Transcript Response of the Innate Immune System to Pathogens: Pattern

Response of the Innate Immune
System to Pathogens:
Pattern Recognition Receptors
What is the Innate Immune Response?
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A universal and evolutionarily conserved mechanism of host defense
against infection
First line of Defense
Predates the adaptive immune response
 Found in all multicellular organisms
 Adaptive only in vertebrates
 Uses receptors and effectors that are ancient in their lineage
 Must provide protection against a wide variety of pathogens
Distinguishes self from non-self perfectly
Defects in innate immunity are very rare and almost always lethal
The Innate Immune Response:
Common Misconceptions
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The innate immune system is an evolutionary rudiment whose
only function is to contain the infection until the “real”
immune response can kick in.
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Adaptive immunity developed because of the inflexibility of
the nonclonal receptors used by the innate immune response.
The innate system cannot cope with the high mutational rate
and heterogeneity of pathogenic organisms.
The Innate immune system instructs the adaptive
immune response to respond to microbial infection
The major decision to respond or not respond to a
particular ligand is decided by the genome-encoded
receptors of the innate immune system
Adapted from Medzhitov and Janeway
Cur. Opin. Immunol. 1997 9:4-9
PAMP
Phagocytosis
Y
PRR
APC
Complement
Endosome
MHC
Pathogen-specific
Antibody
Direct Bactericidal Activity
Phagocytosis
Oxygen burst
Anti-microbial peptides
B7
Y
Naive
T Cell
Inflammatory
and effector
cytokines
Activated
CD40L, FasL, CD30L, CD27L
T Cell
B Cell
Components of Innate and Adaptive
Immunity
Innate Immunity
Adaptive Immunity
Physical Barriers
skin, gut villi, lung cilia,etc
none
Soluble Factors
many protein and
non-protein secretions
Immunoglobulins
(antibody)
Cells
phagocytes, NK cell
eosinophils
T and B lymphocytes
PAMPs: Pathogen Associated Molecular Patterns
PRRs: Pattern Recognition Receptors
Takeda and Akira Genes to Cells (2001) 6:733-742
The NF-kB Family of Transcription Factors
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Eukaryotic transcription factor found in essentially all cell types
First described in 1986 as a nuclear factor required for the
transcription of the immunoglobulin kappa light chain in B cells.
Binds to a 10-bp sequence GGGGYNNCCY
Important component in the inducible expression of many
proteins: cytokines, acute phase proteins, adhesion molecules
The NF-kB signaling system is evolutionarily conserved
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Three NF-kB molecules in Drosophila
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dorsal
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controls dorsal/ventral polarity during development
Regulates antifungal gene expression
dif and relish: regulate expression of antifungal and antibacterial genes
NF-kB exists in the cytoplasm as an inactive
heterotrimer composed of 2 Rel family proteins and an
inhibitory IkB molecule
Stress, infection, or cytokine
IKK
P P
(Ub)n
IkB
p65
p50
26S proteosome
Nuclear
Translocation
Activation of NF-KB
Responsive genes
NF-kB Family Structure
Conservation of
signaling pathways
between flies and
humans
Toll
Pelle
tube
?
?
cactus
kinase
cactus
dorsal
dif
relish
Mutation of genes in the Toll signaling
cascade in Drosophila block the expression of
anti-fungal and anti-bacterial genes
Water
A. fumigatus
E. coli
Signaling to monocytic cells through
the human Toll homolog induces
accessory cell functions
NF-kB Activation
Requirements for the recognition of targets
by the innate immune
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Molecular structures recognized by the immune system
must be shared by large groups of pathogens
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molecular patterns vs. particular structures (antigens) PAMP
PAMPs must be conserved products of microbial
metabolism not subject to antigenic variability
The recognized structures must be absolutely distinct
from self antigens: discrimination of self vs. non-self
Molecules involved in the recognition of
LPS by Cells
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LPS Binding Protein (LBP)
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60-kDa serum glycoprotein that binds with high affinity to LPS
Sequence homology to bactericidal/permeability increasing protein
Acute phase protein secreted by hepatocytes
 Serum levels between 1 and 10 ug/ml in normal human serum
 Concentrations >300 ug/ml in acute phase serum
Critical for rapid responses to small amounts of LPS or gramnegative bacteria and for survival of Salmonella infection
Expression of LBP in hepatocytes is regulated by LPS, IL-1, IL-6
and TNF
Molecules involved in the recognition of
LPS by Cells
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CD14, sCD14
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CD11b/CD18 (Mac1)
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55 kDa GPI-linked protein on the surface on monocytes and PMN
Also found as a soluble protein in serum:sCD14
Each CD14 molecule binds 1 molecule of LPS complexed to LBP
Required for responses of genes to low concentrations of LPS
No cellular signaling activity
Alternative cell surface receptor for LPS
Recognizes LPS at high concentrations
For expression of a full repertoire of LPS-inducible genes, CD14
and CD11b/CD18 must be coordinately engaged to deliver optimal
signaling to the macrophage.
Search for the LPS Gene
Endotoxin hyporesponsive mice
C3H/HeN: Normal LPS response
1. C3H/He 1947
Spontaneous mutation
mid 1960’s
2. C57BL/10Sn
Sometime
After 1953
C3H/HeJ: LPS hyporesponsive
C57BL10/ScCR
LPS hyporesponsive
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January 1998 DNAX reports
the cloning of human
TLR1-5
 No ligands described
 Janeway’s hToll=TLR4
Rock, et al. 1998 PNAS 95:588
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September 1998 Genentech identifies TLR2 as the LPS
signaling molecule by transfection studies
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December 1998 Tularik also identifies TLR2 as LPS
signaling molecule in transfection studies
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Didn’t look at TLR4
TLR4 didn’t work
September 1998: Bruce Buetler’s lab mapped LPS to a
1.2Mb region of chromosome 4. The only intact gene
within this region is TLR4
December 1998: “Defective LPS signaling in C3H/HeJ and
C57BL/10ScCr mice: Mutations in Tlr4 gene” Poltarak et
al. Science 282:2085
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Poltarak et al 1998 Science 282:2085
 A single nucleotide change in the gene for TLR4 renders the
C3H/HeJ mouse non-responsive to LPS
C3HHeN RFHLCLHYRDFI P GCAIAANIIQEGFHK
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C3HHeJ RFHLCLHYRDFI H GCAIAANIIQEGFHK
Hoshino et al 1999 J. Immunol. 162:3749
 TLR4 knockout mice are hyporesponsive to LPS
Dunne and O’Neil 2003 www.stke.org/cgi/content/full/sigtrans;2003/171/re3
Receptor
Agonist(s)
(Pattern Recognition
Receptors)
(Pathogen-Associated
Molecular Patterns)
TLR1
Heterodimerizes with TLR2
TLR2
PGN, some LPS, some LTA,
lipoproteins, AraLAM
TLR3
dsRNA
TLR4
Gram(-) LPS, Taxol, some LTA
TLR5
Flagellin
TLR6
Heterodimerizes with TLR2
TLR7
Imidazoquinoline
TLR9
Bacterial DNA (CpG)
TLR 8,10
Unknown
Akira J.Biol.Chem. 2003 278:38105–38108
How do we explain the Genentech and Tularik findings?
How could TLR4 knockout mice be LPS-nonresponsive but TLR2
transfected cells LPS responsive?
Why were TLR4-transfected cells LPS non-reponsive?
9.0
8.0
In both cases, the LPS
preparations used by the
investigators were contaminated
with bacterial lipopeptides:
TLR2 agonists!
7.0
Fold Induction
1.
6.0
5.0
4.0
3.0
2.0
1.0
0.0
TLR2
Control
TLR2
Sigma
LPS
TLR2
pure
LPS
TLR2
LTA
TLR2
PGN
TLR4
Control
TLR4
Sigma
LPS
TLR4
pure
LPS
0.6000
0.5000
Vector
2.
In Tularik’s experiments with
transfected TLR4, they lacked a
critical accessory protein
required for efficient TLR4
expression and function: MD-2
Relative Light Units
MD2
0.4000
0.3000
0.2000
0.1000
0.0000
Control
LPS
PMA
TLR4
LTA
TLR4
PGN
Common Themes in IL-1, TLR, and IL-18 Signaling
TLR4/4
IL-1RI
IL-1RAcP
CD14
MD-2
MyD88
MyD88
IRAK
IL-18R
MyD88
IRAK
TRAF6
IL-1RAcP
IRAK
TAK1/
NIK
IKK
Complex
IkB
p65 p50
Death domain
TIR domain
NF-kB activation
Nat Immunol. 2002 Apr;3(4):392-8
Nature 2003 420:329
TIRAP=MAL
Nature 2003 420:324
Luke A.J. O’Neil
www.stke.org/cgi/content/full/sigtrans;2003/171/re3
Science. 2003 Aug 1;301(5633):640-3
Barton and Medzhitov Science. 2003 Jun 6;300(5625):1524-5
Common and Distinct Themes in TLR Signaling
TLR4/4
TLR2
CD14
TLR1/6
Rac
MD-2
PI3K
TRIF
PI3K
TIRAP
MyD88
MyD88
IRAK
IRF3
TIRAP
IRAK
TRAF6
TAK1/
NIK
AKT
MAP kinases
IFN-b
Death domain
IKK
Complex
TIR domain
IkB
p65 p50
TLR2Specific
Common
Responses
TLR4Specific
Single ligand-Single response vs. Multiple Ligands-complex response
Ozinsky and Underhill Current Opinion in Immunology 2002, 14:103–110
Other PRRs
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Dectin-1
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Macrophage Mannose Receptor
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C-type lectin that binds b-glucan-containing particles including
zymosan and C. albicans
Cell bound C type lectin that binds sugar molecules on the surface
on many bacteria and viruses.
Macrophage Scavenger Receptor (SR-A)
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Recognize certain anionic polymers and low-density lipoproteins
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4 forms in humans:
Peptidoglycan Recognition Proteins (PGRP)
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PGRP-L: liver
PGRP-Ia and PGRP-Ib: esophagus
PGRP-S: bone marrow
Adjuvants
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Functionally defined as any substance that, when
mixed with antigen, increases the immunogenicity of
the antigen
Injection of unmodified, nonself proteins in the
absence of adjuvant results in tolerance.