Innate Immunity III - Induced Cellular Response.pdf

Innate Immunity III Randal Gregg, PhD Induced Cellular Response harry potter symbol - Bing images defense against the dark arts - Bing images Learning Objectives 1) Identify the physical characteristics and functions of neutrophils, monocytes, macrophages, mast cells, eosinophils, basophils, natu...

Innate Immunity III Randal Gregg, PhD Induced Cellular Response harry potter symbol - Bing images defense against the dark arts - Bing images Learning Objectives 1) Identify the physical characteristics and functions of neutrophils, monocytes, macrophages, mast cells, eosinophils, basophils, natural killer cells, and plasmacytoid dendritic cells. 2) Distinguish between the receptor/ligand interactions leading to phagocytosis of infectious agents or apoptotic cells. 3) Recognize key enzymes and their activities in the phagosome, late phagosome, and phagolysosome and their role in phagocytosis. 4) Identify the timing of release of each neutrophil granule and the effector functions of the contents. 5) Define NETosis and how it contributes to the innate immune response. 6) Identify the pathways of mast cell, eosinophil, and basophil activation and their effector functions. 7) Recall the primary role of plasmacytoid dendritic cells in innate immunity. 8) Identify the mechanisms of natural killer cell activation and effector function and the collaboration with macrophages during a response. 2 Initial cellular innate response K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. 3 Recruitment of neutrophils Neutrophils (polymorphonuclear leukocytes, PMN) • Short-lived (est. 3-5d) phagocytes circulating in the blood o About 70% of white blood cells in the blood  o >70% = neutrophilia = acute bacterial infection and inflammation Reduced neutrophils (neutropenia) = chronic bacterial infection; overgrowth of bacteria and fungi at injury sites • First cells recruited to the infected tissues within 2 hours of pathogen detection • Dominant phagocytic cell within the infected area • Represent the first sign of the inflammatory response • Die in a few hours after tissue entry forming a creamy pus associated with sites of infection 4 Neutrophil infiltration marks the onset of inflammation K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. 5 Receptor ligation triggers phagocytosis MICROBE or PARTICLE Fibronectin* Antigen *Fibronectin in tissues (use β1 integrin to bind for transmigration along chemokine gradient) LPS Phosphotidylserine (PS) Mannosecontaining C3b structures Antibody Integrin Fc receptor LPS Mannose receptor receptor Complement PS receptor receptor Clustering of receptors NEUTROPHIL MACROPHAGE Rho-GTPases Syk (Spleen Tyrosine Kinase) PI3K → PLC → IP3 / Ca2+ PI3K → PKC + ERK 6 Pathogen-associated ligands → Phagocytosis MICROBE Mannose receptor is a nonsignaling receptor and primarily promotes actin reorganization only The other receptors generate signaling for cytokine and prostaglandin/leukotriene production, respiratory burst, and degranulation. Antigen LPS Mannosecontaining structures C3b Antibody Integrin Fc receptor LPS Mannose receptor receptor Complement PS receptor receptor Actin reorganization NEUTROPHIL MACROPHAGE Pro-inflammatory cytokine (IL-1β, IL-6, TNF-α) production and release Prostaglandin / leukotriene production and release Degranulation (Neutrophil) Respiratory burst 7 Dysregulated membrane lipids → Phagocytosis APOPTOTIC HOST CELL Phosphotidylserine (PS) PS flips to the outer leaflet when the cell is undergoing apoptosis for interaction with the PS receptor Antibody Integrin Fc receptor Phosphotidylserine is found on the inner leaflet of the cell membrane in healthy cells LPS Mannose receptor receptor Complement PS receptor receptor Actin reorganization NEUTROPHIL MACROPHAGE Cytokine (TGF-β) production and release (Macrophage) Down-regulation of pro-inflammatory cytokine genes 8 Antimicrobial mechanisms of neutrophils • Neutrophils can destroy microbial pathogens, such as bacteria by degranulation, phagocytosis, and NETosis • During degranulation, antimicrobial proteins are released outside the neutrophil • In phagocytosis, the pathogen is ingested in a vacuole called a phagosome, which then fuses to lysosomes to become the phagolysosome, where the pathogen is destroyed • During NETosis, DNA fibers decorated with histones and granular proteins, such as elastase and myeloperoxidase are released in structures known as neutrophil extracellular traps (NETs) 9 Granule formation correlate with stage of neutrophil development Secretory and Ficolin-1 Mature neutrophil Gelatinase (tertiary) Metamyelocyte Specific (secondary) Myelocyte Azurophilic (primary) Promyelocyte Band neutrophil Myeloblast 10 First granules mobilized after activation Secretory and Ficolin-1 Inflammation high Ca2+ Gelatinase (tertiary) • Reservoir for cell membrane receptors • Integrins, CR1, PRR • Activation and attachment to activated endothelia Specific (secondary) Azurophilic (primary) Form in segmented neutrophils and signify that the cell is fully mature 11 Granules mobilized upon rolling contact with endothelia Extravasation 1) Extracellular matrixdegrading enzymes Secretory and Ficolin-1 Gelatinase elevated Ca2+ o Gelatinase (matrix metallopeptidase-9, MMP9) 2) Arginase 1 Specific (secondary) o Metabolizes Arg → no longer a substrate for nitric oxide synthase (NOS) Azurophilic (primary) o ↓ NO production (competitor for substrate Larginine) – aid closure of tight junctions during extravasation 12 Granules mobilized upon microbe contact Slow pathogen expansion Secretory and Ficolin-1 Gelatinase (tertiary) Specific moderate Ca2+ Azurophilic (primary) 1) Lactoferrin o Sequesters iron in biological fluids o Disrupts microbe membranes = lysis of viruses, bacteria and fungi o ↑ cathepsin G and serine protease activation 2) NGAL (Neutrophil gelatinaseassociated lipocalin) o Binds siderophores generated by microbes 3) Chemotaxis proteins to direct neutrophil trafficking in tissues 13 Granules mobilized upon microbe contact Fuse with phagolysosome Secretory and Ficolin-1 1) Myeloperoxidase Gelatinase (tertiary) Specific (secondary) Azurophilic low Ca2+ o Generate hypochlorous acid (damages nucleic acids) 2) Antimicrobial peptides o Forms pores in microbial membranes 3) Acid hydrolases o Proteases, nucleases, lipases 4) Defensins o Formation of pores in membranes 14 Phagocytosis • Receptor ligation triggers actin reorganization and enclosure around the pathogen until the membrane fuses producing a phagosome • Bacteria that have capsules – Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis, and Klebsiella pneumoniae – are highly resistant to phagocytic uptake and require opsonization using C3b for effective uptake K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. 15 EARLY reactions within the phagosome Exterior of Cell OXYGEN (O2) O2-dependent reactions PRR ligation Cytokines Pentose + NADPH Cell Membrane Glucose + NADP+ Ligation of PRR triggers actions of enzymes in the cell and phagosome membranes o Respiratory Burst  NADPH Oxidase NADP+ Cytosol Cytochrome b558 Electron Phagosome Membrane o NADPH Oxidase (reduced nicotinamide adenine dinucleotide phosphate) or Phox (phagocyte oxidase)  Hydroxyl radicals (.OH, OH-) Superoxide (O2-) SOD Singlet oxygen (1O2) These reactants are known as Reactive Oxygen Species (ROS) + Fe3+ + H+ H2O2 MPO + Cl- Hypochlorous acid (OCl-) Increased uptake of O2 Produces superoxide radicals (O2-) from O2 o SOD (Superoxide Dismutase)  Produces H2O2 and singlet oxygen (1O2) from superoxide o MPO (Myeloperoxidase)  Green heme pigment (colors pus)  Generates hypochlorous acid and hydroxyl radicals (.OH, OH-) 16 EARLY reactions within the phagosome PRR ligation Cytokines Exterior of Cell Arginine + NADPH O2-dependent reactions o iNOS (inducible nitric oxide synthase) Activates OXYGEN (O2) Cell Membrane Citrulline iNOS Redox cofactors FAD FMN Heme BH4 Calmodulin Cytosol Phagosome Membrane  Produces nitric oxide (NO) from Arginine and O2  NO is highly reactive and diffusible, soluble in both lipids and water  NO reacts with O2 and reactive oxygen species such as superoxide to form: • Nitric Oxide (NO) These reactants are known as Reactive NItrogen Species (RNS) NO2 NO2NO3N2O3 ONOO- Reactive Nitrogen Species (RNS) = NO2, NO2-, NO3-, N2O3, ONOO- o ROS/RNS oxidize bases within DNA generating mutations and strand breaks that prevent replication and gene expression o ROS/RNS also inactivate iron-sulfur containing enzymes thereby restricting metabolic pathways FAD = Flavin adenine dinucleotide; FMN = Flavin mononucleotide; BH4 = tetrahydrobiopterin 17 LATE reactions within the phagosome Exterior of Cell Cell Membrane H+ H+ Secretory vesicles fuse with phagosome and deliver the following molecules o Vacuolar ATPase H+ Vacuolar ATPase O2-independent reactions Cytosol Secretory Vesicles  Membrane-embedded proton-pump (ATP)  Pumps H+ ions into the lumen of the phagosome to lower the pH (4-5)  Lower pH reduces microbial enzymatic action and activates acid hydrolases arriving from fused vesicles and upon lysosomal fusion Phagosome Membrane H+ + H+ + H + H H ↓ pH Defensins Cathepsin H o Defensins  Pores in microbial membranes o Cathepsin H  Endopeptidase/aminopeptidase 18 Phagosome-Lysosome fusion Exterior of Cell Cell Membrane H+ H+ H+ Vacuolar ATPase Cytosol Lysosome fusion Phagolysosomal Membrane H+ + H+ + H + H H ↓ pH Acid Hydrolases Lysozyme O2-independent reactions Lysosome membrane fuses with phagosome membrane = phagolysosome o Acid Hydrolases  Lysosomal acid phosphatases  Nucleases  Proteases  Lipases / Phospholipases  Glycosidases  Highly active at low pH (4-5) o Lysozyme  Destroys bacterial cell walls 19 Neutrophil extracellular traps (NET) • NETosis = a type of programmed cell death whereby a NET is generated (distinct from apoptosis and necrosis pathways) • Disintegration of the nuclear and granular membranes and combining of nuclear, granular and cytoplasmic components with corresponding rupture of the plasma membrane • NET composed of primarily of DNA, histones, and proteins from azurophilic, specific and gelatinase granules • NET generation requires ROS/RNS production and the enzymatic activity of NADPH oxidase and MPO • NETs more effective against certain microbes such as S. aureus, S. pneumoniae, C. albicans, and A. fumigatus • Macrophages, mast cells and eosinophils now been shown to produce ET (ETosis) 20 Additional cellular innate responses INFILTRATION FROM BLOOD K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. TISSUE RESIDENT 21 Mast cells • Resident in mucosal and epithelial tissues lining the body surfaces • Operate to maintain tissue integrity, alert the immune system to trauma/infection and promote inflammation as well as wound repair • Present in all vascularized tissues except the CNS and retina o Mast cells regulate vasodilation and vascular permeability • Large granules containing histamine, heparin, proteases, degrading enzymes 22 Mast cells / Basophils • Two methods of activating mast cells (and basophils) 1) PRR binds to PAMP → triggers low level degranulation (eicosanoids and vasoactive amines) then cytokine and chemokine production 2) Antigen binds to IgE of Fcε receptor on surface of the mast cells → full degranulation followed by cytokine and chemokine production 23 Mast cell products amplify inflammation • Histamine o Promotes smooth muscle release of NO to induce vasodilation and shrinks endothelia for increased vascular permeability o Also promotes constriction of airways and mucus secretion (lungs) • Tryptase o Activate metalloproteases (MMP) to remodel the ECM • Leukotrienes (local effects) o Increase vascular permeability and smooth muscle contraction, airway constriction, and mucus secretion (lungs) IL-33 expressed by fibroblasts, endothelial and epithelial cells, macrophages, neutrophils, dendritic cells, B cells, and mast cells; produced at high levels in barrier tissues like the skin and mucus membranes. Stimulates mast cells like PRR and promotes production of IL-33 by the activated mast cells. • Prostaglandins (local effects) o Increase vascular permeability and smooth muscle contraction, airway constriction, and mucus secretion (lungs) 24 Mast cell products amplify inflammation • VEGF (vascular endothelial growth factor) o Operates to promote angiogenesis • IL-3 + IL-5 + GM-CSF o Eosinophil production and activation • IL-4 and IL-13 o T cell responses for IgE production • IL-8 and IL-17 o Promote neutrophil recruitment and activation 25 Plasmacytoid dendritic cells (PDC) • Primarily found in tissues with less than 1% of cells in blood • Morphology resembles that of antibodysecreting plasma cells thus given distinction of plasmacytoid • Express TLR7 and TLR9 to recognize viral nucleic acids (viral infections) • Primary producer of type I interferons (IFNalpha mainly) to induce antiviral state and activate NK cells 26 Natural killer (NK) cells • Circulate in the blood and lymphatics – 5-10% of blood lymphocytes; some presence in tissues • Derived from lymphocyte lineage (along with B and T cells) with two functions: 1) Produce cytokines for enhancing phagocytosis of macrophages (IFN-γ) and promoting wound healing (IL-22) 2) Cytotoxicity of virally-infected and abnormal (cancerous) host cells • NK cells express CD56 , CD16 (Fc receptor) and are CD3negative (not a T cell) • Inflammation is NOT a prerequisite for NK function (i.e. immunosurveillance) • NK deficient patients = persistent viral infections, especially herpesviruses 27 Cytokines can activate NK cells • Cytokines promote NK cell proliferation and mobilization of granules to the point of NK-host cell contact – release of granules dictated by complicated signaling • Cytokines that activate NK cells include: IL-1β, IL-2, IL-12, IL-15, type I IFNs and TNF-α K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. 28 Regulation of NK cell cytotoxicity • Resting and activated NK cells are primed to release granules (default is to be activated) and must receive an inhibitory signal to cancel release o MHC class I molecules are proteins found on ALL nucleated cells o Usually involved in the binding of peptides for display to CD8-expressing T cells o MHC class I molecules serves as an inhibitory ligand for the inhibitory receptors of NK cells → ligation sends an inhibitory signal and prevents NK release of cytotoxins <NK cells INACTIVE> o Viruses infecting tissue cells and cell transformations often reduce the level of MHC class I molecules → no inhibition imposed → granules release cytotoxins → host cell undergoes apoptosis <NK cells ACTIVE> 29 Positive feedback of macrophages and NK cells • During the early response – macrophages produce IL-12 → activate tissue resident and infiltrating NK cells • The activated NK cells release IFN-γ → bound by IFN-γ receptors on the macrophages → increases phagocytosis of engulfed microbes (esp. important for microbes resistant to phagocytosis) o IFN-γ increases activation of NADPH oxidase and iNOS K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. 30 Dual nature of macrophages Differentiation • Monocytes in blood can differentiate into macrophages in tissue via encounter with GM-CSF (granulocyte-macrophage colonystimulating factor) and M-CSF (macrophage colony-stimulating factor) as enter the monocytes enter the tissues Polarization • As the monocytes differentiate into macrophages – other predominant cytokines (dependent upon the response) induce unique gene expression in the differentiating cells • These additional cytokines promote polarization of macrophages to a specific type of response – inflammatory or anti-inflammatory o M1 macrophages  o Induced by GM-CSF + IFN-γ or TLR signals → produce pro-inflammatory cytokines (IL-1β, IL-6, IL-12, TNF-α) M2 macrophages  Produce anti-inflammatory cytokines and ECM proteins to promote tissue repair and remodeling 31 Eosinophils contribute to the innate response • Granulocytes that reside in tissues primarily in the connective tissue underlying the epithelia of the gastrointestinal, respiratory and urogenital tracts; also increase in number during inflammation through recruitment from the blood • Recruited and activated by IL-5 (promotes eosinophilia = bone marrow output of eosinophils is low until IL-5 induces production and increases numbers in the blood) • Granules contain Arg-rich proteins that stain heavily with eosin • Express TLR and can contribute to the response by production of cytokines and release of granules containing proteins that disrupt membranes (ECP, MPB) and degrade DNA (EPO) of parasites primarily o ECP (eosinophilic cationic protein) o MBP (major basic protein) o EPO (eosinophil peroxidase) 32 Eosinophils are “parasite assassins” 33 Concerted action of mast cells, eosinophils and basophils IL-3 (from mast cells and eosinophils) Microbe/particle TLR or Antibody detection Degranulation Recruits and activates MBP Degranulation Degranulation Inflammatory response Major basic protein (MBP) IL-5 (from mast cell) K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. 34 Innate immunity contains an infection until adaptive immunity can eliminate invaders Innate immunity operates to limit the expansion of microbes Adaptive immunity functions in the elimination of microbes Required to activate the adaptive immune response K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. 35 Dendritic cells are tissue resident cells that link innate and adaptive immune responses INNATE IMMUNITY K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. ADAPTIVE IMMUNITY 36 Questions? hogwarts dining hall wallpaper - Bing images 37 Next… Lymphatic System Hogwarts Express Wallpaper (65+ images) (getwallpapers.com) 38