Innate immunity Flashcards
what are the general principles of innate immunity?
- oldest form of immunity - all organisms have a form of innate immunity which has changed on an evolutionary scale
- always available - little exposure required, but no memory
- major form of immunity in infants (period between loss of maternal antibodies and the formation of their own)
- initiates and directs adaptive immunity
- adaptive immunity takes ~4-6 days to develop. Innate immunity is critical in controlling infections before this.
what are the key elements of innate immunity?
- barriers - prevent establishment of infection
- pre-formed mediators - proteins/peptides with broad specificity that damage pathogens, induce inflammation or recruit adaptive immune cells
- innate leukocytes
- recognise and activated by pathogen
- eliminate pathogen
- communicate with other cells to direct adaptive immune response
what is the role of barriers in the innate immune system?
Mechanical barriers help prevent infection; secreted chemicals, anti-microbials and commensals make an unfavourable environment for pathogens
what are the main barriers of the body?
keratinised skin
mucosal membranes:
- GU tract
- GI tract
= respiratory tract
what are the features of keratinised skin?
- generally impermeable unless breached
- keratinocytes produce keratin: tough substance – effective barrier
- sebaceous glands produce sebum – contains fatty acids, defensins
- Shedding – remove microbes from skin surface
- Commensals – deter pathogens from colonising
what are the features of mucosal membranes?
- Mucous – traps pathogens from reaching epithelial lining
- Cilia (respiratory tract) – waft away mucus and the microbes trapped within
- Mucous contains secreted enzymes (e.g. lysozyme in tears and saliva)
- low pH (gut, vagina) – deter pathogens replicating, peristalsis (gut) – expel microbes
- Shedding of epithelia in the gut
- Commensals
what are pre-formed mediators? what are the main ones in the innate immune system?
Proteins/peptides with broad specificity that damage pathogens, induce inflammation or help recruit and activate innate immune cells
- lysozyme
- antimicrobial peptides: defensins, complement
what is lysozyme?
- Present in secretions (tears, saliva, mucous)
- Breaks a bond in peptidoglycan – destabilises the cell wall of gram positive bacteria
- More active against G+ve bacteria, where peptidoglycan is exposed.
what are defensins?
- form of antimicrobial peptide
- 29-47 amino acids
- evolutionarily ancient
- Produced by many epithelial cells and neutrophils
what is the structure of defensins?
Beta-sheet core structure stabilised by 3 conserved intramolecular disulphide bonds
- Two subfamilies (alpha and beta) in humans
- Alpha form already exist in granules of innate cells, whereas beta form is synthesised de novo under infection
how do defensins function?
- cationic, insert into membranes and disrupt lipid bilayers in bacteria, fungi and enveloped viruses (take part of the host membrane with them when they leave the host cell)
what is complement?
-Discovered as a heat-sensitive substance that “complemented” antibodies in killing bacteria
- >20 soluble proteins found in blood and other body fluids
- Components normally inert, but “activated” by presence of pathogens or antibody bound to pathogen
- C3 is the most abundant in the body and is critical for the cascade
- Complement cascade – amplification of reactions
- Originally evolved as part of the innate immune response – ancient form of immune defense
- Provides protection early in infection in the absence of antibodies through other “older” activation pathways
which complement protein is most abundant?
C3
how does complement work?
- Many activated complement components are proteases; act on one another to generate a large and a smaller fragment
- e.g. C3 –> C3b + C3a (b is big fragment, a is small)
what is the central event of all complement activation pathways?
- Cleavage of C3 exposes a reactive thioester bond in C3b, which can bind covalently to adjacent proteins/carbohydrates e.g. on the surface of a pathogen.
- Rapidly inactivated in the fluid phase –> prevents further damage.
- The central event of complement activation is cleavage of C3 by a “C3 convertase
what are the 3 pathways of complement activation?
- classical
- mannose-binding lectin (MBL)
- alternative
what is the classical pathway of complement activation?
- Antibody binding to bacterial surface
- This activates C1 to generate a protease, which activates C4, then C2
- C2 then activates C3 convertase
what is the MBL pathway of complement activation?
- MBL binding to mannose on pathogen surface
- This activates MASP1 which activates MASP2
- MASP2 activates C2, which activates C3 convertase
what is the alternative pathway of complement activation?
- Spontaneously activated by recognition of LPS on surface of bacteria
- Factor B, D and P generate C3 convertase
what is C3 convertase in the classical and MBL pathways of complement?
For Classical and MBL pathway, C3 convertase = C4bC2a complex (from cleavage of C4 and C2 by C1 and MASP2, respectively)
what is C3 convertase in the alternative pathway of complement?
For Alternative pathway, C3 convertase = C3bBb
- some C3b is generated spontaneously in body fluids by a “tickover” mechanism
- If C3b generated binds to LPS on a pathogen surface, factor B binds to C3b
- Factor B is cleaved by factor D → C3 convertase (C3bBb).
- The C3bBb convertase is stabilised by factor P (properdin) and activated (works for longer on the bacterial surface
how can complement amplification occur in classical and MBL complement pathways?
C3b generated by the Classical or MBL pathway can also bind factor B – amplification of complement
what are the later stages of complement activation?
- C3 convertase cleaves C3 to C3a and C3b
- C3 convertase + C3b opsonin –> C5 convertase –> C5a and C5b
- C5b, C6, C7, C8, C9 = membrane attack complex
what are the 3 major biological activities of complement?
- recruitment of phagocytes and inflammation
- opsonisation
- membrane attack complex –> cell lysis
how does complement promote recruitment of phagocytes and inflammation?
C5a (C3a) – recruit phagocytes to site of and infection induce inflammation (chemoattractants, anaphylatoxins)
- Innate cells move towards area of high chemoattractant conc
- Phagocytes have C5a and C3a receptors and undergo chemotaxis in response to C5a/C3a peptide – recruitment of phagocytes out of blood stream and into tissues
- C5a/C3a also act on local blood vessels, increasing blood flow, permeability and phagocyte adhesion.
- C5a/C3a binding to C5a/C3a receptors on mast cells induces release of inflammatory mediators such as histamine – role as anaphylatoxins
which complement protein is the more potent chemoattractant?
C5a is the more potent chemoattractant
how does complement promote opsonisation?
C3b promotes opsonisation and phagocytosis
- Pathogens coated with C3b peptide are recognised by phagocytes with C3b receptors, facilitating binding and phagocytosis
how does complement induce lysis of bacteria?
C5b-C9 form the membrane attack complex, leading to lysis
- Activation of C5b – C9 results in C9 polymerisation, forming a pore in the membrane, disrupting it and killing the pathogen.
- Important for killing gram –ve bacteria
why must complement be strictly regulated?
as it may cause damage to host
how is complement regulated?
Components rapidly hydrolysed and inactivated in fluid phase
Soluble and membrane-bound regulatory proteins, e.g.:
- C1 inhibitor inactivates C1 in classical pathway
- Factor H competes with factor B to inhibit alternative pathway
- carboxypeptidase N inactivates C3a and C5a – halts inflammation
- CD59 on host cells binds C9, preventing MAC formation
how are complement deficiencies implicated in disease?
e.g. age-related macular degeneration (lack Factor H)
e.g. paroxysmal nocturnal hemoglobinuria (lack CD59) – complement attacks red blood cells
why is complement so important?
Complement is particularly important in extracellular bacterial and fungal infections, but may be active against some viruses.
- Defects in C3 – increased susceptibility to pyogenic infections e.g. S.pneumonia
- Defects in C5-C9 – increased susceptibility to Neisseria (Gram–ve) infections
Complement interacts with Adaptive Immune system
- Classical Pathway interacts with antibody
- Aids clearance of immune complexes antigen:antibody
- role in activating B and T cells
inappropriate activation of complement plays a role in some autoimmune diseases and asthma
what is the lineage of leukocytes?
Derived from pluripotent stem cells in the bone marrow, which give rise to two main lineages, one for myeloid cells and one for lymphoid cells.
- Myeloid cells are involved in innate response (except NK cells which derived from lymphoid)
- Lymphoid cells are involved in the adaptive response
what are polymorphonuclear granulocytes? how were they categorised?
Leukocytes with different-shaped nuclei and granules in cytoplasm
Stained the cells with different dyes
- Blue basic dye = basophils
- Acidic red dye (eosin) = eosinophils
- No staining = neutrophils
what are neutrophils?
- Make up most (60-70%) leukocytes in blood
- Released in large numbers from bone marrow
- Live for <24hr in blood
- Life extended on entering tissues in response to chemoattractants
- Receptors for C3b, IgG, IgA
what are the main functions of neutrophils?
Main function is phagocytosis
NETosis:
- Neutrophil extracellular trap = DNA + antimicrobials
- When neutrophils are dying, they release a chromatin net with defensins attached, which can catch bacteria and kill them along with it
what are eosinophils?
- Few in blood (normally 6%), but also beneath mucous surfaces
- Receptors for C3b (can undergo phagocytosis), IgG, IgA, (IgE)
what are eosinophils important for?
- important in defence against multicellular parasites
- They release toxic proteins/ROS onto the surface of the parasite to kill it
- Role in allergy (especially asthma) – when activated inappropriately can cause damage to lungs
how do eosinophils function?
Release toxic proteins and free radicals (ROS which binds to pathogens to cause damage) from their granules
what are basophils? how do they function?
- Very few in blood (1% leukocytes are of this type)
- Receptors for C3a, C5a (chemoattractants), IgE –> Release heparin (increased blood flow) and histamine (inflammatory mediator) from their granules
- Defence against parasites, role in allergy
what are mast cells?
- Restricted to tissues - protect mucosal surfaces.
- Receptors for C3a, C5a (chemoattractants), IgE release histamine etc.
- “Sentinel” cells, defence against parasites, role in allergy
what are macrophages and monocytes?
Macrophages can be derived from monocytes during infection:
- Monocytes occur in blood, while macrophages are found in tissues
-Different tissues have different resident macrophages
- e.g. alveolar macrophages (lung), microglia (brain)
- Long-lived
- Act as “sentinel” cells – often the first to detect infections
- “Big eaters” – one can phagocytose 100 bacteria/cell
- Receptors for C3b (opsonisation), IgG, IgA
- Produce pro-inflammatory mediators
- Can present antigen to T lymphocytes – link innate and adaptive
what are dendritic cells?
- Found in skin and lymphoid tissues
- Finger-like processes called dendrites
- Take up foreign material by phagocytosis or micropinocytosis
- Digest foreign material and display fragments on their cell surface
- Specialised for presenting antigen to T cells (APC)
- Constitutively express high levels of Major Histocompatibility Type II (MHCII) proteins
what is phagocytosis important in killing?
important for bacterial and fungal infection
what leukocytes can undergo phagocytosis?
neutrophils, monocytes, macrophages, dendritic cells
what is the process of phagocytosis?
- Bacterium binds to the surface of phagocytic cell - recognition
- Antibody or complement can aid binding - Phagocyte pseudopods extend and engulf the organism by fusing
- Invagination of phagocyte membrane traps the organism within a phagosome
- A lysosome fuses and deposits enzymes into the phagosome (phagolysosome)
- Enzymes cleave macromolecules and generate ROS, destroying the organism
how do self cells avoid phagocytosis?
Healthy “self” cells express a protein (CD47) which is recognised by phagocytes and prevents phagocytosis – inhibitory signal
what are the key bactericidal agents?
- acidification: pH 3.5-4, bacteriostatic/cidal
- toxic oxygen-derived products: superoxide, hydrogen peroxide, singlet oxygen, hydroxyl radical
- toxic nitrogen oxides
- antimicrobial peptides e.g. defensins
- lysozyme - dissolves cell walls of gram-positive bacteria
- acid hydrolases digest bacteria
- competitors - lactoferrin binds iron, vitamin B12 binding protein
what is the most important phagocyte bactericidal agent?
free radicals:
- ROS and nitrogen oxides
Free radicals are short-lived, contained in the phagosome and are produced following the oxidative burst
what is oxidative burst?
Oxidative burst – transient increase in oxygen consumption following phagocytosis due to activation of a membrane-bound NADPH oxidase.
- NADPH + 2O2 → NADP+ + H+ +2●O2- → H2O2 + Cl- → OH- + HOCl
- Active NADPH oxidase converts oxygen to superoxide ion
- A second enzyme converts superoxide to hydrogen peroxide
- In neutrophils, myeloperoxidase converts hydrogen perixode to hypochlorite ions and hydroxyl radicals
how are nitrogen oxides produced in phagocytes?
- Inducible nitric oxide synthetase in the phagosome membrane can also lead to the production of nitric oxide and other toxic reactive nitrogen species
- arginine + 2O2 → citrulline + NO
what are NK cells?
- Derived from lymphoid progenitor, granules in cytoplasm
- Kill infected host cells
- Recognise “altered-self”
- changes in expression of self-Major Histocompatibility Type I (MHCI) proteins
- detects decrease in MHCI expression
- Receptors for IgG (allows killing of antibody-coated infected host cells)
what are NK cells important in killing?
- Important in viral and some intracellular bacterial infections, especially until adaptive immunity is triggered
- Also active against some cancer cells as these have altered proteins on surface
how do NK cells function?
NK cells induce infected host cells to undergo apoptosis
- Activated NK cells produce a pore-forming protein, perforin, which inserts into the membrane of the infected host cell.
- Polarisation of NK cell granules at the interface between the NK cell and target cell
- Granule contents (“granzymes”) are released into the target cell through the perforin channel to activate apoptosis pathway -> Target cells undergo apoptosis.
- Recently discovered that a granzyme can enter intracellular bacteria, killing them directly.
how are pathogens recognised by the innate immune system?
Pattern recognition receptors (PRRs) recognise invariant structures on pathogens (MAMPs) or damaged cells (DAMPs)
what are MAMPs?
Microbe-associated molecular patterns: MAMPs
- Shared by many microbes
- Distinct from “self” cells/molecules
- Critical for survival/function of pathogens
- Conserved
what are DAMPs?
Damage-associated molecular patterns: DAMPs
- Host components released during injury and cell damage
- Generated in response to injury, heart attack, cancer etcg
give some examples of MAMPs:
Bacteria:
- Gram -ve: Lipopolysaccharides (LPS) (outer membrane)
- Gram +ve: Lipoteichoic acid (thick peptidoglycan)
- Flagellins, unmethylated CpG in bacterial DNA, N-formylated proteins
Fungi: Chitin, beta-glucans,
Viruses: dsRNA,
Protozoa: GPI-linked proteins, mannose-rich glycans
give some examples of DAMPs:
- Fragments of extracellular matrix proteins e.g. fragments of fibronectin
- Exposed phosphatidylserine (component of lipid bilayer which is exposed in apoptosis)
- Mitochondrial components outside the cell
- Uric acid – excess purines lead to buildup of uric acid – causes gout crystals in joints
- DNA
how many MAMP/DAMP ligands can PRRs recognise?
> 1000 DAMP/MAMP ligands
what are the classes of PRRs?
Soluble receptors e.g. Mannose-binding lectin found in fluids to activate complement
Membrane receptors:
- Lectin receptors (binds to carbohydrates found on bacteria)
- Chemotactic receptors
- Toll-like receptors (TLR)
Cytoplasmic receptors: NOD-like receptors (NLRs)
Both membrane and cytoplasmic receptors expressed by immune and non-immune cells e.g. fibroblasts, epithelial cells
what are examples of membrane PRR receptors?
MAMP binding may initiate phagocytosis, chemotaxis or signalling.
- e.g. macrophage mannose receptor (recognises mannose), CD14 on macrophages recognises LPS – both of these binding trigger phagocytosis
CHEMOTACTIC RECEPTORS recognise CHEMOATTRACTANTS
- e.g. f-met-leu-phe receptor recognises N-formylated polypeptides, produced by bacteria
Toll-like receptors or TLRs: sensors that signal the presence of microbial components
- Signals that there is danger and triggers gene expression
what are toll-like receptors?
- Ancient pathogen recognition system discovered in Drosophila
- 10 TLRs in humans, each recognising distinct MAMPs
- TLRs can be on the cell-surface or on endosomal membranes (TLR3)
- The extracellular domain of TLR3 has a horseshoe shape formed by leucine-rich repeats which recognises the MAMP. The inner surface has β-sheet structure and forms the ligand-binding domain
- Ligand-induced dimerization triggers signalling
what are cytoplasmic receptors?
Deal with pathogens that escape into the cytoplasm
- e.g. NOD-like receptors
what are NOD-like cytoplasmic receptors?
important in bacterial infections
- large group of cytoplasmic receptors that recognise bacterial components e.g. peptidoglycan, flagellin
- NOD-like, structurally similar to proteins found in plants (nucleotide-binding oligomerization domains).
- Signal expression of pro-inflammatory cytokines – triggers cells to make cytokines
- They do this by triggering assembly of inflammasomes – multi-subunit complex that cleaves inactive cytokine precursors
how do NLRs or TLRs interact with inflammasomes?
- On binding PAMP, some NLRs signal to nucleus to activate cytokine gene transcription and production of cytokines
- Cytokines are produced in an inactive form to avoid unnecessary activation
- NLRs trigger formation of a large disc-like complex, the inflammasome, which can bind a protease (caspase-1 oligomers) that cleave pro-cytokines into active cytokines.
- Some cytokines are released directly, others are made as inactive precursors that must be cleaved to make the active form
what are RIG-I-like receptors?
- involved in viral infections
- viral sensors that detect viral RNA produced within host cells
- Signal expression of interferons (cytokines specific in dealing with viral infections)
what cells are the first to respond to infection? how do they respond?
Tissue macrophages/mast cells often respond first by releasing inflammatory mediators that increase blood flow and vascular permeability, and chemoattractants that attract phagocytes into the tissues.
- cytokines are also produced in the response
what local response is induced upon infection? why is it important?
Inflammation, triggered by infection and tissue damage, is a critical local response to infection
It allows the phagocytes in blood to gain access to the microbes in tissues
what are the 4 classic signs of inflammation?
Red, swelling, heat, pain
how is inflammation induced?
- Release of inflammatory mediators
- Dilation of local blood vessels
- Increased permeability and blood flow
- Immune cell migration such as neutrophils into inflammatory site
- Pain caused by stimulation of nerve endings which supply tissues
why is inflammation important?
Inflammation ensures that immune cells, defence molecules, coagulation factors etc. reach the site of infection or tissue damage
what cells produce inflammatory mediators?
sentinel cells, damaged cells, microbes
what are examples of inflammatory mediators?
- Lipid mediators e.g. prostaglandins which stimulate dilation of blood vessels and act on pain receptors
- Vasoactive amines e.g. histamine, bradykinin – cause dilation of blood vessels
- Chemoattractants e.g. f-met-leu-phe – help phagocytes move into tissues
- Complement proteins e.g. C5a
- Cytokines e.g. TNF
what is the difference between acute and chronic inflammation?
Acute inflammation: generally beneficial in dealing with infection/injury - transient
Chronic inflammation: caused by chronic infection e.g. TB or other conditions e.g. autoimmune disease, which can be damaging – sustained inflammation
- In TB, the bacterium survives in macrophages and forms a granuloma which triggers chronic inflammation
what is the main role of cytokines?
Cytokines are crucial in orchestrating and controlling immune responses – important in innate and adaptive immunity – can switch off the immune system
- Regulate immune responses by changing cell behaviour or gene expression
what are cytokines?
- 20kD small proteins, >100 identified
- “Hormones” of the immune response
- Most act locally, but can have systemic effects (cytokine storm = damage)
- Can be produced by many cell types in response to immune activation
- Act on cells bearing specific cytokine receptors
- expression of cytokines and their receptors are tightly regulated
how do cytokines act on other cells?
- Cytokine producer is triggered by a stimulus – cytokine transcription
- Cytokine will bind to cytokine receptor on target cell
- This will induce the target cell to undergo various responses to mount an immune response
how are cytokines classified?
grouped into families on basis of structural similarities (but family members may have distinct functions)
- Cytokine receptors for the various subfamilies also tend to have similar structures and to signal in a similar way
what are the cytokine families?
- IL-1 family: most produced as inactive precursors that must be cleaved by inflammasomes, important in inflammation
- Haematopoietin superfamily: includes factors involved in leukocyte differentiation e.g. GM-CSF but also IL-2, IL-4, IL-6 (important in T cell responses) – stimulate leukocyte generation
- Interferons: involved in responses to viruses
- TNF family (e.g. TNFα = tumour necrosis factor): many are transmembrane proteins that are shed, important in inflammation (very potent so is tightly regulated)
- Chemokines: involved in cell movement (e.g. IL-8 a.k.a CXCL8)
what is TNF?
tumour necrosis factor - type of cytokine
- membrane protein, but extracellular region is released by proteolysis and is active as trimer
- induces local inflammation
- at high concs can be dangerous e.g. during sepsis
how are cytokines involved in the early stages of the immune response?
cytokines are secreted by macrophages:
- IL-1 induces inflammation and act on hypothalamus to trigger fever response – activates immune cells and inhibiting growth of pathogens
- TNF triggers acute inflammation
- IL-6 activates T and B cells, as well as triggering fever
- CXC8 causes neutrophils to move out of bloodstream into tissues
- IL-12 triggers NK cells and differentiation of T cells into helper T cells
what are interferons?
intruder alert cytokines:
- Viral infection induces the production of interferons (Interfere with viral replication)
what are the 2 types of interferons?
Type I: IFN-a, IFN-b
- Many cell types make type I interferons after viral infection.
- Induce expression of interferon-stimulated genes (ISGs)
- Some cell types (e.g. dendritic cells) are specialised for this – express high levels of endosomal TLRs e.g. TLR3 (recognise dsRNA) and TLR9
Type 2: IFN-gamma
- modulates immune responses
how are type 1 interferons expressed?
Infected host cell with TLRs will induce expression of IFN-a and IFN-b
what are the functions of type I interferons?
- Induce resistance to viral replication in all cells - Induce expression of endoribonuclease that degrades viral RNA and protein kinase that phosphorylates eukaryotic initiation factor 2, inhibiting protein translation
- Increase MHC class I expression in all nucleated cells - MHCI is required for antigen presentation to cytotoxic T cells, so infected cells are more easily recognised and killed.
- Activate NK cells to kill virally-infected cells.
- Induce chemokines to recruit lymphocytes.
what cells make Type II interferons?
Made by neutrophils, NK cells, T cells
what are the functions of type II interferons?
- Primary role in adaptive immunity
- Increases expression of MHCI and MHCII
- IFN-γ made by T helper cells activates macrophages in responses to intracellular pathogens
- Forms dimers
how does the innate immune system interact with the adaptive immune system?
- Co-evolved and are interdependent
- Innate immune responses initiate adaptive responses (antigen presentation) and different cytokines can “steer” adaptive immune responses by activating different T cell subsets, promoting the production of different antibody classes
- Adaptive responses use elements of innate immunity to eliminate pathogens e.g. classical pathway of complement, activation of macrophages by T cell cytokines, antibodies can help NK cells recognise infected cells, mast cells to respond to parasites
