Innate immunity - innate leukocytes, PRR, MAMP + DAMPs

Question 1

Name the innate leukocytes, where are they derived from?

Answer 1

HSCs : Myeloid + lymphoid stem cells
- Polymorphonuclear leukocytes (PMN)
- neutrophil, eosinophil, basophil, mast cells
- Macrophage, monocyte, dendritic cells
- NK cells
- all derived from HSCs myeloid lineage except NK cells

Question 2

Describe the Polymorphonuclear granulocytes (polymorphs or granulocytes) and how they were disovered

Answer 2

• Basophil,
  
  • found with basic blue dye (→ baso-phil)
• Eosinophil,
  
  • red dye called eosin (eosino-phil)
• Neutrophil
  
  • neutra-phil

Question 3

Describe Neutrophils

Abundance, where they are released and found, how long they live, main functions, interactions, special features

Answer 3

• Make up most (60-70%) leukocytes in blood
  
  • millions made every day and released from bone marrow
• Released in large numbers from bone marrow
• Live for < 24hr in blood
  
  • short lived in blood
• Life extended on entering tissues in response to chemoattractants
  
  • chemoattractants like C5a
  • longer lived in tissue
• Receptors for C3b, IgG, IgA
  
  • opsonisation
• Main function is phagocytosis, but also “NETosis”
  
  • NETosis = when neutrophils are dying they throw out fibers (DNA and histones) to form a “net” which can catch bacteria (contain antimicrobials and e.g. defensins)
  • so even when dying they try kill bacteria
  • neutrophil extracellular trap

Question 4

Describe Eosinophils

Abundance, where they are found, main function, interactions, special features

Answer 4

• Few in blood (normally), but also beneath mucous surfaces
• Receptors for C3b, IgG, IgA, (IgE)
  
  • opsonisation phagocytosis (not very good at phagocytosis)
• Release toxic proteins, free radicals
  
  • upon recognition pathogen → release toxic proteins from granules and free radicals (e.g ROS which can bind to components to pathogens to cause damage)
• Defence against multicellular parasites
  
  • through releasing these toxic proteins and free radicals onto parasite
• Role in allergy (especially asthma)
  
  • if eosinophils activated inappropriately → can cause damage in the lungs

Question 5

Describe Basophils

Answer 5

• Very few in blood. ~1% of white blood cells
• Receptors for C3a, C5a, IgE
  
  • chemoattractants response, and IgE
• Release heparin and histamine
  
  • upon stimulation by pathogen
  • don’t really phagocytose
  • release heparin (increase blood flow) and histamine (inflammatory mediator)
• Defence against parasites, role in allergy
  
  • induce inflammation, increase blood flow
  • increase in Basophils in allergy suffering people.

Question 6

Describe mast cells

Answer 6

• Restricted to tissues - protect mucosal surfaces.
  
  • just under the skin
• similar properties to basophils
• Receptors for C3a, C5a, IgE
• Release histamine etc.
  
  • from their granules
• “Sentinel” cells, defence against parasites, role in allergy
  
  • sentinel as in alert the body

Question 7

What are monocytes? whats the difference between monocytes and macrophages

Answer 7

• In blood → Monocytes
• Macrophages found in tissues
• Upon infectoin → monocytes can/may migrate to site of infection and differentiate into macrophages
• However! macrophages seed into the tissue very early into development (feotus development) and self renew in the tissues
• macrophages more

Question 8

Describe macrophages

Answer 8

Macrophages can be derived from monocytes during infection
- 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” – can phagocytose 100 bacteria/cell
- Receptors for C3b, IgG, IgA
- opsonins
- IgG and IgA help recognition
- Produce pro-inflammatory mediators
- e.g. cytokines
- Can present antigen to T lymphocytes
- important
- antigen-presenting leukocyte → can initiate adaptive immune response

Question 9

Describe Dendritic cells

Answer 9

• Found in skin and lymphoid tissues
• Take up foreign material by phagocytosis/micropinocytosis
  
  • “cell drinking” can drink fluid around cells?
• Digest foreign material and display fragments on their cell surface
• Specialised for presenting antigen to T cells
  
  • efficient at presenting because →
• Constitutively express high levels of Major Histocompatibility Type II (MHCII) proteins

Question 10

Describe the importance and process of phagocytosis

Answer 10

• Phagocytosis
  
  • “professional phagocytes” (neutrophils, monocytes, macrophages, dendritic cells)
    
    • these carry out phagocytosis very effciently
    • many other normal cell types can do phagocytosis like e.g. epithelial cells but not efficiently
  • mainly important in bacterial and fungal infections

1. Bacterium binds to the phagocyte (recognition)
2. Phagocyte pseudopods extend and engulf organism
3. Invagination of phagocyte membrane traps the organism within a phagosome
4. Phagosome vesicle fused with specialised lysosomes present in the phagocyte
   
   1. lyosome (primarily contain free radicals)
   2. phagolysosome

Question 11

What stops phagocytes from phagocytosing self cells

Answer 11

NOTE: Healthy “self” cells express a protein (CD47) which is recognised by phagocytes and prevents phagocytosis.

Question 12

What in phagocytes phagosome/phagolysosome kills the pathogen? i.e. the phagocyte bactericidal agents

Answer 12

• Short-lived, contained in phagosome, produced following “oxidative burst”
• Acidic pH in phagolysosome, usually bacteriostatic but sometimes bactericidal
• Free radicals (very reactive, bind to bacterial proteins and DNA and denature it)
  
  • Toxic oxygen-derived products
    
    • Hydrogen peroxide H2O2, superoxide O2- etc.
  • Toxic nitrogen oxides
    
    • e.g. nitric oxide NO
• Antimicrobial peptides
  
  • e.g. defensins
• enzymes
  
  • e.g.lysozyme and acid hydrolases (
• Competitors
  
  • stop bacteria dividing, bacteria need Fe and B12
  • Lactoferring (competitor) binds iron → stop bacterial growth

Question 13

Describe oxygen-dependent killing and “oxidative burst”

Answer 13

• these are contained in lysosome so not dangerous to our other cells
• Oxygen-dependent killing
• “Oxidative burst” - transient increase in oxygen consumption following phagocytosis due to activation of a membrane-bound NADPH oxidase.
  
  • rapid increase (transient increase) in oxygen consumption (caused by activation of membrane-bound NADPH oxidase (enzyme normally inactive → activated upon phagocytosis) → oxygen dependent killing → converts oxygen to superoxide ion O2- (very reactive) → superoxide dismutase converts superoxide to hydrogen peroxide (bleach)
  • in neutrophils myloperoxidase converts hydrogen peroixed to chlorite ions (bleach that you pour into toilet)
  • very reactive free radicals → can bind to bacterial DNA and proteins and denature them
  • NADPH + 2O2 → NADP+ + H+ +2●O2- → H2O2 + Cl- → OH- + HOCl
• increase in oxygen activated nitric oxide synthetase → causes production of nitric oxide form arginine and oxygen (very reactive)
• 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

Question 14

Describe NK cells

Answer 14

• not phagocytic
• Derived from lymphoid progenitor
  
  • big nucleus and granules
• Kill infected host cells
• Important in viral infections and bacteria that survive inside host cells
• Recognise “altered self”
  
  • recognise changes in expression of self Major Histocompatibility Type I (MHCI) proteins
  • all nucleated cells express MHC I proteins
    
    • self labels
  • virus often interferes with levels of expression of MHC I
  • NK cells can detect the MHC1 expression decrease
• Important in viral and some intracellular bacterial infections, especially until adaptive immunity is triggered
• Receptors for IgG
  
  • allows killing of antibody coated infected host cells
• Also active against some cancer cells
  
  • cancer cells often down regulate MHC I expression to evade immune system
  • NK cells can also detect and kill these

Question 15

How do NK cells kill infected host cells?

Answer 15

• Activated NK cells produce a pore-forming protein, perforin(similar in structure to MAC), which inserts into the membrane of the infected host cell, forming a pore. → contents of granules can enter target cell → granule contents (granzymes) released into the target cell → activate apoptosis pathway → infected host cell die
• Recently discovered that a granzyme can enter intracellular bacteria, killing them directly.
  
  • some granzymes can kill intracellular bacteria

• Polarisation of NK cell granules at the interface between the NK cell and target cell
• Granule contents (“granzymes”) are released into the target cell, activate apoptosis pathway.
• Target cells undergo apoptosis.
• Recently discovered that a granzyme can enter intracellular bacteria, killing them directly.
  
  • some granzymes can kill intracellular bacteria

Question 16

How do immune cells recognise pathogens

Answer 16

Through PRRs and MAMPs

Question 17

What are Pattern Recognition Receptors (PRRs) and Microbe-associated molecular patterns (MAMPs) and DAMPs

Answer 17

• PRRs recognise MAMPs
• The innate immune system recognises structures that are relatively invariant:
• Microbe-associated molecular patterns: MAMPs
  
  • molecules Shared by many microbes
  • Distinct from “self” molecules
  • Critical for survival/function of pathogens (microbe)
  • Conserved (MAMPs conserved amongst pathogens)
• Damage-associated molecular patterns: DAMPs
  
  • alarm signals coming from the bodies own cells → DAMPs
  • generated not only in response to infection but also to injury and heart attack and sometimes cancer
    
    • host components during stress/injury reflecting cell damage → signals to immune system that the damage needs to be repaired
  • Host components released during injury and cell damage

Question 18

Describe some examples of MAMPs in bacteria, fungi and protozoa

Answer 18

• Bacteria
  
  • Gram -ve: (thick peptidoglycan)
    
    • Lipopolysaccharides (LPS)
  • Gram +ve:
    
    • Lipotechoic acid
  • Flagellins
  • unmethylated CpG in DNA
  • N-formylated proteins
    
    • n terminus of bacterial proteins have this
• Fungi
  
  • Chitin
  • Beta-glucans
• Viruses
  
  • dsRNA
• Protozoa (eukaryotes)
  
  • GPI-linked proteins (we have these, but in very low amounts), mannose-rich glycans

Question 19

Give examples of DAMPs

Answer 19

• • Fragments of extracellular matrix proteins (holds cells together)
    
    • e.g. fibronectin
    • broken (fragments) of fibronectin
  • Exposed phosphatidylserine
    
    • component of lipid bilayer, usually found on inside of lipid bilayer, when damaged, flips to outside to say the cell is damaged
    • Phosphatidylserine is normally intracellular but is exposed during apoptosis
  • Mitochondrial components
    
    • mitochondrial are inside cells, if found outside cells → sign of damage
  • Uric acid
    
    • can build up if excess purines (e.g. during stress)
    • can lead to gout
    • Uric acid, a marker of kidney damage and oxidative stress. (Deposition of crystals causes gout.)
  • DNA
    
    • DNA found outside cells detected → signal of damage that needs to be dealt with

Question 20

How many PRRs are there and what are the 4 classes of PRRs

Answer 20

• PRRs Recognise >1000 MAMP/DAMP ligands
  
  • PRRs inherited in DNA, can’t evolve rapidly, but in humans there are PRRs that can recognise more than 1000 MAMPs/DAMPs
• Classes:
  
  • Soluble receptors e.g. Mannose-binding lectin (involved in activating complement - is a PRR)
    
    • can be found in tissue fluids and blood
  • Membrane receptors
    
    • Lectin receptors
      
      • like to bind to carbohydrates on bacteria
    • Chemotactic receptors
    • Toll-like receptors
  • Cytoplasmic receptors (found in cytosol and not on cell membrane → e.g. for virsues and intracellular bacteria)
    
    • NOD-like receptors (NLR)
• Membrane and cytoplasmic receptors expressed by immune and non-immune cells
  
  • e.g. fibroblasts, epithelial cells (because they are usually the first cells a pathogen encounters

Question 21

What happens when a MAMP bind on membrane receptors on macrophages?

Answer 21

• Macrophages express multiple PRR membrane receptors
• MAMP binding may initiate phagocytosis, chemotaxis or signalling.
  
  • e.g. macrophage mannose receptor (recognises mannose)
  • e.g. CD14 (recognises LPS)
  • Mannose receptor and CD14 expressed on macrophage membranes
• CHEMOTACTIC RECEPTORS recognise CHEMOATTRACTANTS
  
  • e.g. f-met-leu-phe receptor (recognises N-formylated polypeptides, produced by bacteria)
  • bacterial components
  • neutrophils, monocytes and macrophages express f-met-leu-phe receptor which recognise N-formylated polypeptides produced by bacteria → acts as chemoattractant for them. Bacterial infection in tissue → move towards concentration gradient of formylated peptide
• Toll-like receptors or TLRs:
  
  • sensors that signal the presence of microbial components.
  • don’t trigger phagocytosis or chemotaxis
  • they signal to the body that there is a danger/infection present → triggers changes in gene expression

Question 22

Describe TLRs

Answer 22

• Cause a change in gene expression i.e. allowing production of cytokines
• 10 TLRs in humans, each recognising distinct MAMPs
• examples:
  
  • TLR1 recognises lipopeptides
  • TLR3 recognises dsRNA
  • TLR4 recognises LPS etc.
• always membrane associated
  
  • Can be on the cell-surface or endosomal membranes!!
• The extracellular domain of TLR3 has a horseshoe shape formed by leucine-rich repeats (recognises MAMP (ligand)).
• The inner surface has β-sheet structure and forms the ligand binding domain (dsRNA in this case)
• Usually 2 subunits need to come together: dimerisation triggers signalling

Question 23

Describe what NOD-like receptors are and their function

Answer 23

Cytoplasmic receptors

NOD-like receptors (NLR)

• large group of cytoplasmic receptors that recognise bacterial components e.g. peptidoglycan, flagellin

function:

• trigger cells to make cytokines, particularly pro-inflammatory cytokines
• signal expression of pro-inflammatory cytokines
• trigger assembly of inflammasomes
  
  • inflammasomes are multisubunit complex that cleaves inactive cytokine precursors into active cytokines

Question 24

Describe the mechanism of action of TLRs, NLRs and inflammaosomes upon MAMP recognition

Answer 24

• Cell recognises MAMP through TLR → induces signalling → changes in gene expression → cytokines produced
• Many cytokines are initially made in an inactive form (important so they aren’t innapropriately produced) these are called pro-cytokines
  
  • they need to be cleaved to become activated cytokines
• Bacterium been taken up by phagocytosis → material from phagolyssome released (MAMPs like LPS) → recognised by NLRs (in cytoplasm), and also signal nucleus to start making different genes? → inflammasome produced
• Inflammasome protein complex needed to process pro-cytokines by cleaving them
• Cytokines produced fully
• NLRs important for bacterial infections
• Receptors in cytoplasm for dealing with viruses
  
  • RIG-I-like receptors detect viral RNA produced within host cells
  • induce different response → induce cells to start making inteferons (sub-type of cytokine)

Question 25

What are RIG-I-like receptors?

Answer 25

RIG-I-like receptors

• viral sensors that detect viral RNA produced within host cells
• signal expression of interferons