TOLL-LIKE

RECEPTOR
(TLR)

Dr. Ema Qurnianingsih, dr., M.Si

Departemen Biokimia Kedokteran FK Unair
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• The innate immune system employs pattern
recognition receptors (PRRs) for initial
detection of microbes.

• Classes of PRRs : Toll-like Receptors (TLR), RIG-

1-like receptors (RLRs), Nod-like receptors
(NLRs), AIM2-like receptors (ALRs), C-type
lectin receptors (CLRs), intracellular DNA
sensors (exp. cGAS)  TLRs were first
identified and well-characterized.

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• PRRs recognizes
– microbe-specific molecular signature 
pathogen-associated molecular patterns (PAMPs)
– Self-derived molecules from damaged cells 
damage-associated molecular patterns (DAMPs)
 Activate down-stream signaling pathways 
induction of innate immune response by
inflamatory cytokines and prime (and
orchestrate) antigen-specific adaptive immune
response.

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 Abbas et al, Basic Immunology 5th ed, 2016

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Figure. Cellular location of innate immune system receptors
 Toll Like Receptors
• Toll-Like Receptors (TLRs) play a critical role in
the early innate immune response to invading
pathogens by sensing microorganism and are
involved in sensing endogenous danger
signals.
• TLRs are evolutionarily conserved receptors
are homologues of the Drosophila Toll
protein, discovered to be important for
defense against microbial infection

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• In Drosophila
melanogaster (fruitfly) :
‘Toll’; mammals: TLR
• Several TLRs
• Role: activate phagocytes
and tissue dendritic cells
(DC)
• Expl.: TLR-4. having bound
LPS, CD14 interacts with
TLRactivation of
transcription factor NFkB
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• TLRs recognize :
– Highly conserved structural motifs known as
pathogen-associated microbial patterns (PAMPs),
which are exclusively expressed by microbial
pathogens, or
– Danger-associated molecular patterns (DAMPs)
that are endogenous molecules released from
necrotic or dying cells.

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• PAMPs include various bacterial cell wall components
such as lipopolysaccharide (LPS), peptidoglycan (PGN)
and lipopeptides, as well as flagellin, bacterial DNA
and viral double-stranded RNA.

• DAMPs include intracellular proteins such as heat

shock proteins as well as protein fragments from the
extracellular matrix.

• Different TLRs are specific for different components of

microbes

• TLRs can serve as heterodimers (TLR1, TLR2, TLR6),

homodimers (TLR4, TLR5?), monomers.

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 Figure.
Structure and
Specificity of
TLRs

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Abbas et al, Basic Immunology 5thed, 2016
 Tabel. Distribusi TLR pada berbagai tipe sel
Tipe TLR Tipe Sel
TLR1 Berada dimana-mana (ubiquitous)

TLR2 DC, PMN cells, monosit

TLR3 DC, NK cells, upregulated pada sel epitel dan endotel

TLR4 Makrofag, PMN cells, DC

TLR5 monosit, immature DC, sel epitel, NK cells, sel T

TLR6 Limfosit B (highly expressed), monosit dan NK cells (ekspresi rendah)

TLR7 Limfosit B, plasmasitoid prekursor sel dendritik

TLR8 Monosit, ekspresi rendah pada sel T dan NK cells

TLR9 plasmasitoid prekursor sel dendritik, sel B, makrofag, PMN, NK, sel
mikroglial
TLR10 Limfosit B, plasmasitoid prekursor sel dendritik

TLR11 Belum teridentifikasi 11

Structure of TLRs
• Type 1 integral membrane
receptors.
• Each TLR is composed of :
1. An ectodomain (N-terminal
ligand recognition domain)
– The N-terminal ectodomains
(ECDs) of TLRs are
glycoproteins with 550–800
amino acid residues
– Ectodomain of TLRs are
constructed at tandem
copies of leucine-rich
repeats (LRRs) 12
Structure of TLRs
– Leucine-rich repeats (LRRs)
of ectodomain is typically
22–29 residues in length
and contains hydrophobic
residues spaced at
distinctive intervals  form
horseshoe structures 
mediate PAMPs recognition
– Mostly, Ligand binding and
dimerization upon it, occurs
on glycan-free surface .

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 • The concave surface is rich in
potential N-glycosylation sites
and probably heavily
glycosylated.
• For example, Asn247 and
Asn413, two residues which are
implicated in glycosylation.
• LRR12 and LRR20 are atypical
LRR motifs containing large
insertions which protrude from
the solenoid.
• According to the symmetrical
assembly model, ligand binding
occurs at the glycan-free surface
involving LRR20
Clin. Microbiol. Rev., 2008 14
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http://www.cell.com/fulltext/S0092-8674(07)01152-X
Structure of TLRs
• Each TLR is composed of :
2. A single
transmembrane helix
domain, contains 20
uncharged, mostly
hydrophobic residues.
3. A C-terminal
cytoplasmic signaling
domain or cytoplasmic
Toll/IL-1 receptor (TIR)
domain  initiates
downstream signaling 16
The signaling domains of TLRs
are known as Toll IL-1
Receptor (TIR) domains
• because they share
homology with the
signaling domains of IL-1R
family members

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 TLRs Family
• TLRs family comprises of 10 members (TLR1 –
TLR10) in humans and 12 members (TLR1 –
TLR 9, TLR11 – TLR 13) in mouse.
• TLR localize :
– To the cell surface
TLR1, TLR2, TLR4, TLR5, TLR6, TLR10(?)
– To intracellular compartments (exp. Endosomes)
TLR3, TLR7, TLR8, TLR9, TLR11, TLR12, TLR13

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• Cell surface TLRs mainly recognize microbial
membrane components, such as lipids,
lipopoteins, lipopolysacharides, proteins
including bacterial flagellin.

• Intacellular TLRs recognize nucleic acids derived

from bacteria and viruses, and also recognize
self-nucleic acids in disease conditions
(autoimmunity).
– Intracellular localization of TLRs is thought to be
critical for ligand recognition as well as for preventing
TLRs from coming into contact with self-nucleic acids,
which could cause autoimmunity 19
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• Based on their sequence homology, vertebrate
TLRs are grouped into 6 subfamilies :
– TLR1/2/6/10
– TLR3
– TLR4
– TLR5
– TLR7/8/9 (not yet well-defined)
– TLR11/12/13 (not yet well-defined)

Botos et al, Structure, 2011

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TLR1/2/6/10

• TLR2 resides on plasma membrane 

responds to lipid-containing PAMPs
(lipoteichioc acid, di- and tri-acylated cysteine-
containing lipopeptides.
– It does this by forming dimeric complexes with
either TLR1 or TLR6 on the plasma membrane.
– TLRs 1 and 2 interact with each other on their
dimerization surfaces via several hydrogen bonds
and hydrophobic interactions in the vicinity of the
binding pockets

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TLR3
• TLR3 recognizes dsRNA, which is produced by most viruses at
some stage in their life cycles and is a potent indicator of viral
infection.
• TLR3 is localized to endosomes where it recognizes dsRNA
• Studies using soluble TLR3-ECD protein or immobilized TLR3-GFP
constructs showed that TLR3-ECD is monomeric in solution but
binds as dimers to 45-bp segments of dsRNA, the minimum
length required for TLR3 binding and activation  In addition,
binding is independent of base sequence and occurs only at pH
6.5 and below.
• Because cells normally contain short (≤25 bp) stretches of dsRNA
for example in miRNA and tRNA hairpins, the inability of TLR3 to
bind dsRNA <40 bp most likely provides an important mechanism
for preventing autoreactive responses against self dsRNA
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Botos et al, Structure, 2011
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Botos et al, Structure, 2011
TLR4 an MD-2
• Lipopolysaccharide (LPS), an essential component of the outer
membrane of Gram-negative bacteria, induces a powerful
inflammatory response that can lead to septic shock and death.
• LPS signals through TLR4 by complexing co-receptor MD-2, which
is anchored by several hydrogen bonds to the lateral and
concave surface of TLR4-ECD and contacts residues from the
LRR2-LRR10 area.
• LPS-binding protein (LBP) and CD14 deliver and load the LPS to
the TLR4-bound MD-2.
• MD-2 is a coreceptor molecule that binds both the extracellular
domain of TLR4 and the hydrophobic portion of LPS .

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Figure 1. (A) A cartoon showing assembly of TLR4–MD2–LPS
complex. The LPS is transferred to MD2 (i) Conjugation of LPS–MD2
with TLR4 (ii) then leads to dimerization of TLR4–MD2, and
formation of TLR4–MD2–LPS complex (iii). (B) After the recognition
of ligand and assembly of TLR4-complex, co-receptors: TIRAP,
MYD88, TRAM and TRIF, are recruited at the intracellular level for
activation of TLR4-signaling resulting into antigen-specific immune
responses. 27
Awasthi, Front. Immunol., 2014
TLR5
• TLR5 is one of the few TLRs that recognize a protein PAMP,
bacterial flagellin.
• It is highly expressed in gut, especially in lamina propria
dendritic cells, where it controls the composition of the
microbiota.
• Expression of TLR5 on IECs regulates the composition and
localization of the intestinal microbiota, preventing diseases
associated with intestinal inflammation  In mice lack of
TLR5  more prone to colitis
(Chassaing et al, AGA journals, 2014)

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TLR Signaling Functions
• Upon PAMPs and DAMPs recognition, TLRs recruit
TIR domain-containing adaptor protein (MyD88 and
TRIF)  initiates signaling cascades leading to the
activation of transcription factors, such as AP-1, NF-
κB and interferon regulatory factors (IRFs), MAP
kinase  result in a variety of cellular responses of
activated phagocytes and other cells, including:
– the production of interferons (IFNs),
– pro-inflammatory cytokines and
– Effector cytokines that direct the adaptive immune
response

Protect host from microbial infection. 29

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Abbas et al, Basic Immunology 5thed, 2016
TLRs Signaling
• Individual TLRs
differentially
recruit members of
a set of TIR
domain-containing
adaptors such as
MyD88, TRIF,
TIRAP/MAL or
TRAM.

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TLRs Signaling
–MyD88 is utilized by
almost all TLRs 
activate NF-kB and
MAPKs  induction of
inflammatory cytokine
genes.

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TLRs Signaling
–TRIF is recruited to TLR3
and TLR4 and promotes
alternative pathway 
activation of IRF3, NF-
kB, MAPKs  induction
of type I IFN and
inflammatory cytokine
genes.

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TLRs Signaling
–TIRAP is a sorting
adaptor that recruits
MyD88 to cell surface
TLRs, such as TLR2 and
TLR4
• Recent study, TIRAP
also participate in
signaling through
endosomal TLRs,
such as TLR9 (in
natural situation,
but not in high level
of TLR9 agonists)
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• Collectively, depending on the
adaptor usage, TLR signaling is
largely divided into two pathways :
– MyD88-dependent pathway
– TRIF-dependent pathway

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 MyD88-dependent
pathway
• After TLR engagement  MyD88
forms complex with IRAK kinases
(Myddosome)  during
Myddosome formation, IRAK 4
activate IRAK1 ( then, auto-
phosphorylated and released
from MyD88)  IRAK1 associates
with TRAF6  poly-
ubiquitination of TRAF6 and TAK1
protein kinase complex 
activation of TAK1  activation
of IKK-complex-NF-kB pathway
and MAPK pathway

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# Lessons from TLR signaling in cCDs and macrophages Kawasaki and Kawai, Frontiers in immunology, 2014
 MyD88-dependent
pathway

• TRAF6 promotes ECSIT

ubiquitination  increased
mitochondrial and cellular
ROS generation

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# Lessons from TLR signaling in cCDs and macrophages Kawasaki and Kawai, Frontiers in immunology, 2014
 MyD88-dependent
pathway

IKK-complex composed of
catalytic subunit IKKalfa,
IKKbeta and regulatory
subunit MEMO.
IKK-complex fascilitate
translocation of NF-kB
(through phosphorylation of
inhibitory protein IκBα
degradation of IκBα)

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# Lessons from TLR signaling in cCDs and macrophages Kawasaki and Kawai, Frontiers in immunology, 2014
 TRIF-dependent
pathway
• TRIF interact with TRAF6 and
TRAF3
•  TRAF6 recruits kinase RIP-1 
RIK-1 interact and activates TAKi
complex  activation of NF-kB
and MAPKs and induction of
inflammatory cytokines
•  TRAF3 recruits IKK-related
kinase TBK1 and IKKi for IRF3
phosphorylation  IRF3 forms
dimer and translocates into
nucleus  induces expression of
type I IFN gene.
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# Lessons from TLR signaling in cCDs and macrophages Kawasaki and Kawai, Frontiers in immunology, 2014
Plasmacytoid DCs are a subset of DCs with the capacity to secrete
vast amounts of type I IFN in response to viral infection

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Kawasaki and Kawai, Frontiers in immunology, 2014
• TLR4 activates both MyD88 pathways and TRIF
pathways  must be balanced
– TRAF3 was shown to be incorporated into MyD88
complex as well as the TRIF complex in TLR4
signaling
– TRAF3 within MyD88 complex is then degraded 
activation of TAK1
 thus, in addition its role in promoting TRIF-
dependent pathway activation, TRAF3 has a role in
inhibiting MyD88-dependent pathway

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• TLR4 activates both MyD88 pathways and TRIF
pathways  must be balanced
– NRDP-1 (ubiquitin ligase) binds and ubiquitinates
MyD88 and TBK1  degradation of MyD88 and
augmenting the activation of TBK1  attenuates
inflammatory cytokine production and induces
preferential type I IFN production,respectively.

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 Co-receptors of TLRs
• Several transmembrane molecules are known
as modulator of TLR signaling pathways :
– CD14
• is co-receptor with TLR4 ( together with MD2 for LPS
recognition)
• Is co-receptor for TLR7 and TLR9
– CD36
• Co-receptor for ox-LDL and amyloid-β peptide

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 Negative Regulators of TLR signaling
• Activation of MyD88-dependent pathway is supressed by
ST2825, SOCS1, Cb1-b
• Activation of TRIF-dependent pathway is supressed by SARM,
TAG
These molecules  associate with MyD88 or TRIF  prevent
them from binding to TLRs or downtream molecules.
• TRAF3 activation is negatively regulated by SOCS3 and DUBA
• TRAF6 is inhibited by A20, USP4, CYLD, TANK, TRIM38, SHP
• TAK1 activation is inhibited by TRIM30α and A20
• NF-kB is supressed by Bcl-3, IκBα, Nurr1, ATF3, PDLIM2
• IRF3 activation is inhibited by Pin1 and RAUL
• The stability of mRNA for cytokines is regulated by miRNAs,
Regnase-1 and TTP
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