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Lecture 4

IMM250- Lecture 4 - Humoral Defences.docx

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University of Toronto St. George
Dana Philpott

IMM250 – Lecture 4 -Innate immunity: Cellular and humoral mediators of defense  Nod-like receptors - cytosolic sensors of bacteria and danger a. NLR  Proteins : detects  MAMPs and DAMPs. b. NLR have two families: i. NOD subfamily – detects peptidoglycan which some into the cytosol ; Nod 1 and Nod 2 ii. NLRP subfamily – in the cytosol; detects DAMPs – high levels of ROS, and K+ efflux  NLRPs detect danger and form the “inflammasome” a. NLRP3 is the sensor, and ASC is the adapter. NLRP3 gets activated behind the adapter. There is a conformational change and then it binds to the adapter. Similar to TLR which gets activated by the adapter. b. NLRP3  Respond to high levels of ROS and K+ efflux. c. N terminus - Pyrin domain | C terminus  LRR domain d. NLRP3 has a Pyrin domain, and so does ASC. ASC binds to NLRP3 through the Pyrin Pyrin domain. e. The domains in the end of the adapter and NLRP3 is important. it helps to bind the proteins and form a complex. Similar to Pyrin domain of TLR. f. Which is similar to domain of TLR  it has a transmembrane domain, and a cytoplasmic domain g. NOD domain – Nucloetide binding domain  The inflammasome activates Caspase 1 into an enzmatically active protein a. Procaspase 1 has a Card domain, just as ASC protein. b. So Pro-caspase I binds to the CARD domain ASC via the Card-Card and form a link. c. Forms the complex within the cell, and auto activates the active form of pPro Caspase I, Caspase 1.  Active Caspase 1 cleaves pro-IL-1β into an active cytokine a. Caspase 1 is the active form and it can then cleave off its target the targets are the pro- form of cytokines. b. It cleaves the end of Pro –IL – I Beta allowing it to be an active cytokine. IL-1beta c. IL-1beta is secreted from the cell and has lots of effects in deriving information. 1. Pathogen breaches barrier and then…. 2. Releases MAMPs 7. Releases MAMPs 3. • Detected by TLR/Nod proteins 8. • Detected by TLR/Nod proteins (NLR)/RLRs (NLR)/RLRs 4. • Activates signal transduction 9. • Activates signal transduction 5. • Activates NFκB, IRF 10. • Activates NFκB, IRF 6. • Controls cytokine production 11. • Controls cytokine production 12. Summary of TLRs, NLRs and RLRs a. TLRs i. Detect MAMPs (eg LPS) ii. Signal transduction to NFκB and IRF iii. Leads to cytokine and interferon production b. RLRs i. Detect MAMPs – mainly nucleic acid from viruses ii. Signal transduction to NFκB and IRF iii. Leads to cytokine and iv. interferon production c. NLRs i. Nod proteins 1. Detect MAMPs(peptidoglycan) ii. Signal transduction to NFκB 1. Leads to cytokine production d. NLRPs i. Detect DAMPs (ROS& K+) ii. Signal transduction to inflammasome activation iii. production of IL-1β (a cytokine - we will talk about this today) 13. Ok, the pathogen has been detected, now what….? a. Innate immune mediators: both cellular and humoral (ie secreted) i. Cells of the innate system b. Killing mechanisms i. Secreted mediators of the innate system c. Killing and/or protective mechanisms d. Pathogen breaches mucosal barrier  releases MAMPS (detected by TLR), NON proteins (detects peptidoglycan), RLR (detects acid mainly from viruses)  activates signal transduction responsible for cell MyD88 binding to piece of TLR to activate TF,  activates TF (NFkB master regulator, driving many reactions specially production of cytokines), IRF (Pretty specific for interferons). e. When cells re infected with pathogens like salmonella, the cells begin to di and on this process they release endogenous DAMPs. High levels of ROS, K+ efflux. Sub family of NLRP family detects DAMPs. It is within the sub family of NLR. It activates signal transduction response. Then it forms the complex called inflammasome  which activates Caspase 1 to control the production and secretion of IL-1B. f. TLR and RLR, NLR i. TLR – MAMP ; LPS, Signal Transduction, NFkB, IRF, leads to cytokines and interferons response ii. RLR – Viruses and, MAMPs (peptidoglycan)  signal transduction NFkB, IRf,  leads to cytokines and interferons production 1. Non proteins : MAMPs – peptodiglycan 2. NLRP – DAMP -> signal transduction pathway  leading to inflammasome 14. General scheme of an innate immune response a. Pathogen with MAMPs damages the epithelium to break through the epithelial barrier b. Epithelial cells ‘activated’ upon contact with microorganism - PRRs are triggered, NFκB, IRFs, inflammasome are activated Mediators of innate immune defense are produced by activated epithelial cells These are cytokines and chemokines that call in cellular re- enforcements!  inflammation c. Epithelial cells – which pathogens comes into contact  releases MAMPs  damages Epethelium  comes into the submucosal surface  PRR gets triggered  IRF, Inflammsome, NFkB pathways turns on d. Production of cytokines, and chemokines calls in for intercellular reinforcement. e. This process calls for inflammation in the person 15. Inflammation a. Heat - Swelling - Redness - Pain - Loss of Function 16. What are Cytokines and Chemokines? a. Cytokine: a category of signaling molecules that, like hormones and neurotransmitters, are used extensively in cellular communication; cytokines generally enhance the ability of our cells to get rid of an invading pathogen - are messenger proteins that tell our body how to get rid of invading pathogen b. Chemokine: a family of small chemotactic cytokines that act to attract other cells to the site where they are produced – class of cytokines, protein molecules, peptides (fes AA chains), chemotactic  attract different cells to the site where they are being produced. c. Features of cytokines and chemokines: d. A diverse collection of soluble proteins and peptides that modulate the behaviour of cells at extremely small concentrations – produced in very very small amounts, but have a huge impact on the cell. e. Act both locally and systemically -they can have an effect on the site where they are being produced, and they can come to the blood and have have systematic effect f. Have many biological activities (pleiotropic effects) - they have many biological acitivites, even though they are just one protein or one peptide. , they have many different functions g. Some examples - Cytokines: Interleukins (1-35 …!)or probably more, Tumor necrosis factor alpha (TNFα), IL-1β i. Chemokines: Interleukin 8 (also known as CXCL8) normal peptide models, found in pretty infected cells , monocyte chemotactic peptide 1 (MCP1) 17. Cytokines and chemokines bring in “effector cells” First step: cellular re-enforcements must escape from blood vessels – get the cellular mediators to the site of infection. a. Cells that make up blood vessels (endothelial cells) underlying epithelium are impermeable to cells in the blood stream and plasma b. Epithelial cells once infected by salmonella produces cytokins and chemokines with a gradient. High concenrtrations near the site close to the infection and lower mediators spreading away. As these mediators are being produced they start have an effect on other cells locally spread around it. These include cells which make up the blood vessels. c. Very close to the epithelial cells are blood vessels, they are actively absorbing stuff from our food from the blood vessels. When mediators are being produced, they can act on the blood vessels. cells which make up this blood vessles are called endothelial cells . It starts to change the endothelial cells so the mediators from the blood vessel can start coming to the site of infection. It prevents a formidable barrier against leakage of stuff from our blood. Prevents other cells from moving across the barrier. d. When The mediators are produced by epithelial cells they start to have an effect on the epithelial cells. i. Dilation – blood vessels become dilated. And blood starts rushing to the site. Because the vessels are starting to get dilated. ii. Endothelial cells lining the Blood vessels start getting gaps. – Permeability of the blood vessels increases and plasma (liquid part of blood) starts to leak out of the blood. Same time, the leukocytes in the blood start to stick to the endothelial cells and tries to move across the endothelial cells and tries to move to the site of inflammation, so they can kill the bacteria coming across the bacteria. 18. What are the cell types brought to the site of infection by this process?? a. White blood cells (leukocytes) i. Leukocytes of the innate system ii. Leukocytes of the adaptive system - called “lymphocytes” (T and B cells) 19. Mechanism of Cell Migration - Tethering and rolling a. If we want the leukocytes to come to the place of inflammation we have to bring them from endothelial cells of the barrier. b. The endothelial cels starts to change their own phenotype, because of the factors excreted from the epithelial cells and start to get sticky for leukocytes. c. Normally, endothelial cells leukocytes roll past them. d. When the cytokines produced by the epithelail cells and starts to act on the endothelial cells, they change their phenotype and start to produce adhesion molecules which catch the leukocytes rolling past the endothelial cells. e. Expresses molecules which are mediated by selectins. Which tether the leukocytes to the surface of the endothelial cells. 20. Migration and diapedesis (also known as extravasation) a. Firm adhesion flattens cell and allows for migration between the endothelial cells Innate effector cells migrate towards site of infection by detecting and following a gradient of chemokine produced by infected epithelial cell. b. Cells have to get through the epithelial cells to the site of infection. This is called diapedesis. They have to push their way through the epithelial cells. c. Leukocytes are binding to the selectins on the endothelial cells d. The leukocytes starts to flatten out and starts pushing pseudopods between the barrier of epithelail cells and tries to get through it. e. The leukocytes then gets into the cell and tries to migrates to the site of infection, and this is done via the gradient of the cytokines and chemokines. Lower [] in the faraway place. Higher [] near the site. f. They move to the infected site and try to clear it. 21. Cellular mediators of defense 22. Cells Of The Immune System 23. Stem cells are cells which can be anything they want to be. In this case, they are hemopoietic stem cells: they have limited ability to diversify itself; pluripotent: they have the potential to be many many dfferent cells. a. Myeloid progenitor – most of the innate cells comes from this. To form cells within the innate system. i. Macrophages, dendritic cell, monocyte, neutrophil, ii. granulocytes: eosinophil, basophil mast cell. b. Common lymphoid progenitor – T & B lymphocytes. Important for adaptive immune response. c. NK cells: even though it is derived from the common lymphoid progenitor, it sits in between innate and adaptive i. Innate because: ability to detect infections and tumor cells and kills them 24. a. I. Phagocytes - Monocyte/Macrophage – cells that eats stuff b. “Big eaters” c. Are called “monocytes” in the blood - roundish cells d. When they reach the tissue they are called “macrophages” - flat with lots of protrusions to seek out pathogens –lots of macrophages can be seen in the mucosal sites.roaming around the mucosal sites, respiratory tract, and genital tract looking for pathogens. Start to flatten out and have lots of protrusions to seek out pathogens.monocytes in their blood, when they come to the tissue it is called macrophages. e. Their job: eat pathogens, kill pathogens and help present chewed up pieces of pathogen to T cells (ie “antigen presentation”), production of soluble mediators 25. “Resident” macrophages have different names a. They all differentiate from blood monocytes b. Liver - Kuppfer cells c. Brain - microglial cells d. Lung - avelolar macrophage e. Bone – osteoclast 26. Phagocytosis 27. 28. Mechanisms by which phagocytes kill pathogens a. Phagasomes quickly becomes acidified – so we have low pH around 3.5-4 in the phagasomes. This could stop bacterial replication ( bacetriostatic) or could kill the bacteria (bactericidal ) b. ROS – ros is delivered into the phagasome thorugh the respiratory burst pathway. Because they are highyly toxic they can kill pathogens directly c. Antimicrobial peptides – defensins and cationic proteins: they can form pores withi the cell membrane of the bacteria and kill them directly, they are contained within the lysozyme and the phagsome d. Enzymes – Lysozyme e. Competitors – lactoferrin – bacteria nee
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