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Innate immunity 2013


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Innate immunity 2013

  1. 1. Exploring the first line of defense: research tools for the innate immune system Any Questions ??? Ask now or contact Technical Support 1-888-503-3187 International customers: Webinar related questions: For Internal Use Only -1- Sample & Assay Technologies
  2. 2. Innate and adaptive immunity Innate Adaptive PAMPs/DAMPs Rapid induction (hours) Short-lived For Internal Use Only Antigenic peptides Slow induction (days) Memory -2- Sample & Assay Technologies
  3. 3. Phases of the innate immune response Recognition Recruitment/Activation Effector Recognize microbial components Macrophage recognition of pathogens inflammation Phagocytosis and microbial killing by PMN and M∅ Germline-encoded receptors Inflammation leads to PMN, monocyte (M∅/DC), NK, eosinophil recruitment via chemokines Cytotoxicity by NK cells Non-clonal (ie, all cells of the same lineage have same receptors) Induction of adaptive immunity by DC and M∅ Non-cytokines or chemokines (complement fragments, fMLP) recruit and activate innate immune cells For Internal Use Only -3- Sample & Assay Technologies
  4. 4. Epithelia, peptides, innate-like lymphocytes Microbial invaders enter the body through skin and mucosa, and are met by several innate defenses before encountering circulating cellular effectors. . Mucus/cilia (sweep out microbes before they can adhere) Chemical defenses β-Defensins (epithelial cells and leukocytes, skin, tongue, respiratory tract) α-defensins (Paneth cell granules in intestine, neutrophil granules) Lysozyme, phospholipase A (saliva, tears) pH and digestive enzymes (stomach) Innate-like lymphocytes are lymphocytes with limited receptor diversity : Intraepithelial γ/δ T lymphocytes – GI B-1 B cells – peritoneal and pleural cavities NK-T cells – thymus, peripheral lymphoid organs . . For Internal Use Only -4- Sample & Assay Technologies
  5. 5. Induction of innate immunity by PRRs Pathogen-associated molecular patterns (PAMPs) . Lipopolysaccharide Flagellin Single- and double-stranded nucleic acids CpG Lipoteichoic acid Zymosan etc… Danger-associated molecular patterns (DAMPs) . HMGB1 Heat shock proteins Pattern recognition receptors (PRRs) . Toll-like receptors (TLRs) NOD-like receptors (NLRs) Mannose receptor Cytosolic DNA sensors RIG1-like receptors (RLRs) For Internal Use Only -5- Sample & Assay Technologies
  6. 6. Cellular and molecular effectors of innate immunity For Internal Use Only -6- Sample & Assay Technologies
  7. 7. Cellular components – phagocytes and natural killer cells Macrophages Long-lived phagocytes Short-lived phagocytes Mannose receptor, scavenger receptors, CD14 Complement receptors Granules (acid hydrolase, myeloperoxidase, defensins, cathepsin G, lysozyme, lactoferrin, elastase, etc) ROS, RNI Respiratory burst Proinflammatory cytokine production (IL-1β, IL-12, TNF-α, IL-6,CXCL8) NETs (neutrophil extracellular traps: chromatin + serine proteases) Produce growth factors for tissue remodeling Arise from circulating monocytes For Internal Use Only NK cells Neutrophils Proinflammatory cytokines (IL-12, TNF-α) -7- Expansion/activation in response to IL-15, IL-12, Type I IFNs Produce IFN-γ, IL-1, and IL-2 Recognize Class I MHC (inhibitory & activating receptors) Cytotoxic cells (via perforin and granzymes, or ADCC) Blood, spleen localization Memory? Sample & Assay Technologies
  8. 8. Cellular components – parasite defense Basophils Eosinophils Helminths, viruses and bacteria (also allergy) Least abundant granulocyte Respond to complement, IgE, TLR stimulation Histamine, proteoglycans, lipid mediators, IL-4, IL17E Produce major basic protein, eicosanoids, histamine, peroxidases, acid phosphatase, growth factors, and cytokines Mast cells Release histamine, heparin, proteases, TNF and other proinflammatory cytokines, eicosanoids, etc. For Internal Use Only Macroparasite and helminth defense, allergy Helminths, viral infection, allergy IgE-activated, matures via IL-3 Nuocytes Recently identified in Type 2 innate response against helminth infection (Neill et al, Nature 2010) Respond to IL-23 and IL-33, produce IL-13 Involved in allergic lung inflammation Activated by IL5, IL-3, and GMCSF -8- Sample & Assay Technologies
  9. 9. Cellular components – dendritic cells “Professional antigen presenting cells (APCs)”, linking innate and adaptive immunity (macrophages and B cells are also considered professional APCs) Present as immature DCs (highly phagocytic) in tissues; upon antigen capture, migrate to lymph nodes and spleen and mature, providing Ag presentation and costimulation to T cells Multiple subsets and sub-subsets based on surface markers, localization, and function Produce IL-12, IL-15, TNF-a, and Type I IFNs, and respond to chemokines through CCR2, CCR5, CCR6, and CCR7 For Internal Use Only -9- Sample & Assay Technologies
  10. 10. Complement cascade Three types: . Classical Alternative Lectin-induced Initiation: . C1q, mannose-binding lectin, or C3b bind microbe surfaces (or C1q binds C-reactive protein) 3 pathways converge with generation of C3 convertase through triggered-enzyme cascades Outcomes: . Opsonization Chemotaxis/activation of leukocytes (C3a, C5a) Membrane-attack complex Results from C3b generation Consists of C5b-9 Effective against Neisseria species For Internal Use Only - 10 - Sample & Assay Technologies
  11. 11. Cytokines and Chemokines Cytokines produced by innate immune cells Type I IFNs, IFN-γ TNF-α IL-1, 6, 8, 10, 12, 15, 18 . Cytokines that enhance/inhibit innate immune activity Enhance: IFN-γ, TNF-a, IL-15, IL-12 Inhibit: IL-10, TGF-beta . Important chemokine receptors CXCR1, 2 (neutrophils) CCR1, 2, 3 (leukocytes) CCR5 (DCs, NK cells, monocytes) CCR6, 7 (DCs) CXCR3 (ligand: CXCL10, monocytes and NK cells) Important chemokines CXCL8 (produced by M∅, recruits PMN) CCL2 (produced by monocytes, fibroblasts, recruits monocytes, DCs, NK cells) . . For Internal Use Only - 11 - Sample & Assay Technologies
  12. 12. Important signaling networks Cytokines JAK/STAT MAPK IRFs PI3K NFkappaB . Chemokines PI3K/AKT MAPK NFkappaB . TLRs . NFkappaB JNK/MAPK IRFs PI3K For Internal Use Only - 12 - Sample & Assay Technologies
  13. 13. Outcomes of an innate immune response Microbial killing . Complement Neutrophil and macrophage microbicidal mechanisms NK cell cytotoxicity against infected cells Type I IFNs induce antiviral state in infected cells Induction of adaptive immunity . Dendritic cells and macrophages present antigen to T cells and provide costimulation Chemokine and cytokine production lymphocyte expansion, activation For Internal Use Only - 13 - Sample & Assay Technologies
  14. 14. Technologies for innate immune research Pathway-focused gene expression analysis Featured publications: Derbigny, W.A. et al. 2012, Infect. Immun. RT2 Profiler PCR Array, Mouse Inflammatory Cytokines Signaling pathway reporter arrays Featured publication: Zughaier, S.M. 2011, J. Leukocyte Biol. Cignal Finder 10-Pathway Reporter Array RT2 Profiler PCR Array, Human TLR and Human Apoptosis Cytokine analysis Featured publication: Rahman, S. et al. 2011 J. Immunol. Multi-Analyte ELISArray RT2 Profiler PCR Array, Mouse IFN-α and IFN-β Response, Mouse Dendritic and Antigen Presenting Cells For Internal Use Only - 14 - Sample & Assay Technologies
  15. 15. RT2 Profiler PCR Arrays 84 of the most relevant genes in biological and disease pathways Gene lists identified through state-of-the-art bioinformatics and text-mining tools Integrated controls for genomic DNA contamination, normalization, and PCR processes Web-based data analysis software at no additional cost Compatible with most real-time PCR instruments Immunity-related pathways: Innate & Adaptive Immune Response Inflammatory Cytokines & Receptors Dendritic & Antigen-Presenting Cells Inflammasomes IFN-α/β Response NFkB Signaling MAPK Signaling PI3K/AKT Signaling (and more… 140+ pathways, including custom arrays) For Internal Use Only - 15 - Sample & Assay Technologies
  16. 16. Application data – which cytokines alter expression after PMA-Ionomycin treatment? Human PBMCs were treated with PMA and ionomycin, and then analyzed using the Common Cytokines RT2 Profiler PCR Array. This volcano plot shows both fold-change and the statistical significance, and demonstrates that 23 genes, including IL-10, IFN-gamma, IL-2, and TNF were upregulated, while IL-1beta and 5 other genes were downregulated in response to treatment. . For Internal Use Only - 16 - Sample & Assay Technologies
  17. 17. How does gene expression change during infection? Characterizing overall responses of gene networks can give a more comprehensive picture of the changes that are occurring. How can you profile the most relevant genes to your system of interest all at once? . Identifying a role for TLR3 in the innate immune response to . Chlamydia muridarum infection in murine oviduct epithelial cells Derbigny, W.A. et al. 2012, Infect. Immun. . Context: . Research aim: to determine how oviduct epithelial cells participate in host defense against Chlamydia. Technique: used Mouse Inflammatory Cytokines RT2 Profiler PCR Array to compare gene expression in infected wild-type or TLR3-deficient OE cells. Significance: demonstrated a role for TLR3 and IFN-β in initiating inflammatory responses against an intracellular bacterial pathogen. For Internal Use Only - 17 - Sample & Assay Technologies
  18. 18. Application data – TLR3 in the innate immune response Compared gene expression changes under the following conditions using the Mouse Inflammatory Cytokines PCR Array: Wild-type cells plus C. muridarum TLR3-deficient cells plus C. muridarum alone TLR3-deficient cells plus IFN-β and C. muridarum TLR3-deficient cells plus IFN-β alone . Results Wild-type infected cells: most chemokines and interleukins, CXCL15, CCR10. Deleting TLR3: diminished response to infection, except CXCL15 effects. CCR9 and Lta showed modest compared to WT cells. IFN-β + infection of TLR3-/-: partially rescued chemokine and Casp1 responses IFN-β sans C. muridarum: still stimulated some chemokine response, but not as much as with infection For Internal Use Only - 18 - Sample & Assay Technologies
  19. 19. Application data – TLR3 in the innate immune response Conclusion TLR3 and IFN-β are major mediators of the inflammatory response to C. muridarum in OE cells, possibly in a cell-type-specific manner (as other studies had showed no involvement of TLR3 in the macrophage response). The ability to compare expression of many pathway-related genes in several different conditions permitted the team to dissect the specific roles of the receptor, the cytokine, and the pathogen in stimulating responses. The CXCL10 findings from the PCR Array, as well as ELISA for CXCL10 and IL-6, prompted the team to examine whether IFN-beta was exerting its effects through enhancement of TLR2 signaling. Indeed, IFN-beta caused upregulation of TLR2 expression in OE cells. RT2 Profiler PCR Arrays provide an excellent tool for identifying which genes in a specific biological pathway are affected by infection, cytokine stimulation, and gene knockdown. For Internal Use Only - 19 - Sample & Assay Technologies
  20. 20. Technologies for innate immune research Pathway-focused gene expression analysis Featured publications: Derbigny, W.A. et al. 2012, Infect. Immun. RT2 Profiler PCR Array, Mouse Inflammatory Cytokines Signaling pathway reporter arrays Featured publication: Zughaier, S.M. 2011, J. Leukocyte Biol. Cignal Finder 10-Pathway Reporter Array RT2 Profiler PCR Array, Human TLR and Human Apoptosis Cytokine analysis Featured publication: Rahman, S. et al. 2011 J. Immunol. Multi-Analyte ELISArray RT2 Profiler PCR Array, Mouse IFN-α and IFN-β Response, Mouse Dendritic and Antigen Presenting Cells For Internal Use Only - 20 - Sample & Assay Technologies
  21. 21. Cignal Reporter Assays & Arrays Functionally verified assays for 45 pathways: Type I IFN IFN-γ NFκB MAPK PI3K/AKT STAT3 TGF-β And more… . Cignal Finder 10-Pathway Arrays: Cancer Immune Signaling Development Stem Cell & Differentiation Nuclear Receptors Stress & Toxicity . Cignal 45-Pathway Array . For Internal Use Only - 21 - Sample & Assay Technologies
  22. 22. Application data: determining the signaling pathways activated in response to cytokine stimulation HeLa cells were reverse transfected with the Immune Response 10-Pathway Cignal Finder Reporter Array. 16 hours after transfection, medium was changed to assay medium. 32 hours after transfection, cells were treated with 5 ng/ml TNFα or left untreated. After 6 hours treatment, dual-luciferase assays were performed. Results are expressed as fold change. . For Internal Use Only - 22 - Sample & Assay Technologies
  23. 23. Which signaling pathways are being triggered? Several signaling pathways can produce innate immune responses. How do you know which are at work in your system? . Neisseria meningitidis capsular polysaccharides induce inflammatory responses via TLR2 and TLR4-MD-2 Zughaier, S. M., J. Leukoc. Biol., March 2011 . . Context: Research aim: determining how CPS-triggered signaling proceeds Human TLR Signaling and Human Apoptosis RT2 Profiler PCR Arrays Cignal Finder 10-Pathway Reporter Array to identify active signaling pathways For Internal Use Only - 23 - Sample & Assay Technologies
  24. 24. Application: Reporter assay in immune response Pathways interrogated: . NFκB PKC/Ca++ (NFAT) Type I IFNs (ISRE) IFN-γ (GAS) MAPK/ERK (SRE) MAPK/JNK (AP-1) TGF-β (SMAD) cAMP-PKA (CRE) C-EBP Glucocorticoid receptor (GRE) Context: . Meningococcal endotoxin (LOS) produces a strong innate immune response through TLR4, but the innate response to another crucial virulence factor, capsular polysaccharide (CPS), was uncharacterized TLR2 and TLR4-MD2 were identified as the CPS receptors on macrophages. TLRs 2 and 4 can signal through NFκB or MAPK pathways. Which ones for CPS? Purified CPS from LOS-deficient strain of N. meningitidis (IpxA mutant) Dosed HEK/TLR2/6 or HEK/TLR4-MD2-CD14 cells with CPS or LOS after transient reverse transfection with reporters Stimulated THP-1 cells with CPS-lpxA, LOS, or Rhizobium LPS (TLR4 ligands) to compare gene induction profiles For Internal Use Only - 24 - Sample & Assay Technologies
  25. 25. Application: Reporter assay in immune response . Findings: Strong NFκB reporter activity in TLR4-MD2 and TLR2-6 stably transfected cells stimulated with serogroup B CPS Milder activity from Type I IFN/ISRE (TLR2-6), MAPK-JNK/AP-1 (both), and MAPERK/SRE (TLR4-MD2). Also observed variation in gene expression profiles between cells stimulated with CPSlpxA vs LOS (including TNF, NOD1, CD40LG, LTA, and CARD6). . Conclusions: Cignal Finder 10-Pathway Reporter Array identified 4 inflammatory signal transduction pathways potentially involved in TLR-mediated recognition of CPS The RT2 Profiler PCR Array determined that CPS induces qualitatively distinct gene expression responses compared to LOS For Internal Use Only - 25 - Sample & Assay Technologies
  26. 26. Technologies for innate immune research Pathway-focused gene expression analysis Featured publications: Derbigny, W.A. et al. 2012, Infect. Immun. RT2 Profiler PCR Array, Mouse Inflammatory Cytokines Signaling pathway reporter arrays Featured publication: Zughaier, S.M. 2011, J. Leukocyte Biol. Cignal Finder 10-Pathway Reporter Array RT2 Profiler PCR Array, Human TLR and Human Apoptosis Cytokine analysis Featured publication: Rahman, S. et al. 2011 J. Immunol. Multi-Analyte ELISArray RT2 Profiler PCR Array, Mouse IFN-α and IFN-β Response, Mouse Dendritic and Antigen Presenting Cells For Internal Use Only - 26 - Sample & Assay Technologies
  27. 27. Multi-Analyte ELISArrays Common Cytokines Inflammatory Cytokines TLR-induced Cytokines I: Viral TLR-induced Cytokines II: Microbial Autoimmunity Th1 / Th2 / Th17 Cytokines Common Chemokines . . . . . . . For Internal Use Only - 27 - Sample & Assay Technologies
  28. 28. Application data – are Th1 or Th2 cytokines being produced? Time-dependent (0, 6, 18, 24, and 48 h) patterns of Th1/Th2 cytokine induction by human peripheral blood mononuclear cells (PBMC) in response to PMA (50 µg/ml) and ionomycin (1 µg/ml) were monitored. The relative amount of each cytokine was profiled at the same time using the ELISArray Kit. . For Internal Use Only - 28 - Sample & Assay Technologies
  29. 29. What comprises the cytokine milieu? Innate immune responses produce a multitude of cytokines, which play distinct roles. Is it possible to efficiently detect several related cytokines simultaneously? . Murine FLT3 Ligand-Derived Dendritic Cell-Mediated Early Immune . Responses Are Critical to Controlling Cell-Free Human T Cell Leukemia Virus Type 1 Infection Rahman, S. et al. J. Immunol. 2011 . Context: . Research aim: to clarify how DCs respond to HTLV-1 infection RT2 Profiler PCR Arrays for IFNα/β Response, Dendritic & Antigen-Presenting Cells Multi-Analyte ELISArray Kit to identify production of multiple cytokines at once in the context of infection For Internal Use Only - 29 - Sample & Assay Technologies
  30. 30. Case study, Rahman et al. Cytokines detected: . IL-2 IL-4 IL-5 IL-6 IL-10 IL-12 IL-13 IL-17A IL-23 IFN-γ TNF-α TGF-β1 Study background . Innate immunity was not thought to have a major part in HTLV-1 control; however, the authors demonstrate a significant role for Flt3L-generated DC and Type I IFN in control of cell-free virus (the type that would be encountered early in infection) Which cytokines are produced in early HTLV-1 infection? Cultured Flt3L-generated BMDC with cell-free virus to analyze cytokines Compared cytokine induction between UV-irradiated and competent virus Profiled gene expression upon DC infection with competent virus. For Internal Use Only - 30 - Sample & Assay Technologies
  31. 31. Case study, Rahman et al. Findings . Multi-Analyte ELISArray – proinflammatory cytokines were secreted by DCs after viral challenge (IL-6, TNF-α, IL-12). Th2 cytokines (IL-4, IL-5, IL-13) were not induced, but IL-10 and TGF-β were secreted as well. RT2 Profiler PCR Arrays IFNα, IFNβ Response: Several IFN-responsive genes were upregulated after viral challenge, as well as signaling molecules DC & Antigen Presenting Cells: Some cytokines and chemokines were upregulated, confirming the ELISArray results, and signaling and antigen uptake/presentation genes were also enhanced. Many key chemokines (CCL2, 7, 8) and receptors (CCR5, CCR3) were downregulated. Conclusions . Multi-Analyte ELISArray Kits allowed detection of multiple cytokines at once, profiling the cytokine response of innate immune cells (in this case, DC) to virus. RT2 Profiler PCR Arrays confirmed the ELISA results and shed light on which chemokines and IFN-responsive genes were being activated or deactivated in response to virus. For Internal Use Only - 31 - Sample & Assay Technologies
  32. 32. Summary The innate immune system comprises a complex network of cellular and molecular components Research tools that allow simultaneous analysis of many immunity-related players at once are an effective way to characterize innate responses to microbes RT2 Profiler PCR Arrays profile expression of 84 genes simultaneously, and are available for over 140 pathways. Many of these are related to innate immunity and host defense, and custom arrays are also available. Cignal Finder Reporter Arrays (10-Pathway and 45-Pathway) permit simultaneous cell-based reporter analysis of several signaling pathways through DNA-based or lentiviral vectors, using either GFP or luciferase. ELISArrays are multiplex cytokine analysis assays using a traditional ELISA format. For Internal Use Only - 32 - Sample & Assay Technologies
  33. 33. Thank You for Attending! If you are interested in trying out these technologies in your research, please visit: . . . . If you have any questions about using our products in your research, please contact us at . Thank you for your time and attention! . For Internal Use Only - 33 - Sample & Assay Technologies