BMS150_PHL3-03_Innate2ON_Win2023_2.pdf

Immunology III Innate Immunity & Inflammation 2 BMS 150 Week 2 Topic Overview The Complement System Video 1; Video 2 Pattern recognition receptors – DAMPs and other PAMPs Video 3 Inflammatory Mediators – part 2 Video 4 Innate Antiviral Responses And The Natural Killer Cell Video 5 Acute Inflammation...

Immunology III Innate Immunity & Inflammation 2 BMS 150 Week 2 Topic Overview The Complement System Video 1; Video 2 Pattern recognition receptors – DAMPs and other PAMPs Video 3 Inflammatory Mediators – part 2 Video 4 Innate Antiviral Responses And The Natural Killer Cell Video 5 Acute Inflammation – part 2 Video 6 Module 1 – The Complement System Describe the general principles that apply to all pathways of the complement cascade Describe the following mechanisms for the alternative, classical, and lectin complement pathways: Recognition of pathogen Formation of the C3 convertase Formation of the C5 convertase Describe the general mechanism of cell lysis by MAC when complement is activated Categorize the role of complement components in: Aiding opsonization Aiding the general process of acute inflammation Describe the possible role of C1q deficiency in autoimmunity Complement A blood-borne molecular defense system that “complements” the immunity provided by antibodies ▪ Complement aids: Phagocytosis → some complement components are opsonins Destruction of microorganisms → the end result of complement activation is formation of a large, antimicrobial protein complex Inflammation → some complement components are potent inflammatory mediators Activated through a tightly-controlled enzyme-triggered cascade ▪ “Cascade” = enzymatic reactions that activate protein effectors in a sequence These proteins could be enzymes or anti-microbial proteins ▪ normally only turned on at certain localized sites Complement 3 Pathways of Complement Activation: Alternative Pathway ▪ Complement component 3 (C3) acts as a pattern-recognition receptor – binds to the surface of a pathogen Mannose-Binding-Lectin (MBL) Pathway ▪ Triggered by binding of MBL (another patternrecognition receptor) to mannose-containing CHO on bacteria/viruses Classical Pathway ▪ When antibodies (Ab) bind to a pathogen, C1q binds to the Fc portion of those antibodies Similar Activation Mechanism Complement – general principles All pathways lead to the production of a stable C3 convertase ▪ C3 convertase cleaves C3 → C3b and C3a ▪ C3b is an important opsonin and also causes progression through the rest of the complement cascade C3b forms a part of the C5 convertase ▪ C5 convertase usually formed when C3b binds to the C3 convertase ▪ C5 convertase cleaves C5 → C5a and C5b ▪ C5b becomes associated with the cell wall/membrane of the microbe and causes lysis as it activates other components of complement (C6 – C9) ▪ C5b+C6+C7+C8+C9 all associate and form a large pore in the microbial membrane and cause lysis Complement – general principles C3a C5a C3 C3b C5 C5b C3 Convertase C5 Convertase Basically, just the C3 Convertase + C3b C6 C7 C8 C9 MAC Complement – an overall view Note the similarities between the classical and lectin pathway Same C3 convertase Same C5 convertase C1q and MBL look very similar The alternative pathway – the first responder C3 forms C3a and C3b spontaneously in the bloodstream, but is degraded quickly under normal conditions ▪ Factor B, a circulating protein, is also spontaneously cleaved to a protein known as Bb ▪ Bb complexes with C3b to form C3bBb ▪ C3bBb can convert C3 → C3a + C3b It’s a C3 convertase ▪ C3bBb is rapidly inactivated in the uninfected host The alternative pathway – the first responder If a bacterium is present, then C3bBb binds to the bacterial membrane, (C3b is a pattern-recognition receptor) ▪ C3Bb bound to the bacterial membrane is a stable C3 convertase As more and more C3b is generated, then more and more C3Bb forms ▪ Another circulating protein, properdin, helps stabilize the entire complex to form the stable C3 and C5 convertases C5 convertase of the alternative pathway: ▪ C3bBbC3b + properdin ▪ Needs to bind to the bacterial membrane to stay stable and keep converting C5 → C5a + C5b The lectin and classical pathways C1q is a complement protein that will bind to the Fc portion of an Ab that is activated (bound to an antigen) Mannose-binding lectin (MBL) is a circulating pattern-recognition receptor ▪ Recognizes mannose residues on bacterial membranes When C1q detects a bound antibody or MBL detects mannose on a membrane: → they bind complementactivating proteins  these proteins cleave C2 and C4 The lectin and classical pathways C3 convertase: ▪ C4b2a ▪ formed from cleavage of C4 and C2 upon C1q or MBL activation As the C3 convertase acts, C3b accumulates and binds to the complex C5 convertase: C4b2aC3b The C5 convertase cleaves C5 → C5a + C5b Complement → cell lysis Membrane Attack Complex (MAC) Generates a pore in lipid bilayer membranes Sequence of events: C5b triggers assembly of complex of C5b, C6, C7 & C8 Upon binding to C7, C8 will insert into membrane Induces polymerization of C9 (n = 10-16) forming pore in membrane Complement → cell lysis The MAC and the pro-inflammatory effects of C5a and C3a can be extremely damaging to cells if they are not tightly regulated (and limited to microbes) There are many proteins that downregulate or degrade complement components FYI – decay-accelerating factor, factor H, Factor I all cause destruction of the C3 convertases FAQs about complement: What else can C3a and C5a do? ▪ They are both cause vasodilation, increased vascular permeability, smooth muscle contraction (i.e. bronchoconstriction) and histamine release from mast cells ▪ C5a is a chemotactic agent for a wide variety of cells (neutrophils & macrophages in particular) Can people have a deficiency of complement? ▪ Yes – it’s a rare cause of immunodeficiency ▪ Inadequate complement proteins (C2, C3, C4, C5, MBL, MAC complex) tend to make patients vulnerable to bacterial infection FAQs about complement: Complement deficiencies cont… ▪ Deficiencies in C1q highly predispose patients to systemic lupus erythematosus (discussed later in immunology) Photosensitivity rash, arthritis of small joints, renal failure, neurological vasculitis, neuropathies, and diverse effects on heart and lungs Prevalence: 50 – 100/100,000 ▪ C1q helps macrophages to clear apoptotic bodies as well as initiate the classical pathway (C1q recognizes phosphatidylserine) Thought that continual presence of self-antigens (especially nuclear material in the extracellular space) from dying cells increases the likelihood of developing autoimmunity C1q may also have complex immunomodulatory roles with Th cells FAQs about complement Why is the alternative pathway the first responder, and the lectin/classical pathways more effective later? ▪ C3 is always present in the bloodstream – it’s constantly being produced by the liver, cleaved, and degraded If a microbe is present, C3b instantly binds (non-specifically) to the cell wall/membrane – if properdin and Bb also bind, then the stable C3 convertase forms very quickly ▪ Mannose-binding lectin does not circulate in high concentrations unless it is secreted by the liver in response to pro-inflammatory signals ▪ Significant quantities of antibodies take days – weeks to produce Complement – need-to-knows How are the 3 pathways of complement activated? What is the C3 convertase for each pathway? ▪ Remember – C3 convertase is the same for the classical and lectin pathway ▪ What is needed to stabilize the C3 convertase in the alternative pathway? What is the C5 convertase for each pathway? ▪ Remember – it’s usually just C3b attached to the C3 convertase What is the role of MAC? How (in general) is it activated? What are the other functions of complement components other than forming MAC? ▪ Relate these to inflammatory processes How can complement deficiency be linked to immunodeficiency and autoimmunity? Module 2 – PRRs, part 2 For TLRs, NLRs, RLRs, and C-lectin receptors: Given a particular pathogen, suggest a logical PAMP-receptor that would detect that pathogen Relate the compartments in which the receptors are found to their ligands Describe the basic signaling mechanisms that lead to cellular activation after PAMP-R activation Describe the immunological consequences of PRR activation Describe the general process of inflammasome activation and the entities that activate the inflammasome Relate inflammasome activation to production of IL-1 and the process of pyroptosis Describe the cells that are key sentinels for pathogen invasion or tissue damage Pattern Recognition Receptors - PAMPs Toll-like receptors have already been discussed – which should you know? ▪ TLR1 – can detect mycobacteria (like TB) and gram-negative bacteria ▪ TLR2 – can detect peptidoglycans – major component of cell wall of gram-positive bacteria ▪ TLR3 – double-stranded RNA → only found in viruses ▪ TLR4 – lipopolysaccharide (LPS), major component of gram negative bacteria Toll-like receptors tend to recognize PAMPs in the ECF or in endosomes (not in the cytosol) Pattern Recognition Receptors - PAMPs C-type Lectin receptors – detect carbohydrate components of many microbes (viruses, fungi, mycobacteria, parasites, some bacteria) ▪ Found on the cell membrane of a wide variety of immune cells (macrophages, dendritic cells, neutrophils, lymphocytes) ▪ These receptors are also found on the cell membrane, so they don’t detect pathogens in the cytosol Pattern Recognition Receptors - PAMPs NOD-like receptors (NLRs) are present in the cytosol of a wide range of immune cells (dendritic cells, neutrophils, macrophages, lymphocytes) and non-immune cells such as epithelial cells ▪ FYI – NOD-1, NOD-2, NLR-3 as examples Most detect bacteria or parasite cell wall components that are present in the cytosol ▪ Some bacteria and parasites reproduce and spend part of their life cycle inside cells ▪ Some NLRs seem to be able to detect viruses as well Like TLRs, activation of NLRs leads to activation of NF-KB and AP-1 Pattern Recognition Receptors - PAMPs RIG-like receptors (RLRs) are present in the cytosol of many (if not all) immune cells as well as non-immune cells (i.e. endothelial, epithelial cells) RLRs detect viral RNA (particularly double-stranded RNA) and activate: ▪ NF-KB ▪ transcription factors (FYI – IRFs) that lead to the production of particular “antiviral” cytokines known as interferons More about interferons in later modules PAMP overview What are the main ligands for each type of major PAMPreceptor? What cellular compartment/structure is the PAMP-receptor found in? ▪ How does this relate to the ligand that it binds? All of these PRRs seem to activate NF-KB ▪ Additional functions → phagocytosis for lectin receptors, interferon production for RIG-like receptors Danger-Associated Molecular Patterns DAMPs DAMPs are molecular “signals” that are present when a cell is damaged We’ve talked about many of these signals before: ▪ Loss of intracellular K+ due to loss of cell membrane integrity ▪ High concentrations of free radicals ▪ Extracellular ATP (leakage from cells due to loss of membrane integrity) ▪ Unfolded proteins (suggested by some studies) DAMPs can also be crystals that “shouldn’t be there” ▪ Cholesterol crystals (cellular damage or oxidized LDL) ▪ Uric acid crystals (gout) DAMP detection Somehow, one NOD-like receptor seems capable of detecting a wide range of DAMPs as well as PAMPs ▪ Known as NLRP3, though other NLRs may be able to do this ▪ How NLRP3 detects so many diverse signals is unclear and an area of intense research When a DAMP activates NLRP3, it associates with caspase 1 and activates it by forming a large molecular complex known as an inflammasome Caspase 1 activation has two major effects: ▪ Activation of pro-IL-1-beta to IL-1-beta ▪ Insertion of a cell membrane pore – gasdermin - into the cell membrane The inflammasome Inflammasomes are a molecular assembly that activates caspase 1 → Conversion of pro-IL-1 to IL-1 Activation of a cellular pore known as gasdermin IL-1 tends to leave the cell via gasdermin pores If enough gasdermin is produced, then the cell (usually a macrophage) will lyse and die → release of more DAMPs This can lead to activation of neighbouring cells This type of cell death is known as pyroptosis https://commons.wikimedia.org/wiki/File:Inflammasome_structure.png DAMP detection IL-1-beta is one of the most important pro-inflammatory cytokines and has a wide range of effects ▪ Released in very large quantities (for a cytokine) by macrophages in response to significant infection or tissue damage IL-1 is generated and released in a two-step process: ▪ Step 1 (priming) – detection of PAMPs or DAMPs → synthesis and storage of large quantities of pro-IL-1 ▪ Step 2 (release) – continued or larger DAMP or PAMP stimuli → NLRP3 activation → caspase 1 activation → conversion of pro-IL-1 to IL-1 → IL-1 release The inflammasome – priming and release of IL-1 FYI: Interesting side-note: IL1 can be its own priming signal activation of the IL-1 receptor can increase proIL-1 production The sentinels – what cells are important DAMP and PAMP detectors? Resident macrophages ▪ In someone without disease or infection, circulating monocytes often enter a wide range of tissues and differentiate to macrophages to serve as a “cellular sentinel” Langerhans cells – dermis and epidermis Kupffer cell – liver Alveolar macrophages - lung Microglia – brain Resident macrophages also found in all connective tissue Spleen (and other secondary lymphoid organs) ▪ These cells express most PAMP and DAMP receptors and can secrete large quantities of pro-inflammatory cytokines (often due to NF-K activation) The sentinels – what cells are important DAMP and PAMP detectors? Endothelial cells – blood and lymph vessels ▪ These lining cells express TLRs and RLRs – when they detect a PAMP they: Increase expression of ICAMs and selectins (what do these do again?) Increase the production of pro-inflammatory cytokines and chemokines Epithelial cells – skin and mucosal surfaces ▪ PRRs tend to result in increased production of local antimicrobial peptides ▪ Chemokines and cytokines can also be produced if more leukocytes need to be recruited from the circulation Module 3 – Inflammatory mediators, part 2 For IL-1, IL-6, and TNF-alpha: Describe the major cellular sources Describe local and systemic functions as they relate to the process of acute inflammation Describe the mechanism of fever generation and the general signaling process for fever induction Describe the source of and general role for the following acute phase reactants: CRP and mannose-binding lectin Ferritin and hepcidin Serum amyloid A The major pro-inflammatory cytokines IL-1, TNF-alpha, IL-6 IL-1 – important cellular sources: ▪ Macrophages/monocytes, dendritic cells, keratinocytes, epithelial cells, endothelial cells TNF-alpha – important cellular sources ▪ Macrophages/monocytes, dendritic cells, mast cells, NK cells, epithelial cells IL-6 – important cellular sources ▪ Macrophages/monocytes, dendritic cells, NK cells, epithelial cells, endothelial cells IL-1, TNF-alpha, IL-6 These cytokines have redundant and pleiotropic effects ▪ Redundant – functions overlap between these cytokines ▪ Pleiotropic – many effects for each cytokine Acute Inflammatory Effect Acts on the hypothalamus → fever IL-1 TNF- IL-6 ✓✓ ✓✓ ✓ Synthesis and release of acute-phase proteins by the liver ✓ ✓ ✓✓ Increased vascular permeability ✓ ✓ ✓ Increased adhesion molecules on vascular endothelium ✓ ✓ X Chemokine production (CXCL-8) ✓ ✓ X Production of IL-6 ✓ ✓ X Hint: just remember the functions IL-6 doesn’t perform Fever and acute inflammation Temperature above 37.7 Celsius ▪ Caused by changing the hypothalamic set-point – your hypothalamus now “thinks” that your normal temperature is higher Mechanisms of higher body temperature: ▪ Peripheral vasoconstriction → blood flow away from the periphery, to the core → less heat loss ▪ Shivering ▪ Increased metabolic rate Benefits to fever? ▪ Adaptive immune mechanisms in general are more effective at higher temperatures Fever and acute inflammation The hypothalamic set-point is altered by increased levels of pro-inflammatory cytokines ▪ IL-1 and TNF- can induce fever at low serum concentrations ▪ IL-6 induces fever at higher (10X) concentrations (less potent) Pro-inflammatory cytokines cause elevation of prostaglandin E2 production by cells in the 3rd ventricle (FYI - area called the OVLT) ▪ PGE2 leads to signaling that changes the hypothalamic setpoint ▪ Why blockers of cyclooxygenase activity (Tylenol, ibuprofen) are effective anti-pyretics Acute phase proteins Elevated levels of inflammatory cytokines – IL-6 in particular - cause the liver to increase the secretion of useful (from an acute inflammation perspective) proteins into the bloodstream ▪ Known as acute phase proteins Major acute phase proteins include: ▪ C-reactive protein (CRP) Opsonin that binds to phosphorylcholine – a component of bacterial cell walls It can also activate C1q, and thus trigger the classical complement cascade when it binds to phosphorylcholine CRP is a common lab measurement ordered to diagnose inflammatory disease Acute phase proteins Major acute phase proteins cont… ▪ Ferritin Binds to serum iron with high affinity – many microbes depend on iron for their metabolism, and ferritin sequesters it from these microbes ▪ Hepcidin Interferes with intestinal transport of iron into the bloodstream – this also sequesters iron from microbes ▪ Mannose-binding lectin (MBL) – we’ve seen this as the PRR that initiates the lectin complement cascade ▪ Serum amyloid protein A (SAA) – complicated molecule Modulates (usually increases) the activation of the inflammasome and TLRs Opsonizes some gram-negative bacteria Module 4 – NK cells and IFNs Differentiate between the function of Type 1 and Type 2 interferons Briefly describe how Type 1 IFNs are produced and how they protect the host from viral infection For NK cells: Briefly describe the process of activation and licensing Describe how NK cells differentiate between virally infected/damaged cells and normal cells Relate the process of NK cell killing to previously discussed concepts in cellular pathology Interferons and the antiviral response 2 major groups: ▪ Group 1 – interferon-alpha (IFN-) and interferon-beta (IFN-) Secreted by macrophages, dendritic cells, and cells that have detected viruses via PRRs Role – to “interfere” with viral replication in a wide variety of cells ▪ Receptors for these Group 1 IFNs found on most cells ▪ Group 2 – interferon-gamma (IFN-) Secreted by Th cells and NK cells Role – activates macrophages and “pushes” the adaptive immune system to a cell-mediated response (more later) Interferons and the antiviral response Type I interferons act in an autocrine and paracrine fashion to provide a rapid, innate protection to viral infection ▪ If Type-1 IFNs and the NK response is inadequate, then we rely on the adaptive immune response Interferons and the antiviral response How do interferons “interfere” with viral replication inside cells? ▪ Inhibit of protein translation in the presence of viral RNA FYI – mediated by protein kinase R (PKR) ▪ Degrading viral mRNA ▪ Inhibition of viral protein assembly FYI – mediated by Mx proteins The net result is that type 1 IFNs reduce the ability of infected cells and the virus to synthesize and assemble proteins The Natural Killer (NK) Cell A specialized cell that is derived from the lymphoid lineage, but has a relatively non-specific way of detecting cancer cells or virally-infected cells ▪ No highly-specific, unique TCR or BCR Large lymphocytes that are activated by Type 1 IFNs and IL-12 ▪ Kill cells that are infected by viruses ▪ Secrete cytokines that predispose the adaptive immune system to adopt a response that relies on cellular effectors rather than antibodies (more later) The NK Cell NK cells survey the body for infected or “stressed”, abnormal cells NK cells have “activating” and “inhibitory” receptors ▪ NK Activating receptors (NKARs) detect molecules expressed on the membrane of cells that are infected by viruses or have developed into cells that may be malignant ▪ NK Inhibitory receptors (NKIRs) detect molecules that are typically expressed by “normal” cells The NK Cell NK cells survey the body for infected or “stressed”, abnormal cells ▪ This surveying increases greatly in the presence of Type I IFNs NK cells have “activating” and “inhibitory” receptors ▪ NK Activating receptors (NKARs) detect molecules expressed on the membrane of cells that are infected by viruses or have developed into cells that may be malignant ▪ NK Inhibitory receptors (NKIRs) detect molecules that are typically expressed by “normal” cells The NK Cell NKARs – examples ▪ NKG2D – an NK receptor that detects “strange” MHC-I molecules on cells More on MHC-I later this week These abnormal MHC-I proteins can not present intracellular antigens and are more typically expressed by virally-infected cells or cells with damaged genomes (i.e. cancer cells) NKIRs - examples ▪ KIR – an NK receptor that detects normal MHC-I molecules on a cell membrane These MHC-I molecules can present antigens NK Cells – the rules they live by Before NK cells can be activated, they need to be licensed to make sure their NKIRs are functional ▪ This is likely done by dendritic cells – protects the body from indiscriminate NK-killing and tissue damage Once they are activated and licensed, they travel to areas of damage/inflammation and “survey” the cells ▪ NKIRs “over-rule” NKARs – if a cell expresses mostly normal MHC-I proteins, for example, then the NK cell does not kill it ▪ If there are many abnormal MHC-I proteins, then the NKARs “over-rule” the NKIRs, and the NK cell kills the abnormal cell NK Cells – general mechanism When NKARs are activated, it kills the cell by: ▪ Receptor-mediated apoptosis via Fas-Fas ligand interaction remember the extrinsic pathway of apoptosis? ▪ Secretion of the proteins perforin and granzyme Perforin pokes holes in the membrane Granzyme directly activates the BH3-only apoptotic protein Bid as well as executioner caspase 3 Module 5 – Innate immunity – time course and appearance Using information from prior lectures, summarize and integrate the events of acute inflammation for a viral vs. a bacterial pathogen, including: How the pathogen is initially detected The immediate signals that develop The cellular and histological findings during the process of acute inflammation Contrast the appearance of acute and chronic inflammation in selected tissues Acute inflammation How do we get from here… To here? ▪ Signaling events? ▪ What cells predominate? ▪ What are the functions of the molecules and the cells? Acute inflammation Given a viral invader: ▪ What PRRs could be activated in the tissue? ▪ What cells are likely detecting the invader? ▪ What signals could they send out – relate to each stage of inflammation: Vasodilation Increased vascular permeability Emigration ▪ Which cells are most likely to be recruited from the circulation? Acute inflammation Given a bacterial invader: ▪ What are the major innate immune defences that can destroy an invader early on (before adaptive immunity)? Molecules? Cells and cellular activities? Acute inflammation Given a viral invader: ▪ What PRRs could be activated in the tissue? ▪ What cells are likely detecting the invader? ▪ What signals could they send out – relate to each stage of inflammation: Vasodilation Increased vascular permeability Emigration ▪ Which cells are most likely to be recruited from the circulation? Acute inflammation Given a viral invader: ▪ What are the major innate immune defences that can destroy an invader early on (before adaptive immunity)? Molecules? Cells and cellular activities? Time course of innate responses Panel A – myocardium one day post-heart attack Panel B – myocardium days postheart attack What are the outcomes of acute inflammation? Acute and chronic inflammation – the lung