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Innate Immunity Importance? - front line of defense - required to instruct adaptive responses

Innate Immunity Importance? - front line of defense - required to instruct adaptive responses Why is it interesting? - the recognition problem - host-pathogen arms race. Innate immunity: Cells – macrophages, neutrophils, dendritic cells, mast cells, NK cells, epithelial cells, etc.

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Innate Immunity Importance? - front line of defense - required to instruct adaptive responses

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  1. Innate Immunity Importance? - front line of defense - required to instruct adaptive responses Why is it interesting? - the recognition problem - host-pathogen arms race

  2. Innate immunity: Cells – macrophages, neutrophils, dendritic cells, mast cells, NK cells, epithelial cells, etc. Immediate destruction of pathogens. Mechanisms (e.g.’s): - low pH (gut) - defensins - complement fixation - phagocytosis, followed by killing mediated by: - antimicrobial peptides, defensins - proteases - ROS, RNS

  3. Questions: 1) What does the innate immune system actually recognize? 2) How are subsequent responses coordinated and controlled?

  4. PAMPs ‘n DAMPs How does the innate immune system recognize pathogens? “Approaching the Asymptote? Evolution and Revolution in Immunology” CA Janeway,Cold Spring Harb. Symp Quant Biol 1989 “The Immunologist’s Dirty little secret” “Pattern Recognition Receptors (PRR).” - Distinguish “infectious non-self from non-infectious self” - Non-clonally distributed, germline encoded - recognize conserved microbial products, required for microbe function, not found in host (“Pathogen-associated molecular patterns” , or PAMPs) - evolutionarily ancient recognition system

  5. Innate immunity instructs adaptive responses microbe cytokines TCR Pattern Recognition receptors MHCII CD28 B7 APCs Innate and adaptive responses are intertwined. (e.g. IFN-gamma activates macrophage killing function)

  6. Toll-like Receptors 1989: Janeway proposes PRRs 1996: Toll shown to be involved in innate immunity in Drosophila 1997: Janeway lab identifies human homologue of Toll (TLR4) 1998 : Beutler lab demonstrates that missense mutation in TLR4 underlies insensitivity to LPS in C3HeJ mouse

  7. TLRs and some of their PAMP ligands: TLR2/1: diacyl lipopeptides TLR2/6: triacyl lipopeptides TLR3: dsRNA TLR4: LPS TLR5: flagelllin TLR7,8: ssDNA TLR9: unmethylated CpG DNA

  8. TLRs and their ligands TLR2 TLR4 (Gay and Gangloff, Ann Rev Biochem 2007) , ssDNA

  9. TLR signalling Kawai and Akira, Semin Immunol 19:24-32 (2007) Outcomes: inflammatory cytokine production, upregulation of APC costimulatory molecules “(toll-like OR tlr*) AND review AND (signalling OR signaling)”->1216 refs. 1462 (toll-like[ti] OR tlr*[ti]) AND review and (signaling OR signalling)-> 580 refs

  10. An Alternative View: The Danger Hypothesis (Polly Matzinger) Not so much non-self as “Danger” that is recognized. Danger signals can be derived from infection, or from damaged/altered self.

  11. Stranger or Danger?

  12. Is the “Danger” model tautological? Recognition of host damage may have arisen in order to see: - damage as a consequence of infection - damage as something that will lead to infection - damage as something that requires similar responses as infection (e.g. leukocyte recruitment)

  13. (Nature 2009) CLEC9A – a C-type lectin Danger receptor? Mincle, another C-type lectin, has also been implicated in responses to necrotic cells (as well as to pathogenic fungi) (Yamasaki et al, Nat. Immunol 2008)

  14. Toll-like receptors are not the only innate PRRs: (Ann Rev Immunol 2005)

  15. “Complexity in action” (Underhill and Ozinsky, Ann Rev Imm 2002) The challenge – to make sense of it all

  16. One source of complexity: cooperation between different receptors.

  17. Receptor cooperation: 1) direct e.g.s?

  18. TLRs use coreceptors TLR4/MD-2 uses CD14 for recognition of LPS. (Also, to a lesser extent CD14 for TLR2). Necessary for TRIF-dependent signaling in response to rough LPS. TLR2/6 and TLR2/1 heterodimerize for recognition of different ligands. TLR2 uses CD36 for recognition of Gm+ve bacteria

  19. FRET Spatial organization of innate immune recognition? Ligand-induced and constitutive association of TLR2 with coreceptors Receptor X TLR2 FRET efficiency (Triantafilou, M et al, J Biol Chem 2006)

  20. 2) intersecting signaling cascades Pi

  21. 3) Indirect or sequential interactions:

  22. Responses due to engaging multiple receptors can be • synergistic • antagonistic • emergent (1+2=C) Three examples of interacting signaling pathways: 1) FcR/TLR: Type II macrophage activation 2) DC-SIGN/TLR: inhibition of TLR responses 3) TLR/TLR : synergistic IL-12 production

  23. 1) FcR cross-linking + LPS -> “type II activated macrophage” IL-10 and IL-12 responses to LPS reversed -> Th2, not Th1 bias (Anderson and Mosser, J. Leuk. Biol. 2002) +LPS

  24. (J Immunol. 2001) Injecting “Type II” activated macrophages into mice protected them from otherwise lethal dose of endotoxin.

  25. Nuclear Ikb-alpha -> inhibition of IL-12 production Mechanism? FcgR TLR PI3K ERK IkB NFkB chromatin remodelling at IL-10 locus

  26. TLR4 TLR2 2) • C-type Lectins: • involved in recognizing both endogenous and exogenous • glycosylated ligands b-glucans HIV, M. Tb ICAM-3 McGreal et al, Curr Op Immunol 2005 (“Dendritic cell-specific ICAM-grabbing non-integrin”)

  27. J. Exp. Med, 2003 Mycobacterial ManLAM binding to DC-SIGN suppresses LPS-induced DC maturation

  28. DC-SIGN TLR Raf Raf-mediated acetylation of NFKB and increased IL-10 production. (Gringhuis et al, Immunity 2007) NFkB

  29. 3) TLRs synergize for IL-12 production Trinchieri and Sher, Nat Rev Imm 2007 IL-12, IL-23 synergistically up-regulated Napolitani, G. et al Nat Imm 2005

  30. Teleology of these interactions? Why [telologically] the synergy in IL-12 production? Napolitani et al proposed a “combinatorial safety code” (a “two-key” model for pathogen detection and IL-12 production) Why does FcR ligation -> increased IL10, decreased IL12? Why does DC-SIGN/TLR coengagement -> increased IL-10?

  31. Do PRRs “code” for targeted pathogen responses? (Underhill and Ozinsky, Curr Op Immunol 2002) Coding or redundancy?

  32. (Jenner and Young, Nat. Rev. Microbiol 2005) Common responses to infection:

  33. (Nat Cell Biol 2006) RAW 264.7 cells: - mouse “macrophage-like” cell line - cell of choice for Alliance for Cellular Signaling large-scale analysis The Question : How complex is the network of signaling pathways in a macrophage? • The approach: • - 22 ligands (TLR agonists, cytokines, and more) • What happens when you add them individually? • What happens when you add them in every possible pairwise combination? • (231 combos [=(222-22)/2])

  34. One at a time, now…. The single ligand screen:

  35. The punchline: (now, red/blue means DDZ values, i.e. non-additivity) (231*147= 33,957 data points)

  36. Unbiased clustering of non-additivity: GCSF, IL10, IL6, RANTES cluster: TLRs + ISO or PGE MIP1alpha, TNFalpha

  37. “A simplified picture of the signalling network”

  38. How many “interaction agents” might there be? (around 40 significant clusters)

  39. Take home points: • “the primary activity of many input ligands is modulation of other • signaling systems rather than direct control over cellular outputs” • 2) “The density of cross-talk demonstrates substantial capacity for encoding • combinatorial complexity in input stimuli, but the clustering of non-additive • response patterns places significant constraints on the mechanistic complexity • of ligand interactions” • In other words, complicated, but not hopelessly so!

  40. signaling pathway network Nat Immunol 10, 327 (April 2009)

  41. (Genome wide RNAi screen in Drosophila) Nat Immunol 10, 327 (2009) Cell as single “mega-network?”

  42. Unbiased, global approaches: • Global readouts? • Global interventions?

  43. Different possibilities for cooperation in vivo (Trinchieri, Nat Rev Imm 2007)

  44. Bruce Beutler lab’s ongoing forward genetic screen: Using ENU mutagenesis, followed by positional cloning. At around 60,000 F1 mice screened as of several years ago. Multiple phenotypic screens, including: ex vivo: TLR signaling screen ((peritoneal mphi) dsDNA screen NALP3 Inflammasome screen control of virus infection (MCMV, adenovirus, flu, Rift Valley Fever) in vivo: susceptibility to MCMV and LCMV DSS-induced colitis

  45. Some hits : (Beutler,B. Immunogenetics 57:385 (2005)

  46. “To date, 282 transmissible mutations that cause discernable phenotypes have been set aside for positional cloning in the Beutler laboratory; 180 mutations have been mapped to chromosomes, and in 152 instances, molecular identification of the causative mutation has been made. These mutations fall within 105 genes. 154 of the mutations studied affect immunity, and about half of the mutations affecting immunity that are cloned prove to be novel in the sense that no such phenotype had been predicted by knockout mutations, or knockouts had not been created. Only about 50% recessive saturation of the genome has been achieved to date in any given screen; therefore, it is expected that many key discoveries of function lie in waiting. “ (Beutler lab webpage: http://www.scripps.edu/genetics/beutler/research.html)

  47. (Nature, 2003) (Nature, 2005) (Nat Imm 2005) (Nat Imm 2006) (PNAS 2006) (Nature Genetics 2007) (Science 2008)

  48. PNAS 2008 PNAS 2009 PNAS 2009

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