博士生課程2(2)-固有免疫模式識別

1、Innate Immunity The most ancient defense Physical & chemical barriers and cellular line Recognition by the innate immune system sets the stage for an effective adaptive immune response.機體在種系發(fā)生和進化機體在種系發(fā)生和進化過程中逐漸形成的一種過程中逐漸形成的一種天然免疫防御功能，構(gòu)天然免疫防御功能，構(gòu)成機體抵御病原生物入成機體抵御病原生物入侵的第一道防線侵的第一道防線. .復習一、固有免疫系統(tǒng)的組成屏障屏障細

2、胞細胞分子分子皮膚黏膜屏障：皮膚黏膜屏障：物理、化學、微生物物理、化學、微生物血血-腦屏障、血腦屏障、血-胸腺屏障胸腺屏障血血-胎屏障、氣胎屏障、氣-血屏障血屏障單核單核-巨噬細胞、中性粒細胞、樹突巨噬細胞、中性粒細胞、樹突狀細胞、狀細胞、T 細胞、細胞、NK細胞、細胞、NKT細胞、細胞、B1細胞、肥大細胞、嗜堿性細胞、肥大細胞、嗜堿性粒細胞和嗜酸性粒細胞等。粒細胞和嗜酸性粒細胞等?？咕?、溶菌酶、急抗菌肽、溶菌酶、急性期蛋白、補體、細性期蛋白、補體、細胞因子和黏附分子、胞因子和黏附分子、Physical, chemical and microbiological barriers of ou

3、r body1、固有免疫屏障固有免疫屏障This may cause inflammation and bleedingNormal Flora competing with Invading Pathogens. Antibiotic treatments disrupt the natural ecology of the colon2、固有免疫細胞、固有免疫細胞 PhagocyteNKILLs（固有樣淋巴細胞）（固有樣淋巴細胞）DC MCBasophil Eosinophil T細胞細胞 NKT細胞細胞 B1細胞細胞Monocyte-macrophageNeutrophilRecogni

4、tion of an infection once it gets past the epithelial barrierPolarization of Tumor-associated macrophages (TAM)分泌分泌 IL-1 IL-6 IL-12 TNF-a a IL-8 GM-CSF細胞因子細胞因子酶酶其它因子其它因子 殺傷殺傷腫瘤細胞腫瘤細胞 抗原抗原呈遞作用呈遞作用前列腺素白三烯補體成分纖維蛋白結(jié)合蛋白凝血因子溶菌酶酸性水解酶賴氨酸酶酯酶膠原蛋白酶彈性纖維蛋白酶免疫調(diào)節(jié)作用免疫調(diào)節(jié)作用吞噬并殺吞噬并殺傷傷病原微生物病原微生物 巨噬細胞的功能Figure 8-19 part

5、 2 of 2Leukocyte recruitment to sites of infection: a multi-step navigation1. Selectins2. Chemokines3. IntegrinsIL-8Interaction between Neutrophils and EndotheliumCellular Adhesion Molecules (CAMs): 1. Mucin-like CAMs2. Selectins3. Integrins4. Ig-superfamily CAMsKiller activatory receptor Killer inh

6、ibitory receptor KIR: KIR2DS,KIR3DS KLR: CD94/NKG2C NKG2D NKp46 NKp30 NKp44NCRKIR2DL,KIR3DLCD94/NKG2ABind class I HLAmoleculesFunctionBind non-class I HLA molecules Receptors associated with killer activation and killer inhibition on NK cells2、固有免疫細胞、固有免疫細胞 PhagocyteNKILLs（固有樣淋巴細胞）（固有樣淋巴細胞）DC MCBaso

7、phil Eosinophil T細胞細胞 NKT細胞細胞 B1細胞細胞Monocyte-macrophageNeutrophil過敏性疾病過敏性疾病抗原的處理與提呈抗原的處理與提呈NUOCYTES REPRESENT A NEW INNATE EFFECTOR LEUKOCYTE THAT MEDIATES TYPE-2 IMMUNITY.NATURE. 2010 MAR 3.Type-2 immunity: responsible for protective immune responses to helminth parasites and the underlying cause o

8、f the pathogenesis of allergic asthma.Type-2 cytokines: interleukin IL-4, IL-5 and IL-13.Nuocytes expand in vivo in response to the type-2-inducing cytokines IL-25 and IL-33, and represent the predominant early source of IL-13 during helminth infection. In the combined absence of IL-25 and IL-33 sig

9、nalling, nuocytes fail to expand, resulting in a severe defect in worm expulsion that is rescued by the adoptive transfer of in vitro cultured wild-type, but not IL-13-deficient, nuocytes. 3、固有性免疫分子指體表分泌液以及血漿和其它體液中能夠識別或攻擊病原體指體表分泌液以及血漿和其它體液中能夠識別或攻擊病原體的可溶性分子。的可溶性分子?？咕目咕?antimicrobial peptides溶菌酶溶菌酶

10、lysozyme急性期蛋白急性期蛋白(acute phase proteins, APP)脂多糖結(jié)合蛋白（脂多糖結(jié)合蛋白（LBP）血清淀粉樣蛋白（血清淀粉樣蛋白（SAP）甘露糖結(jié)合蛋白（甘露糖結(jié)合蛋白（MBP）C反應蛋白等（反應蛋白等（CRP）補體補體 細胞因子和黏附分子細胞因子和黏附分子補體系統(tǒng)補體系統(tǒng)細胞因子和免疫相關(guān)細胞表面分子細胞因子和免疫相關(guān)細胞表面分子二、固有免疫識別病原相關(guān)分子模式(Pathogen-associated molecular patterns, PAMPs)損傷相關(guān)分子模式（damage-associated molecular patterns，DAMPs）模式

11、識別受體（Pattern Recognition Receptors） 病原相關(guān)分子模式(Pathogen-associated molecular patterns, PAMP) ：是病原微生物（尤其是原核生物）表面存在一些人體所沒有的，但可為許多相關(guān)微生物所共享、結(jié)構(gòu)恒定、進化保守的分子結(jié)構(gòu)。PAMP的特征 1.通常為病原微生物所特有,乃天然免疫系統(tǒng)區(qū)分“自己”與“非己（微生物）”的分子基礎(chǔ)。 脂多糖：多數(shù)革蘭陰性菌細胞壁成分； 磷壁酸：多數(shù)革蘭陽性菌胞壁成分； 肽聚糖：革蘭陽性/陰性菌、真菌胞壁成分； 甘露糖：微生物細胞壁上糖蛋白和糖脂成分2.為微生物生存和致病性所必需 PAMP突變或缺

12、失 微生物死亡或微生物對外界環(huán)境適應性3.宿主泛特異性識別的分子基礎(chǔ) PAMP是由一群或一類特定的微生物所共有的恒定結(jié)構(gòu)（如LPS）。 宿主由種系編碼的有限數(shù)量PRR 可察覺任何微生物感染的存在Pattern Lipopolysaccharide (LPS) Lipoteichoic acid Bacterial lipopeptides Peptidoglycan Yeast and gram+ bacteria Bacterial DNA (CpG) Flagellin Terminal mannose/fucose Viral DNA (CpG) ssRNA dsRNAPathogen-

13、AssociatedMolecularPatterns(PAMP)Innate immune recognition of bacterial cell wall componentsGram-negative bacteriaGram-positive bacteria損傷相關(guān)分子模式損傷相關(guān)分子模式（damage-associated molecular patterns，DAMPs）機體自身細胞所釋放的內(nèi)源性分子，即內(nèi)源性危險信號，來源于受損或壞死組織和某些激活的免疫細胞。主要有HMGB1、熱體克蛋白等。PAMP vs DAMPSterile inflammationconserved

14、microbial motifs VS non-microbial signals 模式識別受體（ Pattern Recognition Receptors， PRRs）固有免疫細胞表面、內(nèi)體、溶酶體、細胞質(zhì)中、可識別一種或多種PAMPs或DAMPs的識別分子。PRR甘露聚糖凝集素（甘露聚糖凝集素（MBL）C反應蛋白（反應蛋白（CRP）血清淀粉樣蛋白血清淀粉樣蛋白 (SAP)脂多糖結(jié)合蛋白（脂多糖結(jié)合蛋白（LBP）可溶性：體液和血液可溶性：體液和血液細胞吞噬型：細胞膜細胞吞噬型：細胞膜甘露糖受體（甘露糖受體（MR）清道夫受體（清道夫受體（SR）補體受體（補體受體（CR）Fc受體（受體（FcR

15、）甲酰甲硫氨酰肽受體（甲酰甲硫氨酰肽受體（fMLPR）信號轉(zhuǎn)導型信號轉(zhuǎn)導型細胞膜細胞膜內(nèi)體、溶酶體內(nèi)體、溶酶體細胞質(zhì)細胞質(zhì)TLR1、2、4、5、6、10、11、12、13TLR3、7、8、9NLRs、RLRs、ALRsEXTRACELLULAR/SECRETED PRRsMannose binding lectin/protein (MBP)C-reactive protein (CRP)Serum amyloid protein (SAP)LPS-binding protein (LBP)Acute phase proteinsAcute phase response (APR): the

16、serum changesAPR proteins: their concentrations rose or fell(during the acute phase)Sites of injury or infection signals (proinflammatory cytokines: TNF-a , IL-1, & IL-6 produced by phagocytes) stimulatingLiver: synthesis of APR proteins Increase in the level of C-reactive protein & & & )Involved in

17、 ClottingTwo Secreted PRRs: CRP, MBPSAP made in acute phase liver responseMBLMannose binding lectinLung surfactants A, DFicolins“pattern recognition receptors”; in this case pattern of terminal sugars on cell surfacesRecognizing mannose-containing molecular patterns found on microbes but not on vert

18、ebrate cells directingcomplement attack Mannose-binding lectinMannose binding protein (MBP)lPart of C-type lectin superfamilylAssociates with and activates serine proteases: MASP-1 and MASP-2 lAfter binding to pathogen surface this complex activates lectin pathway of complement system, C2 and C4MB-L

19、ECTINMASP = MBL-associated serine proteaseMBLmannoseMB-LECTINANOTHER VERSION POINTS OUT TOTALITY OF CLEAVED C3 FUNCTIONS lBind to phorphorylcholine (PC) on bacteria, other microorganisms, damaged host cell membranesPC found in teichoic acids, capsular carbohydrates, and lipopolysaccharides Requires

20、Ca+lFunction directly as opsonins (enhancer of phagocytosis)lFunction indirectly by binding to C1q of classical complement pathway and activate complement cascadeC-reactive protein (CRP) and serum amyloid protein (SAP)(belongs to a family of pentameric protein called pentraxins)binding to & phophory

21、lcholine (= ligands) on the cell wall of bacteria & fungi in a reaction activating lysis, opsonization promoting phagocytosis & pathogen clearanceLipid transfer molecule binds to monomeric LPS and to high-affinity LPS receptor named CD14 and, on macrophage, neutrophils, DCsLBP + bactericidal permeab

22、ility increasing protein (BPI) binds LPS on bacteria and then to CD14, a high affinity LPS receptor. See later TLR4LPS-binding protein (LBP)Extracellular factor (LPS) carried by LBP to CD14 where it binds to TLR4 and then MD2 bindsSimplified Version模式識別受體（ Pattern Recognition Receptors， PRRs）固有免疫細胞表

23、面、內(nèi)體、溶酶體、細胞質(zhì)中、可識別一種或多種PAMPs或DAMPs的識別分子。PRR甘露聚糖凝集素（甘露聚糖凝集素（MBL）C反應蛋白（反應蛋白（CRP）血清淀粉樣蛋白血清淀粉樣蛋白 (SAP)脂多糖結(jié)合蛋白（脂多糖結(jié)合蛋白（LBP）可溶性：體液和血液可溶性：體液和血液細胞吞噬型：細胞膜細胞吞噬型：細胞膜甘露糖受體（甘露糖受體（MR）清道夫受體（清道夫受體（SR）補體受體（補體受體（CR）Fc受體（受體（FcR）甲酰甲硫氨酰肽受體（甲酰甲硫氨酰肽受體（fMLPR）信號轉(zhuǎn)導型信號轉(zhuǎn)導型細胞膜細胞膜內(nèi)體、溶酶體內(nèi)體、溶酶體細胞質(zhì)細胞質(zhì)TLR1、2、4、5、6、10、11、12、13TLR3、7、8

24、、9NLRs、RLRs、ALRsMANNOSE RECEPTORThe mannose receptor (MR) is a 175 kDa type I membrane molecule expressed in the mouse by most tissue macrophages and lymphatic and hepatic endothelia.Glycoprotein PRRs recognizelLPS and lipoteichoic acidlIntact G- and G+ bacterialDamaged host cells and tissueslApopto

25、tic and senescent cellslmodified low-density lipoproteinslSix classesScavenger receptors甲酰甲硫氨酰肽受體（甲酰甲硫氨酰肽受體（fMLPR）Staphylococcal Protein A Inhibits Phagocytosis by Blocking Fc果蠅的Toll受體胞漿的功能域與IL-1受體很相像(Toll/IL-1 receptor (TIR) domain)，顯然具有重要的免疫功能。Toll突變后果蠅很容易受霉菌感染。Cell 86:973-83.Toll-like receptor (T

26、LR) Julie A. Hoffmann, Ph.D.Strasbourg, FranceIn 1996, Hoffmanns group Toll functions as a PRR in DrosophilaToll-Like Receptors (TLRs)Total of 13 TLRs have been identified in mammalsHuman (TLRs 1-10)Mouse (TLRs 1-9, 11-13)In general TLRs recognize constituents of microbial cell walls or pathogen-spe

27、cific nucleic acids that are essential to the integrity, function or replication of microbes / viruses that cannot readily be modified.Toll-Like Receptors (TLRs)There are six major clades of TLRs, each recognizing a general class of molecular patterns. Molecular tree of the vertebrate Toll-like rece

28、ptors (TLRs). The evolution of vertebrate Toll-like receptorsToll-Like Receptors (TLRs): Basic ArchitectureEDTMCYTLeader PeptideLeucine Rich Repeats(LRR); BINDINGToll/interleukin 1 receptor (TIR)domain; SIGNALINGStructural organization of human TLRs. ENDOTOXIN-Structural component of the outer leafl

29、et of the outer membrane of Gram negative bacteria-Contains the O-antigenic polysaccharide determinant.-Presence of polysaccharide and lipid components.-Also termed Lipopolysaccharide (LPS).-The lipid component or Lipid A (glycophospholipid) is responsible for the biological activities of LPS (toxic

30、ity / fever)LPS(endotoxin)Outer membranePeptidoglycanInner membraneO-specific chain Core Region Lipid ALPSthe toxic center of LPS The Basic Architecture of LPS Structure of Lipid ALipid A is a glycophospholipid withphosphorylated D-glucosaminesLBP and CD14 the first Endotoxin ReceptorsLPS-Binding Pr

31、otein (LBP)-Plasma protein produced in the Liver.-Delivers LPS from the serum to macrophages.-Enhances the sensitivity of macrophages for LPS.CD14-GPI-linked membrane protein (macrophages).-Also exists in a soluble form recruited to endothelial cells.-Works with LBP to bring LPS to the cell surface.

32、No link between LBP/CD14 and intracellular signaling.However, LPS detection by immune cells results in an intracellular signaling response and production of TNF.Delivery of LPS to TLR4 by lipid transfer proteinsBiologicalActivitiesLBP-CD14Co-receptor(s)MD-2TLR4TLR signaling pathways The generation o

33、f inflammatory cytokines and chemokines The generation of antimicrobial peptidesInitiation Signal-induced assembly of pathway components/involvement of an adaptor moleculeProtein kinase-mediated phosphorylationInitiation of an enzyme cascade: MAP kinase pathway (in many cell types) NFB pathway (a po

34、werful transcriptional factor) generate cytokines, adhesion molecules & other effectors affectthe cell cycle or cellular differentiationConserved pathways in innate immunity in Drosophila and mammals. Other Bacterial PoisonsLipopeptidesPeptidoglycanLipoteichoic AcidUnmethylated DNA OligomersTLR4 Lig

35、andsLipopolysaccharides two grades of purity: - Standard LPS : LPS-EB and LPS-EK are standard preparations of lipopolysaccharide. They are extracted by a phenol-water mixture. LPS-EB and LPS-EK contain other bacterial components, such as lipopeptides, and therefore stimulate both TLR4 and TLR2.- Ult

36、ra-pure LPS : Ultrapure LPS-EB are extracted by successive enzymatic hydrolysis steps and purified by the phenol-TEA-DOC extractionTLR5 LigandsFlagellinTLR6/TLR2 Ligand-MALP-2TLR2 Ligands- Heat-Killed Bacteria- Lipoglycans- Lipopolysaccharide- Lipoteichoic Acids- Peptidoglycans- Synthetic Lipoprotei

37、ns- ZymosanTLR3 LigandsPoly(I:C) a synthetic analog of double-stranded RNA (dsRNA)Poly(A:U)TLR9 Agonists Stimulatory ODNs - CpG ODNs - Control ODNs - Labeled ODNs E.coli DNA - E. coli DNA ef - E. coli ssDNA TLR7 Ligands (human & mouse TLR7) - CL264: Adenine analog- Gardiquimod: imidazoquinoline comp

38、ound- Gardiquimod - Imiquimod: imidazoquinoline compound- Imiquimod VacciGrade NEW - Loxoribine: guanosine analogueTLR8 Ligands (human TLR8 & mouse TLR7)- Single-stranded RNAs - E.coli RNA TLR7/8 Ligands (human, mouse TLR7 & human TLR8)- CL075: thiazoloquinoline compound- CL097: water-soluble R848,

39、imidazoquinoline compound - Poly(dT): thymidine homopolymer phosphorothioate ODN - R848: Imidazoquinoline compoundMyD88-Dependent and independent SignalingTLR4 can signal using both pathwaysTRAM = TRIF-related adaptor moleculeTIRAP = Toll-interleukin 1 receptor (TIR) domain-containing proteinTLR4 si

40、gnaling induces the transcription of both proinflammatory cytokines and IFN- that are required for anti-bacterial and anti-viral immune responses, respectively.Cooperation of Toll-like receptor signals in innate immune defenseNegative regulation of Toll-like receptor 4 (TLR4) signalingThe negative r

41、egulators of TLR signaling pathways NLRs are cytoplasmic bacterial sensors that activate inflammasomesIL-1 family membersCommon sources of interleukin-1 (IL-1) family cytokinesICE: IL-1-converting enzyme, caspase 1Pro-IL-18: 24 kDa and mature IL-18: 18 kDaPro-IL-1beta: 33 kDa and mature IL-1beta: 17

42、 kDapro-caspase-1: 45-kDa mature caspase-1: 20KD 10KDInflammasomes are molecular platforms activated upon cellular infection or stress that trigger the maturation of proinflammatory cytokines such as IL-1 and IL-18 to engage innate immune defenses.THE INFLAMMASOMESActivators of the inflammasome. The

43、 Human NLR Family Members Human and Mouse NLR Family MembersDOMAIN ORGANIZATION OF REPRESENTATIVE NLRsThe NLRs family is composed of 20 members.They contain: a C-terminal leucine-rich repeat domain, LRR a central nucleotide-binding domain, NACHT an N-terminal protein-protein interaction domain compo

44、sed of CARD (caspase activation and recruitment domain) Pyrin domain or Bir (baculovirus inhibitor of apoptosis repeat) domainSchroder and Tschopp Cell 2010NLRP1, NLRP3, IPAF, and AIM2 Inflammasomes What is an inflammasome and what does it do?Activation models and composition of NLRP3 inflammasomeSc

45、hroder and Tschopp Cell 2010PAMPs: MDP, LPS, viral/bacterial RNA, aerolysin, etc.DAMPs: ATP, hyaluronan, uric acid, amyloid- peptide, ROS, silica, asbestos, UVB, etc. The channel model of NLRP3 inflammasome activation.Proposed pathways for NLRP3 activation.A role for mitochondria in NLRP3 inflammaso

46、me activationPathways for activation of the inflammasome by PAMPs and DAMPsThe AIM2 inflammasomeThe role of the inflammasomes in human diseaseThe influence of inflammasome activation on adaptive immunityRLRs 等病毒識別受體等病毒識別受體Host Response to Virus InfectionBarriersAdaptiveResponseInnateResponseVirusRou

47、tes of infection for VirusesRoute of entryMode of TransmissionPathogenDiseaseMucosal surfacesAirwayInhaled dropletsInfluenza virusHantavirusInfluenzaHemorrhagic FeverGastrointestinal tractContaminatedwater or foodPoliovirusRotavirusPolioDiarrheaReproductive tractPhysical contactPapilloma virusHSV-2C

48、ervical CancerGenital herpesExternal epitheliaSkin surfaceDirect contactPapilloma virusWartsInstrumentationI.V. InjectionTransfusionTattooingHIV HBV HCVAIDSHepatitisInsect BitesMosquito bites(Aedes aegypti)FlavivirusTogavirusYellow FeverEncephalitisViral Nucleic Acids double stranded (ds) RNA single

49、 stranded (ss) RNA DNA (hypomethylated or CpG rich)“Some” Viral Proteins - uncommone.g. glycoproteins of Herpesvirus and Respiratory Syncytial VirusViral Pathogen-Associated Molecular Patterns(PAMP) Production of Type 1 (aa) interferons Production of inflammatory mediators: cytokines and chemokines

50、Mechanism: Interferon Response Factor 3/7 (IRF3/7) activation NF B activationTranslocation from cytoplasm to nucleusEngagement of Viral PAMP Receptors on/in Cells cell surface and endosomal localization cell cytoplasm localization extracellular space localizationViral PAMP Receptors = anti-viral sen

51、sors = anti-viral PRRViral PAMP Receptors (anti-viral sensors) TLR - Toll Like Receptors:cell surface &/or endosome Endosomal TLR TLR-9 CpG DNA ( methylated viral DNA) TLR-7/8 ssRNA TLR-3 dsRNA TLR 9 and 7 are major triggers of Type 1 IFN production - particularly by plasmacytoid DC: Cell surface TLR TLR-2 and TLR-4:viral glycoproteins, HCV, RSVThe third member of the RLR family, LGP2, lacks any CARDs and was originally identified as a negative regulator of RLR signaling. Nevertheless, LGP2 an