BMS150 Innate Immunity PDF Spring 2023

Immunology II Innate Immunity & Inflammation BMS 150 Week 1 Innate Immunity—Outline Describe the location and basic mechanism of the major barriers of innate immunity Describe the process of phagocytosis and mechanisms of microbe destruction in macrophages and neutrophils Briefly describe what is me...

Immunology II Innate Immunity & Inflammation BMS 150 Week 1 Innate Immunity—Outline Describe the location and basic mechanism of the major barriers of innate immunity Describe the process of phagocytosis and mechanisms of microbe destruction in macrophages and neutrophils Briefly describe what is meant by the term “pattern-recognition receptor” and “opsonin” For Toll-like receptors: Describe typical ligands and cellular compartments Briefly discuss the signaling cascade that is activated with ligand binding For acute inflammation Briefly describe the steps of the acute inflammatory process and their histologic appearance Relate specific mediators of acute inflammation to the steps of the process Describe the role that acute inflammation plays in protection of the host Innate Immunity Mechanisms of innate immunity discriminate very effectively between host cells and pathogens Innate immune defenses exist in all individuals and act within minutes - hours after an encounter with infectious agent Only when innate defenses are overwhelmed/bypassed/evaded is an adaptive immune response required Barriers ** Chemical Barriers Lysozyme Present in secretions (mucus, tears, milk, saliva) Uses hydrolysis to break apart the peptidoglycan wall à lysis of bacterial cell wall Antimicrobial peptide - defensins Small, heterogeneous, cationic peptides kill Gram-negative and Gram-positive bacteria, some enveloped viruses, fungi Multiple antimicrobial effects: § Destabilize membranes and Pore formation in bacterial cell walls § Proteolytic degradation of bacterial proteins § Inhibit viral binding and entry § Inhibit virus particle assembly Defensins – prototypical AMPs Defensins can act as a chemical barrier when they are secreted by epithelial cells in a variety of mucosal surfaces Defensins and other AMPs (i.e. cathelicidins) are also stored in neutrophil granules and can be released within tissues in response to inflammation § Can kill microbes extracellularly à released when neutrophils die during inflammation § Can kill microbes intracellularly after a cell (i.e. neutrophil) phagocytoses a pathogen Just like many molecules and cells of the immune system, defensins perform a number of roles – not just a chemical barrier Phagocytosis and Phagocytes One of the first lines of defense if microbes do invade tissue Engulf and destroy microorganisms, especially bacteria Key role in innate immunity as they can recognize, ingest and destroy many pathogens without aid of an adaptive immune response § Phagocytosis can also occur after an antibody has bound to an antigen – the antibody can act as a “signal” that triggers efficient phagocytosis Macrophages and neutrophils are the major phagocytes in the body Phagocytic Cells Monocytes & Macrophages: Pro-monocytes (BM)àmonocyte (blood)à macrophage/macrophage-like cells (tissues) Long-lived cells resident within the tissues Neutrophils: Derived from hematopoietic precursors in the BM Non-dividing, short-lived cell type in blood (dominant WBC) Phagocytosis – the basics PRR = Pattern Recognition Receptor Pattern Recognition Evolutionarily conserved mechanism for recognizing common, conserved ‘signs’ of microbial infection, physiological stress, or other damage Recognition is immediate, does not require prior recognition, and activates several arms of the innate (and adaptive) immune response Responses are elicited via the engagement of Pattern Recognition Receptors (PRRs) found on phagocytes, in response to: § Pathogen Associated Molecular Patterns (PAMPs) § Danger Associated Molecular Patterns (DAMPs) Pattern Recognition Receptors Examples of PRRs: § Toll-like receptors § Nod-like receptors § Lectins Elicit responses such as: § Phagocytosis § Cytokine secretion Phagocytosis – the process 1. A pattern-recognition-receptor (PRR) binds to a microbe or bit of debris, OR an opsonin created by another cell binds to the microbe A microbe A bit of debris An opsonin An opsonin is basically a soluble, secreted PRR that enhances the effectiveness of phagocytosis An opsonin coats a microbe, the phagocyte has receptors for parts of that opsonin See table next slide for receptors that trigger phagocytosis PRRs that trigger phagocytosis Receptor Examples Lectin receptors (lectin receptors recognize “carbohydrate patterns”) Mannose receptor, dectin Bacterial, fungal, parasitic receptor cell walls Scavenger receptors SR-A, SR-B LPS, lipoteichoic acid – mostly bacteria Collagen-domain receptors Calreticulin – binds to particular opsonins - FYI Collectins, ficolin – mostly carbohydrate groups on bacteria Complement receptors CR3, CR1 – binds to complement opsonins Mostly bacterial cell walls Fc receptors – receptor Different Fc receptor for for the constant region of each type of Ab – FYI for an antibody now Microbial ligands Almost anything Phagocytosis – the process 2. The microbe is engulfed – the PRR receptors signal the cell membrane to approach, coat and then surround the sites where the receptor is bound Forms a phagosome Mediated by intracellular signalling events and actin polymerization – see diagram PI3 kinase seems to be important here Phagocytosis – the process 3 & 4. Microbe killing – phagosomes fuse with lysosomes as well as (in neutrophils) primary and secondary granules Phagosomes have many molecules that are effective at cellular killing – a little more later Major groups include: Reactive oxygen species “pore”-forming proteins or peptides Hydrolytic enzymes pH changes – i.e. acidic environment of the lysozyme 5. The microbe remnants are either digested and used, or can be excreted from the phagocyte Phagocytosis details à FYI Microbe killing – so many options After the microbe has been phagocytosed, the phagosome will dock with a lysosome and/or neutrophil granules Lysosomes can pretty much break down anything (acid hydrolases) The low pH of a lysosome is also unpleasant for many bacteria See next slide for the NAPDH oxidase complex This complex becomes associated with the membrane of the phagolysosome Uses a large amount of oxygen (respiratory burst) If a particle is too large to phagocytose, macrophages will surround it and “place” their NADPH oxidases close to it to try to kill it Macrophages in particular are also capable of killing cells by inducing the synthesis of nitric oxide at high concentrations Neutrophils have a multitude of pore-forming molecules within their granules – these granules will fuse with the phagosome Microbe killing via neutrophils Neutrophil granules: Defensins are very rich in cysteine Form voltage-dependent pores in bacteria that are permeable to water Cause lysis Cathepsin - a type of protease Cathelicidins – another pore-forming molecule Causes lysis, multitude of different structures Lysozyme – a glycoside hydrolase Doesn’t require an acidic pH Found in a variety of glandular secretions Great at killing gram +(ve) bacteria Lactoferrin – iron-binding protein that interferes with iron metabolism in microbes Microbe killing via neutrophils Neutrophils can do a neat trick – when they’re in an environment with many bacteria (they’re “surrounded”) they can can lyse and release their DNA into the ECF § Known as a NET – a neutrophil extracellular trap § NETs are “sticky” – most bacteria are trapped in the chromatin § Histones are toxic to many bacteria § The granule contents will remain close to the NETs and help with killing bacteria, even after the neutrophil itself is dead Stuck in the NET Phagocytosis – take-aways You need to know the following: § How do phagocytes recognize that something is a “target” for phagocytosis? What’s an opsonin and how are they involved in this process? § How do phagocytes kill a phagocytosed pathogen? What is the role of: Lysosomes Free radicals (how are they produced? Which ones are involved?) Anti-microbial peptides § How can a NET add to host protection beyond phagocytosis? Pattern Recognition Receptors Examples of PRRs: § Lectins § Toll-like receptors § Nod-like receptors Elicit responses such as: § Phagocytosis § Cytokine secretion Toll-Like Receptors Family of 10 cell membrane receptors with variable specificity for a range of pathogens Ligands can include LPS, dsRNA, ssRNA, DNA, Flagellin Figure 3.7 Sensing of LPS by TLR4 on macrophages leads to activation cytokine synthesis Toll-Like Receptors Cytokines secreted in response to TLR’s include: § Inflammatory cytokines (IL-1b, IL-6, CXCL8, IL-12, TNFa) Cytokine – a (small) protein messenger, secreted by a vast array of cells, that can: § Influence the differentiation of a wide variety of cells, including leukocytes § Mediate – activate or inactivate – the activity of many cells, including leukocytes § Increase or decrease the production of a wide variety of stem/hematopoietic cells More cytokine overview in the e-learning module § Interferons Interferon (IFN) alpha, beta, and lambda (IFNa, IFNb, IFNl) Autocrine and paracrine signaling molecules that are effective in activating macrophages, NK cells, and inducing an antiviral state More on interferons in subsequent lectures Consequences of TLR signaling The phenotype of individuals with specific gene mutations/polymorphisms can tell us about the overall importance and function of that gene Example: MyD88 à An essential adaptor in TLR signaling § Patients with MyD88 deficiency: Suffer frequent and severe bacterial infections Antiviral responses generally unaffected § Patients with constitutively active MyD88: Develop various blood disorders and blood cancers: § Overproduction or dysregulated production of IgM § B cell lymphoma, marginal cell lymphoma Nod-Like Receptors Family of intracellular receptors found in the cytoplasm that detect products derived from the intracellular degradation of phagocytosed pathogens (e.g. components of bacterial cell wall) Also recognize DAMPs associated with cellular stress activates expression of inflammatory cytokines Acute inflammation How do we get from here… To here? Steps of Acute Inflammation A. Alteration of vascular caliber - vasodilation Leads to increases in blood flow at the capillary bed due to arteriolar dilation, dilation of precapillary sphincters § Nitric oxide and histamine A variety of prostaglandins (PGI2, PGE2, PGD2) Platelet activating factor (at low concentrations – higher concentrations cause vasoconstriction) Complement § C5a and C3a stimulate histamine release At low concentrations, nitric oxide is a potent vasodilator (why Viagra is a profitable drug) At high concentrations, it’s capable of destroying both microbes and host cells since it’s a free radical Higher concentrations produced by an inducible nitric oxide synthase in macrophages Vasodilation Arterioles and pre-capillary sphincters dilate leading to vastly increased blood flow in inflamed tissue Vasodilation and fluid loss (due to increased permeability) lead to slower blood flow § Known as vascular congestion § This helps with margination of leukocytes Whoa… what do we need to know from that last slide? Prostaglandins and leukotrienes are produced when PLA2 generates arachidonic acid from membrane phospholipids Different types of cyclooxygenases produce different types of prostaglandins from arachidonic acid § Important prostaglandins – PGE2, PGD2, and PGI2 cause vasodilation and increase vascular permeability, important acute inflammatory mediators Different types of 5-lipoxygenase produce different types of leukotrienes from arachidonic acid that seem particularly important in lung tissue § LTB4 – important chemotactic agent § Other LTs – increased vascular permeability and smooth muscle constriction (think asthma) Lipoxins are generated from arachidonic acid by 12lipoxygenase – they decrease inflammation Steps of acute inflammation B. Enhancement of vascular permeability Capillaries and venules become more “leaky” with the release of a number of mediators § § Histamine and serotonin (released by activated platelets, a link between inflammation and clotting) Prostaglandins (PGD2 and PGE2) Leukotrienes (LTC4, LTD4, LTE4) Platelet activating factor C3a and C5a Bradykinin a wide variety of proteins and mediators can enter the interstitial space from the bloodstream Vascular permeability Increased vascular permeability is due to contraction of endothelial cells Occurs mainly in venules Often short-lived Another mechanism is endothelial damage Can be caused by trauma, burns, microbial damage Can also be caused by leukocyte-mediated damage to the endothelium (often longer-lived) Increased transcytosis can also result in leakage of plasma components into the interstitial space Transcytosis? Active, vesicle-mediated transport across the capillary endothelial cell Large molecules can move across the endothelium via: Pinocytosis (caveolin pathway) Receptor-mediated endocytosis Mechanisms of increased vascular permeability Lymphatics As interstitial fluid accumulates during inflammation, pressure increases in the interstitial space and lymphatic drainage increases § Normally only a small amount of interstitial fluid is produced in non-inflamed tissue § Excess fluid, microbes, debris, and leukocytes all migrate into the lymph during inflammation The lymphatic vessels themselves can become inflamed – known as lymphangiitis Leukocyte migration C. Emigration and activation of leukocytes § § Neutrophils, monocytes, eosinophils, and basophils will all migrate from the circulation into inflamed tissue Steps: a) b) c) d) e) Margination Mediated by binding of selectins and Rolling cellular adhesion molecules to their Adhesion respective ligands on leukocytes Diapedesis Chemotaxis of leukocytes to sites of injury or infection Chemokine or cytokine? Cytokine – a (small) protein messenger, secreted by a vast array of cells, that can: § Influence the differentiation of a wide variety of cells, including leukocytes § Mediate – activate or inactivate – the activity of many cells, including leukocytes § Increase or decrease the production of a wide variety of stem/hematopoietic cells Chemokine – structurally-related family of small cytokines that: § Bind to cell-surface receptors (usually leukocytes) § Induce movement of leukocytes along the chemokine concentration gradient § Mediate adhesion of leukocytes for the purposes of: Differentiation Inflammation/migration Chemokines 2 major chemokine families § CXC CXC chemokines attract neutrophils, are angiogenic, and are very similar in structure The “X” indicates the location of a disulphide bond § CC Act on/attract a wide variety of other leukocytes Chemokines - FYI Leukocyte migration C. Emigration and activation of leukocytes § Steps: a) Margination – leukocytes migrate towards vessel wall b) Rolling – formation & dissociation of adhesion bonds between leukocytes and endothelial cells *Activation by chemokines presented on endothelial cells is required before the leukocyte can form stable adhesion Endothelial cell presents a chemokine that stimulates activation of the leukocytes This increases affinity of leukocyte integrin for it’s ligand, allowing stable/tight adhesion bonds to form Leukocyte migration C. Emigration and activation of leukocytes § Steps continued: c) Stable/Tight Adhesion – Formation of tight/stable adhesion bonds between leukocytes and endothelial cells d) Diapedesis/ Transmigration – leukocyte migrates through endothelium e) Chemotaxis of leukocytes to sites of injury or infection Putting it all together Adhesion molecules and their ligands – what are the concepts here? A variety of inflammatory mediators increase the ability of leukocytes to migrate to a target Histamine, Thrombin Rolling Selectin expression by endothelial cells TNF & IL1 ICAM expression by endothelial cells Chemokines Increased integrin affinity Chemotactic agents All of these agents are produced in higher concentrations at sites of cellular damage/pathogen invasion Leukotriene B4 Bacterial products containing N-formyl-methionine Activated complement (particularly C5a) More on this in the e-module Chemokines (IL-8, RANTES, eotaxin) Leukocytes can “follow the breadcrumbs” to the site of pathology via the chemotactic agent concentration gradient For leukocyte emigration: What molecules cause loose adhesion of leukocytes to the vascular endothelium? § What binds to what? How about tight adhesion? § What cells are integrins found on? How about ICAMS? What causes expression of the molecules above? What is the role of a chemokine in: § Tight adhesion? § Migration of a leukocyte to a site of inflammation? What is a chemotactic agent? Name a few