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Antimicrobial peptides in mucosal innate host defense - a model
- Recognition - Patterns on pathogen are recognized by TLRs and other receptors
- Signaling - Signal transduction resulting in activation of transcription factors
- Products - Upregulation of defensin (and other) genes
- Response - Defensins and other inflammatory mediators modulate an inflammatory response
- Differential activation by commensal and pathogenic organisms
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Removal of colonized pathogens in the circulation
- Cell recruit - Neutrophils and macrophages are recruited by cytokines, stimulated by LPS and other signals
- Phagocytosis - These cells phagocytose the organisms signals
- IC Kill - Pathogens are killed intracellularly by a variety of mechanisms
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Macrophages have many different cell-surface receptors by which they ________ and ___________
- recognize and bind to pathogens
- enable phagocytosis and intracellular digestion by fusing phagosome with lysosome to form phagolysosome
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Neutrophils' granules
- Primary/azurophilic
- - lysosomal enzymes
- - myeloperoxidase
- - lysozyme
- - BPI - bactericidal permeability increasing protein (antimicrobial peptide)
- Secondary
- - alkaline phosphatase
- - lysozyme; lactoferrin
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NADPH oxidase
- • Located in plasma membrane
- • Dormant in unstimulated cells
- • Activated by protein phosphorylation
- • Inactive -> Chronic Granulomatous Disease
- - Oxygen and nonoxygen pathways
- - Oxygen pathway: NADPH oxidase is used, involved with hexose monophosphate shunt (HMP shunt)
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Oxygen-dependent antibacterial mechanisms - Respiratory burst:
- Stimulation of HMP shunt pathway
- Production of superoxide, hydrogen peroxide, Hydroxyl radical
- H2O2/chloride/MPO
- Products can also be secreted to kill extracellular bacteria
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Respiratory Burst - 1: Superoxide
- Increase in O2 consumption upon stimulation
- Production of superoxide:
- – 2 O2 + NADPH ---> 2 O2.- +NADP+ +H+
- NADPH oxidase
- – causes increase in HMP pathway
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Respiratory burst - 2: H2O2
- 2O2.- + 2H+ --> H2O2 + O2
- Spontaneous or catalyzed by superoxide dismutase
- Microbicidal and cytotoxic
- Excess of H2O2 scavenged by catalase and glutathione
- Interacts with MPO and Cl-:
- – H2O2 + Cl- --> OCl- + OH-
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Respiratory Burst - 3: Hydroxyl radical
- H2O2 + Fe2+ --> OH + OH.
- Enhanced by lactoferrin (Fe binding)
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Oxygen dependent antibacterial mechanisms - summary
- O2 -> O2- (superoxide ion; by NADPH oxidase)
- O2- -> H2O2 (superoxide dismutase, SOD)
- H2O2 -> ClO-, .OH (peroxidase, iron)
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Myeloperoxidase
- Packaged in azurophilic granules
- – 1-5% of the dry weight of the cell
- Requires heme and glycosylation for activity
- Green in color
- Reacts with H2O2 and halides to form hypohalous acids (HXO; X=Cl, Br, or I)
- MPO-H2O2-halide system in the phagosome
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Oxygen-independent antibacterial mechanisms
- BPI (Bactericidal/Permeability Increasing Protein
- Lysozyme and lactoferrin
- Myeloid Antimicrobial peptides
- –Defensins
- –Cathelicidin
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Bactericidal/Permeability Increasing Protein (BPI)
- found in azurophilic granules
- potent LPS binding and inhibition
- bactericidal against G(-) bacteria
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Lysozyme and Lactoferrin
- Antimicrobial proteins
- Found in secretions including saliva
- Lactoferrin appears to be associated with anti-caries activity
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Myeloid Defensins
- Highly concentrated in azurophilic granules of neutrophils in man, rat, guinea pig, rabbit
- Numerous isoforms in each cell
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Infection stimulates macrophages to
- release cytokines and chemokines, initiating an inflammatory response
- Cytokines -> dilate local small blood vessels
- Endothlium increases adhesion expression, attract leukocytes to periphery of blood vessels.
- Leukocytes extravasate
- Blood clotting
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Activated macrophages secrete a range of important cytokines:
IL-1b, IL-6, CXCL8, IL-12, and TNF-a.
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IL-1b
- Activates vascular endothelium
- Activates lymphocytes
- Local tissue destruction
- Increases access of effector cells
- Systemic: Fever; Production of IL-6
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IL-6
- Lymphocyte activation
- Antibody production
- Systemic: Fever; Induces acute-phase protein production
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CXCL8
- chemokine
- Chemotactic factor recruits neutrophils, basophils, and T cells to site of infection
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IL-12
- Activates NK cells
- Induces the differentiation of CD4 T cells into TH1 cells
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TNF-a
- Activates vascular endothelium and increases vascular permeability, which leads to increased entry of lgG, complement, and cells to tissues and increased fluid drainage to lymph nodes
- Systemic: Fever; Mobilization of metabolites; Shock
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Innate immunity against viruses: NK cells
- Lyse virus-infected cells
- Secrete cytokines (IFN-γ)
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NK cell lysis
- Virus-infected and tumor cell have no or fewer normal MHC I (almost all normal cells have it, except RBC), allowing for recognition by NK cells (disabling inhibitory receptors that inhibit signals from activating receptors)
- Virus-infected cells secrete IFN-a and b - activate NK cells
- Perforins creates pores in target cells
- Granzymes enter through pores and cause apoptosis
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Complement
- Circulating plasma proteins that bind membranes
- Microbial membranes lack inhibitory proteins, and are subjected to lysis
- stimulates influx of neutrophils
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ILCs and _____ are different __________.
- NK cells
- innate lymphoid subgroups
- ILCs more localized; NK tends to move around
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ILC_ is similar to NK cells in that _________.
- 1
- induced by IL-12
- secretes IFN-γ
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Classical pathway via _______, lectin pathway via _____, and alternate pathway via ______, to activate ______.
- Ag:Ab complexes
- lectin (instead of Ab) binding to pathogen surfaces
- pathogen surfaces (absence of Ab)
- complement
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For all pathways, following complement activation:
- recruit immunocompetent and inflammatory cells
- opsonization of pathogens
- kill pathogens
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_______: a key link between the innate immune system and the adaptive immune system
Dendritic cells
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Innate immunity and periodontal disease
- Adult onset periodontitis
- – Genetic component
- – Immunosuppression
- – Introduction of pathogenic bacteria (e.g., P. gingivalis)
- Localized Aggressive Periodontitis
- – Genetic component
- – Most often associated with (commensal) Aa: Bacterial encoded virulence factors
- Papillon-Lefevre Syndrome
- - Lack of mature, active cathelicidin (LL-37)
- - Defect in Cathepsin C, which activates Cathepsin G, which induces maturation of LL-37; predispose to bacterial infection
- Morbus Kostmann
- - Inherited, complete lack of cathelicidin (LL-37) in neutrophils
- - Severe gingivitis and periodontitis
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Innate immunity and LAP
- Aa expresses leukotoxin (which kills leukocytes)
- ~70% of patients exhibit defective neutrophil chemotaxis
- –> reduced capacity to respond to infection
- –> reduced killing capacity of neutrophils
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P. gingivalis LPS is recognized by _____.
TLR2, not TLR4
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Innate immunity effectors are found in ____ sites and in _______ cells.
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Four characteristics of inflammation
pain, heat, redness, swelling
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Innate Host Defenses Against Infection
- Anatomical barriers
- – Mechanical factors
- – Chemical factors
- – Biological factors
- Humoral components
- – Complement
- – Coagulation system
- – Cytokines
- Cellular components
- – Neutrophils
- – Monocytes and macrophages
- – NK cells
- – Eosinophils
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Bordet's experiments leading to the discovery of ____.
- complement
- experiment on lysis of Vibrio cholera w/ serum
- Exp. 1
- - Normal fresh serum (no immunity) -> no lysis
- - Immune fresh serum (w/ immunity) -> lysed; Ab?
- - Heated immune serum -> no lysis
- - Some heat labile components, in addition to Ab, while is heat stable, are needed to lyse.
- Exp. 2
- - Heated immune serum + normal fresh serum -> lysed
- - Some heat labile sub in normal fresh serum "complements" the Ab to lyse.
- - Proof of principle
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Complement
- A complex series of proteins
- Activated in a fixed sequence to non-specifically "complement" the action of Ab
- When activated, all cleaved into a big and a small fragments (split product)
- Sequential lysis -> "proteolytic enzymes" for the next
- The fragments are responsible for their function
- Attach to microbes or Ab bound to microbes
- Inhibited by regulatory proteins on host cells
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All pathway of complement activation converge on ______.
- C3
- Classical, alternative, lectin
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Sequence of complement activation
1, 4, 2, 3, 5-9.
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C1 is composed of:
- C1q: binds to Fc of Ab; hexamer; requires multivalent binding
- C1r: protease, activates C1s
- C1s: protease, lyses C4 and C2
- 750kD in total, heaviest among all complements
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C3 has a serum concentration of
>1mg/ml, highest among all complements
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C1q only binds to IgM when it _______ and IgG when it _____.
- binds to Ag, so the Fc regions of the pentamer are exposed
- binds to pathogen surface so that there are multiple Fc regions adjacent to each other (from different IgG molecules)
- Won't activate C1q when freely moving/soluble
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Steps of classical pathway of complement activation
- C1q binds to Fc region of IgG on Ag surface (can be IgM, too), activates C1r -> C1s
- C4 binds to C1q
- C4a is cleaved off, C4b stays on C1q or binds of pathogen surface
- C2 binds to C4 and C1s cleaves C2a off, C4b2b = C3 convertase
- C3 convertase cleaves C3a off
- C3b binds to pathogen surface or C4b2b, Cab2b3b = C5 convertase
- C5a cleaved off, C5b continues.
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Small pieces cleaved off from complement
esp. C3a and C5a, induce inflammatory acute reaction that mimic serious allergic reaction
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C3b binds to antigen via __________.
- thioester linkage
- thioester concealed inside off C3
- exposed after cleaving off C3a
- hydrolyzed/disabled when no microbe available (fluid phase)
- covalently bonded to surface protein or polysaccharide of microbes
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Components involved in alternative passway
C3, factor B, factor D, Properdin
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Alternative pathway of complement activation
- spontaneous cleavage of C3 -> C3b
- C3b hydrolysis/disabled in fluid phase
- C3b binds to pathogen surface
- factor B binds to C3b-Ag
- cleavage of B -> Bb
- C3bBb = C3 convertase, cleavage of more C3; properdin stabilizes C3bBb
- C3b binds to pathogen surface and C3bBb
- C3bBb3b = C5 convertase, cleavage of C5 -> C5b
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Alternative vs. classical
- C3 convertase: C3bBb vs C4bC2b
- C5 convertase: C3bBbC3b vs C4bC2bC3b
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Late steps of complement activation
- C5 convertase cleaves C5->C5b
- C6,C7 bind to C5b
- C8 binds to C5bC6C7; integrate to membrane and shows some lysis
- this complex binds to poly C9 to form MAC (membrane attack complex)
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Surface receptors for complements
- CR1 (CD35) - many cells; phagocytosis, clearance of immune complexes
- CR2 (CD21) - B cells; corecptors for B cell activation, binds to C3d, cleavage product of C3b
- CR3 (CD11b/18), CR4 (CD11c/18) - monocytes / macrophages; phagocytosis
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C-receptor enhanced phagocytosis by mononuclear phagocyte -
- opsonization
- C3b attached to pathogen surface binds to corresponding receptor on phagocyte surface
- the binding enhances the binding Ag-AbFab-ABFc-FcR
- promotes phagocytosis
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Down-regulators of complement activation
- prevents inappropriate binding leading to self-destruction
- C1 INH -> C1r, C1s
- Factor I + membrane cofactor for protein (MCP, CD46) -> C4b, C3b
- Decay accelerating factor (DAF) -> C3b convertase
- CD59 -> C7, C8
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_____________ displace C2b from C4b; __________ displace Bb from C3b.
- DAF, MCP, or CR1
- DAF or CR1
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Biological Functions of Complement
- Opsonization and Phagocytosis - binding of C3b or C4b to microbe (opsonization), recognition by phagocyte's surface receptor, ingested more efficiently
- Stimulation of Inflammation - C3a, C5a = anaphylatoxins, recruit & activates leukocytes
- Complement-mediated Cytolysis - MAC punches a hole on the microbe membrane -> osmotic lysis
- Immune Clearance (i.e., Solubilization of immune complexes; IC) – important to prevent accumulation in the blood and deposition in vessel walls, which can lead to IC-mediated tissue damage, vasculitis in kidney., via inflammatory cells
- Trapping of IC in germinal centers – C receptors on Follicular Dendritic Cells bind IC and present antigen to B cells during humoral immune responses.
- B cell activation – C3d (cleavage product of C3b) binds to CR2 on B cells, providing a signal to initiate B cell activation during humoral immune responses.
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Complement Deficiencies
- Not life-threatening
- Classical Pathway:
- - C1q, C1r
- - C2 (most common) & C4 -- autoimmune diseases (e.g., SLE, systemic leukoerythroblastosis)
- - C3 -- G(+) infections
- Alternative Pathway:
- - Properdin & Factor D -- G(+) infections
- Membrane Attack Complex (MAC): C5-9 - Neisseria bacterial infections
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