BMS2045 2b Innate Immunity 2 2024 PDF

Theme 2 Innate Immunity Phagocytosis part 2 and Inflammation part 1 THEME LEARNING OUTCOMES: 1. Describe the four main features of an innate immune response 2. Identify the cells and chemical/protein mediators involved in inflammation, including the function of cytokines and chemokines 3. Compare th...

Theme 2 Innate Immunity Phagocytosis part 2 and Inflammation part 1 THEME LEARNING OUTCOMES: 1. Describe the four main features of an innate immune response 2. Identify the cells and chemical/protein mediators involved in inflammation, including the function of cytokines and chemokines 3. Compare the different mechanisms of endocytosis, and describe in detail the processes that take place during phagocytosis 4. Describe the role of Toll-like receptors (TLRs) and other Pattern Recognition Receptors (PRRs) in phagocytosis and in innate immune cell activation 5. Discuss the mechanisms and function of the complement system activation 6. Specify the different cells and killing mechanisms that are involved in cell-mediated innate immunity Innate immunity Comprises four main types of defensive barriers/features: (1) Anatomical (2) Physiological/chemical (3) Phagocytic/endocytic – part 2 (4) Inflammatory – part 1 Stages of Phagocytosis 1 2 3 3 4 (1- Recognition) 2- Ingestion 3- Digestion 4- Exocytosis Antigen presentation Storage Phagocytosis stage (2) ‘Ingestion’ Once activated via receptor clustering after direct or indirect recognition (or more usually a combination of the two), pseudopodia form & surround particle, fuse engulfing particle in membrane-bound vesicle PHAGOSOME This requires energy and cytoskeletal rearrangement Pseudopodia Opsonised yeast particle held by a micropipette tip. The particle is placed in contact with the adhered neutrophil. When firm adherence between cell and zymosan coating is established, the tip is removed, allowing phagocytosis to proceed (www.cardiff.ac.uk/.../ images/micromanip.GIF) Phagocyte receptors bind particle – phagocyte is not activated to undergo cytoskeletal rearrangement unless surface Rs are clustered Force of fluid pushes pseudopodia out around the particle Is now understood that the ER is involved in the formation of phagosome membranes and pseudopodia Phagocytic and endocytic vesicle membranes are recycled Fusion of pseudopodia and formation of the ‘phagolysosome’ (3) Digestion (2) Ingestion & phagosome formation Fusion of lysosomes and/or granules (in neutrophils) with the phagosome…to create the phagolysosome (4) Release, storage or presentation of digested material (debris/Ag) (1) Recognition Phagocytosis stage (3) ‘Digestion’ Takes part in phagolysosome Most mechanisms split into oxygen dependent and oxygen independent Some breakdown products reused, others exocytosed, some ‘presented’ and others stored until cell death Oxygen Independent Acidification Lysozyme Other enzymes Defensins Lactoferrin Cathelicidins S100 proteins Oxygen Dependent Reactive oxygen intermediates Reactive nitrogen intermediates Respiratory burst Oxygen Independent Acidification The phagolysosome acidifies at the same time as lysosome fusion enhancing the activities of many enzymes and inhibiting growth of some pathogens Lysozyme Main enzyme within Lysosomes Mediates digestion of gram +ve bacterial cell walls ? Efficacy, works best in cooperation with other enzymes/systems Other Enzymes Acid hydrolases: phosphatases, sulphatases, glycosidases, deoxyribonucleases Lipases: eg phospholipase A2 Neutral proteases: collagenases, elastase, cysteine proteases Lactoferrin Binds to essential nutrients, inhibiting bacterial and fungal growth Defensins +ve charged polypeptides that bind -ve charged PAMPs e.g. LPS, LTA Electrostatically specific for microbial cells Aggregate to form pores in cytoplasmic membranes Activate complement – classical pathway Most abundant protein in neutrophil granules Cationic proteins Mainly found in Neutrophil granules, some in Eosinophils HMW, Serine proteases, most active at alkaline pH Examples: elastase, cathepsin G, proteinase 3 Damage microbial membranes & proteins Mostly anti-bacterial (cathepsin G also anti-fungal) Tumour necrosis factor alpha (TNFa) Cytokine Secreted (MFs mainly) Cytotoxic to tumour cells Oxygen dependent: The ‘Respiratory burst’ Activated phagocytes produce Reactive Oxygen and Reactive Nitrogen Intermediates (ROI and RNI). These are molecules with unpaired electrons also known as ‘free radicals’ Highly unstable and able to damage proteins, lipids, DNA and cell membranes of microorganisms Oxygen radicals are short-lived but superoxide and H2O2 released in tissues may persist and cause tissue damage RNIs also short-lived but may act with ROIs to give fullest effect Catalase, superoxide dismutase and glutathione are free radical scavengers Reactive Oxygen Intermediates Generation of ROIs take place at low levels beneath plasma membrane in most cells = physiological activity Rapid increase in O2 consumption reflects a ‘burst’ of targeted activity in phagosomes and phagolysosomes Involves cytoplasmic & membrane-associated enzymes: Nicotinamide Adenine Diphosphate (NADPH) -oxidase O2 converted to superoxide, peroxide or hydroxyl anion with unpaired e-s which are highly reactive and damage microbial cell walls, DNA etc Reactive Nitrogen Intermediates ⚫ ⚫ ⚫ ⚫ ⚫ inducible Nitric oxide synthase (iNOS) activated by microbial products and some cytokines eNOS and nNOS constitutively expressed and active in vasculature (endothelial, important for vascular tone) and neurones (NO can act as neurotransmitter) = normal physiological effects Oxidises L-Argenine to yield L-Citrulline and Nitric Oxide (NO) with an unpaired electron Within phagocytes = large ‘burst’ and LOTS of NO produced with potent antimicrobial activity Can combine with superoxide to be even more potent (peroxynitrite) (methylarginine) Overall: Most microbicidal mediators are produced within professional phagocytes (MC/MFs and Neus.) Difference is in location MC/MFs: lysosomes Neus: primary and secondary granules Neutrophils are more likely to kill ingested microorganisms due to higher respiratory burst and higher levels of defensins However, not all pathogens are killed by these mechanisms some escape to the cytoplasm – however, these can be detected by cytoplasmic PRRs and undergo autophagy or succumb to pyroptosis (initiated by the inflammasome formation) Phagocytosis stage (4) – ‘Exocytosis’? Some debris and products are released, a lot are re-used. Heavy metals tend to be ‘stored’ and PAMPs and DAMPs once processed are often presented to the adaptive immune response - more of this in theme 3! Innate immunity Comprises four main types of defensive barriers/features: (1) Anatomical (2) Physiological/chemical (3) Phagocytic/endocytic (4) Inflammatory – part 1 (4) Inflammation Body’s immediate defence reaction to infection or damage 5 hallmarks: rubor (redness) et tumor (swelling) cum calor (heat) et dolor (pain), & loss of function Acute phase response (0-24hours) differs from later inflammation and chronic inflammation Why needed? Events geared towards increasing blood flow, permeability of vasculature – allowing leukocyte migration to aid limiting the spread of infection, tissue damage and to promote healing Involves large number of chemicals & cells acting together Site Activation of immune cells during inflammation MEDIATORS are released from activated tissue Cells (Mast cells/MFs) Systemic effects Local effects Increase expression of adhesion molecules on endothelial cells lining blood vessels & on leukocytes allowing them to attach to endothelium Other mediators affect vascular tone and integrity of endothelial layer allowing attached cells to pass into tissues Stimulate release of granulocytes & monocytes from Bone marrow Hypothalamus – fever Liver – increased Acute Phase Protein production (CRP, SAA, MBL) Migration of leukocytes to site of trauma/infection leading to increased phagocytosis and promotion of healing At site of damage/infection, cells phagocytose toxins, damaged self proteins/cells and pathogens - recognised through binding to cell receptors for foreign molecules (PAMPs and DAMPs) Phagocytosis also stimulated by OPSONINS e.g. Antibody, Lectins/Acute Phase Proteins (such as C-reactive protein, Mannose Binding Lectin and Serum Amyloid A) and proteins of the complement activation pathways (indirect recognition) Organism/toxin destroyed by anti-microbial proteins/peptides (AMPs) enzymes or by respiratory burst in granules/phagolysosomes, Neutrophil Extracellular Traps (NETs) The release of many mediators enhance these effects and affect local blood vessels to allow migration of more leukocytes to the site Inflammatory mediators Include a wide range of proteins, lipids and chemicals: Produced by a variety of cell types, but initially in inflammation by Mast cells, Basophils and MFs at sites of infection or damage Cells express receptors for these mediators Prostaglandins, leukotrienes, thromboxanes, histamine… Cytokines (~58 including 37 interleukins): ‘cytokine’ suggests movement of cells and ‘interleukin’ suggests communication/messaging between white blood cells Chemokines (~45): ‘chemokine’ suggests movement towards a chemical which is an ‘attractant’ (chemo-attractant) Additionally, plasma has 4 interconnected mediator producing systems: kinin, clotting, fibrinolytic and complement Cytokines Many play a significant role in inflammation These include: Interleukin-1, 6, 8, 10, 12 (IL-1, IL-6 etc), Tumour necrosis factor a (TNF-a), Transforming growth factor β (TGF-β) and interferon γ (IFNg) IL-1, IL-6 and TNFα are ‘alarm’ cytokines for the acute phase (or early inflammation) causing local EFFECT STIMULUS and systemic activation of fever, increased vascular permeability, production of Acute Phase Proteins and increased adhesion molecule expression. They are ‘pro-inflammatory’ CYTOKINE PRODUCING CELL TARGET CELL ‘Anti-inflammatory’ cytokines are able to down-regulate responses, include IL-10 and transforming growth factor beta (TGF-β) IL-12 and IL-18 induce the differentiation of pro-inflammatory subset of T cells IL-8 is a potent chemokine for neutrophils IFNγ acts later in the acute phase contributing to chronic inflammation by recruiting Mφs to sites of damage/infection Type I interferons (IFNa and b) have antiviral properties within infected cells NOTE cytokines also play an important role in adaptive immune responses Chemokines Chemoattractants Small proteins – two major families: CC and CXC contain 43 of the 45 defined chemokines Defined by presence of 4 cysteine residues & sequence of amino acids involving first two of these On binding specific receptors, stimulate migration & activation of range of cells towards the source (along a concentration gradient) = Chemotaxis e.g. IL-8, MCP-1 (Monocyte chemotactic protein 1) Chemokine production Other Inflammatory mediators Prostaglandins, Leukotrienes and Thromboxanes All are Eicosanoids = unsaturated fatty acids derived from arachidonic acid Prostaglandins made by enzymes including cyclooxygenases (COX-1, COX-2) – major target of ‘anti-inflammatory’ pain killers Described as having ‘hormone–like’ effects Act on vascular permeability and leukocyte migration to tissues PGE2 acts on hypothalamus to induce fever Histamine Acts to increase vascular permeability and smooth muscle contraction Secreted products of Macrophages Cellular movement during inflammation A wide range of adhesion molecules, selectins and integrins promote binding of leukocytes to endothelium Movement along or between is also regulated by release of chemokines causing chemotaxis Movement (or migration) of white blood cells or fluid from the lumen of blood vessels into tissues = extravasation Also important in lymphocyte homing Overview of Inflammation Site (Kuby, 6e, 2007)