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![Page 1: Toll-like receptors Bridging the innate and adaptive immune responses June 1, 2005 MIMG 261 Stephan Krutzik, Ph.D. skrutzik@mednet.ucla.edu.](https://reader035.fdocuments.in/reader035/viewer/2022062517/56649f065503460f94c1b0fe/html5/thumbnails/1.jpg)
Toll-like receptors
Bridging the innate and adaptive immune responses
June 1, 2005
MIMG 261
Stephan Krutzik, Ph.D.
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• Rapid Response• Dendritic cells, monocytes, NK cells • Pattern recognition receptors-
germ-line encoded– TLRs, mannose and scavenger
• Direct Response for host defense– Phagocytosis– Antimicrobial activity
Cytokines, co-stimulatory molecules
• Slow response• T and B cells• Recognition - initially low affinity
receptors Gene rearrangement
Clonal expansion
• Response - T and B cells with high affinity, very specific receptors and antibodies
• Immunological Memory
Innate Response Adaptive Response
The instructive role of the innate response on adaptive immunity
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Lemaitre, et al. (1996) Cell 86, 973
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DROSOPHILA HOST DEFENSE
• Regulated by Toll receptor family• Evidence for specificity in regulation• Different pathogens, different
response• Toll - Antifungal
18-wheeler - Antibacterial• Secretion of antimicrobial
polypeptides by the fat body• antifungal - drosomycin,
metchnikowan • antibacterial - cecropin, drosocin,
defensin, diptericin, attacin, metchnikowan
Toll 18- Wheeler
Fungus Bacteria
Antimicrobial Genes
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HUMAN TOLL-LIKE RECEPTORS (TLRS)
19%
25%
LRR
Cys-Rich
dToll TLR250 1000
TLR2
TLR6
TLR7
TLR1
TLR5
TLR8
TLR4
TLR3
dToll
TLR9~IL-1R
TIR
TIR=Toll/IL-1R
In 1997, Charles Janeway cloned and sequenced the human homologue to Drosophila Toll
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Immunomodulatory Genes
Cell signaling
TLR2/TLR1TLR2/TLR6
lipoproteins
TLR7
ssRNAimmiquimod
dsRNA
TLR3
LPS
TLR4
flagellin
TLR5
TLR9
CpG DNA
Toll-like receptors (TLRs) and their ligands
TLR8
ssRNA
TLR10-orphan (human)
TLR11-uropathogenic bacteria and protozoa(mouse)PAMPs (Pathogen associated molecular patterns)
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Bell Trends Immunol
Leucine Rich Repeats
• 24-residue repeated sequence with characteristically spaced hydrophobic residues
• 19-25 LRR in the ECD of TLRs
• Involved in ligand recognition
• 6500-8000 A2 v 700 A2 for Ab
TLR extracellular domain
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TLR intracellular domain
TIR domain
Death domain
MyD88
IL-1R
TIR domain
TIR domain
TLR
NFB
TIR=Toll/IL-1R domain
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MyD88 is required for TLR activation
Kawai et al Immunity Volume 11, Issue 1 , 1999, Pages 115-122
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CONSERVED PATHWAYS IN INNATE IMMUNITY
Hoffmann JA, et al. Science. 1999 May 21;284(5418):1313-8
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Kawai et al Immunity Volume 11, Issue 1 , 1999, Pages 115-122
Cells from MyD88 -/- did not produce cytokines but did have delayed activation of NFB
These data suggest that an alternative, MyD88-independent signaling pathway is triggered by TLR4
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Using subtractive hybridization in MyD88-/- macrophages, identified MyD88-independent specific genes (Kawai, Akira)
IP-10, GARG16, IRG1---- have ISRE (IFN-stimulated response element) and NFkB binding sites in promoter
IP-10
GARG16
IRG1
MyD88-/- MyD88-/-
--- Lipid A
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TLR4 signaling leads to the activation of the transcription factor IRF-3 in a MyD88-independent manner
Identified an MyD88-independent pathway that triggers IRF-3
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MyD88-dependent and –independent signaling
Immunomodulatory Genes
MyD88
TIRAP/MAL
MyD88 MyD88
TLR4TLR2/TLR1
TLR2/TLR6
TLR5 TLR7 TLR9
IRAKTRAF6
NFB
IRF-3
TRIF
IFN-
TLR3
TRIF
TRAM
TRAF6
NFB
TBK-1
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Innate Adaptive
TLRs influence both innate and adaptive immune responses
Tissue injury
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In 1884, Metchnikoff published studies on the water-flea Daphnia and its interaction with a yeast-like fungus. He demonstrated the ability of cells of the water-flea, which he termed phagocytes, to engulf the foreign spores. “The spores which reached the body cavity are attacked by blood cells, and- probably through some sort of secretion- are killed and destroyed”.
Metchnikoff’s study of Daphnia
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Metchnikoff’s innate immune system
Thus Metchnikoff had described the key functions of cells of the innate immune system
• rapid detection of microbes• phagocytosis• antimicrobial activity
Through the studies of TLRs, we have a better understanding of how the innate immune system can mediate these events
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Tolls and Phagocytosis
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TLR activation triggers direct host defense against invading pathogens
1. Enhances phagocytosis
Measure GFPInert
microspheres
Blander et al Science 2004
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TLR activation triggers direct host defense against invading pathogens
2. Induces phago-lysosomal fusion
Blander et al Science 2004
Green- E. coli GFP
Red- LysoTracker
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Green- (CFSE) apoptotic cell
Red- LysoTracker
30min 2hr
TLR signaling not required for phago-lysosomal fusion of apoptotic cells
Blander et al Science 2004
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TLRs and antimicrobial pathways
RIPTLR activation
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TLR2 activation induces anti-mycobacteria activity
1. Monocytes infected with M. tb
2. Activated with TLR2/1 ligand +/-
TLR2
3. Measured M. tb CFU
+/- TLR2 L
Human monocyte
CFU
Thoma-Uszynski et al Science 2001
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Doyle et al Immunity, 2002
1. macrophages infected with MHV68
2. Activated with TLR ligands
3. Measured early replication proteins
(CM=conditioned media from TLR stimlulated macrophages)
TLR L
Western blot
MyD88-independent pathways trigger anti-viral activity
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TLR3 and TLR4 induce antiviral responses via IFN
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Immunomodulatory Genes
IRF-3
MyD88
TRIFTIRAP/MAL
MyD88 MyD88
IFN-
TLR3TLR4TLR2/TLR1
TLR2/TLR6
TLR5 TLR7 TLR9
TLR signaling pathways
TRIF
IRAKTRAF6
NFB
TRAM
TRAF6
NFB
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Thus TLRs can mediate key functions of the innate immune system described by Metchnikoff
• rapid detection of microbes• phagocytosis• antimicrobial activity
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TLRs bridge the innate and adaptive immune responses
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TLRs bridge the innate and adaptive immune responses
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Adaptive immune response is dependent on:
1. Density of peptides (Signal 1)
2. Types and levels of co-stimulatory molecules on APC (Signal 2)
3. Types of cytokines secreted by APC (Th skewing)
4. Being kept in check-- Peripheral T cell tolerance controlled by CD80/CD86 levels and Treg cells
TLR activation can influence all 4 factors
TLR
Innate Adaptive
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Signals required for T cell activation
Treg
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TLR
Mature Dendritic cell
immature Dendritic cell
1. Density of peptides (Signal 1)
2. Types and levels of co-stimulatory molecules on APC (Signal 2)
Hertz et al J Immunol 2001
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Increase in levels of MHCII and co-stimulatory molecules enhances antigen presenting capacity
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Signal 3 (Th skewing) can come directly from APC or from surrounding cells/tissue
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Th1 Th2
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TLR activation is important for triggering Th1 immune response
MyD88-/- mice have a deficient Th1 immune response…
Wt
MyD88-/-
Caspase-1 -/-
Mice were immunized with OVA and CFA
Harvest lymph nodes
OVAMeasure proliferation and cytokine production
8 days
Schnare Nat Immunol 2001
CFA=killed mycobacteria in oil and water
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But intact Th2 immune response (antigen specific Th2 immunoglobulin intact)
Mice were immunized with OVA and CFA
Measure serum immunoglobulin levels
Wt
Myd88-/-
Caspase-1 -/-
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MyD88-/- DC do not increase co-stimulatory molecules and do not secrete IL-12 upon TLR activation
Mature Dendritic cell
immature Dendritic cell
IL-12
mycobacteria
Wt
Myd88-/-
Caspase-1 -/-
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T reg cells
CD4+CD25+
Foxp3 expression
Secrete TGF-, IL-6, IL-10
Block T cell proliferation
Thought to be involved in maintaining peripheral tolerance
But may also block pathogen-specific T cell activation
So, there needs to be a way to turn them OFF during infection
TLRs control Treg function
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DC activation via TLRs inhibits Treg cells and allows T cell activation (“Signal 4”)
T cell prolif
T cell prolifTLRL
Pasare et al Science 2003
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DC activation releases Treg block via IL-6
T cell prolif
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IL-6 required for in vivo T cell activation
Mice were immunized with OVA and LPS
Harvest lymph nodes
OVAMeasure T cell activation
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Thus, TLR activation is able to bridge the innate and adaptive immune responses by:
1. Detecting invading microbes
2. Enhancing APC function by increasing levels of MHCII and co-stimulatory molecules
3. Triggering the release of cytokines that skew adaptive immune response
4. Controlling T reg function