Pattern Recognition

Pattern Recognition

1. Innate vs Acquired Immunity

2. T cell activation by DCs requires 2 signals

   • 1) MHC-peptide:TCR 2) cosstimulatory signal (e.g. B7 on DC: CD28 on T cell
     • activation of an antigen specific T cell is determined by the presence of costimulatory molecules on the DC (not present in resting DCs).
     • costimulatory molecules can be induced by recognition of PAMPs or DAMPs by the DC through pathogen-specific receptors such as TLRs

3. Self/Non-self Discrimination

   • Self/non-self hypothesis
     • Burnet and Medawar 1959
       • Burnet = australian virologist/immunologist, developed theory immune tolerance. Director of Walter and Eliza Hall Institute (1944-1965). Won a prize in 1960
     • Ag specific cells which recognise self molecules are eliminated in life, leading to tolerance of self.
       • this leaves only cells which recognise foreign antigens. In this model, specific responses to foreign molecules is just dependent on T and B cells, and the process of tolerance
     • Inconsistent with this hypothesis are:
       • requirement for activated DC to present Ag to T cells
       • requirement for adjuvant in vaccination
         • vaccination with pure protein has little effect bc the acquired immune system does not recognise the antigen as foreign.
           • Adjuvants: aluminium salts, LPS, bacterial DNA + polysaccharides, Freunds adjuvant (emulsion of killed mycobacteria)
             • act as innate immune stimulants
       • not rejected, but built upon in later hypotheses
   • Infectious non-self hypothesis
     • distinction between self and foreign relies on the recognition of characteristic microbial molecules (PAMPs) by the innate immune system (PAMP=pathogen associated molecular pattern)
       • PAMPs recognised by PRRs/TLRs on innate cells
     • activation of DCs by PAMPs upregulates the costimulatory molecules required for T cell activation
     • known that PAMPs are not the only route to DC activation (DAMPS)
   • Danger hypothesis
     • distinction relies on release of cellular molecules which indicate stress, damage, or danger to the cell
     • in support: acquired immunity response to tumours and rejection of grafts in the absence of infectious agents
     • self molecules called DAMPs (danger associated molecular patterns) can act through PRRs that recognise PAMPs
       • Evidence for DAMPs: Monosodium urate crystals
         • cytoplasmic extract behaved as an adjuvant
           • it activated DCs by increasing B7 (CD86) and generated specific CTL against the antigen
             • cytoplasmic extract was fractionated to find an active component - uric acid, active as monosodium urate crystals - note these will only form when outside the cell (indicating cell lysis) due to the elevated Na concentration outside the cell
               • uric acid is normally present at 40-60micrograms/mL in blood, close to the limit of solubility in body fluids (70micrograms/mL). cells have uric acid concentrations between 1-4mg/mL which increase if cell is dying due to DNA and RNA breakdown (uric acid is a purine derivative. therefore when cells lyse, uric acid will form crystals with Na - an abundant ion
                 • cell damage can be caused by injury or infection
       • Examples:
         • Small molecules: monosodium urate, extracellular ATP
         • Intracellular proteins: HMGB1 (high mobility group protein B1)
         • Mitochondrial molecules (similar to bacterial due to endosymbiotic theory of evolution: DNA from mitochondria (not methylated on cytosine), and N-formyl peptides from mitochondria
   • PAMPs:
     • Bacterial Cell wall/surface molecules
       • (GRAM -VE) LPS, lipoprotein, peptidoglycan, (GRAM+VE) lipoteichoic acid, peptidoglycan
       • Flagellin
     • yeast cell wall: "zymosan" beta 1-3 linked glucose
     • DNA: bacterial and viral DNA has unmelthylated CG dinucleotides (170-80% of our CGs are methylated (5-methyl cytosine) and we have lower than random CG frequency)
     • ssRNA: wide range of base modifications in pathogen RNA
     • dsRNA: long dsRNA is foreign to vertebrates + is an intermediate in viral life cycle, may result from txn off both DNA strands of DNA viruses

4. PRRs:

   • named because they recognised repeating patterns characteristic of PAMPs
   • also recognise DAMPs
   • mediate
     • phagocytosis (macrophage mannose receptor in MBL pathway)
     • Chemoattraction (formyl peptide receptors)
     • Production of chemokines and cytokines (inflammation)
     • DC activation, leading to activation of the acquired immune system
   • Examples
   • Localisation
     • Recognition of PAMPs in the cytosol tell the cell that it itself is infected
       • important for viral detection and for bacteria such as Listeria which can live and or inject molecules into the cytoplasm out of the endosome
       • performed by cytosolic TLRs to recognise nucleic acids
     • TLRs on endosome = engulfed microbe (local extracellular infection)
     • TLRs on cell surface = extracellular infection (local)
   • Discoveries
     • 1st discovered in Drosophila as a gene affecting embryonic development (strange morphologies formed) and defence against fungal and bacterial infection as its pathways induces antimicrobial peptides
     • Mammalian TLR
       • shown to stimulate cytokine production
       • a cytoplasmic TLR domain was fused with an extracellular domain that dimerised upon binding. this lead to a protein cascade in the cytoplasm to produce inflammatory molecules
   • Types:
     • Extracellular: Cell membrane
       • TLR5: flagellin
       • TLR6+TLR2: diacyl lipopeptides
       • TLR1+TLR2: triacyl lipopeptides
       • TLR 4: LPS
       • any ligands have been proposed fro TLR2 and 4
     • Intracellular: inside Phagosome/endosome which is required to break down foreign DNA or RNA from the pathogen
       • TLR3: dsDNA
       • TLR7: ssRNA
       • TLR9: unmethylated CG dinucleotides of DNA
         • Bacterial and vertebrate DNA look very similar overall, so specific innate recognition of bacterial DNA was initially very surprising
         • unmethylated CG DNA = CpG DNA, has sequences like AACGTT
           • acts on macrophages, DCs and B cells
           • induces inflammatory cytokines, promotes Ag presentation and induces co-stimulatory molecules on DCs
           • short CpG oligonucleiotides are powerful immune stimulants and are in clinical trials as vaccine adjuvants, treatments for infectious disease and cancer, and as immunomodulators for allergy
   • TLR structure: 2 important domains
     • Leucine rich repeat (LRR) domain involved in ligand recognition
       • has horseshoe shape
       • LRR of TLR3 binds dsRNA directly
     • Toll/interleukin-1 receptor (TIR) domain: cytoplasmic signalling
       • this component is brought together in dimerisation to initiate a signal cascade

5. Nod-like receptors, AIM2 and the inflammasome

   • the inflammasome = a large cytoplasmic protein which recruits and activates the protease caspase 1
     • caspase 1 cleaves pro-IL1beta and pro-IL18 into active inflammatory cytokines
       • IL-1beta
         • causes vasodilation, fever, hypotension, infiltration if inflammatory cells, chemokine and cytokine release, activation of APCs
         • role in inflammatory diseases
         • major sources are macrophages, DCs and monocytes
       • IL-18
         • promotes T cell activation
       • CASPASE-1
         • initially called IL-1beta converting enzyme
         • an inflammatory caspase (others are involved in apoptosis)
           • caspases 8,9 +12 cleave caspases 3 and 7 to induce apoptosis
           • caspases 4 and 5 are also inflammatory and are involved in pyroptosis
         • its pro-domain in what is recruited to the inflammasome, where it is activated and induced to dimerise
     • activation also leads to inflammatory lytic cell death termed pyroptosis
       • activation initiated by a range of PAMPs and DAMPs causing oligomerisation of NLRs (particularly NLRP3 aka NALP3) and/or cytoplasmic DNA recognition by AIM2
         • • AIM2 binds DNA, it recruits ASC, ASC recruits procaspase 1, cleaves to caspase 1 and close proximity results in caspase1 dimerisation to form active caspase 1
             • AIM2 - a cytoplasmic DNA receptor
               • introduction of DNA into the cytoplasm of macrophages activates the inflammasome to increase ILs to cause pyroptosis
             • ASC
               • clusters in inflammasome formation. when tagged with GFP AIM2 and ASC are shown to be spread out in cytoplasm until DNA is added, in which case clustering occurs
   • What activates the inflammasome?
     • DAMPs
       • extracellular ATP: binds P2X7 receptor to increase the potassium influx into the cell, resulting in a change in conformation of the NLRP3 so it can bind ASC to cluster it
       • monosodium urate crystals (also particulate stimulus): macrophages engulf crystals, disrupts phagosome, inflammasome is activated, causing IL-1beta release and continuous inflammation, IL-1 receptor antagonist is a potentially successful treatment (NLRP3
         • present in high levels in gout
     • Particulate Stimuli
       • monosodium urate (NLRP3)
       • alum (used as vaccine adjuvant) (NLRP3)
       • asbestos (NLRP3)
       • silica (NLP3)
       • beta-amyloid (NLRP3)
       • cholesterol crystals (NLRP3)
     • PAMPS
       • Flagellin (NLRC4)
       • cytoplasmic DNA (AIM2)
       • bacterial pore-forming toxins, anthrax lethal toxins (NLRP3)
     • Associated diseases include gout, Alzheimer's, atherosclerosis, diabetes
   • AIM2 inflammasome and Francisella tularensis (FT) innate immure recognition (PAPER)
     • 1. prove that AIM2 is a receptor for DNA using macrophages from gene knockout mice
       • primed cells with LPS to induce the need for an inflammatory event
         • when ASC absent: no inflammatory response, so it is essential for IL release
           • when AIM2 absent: DNA stimuli have no effect, but ATP and flagella have IL release, therefore AIM2 is essential for responses to DNA, so it is a receptor for DNA
     • 2. Prove AIM2 is a receptor that activates caspase 1
       • performed western blot with AIM2 knockout cells
         • found that pro-caspase-1 is found in all cells, but active caspase-1 is only present in WT cells (not AIM2 knockouts)
         • procedure:1. run protein extracts on SDS-PAGE, 2. transfer proteins io nitrocellulose membrane, 3. incubate with soecific Ab to bind protein of interest (active caspase1- and pro-caspase-1), 4. use secondary Ab that binds first Ab. Second Ab will have an enzyme attached that emits light to allow the position of the antibody to be dtermined
     • 3. prove AIM2 is involved in recognition of a cytosilic bacterium
       • AIM2 knockouts had no inflammatory response when infected with FT
     • 4. showed that AIM2 was co-localised to bacterial DNA in infected cells
     • 5. AIM2 + caspase-1 knokouts has the same result, with increased FT growth comparted to WT
       • therefore AIM2 inflammasone decreases bacterial growth and proliferation, as does caspase-1
     • SO, "absent in melanoma 2" =AIM2 is required for innate imune recognition of Francisella tularensis

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