig-
naling downstream of TLRs is facilitated by conformational changes in
their intracellular TIR domains induced following ligand binding, which
leads to recruitment of adaptor proteins that bind to the cytoplasmic
region of TLRs via homotypic TIR-TIR interactions. Currently, 5 TLR
adaptor proteins are known to form these interactions: Myeloid differ-
entiation factor 88 (MyD88), TIR-domain containing adaptor molecule
(TRIF), MyD88-adaptor-like (MAL, also known as TIR domain contain-
ing adaptor protein [TIRAP]), TRIF-related adaptor molecule (TRAM),
and sterile 𝛼- and armadillo-motif-containing protein (SARM).9 Gener-
ally, TLRs transduce signals via 2 major pathways dependent primar-
ily on the adaptors MyD88 or TRIF. MyD88 signaling predominantly
leads to the production of inflammatory cytokines (e.g., TNF, IL-6, IL-
1) and chemokines (e.g., C-C motif ligand 4, CCL4), whereas the TRIF
axis mainly induces the expression of type I IFNs (e.g., IFN𝛼/𝛽) (Fig.
2).7 Whereas TLR3 signals exclusively via the TRIF axis,10 all other
TLRs transduce signaling via a MyD88-dependent mechanism. Among
the family members, TLR4 is unique in terms of its adaptor usage
and translocation. TLR4 signals primarily from the plasma membrane
through MyD88 to activate a strong cytokine response but is subse-
quently internalized into endosomes where it leads to type I IFN pro-
duction via TRIF-dependent signaling, albeit to a lesser extent.11

Received: 23 May 2018 Revised: 27 August 2018 Accepted: 6 September 2018

J Leukoc Biol. 2019;105:339–351. www.jleukbio.org 2018
c Society for Leukocyte Biology 339
340 BALKA AND DE NARDO

LRR domains

TM TM

TIR domains

MyD88 2 x MyD88 DDs

IRAK4
KD 4 x MyD88 DDs
KD
IRAK1/2

4 x IRAK4 DDs

4 x IRAK1/2 DDs

N C
TLR LRR domain TM domain TIR domain
PIP2 binding motif
MAL TIR domain
1 15 35 84 235

MyD88 Death domain TIR domain

1 110 155 296

IRAK4 Death domain Kinase domain

1 95 166 460
TRAF6 binding motifs
IRAK1 Death domain Kinase domain
1 103 198 522 712
TRAF6 binding motifs
IRAK2 Death domain Kinase domain
1 94 196 489 590

F I G U R E 1 Formation of the Myddosome complex by homotypic domain interactions. TLR receptors contain an extracellular leucine-rich repeat
(LRR) domain (shown in blue), a transmembrane (TM) domain (shown in purple) and cytosolic Toll/IL-1R (TIR) domain (shown in green). Following
TLR activation molecules of MyD88 (green shadow), a 296 amino acid protein is recruited to dimeric TLR-TIRs via homotypic TIR domain inter-
actions. Only 2 MyD88 TIR domains are shown for simplicity. The adaptor MAL (not shown) which is required for signaling by a subset of TLRs,
contains a phosphoinositide (PIP2) binding region, allowing MAL to interact with the plasma membrane. Six MyD88 DDs (DD shown in orange)
arranged in 2 rings provides a platform for the recruitment of 2 subsequent layers of 4 IRAK4 and 4 IRAK1/2 DDs, forming the complete Myddo-
some complex. A representation of 1 kinase domain (KD) for IRAK4 (yellow shadow) and IRAK1/2 (red shadow) shows the close proximity of these
kinases in the complex. The C-terminal regions of IRAK1 and IRAK2 contain putative binding motifs (shown in yellow/black) for the recruitment of
TRAF6 and the activation of downstream signaling events

1.1 Pathways downstream of MyD88 IL-1R-associated kinase (IRAK) family to be further recruited to the
receptor complex (Fig. 1).9 This hetero-oligomeric structure compris-
In addition to its TIR domain, which allows recruitment to TLRs fol-
ing molecules of MyD88 and several IRAK members, termed the
lowing their activation, MyD88 also contains an N-terminal death
Myddosome, will be discussed in more detail below. Initially, IRAK4
domain (DD), allowing members of the DD containing serine/threonine
BALKA AND DE NARDO 341

TLR1/2 or TLR 2/6 TLR5 TLR4

Extracellular space

MAL MAL
MyD88
Cytosol
TLR4
IRAK4
IRAK1/2
Endosomes
TLR7 TLR9 TLR3
TRAM
TLR8
TRIF

TRAF6
MAL

TRIF
MyD88 TRAF6
IRAK4
IRAK1/2

TAB2
RIPK1
TAK1 TAB3
TRAF3

IKKα
MAPKs IKKβ NEMO
TBK1
Ub
IRF5 IκBα
AP-1 CREB p65 p50

IRF3

NFκB
AP-1 CREB
p65 p50

IRF5 IRF3

Nucleus

F I G U R E 2 The TLR signaling pathways. TLRs are located on either plasma or endolysosomal membranes where they detect pathogen associated
molecular patterns (PAMPs). Signaling downstream of TLRs is mediated by 2 main adaptor proteins; the MyD88- and/or TRIF-dependent path-
ways. The recruitment of MyD88 to the TIR domain of TLRs induces IRAK4 and IRAK1/2 binding to form the Myddosome. Activation of TRAF6
downstream of MyD88 leads to the activation of NF-𝜅B, IRF5, and the MAPK pathways, inducing the expression of proinflammatory cytokines.
Association of TRIF with TLR3 and internalized TLR4 also leads to expression of proinflammatory cytokines via TRAF6 and RIPK1. TRAF3 also
mediates the expression of type I IFNs following activation of the IFN regulatory factors (IRFs)

is recruited to MyD88 and activated by autophosphorylation of its
central kinase domain (KD), which is thought to then allow for acti-
vation of both IRAK1 and IRAK2. Formation of this signaling plat-
form then induces association and activation of the E3 ubiquitin
(Ub) ligase TNF receptor-associated factor 6 (TRAF6), which simul-
taneously triggers 3 major transduction pathways culminating in the
activation of several critical transcription factors (Fig. 2). Follow-
ing its activation TRAF6 complexes with the TAK1 complex com-
prising TGF-𝛽 activated kinase 1 (TAK1), TAK1-binding protein 1
(TAB1), and TAB2/3, stimulating activation of the I𝜅B kinase (IKK)
complex comprising IKK-𝛼, IKK-𝛽, and NF-𝜅B essential modulator
(NEMO, also known as IKK-𝛾). This facilitates the phosphorylation
and degradation of the inhibitor of NF-𝜅B (I𝜅B𝛼), allowing canoni-
cal NF-𝜅B (comprising subunits p50 and p65) to translocate to the
nucleus. Second, the MAPK pathways are induced downstream of
the TAK1 complex leading to activation of the transcription fac-
tors activator protein-1 (AP-1) and CREB. Third, TRAF6 facilitates
activation of IFN regulatory factor 5 (IRF5)12 via a TAK1-IKK-𝛽
axis.13,14 The activation and cooperative binding of these transcrip-
tion factors to specific gene promoters culminates in production of
potent proinflammatory cytokines and chemokines that elicit an acute
inflammatory response. Of note, in plasmacytoid dendritic cells (pDCs)
exclusively, MyD88 also induces the expression of type I IFNs via
IRAK4/IRAK1-mediated activation of the transcription factor IRF7 in
response to TLR7 and TLR9 activation.15–17
The TIR-adaptor MAL is required for a subset of MyD88-dependent
TLRs (TLR2, TLR4, and TLR9) by facilitating the recruitment of MyD88
to the plasma or endolysosomal membrane by an N-terminal phos-
phatidylinositol (4,5) bisphosphate (PIP2) binding motif (Fig. 1).11,18–21
Additionally, MAL contains large acidic regions on its surface that are
342
able to interact with the predominantly basic surfaces of MyD88-
TIR and TLR4-TIR forming a bridge between these molecules.22 A
single nucleotide polymorphism within the acidic region of MAL
(D96N) leads to loss of MyD88 interaction and impaired cytokine
responses of TLR4 and TLR1/2.22,23 Interestingly, however, addition
of a PIP2 binding motif to MyD88 restores signaling in the absence of
MAL, suggesting that MAL may merely be required to facilitate move-
ment of MyD88 to membranes.20
1.2 Pathways downstream of TRIF
Aside from MyD88-dependent signal transduction, TLR3 and TLR4
also induce a type I IFN transcriptional response from endosomes
following recruitment of the adaptor TRIF to their respective TIR
domains (Fig. 2).24 Whereas TLR3 can directly associate with TRIF,
TLR4 requires the bridging adaptor TRAM to facilitate this receptor-
adaptor interaction.25 The major pathway triggered downstream of
TLR3-TRIF is via recruitment of the E3 Ub ligase TRAF3, which inter-
acts with and activates TRAF family member-associated NF-𝜅B activa-
tor (TANK)-binding kinase 1 (TBK1). This leads to phosphorylation and
dimerization of IRF3, which then translocates into the nucleus to medi-
ate expression of type I IFN genes. A secondary pathway elicited by
the TLR3-TRIF receptor complex is triggered via TRAF6 recruitment,
which interacts with Receptor-interacting serine/threonine-protein
kinase 1 (RIPK1) via their respective RIP homotypic interaction motif
(RHIM) domains, allowing formation of a Ub scaffold for the assem-
bly of the TAK1 complex, which drives canonical NF-𝜅B activation and
the MAPK pathways (Fig. 2). Interestingly, maximal IFN-𝛽 expression is
controlled by a group of transcription factors that bind to positive regu-
latory domains (PRDs) within the IFN-𝛽 promoter region known as the
enhancesome.26 IRF3 dimers (and IRF7 in pDCs) bind to PRD regions I
and III, whereas NF-𝜅B heterodimers bind to PRDII. The AP-1 complex
is also required for maximal transcriptional activation via binding to
PRDIV. This collective of transcription factors facilitates nucleosome
rearrangement, ultimately leading to maximal IFN-𝛽 transcription.
2 THE MAIN PLAYERS IN EARLY
MYD88-DEPENDENT TLR SIGNALING
This section will focus on the primary protein components and their
roles in initiating signal transduction downstream of TLR activation
including, the intracellular TIR domains of the receptors themselves,
the TIR adaptor MyD88, and the IRAK family of effector kinases.
2.1 TLR-TIR domains
The first crystal structures of the TIR domains of TLR1 and TLR2
indicated the prototypical arrangement of 5 𝛽-strands surrounded by
5 𝛼-helices.27 TIR domains also share 3 conserved motifs, (F/Y)DA,
RDXXPG, and FW termed box 1-3, respectively.28 Ligand binding in the
LRR domain of TLRs is thought to induce conformational changes in
TLR dimers that consequently alter the orientation of the intracellular
BALKA AND DE NARDO
TIR domains to allow recruitment of TIR adaptor proteins.29 The BB
loop of the TIR domain, “so called as it” connects the second 𝛽 strand
(𝛽B) to the second 𝛼-helix (𝛼B) contains the box 2 motif and is impor-
tant for interactions between adjacent TIR domains. The C3H/HeJ
mouse strain, vulnerable to Gram-negative bacteria infection and
resistant to LPS, was found to have a proline to histidine mutation at
position 712 (P712H) in exon 3 of the Tlr4 gene within the BB loop
box 2 motif.30,31 Discovery of this mutation not only uncovered TLR4
as the genuine signaling receptor for LPS but further identified the
BB loop as a key region within the TIR domain structure required for
downstream signaling. All MyD88-dependent TLRs similarly share a
conserved proline residue in this BB loop. Mutation of the analogous
proline residues in TLR2 (P681)32 and TLR10 (P674)33 prevents inter-
action with MyD88 and impairs NF-𝜅B reporter activity, suggesting
that conservation of this proline within the TIR domain is a critical
feature. The crystal structure of a TLR10 homodimer showed that
this BB loop formed the interface region connecting the 2 TIR domain
monomers.27 Interestingly, TLR3, the only MyD88-independent TLR,
contains an alanine at this site. However, mutation of this alanine
to a proline (A795P) was shown to convert TLR3 from TRIF- to
MyD88-dependent signaling.34
2.2 MyD88
MyD88 has a tripartite structure containing an N-terminal DD, an
intermediate domain (ID), and C-terminal TIR domain (Fig. 1).35
MyD88 was initially identified as the adaptor that links IRAK1 to
the IL-1R following IL-1𝛽 engagement.35,36 MyD88 adaptor function
was then swiftly established in TLR signaling as a link to downstream
kinases, reflecting the role of the Tube adaptor protein in Drosophila
Toll signaling.37,38 Overexpression of wild-type (WT) MyD88 is suf-
ficient to drive robust NF-𝜅B luciferase activity.35,36,39 However,
overexpression of the C-terminal region (152-296) containing the
TIR domain exhibits a dominant-negative effect, presumably due to
outcompeting WT MyD88 and the inability to recruit downstream
IRAKs.35,36 Key studies using Myd88-deficient mice clearly demon-
strated the requirement of MyD88 for cytokine production induced
downstream of most TLRs,40 IL-1, and IL-18 receptor activation.41
Indeed, Myd88-deficient mice are resistant to LPS-induced sepsis and
lethality due to an inability to activate inflammatory cytokines.40
Surprisingly, although delayed and reduced, the activation of NF-𝜅B
and MAPK pathways were still intact in macrophages from Myd88-
deficient mice following TLR4 activation.40 However, this was subse-
quently explained by the involvement of the TRIF pathway, as signaling
events following LPS treatment were entirely abolished in cells derived
from Myd88/Trif double knockout mice.10
2.3 IRAK4 and the role of its kinase activity
IRAK4, the final member of the IRAK family to be described, was
identified in 2002 based upon its similarity to IRAK1 in a database
search of human complementary DNAs (cDNAs).42 Like IRAK1 and
IRAK2, IRAK4 contains a central active KD (Fig. 1). IRAK4 was
shown to directly interact with MyD88 and IRAK1 via homotypic
BALKA AND DE NARDO
DD-DD interactions, connecting MyD88 to the downstream effec-
tor kinases and leading to activation of IRAK1.42 IRAK4 was sub-
sequently found to be essential for cytokine production following
activation of MyD88-dependent TLRs and the IL-1R through the gen-
eration of Irak4-deficient mice.43 Strikingly, like the Myd88-deficient
mice, Irak4 deficiency also protects mice from LPS lethality and sep-
tic shock by abolishing cytokine responses.43 Of note, multiple human
patients have also been reported to have mutations in IRAK4 that
introduce premature STOP codons leading to truncated and inac-
tive forms of IRAK4 protein.44,45 These patients display an IRAK4
immunodeficient phenotype where induction of cytokines is com-
pletely abolished downstream of TLR stimulation.44,45 Intriguingly,
these patients are only susceptible to a specific subset of pyogenic
bacterial infections in early childhood where the mortality rate is
around 50%.45,46 However, beyond adolescence no deaths have been
reported demonstrating that Myddosome signaling is more critical in
early life.45
Three key publications in 2007 centered around the generation of
Irak4 kinase inactive (KI) knock-in mice by mutating lysine residues 213
and 214 in the ATP binding site to methionine47 and alanine.48,49 These
Irak4-KI mice were protected from septic shock caused by high doses
of LPS or the TLR9 ligand CpG-DNA in a manner similar to Myd88- and
Irak4- knockout mice.47,48 Like Irak4-deficient cells,43 macrophages
isolated from Irak4-KI mice could not induce the phosphorylation of
IRAK1 nor produce TNF or IL-6 cytokine following LPS or CpG DNA
treatment.47–49 However, when examining signaling in Irak4-KI cells,
Kim et al. found that the MAPK components p38 and extracellular
signal-regulated kinase (ERK) were activated to levels comparable to
those from WT cells following TLR activation.47 Conversely, Kawagoe
et al. and Koziczak-Holbro et al. found that the MAPK pathway was
entirely dependent on IRAK4 kinase activity downstream of TLR248
and TLR7.49 However, all groups found only a modest defect in the
degradation of I𝜅B𝛼, and although reduced, NF-𝜅B activation was still
observable.47,48 More recently, using highly specific IRAK4 inhibitors
2 independent groups also showed that NF-𝜅B and MAPK signaling
was mostly intact following TLR1/2, TLR4, and IL-1R activation in
the absence of IRAK4 kinase activity in human and mouse myeloid
cells.50,51 Indeed, an alternative MAPK kinase kinase 3 (MEKK3)-
dependent (TAK1-independent) pathway has been shown to elicit NF-
𝜅B responses in the absence of IRAK4 kinase activity downstream of
TLR activation.52,53 To add further complexity, it appears that human
and mouse cells may have differing requirements for IRAK4 kinase
activity and usage of IRAK1 and IRAK2 in MyD88-dependent TLR
responses.54 Indeed, responses from IRAK4-deficient human cells are
not rescued by reconstitution of murine IRAK4 and vice versa.54
Moreover, the dependency of IRAK4 kinase activity differs in dis-
tinct human cell populations. Use of a selective IRAK4 kinase inhibitor
nicely demonstrated that the kinase activity of IRAK4 is required for
TLR-induced cytokine secretion from human monocytes, whereas in
dermal fibroblasts IRAK4 kinase inhibition had no effect on IL-1𝛽-
mediated cytokine production.55 Reconstitution of IRAK4-deficient
human fibroblasts with IRAK4-KI (K213A/K214A), also restores
NF-𝜅B and AP-1 luciferase activity similarly to WT IRAK4.56 Addi-
tionally, whereas NF-𝜅B and MAPK activation is reported to be only
343
moderately affected following TLR activation in the presence of IRAK4
kinase inhibition in human monocytes, IRF5 activation and subsequent
nuclear translocation is inhibited.14 Because the expression of most
cytokine genes requires cooperative binding of both IRF5 and NF-
𝜅B within promoter regions, the loss of activated IRF5 significantly
reduces cytokine expression.14,57,58 Therefore, it appears the kinase
activity of IRAK4 is essential for activation of IRF5, but potentially
dispensable for NF-𝜅B and MAPK signaling.
2.4 IRAK1 and IRAK2
IRAK1, the first member of the IRAK family to be identified, was
initially described as a kinase recruited to the IL-1R and later found
to interact with TLRs.59,60 Following TLR or IL-1R stimulation, IRAK1
is rapidly recruited to the receptor complex where it is thought to
become phosphorylated by IRAK4. Irak1-deficient mice and cells show
a partial impairment of cytokine production in response to IL-1𝛽 61
and LPS,62 presenting an intermediary phenotype compared to those
lacking IRAK4. IRAK2 was initially identified to be similar to IRAK1
and was shown to induce NF-𝜅B in a dose-dependent manner in
reporter assays, though to a lesser extent.35 Subsequently, IRAK2
was shown to be a pseudokinase requiring activation by IRAK4 due
to an aspartate to asparagine residue change in its catalytic domain,
thus inhibiting its autophosphorylation.63 Interestingly, in response
to TLR stimulation Irak2-deficient mice show a more severe reduction
in cytokine responses compared to Irak1 knockouts.63 Kawagoe et al.
observed that IRAK1 and IRAK2 are essentially redundant in the early
stages of TLR activation; however, IRAK2 is vital for sustained NF-𝜅B
signaling and cytokine production.63 Consequently, the phenotype of
mice lacking both Irak1 and Irak2 resembles the more severe immun-
odeficiency observed in IRAK4 knockouts.43,63 This is likely explained
by the presence of TRAF6 binding motifs within the C-terminal tails
of IRAK1 and IRAK2 that are absent in IRAK4 (Fig. 1).64 To account
for potential artificial protein redundancy generated by the use of
conventional Irak1 or Irak2 single knockout mice, Philip Cohen and
colleagues generated 2 separate knock-in mouse strains expressing
either a KI IRAK1 (D395A) or a mutant IRAK2 (E525A) unable to
interact with TRAF6.65 Similar to findings from deficient mice, the
presence of IRAK1 D395A demonstrated that its kinase activity
was not necessary for expression of cytokine genes in macrophages;
however, it did confirm its absolute requirement for the production of
type I IFNs by pDCs.65 Meanwhile, the use of macrophages expressing
IRAK2 E525A further demonstrated the importance for an interaction
between IRAK2 and TRAF6 for a persistent low-level NF-𝜅B response
to allow for significant levels of proinflammatory cytokine genes in
response to TLR activation.65 Thus importantly, IRAK1 and/or IRAK2
act to link the proximal receptor complex to downstream signaling
pathways through engagement of TRAF6. Whether or not these IRAKs
are simply required to recruit TRAF6 or whether they control its E3
ligase activity remains unclear, although overexpression of IRAK2, but
not IRAK1, has been shown to induce TRAF6 polyubiquitination.66 Of
note, macrophages derived from a TRAF6 E3 Ub ligase inactive mutant
mouse (TRAF6 L74H), demonstrated that the Ub ligase activity of
TRAF6 appears somewhat dispensable for TLR-induced activation of
344
NF-𝜅B and the MAPK pathway, but critical for cytokine production.67
These results were comparable to those found in studies examining the
kinase activity of IRAK4, which suggests these proteins play significant
scaffolding roles in the Myddosome, independent of their respective
enzymatic functions.
3 OUR CURRENT UNDERSTANDING OF
THE MYDDOSOME
The formation of higher-order multiprotein or supramolecular orga-
nizing centers (SMOCs) is now recognized as a fundamental event
in multiple innate immune pathways.68 A common feature of these
oligomeric signaling platforms is the requirement for molecules con-
taining domains within the DD superfamily that comprises the DD,
death effector domain (DED), caspase recruitment domain (CARD),
and pyrin domain (PYD) subfamilies.69 These domains interact via
homotypic interactions to induce large protein platforms. Upon TLR
or IL-1R activation, formation of a MyD88-IRAK4-IRAK1/2 oligomeric
complex, known as the “Myddosome” is required to initiate down-
stream signaling.
The term Myddosome was coined by Nick Gay and colleagues in
2009 following the observation that the DD and ID of MyD88 can form
higher-order structures when combined with IRAK4-DD in solution
at a ratio of 8:4 or 7:4 MyD88:IRAK4.70 A year later the remarkable
DD crystal structure of the Myddosome complex was elegantly solved
by the group of Hao Wu, revealing a complex comprising six MyD88
DDs, 4 IRAK4 DDs, and 4 IRAK2 DDs arranged in a left-hand single-
stranded helical oligomer.71 More precisely the structure revealed 4
layers of DDs arranged in rings; 2 MyD88-DDs atop 4 MyD88-DDs
followed by 2 rings each comprising the 4 DDs of IRAK4 and IRAK2,
respectively (Fig. 1). The Myddosome structure was solved using DDs
from IRAK2, as the authors were unable to be express IRAK1 DDs;
however, IRAK1 is likely to be recruited first following physiologic TLR
activation. Indeed, sequence alignment of IRAK1 and IRAK2 reveals
that key residues required for IRAK2 assembly within the Myddosome,
such as tryptophan 62 and arginine 67, are conserved in IRAK1.71
The DDs complete nearly 4 full revolutions in the helical oligomer,
with 3.7 DDs per turn.71 Recently, exquisite imaging from the group
of Clare Bryant was able to capture Myddosome formation in vivo for
the first time using live single cell imaging of macrophages expressing
GFP-tagged MyD88.72 Upon TLR activation large complexes rapidly
appeared (within just 3 minutes), which correlated to the 6 MyD88
molecules in the crystal structure.72 The Myddosome is thought to
assemble in a hierarchic manner where a MyD88 homo-oligomer is
seeded by TLR-TIR dimers, forming a DD platform for IRAK4 associ-
ation, which then provides a platform for recruitment of IRAK1/2 DDs.
This sequential model of oligomerization is supported by the observa-
tion that DDs of IRAK2 only form stable complexes with complexed
MyD88-IRAK4 DDs and not with the single DDs of either MyD88 or
IRAK4. Furthermore, the ring of IRAK4 molecules in the Myddosome
structure has a high charge complementarity for IRAK2 supporting
sequential recruitment.71 Threonine 66 of IRAK1 is highly conserved
(i.e., Threonine 57 in IRAK2) and has also been shown to be important
BALKA AND DE NARDO
for dimerization of IRAK1 suggesting that this region of IRAK1/2 is
important for the interfaces formed in the Myddosome.73–75 The Myd-
dosome structure also revealed key residues that facilitate the inter-
face between MyD88-DD and IRAK4-DD including arginine 12 (R12)
of IRAK4, which is highly conserved in vertebrate IRAK4.71 Phospho-
rylation of serine 8 within the IRAK4-DD induces a negative charge and
preferentially binds to R12, preventing the binding of R12 to MyD88.76
Importantly, a patient with an arginine to cysteine substitution at posi-
tion 12 (R12C) of IRAK4 suffered from recurrent pyogenic bacte-
rial infections similar to the IRAK4-deficient patients.45,77 The R12C
mutation has also further been shown to disrupt IRAK4 interacting
with MyD88 in vitro.50
Following recruitment of IRAK4 to MyD88, IRAK4 itself associates
via both DD-DD and KD-KD interactions to form the ring of 4 IRAK4
molecules.71,78 The DD-DD interactions between MyD88 and IRAK4
are thought to stabilize the IRAK4-KD interactions as the addition
of MyD88 to full length IRAK4 greatly enhances the autophospho-
rylation of IRAK4 in solution.78 Formation of an IRAK4-DD layer
within the Myddosome enriches the local concentration of IRAK4
stimulating its autophosphorylation.78 Mass spectrometry analysis
revealed that IRAK4 is autophosphorylated at 3 key residues within
its activation loop; T342, T345, and S346,79 with T345 represent-
ing the prototypical phosphorylation site.80 Interestingly, the dimer-
ization or oligomerization of IRAK4 appears to be regulated by its
phosphorylation state. In solution full length IRAK4 and the IRAK4-
KD are able to form dimers when autophosphorylation (as measured
by phospho-T345), but is inhibited by mutation of aspartate 311 to
asparagine (D311N), a key residue in the catalytic loop required for
phosphotransfer.78 Similarly, treatment of full length and IRAK4-KD
with 𝜆-phosphatase induced dimerization of IRAK4, whereas incuba-
tion with ATP and Mg2+ stimulated the autophosphorylation and dis-
sociation of IRAK4 into monomers.78 The crystal structure of IRAK4-
KD dimers stabilized by the D311N mutation shows an enzyme-
substrate arrangement presenting how IRAK4 is activated by inter-
molecular trans-autophosphorylation.78 The prototypical T345 phos-
phorylation site in the activation loop of the “substrate” IRAK4-KD is
positioned within the active site of the “enzyme” IRAK4-KD.78 How-
ever, this activation loop region lacks similarity to IRAK1, the sub-
strate of IRAK4 and hence additional interactions where high surface
area are buried in the dimer interface are required to facilitate IRAK4
autophosphorylation.78 Mutating key residues in these regions such as
R361E, E401K, and K440E reduced the phosphorylation rate of IRAK4,
affected signaling events and reduced expression of the downstream
cytokines TNF and IL-8.78
4 OPEN QUESTIONS IN THE FIELD
The requirement for Myddosome formation to license TLR signal
transduction is now an accepted concept in the field; however, a num-
ber of fundamental questions remain unresolved. Here we will discuss
some of the open questions relating to Myddosome formation, local-
ization, disassembly, and the triggering of early signaling events that
dictate TLR responses (Fig. 3).
BALKA AND DE NARDO 345

High PIP2
concentration

MAL
MyD88 C
IRAK4
IRAK1/2
Destabilized
B Myddosome
A
Low PIP2 P
P P
concentration P
WT MyD88 MyD88 L265P
Loss of MAL
**
***

Signal transduction

F I G U R E 3 Open questions surrounding the Myddosome. (A): The possibility exists that active Myddosome complexes can signal from the
cytosol, uncoupled from TLR-TIR domains. This concept is supported by studies with the B cell lymphoma mutant MyD88 L265P. (B): A potential
model for Myddosome disassembly involves the internalization of the entire TLR complex into endosomes. As the receptor moves from a mem-
brane with a high concentration of PIP2 molecules at the cell surface to a lower concentration of PIP2 at the endosomal membrane, MAL may
detach from the complex. This may disrupt and/or prevent MyD88 interactions with the TIR domain of the receptor and subsequently lead to
dissociation of Myddosome components. (C): An alternative model of disassembly may be driven by intrinsic instability of the active Myddosome
complex. Following post-translational modifications such as the phosphorylation of IRAK1 and its loss from the Myddosome, IRAK4 may become
unstable and dissociate leading to complete disassembling of the complex

4.1 Myddosome formation a helical ring, coinciding with the DD positioning in the crystal struc-
ture of the Myddosome. Loiarro et al. also identified residues E183,
The exact stoichiometry and mechanism of Myddosome formation in
S244 and R288 in the TIR domain that when mutated, prevent MyD88
vivo is still a prominent question in the field, that is, does the complex
homodimerization and recruitment of IRAKs.86 Interestingly, peptides
exist physiologically?
comprising around 30 amino acids that span the region around E183,
Some evidence for cellular assembly of the Myddosome complex
were able to bind to endogenous full length MyD88 causing dimer-
has come from identification of mutations in MyD88 that disrupt
ization disruption and reduced TLR responses.86 The ID of MyD88 is
downstream signaling. Jiang et al. used random germ-line mutagene-
essential for its function, as MyD88 lacking the ID fails to induce sig-
sis that identified an isoleucine to asparagine variant (I179N) that was
naling downstream of TLRs.87 Furthermore, the structure of the Myd-
found to ablate TNF secretion from macrophages in response to all
dosome revealed that the prototypical DD of MyD88 required a region
TLR ligands except those activating TLR2/6 heterodimers or TLR2.81
of the ID to form a complete DD structure.71 Indeed, a splice variant
Residues 195-197 located in the BB loop of MyD88, were also shown
of MyD88 known as MyD88 short (MyD88s) which contains the DD
to be important for MyD88 signaling.82,83 Multiple patients with pyo-
but lacks exon 2, the coding region of the ID, was found to prevent
genic bacterial infections were found to express a variant of MyD88
IRAK1 phosphorylation due to its inability to recruit IRAK4, acting in a
with the mutation R196C (within the BB loop), which fails to bind the
dominant negative manner.87,88 Whereas full length MyD88 is consti-
IL-1R showing severe defects in cytokine production in response to IL-
tutively expressed, MyD88s expression is induced following sustained
1𝛽.84 In addition, Vyncke et al. recently defined 4 key sites in MyD88-
TLR4 or IL-1R activation, acting as a negative feedback mechanism.87
TIR including the regions within the BB loop and 𝛼E, 𝛼D, and 𝛼C’ helices
Residues E52 and Y58 in the MyD88-DD are also important for linking
that are involved in MyD88-TIR oligomerization and interactions with
IRAK4 to the Myddosome.71,89 Patients with a homozygous in-frame
MAL and TLR4.85 Proline 200 of the BB loop inserts between W284
deletion of E52 of MyD88 suffer from invasive bacterial infections and
in the 𝛼E helix and R196 in the BB loop, whereas D195 and D197 of
cannot induce cytokine responses to multiple TLR agonists.84 Collec-
the BB loop also interact with R288 and R291 in the 𝛼E helix forming
tively these MyD88 mutations provide strong evidence for the physi-
an asymmetric MyD88 dimer.85 MyD88-TIRs also form a symmetric
ologic requirements of the binding interfaces formed within the active
dimer, which when alternated with the asymmetric positioning forms
Myddosome structure.
346
Whereas mutational and structural analyses are highly sugges-
tive of its biological relevance, definitive proof that the Myddosome
forms upon TLR activation in living cells has been harder to obtain.
Bryant and colleagues recently showed using live cell imaging tech-
niques, that 6 MyD88 molecules form clusters at the cell membrane
following LPS stimulation, matching the stoichiometry of the crystal
structure of the Myddosome DDs.72 Although 6 molecules of MyD88 is
considered to be optimal, other stoichiometries for MyD88 have been
reported, including 7 or 8 molecules, suggesting that other arrange-
ments may exist.70 However, as a TLR dimer is expected to only form
binding sites for 2 MyD88-TIRs, how 6 or more MyD88 molecules can
spatially be recruited to the receptor and incorporated into the Myd-
dosome remains uncertain. The clustering of multiple TLR dimers has
been considered as a mechanism to provide additional TIR domains
for the recruitment of MyD88 molecules70 ; however, it is thought that
the LRR domains of TLRs are too large and steric hindrance would
prevent the formation of oligomers of TLRs.90 Indeed, recently visu-
alized TLR4 receptor activity in live cells using Halo-tagged TLR4,
demonstrated that following LPS treatment, TLR4 formed dimers at
the plasma membrane as expected, with no formation of higher-order
TLR4 oligomers.72 The crystal structure of MAL revealed that it too
forms a dimer, which when recruited to the TLR-TIR domain could
provide additional platforms for the recruitment of MyD88.22 More
recently MAL has also been shown to form a large macromolecular
structure in vitro assembling into large filaments that can also cou-
ple to TLR4 oligomers.91 Although suggestive of an ability for MAL to
form filaments upon TLR activation, the physiologic relevance of these
structures remains unclear. Additionally, not all TLRs require MAL for
signaling. Nevertheless, it is possible, that only 2 MyD88 molecules are
in direct contact with the TLR-TIR domain and the other molecules of
MyD88 are recruited merely by homo-oligomerization. Interestingly,
Bryant’s group could visualize the formation of so called “super” Myd-
dosomes within cells, corresponding to 2 Myddosome complexes resid-
ing together, especially in the presence of high concentrations of LPS.72
Solving crystal structures of TLR-TIR domains complexed with adap-
tor TIRs (e.g., MAL, MyD88) could provide further important insights
into the assembly of the Myddosome. However, attempts to crystal-
lize these structures have so far been unsuccessful, suggesting that the
interactions are weak and may require multiple connections to stabi-
lize the complex.92
4.2 Localization of active Myddosome complexes
A pertinent question surrounding the Myddosome, is what happens to
the complex following its assembly? Do active Myddosomes stay cou-
pled to TLR-TIR domains or are they released into the cytosol?
Utilizing single-molecule fluorescence microscopy of GFP-tagged
MyD88 expressed in MyD88-deficient macrophages, Bryant and col-
leagues recently showed MyD88 rapidly forms membrane bound
macromolecular complexes, which are removed from the cell surface
within about 10 minutes following LPS stimulation.72 Meanwhile using
more conventional biochemical techniques others have demonstrated
that stable Myddosome interactions can be isolated up to 2 hours
following TLR activation in macrophages.93,94 Together these obser-
BALKA AND DE NARDO
vations suggest that following an initial TIR-driven assembly process
mediated by activation of membrane bound TLR dimers, Myddosomes
may detach from the intracellular TIR domains and be released into the
cytosol. This possibility is strengthened by the finding that a stoichio-
metric mismatch between activated dimeric receptor complexes and
oligomeric Myddosomes exists,72 as well as the presence of potential
transient interactions between TIR domains of TLRs and MyD88,92 as
discussed above (Section 4.1).
This notion is further strengthened by the discovery of a specific de
novo leucine 265 to proline (L265P) mutation within the TIR domain
of MyD88 present in a high number of B-cell human lymphomas, which
leads to constitutive Myddosome complex formation driving aberrant
NF-𝜅B activation and promoting uncontrolled cancer cell survival.95,96
To date MyD88 L265P Myddosomes have not been shown to be reliant
upon coupling to TIR domains of TLRs or IL-1 family receptors. The
L265P mutation induces conformational changes in the loop contain-
ing residues 243-248, which are important for asymmetric MyD88
TIR oligomerization,85 thus the L265P mutation in MyD88 potentially
mimics an active conformation of bound MyD88-TIR domains with
TLR-TIRs providing a platform for IRAK4 recruitment. As survival of
lymphoma cells carrying the MyD88 L265P are completely dependent
on IRAK4 activity, treatment with small molecule inhibitors of IRAK1
and/or IRAK4 have shown efficacy in inducing cancer cell death.95,97
Notably, a recent study from Waldenström macroglobulinemia patient
samples observed that MyD88 L265P Myddosomes can be released
from cancer cells and transported within extracellular vesicles to recip-
ient immune cells where they exist in the cytosolic compartment and
induce signaling.98 In recipient cells MyD88 L265P can further interact
with WT MyD88 molecules to induce amplified inflammatory signal-
ing and potentiate the local cancerous microenvironment.98 The idea
of transfer of oligomeric signaling platforms, such as that shown for
MyD88 L265P Myddosomes, is not new in innate immune signaling.99
For instance, upon inflammasome activation, the large macromolec-
ular complex comprised mostly of the adaptor protein apoptosis-
associated speck-like protein containing a CARD (ASC), can also be
released from dying cells into the extracellular space and transferred
to bystander cells to amplify the immune response. Observations of
cytosolic signaling competent MyD88 L265P Myddosomes supports
the possibility that upon assembly of WT TLR-driven Myddosomes,
these complexes may uncouple from membrane anchored receptors
into the cytosol where they continue to signal (Fig. 3A).
4.3 Models of Myddosome disassembly
Our current understanding of the events succeeding Myddosome for-
mation is narrow. Although it is well-recognized that PRR signaling
pathways are subjected to negative feedback mechanisms to prevent
sustained activation, including degradation of signaling components or
via the induction of negative regulatory molecules,100–102 the question
still remains: how is the Myddosome complex disassembled? Here we
will present evidence for 2 possible models.
One model is that internalization of plasma membrane bound
TLRs drives Myddosome disassembly, which has recently been sug-
gested in the case of TLR472 (Fig. 3B). There is some implication that
BALKA AND DE NARDO
upon activation, TLR2 and TLR4 are recruited to specific cholesterol
and sphingolipid-enriched microdomains within the plasma membrane
called lipid rafts, in order to initiate signaling.103 Lipid rafts have spe-
cific biologic properties that could be crucial for driving effective TLR
signaling. For instance, they are enriched in PIP2 domains, which would
favor the anchoring of MAL (utilized by both TLR2 and TLR4) to lipid
rafts and thus MyD88-dependent signaling and Myddosome forma-
tion at the cell surface. The current belief is that upon LPS activation
TLR4 engages MyD88, initially transducing signals from the plasma
membrane before TLR4 is rapidly internalized into endosomes (within
30 minutes) to facilitate activation of the TRIF-dependent production
of type I IFNs.11 In the case of TLR2, the precise role of its inter-
nalization is less understood, although it may be required for spe-
cific TLR2-mediated responses.104–106 Of note, during endocytosis the
composition of the membrane dramatically changes, with the concen-
tration of PIP2 domains within the forming membrane cavity rapidly
dropping.107 Hence, during TLR2 and TLR4 endocytosis, reductions in
PIP2 levels within membrane microdomains could cause uncoupling
of anchored MAL leading to instability of the Myddosome structure.
However, there are several obvious flaws with this model of disassem-
bly. First, it is not known if other surface TLRs are internalized following
activation (e.g., TLR5); second, MyD88-dependent TLRs expressed on
endosomal membranes (i.e., TLR7/8/9) are not subjected to an inter-
nalization process; and finally, only TLR2, TLR4, and TLR9 pathways
require MAL expression. In further dispute of this model, stable inter-
actions of Myddosome components examined by classical immuno-
precipitation techniques have been reported several hours following
LPS activation,93 suggesting the kinetics of TLR4 internalization and
Myddosome disassembly do not correlate. Furthermore, in the case of
TLR4, endocytosis and subsequent activation of IRF3 and type I IFN
signaling was found to be dependent upon CD14 expression.108 Hence,
if TLR4 endocytosis did indeed mediate complex disassembly, one
might expect Myddosome formation and MyD88-dependent signaling
in responses to LPS to be greatly enhanced in cells lacking CD14. How-
ever, this is not the case, as LPS-induced interactions between MyD88
and IRAK4 and the activation of downstream signaling molecules was
found to be unaffected by the absence of CD14.108
A more likely model is that of structural instability in which the
interactions between Myddosome components are transient and sus-
ceptible to alterations in the activation state of binding partners (Fig.
3C). This model would implicate the phosphorylation states of IRAK
proteins, which may reduce intrinsic protein-protein binding affini-
ties within the Myddosome by altering binding interfaces. Indeed, de-
phosphorylation of full length or the KD of IRAK4 in solution leads to
formation of stable dimeric complexes, whereas the addition of ATP
induces monomeric auto-phosphorylated IRAK4.78 This is highly sug-
gestive that in addition to the DD-DD interactions that organize the
primary complex formation, the KD of IRAK4 plays a critical role in
Myddosome stability. Several early reports noted higher affinity inter-
actions of IRAKs with kinase-inactive versions of IRAK1 or IRAK4 in
overexpression studies.36,42,102,109 Furthermore, luminescence-based
mammalian interactome mapping (LUMIER) was used to examine
protein-protein interactions between MyD88 with a KI form of IRAK4,
and revealed an ∼5-fold greater interaction when compared with
347
WT IRAK4.76 More recently it was shown that chemical inhibition of
IRAK4 kinase activity increases protein-protein interactions within the
Myddosome in primary human dermal fibroblasts stimulated with IL-
1𝛽.50 Similar findings were also reported from mouse macrophages,
where pharmacologic and genetic IRAK4 kinase inhibition resulted in
increased stability of TLR-induced Myddosome complexes.51
Further support for this model comes from the activity of IRAK1. It
has been reported that following its phosphorylation, IRAK1 interacts
with TRAF6 at the plasma membrane, before enabling its translocation
into the cytosol.110 However, whether loss of IRAK1 from the complex
destabilizes the Myddosome remains unclear. Of note, the crystal
structure of the Myddosome DDs shows a high charge complementar-
ity between the layer connecting IRAK4 and IRAK2.71 Therefore, loss
of IRAK2 or IRAK1 would expose the IRAK4 layer to the cytosol, which
may destabilize the complex. Together, these observations suggest
that following the formation of the Myddosome, activation of the
kinase components of the complex dictates its stability and ultimately
drives protein disassociation and signal termination.
5 CONCLUDING REMARKS
The last two decades have equipped us with our current understand-
ing of TLRs as integral components of the innate immune system. A
major development in the field of TLR signaling was the discovery
of the Myddosome complex.70 Solving the crystal structure of the
DDs of this multi-oligomeric signaling platform has provided invalu-
able new insight into MyD88 signaling.71 The Myddosome structure
has substantiated many of the previous findings regarding mutations in
molecules involved in early TLR signaling. A more recent breakthrough
has been the capturing of Myddosomes via advanced imaging tech-
niques that have provided the first direct evidence of the structural
assembly of the complex in living cells.72 A further important observa-
tion of this study was that the output of TLR signaling (i.e., the level of
cytokine produced) is highly dependent on the quantity, size and kinet-
ics at which these complexes form to drive NF-𝜅B activation.72
Although the formation of the Myddosome is now considered “the
mechanism” by which MyD88-dependent TLR and IL-1R signaling ini-
tiates, many aspects regarding its regulation remain unclear. Here we
have discussed our current understanding of the Myddosome and
some of the open questions that remain to be answered. However,
another major gap in our current model of TLR signaling is the pre-
cise physiologic roles of the enzymatic proteins involved in early sig-
nal transduction. Although considerable effort has been dedicated to
answer this question—through the generation of mice expressing spe-
cific enzyme inactivating mutants and the use of selective inhibitors—it
remains frustratingly unclear as to the precise roles of IRAK4, IRAK1,
IRAK2, and TRAF6. As discussed throughout this review it appears that
in most cases these proteins have substantial scaffolding roles within
the Myddosome, in addition to their enzymatic functions. Even in the
absence of IRAK kinase activities, IRAK4 still appears to link the TLR-
TIR-MyD88 complex to IRAK1 and/or IRAK2, which subsequently pro-
vides a platform for TRAF6 binding. Further evidence for this comes
from a recent study that demonstrated that phosphorylation and
348
ubiquitylation of IRAK1 may simply be due to Myddosome-induced
formation of IRAK1 dimers, rather than as a substrate of IRAK4.111
Hence, understanding scaffolding versus enzymatic roles, including the
genuine substrates of the IRAKs, is of fundamental importance to our
understanding of early TLR signaling and may lead to a revaluation of
the current therapeutic strategies targeting the kinase activity of the
IRAKs.112–114 Recently, an interesting drug screening approach iden-
tified a novel compound that directly disrupts MyD88 oligomerization
and subsequently TLR4-induced cytokine secretion in vivo suggesting
that blocking the scaffolding function of Myddosome components may
provide a better therapeutic option.115
A general assumption in the innate immune field is that the struc-
tural assembly and composition of Myddosomes are identical down-
stream of all MyD88-dependent TLRs and IL-1 family receptors. Such
a unified model of the Myddosome is not unreasonable as much of
our understanding of TLRs has come from studies focused specially on
TLR4 or pathways downstream of IL-1R activation. Interestingly, how-
ever, the TIR domains between different TLRs and between TLRs and
the IL-1R show substantial variability that may account for specificity
in signaling outputs. For example, TLR10-TIR shares between 25% and
75% sequence identify with other human TLR TIR domains.116 Fur-
thermore, whereas the TIR domains of TLR1 and TLR2 share 50% iden-
tity there are still large conformational alternations between the two
structures, including the regions of the BB loop, and the 𝛼B and 𝛼D
helices.27 It should be noted, that whereas TIR domain interactions
display variability, the ID of MyD88 may allow enough conformational
freedom and space for the DDs of MyD88 to oligomerize into a more
uniform model of Myddosome structure. However, it is hard to recon-
cile that the Myddosome complex will form with identical conforma-
tion and stoichiometry downstream of all TLRs. In solution, a number
of conformations have been reported,70 whereas imaging has revealed
the formation of so called “super” Myddosome structures compris-
ing the incorporation of multiple Myddosomes.72 Furthermore, in sil-
ico studies have also modelled multiple conformations for interactions
between Myddosome proteins,90 suggesting the complex is dynamic
and/or numerous possible structural arrangements exist. Ultimately, to
better understand specific Myddosomes, we require definitive knowl-
edge of their components through comparative proteomics, as well as
specific complex stoichiometries via additional structural and imaging-
based studies downstream of different TLRs and the IL-1R.
ACKNOWLEDGMENTS
We kindly thank the guest editorial board for the invitation to con-
tribute to this exciting volume of JLB for the Lorne Infection and Immu-
nity meeting 2018. We would also like to acknowledge work from
authors in the field whom we were unable to include in this review.
Finally, we are thankful to C.M De Nardo (Monash University, Aus-
tralia) for proofreading of the manuscript.
REFERENCES
1. Anderson KV, Bokla L, Nusslein-Volhard C. Establishment of dorsal-
ventral polarity in the Drosophila embryo: the induction of polarity
by the Toll gene product. Cell. 1985;42:791–798.
BALKA AND DE NARDO
2. Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA.
The dorsoventral regulatory gene cassette spatzle/Toll/cactus con-
trols the potent antifungal response in Drosophila adults. Cell.
1996;86:973–983.
3. Michel T, Reichhart JM, Hoffmann JA, Royet J. Drosophila Toll is acti-
vated by Gram-positive bacteria through a circulating peptidoglycan
recognition protein. Nature. 2001;414:756–759.
4. Myllymaki H, Valanne S, Ramet M. The Drosophila imd signaling path-
way. J Immunol. 2014;192:3455–3462.
5. Kang JY, Lee JO. Structural biology of the Toll-like receptor family.
Annu Rev Biochem. 2011;80:917–941.
6. Gay NJ, Symmons MF, Gangloff M, Bryant CE. Assembly and local-
ization of Toll-like receptor signalling complexes. Nat Rev Immunol.
2014;14:546–558.
7. De Nardo D. Toll-like receptors: activation, signalling and transcrip-
tional modulation. Cytokine. 2015;74:181–189.
8. De Nardo D. Activation of the innate immune receptors: guardians of
the Micro Galaxy. Adv Exp Med Biol. 2017;1024:1–35.
9. O’Neill LA, Bowie AG. The family of five: tIR-domain-containing
adaptors in Toll-like receptor signalling. Nat Rev Immunol. 2007;7:
353–364.
10. Hoebe K, Du X, Georgel P, et al. Identification of Lps2 as a key
transducer of MyD88-independent TIR signalling. Nature. 2003;424:
743–748.
11. Kagan JC, Su T, Horng T, Chow A, Akira S, Medzhitov R. TRAM cou-
ples endocytosis of Toll-like receptor 4 to the induction of interferon-
beta. Nat Immunol. 2008;9:361–368.
12. Takaoka A, Yanai H, Kondo S, et al. Integral role of IRF-5 in the
gene induction programme activated by Toll-like receptors. Nature.
2005;434:243–249.
13. Lopez-Pelaez M, Lamont DJ, Peggie M, Shpiro N, Gray NS, Cohen P.
Protein kinase IKKbeta-catalyzed phosphorylation of IRF5 at Ser462
induces its dimerization and nuclear translocation in myeloid cells.
Proc Natl Acad Sci U S A. 2014;111:17432–17437.
14. Cushing L, Winkler A, Jelinsky SA, et al. IRAK4 kinase activity
controls Toll-like receptor-induced inflammation through the tran-
scription factor IRF5 in primary human monocytes. J Biol Chem.
2017;292:18689–18698.
15. Uematsu S, Sato S, Yamamoto M, et al. Interleukin-1 receptor-
associated kinase-1 plays an essential role for Toll-like receptor
(TLR)7- and TLR9-mediated interferon-{alpha} induction. J Exp Med.
2005;201:915–923.
16. Honda K, Yanai H, Mizutani T, et al. Role of a transductional-
transcriptional processor complex involving MyD88 and IRF-7 in
Toll-like receptor signaling. Proc Natl Acad Sci U S A. 2004;101:
15416–15421.
17. Kawai T, Sato S, Ishii KJ, et al. Interferon-alpha induction through Toll-
like receptors involves a direct interaction of IRF7 with MyD88 and
TRAF6. Nat Immunol. 2004;5:1061–1068.
18. Fitzgerald KA, Palsson-McDermott EM, Bowie AG, et al. Mal
(MyD88-adapter-like) is required for Toll-like receptor-4 signal trans-
duction. Nature. 2001;413:78–83.
19. Horng T, Barton GM, Flavell RA, Medzhitov R. The adaptor molecule
TIRAP provides signalling specificity for Toll-like receptors. Nature.
2002;420:329–333.
20. Kagan JC, Medzhitov R. Phosphoinositide-mediated adaptor
recruitment controls Toll-like receptor signaling. Cell. 2006;125:
943–955.
21. Gravina HD, Goes AM, Murta SM, Ropert C. MyD88 adapter-
like (Mal)/TIRAP is required for cytokine production by splenic
BALKA AND DE NARDO
Ly6CloTLR2hi but not by Ly6ChiTLR2hi monocytes during Try-
panosoma Cruzi infection. J Biol Chem. 2016;291:23832–23841.
22. Valkov E, Stamp A, Dimaio F, et al. Crystal structure of Toll-like
receptor adaptor MAL/TIRAP reveals the molecular basis for sig-
nal transduction and disease protection. Proc Natl Acad Sci U S A.
2011;108:14879–14884.
23. Nagpal K, Plantinga TS, Wong J, et al. A TIR domain variant of MyD88
adapter-like (Mal)/TIRAP results in loss of MyD88 binding
and reduced TLR2/TLR4 signaling. J Biol Chem. 2009;284:
25742–25748.
24. Yamamoto M, Sato S, Hemmi H, et al. Role of adaptor TRIF in the
MyD88-independent toll-like receptor signaling pathway. Science.
2003;301:640–643.
25. Yamamoto M, Sato S, Hemmi H, et al. TRAM is specifically involved
in the Toll-like receptor 4-mediated MyD88-independent signaling
pathway. Nat Immunol. 2003;4:1144–1150.
26. Thanos D, Maniatis T. Virus induction of human IFN beta gene
expression requires the assembly of an enhanceosome. Cell.
1995;83:1091–1100.
27. Xu Y, Tao X, Shen B, et al. Structural basis for signal transduction by
the Toll/interleukin-1 receptor domains. Nature. 2000;408:111–115.
28. Slack JL, Schooley K, Bonnert TP, et al. Identification of two major
sites in the type I interleukin-1 receptor cytoplasmic region responsi-
ble for coupling to pro-inflammatory signaling pathways. J Biol Chem.
2000;275:4670–4678.
29. Gay NJ, Gangloff M, Weber AN. Toll-like receptors as molecular
switches. Nat Rev Immunol. 2006;6:693–698.
30. Poltorak A, He X, Smirnova I, et al. Defective LPS signaling in
C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science.
1998;282:2085–2088.
31. Qureshi ST, Lariviere L, Leveque G, et al. Endotoxin-tolerant
mice have mutations in Toll-like receptor 4 (Tlr4). J Exp Med.
1999;189:615–625.
32. Underhill DM, Ozinsky A, Hajjar AM, et al. The Toll-like receptor 2
is recruited to macrophage phagosomes and discriminates between
pathogens. Nature. 1999;401:811–815.
33. Hasan U, Chaffois C, Gaillard C, et al. Human TLR10 is a func-
tional receptor, expressed by B cells and plasmacytoid dendritic
cells, which activates gene transcription through MyD88. J Immunol.
2005;174:2942–2950.
34. Verstak B, Arnot CJ, Gay NJ. An alanine-to-proline mutation in the
BB-loop of TLR3 Toll/IL-1R domain switches signalling adaptor speci-
ficity from TRIF to MyD88. J Immunol. 2013;191:6101–6109.
35. Muzio M, Ni J, Feng P, Dixit VM. IRAK (Pelle) family member
IRAK-2 and MyD88 as proximal mediators of IL-1 signaling. Science.
1997;278:1612–1615.
36. Wesche H, Henzel WJ, Shillinglaw W, Li S, Cao Z. MyD88: an
adapter that recruits IRAK to the IL-1 receptor complex. Immunity.
1997;7:837–847.
37. Muzio M, Natoli G, Saccani S, Levrero M, Mantovani A. The
human toll signaling pathway: divergence of nuclear factor kap-
paB and JNK/SAPK activation upstream of tumor necrosis fac-
tor receptor-associated factor 6 (TRAF6). J Exp Med. 1998;187:
2097–2101.
38. Medzhitov R, Preston-Hurlburt P, Kopp E, et al. MyD88 is an adaptor
protein in the hToll/IL-1 receptor family signaling pathways. Mol Cell.
1998;2:253–258.
39. Burns K, Martinon F, Esslinger C, et al. MyD88, an adapter pro-
tein involved in interleukin-1 signaling. J Biol Chem. 1998;273:
12203–12209.
349
40. Kawai T, Adachi O, Ogawa T, Takeda K, Akira S. Unresponsiveness of
MyD88-deficient mice to endotoxin. Immunity. 1999;11:115–122.
41. Adachi O, Kawai T, Takeda K, et al. Targeted disruption of the MyD88
gene results in loss of IL-1- and IL-18-mediated function. Immunity.
1998;9:143–150.
42. Li S, Strelow A, Fontana EJ, Wesche H. IRAK-4: a novel member of the
IRAK family with the properties of an IRAK-kinase. Proc Natl Acad Sci
U S A. 2002;99:5567–5572.
43. Suzuki N, Suzuki S, Duncan GS, et al. Severe impairment of
interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4.
Nature. 2002;416:750–756.
44. Picard C, Puel A, Bonnet M, et al. Pyogenic bacterial infec-
tions in humans with IRAK-4 deficiency. Science. 2003;299:
2076–2079.
45. Ku CL, von Bernuth H, Picard C, et al. Selective predisposition to
bacterial infections in IRAK-4-deficient children: iRAK-4-dependent
TLRs are otherwise redundant in protective immunity. J Exp Med.
2007;204:2407–2422.
46. Picard C, von Bernuth H, Ghandil P, et al. Clinical features and out-
come of patients with IRAK-4 and MyD88 deficiency. Medicine (Balti-
more). 2010;89:403–425.
47. Kim TW, Staschke K, Bulek K, et al. A critical role for IRAK4 kinase
activity in Toll-like receptor-mediated innate immunity. J Exp Med.
2007;204:1025–1036.
48. Kawagoe T, Sato S, Jung A, et al. Essential role of IRAK-4 protein and
its kinase activity in Toll-like receptor-mediated immune responses
but not in TCR signaling. J Exp Med. 2007;204:1013–1024.
49. Koziczak-Holbro M, Joyce C, Gluck A, et al. IRAK-4 kinase activ-
ity is required for interleukin-1 (IL-1) receptor- and toll-like
receptor 7-mediated signaling and gene expression. J Biol Chem.
2007;282:13552–13560.
50. De S, Karim F, Kiessu E, et al. Mechanism of dysfunction of human
variants of the IRAK4 kinase and a role for its kinase activity
in interleukin-1 receptor signaling. J Biol Chem. 2018;293:15208–
15220.
51. De Nardo D, Balka KR, Cardona Gloria Y, Rao VR, Latz E, Mas-
ters SL. Interleukin 1 receptor-associated kinase 4 (IRAK4) plays a
dual role in Myddosome formation and Toll-like receptor signalling.
J Biol Chem. 2018;293:15195–15207.
52. Fraczek J, Kim TW, Xiao H, et al. The kinase activity of IL-1 receptor-
associated kinase 4 is required for interleukin-1 receptor/toll-
like receptor-induced TAK1-dependent NFkappaB activation. J Biol
Chem. 2008;283:31697–31705.
53. Yao J, Kim TW, Qin J, et al. Interleukin-1 (IL-1)-induced TAK1-
dependent versus MEKK3-dependent NFkappaB activation path-
ways bifurcate at IL-1 receptor-associated kinase modification. J Biol
Chem. 2007;282:6075–6089.
54. Sun J, Li N, Oh KS, et al. Comprehensive RNAi-based screening of
human and mouse TLR pathways identifies species-specific prefer-
ences in signaling protein use. Sci Signal. 2016;9:ra3.
55. Cushing L, Stochaj W, Siegel M, et al. Interleukin 1/Toll-like receptor-
induced autophosphorylation activates interleukin 1 receptor-
associated kinase 4 and controls cytokine induction in a cell
type-specific manner. J Biol Chem. 2014;289:10865–10875.
56. Qin J, Jiang Z, Qian Y, Casanova JL, Li X. IRAK4 kinase activ-
ity is redundant for interleukin-1 (IL-1) receptor-associated kinase
phosphorylation and IL-1 responsiveness. J Biol Chem. 2004;279:
26748–26753.
57. Krausgruber T, Saliba D, Ryzhakov G, Lanfrancotti A, Blazek K,
Udalova IA. IRF5 is required for late-phase TNF secretion by human
dendritic cells. Blood. 2010;115:4421–4430.
350
58. Saliba DG, Heger A, Eames HL, et al. IRF5:relA interaction targets
inflammatory genes in macrophages. Cell Rep. 2014;8:1308–1317.
59. Cao Z, Henzel WJ, Gao X. IRAK: a kinase associated with the
interleukin-1 receptor. Science. 1996;271:1128–1131.
60. Croston GE, Cao Z, Goeddel DV. NF-kappa B activation by
interleukin-1 (IL-1) requires an IL-1 receptor-associated protein
kinase activity. J Biol Chem. 1995;270:16514–16517.
61. Thomas JA, Allen JL, Tsen M, et al. Impaired cytokine signaling
in mice lacking the IL-1 receptor-associated kinase. J Immunol.
1999;163:978–984.
62. Swantek JL, Tsen MF, Cobb MH, Thomas JA. IL-1 receptor-associated
kinase modulates host responsiveness to endotoxin. J Immunol.
2000;164:4301–4306.
63. Kawagoe T, Sato S, Matsushita K, et al. Sequential control of Toll-like
receptor-dependent responses by IRAK1 and IRAK2. Nat Immunol.
2008;9:684–691.
64. Ye H, Arron JR, Lamothe B, et al. Distinct molecular mechanism for
initiating TRAF6 signalling. Nature. 2002;418:443–447.
65. Pauls E, Nanda SK, Smith H, Toth R, Arthur JSC, Cohen P. Two phases
of inflammatory mediator production defined by the study of IRAK2
and IRAK1 knock-in mice. J Immunol. 2013;191:2717–2730.
66. Keating SE, Maloney GM, Moran EM, Bowie AG. IRAK-2 partici-
pates in multiple toll-like receptor signaling pathways to NFkap-
paB via activation of TRAF6 ubiquitination. J Biol Chem. 2007;282:
33435–33443.
67. Strickson S, Emmerich CH, Goh ETH, et al. Roles of the TRAF6 and
Pellino E3 ligases in MyD88 and RANKL signaling. Proc Natl Acad Sci
U S A. 2017;114:E3481–E3489.
68. Kagan JC, Magupalli VG, Wu H. SMOCs: supramolecular orga-
nizing centres that control innate immunity. Nat Rev Immunol.
2014;14:821–826.
69. Ferrao R, Wu H. Helical assembly in the death domain (DD) superfam-
ily. Curr Opin Struct Biol. 2012;22:241–247.
70. Motshwene PG, Moncrieffe MC, Grossmann JG, et al. An oligomeric
signaling platform formed by the Toll-like receptor signal transducers
MyD88 and IRAK-4. J Biol Chem. 2009;284:25404–25411.
71. Lin SC, Lo YC, Wu H. Helical assembly in the MyD88-IRAK4-IRAK2
complex in TLR/IL-1R signalling. Nature. 2010;465:885–890.
72. Latty SL, Sakai J, Hopkins L, et al. Activation of Toll-like receptors
nucleates assembly of the MyDDosome signaling hub. Elife. 2018;7.
73. Burns K, Clatworthy J, Martin L, et al. Tollip, a new component of
the IL-1RI pathway, links IRAK to the IL-1 receptor. Nat Cell Biol.
2000;2:346–351.
74. Neumann D, Kollewe C, Pich A, Cao P, Resch K, Martin MU. Threo-
nine 66 in the death domain of IRAK-1 is critical for interaction with
signaling molecules but is not a target site for autophosphorylation. J
Leukoc Biol. 2008;84:807–813.
75. Ross K, Yang L, Dower S, Volpe F, Guesdon F. Identification of threo-
nine 66 as a functionally critical residue of the interleukin-1 receptor-
associated kinase. J Biol Chem. 2002;277:37414–37421.
76. Dossang AC, Motshwene PG, Yang Y, et al. The N-terminal loop of
IRAK-4 death domain regulates ordered assembly of the Myddosome
signalling scaffold. Sci Rep. 2016;6:37267.
77. Hoarau C, Gerard B, Lescanne E, et al. TLR9 activation induces
normal neutrophil responses in a child with IRAK-4 deficiency:
involvement of the direct PI3K pathway. J Immunol. 2007;179:
4754–4765.
78. Ferrao R, Zhou H, Shan Y, et al. IRAK4 dimerization and trans-
autophosphorylation are induced by Myddosome assembly. Mol Cell.
2014;55:891–903.
BALKA AND DE NARDO
79. Cheng H, Addona T, Keshishian H, et al. Regulation of IRAK-4 kinase
activity via autophosphorylation within its activation loop. Biochem
Biophys Res Commun. 2007;352:609–616.
80. Kuglstatter A, Villasenor AG, Shaw D, et al. Cutting Edge: iL-1
receptor-associated kinase 4 structures reveal novel features and
multiple conformations. J Immunol. 2007;178:2641–2645.
81. Jiang Z, Georgel P, Li C, et al. Details of Toll-like receptor:adapter
interaction revealed by germ-line mutagenesis. Proc Natl Acad Sci
U S A. 2006;103:10961–10966.
82. Li C, Zienkiewicz J, Hawiger J. Interactive sites in the MyD88
Toll/interleukin (IL) 1 receptor domain responsible for coupling to the
IL1beta signaling pathway. J Biol Chem. 2005;280:26152–26159.
83. Ohnishi H, Tochio H, Kato Z, et al. Structural basis for the multiple
interactions of the MyD88 TIR domain in TLR4 signaling. Proc Natl
Acad Sci U S A. 2009;106:10260–10265.
84. von Bernuth H, Picard C, Jin Z, et al. Pyogenic bacterial infections in
humans with MyD88 deficiency. Science. 2008;321:691–696.
85. Vyncke L, Bovijn C, Pauwels E, et al. Reconstructing the TIR side
of the Myddosome: a paradigm for TIR-TIR interactions. Structure.
2016;24:437–447.
86. Loiarro M, Volpe E, Ruggiero V, et al. Mutational analysis identifies
residues crucial for homodimerization of myeloid differentiation fac-
tor 88 (MyD88) and for its function in immune cells. J Biol Chem.
2013;288:30210–30222.
87. Janssens S, Burns K, Tschopp J, Beyaert R. Regulation of interleukin-
1- and lipopolysaccharide-induced NF-kappaB activation by alterna-
tive splicing of MyD88. Curr Biol. 2002;12:467–471.
88. Burns K, Janssens S, Brissoni B, Olivos N, Beyaert R, Tschopp J. Inhi-
bition of interleukin 1 receptor/Toll-like receptor signaling through
the alternatively spliced, short form of MyD88 is due to its failure to
recruit IRAK-4. J Exp Med. 2003;197:263–268.
89. Loiarro M, Gallo G, Fanto N, et al. Identification of critical residues of
the MyD88 death domain involved in the recruitment of downstream
kinases. J Biol Chem. 2009;284:28093–28103.
90. Guven-Maiorov E, Keskin O, Gursoy A, et al. The architecture of the
tir domain signalosome in the Toll-like receptor-4 signaling pathway.
Sci Rep. 2015;5:13128.
91. Ve T, Vajjhala PR, Hedger A, et al. Structural basis of TIR-domain-
assembly formation in MAL- and MyD88-dependent TLR4 signaling.
Nat Struct Mol Biol. 2017;24:743–751.
92. Gay NJ, Gangloff M, O’Neill LA. What the Myddosome structure
tells us about the initiation of innate immunity. Trends Immunol.
2011;32:104–109.
93. Bonham KS, Orzalli MH, Hayashi K, et al. A promiscuous lipid-binding
protein diversifies the subcellular sites of toll-like receptor signal
transduction. Cell. 2014;156:705–716.
94. Tan Y, Kagan JC. Biochemical isolation of the Myddosome from
murine macrophages. Methods Mol Biol. 2018;1714:79–95.
95. Ngo VN, Young RM, Schmitz R, et al. Oncogenically active MYD88
mutations in human lymphoma. Nature. 2011;470:115–119.
96. Treon SP, Xu L, Yang G, et al. MYD88 L265P somatic muta-
tion in Waldenstrom’s macroglobulinemia. N Engl J Med. 2012;367:
826–833.
97. Kelly PN, Romero DL, Yang Y, et al. Selective interleukin-1 receptor-
associated kinase 4 inhibitors for the treatment of autoim-
mune disorders and lymphoid malignancy. J Exp Med. 2015;212:
2189–2201.
98. Mancek-Keber M, Lainscek D, Bencina M, et al. Extracellular vesicle-
mediated transfer of constitutively active MyD88(L265P) engages
MyD88(wt) and activates signaling. Blood. 2018;131:1720–1729.
BALKA AND DE NARDO
99. Nguyen TA, Pang KC, Masters SL. Intercellular communication for
innate immunity. Mol Immunol. 2017;86:16–22.
100. Mansell A, Smith R, Doyle SL, et al. Suppressor of cytokine signaling
1 negatively regulates Toll-like receptor signaling by mediating Mal
degradation. Nat Immunol. 2006;7:148–155.
101. Kobayashi K, Hernandez LD, Galan JE, Janeway CA, Jr, Medzhitov R,
Flavell RA. IRAK-M is a negative regulator of Toll-like receptor signal-
ing. Cell. 2002;110:191–202.
102. De Nardo D, Nguyen T, Hamilton JA, Scholz GM. Down-regulation of
IRAK-4 is a component of LPS- and CpG DNA-induced tolerance in
macrophages. Cell Signal. 2009;21:246–252.
103. Ruysschaert JM, Lonez C. Role of lipid microdomains in TLR-
mediated signalling. Biochim Biophys Acta. 2015;1848:1860–1867.
104. Triantafilou M, Manukyan M, Mackie A, et al. Lipoteichoic acid and
toll-like receptor 2 internalization and targeting to the Golgi are lipid
raft-dependent. J Biol Chem. 2004;279:40882–40889.
105. Triantafilou M, Gamper FG, Haston RM, et al. Membrane sorting of
toll-like receptor (TLR)-2/6 and TLR2/1 heterodimers at the cell sur-
face determines heterotypic associations with CD36 and intracellu-
lar targeting. J Biol Chem. 2006;281:31002–31011.
106. Barbalat R, Lau L, Locksley RM, Barton GM. Toll-like receptor 2 on
inflammatory monocytes induces type I interferon in response to
viral but not bacterial ligands. Nat Immunol. 2009;10:1200–1207.
107. Botelho RJ, Teruel M, Dierckman R, et al. Localized biphasic changes
in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis.
J Cell Biol. 2000;151:1353–1368.
108. Zanoni I, Ostuni R, Marek LR, et al. CD14 controls the LPS-
induced endocytosis of Toll-like receptor 4. Cell. 2011;147:
868–880.
109. Wesche H, Gao X, Li X, Kirschning CJ, Stark GR, Cao Z. IRAK-M is a
novel member of the Pelle/interleukin-1 receptor-associated kinase
(IRAK) family. J Biol Chem. 1999;274:19403–19410.
351
110. Qian Y, Commane M, Ninomiya-Tsuji J, Matsumoto K, Li X. IRAK-
mediated translocation of TRAF6 and TAB2 in the interleukin-1-
induced activation of NFkappa B. J Biol Chem. 2001;276:
41661–41667.
111. Vollmer S, Strickson S, Zhang T, et al. The mechanism of activation
of IRAK1 and IRAK4 by interleukin-1 and Toll-like receptor agonists.
Biochem J. 2017;474:2027–2038.
112. Scott JS, Degorce SL, Anjum R, et al. Discovery and optimization
of pyrrolopyrimidine inhibitors of interleukin-1 receptor associated
kinase 4 (IRAK4) for the treatment of mutant MYD88(L265P) diffuse
large B-cell lymphoma. J Med Chem. 2017;60:10071–10091.
113. Seganish WM. Inhibitors of interleukin-1 receptor-associated kinase
4 (IRAK4): a patent review (2012-2015). Expert Opin Ther Pat.
2016;26:917–932.
114. Chaudhary D, Robinson S, Romero DL. Recent advances in the discov-
ery of small molecule inhibitors of interleukin-1 receptor-associated
kinase 4 (IRAK4) as a therapeutic target for inflammation and oncol-
ogy disorders. J Med Chem. 2015;58:96–110.
115. Ippagunta SK, Pollock JA, Sharma N, et al. Identification of Toll-like
receptor signaling inhibitors based on selective activation of hierar-
chically acting signaling proteins. Sci Signal. 2018;11.
116. Nyman T, Stenmark P, Flodin S, Johansson I, Hammarstrom M, Nord-
lund P. The crystal structure of the human toll-like receptor 10
cytoplasmic domain reveals a putative signaling dimer. J Biol Chem.
2008;283:11861–11865.
How to cite this article: Balka KR, De Nardo D. Under-
standing early TLR signaling through the Myddosome. J
Leukoc Biol. 2019;105:339–351. https://doi.org/10.1002/JLB.
MR0318-096R