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Toll like receptors
1. Dr. Tushar B. Patil, MD
Senior Resident
Departmet of Neurology
King George’s Medical University ,
Lucknow, India
2. Introduction
TLRs are germline-encoded pattern recognition receptors
Sense conserved molecular structures produced by
microorganisms
Play essential role in host defence to microbial infection.
Activate intracellular signalling pathways
Induce genes involved in immune responses and
inflammation.
Act as a bridge between Innate and Adaptive immunity by
mediating dendritic cell maturation and activation of
pathogen-specific T lymphocytes.
3. Introduction
TLRs recognize pathogens and generates an immediate defence
response.
Induce cytokines which destroy or limit invading pathogens.
Activation of APCs & expression of MHC and co-stimulatory
molecules like CD40, CD89, CD86 and CD70.
Activation and differentiation of naive T cells into Th1, Th2,
Th3 and Th17 cells or T-regs, facilitating cell mediated
immune responses.
4. Toll: Origin of the word (1985)
The gene in question, when mutated, makes the Drosophila (fruit fly) embryo look
unusual.
The researchers were so surprised that they spontaneously shouted out in German
"Das ist ja toll!" which translates as "That's great!".
[The Nobel Prize in Physiology or Medicine 1995:Edward B. Lewis, Christiane
Nüsslein-Volhard, Eric F. Wieschaus]
10. Cellular Localization of TLRs
TLR1, TLR2, TLR4, TLR5, and TLR6 localized on the cell surface and
recognize microbial membrane components.
TLR3, TLR7, TLR8, and TLR9 expressed within intracellular vesicles and
recognize nucleic acids.
Intracellular vesicles with TLR3, TLR7, TLR8, and TLR9 are localized in
endoplasmic reticulum (ER), endosomes, lysosomes, and endolysosomes.
Intracellular localization important for avoiding contact with ‘‘self’’ nucleic
acids and risk of autoimmunity.
Regulated mechanism is present for TLR mobilization.
11. Structure of TLRs
TLRs are type I membrane
glycoproteins.
Homology in the cytoplasmic
region--- interleukin-1
receptors (IL-1Rs)
superfamily
Extracellular region of TLRs
contains leucine-rich repeat
(LRR) motifs, & IL 1Rs
contains three
immunoglobulin-like domains
12. Structure of TLRs
Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs)
have a conserved cytoplasmic domain, that is known as the
Toll/IL-1R (TIR) domain.
The TIR domain is characterized by the presence of three
highly homologous regions (known as boxes 1, 2 and 3).
18. TLR Signaling Pathway
1]MyD88 (myeloid differentiation primary-response protein 88)
Forms homodimers through DD–DD and TIR-domain–TIR-domain interactions and exists
as a dimer when recruited to the receptor complex.
Functions as adaptor linking TLRs/IL-1Rs with downstream signalling molecules that have
DDs.
2] IRAK family (IL-1-receptor-associated kinases)
Four IRAKs — IRAK1,IRAK2,IRAK4 and IRAK-M identified
The kinase activity of IRAK1 increases strongly following TLR/IL-1R stimulation, and its
kinase domain is essential for signalling through nuclear factor-κB (NF-κB)
3] TRAF6 (tumour-necrosis-factorreceptor- associated factor 6)
Signalling mediator for both the TNF-receptor superfamily and the TLR/IL-1R
superfamily, interacting directly with members of the TNF-receptor superfamily
4] NF-κB (nuclear factor-κB)
Promote expression of genes and synthesis of cytokines
20. TIR-domain-containing MyD88 mediates TLR pathway that activates
IRAKs and TRAF6
Activation of the IKK complex (inhibitor of nuclear factor-κB (IκB)- kinase
complex), which consists of IKK-α, IKK-β and IKK-γ (also known as
IKK1,IKK2 and NF-κB and releases NF-κB from its inhibitor so
NF-κB translocates to the nucleus and induces expression of inflammatory
cytokines.
TIRAP (TIRdomain- containing adaptor protein), is involved in the MyD88-
dependent signalling pathway through TLR2 and TLR4.
TLR3- and TLR4-mediated activation of interferon (IFN)-regulatory factor
3 (IRF3) and the induction of IFN-β are observed in a MyD88-independent
manner.
24. TLR 1
1]PROTECTION AGAINST LEPROSY
SNP in TLR1, 1805T-G, that results in an ile602-to-ser (I602S) substitution
at the junction of the transmembrane and intracellular domains of TLR1.[
Johnson et al. 2007, Misch et al.2008]
2] SUSCEPTIBILITY TO LEPROSY
Association of an asn248-to-ser (N248S) SNP in the TLR1 gene and leprosy
(LPRS5; 613223) in a Bangladeshi population consisting of 842 patients
and 543 controls.[Schuring et al. (2009)]
Homozygosity for S248 significantly associated with leprosy (OR = 1.34)
& heterozygosity was found to be protective against leprosy (OR = 0.78)
Homozygous N248 genotype was equally distributed among patients and
controls
Patients with erythema nodosum leprosum reactions were more likely to
have the N248 allele (68%) than were patients who had no reactions (46%)
25. TLR2
1]Susceptibility to Leprosy
Arg677-to-trp polymorphism (R677W; 603028.0001) in the intracellular domain
of TLR2 in 10 (22%) of 45 Korean lepromatous leprosy patients [Kang and Chae
2001]
R677W leads to poor cellular immune response associated with lepromatous
leprosy.[Bochud et al. 2003]
R677W was undetectable in the Japanese patients [Mikita et al. 2009], similar to
the findings in Indian patients reported by [Malhotra et al. 2005]
Bochud et al. (2008 )analyzed 3 TLR2 polymorphisms in 441 patients and 187
controls in 3 Ethiopian groups.
597C-T SNP was associated with reduced susceptibility to reversal reaction
Homozygous for a 280-bp microsatellite marker had an increased risk of reversal
reaction
26. TLR2
1] Lyme Disease
Monocytes and lymphocytes from healthy subjects produced more TNF
and IFNG, respectively, in response to high concentrations of Borrelia
lysate than did healthy subjects heterozygous for an arg753-to-gln (R753Q)
SNP. [Schroder et al. 2005]
R753Q SNP may protect from development of late-stage Lyme disease due
to reduced signaling via TLR2/TLR1.
2] Tuberculosis
Increased risk of TB in carriers of a nonsynonymous 2258G-A SNP in the
TLR2 gene, which causes the R753Q substitution [Ogus et al. (2004)]
3] Colorectal Cancer
Boraska Jelavic et al. (2006) GT microsatellite repeat polymorphism in
intron 2 of the TLR2 gene in sporadic colorectal cancer patients
Frequency of TLR2 alleles with 20 and 21 GT repeats was decreased &
that of 31 GT repeats was increased in patients versus controls.
27. TLR3
1] Herpes Simplex Encephalitis
Zhang et al. (2007) detected a heterozygous P554S mutation in the
TLR3 gene. The mutation occurred on different TLR3 haplotypes in
the children.
TLR3 is vital for natural immunity to HSV-1 in the CNS and
neurotropic viruses have contributed to the evolutionary maintenance
of TLR3.
2] Age-related macular degeneration
T allele of rs3775291 in the TLR3 gene, which results in a L412F
substitution, is protective against the development of geographic
atrophy or advanced dry age-related macular degeneration
28. TLR4 (The human homolog of Drosophila Toll)
1] ENDOTOXIN HYPORESPONSIVENESS
Arbour et al. (2000) showed that 2 common cosegregating missense mutations
(asp299 to gly and thr399 to ile) that affect the extracellular domain of the TLR4
receptor are associated with blunted response to inhaled lipopolysaccharide in
humans.
Susceptibility to Gm negative sepsis.
2] D299G allele - Lower Levels Of Certain Proinflammatory Cytokines, More
Susceptibility To Severe Bacterial Infections, Lower Risk Of Carotid
Atherosclerosis, And A Smaller Intima-media Thickness In The Common Carotid
Artery. [Kiechl et al. (2002)]
3] gly299 allele of the TLR4 gene was more frequent in colorectal cancer patients
than controls
4] D299G and T399I variants of TLR4 as contributors to susceptibility to age-related
macular degeneration
5] D299G polymorphism– metastasis after breast cancer surgery
6] D299G polymorphism in TLR4 may influence the immunologic component of
anthracycline-based chemotherapy in human cancer.
29. TLR5
1] Susceptibility to Legionnaire disease
C-to-T transition at nucleotide 1174, changed arg392 to a stop codon
(R392X) [
Hawn et al. 2003]
2] Resistance to systemic lupus erythematosus
allele 1174C of TLR5, but not allele 1174T, which encodes the premature
stop codon, was preferentially transmitted to SLE-affected
offspring. [Hawn et al. 2005]
31. TLR 7
Target of investigational agents with antitumor and
antiviral properties
32. FUNCTIONS OF TOLL-LIKE RECEPTORS IN CNS
TLR4 expression in the CNS is necessary to mount an appropriate cytokine
response in the brain in response to systemic LPS exposure
mice with peripheral TLR4-expressing cells, but lacking specific CNS TLR4, were
unable to mount a CNS cytokine response
TLRs may play important roles in cerebral cell proliferation and brain
development.
Inflammation has a strong effect on progenitor cells and reduces adult hippocampal
neurogenesis.
TLR2 deficiency in mice resulted in impaired hippocampal neurogenesis.
absence of TLR4 enhanced proliferation and neuronal differentiation.
Detrimental effects of TLR4 on progenitor cells was shown to be dependent on
prostaglandin E2 receptors.
protective effect on hippocampal neurogenesis by cyclooxygenase inhibitors
TLR8 is also expressed at high levels during brain development, and in cultured
cortical neurons, TLR8 stimulation inhibits neurite outgrowth
33. Immunostaining of cultured microglia and astrocytes for TLR3 and TLR4,
revealed two opposite features.
Both TLR3 and TLR4 were found exclusively localized in vesicular structures
inside microglia and not on the surface of the cells.
With cultured astrocytes, TLR3 and TLR4 were found only on the cellular
surface
Microglial TLRs are crucial as a first line of defence against bacterial or viral
infection.
human astrocytes have been reported to express TLRs 1–5 and TLR 9
TLR signaling in astrocyte can activate the production of a wide range of
neuroprotective and anti-inflammatory mediators rather than merely stimulating
proinflammatory factors.
The preference of astrocyte to express up to 200-fold elevated levels of TLR3
upon activation is puzzling since the only currently known ligand for TLR3 is
dsRNA, which is believed to emerge as an intermediate during viral replication.
34. TLR Signaling Link to Neurotoxicity
TLR Signaling Link to Neurogenesis
TLR Signaling in Neurodegenerative Diseases
35. Neurological diseases with possible link to TLR
pathway
1]Leprosy 9]Bacterial meningitis
2]Herpes Simplex Encephalitis 10] Alzheimer’s Disease
3]Entero and flaviviral 11] Prion Diseases
encephalitis 12] Amyotrophic Lateral
4]Malaria Sclerosis.
5]Toxoplasmosis 13] Parkinson’s Disease
6]Trypanosomiasis 14]Perinatal brain injury
7]Lyme disease 15] Multiple sclerosis
8]Neurocysticercosis and ?????????????????
36. Targeting TLR as Therapeutic Application in AD
TLR activation may modulate glial cell activity in AD.
Recent research suggests the involvement of TLRs 2, 4, 5, 7, and 9 in the
proinflammatory response of microglia toward Aβ, which may be linked to
neurotoxicity
Activation of TLRs 2, 4, and 9 were also linked to both phagocytosis of
the neurotoxic Aβ and to an anti-inflammatory response (TLR9), which
may lead to neuroprotection (Figure 1).
Therefore, elevated expression levels of TLRs 2, 4, and 9, through genetic
modification or toward specific agonists, may be a therapeutic application
in AD.
Use of TLRs 2 and 4 agonist as a specific macrophage activator to increase
the clearance of Aβ in an AD mouse model.
An alternative therapeutic approach may be the reduction of TLR5 and 7,
by using shRNA or specific antagonists
37. Bisdemethoxycurcumin is a natural curcumin, a minor
constituent of turmeric , that enhances phagocytosis and the
clearance of Aβ in cells from most AD patients, and increases
transcription of the MGAT and TLR genes
Administration of CpG, a TLR9 activator, in APP transgenic
mice, resulted in clearance of Aβ from microglial cells.
38. Targeting TLR as Therapeutic Application in Prion Diseases
It has been suggested that TLR9 expression may be linked to the progression of
prion diseases.
Treatment with synthetic oligodeoxynucleotides that contain cytosine phosphate
guanosine (CpG-ODN) motifs, known to bind to TLR9, have been suggested as
possible treatment for prion diseases in a mouse model, by delaying the disease
onset.
Another explanation may be the effect of CpG-ODN on microglia activation that
may lead to prion degradation .
As the activation of TLRs in other amyloidogenic diseases, such as AD, has been
linked to the clearance of neurotoxic amyloid, it may prove to be a potential
therapeutic approach to the prion diseases.
39. Targeting TLR as a Therapeutic Application in Amyotrophic
Lateral Sclerosis.
A potential link between TLR signaling and an increase in
neurotrophic factor secretion from glial cells may prove to be a
therapeutic approach in ALS.
40. Targeting TLR as Therapeutic Application in
Parkinson’s Disease.
α-synuclein immunization in a PD animal model may
ameliorate disease progression.
Targeting mechanisms in which α-synuclein activates TLR
signaling, may open a new horizon for therapeutic application
in PD.
42. Toll-Like Receptor 4: Agonism and
Antagonism
The best characterized ligand for the MD-2/TLR4 complex is lipid A (the
biologically active component of LPS)
Different lipid A structures may be agonists or antagonists at the MD-
2/TLR4 (Walsh et al., 2008).
A synthetic compound CRX-527 is an agonist, but decreasing the
secondary acyl chain length below 6 or increasing it above 14 results in a
loss of agonist activity
Binding of lipid A to MD-2/TLR4 (Raetz et al., 2006) induces structural
rearrangements that trigger oligomerisation of TLR4 and initiate signal
transduction
43. In 1995, a synthetic form of Rhodobacter capsulatus lipid A was generated
that antagonized E. coli lipid A and formed the basis for E5531 (Christ et
al., 1995).
Modification of E5531 generated the stable analog E5564 (eritoran), which
is currently undergoing clinical trials for use in treating Gram-negative
endotoxemia and sepsis
Other antagonists at TLR4 include curcumin, auranofin (an antirheumatic
gold compound), cinnamaldehyde, and acrolein, all of which prevent
homodimerization of TLR4
Small molecules that inhibit MyD88 binding to TLR4 are also emerging
TLR4 agonists are currently being developed as immunomodulators and
adjuvants.
TLR ligands have become a focus in therapeutic studies for their potential
use as adjuvants in vaccine formulations
44.
45. Toll-Like Receptor 2: Agonism and Antagonism
Currently, the major use for compounds that activate TLR2 are
as adjuvants.
The synthetic compounds, such as Pam3CSK4 and MALP-2,
could be developed for adjuvant usage.
Another approach to blocking TLR2 is with a neutralizing
antibody.
One such antibody, T2.5, has been shown to prevent sepsis
induced by TLR2 ligands (Meng et al., 2004)
Furthermore, when T2.5 is used in combination with an anti-
TLR4/MD-2 antibody, it protects mice against sepsis induced
by Salmonella enterica or E. coli when given with antibiotics
46. Toll-Like Receptor 5: Agonism and
Antagonism
M2e is immunogenic component of influenza A
M2e was recently fused with the TLR5 ligand S. typhimurium
flagellin (STF2). The resulting fusion protein can activate cells in
a TLR5-dependent manner and elicits potent antibody responses
in mice.
M2e sequence linked to the TLR5 ligand provides an effective
approach to developing vaccines against wide-spread epidemic
and pandemic influenza
In the case of IBD, TLRs can also amplify inappropriate immune
responses that ultimately cause chronic inflammation.
At low concentrations, flagellin can stimulate TLR5 on CD4 T
cells and enhance the expression of FOXP3, allowing for an
increased suppressive capacity
47. Toll-Like Receptors 7 and 8: Small-Molecule
Targets
Activated by synthetic small-molecularweight compounds of the
imidazoquinoline family, such as resiquimod and imiquimod.
TLR7/TLR8 agonists act as“antiviral agents.”
Imiquimod is the first approved topically active TLR7 agonist.
It is prescribed for treatment of external virus induced skin
lesions, such as the genital and perianal warts resulting from
papillomavirus infection
Therapeutic interest in TLR7/TLR8 for cancer treatment came
about because of the antitumoral activity of TLR7/TLR8 agonists
48. Toll-Like Receptor 9
Only TLR for which a systemically administered specific agonist has shown
substantial evidence of antitumor activity in human clinical Trials
TLR9 has evolved to recognize unmethylated CpG dinucleotides (CpG
ODN) that are prevalent in viral and bacterial DNA
CpG ODNs seem to be the most promising of all adjuvants currently in
preclinical development
ability to help vaccine hyporesponsive populations, such as persons positive
for HIV, to benefit from vaccination
There are also promising results from studies into the adjuvant activity of CpG
ODN for tumor vaccination.
Role of antagonists in SLE?????
52. We have come a long way from the discovery of the first Toll
in the fruit fly.
The intense interest around TLRs, shared by immunologists,
biomedical researchers, and pharmacologists, should surely
yield badly needed therapies for major pathologic conditions.
