1、Toll-like受體研究進展1巨噬細胞吞噬細菌23 The History of EvolutionPlantsAtomsMoleculesLiving cellsMulticellular organismsocietyHuman beingsAnimals4Unicellular organisms Multicellular organismsThe ability of a multicellular organism to defend itself against invasion by pathogens (bacteria, fungi, viruses, etc.) dep

2、ends on its ability to mount immune responses.The first single cell form that created animal life on the planet of LAGA was the B-Prima. The research indicates that life began on the planet of LAGA due to a mysterious and unexplainable explosion 3.8 billion years ago. Gastrotrichs, are very small me

3、tazoans, multi celled organisms. They are related to Rotifers and Nematodes.All metazoans (后生動物) have inborn defense mechanisms that constitute innate immunity.Vertebrates have not only innate immunity but also are able to mount defense mechanisms that constitute adaptive immunity.Metazoans5In addit

4、ion to its role in the early phase of defense, innate immunity in mammals appears to play a key role in stimulating the subsequent, clonal response of adaptive immunity. Pathogen-Associated Molecular Patterns (PAMPs)The surfaces of microorganisms typically bear repeating patterns of molecular struct

5、urePattern Recognition Receptors (PRRs)6歐盟EUP環(huán)保指令 繼WEEE、RoHS指令之后，歐盟另一項主要針對能耗的技術(shù)壁壘指令“用能源產(chǎn)品生態(tài)設(shè)計框架指令”（EUP指令）又將實施。歐盟要求各成員國最遲在2007年8月11日前制定對相關(guān)產(chǎn)品的具體化要求并轉(zhuǎn)化為本國法規(guī)，以確保EUP生態(tài)化設(shè)計指令得以有效運作。 該指令首次將生命周期理念引入產(chǎn)品設(shè)計環(huán)節(jié)中，旨在從源頭入手，在產(chǎn)品的設(shè)計、制造、使用、維護、回收、后期處理這一周期內(nèi)，對用能源產(chǎn)品提出環(huán)保要求，全方位監(jiān)控產(chǎn)品對環(huán)境的影響，減少對環(huán)境的破壞。 十余類相關(guān)產(chǎn)品出口受阻EUP指令所涵蓋的產(chǎn)品范圍非常廣泛。

6、按歐盟EUP指令條文內(nèi)容所示，原則上包括所有投放市場的用能源產(chǎn)品，生成、轉(zhuǎn)換及計量這些能源的產(chǎn)品（不包括運輸工具）；以及用于裝入用能源產(chǎn)品中、并在市場上獨立直接銷售給最終用戶的部件。產(chǎn)品消耗的能源包括電能、固體燃料、液體燃料和氣體燃料。待指令生效后，歐盟委員會將對暖氣與熱水設(shè)備、電動馬達系統(tǒng)、家庭與服務(wù)業(yè)的照明設(shè)備、家用電器、家庭與服務(wù)業(yè)的辦公室設(shè)備、通風與空調(diào)設(shè)備等作出環(huán)境化設(shè)計的實施方法，涵蓋產(chǎn)品所含的原材料、生產(chǎn)方式、使用方式（包括耗水耗能情況）、產(chǎn)品壽命以及產(chǎn)品報廢時的處理方法或可以循環(huán)再造的程度。 75. 何時實施RoHS? 歐盟將在2006年7月1日實施RoHS，屆時使用或含有重金

7、屬以及多溴二苯醚PBDE，多溴聯(lián)苯PBB等阻燃劑的電氣電子產(chǎn)品將不允許進入歐盟市場。6. RoHS具體涉及那些產(chǎn)品？ RoHS針對所有生產(chǎn)過程中以及原材料中可能含有上述六種有害物質(zhì)的電氣電子產(chǎn)品，主要包括： 白家電，如電冰箱，洗衣機，微波爐，空調(diào)，吸塵器，熱水器等，黑家電，如音頻、視頻產(chǎn)品，DVD，CD，電視接收機，IT產(chǎn)品，數(shù)碼產(chǎn)品，通信產(chǎn)品等；電動工具，電動電子玩具醫(yī)療電氣設(shè)備. 8PAMPs and PRRs1989 Charlie JanewayBinding bacteria to PRRs induce activation activation of APCsTwo differ

8、ent types of nonself recognitionA genetically encoded set of PRRs assigned to the cells and molecules of the innate immune systema somatically generated set of receptors expressed by the cells of adaptive immune system9The characteristics of receptors of the innate and adaptive immune systemSpecific

9、 inherited in the genomeExpressed by all cells of a particular type(e.g., macrophages)Trigger immediate responseRecognize broad classes of pathogenEncoded in multiple gene segmentsRequire gene rearrangementClonal distributionAble to recognize a wide variety of molecular structures Innate Immunity Ad

10、aptive immunity10The difference between Innate immunity and adaptive immunityInnate ImmunityAdaptive ImmunityPathogen recognized by receptors encoded in the germlinePathogen recognized by receptors generated randomly (rearrengment receptors)Receptors have broad specificity, i.e., recognize many rela

11、ted molecular structures called PAMPs (pathogen-associated molecular patterns)Receptors have very narrow specificity; i.e., recognize a particular epitopePAMPs are essential polysaccharides and polynucleotides that differ little from one pathogen to another but are not found in the host.Most epitope

12、s are derived from polypeptides (proteins) and reflect the individuality of the pathogen.Receptors are PRRs (pattern recognition receptors)Receptors are B-cell (BCR) and T-cell (TCR) receptors for antigenImmediate responseSlow (3 -5 days) response (because of the need for clones of responding cells

13、to develop -)No memory of prior exposureMemory of prior exposureOccurs in all metazoansOccurs in vertebrates only11Strategies of innate immune recognition(Left) NK cells identify pathogen proteins and discriminate normal cells from virus-infected, allogeneic, or tumor cells. NK cells have two types

14、of receptor for this purpose.(Right) Other innate immune cells-neutrophils, macrophages, and dendritic cells-express pattern recognition receptors, such as TLRs, that recognize PAMPs and are linked to signaling pathways through the adaptor molecules MyD88 or TIRAP. 12Pathogen-Associated Molecular Pa

15、tterns (PAMPs) Pathogens, especially prokaryotes, have molecular structures that are not shared with their host; are shared by many related pathogens; are relatively invariant; that is, do not evolve rapidly (in contrast, for example, to such pathogen molecules as the hemagglutinin(血凝素) and neuramin

16、idase(神經(jīng)酰胺酶) of influenza viruses).Examples: the flagellin of bacterial flagella; peptidoglycan(肽聚糖) of Gram-positive bacteria; lipopolysaccharide (LPS) of Gram-negative bacteria; double-stranded RNA. (Some viruses of both plants and animals have a genome of dsRNA. And many other viruses of both pla

17、nts and animals have an RNA genome that in the host cell is briefly converted into dsRNA). unmethylated DNA (many of the CpG islands in eukaryotes have methyl groups attached). 13Pattern Recognition Receptors (PRRs) There are three groups: secreted molecules that circulate in blood and lymph: a circ

18、ulating protein that binds to the mannose (a monosaccharide) residues found on the surface of many pathogens. This interaction triggers the cleavage of the complement components Example:C4 and C2 leading to the cleavage of C3 and the remaining steps of complement fixation. surface receptors on phago

19、cytic cells that bind the pathogen for engulfment: Macrophages have cell-surface receptors that recognize certain PAMPs, e.g., those containing mannose. When a pathogen covered with polysaccharide with mannose at its tips binds to these, it is engulfed into a phagosome. cell-surface receptors that b

20、ind the pathogen initiating a signal leading to the release of effector molecules (e.g. cytokines), Toll-Like Receptors (TLRs): Macrophages, dendritic cells, and epithelial cells have a set of transmembrane receptors that recognize different types of PAMPs. 14Toll-Like receptors (TLRs)TLRs play a cr

21、itical role in early innate immunity to invading pathogens by sensing microorganisms. These evolutionary conserved receptors, homologues of the Drosophila Toll gene, recognize highly conserved structural motifs only expressed by microbial pathogens, called pathogen-associated microbial patterns (PAM

22、Ps).PAMPs include various bacterial cell wall components such as lipopolysaccharides (LPS), peptidoglycans and lipopeptides, as well as flagellin, bacterial DNA and viral double-stranded RNA. Stimulation of TLRs by PAMPs initiates a signaling cascade that involves a number of proteins, such as MyD88

23、 and IRAK. This signaling cascade leads to the activation of the transcription factor NF-kB which induces the secretion of pro-inflammatory cytokines and effector cytokines that direct the adaptive immune response.15Toll-Like receptors (TLRs)TLRs are transmembrane proteins characterized by an extrac

24、ellular leucine-rich domain and a cytoplasmic tail that contains a conserved region called the Toll/IL-1 receptor (TIR) domain. TLRs are predominantly expressed in tissues involved in immune function, such as spleen and peripheral blood leukocytes, as well as those exposed to the external environmen

25、t such as lung and the gastrointestinal tract.16Toll-Like receptors (TLRs)Ten human and nine murine TLRs have been characterized so far, seven of which have had their ligands identified. TLR2 is essential for the recognition of a variety of PAMPs, including bacterial lipoproteins, peptidoglycan, and

26、 lipoteichoic acids. TLR3 is implicated in virus-derived double-stranded RNA. TLR4 is predominantly activated by lipopolysaccharide. TLR5 detects bacterial flagellinTLR9 is required for response to unmethylated CpG DNA. TLR7 and TLR8 have been shown to recognize small synthetic antiviral molecules F

27、urthermore, in many instances, TLRs require the presence of a co-receptor to initiate the signaling cascade. Current knowledge of the TLRs indicates that these receptors are essential elements in host defense against pathogens by activating the innate immunity a prerequisite to induction of adaptive

28、 immunity. The growing interest on TLRs should bring a more complete understanding of the role of TLR-mediated responses and increase our range of weapons to treat infectious and immune diseases.17Several proteins present in plants, Drosophila, and mammals that respond to pathogen infection 18Toll i

29、n Drosophila development and immunityToll was first identified in the landmark genetic screens of Christiane Nusslein-Volhard and Eric Weischaus for mutations that alter embryonic patterning in Drosophila. The original signal for dorsoventral polarity is maternally derived and is relayed by a signal

30、-transduction pathway into the embryo to produce a nuclear morphogen(成形素) gradient (the members of the classical signal-transduction pathway are indicated in red type). 19Invaders pay the TollThe Toll and Imd pathways regulate the Drosophila antimicrobial innate immune response. Antimicrobial peptid

31、e genes are regulated by the NF-kB-like proteins Dif, Dorsal, and Relish, which are the end targets of two distinct signaling cascades: 1.The Toll pathway, which is principally activated by fungi and Gram-positive bacteria;2. the Imd pathway, which is largely activated by Gram-negative bacteria. PGR

32、P-SA is a circulating protein required for Toll activation in response to Gram-positive bacteria . PGRP-LC is a putative pattern recognition receptor required for the activation of the Imd pathway 20Similar pathway regulate Rel/NF kappa B in mammals and DrosophilaIKK: IB kinase; IRAK: IL-1R-associat

33、ed kinase; Rel: REL homology domain; RIP: receptor-interracting protein; TAK1: TGF-activated kinase; TIR: Toll/IL-1R domain; TRADD: TNFR-associated death-domain-containing protein; TRAF: TNFR-associated factor. ANK: ankyrin domain; DD: death domain; DED: death effector domain; FADD: Fsa-associated d

34、eath-domain-containing protein;21A simple model of the regulation of Drosophila antimicrobial genes22Schematic comparison of the protein architectures of Drosophila and human TLRsThree Drosophila TLRs (Toll, 18w, and the Mst ORF fragment) are arrayed beside the four complete (TLRs 1-4) and one parti

35、al (TLR5) human receptors. Individual LRRs in the receptor ectodomains(外功能區(qū)) are noted by boxes; top and bottom Cys-rich clusters that flank the C- or N-terminal ends of LRR arrays are respectively drawn by apposed half circles. The incomplete chains of DmMst and HuTLR5 are represented by dashed lin

36、es. DRgN: the tobacco disease-resistance gene N product23 The homologies and interconnections between the Toll-like receptors (TLRs) and the type 1 interleukin-1 receptor (IL-1R1) complex. AcP, accessory protein; Ig, immunoglobulin; MyD88, myeloid differentiation factor; R, receptor; TIR, Toll/IL-1

37、receptor.24Comparisons between the signaling pathways in the insect and mammalian IL-1/Toll-like receptor (TLR) pathways (d), Drosophilia homolog; ECSIT, evolutionarily conserved signaling intermediate of Toll; HSP, heat shock protein; IKB, inhibitory subunit B; IKK, I-B kinase; IRAK, IL-1 receptor-

38、associated kinase; LBP, lipopolysaccharide binding protein; LPS, lipopolysaccharide; LTA, lipoteichoic acid; MEKK, mitogen-activated protein extracellular activated receptor kinase kinase; MyD88, myeloid differentiation factor; NF-B, nuclear factor-B; TNF, tumor necrosis factor; TRAF6, tumor necrosi

39、s factor receptor-associated factor 6.25TLR-4 : Toll-like Receptor 4TIRsHuman GeneIsoform AHuman GeneIsoform CMurine GeneORF size2517bp2400bp2505bpPlasmid backbonepUNOpUNOpUNOSubclone withBspHI - NheINcoI - NheIBspHI - NheITLR4 binds LPS (Lipopolysaccharide)LBP, the LPS binding protein conveys LPS t

40、o CD14.CD14 is GPI - anchored on the surface of host mononuclear cells.LPS then comes into contact both with TLR4 and with MD-2, a small protein associated with the TLR4 ectodomain and involved in LPS-mediated signaling.C3H/HeJ mice which bear mutations in the Tlr4 gene exhibit defective LPS signali

41、ng, and TLR4 knockout mice are selectively impaired in their ability to recognize Gram-negative bacteria. 26Currently recognized ligands for Toll-like receptor 4MicroorganismGram-negative bacteriaGram-positive bacteriaMycobacteriaSpirochetesVirusOther ligandsMicrobial productLipopolysaccharide, lipi

42、d ALipoteichoic acidLive Mycobacteria tuberculosisTreponema brennaborense glycolipidsRespiratory syncytial virus protein FHeat shock protein 60, TaxolTIRsTLR-4 : Toll-like Receptor 427CD14, was shown to participates in LPS signaling, leading to NF-kB translocation. This signaling is mediated through

43、 the adaptor protein MyD88 but also through a MyD88-independent pathways that involves the (TIR) domain-containing adapter protein (TIRAP) The intracellular signaling pathways of the Toll-like receptor 4 (TLR4) complex. IKB, inhibitory subunit B; IKK, I-B kinase; IRAK, IL-1 receptor-associated kinas

44、e; LBP, lipopolysaccharide binding protein; LPS, lipopolysaccharide; Mal, myeloid differentiation factor MyD88-like; MyD88, myeloid differentiation factor; NF-B, nuclear factor-B; NIK, nuclear factor-B inducing kinase; PKR, RNA binding protein kinase; TIR, Toll IL-1 receptor; TIRAP, Toll IL-1 recept

45、or adapter protein; Tollip, Toll interactive protein; TRAF6, tumor necrosis factor receptor-associated factor 6.TLR-4 : Toll-like Receptor 4TIRs28TLR2 plays a pivotal role in the recognition of Gram-positive bacteria and Mycobacteria. Pathogen recognition by TLR2 is strongly enhanced by CD14. TLR2 k

46、nockout mice are selectively impaired in their ability to recognize Gram-positive bacteria.TLR-2: Toll-like Receptor 2TIRsThe intracellular signaling pathways of the Toll-like receptor 2 (TLR2) complex. Akt, intracellular protein kinase; IKB, inhibitory subunit B; IKK, I-B kinase; IRAK, IL-1 recepto

47、r-associated kinase; MyD88, myeloid differentiation factor; NF-B, nuclear factor-B; NIK, nuclear factor-B inducing kinase; PG, peptidoglycan; PI3K, phosphatidyl inositol-3-kinase; Rac, Rho-type GTPase; TIR, Toll IL-1 receptor; Tollip, Toll interactive protein; TRAF6, tumor necrosis factor receptor-a

48、ssociated factor 6.29TLR-2: Toll-like Receptor 2Gram-negative bacteriaGram-positive bacteriaMycobacteriaSpirochetesMycoplasmaYeastLPS from various sources; possibly contaminated with peptides (Salmonella, Shigella spp., Escherichia coli). Purified LPS from Leptospira interrogans and Porphyromonas gi

49、ngivalis LPSListeria monocytogenes, Bacillus, Streptococcus spp., Staphylococcus aureus-lipoteichoic acid, peptidoglycan, lipopeptidesHeat-killed Mycobacteria tuberculosis, Mycobacteria avium lipopeptides, lipoarabinomannan, mannosylated phosphatidylinositolBorrelia burgdorferi, Treponema pallidum,

50、Treponema maltophilum (lipopeptides, glycolipids, outer surface protein A)Mycoplasma fermentans (R-MALP, lipopeptides)ZymosanTIRsCurrently recognized ligands for Toll-like receptor 230TLR9 was recently identified as the third member of the mammalian TLR family member responsible for mediating the re

51、cognition of bacterial DNA. The molecular mechanism responsible for the ability to discriminate bacterial DNA from human DNA remained obscure until recent discoveries with TLR9. TLR9-DN mutants had no TNF, IL-12, IL-6 and interferon- response on exposure to oligonucleotides containing CpG motifs fou

52、nd in microbial DNA. Moreover, TLR9 knockout mice are refractory to lethal shock from synthetic oligonucleotides bearing unmethylated CpG motifs that rapidly induce refractory hypotension and lethality in wild-type mice.The intracellular signaling pathways responsible for CpG DNA immunostimulation s

53、hare some similarities with other pattern recognition molecules such as TLR4. TLR-9: Toll-like Receptor 9TIRs31TLR5 is the Toll-like molecule that recognizes flagellin from both Gram-positive and Gram-negative bacteria. Activation of the receptor mobilizes NF-kB and stimulates TNF-a production. Muri

54、ne TLR5 lies within a locus that is associated with susceptibility to Salmonella. Flagellin is critical for bacterial motility and is therefore conserved. Thus it fits well the profile of a relatively invariant PAMP.TLR-5: Toll-like Receptor 5TIRs32TLR3 Toll-like receptor 3 recognizes double-strande

55、d RNA (dsRNA). This molecular pattern is associated with viral infection, because it is produced by most viruses at some point during their replication. TLR3-deficient mice showed reduced responses to polycytidylic acid (poly(I:C), resistance to the lethal effect of poly(I:C) when s ensitized with D

56、-galactosamine, and reduced production of inflammatory cytokines. TLR-3: Toll-like Receptor 3TIRs33TLR1 is expressed ubiquitously and at rather high levels. TLR6 is not as well understood as other members of the TLR family. Studies with TLR6- and TLR2-deficient mice have shown that TLR2 is essential

57、 for the response to bacterial lipoproteins triacylated at the N-terminus cysteine residue. By contrast, mycoplasma macrophage-activating lipopeptide-2 kD (MALP-2) is only diacylated and recognized cooperatively by TLR6 and TLR2. Thus TLR6 appears to discriminate between the N-terminal lipoylated st

58、ructures of MALP-2 and lipopeptides derived from other bacteria. TLR-6/1: Toll-like Receptor 6/1TIRs34TLR7 maybe a receptor for components of viruses, or a molecule generated by an animal during a viral infection. Some imidazoquinolines (咪唑喹啉), which are testing against genital herpes(生殖器皰疹), appear

59、 to activate TLR7. Mice genetically engineered to lack TLR7 didnt have the strong, antivial immune response to imidazoquinolines that normal mice do. When give the drugs, the mutant mice didnt produce the typical inflammatory chemicals, and their immune cells didnt proliferate and mature.TIR7 recogn

60、izes small synthetic immune modifiers including imiquimod, R-848, loxoribine, and bropirimine, all of which are already applied or promising for clinical use against viral infections and cancers. TLR-7: Toll-like Receptor 7TIRs35TLR8 have been shown to recognize small synthetic antiviral moleculesTL