innate_immunity-sr (1).pdf

Dr. Vishy Venketaraman, Professor, COMP, Basic Medical Sciences LECTURE#2 & 3 Introduction to host defense: Slides 1-8: OBJECTIVES: Describe the characteristics and functions of monocytes, macrophages and dendritic cells. Identify the pathways involved in reactive o...

Dr. Vishy Venketaraman, Professor, COMP, Basic Medical Sciences LECTURE#2 & 3 Introduction to host defense: Slides 1-8: OBJECTIVES: Describe the characteristics and functions of monocytes, macrophages and dendritic cells. Identify the pathways involved in reactive oxygen burst and the formation of reactive oxygen metabolites following tissue injury. Compare and contrast the functions of neutrophils, monocytes, eosinophils, and basophils. Describe the phagocytic barrier to infection and identify and describe the function of the phagocytic cells in the body. Differentiate among the three complement pathways: classical, lectin, and alternative. Explain the role of antimicrobial peptides such as defensins or cathelicidins in innate immunity. Explain the role of pattern recognition receptors (PRRs) and their interaction with pathogen associated molecular patterns (PAMPs) in the activation of innate immune cells. Define lysozyme and its biological activity. Compare and contrast innate and adaptive immunity; define antigenicity, immunogenicity, antigen, Describe the characteristics of innate and adaptive immunity. Describe the components of innate immunity. Describe the physical barriers (skin and mucous membrane) Describe the characteristics of neutrophils (primary [myeloperoxidase, lysozyme and a-defensins] and secondary granules [lactoferrin]) 1 Dr. Vishy Venketaraman, Professor, COMP, Basic Medical Sciences Describe the characteristics of monocytes, macrophages and dendritic cells. Describe pattern recognition receptors (TLR-4 and TLR-2) Describe the intracellular mechanisms by which neutrophils and macrophages control intracellular infection. The two main functions of the immune system include: Distinguish self from non-self. Protect the body from invading infectious organisms (parasites, bacteria, fungi and virus) The important components of the immune system include: a) innate immunity b) adaptive immunity Characteristics of innate immunity: 1. It is pre-formed. 2. Active 3. Does not have immunological memory. 4. Innate immunity does not improve over time. 5. Innate immunity is non-specific (can kill wide range of organisms) Innate immunity is often unsuccessful in controlling the infection and hence adaptive immunity kicks in. Components of innate immunity: The innate immune system is made of 1. Barriers like skin and mucous membrane 2. Cells such as neutrophils, monocytes, macrophages, dendritic cells and natural killer cells 2 Dr. Vishy Venketaraman, Professor, COMP, Basic Medical Sciences 3. Antimicrobial peptides and proteins like complement and C-reactive proteins 4. Innate immunity is considered to provide first line defense against infections. However, innate immunity is often unsuccessful in controlling infection when adaptive immunity comes in and controls the infection. Characteristics of adaptive immunity: 1. The onset is slow. 2. Once active it is highly effective. 3. Adaptive immunity is highly specific to a particular organism. 4. It improves over time. 5. Adaptive immunity has immunological memory. Components of adaptive immunity include subset of lymphocytes such as T lymphocytes and B lymphocytes. Lymphocytes are either called T lymphocytes or B lymphocytes based on the organ in which they undergo differentiation. Lymphocytes that undergo differentiation in thymus are called T lymphocytes. Lymphocytes that undergo differentiation in bone marrow are called B lymphocytes. B cells produce antibodies. Antibodies are protein molecules. Antibodies bind to the antigens (derived from the microbes) and neutralize the effects of antigens. T cells produce cytokines or interleukins (IL) which are also proteins. Cytokines, however, do not bind to the antigens but activate (or suppress) the cells of innate and adaptive immunity. Slides 9-15: Innate immunity is preformed and lacks immunological memory. Furthermore, innate immunity does not improve over time. 3 Dr. Vishy Venketaraman, Professor, COMP, Basic Medical Sciences The first line of innate immunity includes barriers such as skin, mucosal membrane, acid pH in the stomach………etc Skin is impermeable and protects the internal organs from microbial invasion. The outer layer of dead cells in the skin is periodically sloughed thereby eliminating the organisms that are bound to the skin. The skin also contains sebaceous glands that secretes sebum and maintains a low pH of 5.5 thus preventing the colonization of bacteria. Secretions of skin and eyes contain lysozyme. This enzyme degrades peptidoglycan layer on the surface of the Gram positive bacteria Vagina in adults is colonized by symbiotic lactobacillus that secretes lactic acid resulting in a low pH of 4.5 thus preventing colonization of microbes. Elimination of lactobacillus due to treatment with antibiotics will increase the risk for candida and other fungal infections due to increased pH. The stomach secretes hydrochloric acid causing a low pH of 2.2 which again creates an unfavorable environment for the organisms to colonize the stomach. Furthermore, stomach panneth cells secrete -defensin that has antimicrobial effects on the pathogen. The coordinated beating of ciliated epithelial cells in the lungs drives the mucosa bound organisms bound to mouth or nostrils leading to expulsion. The respiratory cells secrete -defensin which has antimicrobial effects similar to -defensins. 4 Dr. Vishy Venketaraman, Professor, COMP, Basic Medical Sciences Once the organisms breach the above barriers then the cellular components of the innate immune system contribute to protection against microbial infection Slides 16-18: The cells that participate in innate defenses include neutrophils, macrophages and dendritic cells. Neutrophils have a short life and therefore provide innate defense against acute infections. Neutrophils induce pyogenic response leading to pus formation. Macrophages play an important role in innate defense against both acute and chronic infections. Macrophages and differentiated cells and have a long-life. Dendritic cells like macrophages have a long life. Dendritic cells are capable of both phagocytosis and pinocytosis. Phagocytic cells recognize microbial infection using Pattern recognition receptors such as toll like receptor-4 (TLR-4), TLR-2 and complement receptors. TLR-4 recognizes LPS present in Gram negative bacteria, whereas TLR-2 recognizes peptidoglycan and lipotechoic acid present in Gram positive bacteria. Once they recognize the microbial infection, monocytes and neutrophils bind to the microbes and engulf them. Phagocytosis occurs through special areas on the phagocytic cell surface called clatherin-coated pits. The plasma membrane invaginates and ends of the membrane fuse to form a vacuole called phagosome that contains the ingested organism. The phagosome containing organism then fuse with the granules or lysosomes in the cytoplasm of the phagocytes followed by degranulation or release of granules into the phagosome. As far as the neutrophils are concerned, there are two types of granules, primary and secondary granules. 5 Dr. Vishy Venketaraman, Professor, COMP, Basic Medical Sciences Primary granules contain myeloperoxidase and lysozyme Whereas the secondary granules contain lactoferrin, DNA ase, RNA ase and several different proteases. Monocytes and macrophages do not contain myeloperoxidase. Myeloperoxidase is a green colored enzyme present in the pus and is released from neutrophils indicating ongoing host-inflammatory responses. Slides 19-25: Phagocytes kill intracellular organism by one of the two pathways, oxygen dependent and oxygen independent mechanisms. In the oxygen-dependent mechanisms, perturbation of plasma membrane results in activation of NADPH oxidase that reduces oxygen to singlet superoxide. Superoxide undergoes dismutation to form hydrogen peroxide. Hydrogen peroxide can inhibit the growth of intracellular pathogens. However, some pathogens synthesize superoxide dismutase and catalase that can degrade superoxide and hydrogen peroxide. Hydrogen peroxide can combine with chloride ions in the presence of myeloperoxidase enzyme to produce hypochlorous acid. Hypochlorous acid is a potent antimicrobial agent that can inhibit the growth of intracellular pathogens. Still potent is singlet oxygen which is derived from a reaction between hypochlorous acid and hydrogen peroxide. 6 Dr. Vishy Venketaraman, Professor, COMP, Basic Medical Sciences Macrophages cannot generate hypochlorous acid and singlet oxygen because they lack myeloperoxidase. Macrophages, however, can generate superoxide and hydrogen peroxide. Additionally, macrophages can generate nitric oxide and reactive nitrogen intermediates that are efficient in controlling intracellular pathogens. Nitric oxide is a gas produced from the amino acid L-arginine. The enzyme in macrophages that converts L-arginine to nitric oxide is inducible nitric oxide synthase (NOS2) Nitric oxide is a highly potent microbicidal agent but has short life (gets converted to nitrate and nitrite both lack antimicrobial properties) Oxygen independent control of intracellular pathogens include static or cidal effects on the internalized pathogen mediated by lysosomal enzymes and other antimicrobial peptides. Lysozyme present in the macrophages as well as in the primary granules of the neutrophils can degrade the peptidoglycan on the surface of the Gram positive bacteria. Lactoferrin, an antimicrobial peptide is present in the macrophages and in the secondary granules of the neutrophils sequesters iron and deprives the pathogen from the availability of iron. -defensins present in macrophages and -defensins present in neutrophils have antimicrobial effects. LL37 (cathelicidin) present in macrophages and neutrophils have antimicrobial effects. 7 Dr. Vishy Venketaraman, Professor, COMP, Basic Medical Sciences Other lysosomal enzymes including lipases and DNA ases can damage the bacteria by acting on the cells or DNA. Intracellular contents of the phagocytic cells may be released to the exterior when the organism is extracellular or if the organism is bigger in size to be phagocytosed. The released antimicrobials will act on the microbes and will inhibit the growth. Macrophages express CD14. Macrophages can fuse to form a multi-nucleated cell called giant cell. IL-8 promotes migration of neutrophils from the circulation to the extravascular sites. Slides 26-29: Phagocytic efficiency: Phagocytic efficiency of neutrophils and macrophages can be determined by challenging monocytes with inert particles like latex beads or microorganisms and observing the cells under the microscope after staining for phagocytic uptake. The NADPH oxidase activity can be determined by nitro blue tetrazolium (NBT) staining. During oxidative stress NBT gets reduced and becomes blue in color indicative of NADPH activity and free radical production Chronic granulomatous disease is a rare condition. People suffering from chronic granulomatous disease are deficient in NADPH oxidase resulting in impaired production of ROI and increased susceptibility to infections. 8 Dr. Vishy Venketaraman, Professor, COMP, Basic Medical Sciences Slides 30-39: Complement, a group of serum proteins get activated when there is immune complex formation as a result of antigen -antibody reaction or when there is infection with Gram negative bacteria. Lipopolysaccharide (LPS) is an important constituent of the cell wall of Gram-negative bacteria. Infection with Gram negative bacteria will therefore result in the release of LPS and LPS can activate the complement. Mannose (a polymer of mannan) released during fungal infection or during infection with some Gram-positive bacteria can also activate the complement. There are more than 30 complement factors denoted as C1-C9 and so forth. Complement activation leads to cascade of events resulting in lysis of the bacteria. Complement can get activated by two different pathways a. classical pathway b. alternate pathway c. mannose binding lectin pathway Briefly, in classical pathway, antigen-antibody complex (or immune complex) formation results in activation of C1 followed by C4 and C2. This complex C142 activates C3. This results in sequential activation of C5, C6, C7, C8 and C9. This complex (C5b, 6, 7, 8, 9) is called membrane attack complex. This complex binds to the bacterial cell surface causing lysis of the bacteria. In alternate pathway, infection with gram negative bacteria result in the release of LPS. LPS in the presence of Factor B, D and P (Properdin) acquires C3 convertase activity and directly activates C3 followed by sequential activation of C5, C6, C7, C8, and C9 and lysis of bacterial cells. In mannose lectin pathway, infections with some bacteria or fungus result in the release of mannose than binds to the host protein lectin. This complex activates C4 followed by C2. The C42 complex activates C3. C3 activation results in subsequent 9 Dr. Vishy Venketaraman, Professor, COMP, Basic Medical Sciences activation of other complement factors and formation of membrane attack complex. Complement thereby contributes to innate immunity. Complement as opsonins: Complement along with antibodies enhances the phagocytic ability of the neutrophils and monocytes. Phagocytic cells have receptors for complement and antibodies on their cell surface. When the bacterial surface is coated with complement (or antibodies), then the complement on the bacterial cell surface binds to the corresponding receptors on the macrophage cell surface resulting in easy and enhanced uptake of the bacteria. This process is called opsonization. In this case complement and antibodies are called opsonins. During complement system activation individual components undergo cleavage to form the two dissimilar fractions a and b. Like C3a and C3b, C5a and C5b. The b fraction participates in the cascade of chain reactions leading to the formation of membrane attack complex. The “a” fraction serves as chemotactic agents drawing immune cells from different parts of the body to the site of infection. The C5a component of the complement is also called anaphylotoxin. The C5a component can induce mast cells and basophils to undergo degranulation and release histamines which can enhance vascular permeability. Chemotactic effects and leaky vasculature promote immune cells to leave the circulation and migrate to the extravascular sites or site of infection to mediate inflammatory response. During inflammatory response immune cells release reactive oxygen intermediate, reactive nitrogen intermediates, antimicrobial peptides and proteins leading to necrosis and destruction of host tissue. RBCs have receptors for C1. Complement associated immune complexes in the circulation bind to C1 receptors on the cell surface RBCs. In the liver, macrophages can either ingest the entire RBCs or just complement complex from the RBC. 10 Dr. Vishy Venketaraman, Professor, COMP, Basic Medical Sciences Complement complexes get digested within the intracellular environment of the macrophages. Therefore, complement is important in opsonization and phagocytosis and can serve as an effector molecule in innate defense against microbial infection. Complement deficiency is associated with increased susceptibility to Neisseria infection. However, exacerbation in the activation of complement cascade can lead to leaky vasculature favoring immune cells to leave the circulation and cause inflammation in the extravascular site. Complement activation can be regulated or controlled by inhibitors such as C1 inhibitor, Factor H and Factor I. Factor H inhibits alternate pathway. Whereas Factor I inhibit classical and alternate pathways C1 inhibitor deficiency leads to hereditary angioedema. Human cells are protected from the lytic effects of complement since they express Decay Acceleration Factor (DAF) or CD55 on the cell surface. Binding of C4b or C3b to CD55 will result in inactivation in the C3 convertase activity of C4b and inactivation in the C5 convertase activity of C3b. Slides 40-47: STUDY GUIDE 1. Describe the characteristics of innate and adaptive immunity. 2. Describe the components of innate immunity. Describe the physical barriers (skin and mucous membrane) Describe the characteristics of neutrophils (primary [myeloperoxidase, lysozyme and a-defensins] and secondary granules [lactoferrin]) Describe the characteristics of monocytes, macrophages and dendritic cells. 3. Pattern recognition receptors (TLR-4 and TLR-2) 11 Dr. Vishy Venketaraman, Professor, COMP, Basic Medical Sciences 4. Intracellular mechanisms by which neutrophils and macrophages control intracellular infection. 5. Laboratory methods to determine phagocytosis and chronic granulomatous disease. Oxygen dependent killing mechanism (ROI and RNI) Oxygen independent killing mechanism (lysozyme, lactoferrin, and defensins) 6. Complement and innate immunity. 7. Complement and opsonization. 8. Complement and inflammation. 9. Complement regulation. 10. Complement related disorders. PLEASE REFER TO THE PRACTICE QUESTIONS IN THE POWERPOINT SLIDES Copyright Notice: Copies of documents used in this course were made available under Section 107 of the Copyright Act of 1976, the Fair Use Statute. This material has been made available solely for use in this class and the material may not be further distributed to any person outside the class, whether by copying or by transmission in electronic or paper form. 12