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1、Innate Immune recognitionThreats to the individualProtection from microbial invasionNobel Prize 2011 innate immunityNobel Prize 2011nJules A. Hoffmann: In 1996, found “Toll” gene has a role in the flys immunity to fungal infections. nBruce A. Beutler: in 1998, Bruce A. Beutler and colleagues discove
2、red that the Toll-like receptors (TLRs) act as the principal sensors of infection in mammals. nRalph M. Steinman 1973，Ralph Steinman isolate Dentritic Cell)。 DC can activate T cell. DC is the birdge between innate immunity and adaptive immunity.Toll PathwayDrosophilaThey all deserve the Nobel PrizeR
3、uslan MedzhitovCharles Janeway 19432003 Takashi Fujita, Shizuo Akira PRRTLRRLRYale Univ.Yale Univ.Kyoto Univ., Osaka Univ. pathogen associated molecular patterns, PAMPsConserved pattern found only in pathogens, not in host cells。 (Charles Jenaway 1989，at Cold spring harbor meeting)Typical PAMPs：1、Gl
4、ycans：G- bacteria (lipopolysaccharide, LPS）; G+ bacteria (peptidoglycan)； Bacterial flagellin2、Nucleotide: Virus DNA/RNA.PAMPsGlucanPeptidoglycanLPS Flagellin。pattern recognition receptors, PRR：Receptors, on the surface or inside the cells, for those pathogen associated molecule patterns. Types of P
5、RRs：Toll-like receptors; TLRsRNA sensor（RIG-I, MDA5）DNA sensor（AIM2，DAI）NOD-like receptors； NLRsC-Lectin receptorsOther PRRs PRR-PAMPs engagement activates phagocytesToll like Receptor （TLR）nNature. 1997 Jul 24;388(6640):394-7.nA human homologue of the Drosophila Toll protein signals activation of a
6、daptive immunity.nMedzhitov R, Preston-Hurlburt P, Janeway CA Jr.nSourcenSection of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520-8011, USA.nAbstractnWe report here the cloning and characterization of a human homologue of the Drosophila toll protein (Toll) which has
7、 been shown to induce the innate immune response in adult Drosophila. Like Drosophila Toll, human Toll is a type I transmembrane protein with an extracellular domain consisting of a leucine-rich repeat (LRR) domain, and a cytoplasmic domain homologous to the cytoplasmic domain of the human interleuk
8、in (IL)-1 receptor. Both Drosophila Toll and the IL-1 receptor are known to signal through the NF-kappaB pathway. We show that a constitutively active mutant of human Toll transfected into human cell lines can induce the activation of NF-kappaB and the expression of NF-kappaB-controlled genes for th
9、e inflammatory cytokines IL-1, IL-6 and IL-8, as well as the expression of the co-stimulatory molecule B7.1, which is required for the activation of naive T cells.PUBMED search “Toll like receptor” 24700 papers1-1) Cellular distribution of TLRsCell membraneEndosome membranePRRs in PMN1-2) TLRs expre
10、ssion in cellsImmune cells and some epithelia cells express TLRs TLRs expressed in immune cells1-3) Structure of TLRs* Homo-or heterodimers, or together with other molecules 1-4) TLRs signaling pathway：MyD88 dependent pathway: All TLRs except 3 MyD88 independent pathway:TLR3; part of TLR4effector ge
11、nesTLRs signaling pathway：TLRs signaling pathway：1.5) Negative regulation of TLRs signaling1-6) Role of TLRs in immune responsenInnate immunity:Activate phagocytosis and killing: regulated expression of genes of phagocytosis Promoting secretion of cytokines/ chemokines : IL1, IL6, TNFa, IFNs, CXCLs,
12、 CCLs (recruit other immune cells)Promote secretion of anti-peptide: eg: Defensins1-6) Role of TLRs in immune responsenAdaptive immunity:TLRs induce DC maturation: cytokine secretion; costimulatory molecules - DC activation - present antigen to activate T cellsTLRs signaling influences Th cell diffe
13、rentiation: to influence the differentiation of T cells: Th1, Th2, Th17TLRs induce Treg activation: regulatory T cells Treg express TLR4/5/7/8, TLR signal directly involved in the biological function of TregB cell activation (eg: CpG DNA TLR9 ligands may be used as adjuvant)2-1）RNA sensors（TLRs and
14、RLRs）RIG-I Like Receptors（RLR）：RIG-IMDA-5LGP2。Fujita TNat Immunol. 2004 J Immunol 20052-2） structure of RIG-IRIG-I 结合 5-ppp dsRNAMAVS： adapter for RIG-InSeth, R.B., Sun, L., Ea, C., and Chen, Z.J., Identification and characterization of MAVS: a mitochondrial antiviral signaling protein that activate
15、s NF-B and IRF3. Cell, 122:669-682, 2005 Prion-like protein fight off viruses: MAVS, Cell 2011University of Texas SouthwesternHHMI2-3）RIG-1/MDA-5 signaling pathway2-4） regulator of RIG-I/MDA-5 Ubiquitin (泛素）对泛素）对PRR信号的调节信号的调节泛素由泛素由76个氨基酸残基组成，其中包括个氨基酸残基组成，其中包括7个赖氨酸残基个赖氨酸残基(K), 其其C末端可与末端可与底物的赖氨酸残基形成异肽
16、键，从而引起底物的赖氨酸残基形成异肽键，从而引起底物泛素化底物泛素化。泛素的。泛素的K11、K29、K48和和K63均能参与形成泛素与泛素间的异肽键均能参与形成泛素与泛素间的异肽键 (Isopeptide bond)。30Ubiquitination for RIG-I signalingK63泛素化：激活K48泛素化：降解Back3-1） DNA sensor DNA sensor： TLR9； Pol III； DAI； AIM24-1） NOD like receptors（NLRs）NLRs： C端：亮氨酸重复序列 （LRR）中间：NACHT： 寡聚体化，活化N端：CARD，PYD：相
17、互作用4-2）NLRs activtes inflammasomePAMP通过NLRs激活炎症反应NLRC4 inflammasome 邵峰北京生命科学研究所Shao F. (2011) Nature, 477, 596600. 5-1）C-Lectin receptor familynDectin-1， Mannose Receptors （MRs）5-2） The fungal pattern receptor： Dectin-1One such subfamily is the Dectin-1 clusterwhich is primarily, but not exclusively
18、, expressed by myeloidcells macrophages, dendritic cells (DC) and neutrophils。5-2） The fungal pattern receptor： Dectin-1BackDectin Signaling Pathway葡聚糖甘露聚糖甘露聚糖6-2）other PRRsn清道夫受体（Scavenger receptors）: 结合脂类，LPS等n甲酸基多肽受体（Formyl peptide receptor)：结合含甲酸基的特殊氨基酸结构n补体受体（Complement receptor）：Cross talk of
19、PRR signalingCross talk of PRR signalingPathogen evasion of PRR signaling pathwayneg: HCV interact with PRRsFig1: structure of HCVHCV interact with PRRsPaper Discussion （1）： Role of TLR7 in WNV infection (Town T et al., 2009 Immunity; IF20)ssRNA dsRNA (during replication) Fig 1: Increased Susceptibi
20、lity of Tlr7/ and Myd88/ Mice, but Not Tlr9/ Mice, after West Nile Virus ChallengeWT: 50%死亡TLR7-/-: 90%死亡TLR7 ：抗病毒作用Fig 2. Tlr7- and Myd88-Dependent Viral Load and Innate Immune Cytokine Responses after West Nile Virus Challenge：Viral Load: Cytokine： IL23, IL12 Fig 3：Immune Cell Homing to WNV-Infect
21、ed Cells In Vivo depends on TLR7Green： WNV （More in TLR7 -/-）Red： Immune Cells (Less in TLR6 -/-)Immune Cell HomingHoming的意义：1、淋巴细胞得以合理的再分布及补充；2、增加抗原和淋巴细胞的接触机会，产生有效的免疫应答。(免疫细胞有效的迁移到受感染的部位)。TLR7 dependant macrophage homingLess cell infiltrationFig 4. Macrophage Homing to West Nile Virus Is IL-23 Sign
22、aling DependentMacrophageVirusHoming IssueBrain section stainingSummary: TLR7 mediated anti-WNV functionImmune Cell HomingVirus ClearancePaper discussion (2): Caspase-12 controls West Nile Caspase-12 controls West Nile virus infection via the viral RNA receptor RIG-I. virus infection via the viral R
23、NA receptor RIG-I. (Wang P. et al., Nature Immunology 2010)(Wang P. et al., Nature Immunology 2010)Caspase 12 -/- :Less survival;Severe neurological symptoms1) Viral load in Caspase 12-/- mice 2) INFb production in Caspase12 -/- mice3) Caspase12 interacts with RIG-I/TRIM25 4) Less Ub-RIG-I in Caspas
24、e 12-/- miceUb- RIG-I Total RIG-ITotal TRIM25Story Summary: Caspase12 is required for RIG-I Ub IFNb production （Caspase 12 的抗病毒作用）Casp12eg: IFN productionPaper Discussion （3）： Toll-like receptor 3 mediates West Nile virus entry into the brain causing lethal encephalitis （TLR3 helps the virus?）Wang T
25、. et al., 2004 Nature MedicineWT: 0% survivalTLR3-/-: 40% survivalTLR3的存在 增强了病毒感染2） TLR3-/- mice have viral load in blood; but TNFa, IL63) TLR3-/- have viral in brain （TLR3-/- 血液里virus多，但virus不能有效进入大脑。）4) Reduced brain tissue damage in TLR3-/- mice炎性细胞渗入减少5） BBB permeability increased in WT but in T
26、LR3-/- mice after WNV infection 血脑屏障被打开血脑屏障相对完好Virus 难以进入TLR3-/- 小鼠脑部。6） TNFa is important for BBB breakdown1） TNFa -/-鼠，WNV感染死亡率下降2） IL6-/- 鼠，没差异3）TNFa-/- 鼠， BBB保持相对完好TNFa是破坏BBB的细胞因子Summary： TLR3 helps WNV enter the brain via TNFa* 免疫分子往往是把双刃剑血液- 大脑Paper discussion 4: TLR and AgingJ Immunol. 2010 M
27、ar 1;184(5):2518-27. Panda A, et al， Yale University School of Medicine Age-associated decrease in Age-associated decrease in TLR function in primary human function in primary human dendritic cells predicts influenza vaccine response.dendritic cells predicts influenza vaccine response.Fig1. TLR expr
28、ession in DCsFig 2. less cytokine production in oldFig 3. Vaccine efficiency in young and old populationVaccine efficiencyYoungoldResponses to influenza vaccination in older adults and young adults.Paper 4 Summaryn1. Less TLR expression in oldn2. TLR signaling are less responsive in oldn3. Defect of
29、 TLR signaling resulted in vaccine failure in old Paper Discussion 5:DDX24 Negatively Regulates Cytosolic RNA-MediatedInnate Immune Signaling DDX24 interact with FADDIFN induce DDX24 expressionDDX24 inhibit IFN productionSilence DDX24， IFN production increasedFigure 3 SiRNA-mediated knockdown of DDX
30、24 enhances dsRNA induced RLR signaling.(G) Gene array indicating most up-regulated genes in MEFtreated with poly I:C at 3 hours and 9 hours VS non treatment. Data from (A)(B)(C)(D)(E)(F) are presented as means6s.e. from three independentexperiments. * indicates P,0.05. * indicates P,0.01.doi:10.137
31、1/journal.ppat.1003721.g003Figure 4. SiRNA-mediated knockdown of DDX24 inhibits VSV replication.Figure 4. SiRNA-mediated knockdown of DDX24 inhibits VSV replication.(F) Loss of hDDX24 affects VSV replication in HUVECs. HUVECs transfected with ns or hDDX24 siRNA for 72 hours were infected byVSV-luc a
32、t M.O.I. = 0.1 or 1. Eight hours and 24 hours post infection, plaque assays were performed using supernatants from infected MEFs. HUVECcell lysates were analyzed by immunoblotting with the indicated antibodies to ensure the knockdown of hDDX24. Data from (A)(B)(E)(F) arepresented as means6s.e. from
33、three independent experiments. * indicates P,0.05. * indicates P,0.01.Figure 5. DDX24 can sequester RLR activator RNA(H) DDX24 inhibits dRIG-I-, dMDA5, IPS-1 andTBK-1 mediated IFNbpromoter activation. Figure 6. DDX24 interacts with RIP1 and disrupts RLRs activation of IFN-dependent transcription factor IRF7(D) DDX24s effect on RIG-I dependent IFNa4 promoter activation. (G)(H) DDX24 disrupts RIP1-IRF7 interaction. 总结： 固有免疫识别的分子机制n1） 固有免疫识别分子（PRRs）也有它特殊的“特异性”- 一类特殊的DNA，RNA或者蛋白质，糖，脂 我们称为“泛特异性”-（但相对于适应性免疫中Ag-Ab反应，它没有那种一对一的特异性。）n2）识别后启动下游的抗病原体反应：增强吞噬，促进抗原加工递呈，激活适应性免疫系统等n3）免疫分子的双重作用：介导免疫清除和/或免疫损伤
