Toll-like receptors
Bridging the innate and adaptive immune responses
June 1, 2005
MIMG 261
Stephan Krutzik, Ph.D.
• 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
Lemaitre, et al. (1996) Cell 86, 973
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
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
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)
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
TLR intracellular domain
TIR domain
Death domain
MyD88
IL-1R
TIR domain
TIR domain
TLR
NFB
TIR=Toll/IL-1R domain
MyD88 is required for TLR activation
Kawai et al Immunity Volume 11, Issue 1 , 1999, Pages 115-122
CONSERVED PATHWAYS IN INNATE IMMUNITY
Hoffmann JA, et al. Science. 1999 May 21;284(5418):1313-8
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
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
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
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
Innate Adaptive
TLRs influence both innate and adaptive immune responses
Tissue injury
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
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
Tolls and Phagocytosis
TLR activation triggers direct host defense against invading pathogens
1. Enhances phagocytosis
Measure GFPInert
microspheres
Blander et al Science 2004
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
Green- (CFSE) apoptotic cell
Red- LysoTracker
30min 2hr
TLR signaling not required for phago-lysosomal fusion of apoptotic cells
Blander et al Science 2004
TLRs and antimicrobial pathways
RIPTLR activation
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
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
TLR3 and TLR4 induce antiviral responses via IFN
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
Thus TLRs can mediate key functions of the innate immune system described by Metchnikoff
• rapid detection of microbes• phagocytosis• antimicrobial activity
TLRs bridge the innate and adaptive immune responses
TLRs bridge the innate and adaptive immune responses
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
Signals required for T cell activation
Treg
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
Increase in levels of MHCII and co-stimulatory molecules enhances antigen presenting capacity
Signal 3 (Th skewing) can come directly from APC or from surrounding cells/tissue
Th1 Th2
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
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 -/-
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 -/-
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
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
DC activation releases Treg block via IL-6
T cell prolif
IL-6 required for in vivo T cell activation
Mice were immunized with OVA and LPS
Harvest lymph nodes
OVAMeasure T cell activation
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