1_Innate Immunity

1_Innate Immunity

1. Sensing danger via:

   • sensing something not present on self
     • PAMP (cellular mediator)
       • recognised by pathogen recognition receptors (PRRs) -> most PRRs = TLRs
         • initiate an inflammatory response and leukocyte recruitment and antimicrobial response for pathogen destruction
     • Classical and MBL complement activation (soluble mediators)
     • activating signal initiates microbial destruction
   • sensing absence of something normally present on self
     • NK cell activation (cellular mediator)
     • alternative complement activation (soluble mediators)
     • an inhibitory signal that usually prevents host destruction
   • soluble mediators
     • Antimicrobial peptides: primarily in epithelial cells, have direct antimicrobial action: alpha and beta defensins
     • Complement system
       • Classical Pathway
         • Ab binds Ag on the cell surface, Ab clusters form to activate complement system
       • Mannose-Binding Lectin Pathway
         • MBL bins mannose + fructose with high affinity and the correct spacing to trigger activation of the complement system
       • Alternative Pathway
         • the slow spontaneous hydrolysis of C3 enables the complex (of C3 derived molecules) to bind to host or pathogen cells. host cells have regulatory proteins to inhibit C3 binding, but pathogens lacking these inhibitory proteins are targeted + complement system is activated
           • C3 convertase produces peptides:
             • C5a: inflammatory cell recruitment
             • C3b: onsonisation
             • C9 - Membrane-attack complex, creates a pore in the membrane for direct killing f the pathogen
     • Chemokines and cytokines = proteins secreted from a range of cell types that act locally and systemically to increase vacular permeability leukocyte recruitment, body temp, Ag presentation + acute phase response
     • lysozyme: macrophage product to degrade peptidoglycan
     • calgranulins: neutrophil products w anti bacterial and anti fungal properties
     • Pentraxins: e.g. CRP - enhance phagocytosis, range of functions

2. Epithelium

   • a tissue containing layers of cells lining the skin and inside cavities and structures in the body
   • function: exchange of nutrients, gas, waste products, water
   • function: in the innate immune system: a physical barrier, protection, constantly challenged
   • Pathogens can evade the epithelium
     • spores in the airway
     • inhaled droplet
     • skin abrasions, insect bites, puncture wounds
       • highlights importance of wound healing
     • so protectiive cytokines, complement system + antimicrobial peptides are required
   • protected by normal flora, phagocytes (esp in lungs) + local chemical factors

3. Immune response

   • infection
     • chemotactic and vasoactive factors to increase blood flow and capillary permeability -> influx of fluid and cells -> phagocytes migrate to site of inflammation (chemotaxis) -> phagocytes and antibacterial molecules increase to destroy bacteria. effector cells = non-specific (0-4hrs)
       • early induced response: activation of effector cells (4-96hrs)
         • Adaptive immure response: Clonal expansion and differentiation to effector cells (>96hrs)
   • TIMING is everything
     • immediate response = neutrophils (hrs)
     • delayed response = macrophages (days) which clear away pathogens and dead neutrophils

4. cells

   • Innate Lymphoid Cells (ILCs)
     • 3 distinct groups: depending on cytokine profiles
       • ILC class 1: NK cells
         • associated with inflammatory conditions such as IBD
       • ILC class 2: target larger pathogens
         • associated with asthma
       • ILC class 3 may be associated with IBD
     • role in pathogen clearance and inflammation
   • Myeloid cells
     • neutrophils (phagocytosis)
     • mast cells (histamine granule release -> inflammation)
     • macrophage (phagocytosis and Ag presentation)
     • Dendritic Cell (DC) - Ag uptake (peripheral role) and Ag presentation (in LN)
   • RECRUITMENT
     • 1. Rolling adhesion: infection triggers chemokine release + upregulation of selectins on endothelial cells. selectins bind carbohydrate groups on leukocytes (this is the problem step in LAD2 due to faulty upregulation of selectin)
       • 2. Firm adhesion: ICAM-1 on endothelial cells binds integrin (e.g. LFA-1 leukocyte functional antigen) on leukocyte
         • 3. Diapedesis/extravasation: leukocyte crosses the endothelial cell wall
           • 4. Migration: along a chemokine gradient to inflammatory site (cytokines bind GPCRs to direct cells in a given direction along their gradient

5. CELLULAR MEDIATORS OF INNATE IMMUNITY

   • Extracellular Pathogens
     • Neutrophils
       • abundant, short lived cell in human blood
       • Granulocyte/polymorphonuclear leukocyte
       • bacterial destruction (phagocytosis)
       • for small extracellular pathogens
     • Eosinophils + Basophils
       • large extracellular pathogens
       • Granulocyte/polymorphonuclear leukocytes
       • IgE dependent - activated by crosslinking of high affinity IgE receptors
       • release toxic proteins (eosinophil cationic protein), proteases (MMPs), and inflammatory mediators (histamine + leukotrienes that are preformed within cells)
       • eosinophils destroy the pathogen by surrounding it and exocytosing proteases and eosinophil cationic protein products to kill the large pathogen
     • Mast Cells
       • present all throughout the body
       • has granules + a normal nucleus
       • function is similar to eosinophils and basophils
       • expulsion of pathogen
       • release histamine + leukotrienes to drive an allergic reesponse
         • weepy eyes, sneezing, vomiting (increased secretions) to drive expulsion of microbes
       • activity deoendent ong IgE - made in small quantities, so it is quickly bound by high affinity Fc receptors on mast cells and eosinophils, when Ag binds IgE, the cell is activated
     • Neutrophil Extracellular Traps (NETs)
       • released by neutrophils to ensnare and destroy microbes (small) in a coordinated way
       • made of chromatin and antimicrobial products (e.g. elastase)
         • elastase kills the microbe
         • chromatin immobilises the microbe to limit speed
       • macrophages can also produce extracellular traps, but not as well as nautrophils. not that macrophages are better at producing producing degradative enzymes
   • Intracellular Pathogens
     • NK Cell
       • clearance of cytoplasmic pathogens (e.g. viruses) and tumour cells
       • destroy infected cells through detection of altered or absent MHC1
       • activated by cytokines produced by innate immune cells
       • Anti-viral response
         • infected cells release type 1 interferons (interferon alpha and interferon beta)
           • induces resistance to viral replication
             • activates NK cells to kill the cells infected by the virus
           • increase MHC1 and antigen presentation
             • many viral pathogens down regulate MHC 1 to evade the host
               • these cells are also targeted by NK cells
                 • Apoptosis initiated
                 • NK cells have inhibitory receptors (have immunoreceptor tyrosine-based inhibitory motifs(ITIMs) - recruit tyrosine phosphatase to prevent activation) to which MHC1 binds to prevent healthy cells being targeted. If MHC1 is low, the inhibitory receptor is unbound -> activation of virally infected vells
                 • n.b. an activating receptor (e.g. DAP10 or DAP12) must be bound
                   • DAP10 + DAP12 have immunoreceptor tyrosine based activation motifs (ITAMS) to recruit kinases and activate the NK cell
       • mediators of antibody dependent cell-mediated cytotoxicity
         • Ab binds Ag on target cells
         • Fc gamma receptor(FC receptor 3) recognises Ab -> NK cell binds
         • Fc receptor crosslinks activate NK cell
         • Perforins and granzymes released
           • causes pores to form -> cell lysis/death
     • MACROPHAGES
       • long lived cells present in all tissues, sentinels of innate immunity
       • Phagocytes
         • opsonic
           • Ab binds pathogen
             • Fc Receptors cross link
               • Aids Phagocytosis
           • allows capture of non-opsonic-resistant pathogens
           • Indirect recognition
         • non opsonic
           • macrophage recognises motifs of micro-organisms directly via scavenger receptors or C-type lectin receptors (direct recognition of pathogen)
       • Role in clearing pathogens present within intracellular vesicles
       • Mediators of inflammatory responses
       • antimicrobial pathways
         • note many are similar to those in neutrophils
         • Immediate (30mins)
           • Reactive oxygen species
             • inactivating key metabolic enzymes required for microbial survival
             • destroying protein structure (cysteine oxidation)
             • lipid + DNA oxidation => cytotoxicity
             • indirectly increases solubilisation of cationic proteases delivered to phagosome
           • Proteases
             • destroy microbe's proteins
           • acidification of phagosome
             • microbe cannot survive in the acidic conditions
         • Delayed (8hrs)
           • Anti-microbial peptide production
           • nutrient depletion e.g. iron depletion
           • Nitric Oxide production
             • slower than ROS because because NO synthase must be first described. Acts to form reactive nitrogen species
             • acts like ROS + has synergistic effects with ROS
               • formed are MPO (contains copper- gives infected fluid a green colour) and bleach (HOCl) which also destroy microbes
           • Last Resort: cell death
             • the inflammatory pathway this occurs by necrosis
             • releases pathogen to site of inflammation to be targeted by the assembled cells
     • Most cells
       • can mount an anti-viral response by producing type 1 interferons
         • can thus induce expression of genes that enable degradation of viral RNA (RNAse L) and inhibition of protein translocation and thus viral replication

6. Phagocytosis

   • specific form of endocytosis in which specialised cells take up particulate matter
   • particles taken up into a phagosome
   • phagosome fuses with lysosome to form phagolysosome, contents are destroyed here
   • macrophages and neutrophils are professional phagocytes

7. Opsonins

   • agents which coat a particle to enhance phagocytosis. e.g. Ab, C3b
   • Macrophages have specific opsonic receptors, FcRs, CRs that bind opsonins

8. PATHOGEN STRIKES BACK

   • resides in vesicles and avoids fusion with lysosome
   • exit vesicle and exist in the cytoplasm (listeria)
     • must trigger vesicular lysis
     • must then avoid autophagy (a TLR inducable event to recycle the contents of the host cell)
   • Survive in the acidic conditions of the phagosome
   • avoid metals e.g. zinc
     • the host cell can mobilise toxic levels of metals (toxic to the pathogen)
   • Salmonella
     • encodes Type 3 secretion system (SPI encoded)
       • delivers pathogen proteins to the host cytoplasm
         • increased uptake of the bacteria into the cell
         • targets antimicrobial feature of the host cell
     • SPI-2 encodes proteins to mislocate iNOS and NADPH oxidase
       • less ROS and NOS present to target bacteria
     • Host TLR binding activates virulence genes SPI-2 and SPI-2

9. Genetic Diseases of Innate Immunity

   • COMPLEMENT DEFICIENCIES
     • increases susceptibility to bacterial infection
       • specially to Neisseria, which is able to evade adaptive immunity (up to 10,000x risk
     • Classical Pathway: a C1 deficiency
       • immune complex disease
       • autoimmune phenotype because Ag-Ab complexes cannot be efficiently cleared and they can activate other pathways of the complement system
     • Mannose-binding-lectin pathway and Alternative pathway
       • MBL deficiency
         • usually in childhood (bacterial infections)
       • Alternative - no immune complex disease, but bacterial infection rates increase
   • Phagocyte deficiencies
     • Chronic granulomatous disease
       • intracellular and extracellular infection +granulomas form to contain the infection
       • NADPH system is not working, therefore ROS to kill pathogen is decreased
     • Glucose - Phosphate dehydrogenase deficiency
       • decreased NADPH, so decreased ROS, so decreased respiratory burst leads to chronic infection
     • myeloperoxidase deficiency
       • this is the enzyme for ROS production, so decreased ROS, so defective intracellular killing
     • Chediak-Higashi Syndrome
       • decreased enzymes required to kill bacteria and decreased phagosome/lysosome fusion
     • Leukocyte adhesion deficiency
       • Neutrophils fail to migrate to the site of infection
       • LAD-1: defect in beta integrin chain, so defective LFA-1, so defective binding of leukocyte to ICAM-1 of endothelial cell, so no firm adhesion, so decreased extravasation
       • recurrent and persistant pyogenic bacterial infections as well as poor wound healing are associated
       • LAD-2
         • ligands on leukocytes are faulty, so rolling adhesion to selectins of endothelial cells is decreased
   • TLR signalling deficiencies
     • e.g. MyD88 and IRAK-4 (serine threonine kinase) molecules of the TLR pathway are deficient, therefore inflammatory cytokines are not given the signal to be transcribed
     • lethal without antibiotic therapy during childhood, in adulthood other immune responses are enough to compensate

10. Inflammation

    • Acute
      • body's immediate response to infection, injury, or any perturbation to homeostasis
      • vasodilation occurs, WBCs move into inflammatory site
      • plasma and soluble mediators leak into inflammatory site
      • characterised by pain, swelling, redness and warmth
      • once the injury is repaired and infection is cleared there is inflammation resolution
      • disease state - sepsis can occur
        • severe systemic sepsis -> 30-50% fatality
        • trauma event
    • Chronic
      • long term inflammation due to long term stimulation -> tissue destruction and repair attempts
      • macrophages and lymphocytes (mononuclear) come in
      • abnormal repair - fibrosis and angiogenesis -> leads to decreased functionality and increased damage
      • can occur with a sterile agent, does not need to be infectious
      • associated diseases
        • rheumatoid arthritis, Crohn's disease + atherosclerosis
    • PRR+Inflammatory disease
      • PAMPs induce inflammation
      • Endogenous factors
        • made in disease state, e.g. inappropriate cell death -> cholesterol crystals, nucleic acids
          • activate NLRs to induce an inappropriate inflammatory response
        • DAMPs

11. LINK to ADAPTIVE IMMUNITY

    • pathogen may evade the innate immune system
      • adaptive immune system has T and B cells to make new specific Ab to target the pathogen with opsonisation
        • macrophages, eosonophils, basophils, mast cells and neutrophils of the innate system can not target the pathogen
          • Adaptive immune system is activated by innate immune system but also feeds back to innate system to increase its response