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1、1,Innate Immune recognition,2,Threats to the individual,3,Protection from microbial invasion,4,5,Nobel Prize 2011 innate immunity,6,Nobel Prize 2011,Jules A. Hoffmann: In 1996, found “Toll” gene has a role in the flys immunity to fungal infections. Bruce A. Beutler: in 1998, Bruce A. Beutler and col
2、leagues discovered that the Toll-like receptors (TLRs) act as the principal sensors of infection in mammals. Ralph M. Steinman 1973，Ralph Steinman isolate Dentritic Cell)。 DC can activate T cell. DC is the birdge between innate immunity and adaptive immunity.,7,Toll Pathway,Drosophila,8,They all des
3、erve the Nobel Prize,Ruslan Medzhitov,Charles Janeway 19432003,Takashi Fujita, Shizuo Akira,PRR,TLR,RLR,Yale Univ.,Yale Univ.,Kyoto Univ., Osaka Univ.,9,pathogen associated molecular patterns, PAMPs Conserved pattern found only in pathogens, not in host cells。 (Charles Jenaway 1989，at Cold spring ha
4、rbor meeting) Typical PAMPs： 1、Glycans：G- bacteria (lipopolysaccharide, LPS）; G+ bacteria (peptidoglycan)； Bacterial flagellin 2、Nucleotide: Virus DNA/RNA.,10,PAMPs,GlucanPeptidoglycanLPS Flagellin 。,11,pattern recognition receptors, PRR： Receptors, on the surface or inside the cells, for those path
5、ogen associated molecule patterns. Types of PRRs： Toll-like receptors; TLRs RNA sensor（RIG-I, MDA5） DNA sensor（AIM2，DAI） NOD-like receptors； NLRs C-Lectin receptors Other PRRs,12,PRR-PAMPs engagement activates phagocytes,13,Toll like Receptor （TLR）,Nature. 1997 Jul 24;388(6640):394-7. A human homolo
6、gue of the Drosophila Toll protein signals activation of adaptive immunity. Medzhitov R, Preston-Hurlburt P, Janeway CA Jr. Source Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520-8011, USA. Abstract We report here the cloning and characterization of a human
7、 homologue of the Drosophila toll protein (Toll) which has 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 h
8、omologous to the cytoplasmic domain of the human interleukin (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-kapp
9、aB and the expression of NF-kappaB-controlled genes for the 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 papers,14,1-1) Cellular distribution
10、of TLRs,Cell membrane,Endosome membrane,15,PRRs in PMN,16,1-2) TLRs expression in cells,Immune cells and some epithelia cells express TLRs,17,TLRs expressed in immune cells,18,1-3) Structure of TLRs,* Homo-or heterodimers, or together with other molecules,19,1-4) TLRs signaling pathway：,MyD88 depend
11、ent pathway: All TLRs except 3 MyD88 independent pathway: TLR3; part of TLR4,effector genes,20,TLRs signaling pathway：,21,TLRs signaling pathway：,22,1.5) Negative regulation of TLRs signaling,23,1-6) Role of TLRs in immune response,Innate immunity: Activate phagocytosis and killing: regulated expres
12、sion of genes of phagocytosis Promoting secretion of cytokines/ chemokines : IL1, IL6, TNFa, IFNs, CXCLs, CCLs (recruit other immune cells)Promote secretion of anti-peptide: eg: Defensins,24,1-6) Role of TLRs in immune response,Adaptive immunity:TLRs induce DC maturation: cytokine secretion; costimu
13、latory molecules - DC activation - present antigen to activate T cellsTLRs signaling influences Th cell differentiation: 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
14、function of TregB cell activation (eg: CpG DNA TLR9 ligands may be used as adjuvant),25,2-1）RNA sensors（TLRs and RLRs）,RIG-I Like Receptors（RLR）： RIG-I MDA-5 LGP2 。,Fujita T,Nat Immunol. 2004 J Immunol 2005,26,2-2） structure of RIG-I,RIG-I 结合 5-ppp dsRNA,27,MAVS： adapter for RIG-I,Seth, R.B., Sun, L
15、., Ea, C., and Chen, Z.J., Identification and characterization of MAVS: a mitochondrial antiviral signaling protein that activates NF-B and IRF3. Cell, 122:669-682, 2005,Prion-like protein fight off viruses: MAVS, Cell 2011,University of Texas Southwestern HHMI,28,2-3）RIG-1/MDA-5 signaling pathway,2
16、9,2-4） regulator of RIG-I/MDA-5,30,Ubiquitin (泛素）对PRR信号的调节,泛素由76个氨基酸残基组成，其中包括7个赖氨酸残基(K), 其C末端可与底物的赖氨酸残基形成异肽键，从而引起底物泛素化。泛素的K11、K29、K48和K63均能参与形成泛素与泛素间的异肽键 (Isopeptide bond)。,30,31,Ubiquitination for RIG-I signaling,K63泛素化：激活 K48泛素化：降解,Back,32,3-1） DNA sensor,DNA sensor： TLR9； Pol III； DAI； AIM2,33,4-
17、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：相互作用,34,4-2）NLRs activtes inflammasome,PAMP通过NLRs激活炎症反应,35,NLRC4 inflammasome,邵峰 北京生命科学研究所 Shao F. (2011) Nature, 477, 596600.,36,5-1）C-Lectin receptor family,Dectin-1， Mannose Receptors （MRs）,37,5-2） The fungal pattern
18、 receptor： Dectin-1,One such subfamily is the Dectin-1 clusterwhich is primarily, but not exclusively, expressed by myeloid cells macrophages, dendritic cells (DC) and neutrophils。,38,5-2） The fungal pattern receptor： Dectin-1,Back,39,Dectin Signaling Pathway,葡聚糖,甘露聚糖,甘露聚糖,40,6-2）other PRRs,清道夫受体（Sc
19、avenger receptors）: 结合脂类，LPS等 甲酸基多肽受体（Formyl peptide receptor)：结合含甲酸基的特殊氨基酸结构 补体受体（Complement receptor）：,41,Cross talk of PRR signaling,42,Cross talk of PRR signaling,43,Pathogen evasion of PRR signaling pathway,eg: HCV interact with PRRs,Fig1: structure of HCV,44,HCV interact with PRRs,45,Paper Dis
20、cussion （1）： Role of TLR7 in WNV infection (Town T et al., 2009 Immunity; IF20),ssRNA dsRNA (during replication),46,Fig 1: Increased Susceptibility of Tlr7/ and Myd88/ Mice, but Not Tlr9/ Mice, after West Nile Virus Challenge,WT: 50%死亡 TLR7-/-: 90%死亡 TLR7 ：抗病毒作用,47,Fig 2. Tlr7- and Myd88-Dependent V
21、iral Load and Innate Immune Cytokine Responses after West Nile Virus Challenge：Viral Load: Cytokine： IL23, IL12 ,48,Fig 3：Immune Cell Homing to WNV-Infected Cells In Vivo depends on TLR7,Green： WNV （More in TLR7 -/-） Red： Immune Cells (Less in TLR6 -/-),49,Immune Cell Homing,Homing的意义： 1、淋巴细胞得以合理的再分
22、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)。,50,TLR7 dependant macrophage homing,Less cell infiltration,51,Fig 4. Macrophage Homing to West Nile Virus Is IL-23 Signaling Dependent,Macrophage,Virus,Homing Issue,Brain section staining,52,Summary: TLR7 mediated anti-WNV function,Immune Cell Hom
23、ing,Virus Clearance,53,Paper discussion (2): Caspase-12 controls West Nile virus infection via the viral RNA receptor RIG-I. (Wang P. et al., Nature Immunology 2010),Caspase 12 -/- : Less survival; Severe neurological symptoms,54,1) Viral load in Caspase 12-/- mice,55,2) INFb production in Caspase12
24、 -/- mice,56,3) Caspase12 interacts with RIG-I/TRIM25,57,4) Less Ub-RIG-I in Caspase 12-/- mice,Ub- RIG-I,Total RIG-I,Total TRIM25,58,Story Summary: Caspase12 is required for RIG-I Ub IFNb production （Caspase 12 的抗病毒作用）,Casp12,eg: IFN production,59,Paper Discussion （3）： Toll-like receptor 3 mediates
25、 West Nile virus entry into the brain causing lethal encephalitis （TLR3 helps the virus?）Wang T. et al., 2004 Nature Medicine,WT: 0% survival TLR3-/-: 40% survival TLR3的存在 增强了病毒感染,60,2） TLR3-/- mice have viral load in blood; but TNFa, IL6,61,3) TLR3-/- have viral in brain （TLR3-/- 血液里virus多，但virus不能
26、有效进入大脑。）,62,4) Reduced brain tissue damage in TLR3-/- mice,炎性细胞渗入减少,63,5） BBB permeability increased in WT but in TLR3-/- 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。,64,6） TNFa is important for BBB breakdown,1） 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
27、BBB的细胞因子,65,Summary： TLR3 helps WNV enter the brain via TNFa,* 免疫分子往往是把双刃剑,血液- 大脑,66,Paper discussion 4: TLR and Aging,J Immunol. 2010 Mar 1;184(5):2518-27. Panda A, et al， Yale University School of Medicine Age-associated decrease in TLR function in primary human dendritic cells predicts influenza
28、vaccine response.,Fig1. TLR expression in DCs,67,Fig 2. less cytokine production in old,68,Fig 3. Vaccine efficiency in young and old population,Vaccine efficiency Youngold,Responses to inuenza vaccination in older adults and young adults.,69,Paper 4 Summary,1. Less TLR expression in old 2. TLR sign
29、aling are less responsive in old 3. Defect of TLR signaling resulted in vaccine failure in old,70,Paper Discussion 5:DDX24 Negatively Regulates Cytosolic RNA-MediatedInnate Immune Signaling,71,72,DDX24 interact with FADD,73,IFN induce DDX24 expression,74,DDX24 inhibit IFN production,75,Silence DDX24
30、， IFN production increased,76,Figure 3 SiRNA-mediated knockdown of DDX24 enhances dsRNA induced RLR signaling.,(G) Gene array indicating most up-regulated genes in MEF treated 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 i
31、ndependent experiments. * indicates P,0.05. * indicates P,0.01. doi:10.1371/journal.ppat.1003721.g003,77,Figure 4. SiRNA-mediated knockdown of DDX24 inhibits VSV replication.,78,Figure 4. SiRNA-mediated knockdown of DDX24 inhibits VSV replication.,(F) Loss of hDDX24 affects VSV replication in HUVECs
32、. HUVECs transfected with ns or hDDX24 siRNA for 72 hours were infected by VSV-luc at M.O.I. = 0.1 or 1. Eight hours and 24 hours post infection, plaque assays were performed using supernatants from infected MEFs. HUVEC cell lysates were analyzed by immunoblotting with the indicated antibodies to en
33、sure the knockdown of hDDX24. Data from (A)(B)(E)(F) are presented as means6s.e. from three independent experiments. * indicates P,0.05. * indicates P,0.01.,79,Figure 5. DDX24 can sequester RLR activator RNA,80,(H) DDX24 inhibits dRIG-I-, dMDA5, IPS-1 andTBK-1 mediated IFNbpromoter activation.,81,Fi
34、gure 6. DDX24 interacts with RIP1 and disrupts RLRs activation of IFN-dependent transcription factor IRF7,82,(D) DDX24s effect on RIG-I dependent IFNa4 promoter activation.,83,(G)(H) DDX24 disrupts RIP1-IRF7 interaction.,84,总结： 固有免疫识别的分子机制,1） 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。） 2）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 3）免疫分子的双重作用：介导免疫清除和/或免疫损伤,