The document summarizes Toll-like receptors (TLRs) and their role in innate immunity. It discusses how TLRs were first discovered in invertebrates and then found to have vertebrate homologs. TLRs recognize pathogen-associated molecular patterns and trigger signaling pathways that induce antimicrobial responses. There are 10 TLRs in humans that recognize distinct microbial ligands. TLR signaling involves adaptor proteins and transcription factors that activate genes encoding inflammatory cytokines, chemokines, and interferons.

1. TLR
Dr Sitaram Swain

2. Microbes induce a broad spectrum of cellular innate
immune responses by a wide variety of cell types In
addition to triggering their own uptake and killing by
phagocytic cells.
Several families of pattern recognition receptors (PRRs)
play major roles in innate immunity. These PRRs bind to
PAMPs as well as to some endogenous (self) DAMPs and
trigger signal-transduction pathways that turn on
expression of genes with important functions in innate
immunity.
Among the proteins encoded by these genes are
antimicrobial molecules such as antimicrobial peptides
and interferons, chemokines and cytokines that recruit and
activate other cells, enzymes such as iNOS that generate
antimicrobial molecules, and proinflammatory mediators
(i.e., components that promote inflammation).

3. Toll-Like Receptors Recognize Many
Types of Pathogen Molecules
Toll-like receptors (TLRs) were the first family of PRRs to be
discovered and are still the best-characterized in terms of their
structure, how they bind PAMPs and activate cells, and the extensive
and varied set of innate immune responses that they induce.
Discovery of Invertebrate Toll and Vertebrate Toll-like Receptors
In the 1980s, in Germany it was discovered that Drosophila fruit fly
embryos could not establish a proper dorsal-ventral (back to front)
axis if the gene encoding the Toll membrane protein is mutated.
(The name “Toll” comes from German slang meaning “weird,”
referring to the mutant flies’ bizarrely scrambled anatomy.)
For their subsequent characterization of the toll and related
homeobox genes and their role in the regulation of embryonic
development, Christiane Nusslein-Volhard, Eric Wieschaus, and
Edward B. Lewis were awarded the Nobel Prize in Physiology and
Medicine in 1995.

4. Many mutations of the toll gene were generated, and in
1996 Jules Hoff man and Bruno Lemaitre discovered that
mutations in toll made flies highly susceptible to lethal
infection with Aspergillus fumigatus, a fungus to which
wild-type flies were immune .
This studies showing that Toll and related proteins are
involved in the activation of innate immune responses in
invertebrates.
For his pivotal contributions to the study of innate
immunity in Drosophila, Jules Hoff man was a co-winner of
the 2011 Nobel Prize in Physiology and Medicine.

5. Impaired innate immunity in fruit flies with a mutation in the Toll pathway. Severe
infection with the fungus Aspergillus fumigatus (yellow) results from a mutation in
the signaling pathway downstream of the Toll pathway in Drosophila that
normally activates production of the antimicrobial peptide drosomycin.

6. Characterization of the Toll protein surprisingly revealed
that its cytoplasmic signaling domain was homologous to
that of the vertebrate receptor for the cytokine IL-1 (IL-1R).
Through a search for human proteins with cytoplasmic
domains homologous to those of Toll and IL-1R, in 1997
Charles Janeway and Ruslan Medzhitov discovered a
human gene for a protein similar to Toll that activated the
expression of innate immunity genes in human cells.
Appropriately, this and other vertebrate Toll relatives
discovered soon thereafter were named Toll-like receptors
(TLRs).

7. Through studies with mutant mice, in 1998 Bruce Beutler obtained the
important proof that TLRs contribute to normal immune functions in
mammals. Mice homozygous for a mutant form of a gene called lps
were resistant to the harmful responses induced by lipolysaccharide
(LPS; also known as endotoxin), a major component of the cell walls of
Gram negative bacteria.
In humans, a buildup of endotoxin from severe bacterial infection can
induce too strong of an innate immune response, causing septic
shock, a life-threatening condition in which vital organs such as the
brain, heart, kidney, and liver may fail.

8. Beutler found that the defective mouse lps gene
encoded a mutant form of one TLR, TLR4, which differed
from the normal form by a single amino acid so that it no
longer was activated by LPS.
TLR4 is the cellular innate pattern recognition receptor
that recognizes LPS and earned Beutler a share of the
2011 Nobel Prize.
Invertebrates use receptors to respond to pathogens,
also found in vertebrates, and that one of these receptors
is responsible for LPS-induced innate immune responses.

9. TLRs and Their Ligands
TLRs 1-10 are conserved between mice and humans,
although TLR10 is not functional in mice, while TLRs 11-13
are expressed in mice but not in humans. Each TLR has a
distinct repertoire of specificities for conserved PAMPs; the
TLRs and some of their known PAMP ligands .
Biochemical studies have revealed the structure of several
TLRs and how they bind their specific ligands.
TLRs are membrane-spanning proteins that share a
common structural element in their extracellular region
called leucine-rich repeats (LRRs); multiple LRRs make up
the horseshoe-shaped extracellular ligand-binding domain
of the TLR polypeptide chain .

11. When TLRs bind their PAMP or DAMP ligands via their
extracellular LRR domains, they are induced to dimerize.
In most cases each TLR dimerizes with itself, forming a
homodimer, but TLR2 forms heterodimers by pairing with
either TLR1 or TLR6.

12. Structures of TLR2/1 with a bound lipopeptide and TLR3 with
dsRNA are the characteristic “m”-shaped conformation of TLR
dimers is apparent.
TLRs exist both on the plasma membrane and in the membranes of
endosomes and lysosome lysosomes; their cellular location is
tailored to enable them to respond optimally to the particular
microbial ligands they recognize.
TLRs that recognize PAMPs on the outer surface of extracellular
microbes are found on the plasma membrane, where they can
bind these PAMPs and induce responses.
TLRs that recognize internal microbial components that have to be
exposed by the dismantling or degradation of endocytosed
pathogens— nucleic acids in particular—are found in endosomes
and lysosomes.
Unique among the TLRs, TLR4 has been shown to move from the
plasma membrane to endosomes/ lysosomes after binding LPS or
other PAMPs. it activates different signaling pathways from the two
locations.

13. In addition to microbial ligands, TLRs also recognize
DAMPs, endogenous (self) components released by
dead/ dying cells or damaged tissues.
Among the DAMPS recognized by plasma membrane
TLRs are heat shock and chromatin proteins, fragments of
extracellular matrix components (such as fibronectin and
hyaluronin), and oxidized low density lipoprotein and
beta amyloid.
While self nucleic acids usually do not activate the
intracellular PRR, under certain circumstances (such as
when bound by anti-DNA or antichromatin antibodies in
individuals with the autoimmune disease lupus
erythematosus) they can be endocytosed and activate
endosomal/lysosomal TLRs, contributing to the disease .

14. Signaling Through TLRs: Overview
Signaling through TLRs utilizes many of the principles and
some of the signaling molecules, along with some unique to
pathways activated by TLRs (and by other PRRs).
An important example of a shared component is the
transcription factor NF-kB. NF-kB is key for inducing many
innate and inflammatory genes, including those encoding
defensins; enzymes such as iNOS; chemokines; and
cytokines such as the proinflammatory cytokines TNF-alpha,
IL-1, and IL-6, produced by macrophages and dendritic cells.
There are also TLR-specific signaling pathways and
components that induce the expression of subsets of
proteins, some of which are particularly effective in
combating the type of pathogen recognized by the particular
TLR(s).

15. The generation of RNS requires the transcriptional activation of the
gene for the enzyme inducible nitric oxide synthase (iNOS, or NOS2.
Expression of iNOS is activated by microbial components binding to
various PRRs. iNOS oxidizes L-arginine to yield L-citrulline and nitric
oxide (NO), a potent antimicrobial agent.
Collectively the ROS and RNS are highly toxic to phagocytosed
microbes due to the alteration of microbial molecules through
oxidation, hydroxylation, chlorination, nitration, and S-nitrosylation,
along with formation of sulfonic acids and destruction of iron-sulfur
clusters in proteins.
One example of how these oxidative species may be toxic to
pathogens is the oxidation by ROS of cysteine sulfhydryls that are
present in the active sites of many enzymes, inactivating the enzymes.
ROS and RNS also can be released from activated neutrophils and
macrophages and kill extracellular pathogens.

17. An important example is expression of the potent antiviral Type 1
interferons, IFNs-alpha and beta, induced by pathways downstream
of the TLRs that bind viral components. Activation of the interferon
regulatory factors (IRFs) is essential for inducing transcription of the
genes encoding IFN-alpha and beta. Combinations of transcription
factors contribute to inducing the expression of many of these
genes; examples include combinations of NF-kB, IRFs, and/or
transcription factors downstream of MAP kinase (MAPK) pathways ,
such as AP-1, that can be activated by signaling intermediates
downstream of certain TLRs.
The particular signal transduction pathway(s) activated by a TLR
dimer following PAMP binding are largely determined by the TLR
and by the initial protein adaptor that binds to the TLR’s
cytoplasmic domain. TIR domain (from Toll/IL-1 receptor), referring
to the similarity noted earlier between the cytoplasmic domains of
TLRs and IL-1 receptors .TIR domains of all TLR dimers serve as
binding sites for the TIR domains of adaptors that activate the
downstream signaling pathways.

18. Signaling from IL-1 Receptors
Productive ligand binding to the extracellular portion of the IL-1
receptor leads to a conformational alteration in its cytoplasmic
domain. This structural alteration in the receptor leads to a series of
downstream signaling events.
First, binding of the adapter protein MyD88 to the occupied receptor
allows recruitment to the receptor complex of one or more members
of the IL-1 Receptor Activated Kinase (IRAK) protein family.
IRAK-4, is activated by autophosphorylation and phosphorylates its
fellow IRAKs, resulting in the generation of binding sites for TNF
Receptor Associated Factor 6 (TRAF6), which is associated with a
ubiquitin-ligase complex capable of generating polyubiquitin chains.
The IRAK-TRAF6 complex now dissociates from the receptor and
interacts with a preformed cytosolic complex made up of the kinase
TGF beta Associated Kinase 1 (TAK1) and two TAK1-Binding
proteins, TAB1 and TAB2. Binding of polyubiquitin chains to the TAB
proteins in the TAK1 complex activates it.

19. The TAK1 complex now performs two functions. It phosphorylates and
activates the IKK complex, leading to the destruction of IB and the
resultant activation of the transcription factor NF-B (see Figure 3-17).
In addition, TRAF6 also plays a role in IKK activation by providing
ubiquitination sites to which the NEMO component of IKK can bind,
resulting in its further activation. TAK1 also activates downstream
members of the MAP kinase cascade, which in turn activate the AP-1
transcription factor (see Figure 3-16).
Binding of IL-1 family cytokines to their receptors thereby leads to a
global alteration in the transcription patterns of the affected cells, which
in turn results in the up-regulation of proinflammatory cytokines and
adhesion molecules.

22. The two key adaptors are MyD88 (Myeloid differentiation
factor 88) and TRIF (TIR-domain-containing adaptor-inducing
IFN- factor). Most TLRs, whether found on the cell surface or
in endosomes/lysosomes, bind the adaptor protein MyD88
(activating MyD88-dependent signaling pathways).
In contrast, TLR3 binds the alternative adaptor protein TRIF
(activating TRIF dependent signaling pathways). TLR4 is
unique in binding both MyD88 (when it is in the plasma
membrane, signaling its endocytosis) and TRIF (when it is in
endosomes, after internalization).
The presence of two additional adaptors associated with
most TLRs: TIRAP (TIR-domain containing adaptor protein)
and TRAM (TRIF-related adaptor molecule). They are TIR
domain-containing adaptors that serve as sorting receptors:
TIRAP helps to recruit MyD88 to TLRs 2/1, 2/6, and 4, and
TRAM helps to recruit TRIF to both TLR3 and
endosomal/lysosomal TLR4.

23. MyD88-Dependent Signaling Pathways
After associating with a TLR dimer following ligand binding,
MyD88 initiates a signaling pathway that activates the NF-B
and MAPK pathways by essentially the same pathway as that
activated by IL-1. As shown for plasma membrane TLRs 2/1, 4,
and 5, MyD88 recruits and activates several IRAK protein
kinases, which then bind and activate TRAF6.
TRAF6 ubiquitinates NEMO and TAB proteins, leading to the
activation of TAK1, which then phosphorylates the IB kinase
(IKK) complex. Activated IKK then phosphorylates the
inhibitory IB subunit of NF-B, releasing NF-B to enter the
nucleus and activate gene expression.
TAK1 does double duty in this TLR signaling cascade. After
separating from the IKK complex, it activates MAPK signaling
pathways that result in the activation of transcription factors
including Fos and Jun, which make up the AP-1 dimer.

25. In addition to activating NF-B and MAPK pathways via the
MyD88-dependent pathway, the endosomal TLRs 7, 8, and 9,
which bind microbial nucleic acids (especially viral RNA and
bacterial DNA), also trigger pathways that activate IRFs.
As shown in Figure 5-13, when triggered by these TLRs, the
MyD88/IRAK4/TRAF6 complex activates a complex
containing TRAF3, IRAK1, and IKK, leading to the
phosphorylation, dimerization, activation, and nuclear
localization of IRF7. IRF7 induces the transcription of genes
for both Type 1 interferons, IFN alpha and beta, which have
potent antiviral activity.
Thus different TLRs may differentially activate distinct
transcription factors (NF-B, certain IRFs, and/or those activated
by MAPK pathways), leading to variation in which genes are
turned on to help protect us against the invading pathogens.

26. TRIF-Dependent Signaling Pathways
For the two endosomal TLRs that recruit the TRIF
adaptor instead of MyD88—TLR3 and endosomal
TLR4— the downstream signaling pathways differ
somewhat from those activated by MyD88.
TRIF recruits the RIP1 protein kinase that in turn
recruits and activates TRAF6, which then initiates the
same steps as in the MyD88-dependent pathway.

27. TLR3 also activates PI3K, which enhances
MAPK pathway activation. In addition, TRIF
activates TRAF3 and a complex of TBK-1 and
IKK, which phosphorylates and activates IRF7
and a different IRF—IRF3.
IRF3 and IRF7 dimerize and move into the
nucleus and (together with NF-B and AP-1)
induce the transcription of the IFN genes.
Thus, all of the intracellular TLRs that bind viral PAMPs
in an infected cell induce the synthesis and secretion
of Type I interferons, which feed back to potently
inhibit the replication of the virus in that infected cell.

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