How Drosophila has helped understanding innate immunity? An evolutionary perspective on Toll-like receptors

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1 How Drosophila has helped understanding innate immunity? An evolutionary perspective on Toll-like receptors by Bruno Lemaitre Global Health Institute Ecole Polytechnique Fédérale de Lausanne

. - Immediately active Adaptive Immunity Uniquely in vertebrates Genetic

2 Innate vs Adaptive Immunity Innate Immunity - Conserved throughout evolution - Phagocytosis, NK, antimicrobial peptides, Complement.. - Immediately active Adaptive Immunity Uniquely in vertebrates Genetic rearrangements of somatic genes allows for millions of combinations of Antigen receptors (BCR, Immunoglobulins, TCR) Requires priming

3 Innate Recognition mechanism: The Janeway hypothesis Innate immunity is activated by conserved microbial patterns (pathogen-associated molecular patterns, PAMPs, note: they are also found on non-pathogens) PAMPs are recognized by dedicated Pattern-Recognition Receptors (pattern recognition proteins, PRRs) Recognition of PAMPs through PRRs is a prerequisite for activation of most immune reactions This explains why adjuvants had to be used to induce reaction against most antigens (immunologists dirty little secret ), adjuvants provided the PAMPs, for example Mycobacterium tuberculosis) C. Janeway ( ) Janeway C. Cold Spring Harb Symp Quant Biol 1989; R Medzhitov, CA Janeway Jr - Cell, 1997

4 Innate Recognition mechanism: The Janeway hypothesis C. Janeway ( ) Janeway C. Cold Spring Harb Symp Quant Biol 1989; R Medzhitov, CA Janeway Jr - Cell, 1997

5 Which molecules mediate patternrecognition? Bacteria PAMPs (LPS, peptidoglycans, lipoproteins, dsrna) PRRs (?) Immune cells cytoplasm nucleus

6 «Toll-like receptors» (TLRs), mediators of innate immune response Microbial elicitors (LPS, dsrna, lipoproteins ) Extracellular LRR: Leucin-Rich-Repeats TIR : Toll/IL1R domain Intracellular Activation of immune genes

7 The systemic immune response of Drosophila: The road to Toll

9 Induction of antimicrobial activity by immune challenge Antimicrobial activity Control Time after bacterial injection (h)

10 Hans G. Boman ( ) Antimicrobial activity due to the production of Antimicrobial peptides Control Time after bacterial injection (h)

11 The systemic antimicrobial response: Effectors Drosomycin Fungi Metchnikowin nd septic injury Defensin Gram-positive bacteria Massive production of antimicrobial peptides by the fat body Cecropin Drosocin Gram-negative bacteria Attacin Diptericin nd nd

12 Organisation of the diptericin gene promoter enhancer -1kb Coding sequence κb -RE Kappler et al., EMBO J 1993 Engström et al JBC 1993

The transcription factors NF-κB - Discovered in 1986 - Cloned in

Activated by IL1 and TNF-α - Regulate many inflammatory genes Sen

13 The transcription factors NF-κB - Discovered in Cloned in 1990 IL1 TNFα TNF-R David Baltimore - Related to c-rel IL1-R - Activated by IL1 and TNF-α - Regulate many inflammatory genes Sen and Baltimore Cell, 1986; Ghosh et al 1990; Kieran et al.,cell. 1990

14 Drosophila Rel/NF-κB and IκB proteins Rel domain Dorsal NLS 678 Dif NLS 667 Relish NLS 972 PEST Cactus Ac Ankyrin repeats PEST 500 Steward et al, Science 1987; Ip et al Cell 1993; Dushay et al PNAS 1996

15 The Toll pathway in Drosophila development Kathryn V. Anderson Anderson, K. V. & Nusslein-Volhard, 1984 Anderson, K. V., et al., Cell 1985 Hashimoto, et al., Cell 1988

16 The Toll pathway in Drosophila development Kathryn V. Anderson Anderson, K. V. & Nusslein-Volhard, 1984 Anderson, K. V., et al., Cell 1985 Hashimoto, et al., Cell 1988

17 Conserved signaling pathways control Dorsal and NF-κB nuclear translocation Drosophila dorso-ventral patterning spätzle Toll-R tube pelle cactus dorsal dorsal Mammalian immune response Transcriptional activation IL-1 IL1-R IRAK I-κB NF-κB Membrane NF-κB Transcriptional activation Extracellular Cytoplasm Nucleus Gay, N. & Keith, F. Nature Schneider et al., Genes Dev. 1991

18 Conserved signaling pathways control Dorsal and NF-κB nuclear translocation Drosophila dorso-ventral patterning spätzle Toll-R tube pelle cactus dorsal dorsal Mammalian immune response Transcriptional activation IL-1 IL1-R IRAK I-κB NF-κB Membrane NF-κB Transcriptional activation Extracellular Cytoplasm Nucleus Gay, N. & Keith, F. Nature Schneider et al., Genes Dev. 1991

19 Induction of genes encoding antimicrobial peptides in Toll deficient mutants wildtype Toll - - 1h 6h 24h - 1h 6h 24h Drosomycin (antifungal) Diptericin (antibacterial) Rp49 -, control h, hours after infection Lemaitre et al., Cell 1996

20 Fungi Regulation of the systemic antimicrobial response Gram + bacteria Yeast Gram - bacteria Toll Fat body cells Imd DIF/DORSAL RELISH κb κb κb κb κb κb Drosomycin Diptericin Lemaitre et al., PNAS 1995, Lemaitre et al., Cell 1996

21 Fungi Regulation of the systemic antimicrobial response Gram + bacteria Yeast Gram - bacteria SPZ Toll Fat body cells Imd DIF/DORSAL RELISH κb κb κb κb κb κb Drosomycin Diptericin Lemaitre et al., PNAS 1995, Lemaitre et al., Cell 1996

22 Drosophila immune responses are adapted to the pathogen: B. bassiana Natural infection by B. bassiana OR R Tl - Tl D C 1d 2d 3d 4d 5d 6d 7d 8d 1d 2d 3d C SI OR R Drom Metch Dipt CecA Rp49 Lemaitre et al. PNAS 1997

A constitutively active form of TLR4 induces the expression of cytokines and co-stimulatory molecules htoll = TLR4 LRR CD4 Ig domain C. Janeway & R.

23 A constitutively active form of TLR4 induces the expression of cytokines and co-stimulatory molecules htoll = TLR4 LRR CD4 Ig domain C. Janeway & R. Medzhitov TIR : Toll-IL1R domain Cells expressing a chimeric construct CD4-hTOLL induce the expression of genes encoding IL-1, IL-8, B7.1 via NF-κB =>striking similarities between vertebrate and insect immune responses (Toll/NF-κB) Role in the activation of adaptative immune response Medzhitov et al., (1997) Nature 388:

24 Mutations in TLR4 block the host response to LPS LPS Macrophages TNF Cytokines Other cells (Granulocytes) PAF Kinins Leukotriens Reactive oxygen species Proteases NO... Septic Shock Mouse lps locus : * Higher susceptibility to Gram negative bacterial infection * Higher resistance to endotoxin (LPS) The mutation lps affects the TLR4 receptor Poltorak et al., (1998) Science 282; ; Quershi et al., (1999) J Exp Med, 189; B. Beutler C3H/Hej : point mutation in the TLR4 gene (codominant)) C57BL/10ScCr : deletion of the TLR4 gene (recessive) Production of TLR4 -/- confirms these observations Hoshino et al., (1999) J immunol, 162; S. Akira

25 TLRs Today

26 Toll-like receptors (TLRs) and their respective ligands Extracellular medium Phagosome Signalisation Cytoplasm NF-κB

27 Different cellular localization of TLRs The nucleic acid sensor TLRs are located in the endosome

Binding of a Tri-Acylated Lipopeptide Jin et al.

28 Molecular Basis of TLR-Ligand Interaction Crystal Structure of the TLR1- TLR2 Heterodimer Induced by Binding of a Tri-Acylated Lipopeptide Jin et al., Cell Derived from Ishii and Akira, Cell host and Microbes 2008

29 TLRs activate distinct signaling cascades via the recruitment of TIR domain adaptors

Roles of TLRs Regulate NF-κB, MAPK and IRF pathways (production of type I interferons) - inflammation innate immunity Induce immune effectors : - regulation of antimicrobial peptides Antigen

30 Roles of TLRs Regulate NF-κB, MAPK and IRF pathways (production of type I interferons) - inflammation innate immunity Induce immune effectors : - regulation of antimicrobial peptides Antigen Presenting cells Activate dendritic cell maturation : - Secretion of cytokines - Expression of co-stimulatory molecules - Expression of CMH-II - Modify adhesive properties - Induce migration to secondary lymphoid organs T Lymphocytes

31 Relevance of TLRs in mouse and Human host defense * TLR and Myd88 deficient mice are viable but exhibit an increased susceptibility to experimental infections=> point to an important role of TLRs in host defense * Human patients with deficiency for IRAK4 or MYD88 suffer from life-threatening infections caused by pyogenic bacteria (Picard et al., Science 2003; von Bernuth H et al., Science 2008). * Human patients with TLR3 deficiency present a predisposition to Herpes Simplex Virus-1 infection (Zhang et al., Science 2007).

Different families of pattern-recognition receptors in mammals Microbes are sensed by distinct Pattern-recognition receptors that activate innate immune responses and orient the adaptive response.

32 Different families of pattern-recognition receptors in mammals Microbes are sensed by distinct Pattern-recognition receptors that activate innate immune responses and orient the adaptive response. This response is influenced by the nature of the infectious agent (determined by its PAMPs), the route of infection, the tissue that is infected, as well as the localization of the infectious agent.

33 Current questions in the field of Pattern recognition In vivo imaging of PRR recognition, intracellular trafficking Microbial recognition beyond PRR recognition (microbial and host factors influencing recognition) Negative regulation of PRR Role of PRR in mucosal immunology and microbiotae control Activation of PRRs by endogenous ligands- synergy with danger signals (and damage) Relevance of PRRs during natural course of infection Impact on other facets of the immune systems Roles of PRRs in Humans and implication in auto immune diseases

34 TLRs: an evolutionary perspective Microbial elicitors (LPS, PG ) Extracellular LRR: Leucin-Rich-Repeats TIR : Toll/IL1R domain Intracellular Activation of immune genes

35 Proteins of the Toll-IL1R family TOLL/TLR IL-1R/IL-18R Extracellular LRR Ig R proteins TIR TIR TIR DD MyD88 LRR: Leucin-Rich-Repeats Ig : Immunoglobulin-domain DD : Death-Domain NBS : Nucleotide-Binding-Site TIR : Toll-IL1R domain TIR NBS LRR Intracellular 30

36 Phylum/Subphylum Metazoans Eumetazoans Bilateria Deuterostomes Chordates Vertebrates Questions: History and origins of human TLRs? Urochordates Cephalochordates Echinoderms Protostomes Platyhelminthes Annelids Molluscs Origin of PRR function for TLRs? Ancestral function of the TLR-NF-κB pathway? Origins of TLRs and NF-κB modules? Nematods Arthropods Cnidarians Poriferans Choanoflagelate Fungi Plants

37 Deciphering the history of TLRs in the animal kingdom: the tools - Genome sequence Gene number, TLR gene chromosomal location, homology => trees (homologous/paralagous) polymorphisms among populations and species - Functional data loss-of-function mutations (rescue) RNAi, morpholino polymorphisms - Expression data expression profile, immune induction, => Elaboration of scenarios Confronted to animal phylogeny (and history in humans)

38 Phylum/Subphylum Metazoans Eumetazoans Bilateria Deuterostomes Chordates Vertebrates Questions: History and origins of human TLRs? Urochordates Cephalochordates Echinoderms Protostomes Platyhelminthes Annelids Molluscs Origin of PRR function for TLRs? Ancestral function of the TLR-NF-κB pathway? Origins of TLRs and NF-κB pathways? Nematods Arthropods Cnidarians Poriferans Choanoflagelate Fungi Plants

39 Human Toll-like receptors (TLRs): the Repertoire Extracellular medium Phagosome Signalisation Cytoplasm 10 TLRS+ 1 pseudogene (TLR11) NF-κB

display higher evolutionary flexibility -High rate of stop codons in TLR5 (up to 23% in Europe and South Asia)=> a largely redundant

40 Evolutionary dynamic of human TLRs Sequence analysis of TLR genes in a panel of 158 healthy individuals indicates : - intracellular TLRs have been subjected to strong purifying selection => essential and non-redundant roles -TLR1, TLR2, TLR4, TLR5,TLR6 and TLR10 display higher evolutionary flexibility -High rate of stop codons in TLR5 (up to 23% in Europe and South Asia)=> a largely redundant role of TLR5 - TLR10-TLR1-TLR6 gene cluster show signs of positive selection in Europeans and East-Asians Barreiro et al Plos Genetic 2009

41 Evolution of TLR family members in vertebrates TLRs/species (20 in Xenopus) -Six major families: -TLR1/2/6/10, -TLR3, -TLR4, -TLR5, -TLR7/8/9, -TLR11 -TLR1,2,3,4,5,7,8,9 are found in fishes -Evidence for a PRR Function *in fishes (TLR3/DsRNA; TLR5/flagellin) *in chicken (TLR2/lipoprotein) Roach et al., PNAS 2005; Oshiumi et al.; Current Genomics 2008; Rebl et al., 2009

42 Evolution of TLR family members in vertebrates: Species adaptation Roach et al., PNAS 2005; Oshiumi et al.; Current Genomics 2008; Rebl et al., 2009

43 Evolution of TLR family members in vertebrates: Species adaptation -Expansion and variation of TLR1/TLR2 family member Duplication of TLR2 and 1 in Chicken TLR14 in amphibians and fishes Roach et al., PNAS 2005; Oshiumi et al.; Current Genomics 2008; Rebl et al., 2009

44 Evolution of TLR family members in vertebrates: Species adaptation TLR11, 12 and 13 in mouse -Presence of non-primate TLRs *TLR11, 12 and 13 in mouse *TLR22 (fish and amphibian) *TLR21 (fish, amphibian, birds) [TLR22 is cell surfacetlrs recognizing long dsrna] TLR21, 22 in fishes and amphibians Roach et al., PNAS 2005; Oshiumi et al.; Current Genomics 2008; Rebl et al., 2009

45 Evolution of TLR family members in vertebrates: Species adaptation -No TLR4 in Fugu and Puffer fish -D. rerio TLR4 is Negative regulator -Absence of TLR4 in some fish species Roach et al., PNAS 2005; Oshiumi et al.; Current Genomics 2008; Rebl et al., 2009

46 Evolution of TLR family members in vertebrates: Species adaptation - A soluble TLR5 Roach et al., PNAS 2005; Oshiumi et al.; Current Genomics 2008; Rebl et al., 2009

47 The TLR Repertoire of the vertebrate ancestor Echinoderms Cephalochordata Independent development of adaptive immune systems in craniates Chordates Urochordata VLRs Craniates Lampreys Vertebrate TCRs, BCRs

48 The TLR Repertoire of the vertebrate ancestor Echinoderms Cephalochordata Chordates Urochordata Craniates The Japonese Lamprey (Lethentheron japonicum) - 16 TLRs * 4 TLR24 (TLR1/2 type) * 4 TLR14 (TLR1/2 type) * 3 TLR21 * 1 TLR22 * 1 TLR3, * 1TLR5, * 1TLR7/8 Lampreys Vertebrate

The TLR Repertoire of the vertebrate ancestor (Cambrian period) -Conservation of most TLRs across the vertebrate phylum =>Strong conservation of PAMPs recognition - The star like tree of TLRs suggest

49 The TLR Repertoire of the vertebrate ancestor (Cambrian period) -Conservation of most TLRs across the vertebrate phylum =>Strong conservation of PAMPs recognition - The star like tree of TLRs suggest that each TLR family was generated at the same time -Existence of species adaptation compatible with adaptation to pathogen pressure -The adaptive immune system has emerged in the context of a pre-existing TLR network Lipoprotein LPS Flagelin TLR2 TLR4 TLR3 TLR7 TLR8 TLR9 TLR5 TLR22 viral dsrna? TLR21 Ancestor of Vertebrates

50 Phylum/Subphylum Metazoans Eumetazoans Bilateria Deuterostomes Chordates Vertebrates Questions: History and origins of human TLRs? Urochordates Cephalochordates Echinoderms Protostomes Platyhelminthes Annelids Molluscs Origin of PRR function for TLRs? Ancestral function of the TLR-NF-κB pathway? Origins of TLRs and NF-κB pathways? Nematods Origin of TLR-related molecules Arthropods Cnidarians Poriferans Choanoflagelate Fungi Plants

51 TLRs in invertebrate deuterostomes: Urochordates Kasamatsu et al., DCI 2010, Satake and Sasaki Zool. Science 2010; Yuan et al., mol. Immunol. 2009; Hibino et al., Dev. Biol 2006 Echinoderms Cephalochordata Chordates Urochordata Lampreys Craniates Vertebrate

52 TLRs in invertebrate deuterostomes: Urochordates Kasamatsu et al., DCI 2010, Satake and Sasaki Zool. Science 2010; Yuan et al., mol. Immunol. 2009; Hibino et al., Dev. Biol 2006 Echinoderms Cephalochordata Chordates Urochordata Lampreys Craniates Vertebrate The Ascidian Ciona intestinalis - 2 TLRs - distantly related to vertebrate types - Expressed in the digestive tract and hemocytes - present at both the membrane and endosomes - Activate NF-κB in response to multiple ligands

53 Expansion of TLRs in Cephalochordates Kasamatsu et al., DCI 2010, Satake and Sasaki Zool. Science 2010; Yuan et al., mol. Immunol. 2009; Hibino et al., Dev. Biol 2006 The Amphoxius: Branchiostoma floridae - 48 TLRs (36 vertebrate type, 12 insect type) - Evidence for an immune role (BfTLR1 is induced upon LPS) Add a tree

Biol 2006 The Echinoderms: the Purple Sea Urchin (Strongylocentrotus purpuratus) - 224 TLRs (213 NLRs, >300 SCR) -

54 Massive Explosion of TLRs in Echinoderms Kasamatsu et al., DCI 2010, Satake and Sasaki Zool. Science 2010; Yuan et al., mol. Immunol. 2009; Hibino et al., Dev. Biol 2006 The Echinoderms: the Purple Sea Urchin (Strongylocentrotus purpuratus) TLRs (213 NLRs, >300 SCR) - no functional data - 211/222 are specific to the sea urchin lineage - more related to vertebrate TLRs except 3 related to the insect type -No expansion of NF-κB pathway members Add a tree

55 Evolution of TLRs in Deuterostomes: Conclusions - Strong conservation of TLRs in Agnathes - Probable diversification of vertebrate TLRs from a small pool of multifunctional TLRs - Expansion of lineage specific TLRs in Echinoderms and Cephalochordates - The lineage of mammalian TLRs cannot be followed before Agnathes due to important species diversifications

56 Phylum/Subphylum Metazoans Eumetazoans Bilateria Deuterostomes Chordates Vertebrates Questions: History and origins of human TLRs? Urochordates Cephalochordates Echinoderms Protostomes Platyhelminthes Annelids Molluscs Origin of PRR function for TLRs? Ancestral function of the TLR-NF-κB pathway? Origins of TLRs and NF-κB pathways? Nematods Arthropods Cnidarians Poriferans Choanoflagelate Fungi Plants

57 Phylum/Subphylum Metazoans Eumetazoans Bilateria Deuterostomes Chordates Vertebrates Questions: History and origins of human TLRs? Urochordates Cephalochordates Echinoderms Protostomes Platyhelminthes Annelids Molluscs Origin of PRR function for TLRs? Ancestral function of the TLR-NF-κB pathway? Origins of TLRs and NF-κB pathways? Nematods Arthropods Cnidarians Analysis in Protostomians - Ecdysozoan - Lophotrochozoan Poriferans Choanoflagelate Fungi Plants

58 Ecdysozoan Tolls versus Vertebrate TLRs Human Drosophila C. elegans Number 10 TLRs 9 Toll (5-12 in insects) 1 Toll Type v-type insect type (except Toll9) insect -type Ligands PAMPS Spätzle (endogenous)? Role Immunity Immunity & Development Development Signaling NF-κB, IRF NF-κB and NF-κB Independant No NFexternal capping Ectodomain internal capping Ectodomain

Lophotrochozoan TLRs and the quest for the TLR ancestral function Annelids: - The polychaete Capitella capitata -105 TLRs, mostly p-typetlrs - An NF-κB pathway -The leech Helobdella -16 TLRs with

59 Lophotrochozoan TLRs and the quest for the TLR ancestral function Annelids: - The polychaete Capitella capitata -105 TLRs, mostly p-typetlrs - An NF-κB pathway -The leech Helobdella -16 TLRs with capping -The parasite Shistosoma mansoni -No TLRs Conclusions: - A TLR-NF-κB cassette was present at the origin of Bilaterians. -The role of TLRs in the bilaterian ancestor cannot be assessed in the absence of functional data in lophotrochozoans - Role of TLR in innate immunity in insects and vertebrate: Conservation or independent co-optation?

60 Phylum/Subphylum Metazoans Eumetazoans Bilateria Deuterostomes Chordates Vertebrates Questions: History and origins of human TLRs? Urochordates Cephalochordates Echinoderms Protostomes Platyhelminthes Annelids Molluscs Origin of PRR function for TLRs? Ancestral function of the TLR-NF-κB pathway? Origins of TLRs and NF-κB modules? Nematods Arthropods Cnidarians Poriferans Choanoflagelate Fungi Plants NO TLRs

61 Phylum/Subphylum Metazoans Eumetazoans Bilateria Deuterostomes Chordates Vertebrates Questions: History and origins of human TLRs? Urochordates Cephalochordates Echinoderms Protostomes Platyhelminthes Annelids Molluscs Origin of PRR function for TLRs? Ancestral function of the TLR-NF-κB pathway? Origins of TLRs and NF-κB modules? Nematods Arthropods Cnidarians Analysis in - Cnidarians - Poriferans Poriferans Choanoflagelate Fungi Plants NO TLRs

62 Early origins of TLRs: the Cnidaria phylum The Sea anemone Nematostella vectensis - 1 true TLR (TIR-TM-LRR) - 3 IL1-R (TIR-TM-IG) - Most NF-κB pathway components Hemmerich et al Trends in Immunol. 2010; Gauthier et al., Evol and Dvt 2010

63 Early origins of TLRs: the Porifera phylum The Desmoponges Amphimedon queenslandica - 2 «IL1-R»related receptor (TIR +TM+IG) - Most NF-κB components -Expression pattern compatible with a role in development Gauthier et al, Evolution and Development 2010

Early origins of TLRs: the Porifera phylum The Desmoponges Amphimedon queenslandica - 2 «IL1-R»related receptor (TIR +TM+IG) - Most NF-κB components -Expression pattern compatible with a

64 Early origins of TLRs: the Porifera phylum The Desmoponges Amphimedon queenslandica - 2 «IL1-R»related receptor (TIR +TM+IG) - Most NF-κB components -Expression pattern compatible with a role in development No true TLR in the porifera phylum: The combination of TIR and LRR domains on the same protein would appear in Eumetazoan Gauthier et al, Evolution and Development 2010

65 Origin of TLRs Phylum/Subphylum Metazoans Eumetazoans Bilateria Deuterostomes Chordates Vertebrates Urochordates Cephalochordates Echinoderms Protostomes Platyhelminthes Annelids Molluscs Nematods Origins of TLRs and NF-κB pathways? - TLR genes are restricted to Eumetazoans and probably originated at the dawn of animal evolution more than 600 million years ago. - Ancient origin of the TLR/NF-κB signalling module in animals. - Basal Metazoans have a sophisticated immune toolkit (NLR, C3) Origin of TLR-related molecules Arthropods Cnidarians Poriferans Choanoflagelate Fungi Plants NO TLRs

66 Origin of TLRs Origin of TLR-related molecules Phylum/Subphylum Metazoans Eumetazoans Bilateria Deuterostomes Chordates Vertebrates Urochordates Cephalochordates Echinoderms Protostomes Platyhelminthes Annelids Molluscs Nematods Arthropods Cnidarians Poriferans Choanoflagelate Fungi Plants Ancestral function of TLRs -Three functions of TLR: *regulation of innate immunity mostly through NF-κB in insects and vertebrates *developmental role through NF-κB signalling in insects cell adhesion and development, independently of NF-κB in both insects and nematodes - The ancestral role of TLRs is not known - It is probable that Toll-mediated humoral immune response in insects and TLR mediated immune signalling in vertebrates result from convergent evolution.

67 Speculation:Two scenarios for TLR evolution An ancestral role for TLR In Pattern-recognition Parallel evolution of TLR immune functions

68 Speculation:Two scenarios for TLR evolution Phylum/Subphylum An ancestral role for TLR In Pattern-recognition Metazoans Eumetazoans Bilateria Deuterostomes Chordates Vertebrates Supports : -An ancestral role for TLR as PRR in sponges and cnidaria makes sense - Variation in TLRs would be due to gene loss and lineage diversification - The roles of Toll in Drosophila and nematod development are novel adaptations -A number of preliminary experiments suggest an immune role for basal metazoan TLRs Origin of TLRs Origin of TLR-related molecules Urochordates Cephalochordates Echinoderms Protostomes Platyhelminthes Annelids Molluscs Nematods Arthropods Cnidarians Hemmrich et al, Trend Immunol 2010 Poriferans Choanoflagelate

69 Speculation:Two scenarios for TLR evolution Parallel evolution of TLR immune functions Supports : -Most TIR domain proteins belong to species specific branches -Drosophila and mammalian TLRs immune function have been co-opted - The IG-TIR (IL1-R) (TLR) domain combination has probably evolved twice Zhang et al., Immunogenetic 2010

70 Speculation:Two scenarios for TLR evolution Parallel evolution of TLR immune functions Supports : -Most TIR domain proteins belong to species specific branches -Drosophila and mammalian TLRs immune function have been co-opted - The IG-TIR (IL1-R) (TLR) domain combination has probably evolved twice -Possible if protein domain combinations are constraint (supported by the limited number of protein architecture (eg TIR with LRR, Ig, DD and SAM) -Possibility that domain architectures are created several times Zhang et al., Immunogenetic 2010

71 Retracings the evolution of TLR+NF-κB Pathway Gauthier et al, 2010

72 Key References

73 Phylum/Subphylum Metazoans Eumetazoans Bilateria Deuterostomes Chordates Vertebrates Representative species Characterized Function Homo sapiens Immunity Mus musculus Immunity Urochordates Cephalochordates Ciona savigny Branchiostoma floridae?? Echinoderms Protostomes Platyhelminthes Strongylocentrotus purpuratus? Schistosoma mansoni Annelids Capitella sp. I? Origin of TLRs Molluscs Nematods Euprymna scolopes Lottia gigantea Caenorhabditis elegans? Development Origin of TLR-related molecules Arthropods Drosophila melanogaster Tachypleus tridentatus Litopenaeus vannamei Development and immunity?? Cnidarians Poriferans Fungi Plants? Nematostella vectensis Acropora millepora? Hydra magnipapillata? Amphimedon queenslandica? Suberites domoncula? Candida albicans Arabidopsis thaliana no TLR TLR-related multiple cysteine cluster TLR single cysteine cluster TLR

74 Domain architecture evolution of Pattern recognition receptors -The TIR domain is present in several group of proteins with other domains (LRR, IG, DD, SAM) - separation in two groups (IG-TIR ou LRR-TIR) - Many TIR domain proteins belong to species specific branches -Either the IG-TIR or the LRR-TIR domain combination has evolved independantly more than once -Drosophila and mammalian TLRs have evolved independtly (except Toll9) Zhang et al., Immunogenetic 2010

75 The conservation of NLR and TLR receptors from (at least) cnidarians to mammals highlights the ancient evolutionary history of these innate immunity families. The presence of multiple proteins with similar domain architectures creates the impression that all these proteins and, by extension the specific pathways in which they participate, could have been present in ancestral species. However, we show here that the appearance of conservation hides a very complex evolutionary history of these receptor families, which underwent massive species-specific expansions and independently evolved identical domain architectures. Also, studies for TLRs and other innate immune related protein families between arthropods and vertebrates reach similar conclusions that members of innate immune systems could have evolved independently (Hughes 1998; Hughes and Piontkivska 2008), which reinforces our parallel evolution hypothesis. Both NACHT and TIR domain are protein protein interaction domains that contribute to signal transduction, and this functional class of proteins was called promiscuous because of their tendency to associate with different domains (Basu et al. 2008). When compared with the list of the top 215 highly promiscuous domains in eukaryotes (Basu et al. 2008), it turned out not only the NACHT and TIR domain themselves, but also the domains they associate with, such as Death, CARD and Ig domains, are on that list. However, only a small fraction of the possible domain combinations actually exist in nature, suggesting that domain architectures are under strong evolutionary selection (Han et al. 2007). For example, both amphioxus and sea anemone have Ig TIR domain-containing sequences, the same architecture as IL-1R family members in vertebrates. These sequences are likely reinvented in various animal lineages by parallel evolution. The most interesting observation is that such massive expansions and domain shuffling only resulted in a relatively small number of protein architectures. Clearly, the number of possible solutions must be limited by functional considerations that act as constraints. The presence of such constraints limiting the number of functional domain combinations provides a possible alternative explanation for the conservation of domain architectures in eukaryotes, where the majority of the genomic proteins are multidomain proteins (Han et al. 2007), but only a small fraction of all possible domain combinations are present. that domain architecture reinvention is a more common phenomenon than previous thought (Forslund et al. 2008). These authors suggested that between 5.6% and 12% of all domain architectures could have been created more than once in different genomes.

76 Ecdysozoans: Multiple functions of insect Tolls Diptera: D. melanogaster - 9 Tolls, Toll9 is related to vertebrate type TLRs, other are divergent - Toll-1 (and possibly Toll9) are involved in both innate immunity - Toll-1 is not a PRR but is activated by an endogenous ligand, Spazle - Toll 1 have Tir dependent or TIR independant (cell adhesion )function - Toll2-8 have non-immune and NF-κB independant function, (development, neurogenesis, cell adhesion) Other insects - 5 in (Apis mellifera) to 12 (Aedes Egypti) Tolls - The immune role seems conserved Nematod: C. elegans - 1 Toll (Tol-1) with developmental function - Absence of NF-κB pathway

The conservation of NLR and TLR receptors from (at least) cnidarians to mammals highlights the ancient evolutionary history of these innate immunity families.

77 The conservation of NLR and TLR receptors from (at least) cnidarians to mammals highlights the ancient evolutionary history of these innate immunity families. => These proteins and, by extension the specific pathways in which they participate, could have been present in ancestral species. However, we show here that the appearance of conservation hides a very complex evolutionary history of these receptor families, which underwent massive species-specific expansions and independently evolved identical domain architectures=> parallel evolution Two factors could supports the hypothesis of parallel evolution: -domain architecture reinvention is a more common phenomenon than previous thought (Forslund et al. 2008). These authors suggested that between 5.6% and 12% of all domain architectures could have been created more than once in different genomes -The number of possible combination of domain must be limited by functional considerations that act as constraints. This is supported by the observation that only a small fraction of the possible domain combinations actually exist in nature, suggesting that domain architectures are under strong evolutionary Selection. that.

78 TLR Evolution I: functional diversification Development (Nematods, insects) Immunity (Insects, vertebrates)

79 TLR Evolution II: Origin and evolution of TLR genes 10 TLRs >

80 TLR Evolution: Conclusions - TLR genes are restricted to Eumetazoans and probably originated at the dawn of animal evolution more than 700 million years ago. - With rare exception, both TLR and NF-κB genes are found in sequenced animal genomes pointing to the ancient origin of the TLR/NF-κB signalling module. -lineage-specific protein family expansion and diversification of TLR (e.g. echinoderms) - Three distinct functions of TLR have been identified so far: i) TLRs are essential during host immune responses through NF-κB signalling in insects and vertebrates; ii) they contribute to normal patterning and organogenesis during development through NF-κB signalling in insects and iii) they contribute to cell adhesion during embryonic development, apparently independently of NF-κB activation in both insects and nematodes. - It is probable that Toll-mediated humoral immune response in insects and TLR mediated immune signalling in vertebrates result from convergent evolution. - Recurrent use of similar protein module (TIR domain and LRR motifs) and signalling pathway (NF-κB) in immune response is observed in phylogenetically distant lineages. Future challenges: - Analyse TLR function in invertebrate deuterostomes, lophocotrozoan and cnidarian model organisms. - Further dissect the NF-κB independent role of TLRs during development.

81 Sensing of Microbial Signatures by Multiple Host Innate Immune Receptors

82 TLRs activate distinct signaling cascades via the recruitment of TIR domain adaptors

83 Lipoprotein TLR2 LPS Flagelin TLR4 TLR5 TLR22 viral dsrna TLR2 TLR2 TLR5 TLR22 TLR3 TLR7 TLR8 TLR9? TLR21 TLR3 TLR7 TLR8 TLR9 TLR21 Ancestor of Vertebrates Gain of new TLRs Loss of non-mammalian TLRs TLR3 TLR7 TLR8 TLR9 TLR2 TLR2 TLR4 TLR5 Mammals Teleosts Gain of new TLRs Loss of mammalian TLRs after Oshiumi et al. Current Genomics, 2008, Vol. 9, No. 7

84 TLRs: differences between Drosophila and mammals RECOGNITION / MODE OF ACTIVATION - Mammalian TLRs are «Pattern Recognition Receptors» directly involved in the sensing of PAMPs - In Drosophila, Toll is activated by an endogenous ligand, Spaztle, which is activated by a cascade of serine proteases. Microbial recognition occurs upstream of this proteolytic cascade and involves secreted pattern recognition receptors (PGRPs, GNBPs) FUNCTION * In mammals, TLRs activate NF-κB, MAPK, and IRF cascades that modulate the activity of the immune system (cytokines, co-stimulatory factors) while in Drosophila, the Toll pathway regulates genes encoding effectors (antimicrobial peptides). * Drosophila Tolls play a role in development.

85 TLRs and activation of innate immune response of mammals : conclusions * FUNCTIONS : - TLRs are important pattern-recognition receptors that sense infectious agents and activate innate immune response: they play a role of sentinels of the innate immune system - TLRs links innate to adaptive immune response * LIGANDS : - TLRs are activated by different classes of elicitor derived from bacteria and virus as well as endogenous factors. - There is evidence for direct interactions between TLRs and microbial molecules with the help of co-factors - Cooperation of TLR1 and TLR6 with TLR2 determine new recognition specificity. * MODE OF ACTION : TLRs modulate the transcription of different sets of immune genes via the activation of distinct signaling cascades The TLR - NF-kB is an ancient signaling cascade co-opted in the immune response of both insects and mammals

86 Innate Recognition mechanism: The Janeway hypothesis Innate immunity is activated by conserved microbial patterns (pathogen-associated molecular patterns, PAMPs, note: they are also found on non-pathogens) PAMPs are recognized by dedicated Pattern-Recognition Receptors (pattern recognition proteins, PRRs) Recognition of PAMPs through PRRs is a prerequisite for activation of most immune reactions This explains why adjuvants had to be used to induce reaction against most antigens (immunologists dirty little secret ), adjuvants provided the PAMPs, for example Mycobacterium tuberculosis) C. Janeway ( )

87 Innate Recognition mechanism: The Janeway hypothesis Innate immunity is activated by conserved microbial patterns (pathogen-associated molecular patterns, PAMPs, note: they are also found on non-pathogens) PAMPs are recognized by dedicated Pattern-Recognition Receptors (pattern recognition proteins, PRRs) Recognition of PAMPs through PRRs is a prerequisite for activation of most immune reactions This explains why adjuvants had to be used to induce reaction against most antigens (immunologists dirty little secret ), adjuvants provided the PAMPs, for example Mycobacterium tuberculosis) Other Innate Recognition mechanisms C. Janeway ( ) - Recognition of damage caused by pathogens (Danger signals (Matzinger P., 1994) - Recognition by absence of «self» (ex. NK cells, Complement) - Recognition of virulence factors (gene for gene)- the Guard model Janeway C. Cold Spring Harb Symp Quant Biol 1989; R Medzhitov, CA Janeway Jr - Cell, 1997

88 How do Drosophila sense bacterial infections? bacteria Pathogen-associated molecular pattern Peptidoglycan (PGN) Pattern recognition receptor Peptidoglycan Recognition Proteins Janeway, 1989

89 How do Drosophila sense bacterial infections? bacteria Pathogen-associated molecular pattern Peptidoglycan (PGN) Pattern recognition receptor PGRP domain Peptidoglycan Recognition Proteins 13 genes in Drosophila Related to bacterial T7 lysozymes Janeway, 1989

90 How do Drosophila sense bacterial infections? bacteria Pathogen-associated molecular pattern Peptidoglycan (PGN) Pattern recognition receptor PGRP domain Peptidoglycan Recognition Proteins 13 genes in Drosophila Related to bacterial T7 lysozymes Recognition PGRP Amidase PGRP (PGN degradation) Janeway, 1989

91 Bacteria mediates Toll and Imd pathway activation through distinct PGRPs Gram positive bacteria Gram negative bacteria GNBP-1 PGRP-SA Serine protease Spätzle Toll PGRP-LC Imd DIF/DORSAL RELISH κb κb κb Drosomycin κb κb κb Diptericin Michel et al, 2001; Choe et al, Science 2002; Gottar et al Nature 2002; Ramet et al Nature 2002

92 Peptidoglycan (PGN) as a bacterial elicitor (DiAminoPimelic acid) TCT Tracheal CytoToxin GlcNAc - N-acteylglucosamine Lysine type PGN, multilayered at bacterial surface MurNAc - N-acetylmuramic acid (Bacillus sp. have DAP type)

93 DAP and Lys type peptidoglycan activate distinct pathways Lys type peptidoglycan DAP type peptidoglycan GNBP-1 PGRP-SA Spätzle Toll PGRP-LCx Imd DIF/DORSAL RELISH κb κb κb Drosomycin κb κb κb Diptericin

94 Lim et al., 2006; Chang et al., Science 2006

95 The Toll and Imd pathways: a paradigm of innate immunity

96 Mammals Drosophila Mammals Toll pathway Imd pathway TNF-R1 pathway IL1-R pathway TNF-α IL1-R Hemolymph Nec Proteases Toll Spaetzle TNF-R1 MyD88 TAB2 TAK1 TAB1 TIR TIR DD DD IRAK TRAF6 Cytoplasm dmyd88 Tube TIR TIR DD DD? DD Pelle Imd? dtak1 DD dfadd DD D D Dredd E E D D RIP TRAF2 MEKK3 TRADD DD DD DD DD DD DD FADD DD D ProCaspase-8 E D D E D IKKγ IKKβ IκB ANK Rel Rel Rel ANK p50 RelA p105 Cactus ANK Rel Rel Dorsal DIF dmikkγ dmikkß ANK Rel ANK Relish IKKγ IKKβ IκB ANK Rel Rel Rel ANK p50 RelA p105 Caspase-8 Effector caspases (Caspase-3) Apoptosis Rel Rel p50 RelA κb κb κb Immunity genes Anti-apoptotic genes Noyau Rel Dorsal κb Rel Rel DIF Relish κb κb Antimicrobial peptide genes p50 κb Rel Rel RelA κb κb Immunity genes Anti-apoptotic genes

97 Micro-organisms are distinguished by separate recognition modules Spätzle

November(2015( 1( anus( DEUTEROSTOME+ mouth+ PROTOSTOME+ VERTEBRATES+ GNATHOSTOMES( Petromyzon( AGNATHANS( HEMICHORDATES+ ECHINODERMS+

November(2015( 1( anus( DEUTEROSTOME+ mouth+ PROTOSTOME+ VERTEBRATES+ GNATHOSTOMES( Petromyzon( AGNATHANS( HEMICHORDATES+ ECHINODERMS+ November2015 1! At some stage of evolution one or more species must have made the momentous discovery that a very convenient source of food consists of the tissues of other organisms. And whatever the