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1、Innate Immunity,monocyte,macrophage,bacteria,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.,机体在种系发生和进化过程中逐渐形成的一种天然免疫防御功能，构成机体抵御病原生物入侵的第一道防线.,复习,一、固有免疫系统的组成,屏障 细胞 分子,皮肤黏膜屏障：物理、化学
2、、微生物 血-脑屏障、血-胸腺屏障 血-胎屏障、气-血屏障,单核-巨噬细胞、中性粒细胞、树突状细胞、T 细胞、NK细胞、NKT细胞、B1细胞、肥大细胞、嗜碱性粒细胞和嗜酸性粒细胞等。,抗菌肽、溶菌酶、急性期蛋白、补体、细胞因子和黏附分子、,Physical, chemical and microbiological barriers of our body,1、固有免疫屏障,This may cause inflammation and bleeding,Normal Flora competing with Invading Pathogens. Antibiotic treatments
3、disrupt the natural ecology of the colon,2、固有免疫细胞,Phagocyte NK ILLs（固有样淋巴细胞） DC MC Basophil Eosinophil, T细胞 NKT细胞 B1细胞,Monocyte-macrophage Neutrophil,Recognition of an infection once it gets past the epithelial barrier,Polarization of Tumor-associated macrophages (TAM),分泌,IL-1,IL-6,IL-12,TNF-,a,IL-8
4、,GM-CSF,细胞因子,酶,其它因子,杀伤,肿瘤细胞,抗原,呈递作用,前列腺素,白三烯,补体成分,纤维蛋白,结合蛋白,凝血因子,溶菌酶,酸性水解酶,赖氨酸酶,酯酶,胶原蛋白酶,弹性纤维蛋白酶,免疫调节作用,吞噬并杀伤,病原微生物,巨噬细胞的功能,Figure 8-19 part 2 of 2,Leukocyte recruitment to sites of infection: a multi-step navigation,1. Selectins 2. Chemokines 3. Integrins,IL-8,Interaction between Neutrophils and En
5、dothelium,Cellular Adhesion Molecules (CAMs): Mucin-like CAMs Selectins Integrins Ig-superfamily CAMs,Killer activatory receptor,Killer inhibitory receptor,KIR: KIR2DS,KIR3DS KLR: CD94/NKG2C NKG2D NKp46 NKp30 NKp44,NCR,KIR2DL,KIR3DL CD94/NKG2A,Bind class I HLA molecules,Function,Bind non-class I HLA
6、 molecules,Receptors associated with killer activation and killer inhibition on NK cells,2、固有免疫细胞,Phagocyte NK ILLs（固有样淋巴细胞） DC MC Basophil Eosinophil, T细胞 NKT细胞 B1细胞,Monocyte-macrophage Neutrophil,过敏性疾病,抗原的处理与提呈,Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity. Natur
7、e. 2010 Mar 3.,Type-2 immunity: responsible for protective immune responses to helminth parasites and the underlying cause of 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,
8、 and represent the predominant early source of IL-13 during helminth infection. In the combined absence of IL-25 and IL-33 signalling, 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-defici
9、ent, nuocytes.,3、固有性免疫分子,指体表分泌液以及血浆和其它体液中能够识别或攻击病原体的可溶性分子。,抗菌肽 antimicrobial peptides 溶菌酶 lysozyme 急性期蛋白(acute phase proteins, APP) 脂多糖结合蛋白（LBP） 血清淀粉样蛋白（SAP） 甘露糖结合蛋白（MBP） C反应蛋白等（CRP） 补体 细胞因子和黏附分子,补体系统,细胞因子和免疫相关细胞表面分子,二、固有免疫识别,病原相关分子模式(Pathogen-associated molecular patterns, PAMPs) 损伤相关分子模式（damage-as
10、sociated molecular patterns，DAMPs） 模式识别受体（Pattern Recognition Receptors）,病原相关分子模式(Pathogen-associated molecular patterns, PAMP) ：是病原微生物（尤其是原核生物）表面存在一些人体所没有的，但可为许多相关微生物所共享、结构恒定、进化保守的分子结构。 PAMP的特征 1.通常为病原微生物所特有,乃天然免疫系统区分“自己”与“非己（微生物）”的分子基础。 脂多糖：多数革兰阴性菌细胞壁成分； 磷壁酸：多数革兰阳性菌胞壁成分； 肽聚糖：革兰阳性/阴性菌、真菌胞壁成分； 甘露糖：微
11、生物细胞壁上糖蛋白和糖脂成分 2.为微生物生存和致病性所必需 PAMP突变或缺失 微生物死亡或微生物对外界环境适应性 3.宿主泛特异性识别的分子基础 PAMP是由一群或一类特定的微生物所共有的恒定结构（如LPS）。 宿主由种系编码的有限数量PRR 可察觉任何微生物感染的存在,Pathogen- Associated Molecular Patterns (PAMP),Innate immune recognition of bacterial cell wall components,Gram-negative bacteria,Gram-positive bacteria,损伤相关分子模式
12、（damage-associated molecular patterns，DAMPs）,机体自身细胞所释放的内源性分子，即内源性危险信号，来源于受损或坏死组织和某些激活的免疫细胞。主要有HMGB1、热体克蛋白等。,PAMP vs DAMP,Sterile inflammation,conserved microbial motifs VS non-microbial signals,模式识别受体（ Pattern Recognition Receptors， PRRs） 固有免疫细胞表面、内体、溶酶体、细胞质中、可识别一种或多种PAMPs或DAMPs的识别分子。,PRR,甘露聚糖凝集素（MB
13、L） C反应蛋白（CRP） 血清淀粉样蛋白 (SAP) 脂多糖结合蛋白（LBP）,可溶性：体液和血液,细胞吞噬型：细胞膜,甘露糖受体（MR） 清道夫受体（SR） 补体受体（CR） Fc受体（FcR） 甲酰甲硫氨酰肽受体（fMLPR）,信号转导型,细胞膜 内体、溶酶体 细胞质,TLR1、2、4、5、6、10、11、12、13,TLR3、7、8、9,NLRs、RLRs、ALRs,EXTRACELLULAR/SECRETED PRRs,Mannose binding lectin/protein (MBP) C-reactive protein (CRP) Serum amyloid protein
14、 (SAP) LPS-binding protein (LBP),Acute phase proteins,Acute phase response (APR): the serum changes APR proteins: their concentrations rose or fell,(during the acute phase),Sites of injury or infection signals (proinflammatory cytokines: TNF- , IL-1, & IL-6 produced by phagocytes) stimulating Liver:
15、 synthesis of APR proteins Increase in the level of C-reactive protein & Mannose-binding lectin/MBL & Serum amyloid protein/SAP & fibrinogen),Involved in Clotting,Two Secreted PRRs: CRP, MBP,SAP made in acute phase liver response,Mannose binding lectin Lung surfactants A, D Ficolins “pattern recogni
16、tion receptors”; in this case pattern of terminal sugars on cell surfaces,Recognizing mannose-containing molecular patterns found on microbes but not on vertebrate cells directing complement attack,Mannose-binding lectin,Mannose binding protein (MBP) Part of C-type lectin superfamily Associates with
17、 and activates serine proteases: MASP-1 and MASP-2 After binding to pathogen surface this complex activates lectin pathway of complement system, C2 and C4,MB-LECTIN MASP = MBL-associated serine protease MBL mannose,MB-LECTIN,ANOTHER VERSION POINTS OUT TOTALITY OF CLEAVED C3 FUNCTIONS,Bind to phorpho
18、rylcholine (PC) on bacteria, other microorganisms, damaged host cell membranes PC found in teichoic acids, capsular carbohydrates, and lipopolysaccharides Requires Ca+ Function directly as opsonins (enhancer of phagocytosis) Function indirectly by binding to C1q of classical complement pathway and a
19、ctivate complement cascade,C-reactive protein (CRP) and serum amyloid protein (SAP),(belongs to a family of pentameric protein called pentraxins) binding to polysaccharide & phophorylcholine (= ligands) on the cell wall of bacteria & fungi in a calcium-dependent reaction activating complement system
20、 lysis, opsonization promoting phagocytosis & pathogen clearance,Lipid transfer molecule binds to monomeric LPS and to high-affinity LPS receptor named CD14 and, on macrophage, neutrophils, DCs LBP + bactericidal permeability increasing protein (BPI) binds LPS on bacteria and then to CD14, a high af
21、finity LPS receptor. See later TLR4,LPS-binding protein (LBP),Extracellular factor (LPS) carried by LBP to CD14 where it binds to TLR4 and then MD2 binds,Simplified Version,模式识别受体（ Pattern Recognition Receptors， PRRs） 固有免疫细胞表面、内体、溶酶体、细胞质中、可识别一种或多种PAMPs或DAMPs的识别分子。,PRR,甘露聚糖凝集素（MBL） C反应蛋白（CRP） 血清淀粉样蛋白
22、 (SAP) 脂多糖结合蛋白（LBP）,可溶性：体液和血液,细胞吞噬型：细胞膜,甘露糖受体（MR） 清道夫受体（SR） 补体受体（CR） Fc受体（FcR） 甲酰甲硫氨酰肽受体（fMLPR）,信号转导型,细胞膜 内体、溶酶体 细胞质,TLR1、2、4、5、6、10、11、12、13,TLR3、7、8、9,NLRs、RLRs、ALRs,MANNOSE RECEPTOR The mannose receptor (MR) is a 175 kDa type I membrane molecule expressed in the mouse by most tissue macrophages a
23、nd lymphatic and hepatic endothelia.,Glycoprotein PRRs recognize LPS and lipoteichoic acid Intact G- and G+ bacteria Damaged host cells and tissues Apoptotic and senescent cells modified low-density lipoproteins Six classes,Scavenger receptors,甲酰甲硫氨酰肽受体（fMLPR）,Staphylococcal Protein A Inhibits Phago
24、cytosis by Blocking Fc,果蝇的Toll受体胞浆的功能域与IL-1受体很相像(Toll/IL-1 receptor (TIR) domain)，显然具有重要的免疫功能。 Toll突变后果蝇很容易受霉菌感染。Cell 86:973-83.,Toll-like receptor (TLR),Julie A. Hoffmann, Ph.D. Strasbourg, France,In 1996, Hoffmanns group Toll functions as a PRR in Drosophila,Toll-Like Receptors (TLRs) Total of 13
25、TLRs have been identified in mammals Human (TLRs 1-10) Mouse (TLRs 1-9, 11-13) In general TLRs recognize constituents of microbial cell walls or pathogen-specific nucleic acids that are essential to the integrity, function or replication of microbes / viruses that cannot readily be modified.,Toll-Li
26、ke Receptors (TLRs),There are six major clades of TLRs, each recognizing a general class of molecular patterns.,Molecular tree of the vertebrate Toll-like receptors (TLRs).,The evolution of vertebrate Toll-like receptors,Toll-Like Receptors (TLRs): Basic Architecture,Structural organization of human
27、 TLRs.,ENDOTOXIN,-Structural component of the outer leaflet 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 (glycophospholipi
28、d) is responsible for the biological activities of LPS (toxicity / fever),LPS (endotoxin),Outer membrane,Peptidoglycan,Inner membrane,O-specific chain Core Region Lipid A,LPS,the toxic center of LPS,The Basic Architecture of LPS Structure of Lipid A,Lipid A is a glycophospholipid with phosphorylated
29、 D-glucosamines,LBP and CD14 the first Endotoxin Receptors,LPS-Binding Protein (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 r
30、ecruited to endothelial cells. -Works with LBP to bring LPS to the cell surface. 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 proteins,Biolo
31、gical Activities,LBP-CD14 Co-receptor(s),MD-2,TLR4,TLR signaling pathways, The generation of inflammatory cytokines and chemokines The generation of antimicrobial peptides,Initiation,Signal-induced assembly of pathway components/involvement of an adaptor molecule,Protein kinase-mediated phosphorylat
32、ion,Initiation of an enzyme cascade: MAP kinase pathway (in many cell types) NFB pathway (a powerful transcriptional factor), generate cytokines, adhesion molecules & other effectors, affect the cell cycle or cellular differentiation,Conserved pathways in innate immunity in Drosophila and mammals.,O
33、ther Bacterial Poisons,Lipopeptides,Peptidoglycan,Lipoteichoic Acid,Unmethylated DNA Oligomers,TLR4 Ligands Lipopolysaccharides 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 co
34、ntain other bacterial components, such as lipopeptides, and therefore stimulate both TLR4 and TLR2. - Ultra-pure LPS : Ultrapure LPS-EB are extracted by successive enzymatic hydrolysis steps and purified by the phenol-TEA-DOC extraction,TLR5 Ligands Flagellin,TLR6/TLR2 Ligand -MALP-2,TLR2 Ligands -
35、Heat-Killed Bacteria - Lipoglycans - Lipopolysaccharide - Lipoteichoic Acids - Peptidoglycans - Synthetic Lipoproteins - Zymosan,TLR3 Ligands Poly(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.
36、coli DNA ef - E. coli ssDNA,TLR7 Ligands (human & mouse TLR7) - CL264: Adenine analog - Gardiquimod: imidazoquinoline compound - Gardiquimod - Imiquimod: imidazoquinoline compound - Imiquimod VacciGrade NEW - Loxoribine: guanosine analogue TLR8 Ligands (human TLR8 & mouse TLR7) - Single-stranded RNA
37、s - E.coli RNA TLR7/8 Ligands (human, mouse TLR7 & human TLR8) - CL075: thiazoloquinoline compound - CL097: water-soluble R848, imidazoquinoline compound - Poly(dT): thymidine homopolymer phosphorothioate ODN - R848: Imidazoquinoline compound,MyD88-Dependent and independent Signaling,TLR4 can signal
38、 using both pathways,TRAM = TRIF-related adaptor molecule TIRAP = Toll-interleukin 1 receptor (TIR) domain-containing protein TLR4 signaling induces the transcription of both proinflammatory cytokines and IFN- that are required for anti-bacterial and anti-viral immune responses, respectively.,Cooper
39、ation of Toll-like receptor signals in innate immune defense,Negative regulation of Toll-like receptor 4 (TLR4) signaling,The negative regulators of TLR signaling pathways,NLRs are cytoplasmic bacterial sensors that activate inflammasomes,IL-1 family members,Common sources of interleukin-1 (IL-1) fa
40、mily cytokines,ICE: IL-1-converting enzyme, caspase 1,Pro-IL-18: 24 kDa and mature IL-18: 18 kDa Pro-IL-1beta: 33 kDa and mature IL-1beta: 17 kDa pro-caspase-1: 45-kDa mature caspase-1: 20KD 10KD,Inflammasomes are molecular platforms activated upon cellular infection or stress that trigger the matur
41、ation of proinflammatory cytokines such as IL-1b and IL-18 to engage innate immune defenses.,THE INFLAMMASOMES,Activators of the inammasome.,The Human NLR Family Members,Human and Mouse NLR Family Members,DOMAIN ORGANIZATION OF REPRESENTATIVE NLRs,The NLRs family is composed of 20 members. They cont
42、ain: a C-terminal leucine-rich repeat domain, LRR a central nucleotide-binding domain, NACHT an N-terminal protein-protein interaction domain composed of CARD (caspase activation and recruitment domain) Pyrin domain or Bir (baculovirus inhibitor of apoptosis repeat) domain,Schroder and Tschopp Cell
43、2010,NLRP1, NLRP3, IPAF, and AIM2 Inammasomes,What is an inflammasome and what does it do?,Activation models and composition of NLRP3 inflammasome,Schroder and Tschopp Cell 2010,PAMPs: MDP, LPS, viral/bacterial RNA, aerolysin, etc. DAMPs: ATP, hyaluronan, uric acid, amyloid-b peptide, ROS, silica, a
44、sbestos, UVB, etc.,The channel model of NLRP3 inflammasome activation.,Proposed pathways for NLRP3 activation.,A role for mitochondria in NLRP3 inflammasome activation,Pathways for activation of the inflammasome by PAMPs and DAMPs,The AIM2 inammasome,The role of the inflammasomes in human disease,Th
45、e influence of inflammasome activation on adaptive immunity,RLRs 等病毒识别受体 Host Response to Virus Infection,Viral Nucleic Acids double stranded (ds) RNA single stranded (ss) RNA DNA (hypomethylated or CpG rich) “Some” Viral Proteins - uncommon e.g. glycoproteins of Herpesvirus and Respiratory Syncytia
46、l Virus,Viral Pathogen-Associated Molecular Patterns (PAMP),Production of Type 1 () interferons Production of inflammatory mediators: cytokines and chemokines Mechanism: Interferon Response Factor 3/7 (IRF3/7) activation NFB activation Translocation from cytoplasm to nucleus,Engagement of Viral PAMP
47、 Receptors on/in Cells,cell surface and endosomal localization cell cytoplasm localization extracellular space localization,Viral PAMP Receptors = anti-viral sensors = anti-viral PRR,Viral PAMP Receptors (anti-viral sensors),TLR - Toll Like Receptors:cell surface &/or endosome Endosomal TLR TLR-9 Cp
48、G 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, RSV,The third member of the RLR family, LGP2, lacks any CARDs and was originally identified as
49、 a negative regulator of RLR signaling. Nevertheless, LGP2 and its ATPase activity were dispensable for the responses to synthetic RNA ligands for MDA5 and RIG-I. LGP2 facilitates viral RNA recognition by RIG-I and MDA5 through its ATPase domain.,PNAS January 26, 2010 vol. 107 no. 4 1512-1517,Virus recognized by NLRs.,Viral PAMP Viral RNA, DNA, Protein,Viral PAMP R