Advanced Lectures of Molecular Medicine Part I: Immunology PDF

Advanced Lectures of Molecular Medicine Part I: Immunology Christian Bogdan Professor of Medical Microbiology and Infectious Disease Immunology Mikrobiologisches Institut – Klinische Mikrobiologie, Immunologie und Hygiene Friedrich-Alexander Universität Erlangen-Nürnberg und Universitätsklinikum E...

Advanced Lectures of Molecular Medicine Part I: Immunology Christian Bogdan Professor of Medical Microbiology and Infectious Disease Immunology Mikrobiologisches Institut – Klinische Mikrobiologie, Immunologie und Hygiene Friedrich-Alexander Universität Erlangen-Nürnberg und Universitätsklinikum Erlangen, Erlangen, Germany Agenda: Innate Immunity  Dermal and epithelial barriers  Myeloid cells  Leukocyte chemotaxis receptors  Soluble factors: complement, chemokines, cytokines  Pattern recognition receptors  Antimicrobial effector functions of phagocytes  Natural killer cells and innate lymphoid cells  Principals of innate antiviral responses Innate immune response: stages  Tissue damage  Epithelial response not essentially belong to immune system but are equally imp  Attraction and transmigration of leukocytes (e.g. PMN*, natural killer cells)  Pathogen recognition by recruited PMNs and resident myeloid cells (macrophages [Mφ], dendritic cells [DC])  Activation of recruited PMNs and resident myeloid cells: a) phagocytosis b) antimicrobial effector mechanisms c) production of cytokines d) initiation of adaptive immune response final aim is to:  Killing of pathogen, control of infection  Tissue repair PMN(G) = polymorphonuclear neutrophilic granuloyctes = neutrophils  Innate immune response: Epithelial barrier eg is a person with burns - more susceptible to pathogen attack  Physical barrier: Epithel cell layer, skin epithelial - inherent antimicrobial protection  Killing of microbes: Antimicrobial peptides released by epithelial cells (e.g. defensins, cathelicidin) very imp for starting cytokinetic cascades both skin and gut  Killing of microbes, immunoregulation (cytokines, macrophage activation, lysis of infected cells): a) intraepithelial T cells (α/β or γ/δ) b) innate lymphoid cells (e.g. ILC2) Innate immune response: Epithelial barrier damage in the cell layer causes subcutaneous layer) blood From: MacPhearson and Austyn, Exploring Immunology 2012 Innate immune response: Epithelial barrier From: MacPhearson and Austyn, Exploring Immunology 2012 Function of mast cells From: MacPhearson and Austyn, Exploring Immunology 2012 Agenda: Innate Immunity  Dermal and epithelial barriers  Myeloid cells  Leukocyte chemotaxis receptors  Soluble factors: complement, chemokines, cytokines  Pattern recognition receptors  Antimicrobial effector functions of phagocytes  Natural killer cells and innate lymphoid cells  Principals of innate antiviral responses Myeloid cells  Granulocytes - neutrophils - eosinophils - basophils  (circulating blood) monocytes  Resident tissue macrophages (MΦ) they are named based on the location - CNS: microglia - Liver: Kupffer cells - Gut: intestinal macrophages - Lung: alveolar macrophages - Spleen: red pulp macrophage, marginal zone macrophage, metallophilic macrophage  Conventional dendritic cells (cDC) iftheytheyarearehighly immature (not exposed to pathogens), phagocytic)  Plasmacytoid dendritic cells (pDC) Elie Metchnikoff (1845-1916) "Über eine Sproßpilzkrankheit der Daphnien. when it is infected using a Beitrag zur Lehre über den Kampf der bacteria , u can see under the microscope, u can see Phagozyten gegen Krankheitserreger" how phagozytic activity of l eucocytes. (Virchow´s Archiv für Pathologische Anatomie und Physiologie und für Klinische Medizin 1884, Vol. 96: 177-195) Daphnia (Wasser"flöhe") Phylum: Arthropoda Class: Crustacea Order: Cladocera migration phagocytosis Metchnikoviella degradation bicuspicata (Ascomyces)  Professional phagocytes Polymorphonuclear granulocytes (neutrophils) Blood monocytes Tissue macrophages (e.g. alveolar macrophages, Kupffer cells, osteoclasts….) Immature dendritic cells Neutrophils what happens during phagocytosis? reactive oxygen intermediates NET = neutrophil extracellular traps Cytokines Immunoregulation Chemokines neutrophils produce cytokines and chemokines in a per cell basis Modified from: MacPhearson and Austyn, Exploring Immunology 2012  The classical view on macrophages (until 2000)  Tissue-macrophages are derived from circulating monocytes  Non-replicative tissue cells  Primarily strong phagocytic activity („professional phagocyte“)  Non-specific uptake of invading pathogens  enzymatic and oxidative antimicrobial activity: degradation of killed pathogens and waste, e.g. old erythrocytes, cell debris („garbage man of the body“)  Activation by lymphocyte products  Tissue repair and remodelling, maintenance of homeostasis  Important for development (e.g. bone, fingers) embryonic development only possible with macrophages Changes of paradigms in macrophage research (2000-2016)  Macrophage ontogenesis and development: - Prenatal: tissue macrophages of adults originate from the yolk sac or the fetal liver - Postnatal: tissue macrophages of adults can also originate from circulating resting monocytes, which transmigrate vessels as inflammatory macrophages  Macrophage replication: differentiated, inflammatory macrophages have the capacity to replicate (“self renewal”)  Macrophage markers: transcriptome analysis has revealed a much more complex set of markers found on macrophages it is possible to dissect the population of macrophages in subsets  Macrophage activation statuses: transcriptional analyses revealed a whole range of different activation statuses depending on the eliciting stimulus. The different macrophage activation statuses show a high degree of plasticity.  “Trained immunity” of myeloid cells: memory-like function of myeloid cells based on epigenetic remodelling  Development of Macrophages and dendritic cells Modified from: Lavin and Merad, Adult Myelopoesis Cancer Immunol Research 2014 in the bone marrow Myelopoeseis in the embryo HSC = hämatopoetische Stammzelle CMP = Common Myeloid Precursor MDP = Monozyte-Dendritic cell Precursor CDP = Commited Dendritic Cell Precursor cMoP = common Monozyte Precursor CSF-1 = Colony Stimulating Factor-1 (M-CSF) embryonic macrophages are originated from yolk sac and liver Yolk sac Fetal liver Spleen transformation to Mikroglia (Red pulp) resident macrophages Langerhans Alveolar Inflammatory Intestinal StudOn Dendritic Splenic macro- cells Macrophages Macrophages 5.-8.Std./24 Agenda: Innate Immunity  Dermal and epithelial barriers  Myeloid cells  Leukocyte chemotaxis receptors  Soluble factors: complement, chemokines, cytokines  Pattern recognition receptors  Antimicrobial effector functions of phagocytes  Natural killer cells and innate lymphoid cells  Principals of innate antiviral responses Attraction of leukocytes by microbes Neutrophils sense chemotactic Microorganism-associated molecular patterns (MAMPs) PAMPs - pathogen associated... Bloes et al., Nat. Rev. Immunol. 2015 Attraction of leukocytes by microbes Bacterial products are sensed by G-protein-coupled receptors (GPR) of neutrophils these can be used to trigger cytokinesis phenol soluble modulins CoNS, cagulase-negative staphylococci; FPR: formyl peptide receptor; PSM: phenol soluble modulin; SCFA, short chain fatty acids Bloes et al., Nat. Rev. Immunol. 2015 Vascular binding and transmigration of PMNs transmigration is complex- because of blood flow, the binding is difficult to the vessels (paracellular process!) Taken from: MacPhearson and Austyn, Exploring Immunology 2012 Vascular binding and transmigration of PMNs Dermal Taken from: microvasculature Hickey et al., Nat. Rev. Immunol. Vol. 9, 364 ff., 2009 named after the sites they are expressed E-selectin = endothelial leukocyte adhesion molecule 1 (ELAM-1; CD62E) ICAM-1 = intercellular adhesion molecule 1 (CD54) LFA-1 = leukocyte function-associated antigen 1 (αLβ2 integrin; CD11a/CD18) PSGL-1 = P(latelet)-selectin glycoprotein ligand 1 MAC-1 = macrophage antigen 1 (αMβ2 integrin; CD11b/CD18) many of them are used as markers Agenda: Innate Immunity  Dermal and epithelial barriers  Myeloid cells  Leukocyte chemotaxis receptors  Soluble factors: complement, chemokines, cytokines  Pattern recognition receptors  Antimicrobial effector functions of phagocytes  Natural killer cells and innate lymphoid cells  Principals of innate antiviral responses Cytokines and chemokines  Cytokines (e.g. interleukins, interferons, growth factors) the orgin of the name interleukin -mediators which work between leukocyctes  Chemokines = cytokines with primarily (but not exclusively) chemoattractant function Functions: Autocrine Paracrine Cell activation Cell inhibition Cell attraction Cell growth Release of stimulatory or inhibitory products Cytokine families From: Abbas/Lichtman/Pillai, Cellular and Molecular Immunology, 7th ed. 2012 why do different cytokines have diff effects or which does some cytokines have diff effects on diff sites- cytokines bind on diff sites Type I cytokine receptors: Type II cytokine receptors:  Dimers or trimers  Dimers  Unique ligand binding chain  One ligand binding chain  One or more signal transducing chains  One signal-transducing chain  Conserved cysteines  Conserved cysteins  Trp-Ser-X-Trp-Ser-motif (WSXWS)  No WSXWS-motif no need to learn all the names by heart Cytokine families They all use the common gamma chain for signalling From: Abbas/Lichtman/Pillai, Cellular and Molecular Immunology, 7th ed. 2012 GM-CSF = granulocyte-monocyte –colony stimulating factor IL = interleukin LIF = leukemia inhibitory factor CNTF = ciliary neurotrophic factor Chemokines and chemokine receptors Nomenclature based on their structure don´t by heart Gemma E. White et al. Pharmacol Rev 2013;65:47-89 Chemokines and immune cell migration where do to find chemokines? why do lymph nodes swell up during a infection? during infection - mucosa along with bacterial particles are drained to next lying lymph node From: Albert Zlotnik, Amanda M. Burkhardt & Bernhard Homey Nat Rev Immunology 11, 597-606, 2011 Complement system: cascade of activating processes Pathways of activation Taken from: MacPhearson and Austyn, Exploring Immunology 2012 Complement system: Functions Alternative Classical Lectin pathway pathway pathway Antibodies Mannose- Initiation of Bacterium Binding protein complement activation Effector functions C3a: Chemoattractant Early steps of Inflammation complement activation C3b:via complement and activation C3b binds to an Bakterium Opsonisation and phagocytosis C5a: Late steps of Chemoattractant complement Inflammation activation Membrane Attack Complex C5b-C9: Modified from: Abbas/Lichtman/Pillai, Cellular and Molecular Immunology, 7th ed. Cell-Lysis 2012 Agenda: Innate Immunity  Dermal and epithelial barriers  Myeloid cells  Leukocyte chemotaxis receptors  Soluble factors: complement, chemokines, cytokines  Pattern recognition receptors  Antimicrobial effector functions of phagocytes  Natural killer cells and innate lymphoid cells  Principals of innate antiviral responses Immune cell receptors Taken from: MacPhearson and Austyn, Exploring Immunology 2012  Pathogen recognition receptors: Recognition of pathogens and antigens in different locations phagocyte extracellular (e.g. macrophage, dendritic cell) intraphago- (lyso)somal cytosolic Infectious pathogens Pathogen recognition receptors in different locations  Pathogen recognition receptors also used for scavenging lipid particles - LDLP particles Taken from: MacPhearson and Austyn, Exploring Immunology 2012  Recognition of infectious pathogens: Signalling of pathogen recognition receptors Microbial ligand Sensing receptor („pathogen-associated molecular („pattern recognition receptor“, PRR) pattern“, PAMP), e.g. LPS e.g. Toll-like receptor 4 Cell membrane Adaptor-Molecule (e.g. MyD88) Innate immune cell (e.g. macrophage) Various kinases phosphorylating the transcription factors (e.g. IRAK1-Kinase, cytosol IKKα,β,γ) Transcription factors (e.g. NF-κB) nucleus Protein product Promotor Gene for inflammatory cytokine (e.g. IL-1, TNF, IL-6) Recognition of pathogens (II): Toll-like receptors (TLR) vs. Non-TLR-receptors node like receptors Modified from: Abbas/Lichtman 2012  Inflammation via activation of TLR and inflammasome Pathogenic bacteria, PAMPs Extracellular ATP K+ Efflux Reactive oxygen species (ROS) Endogenous crystals (Urate, Ca-Pyro- same mechanism is observed in case of non-infectious inflammation phosphate, Amyloid) - like gout for example Exogenous crystals (Asbestos, Silicate, Cholesterol, Al(OH)3) NLRP3 Viral DNA Inflammasome Peptidoglycan can cleave the precursor of IL 1 Modified from: Abbas/Lichtman 2012 Acute inflammation  Toll-like receptors for each structure of bacteria or pathogen, we have a receptor Protozoa Bacteria Viruses and fungi Cell wall bacterial Bact. Uropath. dsRNS RSV ssRNS virale Cell wall T. gondii components fagella DNA E. coli polyIC F-Protein siRNA DNS Zymosan Profilin Imiquimod GIPLs Lipoprotein LPS Flagellin CpG Protein CpG resiquimod Lipopeptide Glykolipids LAM S. Typhi Peptidoglykan Flagellin Porin NF-κB-Activation NF-κB-Activation NF-κB-Activation IRF3/7 Activation Proinflammatory Typ I Interferons Proinflammatory cytokines Proinflammatory cytokines modif. from West et al. Nature 2006 end of the cascade is always monomorphic cytokines  Pattern recognition receptors will be stimulated by both PAMPs and DAMPs PAMPs = Pathogen-associated molecular patterns (exogen) DAMPs = „Danger“-associated molecular patterns (endogen) sepsis-like inflammatory syndrome - heavy tissue damage releases mitochondria which habve bacterial origin. Examples for DAMPs: This means that the DNA binds to TLR causing the relase of proinflammatory reactions  Degradation products of extracellular matrix (e.g. LMW- hyaluronan)  Released intracellular proteins (heat-shock proteins, histones, HMGB-proteins)  Released host cell DNA (genomic, mitochondrial)  Crystals (urate, cholesterols, amyloid) Agenda: Innate Immunity  Dermal and epithelial barriers  Soluble factors: complement, chemokines, cytokines  Myeloid cells  Leukocyte chemotaxis receptors  Pattern recognition receptors  Antimicrobial effector functions of phagocytes  Natural killer cells and innate lymphoid cells  Principals of innate antiviral responses  Antimicrobial Effector Mechanisms of Phagocytes constitutive/ (cytokine-, TLR-) rapidly available inducible Oxygen- enzymes (e.g. lysozyme, acidification independent esterase, gelatinase) arginine depletion antimicrobial peptides (arginase) (defensins, BPI, serprocidin) iron chelators and proteins (histones) (lipocalin-2) iron chelators (lactoferrin) every living cell is dependent on Fe. Fe is a component of many enzymes, small GTPases hormones etc Mn2+/Zn2+ chelators itaconic acid (calprotectin) iron chelators perforin-2 TNF Oxygen- NADPH-Oxidase (phox) inducible NO synthase dependent Myeloperoxidase (MPO) (iNOS, NOS2) Haber-Weiss-reaction NADPH oxidase (phox) catalytic antibodies tryptophan depletion (IDO) DNA webs („neutrophil extracellular traps“, NETs) Phagocytosis - endocytosis Taken from: MacPhearson and Austyn, Exploring Immunology 2012  Opsonisation: improved phagocytosis via antibodies or complement enhanced phagocytosis Bacterium IgG FcR Antibody- Ag mediated C3b(i) CR1(3) Complement- mediated PAMP Phagocyte Antibody- and complement- Ag mediated IgG C3b CR1 or IgM Antimicrobial Activity of Phagocytes: Modes of Action direct ROI/RNI-mediated killing (iNOS, phox, MPO, catalytic ab): - nitrosylation, nitration - (lipid) peroxidation most common - DNA strand breaks - formation of membrane pores enzymatic digestion (lysozyme, proteases, acid pH) pore-forming peptides (e.g. defensins; perforin-2) trapping and enzymatic killing (e.g. neutrophil extracellular traps) nutritional depletion (arginase, iNOS, IDO, iron-chelators) Antimicrobial mechanisms of GDP- granulocytes CARD9 GDI Oxidative / Nitrasoidase Stress Rac GEF Phagocytic cup Phagosome p67 _ HWR/Fe 3+ GTP- O2 NADPH OH- OH. Rac OH. O3 1O SOD P67* gp91 2 H2O2 O2 - Cat p40 p47* p22 H2O + O2 O2 ClO- active Cl-/MPO p40 Primary (azurophilic) p47 granules gp91 α-defensins, BPI, seprocidins (cathepsin G, elastase, proteinase 3, azuricidin/ CAP37), MPO Nitroxosome p22 iNOS Secondary (specific) and L-Arg + O2 L-Cit + NO tertiary (gelatinase) granules inactive lactoferrin, lipocalin, lysozyme, LL37 MMP8, 9 und 25 matrix metaloprotease Antimikrobielle Abwehrmechanismen von Phagozyten (Neutrophile Granulozyten) pumping out Fe Ferretin - storage of Fe Uribe-Querol et al., Frontiers Immunol. 2017  NOS - Chemical Reaction iNOS FAD, FMN, heme, THB L-Arginine Nω-hydroxy- L-Citrullin NO L-Arginine 5-electron-oxidation of the =NH2-group of arginine  Regulation of iNOS Expression mRNA-synthesis mRNA-stability protein synthesis protein stability L-Arginine + LPS CAT + IL-4, IL-10, IL-13, + IFNγ, TNF, IFNαβ, TGFβ, LPS LPS L-Arg - - IFNγ TGFβ, IL-4, IL-13, (IFNαβ, LPS) calmodulin NADPH FAD Arginase iNOS FMN THB Ornithine + urea NO + citrullin ODC OAT putrescine proline spermidin collagen antimicrobial activity spermin synthesis tumoricidal activity signaling and immunoregulation iNOS↓ proinflamma- cytotoxicity/tissue destruction tory cytokines ↓ Mode of iNOS/NO action Macrophage Indirect antimicrobial effects of NO Host cell apoptosis Autophagosomal degradation of bacteria Fpn1-mediated iron deprivation Nrf2 Phagosolysosomal fusion Dispersion of bacterial biofilms Fe2+ Immunoregulatory effects phago- NO NOS2 L-arg lysosome Fpn1 Direct antimicrobial effects of NO Disruption of [FeS] clusters Blockade of Krebs cycle Inhibition of pathogen proliferation Inactivation of virulence factors Fe2+ Indirect antimicrobial Direct antimicrobial Intracellular pathogen effects of NO effects of NO (e.g. S. enterica Typhimurium)  Formation of ROI and RNI – Interaction of PHOX, MPO and iNOS enzyme MPO iNOS Phox (co-)substrates H2O2, Cl- L-arginine, O2, NADPH O2, NADPH key products HOCl Citrulline O2-.NO SOD.OH NO2- ONOO- H2O2 1O 2 Fe2+ Fe3+ NO2Cl S-nitroso- (Fenton)(Haber-Weiss).NO thiols 2 Cl2 OH- OH-.OH.OH effects oxidation S-nitros(yl)ation tyrosine oxidation of proteins (examples) chlorination disruption of nitration and lipids tyrosine nitration FeS- or ZnS nucleoside DNA strand breaks clusters nitration DNA mutation Antimicrobial Effector Mechanisms of Phagocytes constitutive/ (cytokine-, TLR-) rapidly available inducible Oxygen- enzymes (e.g. lysozyme, acidification independent esterase, gelatinase) arginine depletion antimicrobial peptides (arginase) (defensins, BPI, serprocidin) iron chelators and proteins (histones) (lipocalin-2) iron chelators (lactoferrin) small GTPases Mn2+/Zn2+ chelators itaconic acid (calprotectin) iron chelators perforin-2 TNF Oxygen- NADPH-Oxidase (phox) inducible NO synthase dependent Myeloperoxidase (MPO) (iNOS, NOS2) Haber-Weiss-reaction NADPH oxidase (phox) catalytic antibodies tryptophan depletion (IDO) DNA webs („neutrophil extracellular traps“, NETs)  Neutrophil extracellular traps (NETs, DNS-Netze) Pus under microscope DNA web these nets have antimicrobial activity DNA decorated with histone , elastase Cell death called necrosis NETosis of neutrophils: NET formation and cell death CatS cathepsin S, citH3 citrullinated histone H3, C5aR complement component 5a receptor, DOCK dedicator of cytokinesis proteins, ERK extracellular signal-regulated kinases, HOCl hypochlorous acid, HOCSN hypothiocyanous acid, MASPK mitogen-activated protein kinases, MPO myeloperoxidase, mTOR mammalian target of rapamycin, NE neutrophil elastase, NFκB nuclear factor kappa-light-chain-enhancer of activated B cells, OXPHOS oxidative phosphorylation, PAD4 peptidylarginine deiminase 4, PI3K phosphoinositide-3-kinase, PKC protein kinase C, SK3 small conductance calcium-activated potassium channel 3, SOD superoxide dismutase, TLR4 toll-like receptor 4. From: Stoiber et al., Biomolecules Vol. 5, 702-7023, 2015  NETosis of neutrophils: NET formation and cell death fMLP = N-Formylmethionyl-leucyl- phenylalanine NE = neutrophil elastase MPO = myeloperoxidase NET = neutrophil extracellular trap PLC = phospholipase C PAD4 = peptidyl (or protein) arginine deiminase TLR4 = Toll-like receptor 4 NETosis requires  Reactive oxygen species  Histone deimination by PAD4 From: Dwivedi et al., Ann.Rheumat. Dis. 2014 Antimicrobial effector mechanisms of neutrophils From: Hickey and Kubes, Nat. Rev. Immunol. Vol. 9, 364 ff. (2009) Role of antibodies in innate immunity Taken from: MacPhearson and Austyn, Exploring Immunology 2012 Agenda: Innate Immunity  Dermal and epithelial barriers  Soluble factors: complement, chemokines, cytokines  Myeloid cells  Leukocyte chemotaxis receptors  Pattern recognition receptors  Antimicrobial effector functions of phagocytes  Natural killer cells and innate lymphoid cells  Principals of innate antiviral responses  Activation and function of natural killer cells (NK cells) Activating NK- Activating Receptor Ligand NK + Target cell cell* - Inhibitory NK Inhibitory cell receptor Ligand (e.g. MHC class I) *e.g. cytotoxic granules a) Virus-infected cell b) Tumor cell  Activation and function of natural killer cells (NK cells): Antibody-dependent cellular cytotoxicity (ADCC) Activating NK- IgG Antigen Receptor (FcR [CD16]) NK + Target cell cell* - Inhibitory NK- Inhibitory Receptor Ligand (e.g. MHC class I) Ag *e.g. cytotoxic granules a) Virus-infected cell b) Tumor cell  NK cell – target cell interaction: Normal self vs. missing self vs. altered self Diefenbach and Raulet, CurrOpinImmunol 2003 Target cell lysis by NK cells Taken from: MacPhearson and Austyn, Exploring Immunology 2012  Dual function of NK cells in innate immunity Macrophage Activation via IFN-γ Taget cell lysis via granule exocytosis or death receptor ligation Taken from: MacPhearson and Austyn, Exploring Immunology 2012 Agenda: Innate Immunity  Dermal and epithelial barriers  Soluble factors: complement, chemokines, cytokines  Myeloid cells  Leukocyte chemotaxis receptors  Pattern recognition receptors  Antimicrobial effector functions of phagocytes  Natural killer cells and innate lymphoid cells  Principals of innate antiviral responses Innate lymphoid cells (ILCs) From: Artis and Spits, Nature Vol. 517, 293ff, 2015 Innate lymphoid cells (ILC) versus T cells Many similarities between ILCs and CD4+ T helper cells but no somatic recombination no MHC-dependent antigen recognition T-bet no separate memory populations known to date Walker et al, Nat Rev Immunol 2013 Innate lymphoid cells (ILCs) From: Artis and Spits, Nature Vol. 517, 293ff, 2015 Agenda: Innate Immunity  Dermal and epithelial barriers  Soluble factors: complement, chemokines, cytokines  Myeloid cells  Leukocyte chemotaxis receptors  Pattern recognition receptors  Antimicrobial effector functions of phagocytes  Natural killer cells and innate lymphoid cells  Principals of innate antiviral responses Type I interferon action From: Abbas/Lichtman/Pillai, Cellular and Molecular Immunology, 7th ed. 2012 Interaction innate and adaptive immunity Taken from: MacPhearson and Austyn, Exploring Immunology 2012