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

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

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

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

  2. 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

  3. 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.

  4. 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

  5. Adapted from Medzhitov and Janeway Cur. Opin. Immunol. 1997 9:4-9 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

  6. Componentsof 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

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

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

  9. 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 • difandrelish: regulate expression of antifungal and antibacterial genes

  10. Stress, infection, or cytokine p50 p65 P P NF-kB exists in the cytoplasm as an inactive heterotrimer composed of 2 Rel family proteins and an inhibitory IkB molecule IKK (Ub)n IkB 26S proteosome Nuclear Translocation Activation of NF-KB Responsive genes

  11. NF-kB Family Structure

  12. Toll Pelle tube ? ? cactus kinase dorsal dif relish cactus Conservation ofsignaling pathwaysbetween flies and humans

  13. 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

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

  15. 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

  16. 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

  17. 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 fullrepertoire of LPS-inducible genes, CD14 and CD11b/CD18must be coordinately engaged to deliver optimal signaling tothe macrophage.

  18. 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

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

  20. September 1998 Genentech identifies TLR2 as the LPS signaling molecule by transfection studies • Didn’t look at TLR4 • December 1998 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

  21. 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

  22. Dunne and O’Neil 2003 www.stke.org/cgi/content/full/sigtrans;2003/171/re3

  23. Akira J.Biol.Chem. 2003 278:38105–38108

  24. 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?

  25. 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

  26. p50 p65 TIR domain IkB Common Themes in IL-1, TLR, and IL-18 Signaling TLR4/4 IL-18R IL-1RAcP IL-1RI MD-2 CD14 IL-1RAcP MyD88 MyD88 MyD88 IRAK IRAK TRAF6 IRAK TAK1/ NIK IKK Complex Death domain

  27. NF-kB activation

  28. Nat Immunol. 2002 Apr;3(4):392-8

  29. Nature 2003 420:329 TIRAP=MAL Nature 2003 420:324

  30. Luke A.J. O’Neil www.stke.org/cgi/content/full/sigtrans;2003/171/re3

  31. Science. 2003 Aug 1;301(5633):640-3

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

  33. p50 p65 TIR domain IkB Death domain Common and Distinct Themes in TLR Signaling TLR4/4 MD-2 CD14 TLR2 TLR1/6 Rac PI3K TRIF PI3K TIRAP IRF3 MyD88 TIRAP MyD88 IFN-b IRAK IRAK TRAF6 TAK1/ NIK AKT MAP kinases IKK Complex

  34. Common Responses TLR4- Specific TLR2- Specific

  35. Single ligand-Single response vs. Multiple Ligands-complex response Ozinsky and Underhill Current Opinion in Immunology2002, 14:103–110

  36. Other PRRs • Dectin-1 • 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

  37. 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.

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