Innate immunity - barriers & complement

Question 1

Name 5 general principles of innate immunity

Answer 1

• Oldest form of immunity
  
  • Elements shared by plants, insects, mammals
  • Changes on an evolutionary timescale
• Always available
  
  • Prior exposure not required
  • Little/no ‘memory’
• Major form of immunity in young children
  
  • Period between loss of maternal antibodies/formation of their own
• Crucial in initiating and directing adaptive immune responses.
• Adaptive immunity takes ~ 4-6 days to develop. Innate immunity is critical in controlling infections before this.

Question 2

What are the 3 elements of innate immunity?

Answer 2

Barriers

• prevent establishment of infection

Pre-formed mediators

• proteins/peptides with broad specificity that damage pathogens or induce inflammation or help recruit and activate innate immune cells

Innate immune cells

• recognise and are activated by pathogen
• eliminate pathogen
• communicate with other cells
• activate and “steer” adaptive immune response

Question 3

What are the 4 regions in which we have barriers

Answer 3

Skin, Gi tract, UG tract & Respiratory tract

Question 4

What are the 3 types of barriers?- describe them briefly

Answer 4

• Physical
  
  • epithelial cells joined by tight junctions, flow of air or fluid, cilia (move mucus)
• Chemical
  
  • Fatty acids, low pH enzymes (pepsin), Lysozyme, antibacterial peptides (defensins)
• Microbiological
  
  • commensals (microbiota)

Question 5

Which regions would you find the following barriers

• Physical
  
  • epithelial cells joined by tight junctions
  • flow of air or fluid
  • cilia (move mucus)
• Chemical
  
  • Fatty acids
  • low pH
  • enzymes (pepsin),
  • Lysozyme,
  • antibacterial peptides (defensins)
• Microbiological
  
  • commensals (microbiota)

Answer 5

• Physical
  
  • epithelial cells joined by tight junctions: S + GI + UG + Respt tract
  • flow of air or fluid: Skin + GI + UG + Resp tract
  • cilia (move mucus): Resp tract
• Chemical
  
  • Fatty acids: Skin
  • low pH: GI + UG
  • enzymes (pepsin): GI + UG
  • Lysozyme: Resp tract
  • antibacterial peptides (defensins): Skin + GI + UG + Resp tract
• Microbiological
  
  • commensals (microbiota): Skin + GI + UG + Resp tract

Question 6

What are the 3 physicalbarriers that create the 3 types of physical barriers

Answer 6

• Keratinized skin generally impermeable (in brown)
  
  • keratinocytes produce keratin (hard substance), sebum -contains fatty acids, defensins
  • Shedding skin, frees microbes
  • Commensals on skin
• Mucous membranes largest interface with environment, semi-permeable
  
  • GI, UI tracts, respiratory tract
  • mucous, cilia (respiratory tract), secreted enzymes (e.g. lysozyme in tears and saliva)
    
    • CF patients more prone to respiratory and GI infections (mucus effected)
    • in mucus → enzymes
  • low pH (gut, vagina), peristalsis (gut) (movement of the gut)
  • Shedding of epithelia (particularly in infection increase in shedding as well)
• Commensals

Question 7

Name 3 examples of pre-formed mediators

Answer 7

Lysozyme, Antimicrobial peptides (e.g. defensins), Complement

Question 8

Describe Lysozyme function i.e. where are they found, what do they do

Answer 8

• present in secretions (tears, saliva, mucous etc.)
• Breaks a bond in peptidoglycan. More active against G+ve bacteria, where peptidoglycan is exposed.
• cleaves peptidoglycan bonds → destabilises → Gram positive bacteria therefor more susceptible to lysozymes

Question 9

Describe antimicrobial peptides such as defensins: structure, function, who produces

Answer 9

• 29-47 AA in length, found in very primitive organisms
• Beta-sheet core structure stabilised by 3 conserved intramolecular disulphide bonds. 2 subfamilies (alpha and beta) in humans (not needed to learn)
  
  • alfa form are pre-formed in granules of innate immune cells.
  • Beta form have to be synthesised de-novo upon infection
• produced by many epithelial cells and neutrophils
• way of function:
  
  • cationic, disrupt lipid bilayers in bacteria, fungi and enveloped viruses
  • can insert into bacterial membranes to disrupt lipid bilayer

Question 10

Describe the basic principles of complement

Answer 10

• > 20 soluble proteins found in blood and other body fluids
• Components normally inert, but “activated” by presence of pathogens or antibody bound to pathogen
• pro-enzyme (inert) acted upon stimulus → turns into enzyme
• cascade of reactions → amplification of enzymes
• Originally evolved as part of the innate immune response
• Provides protection early in infection in the absence of antibodies through other “older” activation pathways

Question 11

Describe the principles of the complement cascade: Central event, cleavage of c3 does what?

Answer 11

• Many activated complement components are proteases; act on one another to generate a large and a smaller fragment:
  
  • proteases are enzymes that cleave proteins
• C3 → C3b + C3a
• Cleavage of C3 exposes a reactive thioester in C3b, which can bind covalently to adjacent proteins/carbohydrates e.g. on the surface of a pathogen.
  
  • (rapidly inactivated in the fluid phase). i.e. C3b can be made without infection by random chance but quickly inactivated if it doesn’t bind to anything
  • C3b (bigger fragment) C3a (smaller fragment)
• The central event of complement activation is cleavage of C3 by a “C3 convertase”.
• 3 pathways of complement activation, initiated in different ways, but all generate a C3 convertase.

Question 12

What 3 ways can complement be activated? and how are they similar?

Answer 12

• The central event of complement activation is cleavage of C3 by a “C3 convertase”.
• 3 pathways of complement activation, initiated in different ways, but all generate a C3 convertase.
  1. Classical pathway
  2. Mannose-binding lectin (MBL) pathway
  3. Alternative pathway

Question 13

Describe the classical pathway of activation of complement

Answer 13

Antibody binds bacterial surface → C1 interacts with antibbody Fc region → activates C1 → serine protease which acts on C4 which acts on C2 → C3 convertase
C4b + C2a (is the C3 convertase) (from cleavage of C4 and C2 by C1)
NB! dependent on adaptive immune system antibodies!
C1 -> C4 -> C2 -> C3 -> C5 -> C6 -> C7 -> C8 -> C9

Question 14

Describe the Mannose-binding lectin pathway of activation of complement

Answer 14

• binding of lectin to bacterial surface acts MASP1 → acts on MASP2 → C3 convertase
  C4b + C2a (is the C3 convertase) (from cleavage of C4 and C2 by MASP2)
• MASP = MBL associated serine protease
• Lectin (Mannose-binding lectin) to bacterial surfaces
• Lectin is a molecule that loves to bind to carbohydrate. MBL loves to bind to mannose residues which are often found on surface of bacteria. Other lectins discovered but MBL is the main one. (research this!)
• MASP = MBL associated serine protease

Question 15

Describe the alternative pathway of activation of complement

Answer 15

• some C3b is generated spontaneously in body fluids by a “tickover” mechanism
  
  • tickover mechanism = even without infection
  • during infection, some C3b generated can bind to bacterial membranes to LPS in particular (through reactive thioesters in C3b
• C3b + Bb
  
  • If C3b generated binds to LPS on a pathogen surface → factor B binds
  • Factor B is cleaved by Factor D → generates Bb which is connected to C3b → C3bBb → acts as a C3 convertase
  • Normally C3bBb is quiet easily inactivated → Factor P (properdin) stabilises C3bBb enzyme (allows it to work longer on the bacterial surface)
  • C3b generated by the Classical or MBL pathway can also bind factor B → amplification of complement activation!
• complement interacts with LPS on bacterial surface → factors which are found at high concentration in body fluids called factor B and Factor D, which are stabilised by factor P → c3 convertase made
• C3 convertase = C3b + Bb
• C3b: Cleavage of C3 exposes a reactive thioester in C3b, which can bind covalently to adjacent proteins/carbohydrates e.g. on the surface of a pathogen.
  (rapidly inactivated in the fluid phase). i.e. C3b can be made without infection by random chance but quickly inactivated if it doesn’t bind to anything
  C3b (bigger fragment) C3a (smaller fragment)

Question 16

Describe what C3 convertase is, i.e. what its made up of

Answer 16

• For Classical and MBL pathway, C3 convertase = C4bC2a
  
  • (from cleavage of C4 and C2 by C1 and MASP2, respectively)
  • C4b + C2a (is the C3 convertase)
• For Alternative pathway, C3 convertase = C3bBb.
  
  • some C3b is generated spontaneously in body fluids by a “tickover” mechanism
    
    • even without infection: tickover mechanism.
    • during infection some C3b generated can bind to bacterial membrane to LPS in particular
  • C3b + Bb
    
    • Factor B can now bind to C3b (as C3b bound to LPS) on bacterial surface
    • Factor D cleaves Factor B → generates Bb which is connected to C3b → C3bBb → acts as a C3 convertase
    • Normally C3bBb is quiet easily inactivated → Factor P (properdin) stabilises C3bBb enzyme (allows it to work longer on the bacterial surface)
• if C3b generated binds to LPS on a pathogen surface, factor B binds
• Factor B is cleaved by factor D → C3 convertase (C3bBb).
• the C3bBb convertase is stabilised by factor P (properdin)
• C3b generated by the Classical or MBL pathway can also bind factor B → amplification of complement activation!

Question 17

Draw a diagram showing the mechanism of action of the 3 different pathways of complement activation

Answer 17

look at lecture 2 notes

Question 18

What happens after C3 convertase is generated?

Answer 18

• C3 convertase on surface of bacterium (through either method)
• C3 → C3a + C3b
• C3 convertase + C3b = C5 convertase → C5a + C5b → activation of rest of complement → MAC (membrane attack complex)

Question 19

What are the 3 major biological activites of complement

Answer 19

1- C5a (C3a) - recruit phagocytes and induce inflammation
2- C3b promotes opsonization
3- C5b-C9 form the Membrane Attack Complex (MAC), leading to lysis

Question 20

Describe the function of C5a (C3a)

Answer 20

• recruit phagocytes to site of infection and induce inflammation
  
  • Recruitment of phagocytes = acts as chemoattractant
  • Induction of inflammation = anaphylatoxins
  • a.k.a chemoattractants, “anaphylatoxins”
  • C3a less potent than C5a
• infection in tissues → C5a generated → phagocytes like neutrophils have receptors on their surface which can bind to C5a or C3a → phagocytes undergo chemotaxis → enter site
• 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.
  
  • mast cells (found under mucus surfaces) respond to binding C5a/C3a by releasing inflammatory mediators like histamine
  • can cause body wide inflammation in anaphylaxis in allergic people to e.g. bee sting
  • otherwise very beneficial

Question 21

Describe waht C3b does

Answer 21

• on its own C3b promots phagocytosis of bacteria or fungi
  
  • does this by coating bacteria
  • Phagocytes like neutrophils have receptors for C3b on surface → recognise coated bacteria more efficiently → phagocytosis
    
    • to make tasty

Question 22

Describe what C5b-C9 do

Answer 22

• • C5b-C9 form the MAC → induces polymerisation of C9 → forms pores into pathogen causing cell 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 negative bacteria

Question 23

Describe the ways in which complement is regulated

Answer 23

• “Double-edged sword” - regulation needed to prevent damage to host:
  
  • components (enzymes) rapidly hydrolysed in fluid phase
  • soluble and membrane bound regulatory proteins ( a few examples)
    
    • E.g
      
      • C1 inhibitor inactivates C1 (in classical pathway)
      • Factor H inhibits alternative pathway
      • carboxypeptidase N (enzyme) inactivates C3a and C5a (to ensure inflammation for too long)
      • CD59 on host cells binds C9, preventing MAC formation
• deficiencies in inhibitors increase risk of some diseases
  
  • e.g. - age-related macular degeneration (lack Factor H)
    
    • macular degeneration → major cause of blindness in elder people
    • Factor H → inaaproriate activation og alternative pathway
  • e.g. - paroxysmal nocturnal hemoglobinuria (lack CD59)
    
    • form of anemia
    • lack of CD59 → more MAC? may kill own blood cells??

Question 24

Describe the importance of complement

Answer 24

• Complement is particularly important in extracellular bacterial and fungal infections, but may be active against some viruses.(mainly enveloped viruses)
  
  • Defects in C3 – increased susceptibility to pyogenic infections e.g. S.pneumonia
    
    • puss forming = pyogenic
  • Defects in C5- C9 – increased susceptibility to Neisseria infections
    
    • gram negative bacteria → MAC down → less potent for gram negative
• Complement interacts with Adaptive Immune system
  
  • Classical Pathway → acts with antibodies
  • aids clearance of immune complexes (antigen:antibody)
    
    • immune complexes
  • role in activating B and T cells
• Complement plays a role in some autoimmune diseases and asthma
  
  • antigen which triggered autoimmune response is always there → complement always activated etc.