INNATE IMMUNITY I Abul K. Abbas: Basic Immunology page 23-46 (fig 3,4,5, 10, 13, are not required)

INNATE IMMUNITY I Abul K. Abbas: Basic Immunology page (fig 3,4,5, 10, 13, are not required)

Innate immunity Cells, components and their functions Recognition
Killing Other effector functions The innate immune system provides the first line of host defense against microbes. The mechanisms of innate immunity exist before exposure to microbes. The cellular components of the innate immune system include epithelial barriers, leukocytes (neutrophils, macrophages, NK cells, lymphocytes with invariant antigen receptors, and mast cells). Innate immunity serves three important functions: Innate immunity is the initial response to microbes that prevents, controls, or eliminates infection of the host by many microbes. Innate immune mechanisms recognize the products of damaged and dead host cells and serve to eliminate these cells and to initiate the process of tissue repair. Innate immunity to microbes stimulates adaptive immune responses and can influence the nature of the adaptive responses to make them optimally effective against different types of microbes.

Monocytes/ Macrophages Dendritic cells Granulocytes NK cells Mast cells Complement components Recognition Killing Other effector functions

WHITE BLOOD CELLS IN THE SMEAR OF HUMAN PERIPHERAL BLOOD
eosinophil granulocyte neutrophil granulocyte MONOCYTE neutrophil granulocyte LYMPHOCYTE LYMPHOCYTE basophil granulocyte

DISTRIBUTION OF BLOOD CELLS AND LYMPHOCYTE SUBTYPES
Percentage Cell number/L WHITE BLOOD CELLS leukocytes 4.8 – 10.8 x 109 neutrophil granulocytes 40 – 74 1.9 – 8 x 109 eosinophil 0.1 – 5 0.01 – 0.6 x 109 basophil 0.l – 1.5 0.01 – 0.2 x 109 lymphocytes 19 – 41 0.9 – 4.4 x 109 monocytes 3.4 – 9 0.16 – 0.9 x 109 RED BLOOD CELLS erithrocytes 4.2 – 6.1 x 1012 PLATELETS thrombocytes x 109

One of the cells which initiate the immune response
MONOCYTES origin: pluripotent cells of the bone marrow myeloid progenitors size: µm - nucleus: bean-shaped localization: circulation out of circulation: macrophage TISSUE - VENTRICLE MACROPHAGES phagocytic cells antigen presenting cells (APC) main types (based on tissue localization): microglia (brain) Kuppfer-cells (liver) histiocytes (connective tissue) osteoclasts (bone) alveolar macrophages (lung) Cells that have specialized phagocytic functions, primarily macrophages, are the first line of defense against microbes that breach epithelial barriers. - function: in cellular and humoral immun response One of the cells which initiate the immune response 6

DCs are professional antigen presenting cells (APC)
DENDRITIC CELLS origin: myeloid or lymphoid progenitors localization: the immature dendritic cell migrates from the circulation into the tissues and upon pathogen uptake it differentiates to a mature dendritic cell and migrates to the draining lymph node and transports the antigen from the periphery to the secondary lymphatic organs DCs are professional antigen presenting cells (APC) One of the cells which initiate the immune response types : a) myeloid DCs: - Langerhans cells (mucosa, skin) - intersticial DCs (liver, spleen, etc.) b) lymphoid DCs: - thymic DCs - plasmacytoid DCs (pDC) 7

NEUTROPHIL GRANULOCYTES BASOPHIL GRANULOCYTES EOSINOPHIL GRANULOCYTES
highest number in blood (68% of circulating leukocytes, 99% of circulating granulocytes) phagocyting cells - They are not present in healthy tissues tissue damage, migration, elimination of pathogens (enzymes, reactive oxygen intermediers) main participants in inflammatory processes BASOPHIL GRANULOCYTES 1% of circulating leukocytes large granules in the cytoplasm nucleus with 2 lobes mast cells, histamin, allergic reactions high affinity IgE receptors against parasites EOSINOPHIL GRANULOCYTES against parasites 2-3% of leukocytes allergic reactions 8

MAST CELLS origin: pluripotent cells of the bone marrow
myeloid progenitors localization: - absent in circulation - differentiate in tissues - especially around small vessels function: - upon activation they regulate the permeability of the vessels with their secreted molecules - native and adaptive immunity - allergic reactions (cell surface FceRI receptors) - main types: a) mucosal b) connective tissue Mast cells are present in the skin and mucosal epithelium and rapidly secrete proinflammatory cytokines and lipid mediators in response to infections and other stimuli. 9

NK CELLS (natural killer)
- origin: - pluripotent cells of the bone marrow lymphoid progenitors - bigger than lymphocytes several granules in their cytoplasm have no antigen binding receptors („null cells”) participants of native immunity Natural killer (NK) cells are lymphocytes distinct from T and B cells that play important roles in innate immune responses mainly against intracellular viruses and bacteria. The term natural killer derives from the fact that these cells are capable of performing their killing function without a need for clonal expansion and differentiation, which is required for effector responses of the immune system's other killer cells, the cytotoxic T lymphocytes (CTLs). NK cells distinguish infected and stressed cells from healthy cells, and NK cell activation is regulated by a balance between signals that are generated from activating receptors and inhibitory receptors. Most NK cells express inhibitory receptors that recognize class I major histocompatibility complex (MHC) molecules, which are cell surface proteins normally expressed on almost all healthy cells in the body. The effector functions of NK cells are to kill infected cells and to activate macrophages to destroy phagocytosed microbes

Professional phagocytic cells Professional antigen presenting cells
macrophages neutrophyl granulocytes dendritic cells the phagocytosed cells or molecules may modify the functions of the cell phagocytosis followed by enzymatic degradation Professional antigen presenting cells macrophages B lymphocytes dendritic cells they express MHCII molecules the protein degradation products (peptides) can be presented to T lymphocytes by MHC molecules APCs function to display antigens for recognition by T lymphocytes and to promote the activation of lymphocytes. The cells that perform the majority of effector functions of innate and adaptive immunity are phagocytes (including neutrophils and macrophages), 11

The localization of blood cells
NK cells

Macrophage, dendritic cell – act as TISSUE SENSORS
Neutrophil granulocytes, complement, NK cells – migrate from the blood to the SITE OF INFECTION 13

THE TWO ARMS OF THE IMMUNE SYSTEM
Monocytes, Macrophages, Dendritic cells, Granulocytes, NK cells and Complement components Monocytes, Macrophages, Dendritic cells, Granulocytes, NK cells and Complement components B and T cells Defense against microbes is mediated by the early reactions of innate immunity and the later responses of adaptive immunity. Innate immunity (also called natural or native immunity) provides the early line of defense against microbes. It consists of cellular and biochemical defense mechanisms that are in place even before infection and are poised to respond rapidly to infections. These mechanisms react to microbes and to the products of injured cells, and they respond in essentially the same way to repeated infections. The principal components of innate immunity are (1) physical and chemical barriers, such as epithelia and antimicrobial chemicals produced at epithelial surfaces; (2) phagocytic cells (neutrophils, macrophages), dendritic cells, and natural killer (NK) cells; (3) blood proteins, including members of the complement system and other mediators of inflammation; and (4) proteins called cytokines that regulate and coordinate many of the activities of the cells of innate immunity. The mechanisms of innate immunity are specific for structures that are common to groups of related microbes and may not distinguish fine differences between microbes. In contrast to innate immunity, there are other immune responses that are stimulated by exposure to infectious agents and increase in magnitude and defensive capabilities with each successive exposure to a particular microbe. Because this form of immunity develops as a response to infection and adapts to the infection, it is called adaptive immunity. The defining characteristics of adaptive immunity are exquisite specificity for distinct molecules and an ability to "remember" and respond more vigorously to repeated exposures to the same microbe. The adaptive immune system is able to recognize and react to a large number of microbial and nonmicrobial substances. In addition, it has an extraordinary capacity to distinguish between different, even closely related, microbes and molecules, and for this reason it is also called specific immunity. It is also sometimes called acquired immunity, to emphasize that potent protective responses are "acquired" by experience. The main components of adaptive immunity are cells called lymphocytes and their secreted products, such as antibodies. Foreign substances that induce specific immune responses or are recognized by lymphocytes or antibodies are called antigens. Innate and adaptive immune responses are components of an integrated system of host defense in which numerous cells and molecules function cooperatively. The mechanisms of innate immunity provide effective initial defense against infections. However, many pathogenic microbes have evolved to resist innate immunity, and their elimination requires the more powerful mechanisms of adaptive immunity. There are many connections between the innate and adaptive immune systems. The innate immune response to microbes stimulates adaptive immune responses and influences the nature of the adaptive responses. Conversely, adaptive immune responses often work by enhancing the protective mechanisms of innate immunity, making them capable of effectively combating pathogenic microbes. 14

Cells of the innate immune system: Macrophages:
Macrophages are constitutively present in tissues and recognize microbes that enter these tissues and respond rapidly to these microbes. They initiate the immune response. These cells are phagocytes (eliminate the pathogens) Activate the innate immune response (by secreted proteins, called cytokines) Activate the adaptive immune system. Macrophages serve as APCs that display antigens and activate T lymphocytes Dendritic cells are constitutively present in tissues and recognize rapidly microbes that enter these tissues. Initiate the immune response. They have phagocytic capabilities, migrate to lymph nodes, and display microbial antigens to T lymphocytes,professional antigen presenting cells (APC) Neutrophil granulocytes are phagocytes, their main function is to eliminate the pathogens They appear only in the circulation under normal condition. Main actors in inflammatory processes. Macrophages: A major function of macrophages in host defense is to ingest and kill microbes. The mechanisms of killing, include phagocytosis (proteolytic digestion) and the enzymatic generation of reactive oxygen and nitrogen species that are toxic to microbes. Activated macrophages secrete proteins, called cytokines, that bind to signaling receptors on other cells and thereby instruct those cells to respond in ways that contribute to host defense. Macrophages serve as APCs that display antigens to and activate T lymphocytes. This function is important in the effector phase of T cell-mediated immune responses Another important function of macrophages is to promote repair of damaged tissues by stimulating new blood vessel growth (angiogenesis) and synthesis of collagen-rich extracellular matrix (fibrosis). This function is mediated by certain cytokines secreted by the macrophages that act on various tissue cells. Macrophages also recognize and engulf apoptotic cells before the dead cells can release their contents and induce inflammatory responses. Throughout the body and throughout the life of an individual, unwanted cells die by apoptosis, as part of many physiologic processes, such as development, growth, and renewal of healthy tissues, and the dead cells must be cleaned up by macrophages. Macrophages are activated to perform their functions: by recognizing many different kinds of microbial molecules, as well as abnormal appearence of host molecules so called danger signals . These various activating molecules bind to specific signaling receptors located on the surface of or inside the macrophage. These receptors are PRR Pattern recognition receptors. Macrophages are also activated when receptors on their plasma membrane bind opsonins on the surface of microbes. Opsonins are substances that coat particles for phagocytosis. Examples of these opsonin receptors are Fc receptors In adaptive immunity, macrophages are activated by secreted cytokines and membrane proteins made by T lymphocytes Neutrophils Neutrophils are the most abundant population of circulating white blood cells, are always present in the blood and can be quickly delivered anywhere in the body. Neutrophils may migrate to sites of infection within a few hours after the entry of microbes. (mainly due to citokines produced by macrophages) Primary function is to identify and destroy microbes. These cells can eliminate the pathogens by phagocytosis or the release of their granules, which contain Dendritic cells Dendritic cells are the most important APCs for activating naive T cells, and they play major roles in innate responses to infections and in linking innate and adaptive immune responses. Similar to macrophages, dendritic cells express receptors that recognize molecules typically made by microbes and not mammalian cells. In response to activation by microbes dendritic cells become mobile, migrate to lymph nodes, and display microbial antigens to T lymphocytes. Thus, these cells function in both innate and adaptive immune responses and are a link between these two components of host defense. 15

Pattern recognition (PAMP) Danger signal (DAMP) NK cell Opsonization Killing Other effector functions

Innate immunity as a first line of defense
Innate immune cells recognize frequently found structures of pathogens, these are not found in human cells! Examples: duple stranded RNA bacterial cell wall components bacterial flagellin…. The specificities of innate immune recognition have evolved to combat microbes and are different from the specificities of the adaptive immune system in several respects. The innate immune system recognizes molecular structures that are characteristic of microbial pathogens but not mammalian cells. The microbial substances that stimulate innate immunity are called pathogen-associated molecular patterns (PAMPs). Different classes of microbes (e.g., viruses, gram-negative bacteria, gram-positive bacteria, fungi) express different PAMPs. These structures include nucleic acids that are unique to microbes, such as double-stranded RNA found in replicating viruses and unmethylated CpG DNA sequences found in bacteria; features of proteins that are found in microbes, such as initiation by N-formylmethionine, which is typical of bacterial proteins; and complex lipids and carbohydrates that are synthesized by microbes but not by mammalian cells, such as lipopolysaccharide (LPS) in gram-negative bacteria, lipoteichoic acid in gram-positive bacteria, and mannose-rich oligosaccharides found in microbial but not in mammalian glycoproteins. In actuality, there are only a limited number of fundamental differences between microbial molecules and the molecules that higher organisms produce. Thus, the innate immune system has evolved to recognize only a limited number of molecules, most of which are unique to microbes, whereas the adaptive immune system is capable of recognizing a much wider array of foreign substances whether or not they are products of microbes. Recognition is inevitable monocytes, macrophages, dendritic cells, granulocytes (NK cells, T cells, B cells, somatic cells)

PAMP Pathogen-associated molecular patterns
The innate immune system uses cell-associated pattern recognition receptors, present on plasma and endosomal membranes and in the cytoplasm, to recognize structures called pathogen-associated molecular patterns (PAMPs), which are shared by microbes, are not present on mammalian cells, and are often essential for survival of the microbes, thus limiting the capacity of microbes to evade detection by mutating or losing expression of these molecules. In addition, these receptors recognize molecules made by the host but whose expression or location indicates cellular damage; these are called damage-associated molecular patterns (DAMPs). PAMP Pathogen-associated molecular patterns PRR Pattern recognition receptors

PAMPs- Pathogen associated molecular patters
Structures on pathogens recognized by the innate cells Only some example most be known! Abbreviations: lipopolysaccharide (LPS), lipoteichoic acid (LTA), lipoproteins (LP), glycophosphatidylinositol (GPI). PRRs (Pattern recognition receptors) PRRs on innate immune cells generally recognize structures shared by many different pathogens, these are the pathogen associated molecular patterns (PAMPs) and alterations on human cells that are commonly induced by the presence of pathogens.

Intracellular and extracellular receptors
Recognition is mediated by several PRR receptor families with overlapping functions PRR types TOLL RIG like receptors NOD Scavenger receptors C-type lectin receptors Mannose recognizing receptors PRRs (Pattern recognition receptors) PRRs on innate immune cells generally recognize structures shared by many different pathogens, these are the pathogen associated molecular patterns (PAMPs) and alterations on human cells that are commonly induced by the presence of pathogens. TLRs, present on the cell surface and in endosomes, are the most important family of pattern recognition receptors, recognizing a wide variety of ligands, including bacterial cell wall components and microbial nucleic acids. NOD-like receptors (NLRs) are a family of more than 20 different cytosolic proteins, some of which sense cytoplasmic PAMPs and DAMPs and recruit other proteins to form signaling complexes that promote inflammation. RIG-like receptors (RLRs) are cytosolic sensors of viral RNA that respond to viral nucleic acids by inducing the production of the antiviral type I interferons. Receptors that recognize carbohydrates on the surface of microbes facilitate the phagocytosis of the microbes and stimulate subsequent adaptive immune responses. These receptors belong to the C-type lectin family, so called because they bind carbohydrates (hence, lectins) in a Ca++-dependent manner (hence, C-type). Scavenger receptors comprise a structurally and functionally diverse collection of cell surface proteins that were originally grouped on the basis of the common characteristic of mediating the uptake of oxidized lipoproteins into cells.There is a wide range of molecular structures that bind to each scavenger receptor, including LPS, lipoteichoic acid, nucleic acids, β-glucan, and proteins. Mannose receptor. One of the most studied membrane C-type lectins is the mannose receptor (CD206), which is involved in phagocytosis of microbes. This receptor recognizes certain terminal sugars on microbial surface carbohydrates, including d-mannose, l-fucose, and N-acetyl-d-glucosamine. These terminal sugars are often present on the surface of microorganisms, whereas eukaryotic cell carbohydrates are most often terminated by galactose and sialic acid. Thus, the terminal sugars on microbes can be considered PAMPs. Intracellular and extracellular receptors

Additional PRRs: Lectins: receptors ( or plasma proteins) that bind to carbohydrates. On macrophages one finds mannose rec. And glucan receptors. Scavenger receptor binds various substrates generally negatively charges (sulfated polysaccharides, nucleic acids, lipoteic acid bound in the cell wall of Gram positive bacteria) PRRs (Pattern recognition receptors) PRRs on innate immune cells generally recognize structures shared by many different pathogens, these are the pathogen associated molecular patterns (PAMPs) and alterations on human cells that are commonly induced by the presence of pathogens. N-Formyl met-leu-phe receptors, including FPR and FPRL1 expressed by neutrophils and macrophages, respectively, recognize bacterial peptides containing N-formylmethionyl residues and stimulate directed movement of the cells. Because all bacterial proteins and few mammalian proteins (only those synthesized within mitochondria) are initiated by N-formylmethionine, FPR and FPRL1 allow phagocytes to detect and respond preferentially to bacterial proteins.

Protein glycosilation is highly differnt in species
Eukaryotes Prokaryotes Mannose Glucosamine Galactose Mannose Siallic acid Bacterium Mannose Mannose receptors Macrophage / Dendritic cell

Danger signal

Danger signal! The innate immune system also recognizes molecules that are released from damaged or necrotic cells. Such molecules are called damage-associated molecular patterns (DAMPs). The innate immune system also recognizes endogenous molecules that are produced by or released from damaged and dying cells. These substances are called damage-associated molecular patterns (DAMPs). DAMPs may be produced as a result of cell damage caused by infections, but they may also indicate sterile injury to cells caused by any of myriad reasons, such as chemical toxins, burns, trauma, or decreased blood supply. DAMPs are generally not released from cells dying by apoptosis. In some cases, healthy cells of the immune system are stimulated to produce and release DAMPs, which enhances an innate immune response to infections.

Soluble mediators

Killing of the cells infected with intracellular pathogens
KAR KIR KIR – Killer Inhibitory Receptor association to MHC I KAR – Killer Activatory Receptor NK Target MHC+ NK KAR KIR Target MHC- The activity of NK cells is enhanced by activatory receptors Inhibitory receptors block NK cell activity. Self cells are protected by inhibitory receptors. Infection or tumors may increase the amount of activation and/or decrease the efficacy of inhibition Inhibition of lysis lysis NK cells are lymphocytes that defend against intracellular microbes by killing infected cells and providing a source of the macrophage-activating cytokine IFN-γ. NK cell recognition of infected cells is regulated by a combination of activating and inhibitory receptors. Inhibitory receptors recognize class I MHC molecules, because of which NK cells do not kill normal host cells but do kill cells in which class I MHC expression is reduced, such as virus-infected cells. NK cells distinguish infected and stressed cells from healthy cells, and NK cell activation is regulated by a balance between signals that are generated from activating receptors and inhibitory receptors. There are several families of these receptors. These receptors recognize molecules on the surface of other cells and generate activating or inhibitory signals that promote or inhibit NK responses. In general, the activating receptors recognize ligands on infected and injured cells, and the inhibitory receptors recognize healthy normal cells. When an NK cell interacts with another cell, the outcome is determined by the integration of signals generated from the array of inhibitory and activating receptors that are expressed by the NK cell and that interact with ligands on the other cell. Most NK cells express inhibitory receptors that recognize class I major histocompatibility complex (MHC) molecules, which are cell surface proteins normally expressed on almost all healthy cells in the body . 28

OPSONIZATION Opsonization facilitates and accelerates the recognition of the pathogen by phagocytes. Opsonins form a bridge and connect the pathogen with the phagocyte. Main opsonins: Antibodies Complement fragments Acute-phase proteins The process of coating particles for subsequent phagocytosis is called opsonization, and the molecules that coat microbes and enhance their phagocytosis are called opsonins. For example when several antibody molecules bind to a microbe, an array of Fc regions is formed projecting away from the microbial surface. Their Fc regions bind to a high-affinity receptor for the Fc regions of γ heavy chains, called FcR, which is expressed mainly on neutrophils and macrophages. The phagocyte extends its plasma membrane around the attached microbe and ingests the microbe into a vesicle called a phagosome, which fuses with lysosomes. The binding of antibody Fc tails to FcR also activates the phagocytes, because the FcR contains a signaling chain that triggers numerous biochemical pathways in the phagocytes. The activated neutrophil or macrophage produces, in its lysosomes, large amounts of reactive oxygen species, nitric oxide, and proteolytic enzymes, all of which combine to destroy the ingested microbe. Antibody-mediated phagocytosis is the major mechanism of defense against encapsulated bacteria, such as pneumococci. The polysaccharide-rich capsules of these bacteria protect the organisms from phagocytosis in the absence of antibody, but opsonization by antibody promotes phagocytosis and destruction of the bacteria. The spleen contains large numbers of phagocytes and is an important site of phagocytic clearance of opsonized bacteria. This is why patients who have undergone splenectomy for traumatic rupture of the organ are susceptible to disseminated infections by encapsulated bacteria.

Pathogen recognition by innate immune system
Directly via PRR Indirectly via opsonization

Killing Phagocytosis Soluble mediators Complement system NK cells Other effector functions

Professional phagocytic cells MACROPHAGES DENDRITIC CELLS NEUTROPHILS
And mast cells

Intracellular killing
PHAGOCYTOSIS NEUTROPHIL GRANULOCYTE – killing MACROPHAGE – killing, antigen presentation DENDRITIC CELLS – killing, antigen presentation Phagocyte PRR Degradation ACTIVATION Bacterium Uptake Intracellular killing Antigen presentation T cell ACQUIRED IMMUNITY Neutrophils and macrophages that are recruited into sites of infections ingest microbes into vesicles by the process of phagocytosis and destroy these microbes . Neutrophils and macrophages express receptors that specifically recognize microbes, and binding of microbes to these receptors is the first step in phagocytosis. Activated neutrophils and macrophages kill phagocytosed microbes by the action of microbicidal molecules in phagolysosomes . When neutrophils and macrophages are strongly activated, they can injure normal host tissues by release of lysosomal enzymes, ROS, and nitric oxide. hours The amount of internalized particles is limited

Extracellular pathogen phagocytosis
and killing Phagocytosis and intracellular killing of microbes. Macrophages and neutrophils express many surface receptors that may bind microbes for subsequent phagocytosis; select examples of such receptors are shown. Microbes are ingested into phagosomes, which fuse with lysosomes, and the microbes are killed by enzymes and several toxic substances produced in the phagolysosomes. The same substances may be released from the phagocytes and may kill extracellular microbes (not shown). iNOS, Inducible nitric oxide synthase; NO, nitric oxide; ROS, reactive oxygen species. Innate Immunity : The Early Defense Against Infections Abbas, Abul K., MBBS, Basic Immunology: Functions and Disorders of the Immune System, Chapter 2, 23-48 Copyright © Copyright © 2014, 2011, 2009, 2006, 2004, 2001 by Saunders, an imprint of Elsevier Inc.

Soluble mediators are released mainly from macrophages and granulocytes are responsible for killing of extracellular pathogens ROS - reactive oxigen species NO - nitric oxide destructive enzymes antimicrobial substances Reactive oxygen species. Activated macrophages and neutrophils convert molecular oxygen into reactive oxygen species (ROS), which are highly reactive oxidizing agents that destroy microbes (and other cells). The primary free radical-generating system is the phagocyte oxidase system. Phagocyte oxidase is a multisubunit enzyme that is assembled in activated phagocytes mainly in the phagolysosomal membrane. Phagocyte oxidase is induced and activated by many stimuli, including IFN-γ and signals from TLRs. The function of this enzyme is to reduce molecular oxygen into ROS such as superoxide radicals, with the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) acting as a cofactor. Superoxide is enzymatically dismutated into hydrogen peroxide, which is used by the enzyme myeloperoxidase to convert normally unreactive halide ions into reactive hypohalous acids that are toxic for bacteria. The process by which ROS are produced is called the respiratory burst because it occurs during oxygen consumption (cellular respiration). Although the generation of toxic ROS is commonly viewed as the major function of phagocyte oxidase, another function of the enzyme is to produce conditions within phagocytic vacuoles that are necessary for the activity of the proteolytic enzymes discussed earlier. The oxidase acts as an electron pump, generating an electrochemical gradient across the vacuole membrane, which is compensated for by movement of ions into the vacuole. The result is an increase in pH and osmolarity inside the vacuole, which is necessary for elastase and cathepsin G activity. A disease called chronic granulomatous disease is caused by an inherited deficiency of one of the components of phagocyte oxidase; this deficiency compromises the capacity of neutrophils to kill certain species of gram-positive bacteria (see Chapter 20). Nitric oxide. In addition to ROS, macrophages produce reactive nitrogen species, mainly nitric oxide, by the action of an enzyme called inducible nitric oxide synthase (iNOS). iNOS is a cytosolic enzyme that is absent in resting macrophages but can be induced in response to microbial products that activate TLRs, especially in combination with IFN-γ. iNOS catalyzes the conversion of arginine to citrulline, and freely diffusible nitric oxide gas is released. Within phagolysosomes, nitric oxide may combine with hydrogen peroxide or superoxide, generated by phagocyte oxidase, to produce highly reactive peroxynitrite radicals that can kill microbes. The cooperative and redundant function of ROS and nitric oxide is demonstrated by the finding that knockout mice lacking both iNOS and phagocyte oxidase are more susceptible to bacterial infections than single phagocyte oxidase or iNOS knockout animals are. Proteolytic enzymes. Activated neutrophils and macrophages produce several proteolytic enzymes in the phagolysosomes that function to destroy microbes. One of the important enzymes in neutrophils is elastase, a broad-spectrum serine protease known to be required for killing many types of bacteria. Another important enzyme is cathepsin G. Mouse gene knockout studies have confirmed the essential requirement for these enzymes in phagocyte killing of bacteria.

Extracellular pathogen by phagocytosis
and soluble mediators

Killing Phagocytosis Soluble mediators Complement system activation function NK cells Other effector functions

Initiation of complement response
Alternative pathway - constitutively active - inhibited by self structures Classical patway - activated by antibodies/immuncomplexes Lectin pathway - mannose binding lektin (MBL) or – acute-phase protein activates it The complement system consists of several plasma proteins that work together to opsonize microbes, to promote the recruitment of phagocytes to the site of infection, and in some cases to directly kill the microbes (Fig. 4-9). Complement activation involves proteolytic cascades, in which an inactive precursor enzyme, called a zymogen, is altered to become an active protease that cleaves and thereby induces the proteolytic activity of the next complement protein in the cascade. As the cascade proceeds, the enzymatic activities result in tremendous amplification of the amount of proteolytic products that are generated. These products perform the effector functions of the complement system. Other proteolytic cascades include the blood coagulation pathways and the kinin-kallikrein system that regulates vascular permeability. The complement system consists of serum and cell surface proteins that interact with one another and with other molecules of the immune system in a highly regulated manner to generate products that function to eliminate microbes. Complement proteins are plasma proteins that are normally inactive; they are activated only under particular conditions to generate products that mediate various effector functions of complement. Several features of complement activation are essential for its normal function. The complement system is activated by microbes and by antibodies that are attached to microbes and other antigens. The mechanisms of initial activation are described later. Activation of complement involves the sequential proteolysis of proteins to generate enzyme complexes with proteolytic activity. Proteins that acquire proteolytic enzymatic activity by the action of other proteases are called zymogens. The process of sequential zymogen activation, a defining feature of a proteolytic enzyme cascade, is also characteristic of the coagulation and kinin systems. Proteolytic cascades allow tremendous amplification because each enzyme molecule activated at one step can generate multiple activated enzyme molecules at the next step. The products of complement activation become covalently attached to microbial cell surfaces or to antibodies bound to microbes and to other antigens. In the fluid phase, complement proteins are inactive or only transiently active (for seconds), and they become stably activated after they are attached to microbes or to antibodies. Many of the biologically active cleavage products of complement proteins also bind covalently to microbes, antibodies, and tissues in which the complement is activated. This characteristic ensures that the full activation and therefore the biologic functions of the complement system are limited to microbial cell surfaces or to sites of antibodies bound to antigens and do not occur in the blood. Complement activation is inhibited by regulatory proteins that are present on normal host cells and absent from microbes. The regulatory proteins are an adaptation of normal cells that minimize complement-mediated damage to host cells. Microbes lack these regulatory proteins, which allows complement activation to occur on microbial surfaces. The classical pathway, so called because it was discovered first, uses a plasma protein called C1q to detect antibodies bound to the surface of a microbe or other structure (Fig. 4-10). Once C1q binds to the Fc portion of the antibodies, two associated serine proteases, called C1r and C1s, become active and initiate a proteolytic cascade involving other complement proteins. The classical pathway is one of the major effector mechanisms of the humoral arm of adaptive immune responses (see Chapter 12). Because IgM natural antibodies are very efficient at binding C1q, the classical pathway also participates in innate immunity. In addition, other innate immune system soluble proteins called pentraxins, discussed later, can also bind C1q and initiate the classical pathway. The alternative pathway, which was discovered later but is phylogenetically older than the classical pathway, is triggered when a complement protein called C3 directly recognizes certain microbial surface structures, such as bacterial LPS. C3 is also constitutively activated in solution at a low level and binds to cell surfaces, but it is then inhibited by regulatory molecules present on mammalian cells. Because microbes lack these regulatory proteins, the spontaneous activation can be amplified on microbial surfaces. Thus, this pathway can distinguish normal self from foreign microbes on the basis of the presence or absence of the regulatory proteins. The lectin pathway is triggered by a plasma protein called mannose-binding lectin (MBL), which recognizes terminal mannose residues on microbial glycoproteins and glycolipids, similar to the mannose receptor on phagocyte membranes described earlier (see Fig. 4-10). MBL is a member of the collectin family (discussed later) with a hexameric structure similar to the C1q component of the complement system. After MBL binds to microbes, two zymogens called MASP1 (mannan-binding lectin-associated serine protease) and MASP2, with similar functions to C1r and C1s, associate with MBL and initiate downstream proteolytic steps identical to the classical pathway. 40

THE FUNCTION of COMPLEMENT SYSTEM
The complement system is a set of plasma proteins that act in a cascade to attack and kill extracellular pathogens to recruit inflammatory cells to opsonizate pathogens to remove immunecomplexes

ACTIVATION OF THE COMPLEMENT SYSTEM
COMPLEMENT ACTIVATION RECRUITMENT OF INFLAMMATORY CELLS OPSONIZATION OF PATHOGENS DIRECT KILLING OF PATHOGENS Clearence of Immune complexes The principal effector functions of the complement system in innate immunity and specific humoral immunity are to promote phagocytosis of microbes on which complement is activated, to stimulate inflammation, and to induce the lysis of these microbes. Phagocytosis, inflammation, and stimulation of humoral immunity are all mediated by the binding of proteolytic fragments of complement proteins to various cell surface receptors, whereas cell lysis is mediated by the MAC. FACILITATING PHAGOCYTOSIS Immunecomplex removal

Complement-dependent
COMPLEMENT ACTIVATION Bacterium COMPLEMENT Lectin pathway Alternative pathway Lysis of bacteria Complement-proteins Inflammation Chemotaxis Complement-dependent phagocytosis The complement system includes several plasma proteins that become activated in sequence by proteolytic cleavage to generate fragments of the C3 and C5 proteins, which promote inflammation, or opsonize and promote phagocytosis of microbes. Complement activation also generates membrane pores that kill some types of bacteria. The complement system is activated on microbial surfaces and not on normal host cells because microbes lack regulatory proteins that inhibit complement. In innate immune responses, complement is activated mainly spontaneously on microbial cell surfaces and by mannose-binding lectin to initiate the alternative and lectin pathways, respectively. Antigen + Antibody ACQUIRED IMMUNITY Few minutes – 1 hour Enzymes get fragmented, complement activity can be exhausted

MEMBRANE ATTACK COMPLEX (MAC)
live and dead bacteria MAC in the cell membrane Pore formation  osmotic lysis of pathogens

Killing of the cells infected with intracellular pathogens
KAR KIR KIR – Killer Inhibitory Receptor association to MHC I KAR – Killer Activatory Receptor NK Target MHC+ NK KAR KIR Target MHC- The activity of NK cells is enhanced by activatory receptors Inhibitory receptors block NK cell activity. Self cells are protected by inhibitory receptors. Infection or tumors may increase the amount of activation and/or decrease the efficacy of inhibition Inhibition of lysis lysis NK cells distinguish infected and stressed cells from healthy cells, and NK cell activation is regulated by a balance between signals that are generated from activating receptors and inhibitory receptors. There are several families of these receptors. These receptors recognize molecules on the surface of other cells and generate activating or inhibitory signals that promote or inhibit NK responses. In general, the activating receptors recognize ligands on infected and injured cells, and the inhibitory receptors recognize healthy normal cells. When an NK cell interacts with another cell, the outcome is determined by the integration of signals generated from the array of inhibitory and activating receptors that are expressed by the NK cell and that interact with ligands on the other cell. Most NK cells express inhibitory receptors that recognize class I major histocompatibility complex (MHC) molecules, which are cell surface proteins normally expressed on almost all healthy cells in the body . 46

Adaptive components are also able to activate NK cells
ADCC-Antibody Dependent Cell Cytotoxicity Activating NK cells through FcR on NK cells recognizing pathogen-bound Antibodies During an infection, the adaptive immune system produces IgG1 and IgG3 antibodies that specifically bind to the infecting microbes and their antigens on infected cells, and FcR on NK cells can bind to the Fc parts of these antibodies. As a result, FcR generates activating signals, through the associated signaling partners, and the NK cells may kill the infected cells that have been coated with antibody molecules. This process is called antibody-dependent cell-mediated cytotoxicity; it is an effector function of adaptive immunity.

RECOGNITION OF ALTERED HOST CELLS
ACTIVATION OF NATURAL KILLER CELLS NK-CELLS Virus-infected cell PRR RECOGNITION ACTIVATION Lysis of infected cell RECOGNITION OF ALTERED HOST CELLS Kinetics of the activity of the complement system and NK cells in virus infection IFN IL-12 Complement system NK-cells days Relatív szint/aktivitás

Killing Other effector functions Acute inflammation Localized effects Systemic effects anti-viral response (interferons) opsonization antigen prezentation

ACUTE INFLAMMATION A rapid response to an injurious agent that serves to deliver leukocytes and plasma proteins to the site of injury. The major way by which the innate immune system deals with infections and tissue injury is to stimulate acute inflammation, which is the accumulation of leukocytes, plasma proteins, and fluid derived from the blood at an extravascular tissue site of infection or injury. Typically, the most abundant leukocyte that is recruited from the blood into acute inflammatory sites is the neutrophil, but blood monocytes, which become macrophages in the tissue, become increasingly prominent over time and may be the dominant population in some reactions. Among the important plasma proteins that enter inflammatory sites are complement proteins, antibodies, and acute-phase reactants. The delivery of these blood-derived components to the inflammatory site is dependent on reversible changes in blood vessels in the infected or damaged tissue. These changes include increased blood flow into the tissue due to arteriolar dilation, increased adhesiveness of circulating leukocytes to the endothelial lining of venules, and increased permeability of the capillaries and venules to plasma proteins and fluid. All these changes are induced by cytokines and small-molecule mediators initially derived from resident cells in the tissue, such as mast cells, macrophages, and endothelial cells, in response to PAMP or DAMP stimulation. As the inflammatory process develops, the mediators may be derived from newly arrived and activated leukocytes and complement proteins.

TRIGGERS OF ACUTE INFLAMMATION:
Infections Trauma Physical and Chemical agents (thermal injury, irradiation, chemicals) Tissue necrosis Foreign bodies (splinters, dirt, sutures) Hypersensitivity or autoimmune reactions MAJOR COMPONENTS OF INFLAMMATION: Vascular response: Increased vascular diameter  Increased flood flow. Endothelial cell activation increased permeability that permits plasma proteins and leukocytes to leave the circulation and enter the tissue  edema increased expression of cell adhesion molecules e.g. E-selectin, ICAM Cellular response: Migration of leukocytes (diapedesis/extravasation), accumulation, effector functions

Recognition of PAMP or DAMP induce inflammation
Cytokines of innate immunity. A, Dendritic cells and macrophages responding to microbes produce cytokines that stimulate inflammation (leukocyte recruitment) and activate natural killer (NK) cells to produce the macrophage-activating cytokine interferon-γ (IFN-γ). Several cytokines produced mainly by activated macrophages mediate inflammation. TNF and IL-1 activate endothelial cells, stimulate chemokine production, and increase neutrophil production by the bone marrow. IL-1 and TNF both induce IL-6 production, and all three cytokines mediate systemic effects, including fever and acute-phase protein synthesis by the liver. IL-12 stimulates production of the macrophage-activating cytokine IFN-γ by NK cells and T cells. These cytokines function in innate immune responses to different classes of microbes, and some (IL-1, IL-6, IL-12) modify adaptive immune responses that follow the innate immune response.

INFLAMMATION – ACUTE- PHASE RESPONSE
Reminder neutrophil TNF- DANGER SIGNAL ACTIVATION PRR Bacterium LPS cytokines TNF- IL-1 IL-6 Few hours ACUTE PHASE RESPONSE NK-cell IL-12 macrophage IFN hrs Plasma level LPS (endotoxin) (Gram(-) bacteria) TNF- IL-1 IL-6 Kinetics of the release of pro-inflammatory cytokines in bacterial infection

TNF, IL-1, and IL-6 produced during the innate immune response to infection or tissue damage have systemic effects that contribute to host defense and are responsible for many of the clinical signs of infection and inflammatory disease . TNF, IL-1, and IL-6 all act on the hypothalamus to induce an increase in body temperature (fever), and these cytokines are therefore called endogenous pyrogens . IL-1, TNF, and IL-6 induce hepatocytes to express acute-phase reactants, including CRP, SAP, and fibrinogen, which are secreted into the blood. In severe infections, TNF may be produced in large amounts and causes systemic clinical and pathologic abnormalities. If the stimulus for cytokine production is sufficiently strong, the quantity of TNF may be so large that it enters the blood stream and acts at distant sites as an endocrine hormone. Acute inflammation may cause tissue injury because the effector mechanisms that phagocytes use to kill microbes are also highly toxic to host tissues.

Killing Other effector functions acute inflammation anti-viral response (interferons) opsonization antigen prezentation

VIRUS INDUCED TYPE I INTERFERON PRODUCTION
Type I IFN receptor IFN response Virus IFN- IRF-3 NFB AP-1 IRF-3 IFN- paracrine IFN- IRF-7 autocrine TLR3 binds dsRNA, IRF3 gets phosphorylated, dimerizes and translocate into the nucleus. IFN-beta expression is induced. Autokrine and paracrine effects. The major way by which the innate immune system deals with viral infections is to induce the expression of type I interferons, whose most important action is to inhibit viral replication. Type I interferons are a large family of structurally related cytokines that mediate the early innate immune response to viral infections. Infected cell IFN response IFN- subtypes IRF: interferon regulatory factor 56

Induction of interferons
virus-infected cell will release interferons causing nearby cells to heighten their anti-viral defenses. are produced by different (~all) cell types usually in response to the entry of a virus, which has the property of inhibiting virus replication

EFFECTS OF TYPE I INTERFERONS NATURAL INTERFERON PRODUCING CELLS – IPC
vírus Type I interferons, signaling through the type I interferon receptor, activate transcription of several genes that confer on the cells a resistance to viral infection, called an antiviral state. Type I interferons cause sequestration of lymphocytes in lymph nodes, thus maximizing the opportunity for encounter with microbial antigens. Type I interferons increase the cytotoxicity of NK cells and CD8+ CTLs and promote the differentiation of naive T cells to the TH1 subset of helper T cells. Type I interferons upregulate expression of class I MHC molecules and thereby increase the probability that virally infected cells will be recognized and killed by CD8+ CTLs. Plasmacytoid dendritic cells produce 1000x more type I interferon than other cells NATURAL INTERFERON PRODUCING CELLS – IPC After viral infection they are accumulated at the T cell zone of the lymph nodes

RECOGNITION OF ALTERED HOST CELLS
ACTIVATION OF NATURAL KILLER CELLS NK-CELLS Virus-infected cell PRR RECOGNITION ACTIVATION Lysis of infected cell RECOGNITION OF ALTERED HOST CELLS

Killing Other effector functions acute inflammation anti-viral response (interferons) opsonization antigen presentation

OPSONIZATION Opsonization facilitates and accelerates the recognition of the pathogen by phagocytes. Opsonins form a bridge and connect the pathogen with the phagocyte. Main opsonins: Antibodies Complement fragments Acute-phase proteins The process of coating particles for subsequent phagocytosis is called opsonization, and the molecules that coat microbes and enhance their phagocytosis are called opsonins. When several antibody molecules bind to a microbe, an array of Fc regions is formed projecting away from the microbial surface. If the antibodies belong to certain isotypes (IgG1 and IgG3 in humans), their Fc regions bind to a high-affinity receptor for the Fc regions of γ heavy chains, called FcγR, which is expressed on neutrophils and macrophages. The phagocyte extends its plasma membrane around the attached microbe and ingests the microbe into a vesicle called a phagosome, which fuses with lysosomes. The binding of antibody Fc tails to FcγR also activates the phagocytes, because the FcγR contains a signaling chain that triggers numerous biochemical pathways in the phagocytes. The activated neutrophil or macrophage produces, in its lysosomes, large amounts of reactive oxygen species, nitric oxide, and proteolytic enzymes, all of which combine to destroy the ingested microbe. Antibody-mediated phagocytosis is the major mechanism of defense against encapsulated bacteria, such as pneumococci. The polysaccharide-rich capsules of these bacteria protect the organisms from phagocytosis in the absence of antibody, but opsonization by antibody promotes phagocytosis and destruction of the bacteria. The spleen contains large numbers of phagocytes and is an important site of phagocytic clearance of opsonized bacteria. This is why patients who have undergone splenectomy for traumatic rupture of the organ are susceptible to disseminated infections by encapsulated bacteria.

Killing Other effector functions Acute inflammation Anti-viral response (interferons) Opsonization Antigen presentation

Professional phagocytic cells macrophages neutrophyl granulocytes
dendritic cells Professional antigen presenting cells macrophages B lymphocytes dendritic cells they express MHCII molecules the protein degradation products (peptides) can be presented to T lymphocytes by MHC molecules APCs function to display antigens for recognition by T lymphocytes and to promote the activation of lymphocytes. The cells that perform the majority of effector functions of innate and adaptive immunity are phagocytes (including neutrophils and macrophages), 63

INNATE IMMUNITY I Abul K. Abbas: Basic Immunology page 23-46 (fig 3,4,5, 10, 13, are not required)

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INNATE IMMUNITY I Abul K. Abbas: Basic Immunology page 23-46 (fig 3,4,5, 10, 13, are not required)

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Presentation on theme: "INNATE IMMUNITY I Abul K. Abbas: Basic Immunology page 23-46 (fig 3,4,5, 10, 13, are not required)"— Presentation transcript:

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