Immunology Exam 3

What are some of the mechanisms by which intracellular microbes resist phagocyte killing?

- Interfere with phagocytic engulfment by scavenging/inhibiting/degrading opsonins - Modify surface to degrade or shield against antimicrobial factors - Counteract acid accumulation - Produce enzymes, such as catalase, that detoxify/neutralize ROS/RNS - Express enzymes that prevent recruitment of the protein complexes that synthesize ROS/RNS - Overcome reduced iron levels by secreting siderophores - Mount stress response to dispose of and replace damaged proteins

Which types of TLRs are at the plasma membrane? What about the intracellular membranes?

- TLRs that recognize bacterial and fungal cell wall components, such as TLR4, TLR5, and the heterodimers TLR2/TLR1 and TLR2/TLR6, are localized to the plasma membrane * plasma is even and prime: 1/2, 2/6, 4, 5 -bacterial lipopeptides detected by heterodimers TLR1:TLR2 and TLR2:TLR6 (hetero people are fat/happy: lipo and pep) each heterodimer has a different ligand specificity*** TLR1:TLR2 detects triacyl lipopeptides (1 plus 2 = tri) TLR2:TLR6 detects diacyl lipopeptides (both divisible by di) - bacterial peptidoglycans detected by TLR2 (primary pep wants 2 be happy) - LPS detected by TLR4 (lips four letter word) - bacterial flagellin detected by TLR5 (5 looks like a wiggly flagella) - TLRs recognizing nucleic acids (TLR3, TLR7, TLR8, TLR9) are primarily or completely contained in intracellular membranes (phagolysosome or endosome compartment) * intracellular is odd plus 8 (8 is contained): 3, 7, 8, 9 -dsRNA detected by TLR3 (d = delta = three) - ssRNA detected by TLR7 and TLR8 (s looks like 8. there are two s's so 8 get backup from 7) - CpG DNA detected by TLR9 (nien a certain type of godly DNA...) Another thing: TLRs depend on other CO-RECEPTORS for full ligand sensitivity. (the toll needs co-workers to catch everyone) Example: TLR4 recognition of LPS requires MD-2 (you need an MD 2 do your lips)

How does the specificity of innate immunity differ from that of adaptive immunity?

-innate immune effector cells and molecules are fully functional even before infection or are rapidly activated by microbes - innate: 0-12 hours, adaptive 1-5 days - innate immune mechanisms eliminate damaged cells and initiate tissue repair (for both infected and sterile cells) - innate stimulates adaptive responses and can influence the nature of the adaptive responses to make them optimally effective against different types of microbes Specificity Innate - for structures shared by classes of microbes ("molecular patterns") - often essential for survival of the microbe - different microbes but identical mannose receptors Adaptive - for structural detail of microbial molecules (antigens); may recognize nonmicrobial antigens - different microbes AND distinct antibody molecules

CR2 Functions to Enhance B Cell Activation

1. Complement receptor 2 (CR2) is expressed on B cells 2. Binds the cleavage products of C3b, called C3d, C3dg and iC3b, which are generated by factor I- mediated proteolysis Microbial antigens that have bound these cleavage products can simultaneously engage both CR2 and the BCR on the surface of a B cell This leads to the initiation of signaling cascades that enhance the responses of B cells to antigen In humans, CR2 is the cell surface receptor for Epstein-Barr virus, a herpesvirus that causes infectious mononucleosis and is also linked to several malignant tumors

List two consequences of complement deficiencies?

1. Deficiency of C1 INH: Autosomal Dominant Disease Called Hereditary Angioedema - Clinical manifestations include intermittent acute accumulation of edema fluid in the skin and mucosa - Causes abdominal pain, vomiting, diarrhea, and potentially life-threatening airway obstruction - Plasma levels of C1 INH protein are sufficiently reduced (<20-30% of normal) that activation of C1 by immune complexes is not properly controlled and increased breakdown of C4 and C2 occurs -C1 INH is an inhibitor of other plasma serine proteases besides C1, including kallikrein and coagulation factor XII and both activated kallikrein and factor XII can promote increased formation of bradykinin - Treatment: Recombinant C1 inhibitor 2. Deficiency in C1q, C2 or C4 - More than 50% of patients with C1q, C2 and C4 deficiencies develop lupus - Important role in clearing apoptotic bodies containing fragmented DNA - Apoptotic bodies may be a source of nuclear antigen that trigger autoantibody responses - Complement proteins also regulate antigen-mediated signals received by B cells; in their absence, self antigens may not induce B cell tolerance, and autoimmunity results - Deposition of immune complexes in tissue results in inflammation and tissue damage

How do phagocytes ingest and kill microbes?

1. Phagocytic cells facilitate the removal of aged red blood cells, necrotic tissues and foreign substances from the circulation 2. Homeostatic functions are increased by a variety of activating stimuli after tissue injury or during infection 3. Cells express pattern recognition receptors that identify pathogen-associated or damage-associated molecular patterns expressed by microbial pathogens or during cellular stress, respectively 4. These receptors 'instruct' phagocytes to engulf (phagocytosis) and destroy through the generation of a respiratory burst (promote inflammation) Interaction can be through pattern recognition receptors (e.g. TLR) or through receptors that recognize opsonins Phagocytic activation: - Enhances rate of phagocytosis - Enhances production of toxic reactive oxygen species (antimicrobial) - Enhances production of nitric oxide(antimicrobial) - Enhances phagosome-lysosome fusion - Increases number of MHC class II molecules - Secretion of IL-12: differentiation of CD4 T cells phagocytes include 1. Neutrophils - phagocytosis - reactive oxygen and nitrogen species - antimicrobial peptides (anti = no = n for. neutrophil) 2. Macrophages - phagocytosis - inflammatory mediators - antigen presentation - reactive oxygen and nitrogen species - cytokines - complement proteins (come from monocytes: bone marrow stem cell to blood monocyte to tissue macrophage then activation)

Antiviral state

1. inhibition of viral protein synthesis 2. degradation of viral RNA 3. inhibition of viral gene expression and virion assembly

What are the mechanisms by which the epithelium of the skin prevents the entry of microbes?

1. physical barrier by intact epithelial surface 2. epithelial cells produce antimicrobial chemicals (ex. defensins) that further impede entry of microbes 3. locally produced antibiotics 4. intraepithelial lymphocytes loss of the integrity of these epithelial layers by trauma or other reasons predisposes an individual to infections Innate mechanisms by organ or tissue: - Skin: antimicrobial peptides, fatty acids in sebum - Mouth and upper alimentary canal: enzymes, antimicrobial peptides, and sweeping of surface by directional flow of fluid toward stomach - Stomach: low pH, digestive enzymes, antimicrobial peptides, fluid flow to large intestine - Large intestine: normal intestinal flora compete with invading microbes, fluid/feces expelled from rectum - Airway and lungs: cilia sweep mucus outward, coughing, sneezing expel mucus, macrophages in alveoli of lungs

Other Functions - Recap compliment system

By binding to antigen-antibody complexes, complement proteins promote their clearance by phagocytes Immune complexes with attached C3b are bound to CR1 on erythrocytes, and the complexes are cleared by phagocytes in the liver Opsonized antigens are also bound by follicular dendritic cells (FDCs) in the germinal centers of lymphoid organs. FDCs display antigens to B cells and this process is important for the selection of high-affinity B cells Cleavage products of C3b bind to CR2 on B cells and facilitate B cell activation and the initiation of humoral immune responses Local complement activation has a role in T cell activation and function either directly through stimulating complement receptor- mediated signaling events in T cells or indirectly through modulating antigen presenting cell function

Anaphylatoxins

C3a and C5a act on specific receptors on endothelial cells and mast cells When produced in large amounts or injected systemically, they induce a generalized circulatory collapse, producing a shock-like syndrome Such a reaction is termed anaphylactic shock, and these small fragments are hence called anaphylatoxins C3a and C5a are cleaved by carboxypeptidase N to produce a desArg variant, which has reduced biological activity

Pathway initiations

Classical pathway of activation - Recognized by C1q: The overall scheme of the classical pathway is similar to the lectin pathway, except that it is initiated by a different protein (C1 complex = C1(C1qr2s2)) The C1 complex is composed of a large subunit (C1q), which acts as the pathogen sensor, and two serine proteases (C1r and C1s) When two or more of the globular heads of C1q interact with a ligand, this causes a conformational change in the C1r:C1s complex, which leads to activation of an autocatalytic enzymatic activity in C1r Active C1r cleaves and activates C1s Activated C1s cleaves the next protein in the cascade, C4 Complement protein C1 binds to the CH2 domains of IgG or the CH3 domains of IgM that have bound antigen Among IgG antibodies, IgG3 and IgG1 are more efficient activators of complement than other subclasses IgM is a potent activator of complement Only antibodies bound to antigens, and not free circulating antibodies can initiate classical pathway activation. Each C1q molecule must bind to at least 2 immunoglobulin heavy chains to be activated and each monomeric immunoglobulin Fc region has only one C1q-binding site Lectin pathway of activation - Recognized by proteins such as mannose binding lectin Alternative pathway of activation - Triggered by spontaneous hydrolysis of C3 Three pathways converge at the central and most important step in complement activation (formation of C3 convertase) There are several types of C3 convertase (depends on the complement pathway activated; C4bC2a and C3bBb) Each is a multi-subunit protein with protease activity that cleaves C3 into C3b and C3a

Important for Phagocytosis, Immune Complex Clearance and B Cell Responses

Complement Opsonins. Cells interact with complement fragments via complement receptors (CR)

How is the complement system activated?

Complement activation depends on the generation of two proteolytic complexes: the C3 convertase, which cleaves C3 into two proteolytic fragments called C3a and C3b; and the C5 convertase, which cleaves C5 into C5a and C5b 3 Activation Pathways: i. Alternative Pathway - occurs all the time, spontaneous hydrolysis of C3 ii. Classical Pathway - d/t IgG + IgM 1. C1 complex iii. Lectin Pathway - recognition of specific carbohydrates (e.g. mannose-binding lectin) found on microbes iv. All three merge @ C3 convertase activation 1. Different C3 convertases dependent on pathway (e.g. C4bC2a + C3bBb) 2. C3 convertases are multi-subunit proteins w/ protease activity that cleaves C3 into C3b + C3a Key Points of activation pathways a. Amplification of C3 cleavage d/t the ability of alternative pathway C3 convertase to be formed from C3b generated from any of the classical, lectin, or alternative pathways b. C3b + C4b - cleavage induces conformational changes which expose thioester domains i. Covalently attch't to cell surfaces via NH3 or OH groups of surface proteins OR polysaccharides = amide or ester bonds ii. Lack of bonding = quick hydrolysis + inactive proteins - The C3 (and C4) proteins contain a reactive thioester bond that is buried in a region of the protein - When C3 (C4) is cleaved, the C3b (C4b) molecule undergoes aconformational change and the thioester domain is exposed - C3b (C4b) may become covalently attached to the surface of cells through the thioester domain, which reacts with amino or hydroxyl groups of cell surface proteins or polysaccharides to form amide or ester bonds - If the bonds are not formed, the C3b (C4b) reactive thioester bond is quickly hydrolyzed rendering the protein inactive

Compliment regulation

Complement activation needs to be regulated for two reasons: 1. Low level complement activation goes on spontaneously, and if activation is allowed to proceed, the result can cause damage tonormal cells and tissues 2. Even when complement is activated where needed, such as on microbial cells, it must be controlled because degradation products of complement proteins can diffuse to adjacent cells and injure them Different regulatory mechanisms inhibit the formation of C3 convertases in the early steps of complement activation, break down and inactivate C3 and C5 convertases, and inhibit formation of the MAC in the late steps of the complement pathway C1 INH is a serine protease inhibitor (serpin) that mimics the normal substrates of C1r and C1s C1 INH becomes a target of the enzymatic activity of the bound C1r2-C1s2 C1 INH is cleaved by and becomes covalently attached to C1r2-C1s2 and as a result the C1r2-C1s2-C1 INH dissociates from C1q C1 INH prevents the accumulation of enzymatically active C1r2-C1s2 in the plasma and limits the time for which active C1r2-C1s2 is available to activate subsequent steps in the complement cascade Proteolytic activity of C1r and C1s is inhibited by a plasma protein called C1 inhibitor (C1 INH) If C3b is deposited on the surface of normal host cells, it may be bound to several membrane proteins, including MCP, CR1, DAF and plasma protein Factor H If C4b is deposited on the surface of normal host cells it may be bound by MCP, CR1, DAF and plasma protein C4BP By binding to C3b or C4b these proteins competitively inhibit the binding of other components of the C3 convertase, such as Bb and C2a, thus blocking further progression of the complement cascade Factor H inhibits binding of only Bb to C3b and thus is a regulator of the alternative but not classical pathway Factor I circulates in an active form but can cleave C3b and C4b only when bound to a cofactor MCP, Factor H, C4-BP and CR1 all serve as cofactors for Factor I-mediated cleavage of C3b (and C4b) Catalyzes the cleavage of C3b or C4b into inactive products, which do not participate in complement activation but are recognized by receptors on phagocytes and B cells - Cleaves C3b into iC3b and then into C3dg - Cleaves C4b into C4c and C4d Breakdown of C3: Formation of C3 opsonins

CR1 receptor functions to promote phagocytosis

Complement receptor 1 (CR1) is a high affinity receptor for C3b and C4b Phagocytes use this receptor to bind and internalize particles opsonized with C3b or C4b The binding of C3b- or C4b- coated particles to CR1 also transduces signals that activate the antimicrobial mechanisms of phagocytes, especially when the Fcγ receptor is simultaneously engaged by antibody-coated particles CR1 on Erythrocytes Binds C3b and C4b on Circulating Immune Complexes 1. CR1 on erythrocytes binds circulating immune complexes with attached C3b and C4b and transports the complexes to the liver and spleen 2. Phagocytes remove the immune complexes from the erythrocytes and the erythrocytes continue to circulate

Describe danger signals and how the innate immune system activates the adaptive immune system?

Danger signals, aka alarmins or DAMPS may be produces as a result of cell damage caused by infections, but they may also indicate sterile injury to cells caused by chemical toxins, burns, trauma, or decreased blood supply. Examples: 1. Stress-induced proteins: HSPs (heat shock protein) 2. Crystals: monosodium urate 3. Nuclear proteins: HMGB1 (high-mobility group box 1)

Which Ig will activate the classical compliment system?

Even though circulating IgM is pentameric, it does not bind C1q because the Fc regions of free IgM are in a configuration that is inaccessible to C1q Binding of IgM to an antigen induces a conformational change that exposes the C1q binding site in the Fc region Because of its pentameric structure, a single IgM-antigen complex can bind at least two C1q globular heads Because each IgG molecule has only one Fc region, multiple IgG molecules must be brought close together before C1q can bind, and multiple IgG antibodies are brought together only when they bind to a multivalent antigen

MAC formation

Formation of MAC is Inhibited by a Membrane Protein Called CD59 CD59 is a glycosylphosphatidylinositol (GPI)-linked protein expressed on many cell types Incorporates itself into assembling MACs after membrane insertion of C5b-8; thereby inhibiting the addition of C9 molecules

Draw a diagram of the complement system?

Function: opsonization of microbes + recruitment of phagocytes; may directly kill microbes Collection of membrane-bound + fluid phase proteins w/in blood, lymph, and IF i. Fluid phase to inactive or transiently active (≤ 5 secs) 1. Stable activation after attch't to microbes, Abs or dying cells Produced by most cell of body, but most synthesis occurs in liver Cascade allows tremendous amplification because each enzyme molecule activated at one step can generate multiple activated enzyme molecules at the next step Some of the products of complement activation become covalently attached to microbial cell surfaces (C3b, C4b) Complement activation is inhibited by regulatory proteinsthat are present on normal host cells and absent from microbes Sequential Proteolysis - induction of proteolytic cascades: zymogens to active proteases i. Cleave + induce proteolytic activity of next complement protein in cascade ii. Activation amplification iii. Some activation products form covalent bonds with microbial surfaces (e.g. C3b, C4b) Inhibited by regulator proteins on normal host cells + absent from microbes i. Regulation targets C3 activation, the convergence of all 3 activation pathways Nomenclature i. Products of cleave reactions designated by adding lower-case letter as suffix 1. Ex: C3 cleavage into C3a + C3b a. Larger fragments always denoted as "b" and binds b. Exceptions i. C2 into a is bigger + binds c. C1 complex i. C1q + C1r + C1s subunits ii. C1q - sensor protein iii. C1r + C1s serine proteases are inactive + only active upon binding of C1q to 2 Fc regions iv. Does NOT create cleavage products upon binding

What are the functions of the complement system, and what components of complement mediate these functions?

Function: opsonization of microbes + recruitment of phagocytes; may directly kill microbes. In addition, products of complement activation facilitate the activation of B cells and the production of antibodies Effector Functions: C3 1. C3a: inflammation 2. C3b: opsonization + phagocytosis C5 1. C5a: inflammation Membrane Attack Complex (MAC): lysis of microbes. C5 convertases generated by the alternative, classical, or lectin pathway initiate activation of the late components of the complement system, which culminates in formation of the membrane attack complex (MAC) Late components C5 convertase - Initiate activation of late components (cleavages of C5, C6, C7, C8, C9n) = formation of membrane attack complex (MAC) - C3bBbC3b - generated by alternative pathway - C4bC2aC3b - generated by classical + lectin pathways - Bb + C2a - serine protease activity - C5 into C5b + C5a C5b remains bound to complement proteins deposited on cell surface - C6, C7, C8, C9 do NOT have enzymatic activity - C5b possesses transient binding ability for C6 + C7 C7 is hydrophilic = inserts into PM - High affinity for C8 - C5bC6C7C8 complex (aka C5b-8) has limited ability to lyse cells Binding of multiple C9 proteins to C9 polymer = active MAC (aka C5b-9n) MAC forms pore in the PM = cell lysis

Paroxysmal Nocturnal Hemoglobinuria (PNH)

Impaired Synthesis of CD59 and DAF Can Lead to Paroxysmal Nocturnal Hemoglobinuria CD59 and DAF are both linked to the cell surface by aglycosylphosphatidylinositol (GPI) tail One of the enzymes involved in the synthesis of GPI tails is encoded on the X chromosome In people with a mutation in the gene encoding this enzyme, both CD59 and DAF fail to function. This causes the disease paroxysmal nocturnal hemoglobinuria Characterized by recurrent episodes of intravascular red blood cell lysis. Leads to chronic hemolytic anemia andvenous thrombosis Tendency for intravascular hemolysis to occur at night Acute treatment includes steroids and transfusions Long term prophylaxis includes warfarin, complement inhibition (eculizumab = anti-C5)

What is the role of MHC molecules in the recognition of infected cells by NK cells, and what is the physiologic significance of this recognition?

In general, the activating receptors recognize ligands on infected and injured cells, which need to be eliminated, and the inhibitory receptors recognize healthy normal cells, which need to be preserved. MHC Class I will be present on healthy host cells, thus inhibiting NK cell activation. - Receptors recognize a heterogeneous group of ligands - Some ligands may be expressed on normal cells and some are expressed mainly on infected, stressed or transformed cells - The activating receptor FcγRIIIA, is a low-affinity receptor for IgG antibodies > important for ADCC (antibody- dependent cell-mediated cytotoxicity) (FcγRIIIA = F-uckyouRIIIAn = KILL) - Most NK cells express inhibitory receptors that recognize MHC I, which are expressed on all healthy nucleated cells - The ability of NK cells to become activated by host cells that lack MHC I has been called recognition of missing self Early in the course of a viral infection, NK cells are expanded and activated by IL-12 and IL-15, and they kill infected cells before antigen- specific CTLs can become fully active NK cells may also be important later in the course of viral infection by killing infected cells that have escaped CTL-mediated immune attack by reducing expression of class I MHC molecules

What is the inflammasome, and how is it stimulated?

Inflammasome is a complex formed following the activation of NLRP receptors by cytosolic PAMPs and DAMPs. Specific inducers: - microbial products (flagellin, muramyl peptide, LPS, pore-forming toxins, bacterial and viral RNA) - environmentally (asbestos, silica) or endogenously derived crystals (monosodium urate in gout, cholesterol in atherosclerosis) - extracellular ATP, reactive oxygen species, reduction in cytosolic potassium ion levels Cascade: After binding a ligand, multiple NLRP3 proteins interact to form an oligomer NLRP3 oligomers bind an adaptor protein called ASC Adaptor binds an inactive precursor form of the enzyme caspase-1 Caspase-1 becomes active only after recruitment to the inflammasome complex Caspase cleaves inactive cytoplasmic precursor of cytokines IL-1β and IL-18 Active forms of IL-1β and IL- 18 leave the cell and perform various pro-inflammatory functions 1. Ligand binds to NLRP 2. multiple NRLP3 proteins combine to form an oligomer 3. NLRP3 oligomer is a loner, but it ASCs for help by binding to an adaptor protein called ASC. 4. ASC helps by binding inactive precursor form of enzyme caspase-1 (who is violent) above is triggered by: pathogenic bacteria extracellular ATP, or bacterial products, crystals, K+ efflux, reactive oxygen species) occurs in cytosol 5. since caspase-1 is violent (therefore pyrogenic) it becomes active ONLY after gaining access to inflammasome complex. 6. now caspase-1 can cleave (axe) pro-IL 1B and pro-IL 18, (which were released from nucleus into cytosol following gene transcription by innate signals, such as TLRs) 7. IL-1B and IL-18 are now active and secreted from the cells to wreak havoc via pro-inflammatory functions, such as acute inflammation

What are examples of microbial substances recognized by the innate immune system, and what are the receptors for these substances?

Innate system recognizes a relatively limited set of molecular structures (1000 products of microbes and damaged host cells). What is recognized from the microbial products are shared by classes of microbes: microbial-associated molecular patterns (MAMPS) or pathogen-associated molecular patterns (PAMPS). Come in four categories: 1. Nucleic acids - ssRNA (single-stranded RNA): Virus [RIG-like receptors] - dsRNA (double-stranded RNA): Virus [RIG-like receptors] - CpG (cytosine-guanine-rich oligonucleotide): Virus, Bacteria 2. Proteins - Pilin: Bacteria - Flagellin: Bacteria 3. Cell wall lipids - LPS (lipopolysaccharide): Gram-negative bacteria TLR4 (gram-negative bacteria). *lips four letter word - Lipoteichoic acid: Gram-positive bacteria TLR2. *discoTECH makes you happy, you want 2 be happy (positive) 4. Carbohydrates - Mannan: Fungi, Bacteria - Glucans: Fungi [C-type lectin-like receptors: Dectin] cellular receptors, pattern recognition receptors (PRRs), are present in different locations in the cells and soluble molecules in the blood and mucosal secretions recognize PAMPS and DAMPS. PRRs are expressed by: 1. phagocytes 2. dendritic cells 3. epithelial cells that form barrier between body and external environment 4. many other types of cells that occupy tissue and organs How are PRRs expressed? - on cell surface - in phagocytic vesicles - in the cytosol of various cell types How do PRRs work? They activate signal transduction pathways that promote the antimicrobial and pro-inflammatory functions of the cells in which they are expressed.

NOD-like receptors (NLRs)

Location: cytosol of - phagocytes - epithelial cells - others DETECT PEPTIDES DERIVED FROM PEPTIDOGLYCAN: These peptides are released from intracellular or extracellular bacteria; in the latter case, their presence in the cytosol requires specialized bacterial mechanisms of delivery of the peptides into host cells Examples: - NOD1/2: bacterial cell wall peptidoglycans - NLRP family (inflammasomes): intracellular crystals (urate, silica); changes in cytosolic ATP and ion concentrations; lysosomal damage Three subfamilies, members of which use different effector domains to initiate signaling: 1. NOD1: - expressed in the cytosol of several cell types including mucosal epithelial cells and phagocytes. - Respond to bacterial cell wall peptidoglycans - Detects D-glutamyl-meso-diaminopimelic acid (iE- DAP) which is found primarily in Gram-negative bacteria and select groups of Gram-positive bacteria (e.g. Listeria and Bacillus) (thing one for example (ie) DABS to say hello, which is a negative behavior. however, some positive impression to listeria and bacillus (bc they naughty)) 2. NOD2: - expressed in the cytosol of several cell types including mucosal epithelial cells and phagocytes. - Respond to bacterial cell wall peptidoglycans (thing one and thing two are peppy with glee). - Highly expressed in intestinal Paneth cells, where it stimulates expression of antimicrobial substances called defensins (nod twice, pan around the room, then reach for intestines as defense) - Detects muramyl dipeptide (MDP) which is ubiquitously present in bacterial peptidoglycan (thing two looks in the mirror, his pep has died, so he looks for more pep and glee) 3. NLRP: INFLAMMASOME! respond to cytosolic PAMPs and DAMPs by forming complexes called INFLAMMASOMES, which generate active forms of the inflammatory cytokines IL-1β and IL-18 (at age 18 I'L let you smoke) Induced by a wide variety of cytoplasmic stimuli: - microbial products (flagellin, muramyl peptide, LPS, pore-forming toxins, bacterial and viral RNA) - environmentally (asbestos, silica) or endogenously derived crystals (monosodium urate in gout, cholesterol in atherosclerosis) - extracellular ATP, reactive oxygen species, reduction in cytosolic potassium ion levels

Toll-like receptors (TLRs)

Location: plasma membrane and endosomal membranes of -dendritic cells - phagocytes - B cells - endothelial cells - many others OVERALL MEMBRANE BOUND Examples: - TLRs 1-9 PAMP/DAMP Ligands: various microbial molecules including bacterial LPS and peptidoglycans, viral nucleic acids - in vertebrate and invertebrates - recognize products of a wide variety of microbes as well as molecules expressed or released by stressed/dying cells - conserved structurally - multiple leucine-rich repeats in extracellular domain - contain conserved TIR (Toll-IL-1 receptor) intracellular domain for signaling Mammalian TLRs are involved in responses to wide variety of molecules expressed by microbial but not healthy mammalian cells. Ligands recognized are structurally diverse and include products of all classes of microbes (tolls want money so widen their consumer population) BINDING of ligand to TLR triggers a signaling cascade and ultimately transcription factors, which induce the expression of genes whose products are important for inflammatory and antiviral responses. Moreover, combined activation of TLRs produces different specific responses. ***Major gene activators are NF-kB and MAP kinases

List the different types of macrophages and their specialist functions?

Macrophages are subcategorized based upon their anatomical location and functional properties. Tissue resident macrophages: less flexible programming determined during ONTOGENY (specific transcription factors and epigenetic modifications direct lineage commitment) - osteoclasts (bone): bone resorption - microglia (brain) - alveolar macrophages (lung): elimination of dust, allergens, and microorganisms - histiocytes (interstitial connective tissue) - kupffer cells (liver) (cut her or let her live): clearance of pathogens and toxins - subcapsular sinusoidal macrophages and medullary macrophages (lymph node): antigen capture and presentation to B cells) - intestinal macrophage: recognition and removal of enteric pathogens, tolerance to food antigens and microbiota - dermal macrophage (langerhans cell): clearance of senescent red blood cells by red pulp macrophages. clearance of blood-born particulate antigens by marginal zone macrophages Macrophage activation phenotypes: flexible; driven by microenvironmental signals (cytokines, transcription factors and epigenetic changes modulate phenotypic and functional plasticity) - Classically activated macrophages (M1 macrophages); mediate host defense and antitumor immunity- pro-inflammatory. Induced by NK cells/T cells + IFN-y. Promotes Th1-Th17 immunity, host defense, anti tumor immunity - Alternatively activated macrophages (M2 macrophages); suppressors and regulate wound healing- anti-inflammatory. Induced by IL-4 and IL-13 (lets two the math: 1+3 = 4). regulate wound healing, suppress T cell response, suppress host defense, suppress anti tumor immunity... seems bad to me? - Regulatory macrophages; secrete IL-10. Induced by Toll ligands and immune complexes. Anti-inflammatory. (on highway 10 there is a toll that controls inflammation) - Tumor-associated macrophages (TAMs); suppress antitumor immunity. regulate wound healing, suppress T cell response, suppress host defense, suppress anti tumor immunity... seems bad to me? - Myeloid-derived suppressor cells (MDSCs); functionally similar to tumor- associated macrophages. populations expand during cancer inflammation infection. regulate wound healing, suppress T cell response, suppress host defense, suppress anti tumor immunity... seems bad to me? Activation: - Macrophages are activated by microbial products such as LPS and by natural killer cell-derived IFNγ - Macrophages are activated by stimuli from T cells (CD40 ligand and IFNγ) - Macrophages also may be activated by other signals to promote tissue repair and fibrosis (not shown)

List the differences between the roles of neutrophils and macrophages?

Macrophages develop from blood monocytes originally coming from bone marrow stem cells. Upon the monocyte leaving the blood and entering tissue, macrophage can be activated. Once activated, it will release mediators: - prostaglandins - leukotrienes - platelet-activating factor - cytokines • Long-lived (weeks to months) • Present in tissues (when mature) • Able to migrate from tissues back into lymph nodes • Oxygen-dependent killing not vigorous • Conditions can be made amenable for intracellular growth of pathogen Pathogens can potentially: - Circumvent respiratory burst - Break out of phagosome - Prevent phagolysosome fusion - Resist granule contents Neutrophils • Short-lived (days) >>> PUS • Circulating • Respond to conditions simulating inflammation, leaving circulation never to return • In response to infections, production from bone marrow increases rapidly • Vigorous respiratory burst during phagocytosis; oxygen-dependent killing lethal • Hostile environment for intracellular pathogens Pathogens can potentially: - Resist phagocytosis - KILL OR BE KILLED!!! (nasty bitches luv)

C5a, C4a, and C3a Stimulate Inflammatory Responses

Microbes Opsonized with C3b, iC3b or C4b Interact with Specific Receptors on Macrophages and Neutrophils: C3b and C4b bind to CR1 iC3b binds to CR3 and CR4 By itself, CR1 is inefficient at inducing the phagocytosis of C3b-coated microbes, but its ability is enhanced if the microbes are coated with IgG antibodies that simultaneously bind to Fcγ receptors Macrophage activation by the cytokine IFN-γ also enhances CR1-mediated phagocytosis C3b- and iC3b-dependent phagocytosis of microbes is a major defense mechanism against infections C5a, C4a, and C3a Stimulate Inflammatory Responses All three peptides bind to mast cells and inducedegranulation, with the release of vasoactive mediators These peptides are called anaphylatoxins because the mast cell reactions they trigger are characteristic of anaphylaxis C5a is the most potent mediator of mast cell degranulation, C3a is about 20-fold less potent, and C4a is about 2,500-fold less potent In neutrophils, C5a stimulates motility, firm adhesion to endothelial cells and, at high doses, stimulation of therespiratory burst and production of reactive oxygen species C5a may act directly on vascular endothelial cells and induce increased vascular permeability and the expression of P- selectin, which promotes neutrophil binding

Respiratory Burst

O2 to H2O2 Radicals: a molecule with one or more unpaired electron - superoxide: O2*- - hydroxyl: *OH Non-Radicals: reactive intermediates of oxygen that harm biomolecules and contribute to disease states - hydrogen peroxide: H2O2 Reactive nitrogen species: - nitric oxide: NO

Combined activation of TLR Example: A microbe activates TLRs 4 and 5, what could you deduce from this information?

TLR4 is activated by LPS, which is gram-negative bacteria. TLR5 is activated by flagella. Therefore, the microbe is most likely a gram-negative, flagellated bacterium.

A microbe activates TLR5 and TLR2/TLR6, what do you know?

TLR5: flagella TLR2/TLR6: diacyl lipopeptide gram-positive

What are the roles of the cytokines TNF, IL-12, and type I interferons in defense against infections?

TNF: Tumor Necrosis Factor alpha (TNF alpha), is an inflammatory cytokine produced by macrophages/monocytes during acute inflammation and is responsible for a diverse range of signalling events within cells, leading to necrosis or apoptosis. The protein is also important for resistance to infection and cancers. IL-12 is secreted following phagocytic activity, induces differentiation of CD4 T cells Both TNF and IL-12 are cytokines released by macrophages (which were activated by microbial products on toll-like receptors, such as LPS, or by natural killer cell- derived IFN-y). They enhance inflammation and adaptive immunity. The innate immune system deals with viral infections by inducing the expression of type I interferons, which inhibits viral replication The most important type I interferons in viral defense are IFN-α and IFN-β Most potent stimuli for type I interferon synthesis are viral nucleic acids Recognized by PRRs in the cytosol and endosomal vesicles (e.g. TLR 3, 7, 8 and 9!) Type I interferons are secreted from cells and act on other cells to prevent the spread of viral infection Viral nucleic acids stimulate pattern recognition receptors and activate the IRF family of transcription factors, which induce type I interferon gene expression Virus (endocytosis) - virus enters cytosol and begins replication - nucleic acid "leakage" into nucleus - PAMP (dsRNA) detected by PRRs like TLR3 in endosomal membrane - PRRs activate IRF family of transcription factors, inducing type I interferon gene expression - IFN-a and IFN-B created - Type I interferon signaling through the interferon receptor activates transcription of several genes that confer on the cells a resistance to viral infection called an !antiviral state! - Virally infected cells secrete interferon that act on and protect neighboring cells that are not yet infected (paracrine action) - Virally infected cells secrete interferon that inhibit viral replication in the same cell (autocrine action) - Induce expression of genes whose products enhance the cell's susceptibility to CTL- mediated killing (e.g. presentation of peptide-class I MHC complexes) - Increase cytotoxicity of natural killer cells

Alternative Pathway Recognition + Activation

The alternative pathway can be initiated by spontaneous hydrolysis and activation of the complement component C3, which can then bind directly to microbial surfaces as C3b Spontaneous hydrolysis (aka tickover) i. C3 to C3(H2O) C3(H2O) binds to Factor B i. Factor B into Ba + Bb d/t cleavage by Factor D ii. C3(H2O)Bb = fluid-phase C3 convertase C3 into C3a + C3b via C3 convertase i. C3b has internal thioester bond - some covalently bonds to Ag-Ab complex or adj cell surface ii. C3b undergoes conformational change - exposure of Factor B plasma protein iii. C3b binds to Factor B 1. Factor B à Ba + Bb d/t cleavage by Factor D 2. C3bBb = alternative pathway C3 convertase iv. C3b formed via classical or lectin pathways can also bind to Factor B 1. C3bBb = alternative pathway C3 convertase a. AP C3 convertase can be generated from C3b generated from ALL THREE pathways 2. Amplification of C3 cleavage à C3b formation v. C3b formed via alternative pathway can bind to alternative pathway C3 convertase 1. C3bBbC3b = alternative pathway C5 convertase When C3b undergoes its post-cleavage conformational change, a binding site for a plasma protein called Factor B is also exposed Factor B binds to the C3b protein that is now covalently tethered to the surface of a microbial or host cell Bound factor B is in turn cleaved by a plasma serine protease called Factor D, releasing fragments Ba and Bb Bb remains attached to C3b forming the alternative pathway C3 convertase C3bBb When C3b is generated by the alternative, classical or lectin pathway, it can form a complex with Bb, and this complex (C3 convertase) is able to cleave more C3 >AMPLIFICATION Some of the C3b molecules generated by the alternative pathway C3 convertase bind to the C3 convertase itself forming the alternative pathway C5 convertase C3bBbC3b

Classical Pathway Recognition + Activation (Complement System)

The classical pathway is initiated when complement component C1, which is comprised of a recognition protein (C1q) associated with two proteases (C1r and C1s), recognizes antibodies (IgM, IgG) already bound to a pathogen C1 complex - initiator; serine protease - Inactive "closed umbrella"; C1r + C1s are hidden to prevent activation - Active "open umbrella" requires TWO globular heads bound to either IgG or IgM bound to Ags 1. IgG3 + IgG1 - are the most efficient activators of complement. Unbound monomer is insufficient for C1 binding. Multiple monomers bound to multivalent Ag is sufficient for C1 binding. 2. IgM - pentamer. Unbound pentamer prevents C1 access to Fc region. Bound pentamer undergoes conformational changes exposing Fc regions. A single pentamer contains sufficient Fc regions for C1 binding. 3. C1r - autocatalytic enzyme activity: cleaves C1s 4. C1s - cleaves C4 + C2 C4 into C4a + C4b via C1s C4b has internal thioester bond - some covalently bonds to Ag-Ab complex or adj cell surface C2 complexes w/ some C4b molecules - C2 into C2a + C2b via C1s - C4bC2a complex = C3 convertase C3 complexes with some C4b + C2a molecules - C3 into C3a + C3b via C3 convertase - C3b has internal thioester bond - some covalently bonds to Ag-Ab complex or adj cell surface - Some C3b molecules bind to C3 convertase - C4bC2aC3b complex = C5 convertase

Lectin Pathway Recognition + Activation

The lectin pathway is initiated by soluble carbohydrate binding proteins (e.g. mannose binding lectin) that bind to particular carbohydrate structures on microbial surfaces Mannose Binding Lectin (MBL) - initiator i. Complexes with MBL-associated serine proteases 1. MSAP-1 + MSAP 2; inactive zymogens ii. Binding to pathogens - conformational change of MASP - activation C4 to C4a + C4b via activated MASP i. C4b has internal thioester bond - some covalently bonds to Ag-Ab complex or adj cell surface C2 complexes w/ some C4b molecules i. C2 to C2a + C2b via MASP 1. C2a is active serine protease ii. C4bC2a complex = C3 convertase C3 complexes with some C4b + C2a molecules i. C3 to C3a + C3b via C3 convertase ii. C4bC2aC3b complex = C5 convertase

Cytosolic (intracellular) Receptors

Three major classes: 1. NOD-like receptors 2. RIG-like receptors 3. Cytosolic DNA sensors Induced by a wide variety of cytoplasmic stimuli that are often associated with infections and cell stress. - microbial products - environmentally (e.g., asbestos) or endogenously derived crystals (e.g., monosodium urate) - reduction in cytosolic potassium ion concentrations Linked to signal transduction pathways that promote inflammation or type I interferon production

Terminal Pathway and last steps

Up to 1000 molecules of C3b can bind in the vicinity of a single active C3 convertase (C4bC2a or C3bBb) Surface bound C3 convertase deposits large numbers of C3b fragments on a pathogen surface and generates C5 convertase activity In the classical and lectin pathways, a C5 convertase is formed by the binding of C3b to C4bC2a (= C3 convertase) to yield C4bC2aC3b (= C5 convertase) In the alternative pathway, a C5 convertase is formed by the binding of C3b to C3bBb (= C3 convertase) to yield C3bBbC3b (= C5 convertase) C5 binds to an acceptor site on C3b, and is thus rendered susceptible to cleavage by the serine protease activity of C2a or Bb This reaction generates C5b and C5a last steps: C5 convertase cleaves C5 into C5a and C5b C5b remains bound to the complement proteins deposited on the cell surface The remaining components of the complement cascade C6, C7, C8, C9 do not have enzymatic activity C5b transiently maintains a conformation capable of binding the next proteins in the cascade, C6 and C7 C7 is hydrophobic and inserts into the lipid bilayer of cell membranes which becomes a high affinity receptor for C8 C5b,6,7,8 (C5b-8) has a limited ability to lyse cells Formation of a fully active MAC is accomplished by binding to C9 C9 is a serum protein that polymerizes at the site of bound C5b-8 to form pores in plasma membrane (C5b,6,7,8,9n = C5b- 9n)

Bactericidal agents produced or released by phagocytes upon ingestion of microbes

V-ATPase - Impedes microbial growth by making lumen acidic - Favors activity of many hydrolytic enzymes that have acidic pH optima - Facilitates generation of superoxide NADPH oxidase - ROS production - Impairs microbial metabolism Inducible nitric oxide (iNOS) synthase - RNS production - Impairs microbial metabolism Antimicrobial proteins/peptides - Scavengers (lactoferrin) - Disrupts integrity (defensin, lysozyme, lipases, proteases) - Degrades (hydrolases, peptidases)

Environmentally derived crystal

asbestos: induces cytosolic receptor that may lead to inflammation or type I interferon production

Why is the complement system effective against microbes but does not react against host cells and tissues?

innate system will recognize host components released or generated after tissue injury: damage-associated molecular patterns (DAMPS, also known as alarmins or danger signals). 1. Triggered by signals not normally found on host cells (e.g. microbial carbohydrates, attached antibodies) 2. Reactive thioester on C3b and C4b are labile (i.e. easily inactivated) 3. Multiple steps, each under tight control by proteins 4. Presence of host regulatory proteins: - Fluid phase (CI inhibitor, Factor H, C4-Binding Protein (C4-BP)) - Membrane-bound (Decay Accelerating Factor (DAF), Membrane Cofactor Protein (MCP), Complement Receptor 1 (CR1), CD59)

endogenously derived crystals

monosodium urate: induces cytosolic receptor that may lead to inflammation or type I interferon production

List NK cell killing mechanisms?

• Innate lymphoid cell (subtype 1) • CONTROLS: - Tumor cells - Viral infections - Intracellular bacterial infections - Stressed cells • FUNCTIONS: - Cytotoxicity (kill other cells) - Cytokine production (recruit and activate or potentially suppress other cells) - Main antiviral cytokine, interferon-gamma (IFNγ) - NK cells kill host cells infected by intracellular microbes, thus eliminating reservoirs of infection - NK cell-mediated cytotoxicity is essentially the same as that of CD8+ CTLs - NK cells respond to IL- 12 produced by macrophages and secrete IFNγ, which activates the macrophage to kill phagocytosed microbes - NK cells distinguish infected and stressed cells from healthy cells - NK cell function is regulated by a balance between signals that are generated from activating receptors and inhibitory receptors - Receptors recognize molecules on the surface of other cells and generate activating or inhibitory signals that promote or inhibit NK responses: if MHC Class I is present on cell, NK will recognize as self and not be activated - Activating receptors stimulate protein kinases that phosphorylate downstream signaling substrates (K in kinase for Kill) - Inhibitory receptors stimulate phosphatases that counteract the kinases ("its ME" - MHC Class I... Phase away by stimulating phosphatases) - Large number of activating and inhibitory receptors expressed on different NK cells

How do innate immune responses enhance adaptive immunity?

• Small populations of lymphocytes express antigen receptors that are structurally the same as those of T or B cells, but these receptors have very little diversity - Natural killer T cells (NKT) - Intraepithelial T cells - γδ T cells - B-1 cells (TI-antigens) Natural Killer T Cells - Invariant NKT (iNKT) and diverse NKT - Exist in a 'poised effector' state; able to respond in an innate-like manner to danger signals and proinflammatory cytokines - Express αβ TCR that preferentially use a single TCRα gene (Vα24- Jα18 in humans) and very restricted Vβ gene segments (mostly Vβ11 in humans) - Unlike 'conventional' T cells which recognize peptide antigens bound to MHC I or II, iNKT cells recognize self and foreign lipid antigens displayed on CD1d molecules - CD1d is a conserved, non-polymorphic MHC class I-like molecule - Member of a larger family that includes the CD1a, CD1b, and CD1c molecules - All present lipids rather than peptides to non- MHC-restricted T cells - CD1d is expressed by many cells of hematopoietic origin, including dendritic cells, macrophages, granulocytes, and B cells Two signals are involved in the physiological activation of iNKT cells: a T cell receptor (TCR) signal provided by a lipid-CD1d complex; and a cytokine signal that depends on the constitutive expression of certain cytokine receptors by iNKT cells iNKT cell activation = rapid production of cytokines The innate immune response provides signals that function in concert with antigen to stimulate the proliferation and differentation of antigen-specific T and B cells The activation of lymphocytes requires two distinct signals, the first being antigen and the second being molecules that are produced during innate immune responses to microbes or injured cells - The requirement for antigen (signal 1) ensures that the immune response is specific - The requirement for additional stimuli triggered by innate immune reactions to microbes (signal 2) ensures that adaptive immune responses are induced when there is a dangerous infection and NOT when lymphocytes recognize harmless antigens, including self antigens Examples of second signals: - costimulators (for T cells) - cytokines (for both T and B cells) - complement breakdown products (for B cells)