1. Response of the Innate Immune System to Pathogens: Pattern Recognition Receptors

3. What is the Innate Immune Response?
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

4. The Innate Immune Response: Common Misconceptions
The innate immune system is an evolutionary rudiment whose only function is to contain the infection until the “real” immune response can kick in.
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.

5. 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

7. Y Y Phagocytosis APC PAMP PRR B Cell Complement
Adapted from Medzhitov and Janeway
Cur. Opin. Immunol :4-9
PAMP
Phagocytosis Y
PRR
APC
Complement
Endosome
Direct Bactericidal Activity
Phagocytosis
Oxygen burst
Anti-microbial peptides
MHC B7
Pathogen-specific
Antibody Y
Naive
T Cell
Inflammatory
and effector
cytokines
Activated
T Cell
CD40L, FasL, CD30L, CD27L
B Cell

8. 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

9. PAMPs: Pathogen Associated Molecular Patterns
PRRs: Pattern Recognition Receptors

10. Takeda and Akira Genes to Cells (2001) 6:733-742

11. The NF-kB Family of Transcription Factors
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
Three NF-kB molecules in Drosophila
dorsal
controls dorsal/ventral polarity during development
Regulates antifungal gene expression
dif and relish: regulate expression of antifungal and antibacterial genes

12. 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

13. NF-kB Family Structure

14. Conservation of signaling pathways between flies and humans
Toll
Pelle
tube ?
cactus
kinase
dorsal
dif
relish
Conservation of signaling pathways between flies and humans

16. 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

20. Signaling to monocytic cells through the human Toll homolog induces accessory cell functions
NF-kB Activation

21. Requirements for the recognition of targets by the innate immune
Molecular structures recognized by the immune system must be shared by large groups of pathogens
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

22. Molecules involved in the recognition of LPS by Cells
LPS Binding Protein (LBP)
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 gram-negative bacteria and for survival of Salmonella infection
Expression of LBP in hepatocytes is regulated by LPS, IL-1, IL-6 and TNF

23. Molecules involved in the recognition of LPS by Cells
CD14, sCD14
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
CD11b/CD18 (Mac1)
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.

24. Search for the LPS Gene Endotoxin hyporesponsive mice
C3H/HeN: Normal LPS response
1. C3H/He 1947
Spontaneous mutation
mid 1960’s
C3H/HeJ: LPS hyporesponsive
Sometime
2. C57BL/10Sn
C57BL10/ScCR
LPS hyporesponsive
After 1953

25. January 1998 DNAX reports the cloning of human TLR1-5
No ligands described
Janeway’s hToll=TLR4
Rock, et al PNAS 95:588

26. September 1998 Genentech identifies TLR2 as the LPS signaling molecule by transfection studies
Didn’t look at TLR4
December Tularik also identifies TLR2 as LPS signaling molecule in transfection studies
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

27. 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
C3HHeJ RFHLCLHYRDFI H GCAIAANIIQEGFHK
Hoshino et al 1999 J. Immunol. 162:3749
TLR4 knockout mice are hyporesponsive to LPS

28. Dunne and O’Neil 2003 www. stke
Dunne and O’Neil

29. Receptor Agonist(s) TLR1 TLR2 TLR3 TLR4 TLR5 TLR6 TLR7 TLR9 TLR 8,10
(Pattern Recognition Receptors)
Agonist(s)
(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
TLR7
Imidazoquinoline
TLR9
Bacterial DNA (CpG)
TLR 8,10
Unknown

30. Akira J.Biol.Chem :38105–38108

31. How do we explain the Genentech and Tularik findings
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?

32. In both cases, the LPS preparations used by the investigators were contaminated with bacterial lipopeptides: TLR2 agonists!
In Tularik’s experiments with transfected TLR4, they lacked a critical accessory protein required for efficient TLR4 expression and function: MD-2

33. Common Themes in IL-1, TLR, and IL-18 Signaling
IL-18R
IL-1RI
IL-1RAcP
CD14
MD-2
IL-1RAcP
MyD88
MyD88
MyD88
IRAK
IRAK
TRAF6
IRAK
TAK1/
NIK
IKK
Complex
IkB
p65
p50
Death domain
TIR domain

34. NF-kB activation

35. Nat Immunol Apr;3(4):392-8

36. Nature :329
TIRAP=MAL
Nature :324

37. Luke A.J. O’Neil

38. Science Aug 1;301(5633):640-3

39. Barton and Medzhitov Science. 2003 Jun 6;300(5625):1524-5

40. Common and Distinct Themes in TLR Signaling
CD14
MD-2
TLR2
TLR1/6
Rac
PI3K
TRIF
PI3K
TIRAP
MyD88
IRF3
MyD88
TIRAP
IFN-b
IRAK
IRAK
TRAF6
TAK1/
NIK
AKT
TIR domain
Death domain
MAP kinases
IKK
Complex
IkB
p65
p50

41. Common
Responses
TLR2-
Specific
TLR4-
Specific

42. Single ligand-Single response vs. Multiple Ligands-complex response
Ozinsky and Underhill Current Opinion in Immunology 2002, 14:103–110

43. Other PRRs Dectin-1 Macrophage Mannose Receptor
C-type lectin that binds b-glucan-containing particles including zymosan and C. albicans
Macrophage Mannose Receptor
Cell bound C type lectin that binds sugar molecules on the surface on many bacteria and viruses.
Macrophage Scavenger Receptor (SR-A)
Recognize certain anionic polymers and low-density lipoproteins
Peptidoglycan Recognition Proteins (PGRP)
4 forms in humans:
PGRP-L: liver
PGRP-Ia and PGRP-Ib: esophagus
PGRP-S: bone marrow

44. Adjuvants
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.