Micro 3.pdf

MICROBIOLOGY AND IMMUNOLOGY (PBBS505A&B) Innate Immunity Fabio Re, PhD [email protected]  Learning Objectives A. To know the cellular and soluble components of the innate immune system B. To know the role of phagocytosis in the r...

MICROBIOLOGY AND IMMUNOLOGY (PBBS505A&B) Innate Immunity Fabio Re, PhD [email protected]  Learning Objectives A. To know the cellular and soluble components of the innate immune system B. To know the role of phagocytosis in the response to infection C. To know the receptors used to recognize infection and danger and the pathway they activate D. To understand how viruses are recognized and know the antiviral mechanisms of innate immunity (or Adaptive) Pathogen detection by PRR Activation of Adaptive Immunity  Innate Immunity First Line of Defense Innate Immunity  Innate Immunity Physical and Chemical Barriers Physical barriers include skin and mucus membranes (Mucus/cilia to remove particles (lung, intestines) Chemical barriers Lysozyme: hydrolize peptidoglycans. Produced by neutrophils. Found in tears, saliva Phospholipase2A: hydrolyzes membrane phospholipids Antimicrobial peptides. Short peptides (29-35 amino acids long), cationic (positive charge- interact with negatively charged bacterial membrane) or amphipathic structure (insert into membranes). Destabilize membrane integrity Acid pH (stomach), bile salts Biological barriers Commensal (non-pathogenic) bacterial flora (microbiota). Competes with pathogens.  Recognition of an infection once it gets past the epithelial barrier Microbicidal mechanisms Soluble innate immune recognition elements (collectins, ficolins, Complement activation) Sentinel innate immune cells of tissues: neutrophils, macrophages, mast cells, NK cells, and dendritic cells Phagocytosis Toxic products: Nitric oxide: iNOS2 Hydrogen peroxide Superoxide anions O2- NADPH oxidase in neutrophils (respiratory burst) Leukocytes of Innate Immunity  Leukocytes of Innate Immunity Polymorphonuclear Leukocytes Neutrophils (Granulocytes) 40-70% of all white blood cells. First to arrive at the infection site Short lived. 1011 are produced daily Highly phagocytic. Highly microbicidal. Produce respiratory burst. Contain lytic granules that fuse with phagosome and discharge content in it Essential for resistance to bacterial and fungal infections Monocytes, Macrophages Circulating monocytes (2-10%) give rise to macrophages Long lived. Phagocytic. Respiratory burst. Lytic granules Produce several cytokines in response to microbial products Mast cells Reside in connective tissue Cytoplasmic granules contain inflammatory mediators Protection from parasites Critically involved in allergic reactions Dendritic cells NK Cells Antigen processing and presentation Lysis of virus-infected cells Costimulation of T lymphocytes Production of Interferon Production of cytokines Macrophage activation  Innate Cellular Components Cell Functions Neutrophils Phagocytosis and intracellular killing Inflammation and tissue damage Macrophages Phagocytosis and intracellular killing Extracellular killing of infected or altered self targets Tissue repair Dendritic cells Antigen presentation and co-stimulation for activation of antigen-specific T cells NK cells Killing of virus-infected and altered self targets Mast cells Rapid production of inflammatory mediators. Allergy  Phagocytosis: engulfment of large particle Cell Types: Macrophage, Granulocytes, Dendritic cells Phagocytosis is mediated by phagocytic receptors and opsonins Opsonization: Coat the particle and render it more recognizable and “eatable” Opsonin: Complement components (C3b), Antibodies Phagocytic receptors Receptors for opsonins (complement receptors, Fc receptors) Pattern recognition receptors (Scavenger Receptors, Mannose receptor, etc.). Bind to many microbial ligands Receptors for apoptotic cells Functions: Destruction of pathogen Exposure of microbial products Antigen presentation Removal necrotic/apoptotic cells Phagosome-Lysosome fusion  Opsonization Opsonins (complement proteins or antibodies) coat bacteria and promote phagocytosis mediated by opsonin receptors  Chediak-Higashi syndrome § Chediak-Higashi syndrome is a rare, autosomal recessive disorder characterized by recurrent bacterial infections including pyogenic infections, oculocutaneous albinism, progressive neurologic abnormalities, mild coagulation defects § Mutation of a lysosomal trafficking regulator prevents lysosome-phagosome fusion Lysosome fusion  Phagocytosis and Production of Toxic Radicals Phagocytosis is coupled to the “Respiratory Burst”: production of highly reactive radicals that are very toxic to microbes Phagocyte oxidase (NADPH oxidase): makes reactive oxygen intermediates (superoxide anion, hydrogen peroxide) Myeloperoxidase: hypochlorous acid Inducible Nitric oxide synthase (iNOS): makes reactive nitrogen intermediates (NO) SOD Superoxide  Chronic Granulomatous Disease (CGD) Genetic disease caused by mutations in NADPH oxidase enzyme complex of neutrophils most common form is X-linked recurrent infections, particularly by intracellular bacteria, pneumonia, abscesses, arthritis granulomas form due to inability to kill phagocytosed bacteria  Oxygen-Independent Killing in the Phagolysosome Effector Molecule Function Antimicrobial peptides Damage to microbial membranes Lysozyme Hydrolyses peptidoglycans in the cell wall Lactoferrin Deprives pathogens of iron Hydrolytic enzymes (proteases) Digests killed organisms  Microbial Evasion of Phagocytosis Some bacteria employ strategies to avoid engulfment by phagocytes Masking bacterial surface Carbohydrate capsule, M protein and fimbriae Prevent antibody recognition. Blocks opsonization/phagocytosis (Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis) Inhibition of acting polymerization and cytoskeleton remodeling (Yersinia injects YopH, a Tyr- phosphatase) Survival Inside of Cells Block phagosome-lysosome fusion and colonize the phagosome (Mycobacterium tuberculosis, Salmonella). Protection from oxidative damage. Escape from phagosome and cytoplasm colonization (Listeria monocytogenes, Francisella tularensis, Shigella) Destruction & Recognition of Pathogens Figure 1-14  Pattern Recognition Receptors Recognition of Pathogens & Danger Most microorganisms express repeating patterns of molecular structures termed Pathogen Associated Molecular Patterns (PAMPs). Infected, stressed, or necrotic host cells release various molecules that act as Danger Associated Molecular Patterns (DAMPs) Innate immune cells express several Pattern Recognition Receptors (PRRs) that are capable of recognizing PAMP and DAMP PAMP are invariant between microorganisms of a given class. Limited number of germ-line encoded PRR can detect infection by many microbe types Examples of Pattern Recognition Receptors: Mannose-Binding Lectin (MBL) Macrophage Mannose Receptor Scavenger Receptors Toll-like Receptors (TLRs) Nod-like Receptors (NLRs) Rig-like Receptors (RIG-I, MDA-5)  Activation of PRR triggers the Inflammatory Response Activation of TLRs, NLRs and RLR results in production of proinflammatory cytokines, chemokines, inflammatory mediators, and co-stimulatory molecules Cytokines Soluble molecules that stimulate inflammation and phagocytosis/killing of pathogens – TNFa and IFNg activate macrophage and PMN phagocytosis and killing – IFNa/b activates anti-viral mechanisms – IL-1b and IL-6 stimulates inflammation and fever Chemokines Large family of chemotactic cytokines, induce directed chemotaxis of cells. Attract cells to infection site and induce migration of DC to lymph nodes  Toll-Like Receptors (TLR) Recognition of microbial products on the cell surface or endosomal compartments ssRNA Humans express 10 TLR TLR evolved to recognize microbial products (PAMP) derived from virus, bacteria, fungi, and protozoa (self/non- self discrimination) TLR are expressed on the cell surface or endosomal compartments TLR also recognize endogenous molecules, “danger NF-κB signals” (DAMP) Pro-inflammatory mediators  Toll-Like Receptors link innate and adaptive immunity Signal the presence of microbes Initiate inflammation o Cytokines/chemokines production o Adhesion molecules upregulation Initiate and instruct adaptive immune response 1. Ag presentation (DC maturation) 2. Induction of co-stimulatory molecules 3. Induction of cytokines that guide Th cell polarization  Rig-like Receptors (RLR) cytoplasmic recognition of viral genomes RLR recognize ssRNA and dsRNA that constitute the genome of viruses and induce production of proinflammatory cytokines and interferons Viral Genomes ssRNA, dsRNA  cGAS/STING Cytoplasmic recognition of DNA Chronic activation of cGAS/STING, due to presence of host DNA in the cytosol, can lead to autoimmune disorders (Aicardi–Goutières syndrome)  Nod-like Receptors (NLR) Cytoplasmic recognition of microbial products and “danger signals” NLR are part of a multiprotein complex called Inflammasome Inflammasome activation leads to activation of caspase-1 and production of the proinflammatory cytokines IL-1b and IL-18 Sharma D and Kanneganti TD. J. Cell Biol. Vol. 213 No. 6 617–629  NLR in human Diseases NLR genes mutated in human diseases: NLRP3: Cryopyrin-associated periodic syndrome (CAPS) is a spectrum of autoinflammatory syndromes (Muckle-Wells syndrome, Familial cold autoinflammatory syndrome, Chronic infantile neurological cutaneous and articular syndrome). More than 40 mutations. Autosomal dominant. Gain of function. Recurrent systemic inflammation. Periodic fever, skin rashes, amyloidosis. Production of IL-1b and other cytokines and acute phase proteins in absence of infection. IL-1ra treatment reverse symptoms. Nod2: Crohn’s disease (subtype of inflammatory bowel diseases) Blau syndrome (skin rashes, uveitis and recurrent arthritis) Early-onset sarcoidosis (granulomatous inflammation). Mutations are gain of function. Chronic inflammation of mucosal barriers. High level of proinflammatory cytokines. Pattern Recognition Receptors Summary  Endogenous “Danger Signals” and Sterile Inflammation Infected cells, stressed tumor cells, necrotic cells release molecules that are recognized by PRR leading to inflammatory responses in absence of infection. Aggregates of insoluble proteins, lipids, or crystals also activate PRR. Examples: Uric acid crystals in articular joints activate NLR leading to IL-1b secretion (gout) Cholesterol crystals in the artery plaque activate NLR leading to IL-1b secretion (atherosclerosis) Amyloid-b fibrils deposited in the brain activate NLR leading to IL-1b secretion (Alzheimers’) Oxidized low density lipoproteins activate TLR (atherosclerosis) If genomic DNA released by necrotic cells is not promptly degraded it may activate TLR9 or cGAS/STING contributing to development of autoimmunity  Innate recognition of viruses TLR (endosome detection) RIG-like receptors, NLR, (cytoplasmic detection) NK cells (detect signs of infection on cells) Innate anti-viral effector mechanisms Interferon response NK cells  Viruses induce Interferon-a/b production Interferons are soluble cytokines Termed interferons because they “interfere” with viral replication Type I interferons: IFNb and IFNa (13 genes). Different from IFNg (type II) IFN-a/b produced by many cell types following viral infection (but also during bacterial infections) Induced by viral products (dsRNA, ssRNA, viral genomic DNA) recognized by TLR3, TLR7/8, TLR9, RIG-I, cGAS FUNCTIONS Induce expression of host cell proteins that inhibit viral replication and create an “antiviral state” Act on infected cells and bystander uninfected cells Activate NK cells to lyse infected cells and to secrete cytokines Activate adaptive immunity  Evasion of Innate Immunity by Coronavirus Detection of ssRNA and dsRNA by TLR7/8 and TLR3 is blocked Detection of ssRNA by RIG-I/MDA5 is blocked Synthesis and Type I IFN and signaling through IFN receptor is blocked  Natural Killer cells First identified for having the ability to lytically kill certain tumor cell lines without prior sensitization Lymphoid origin but do not express T Cell Receptors. Develop in bone marrow. 10- 15% of all leukocytes. Control virus infection until adaptive immunity (CD8 T cells) takes over. Do not clear the virus. Also involved in protection against intracellular bacteria and protozoa (Listeria, Leishmania). Tumor surveillance. NK cells are ready to kill. No expansion, Ag-specific recognition, or acquisition of effector functions required Kill target cells using the same granules as T cells but do not require prior activation Lytic granules contains perforin and granzymes which penetrate target cell membrane and induce programmed cell death Activated NK cells secrete IFNg that acts on macrophages to increase microbial phagocytosis and killing NK are activated by type I IFN, IL-12, and IL-15 (increased killing) Do not recognize PAMP. Rather, recognize changes on the surface of infected/tumor cells. “missing/altered self”  NK cell receptors NK cells use two classes of receptors to determine whether certain self-ligands are normally expressed. Upon the interaction with a host cell, it is the balance between inhibitory and activation signals that determines whether the NK cell will kill the host cell. Inhibitory receptors recognize MHC-I. Their function is to prevent the lysis of cells that express MHC-I normally. Consequently, when target cells lose the expression of self-MHC-I, which is a common feature of infected or transformed cells, NK cells fail to receive inhibitory signals, shifting the balance towards NK cell activation ("missing-self recognition"). Activating receptors. Infected or stressed host cells induce the expression of endogenous self-molecules that serve as ligands for potent activating NK cell receptors (”altered-self recognition"). Importantly, many tumor cells express such ligands constitutively. Changes in surface glycoproteins is also recognized. Upon the interaction with a host cell, it is the balance between inhibitory and activating signals that determines whether the NK cell will kill the host cell.  NK “missing self” Killing No Lysis Lysis of target cell NK cells posses small granules in their cytoplasm containing perforin and the proteases granzymes. Upon release in close proximity to a cell slated for killing, perforin forms pores in the cell membrane of the target cell, creating an aqueous channel through which the granzymes and associated molecules can enter, inducing either apoptosis or osmotic cell lysis.  Antibody-dependent cellular cytotoxicity (ADCC) Infected cells are opsonized with antibodies Antibodies bound to antigens can be recognized by FcγRIII (CD16) receptors expressed on NK cells, resulting in NK activation, release of cytolytic granules and induction of apoptosis. This is a major killing mechanism of some monoclonal antibodies like rituximab (Rituxan), ofatumumab (Azzera)  Innate Lymphoid Cells ILC Innate lymphoid cells (ILCs) are recently discovered cells that promote functions associated with T helper cells, but do not share their surface markers (e.g., T cell receptors) ILC1 group includes Natural Killer (NK) cells and ILC1 cells, which are both known for their IFN-γ production, tumor surveillance, and inflammation ILC2 cells produce IL-5 and IL-13 and promote anti-helminth responses and allergic lung inflammation. ILC3s are a subclass typically found in mucosal tissue like the intestinal tract, interacting with microbiota The biology of innate lymphoid cells. Nature 517, 293–301  Summary Physical and chemical barriers protect from infections and are part of innate immunity Complement cascade leads to opsonization and lysis of pathogen and inflammation Phagocytosis and “respiratory burst” are effective microbicidal mechanisms Pattern Recognition Receptors (PRR) recognize microbial products and danger signals and trigger production of cytokines, inflammatory mediators, and co-stimulatory molecules Type I interferons induce an antiviral state in the target cells that prevents viral replication and infection NK cells recognize changes on the surface of infected cells and kill these cell using lytic granules “Danger signals” released by necrotic host cells may trigger: A. Phagocytosis B. Complement fixation C. Opsonization D. NK cells activation E. Sterile inflammation 0% 0% 0% 0% 0% ion sis n on ion tio yto at ati at niz ixa oc tiv m so tf ag ac lam Op en Ph lls m inf ce ple rile NK m Ste Co 8 Macrophages isolated from a patient are unable to produce cytokines when stimulated in vitro with gram-negative lipopolysaccharide (LPS). Which of these genes may be mutated in this patient? A. Complement C3 B. TLR4 C. NLRP3 D. Myeloperoxidase (MPO) 0% 0% 0% 0% 0% E. Lysozyme 3 R4 e RP 3 m TL tC ) PO NL zy en so (M m Ly ple se ida m Co ox er lop ye M 8  Genetic testing reveals that a patient who experiences recurrent bacterial infection is deficient in a major complement component and as a consequence: A. His/her leukocytes cannot phagocytose bacteria efficiently B. His/her leukocytes are unable to recognize bacterial components released into the cytoplasm C. His/her leukocytes cannot produce cytokines and other inflammatory mediators in response to bacterial products D. 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