Lecture 16: Innate Immunity I: Immediate Response

• Innate immunity provides an immediate response through recognition of pathogens, recruitment of immune cells, removal/killing of foreign material/pathogens and regulation/activation of the adaptive immune system

Physical Barriers

• Physical barriers include epithelial cells, mucous membranes, and flushing/directional flow
• Epithelial cells that line the digestive, respiratory, and urogenital tracts form a physical barrier between environmental microbes and the host.
• Tight junctions block microbial passage.
• The stratum corneum flakes off, removing adherent microbes.
• Mucus captures pathogens and foreign agents, which are then removed by ciliary action in the bronchial tree along with sneezing and coughing or bodily functions like peristalsis in the gut, vomiting, and diarrhea.

Chemical Barriers

• Chemical barriers include defensins, pentraxins, lysozyme, lipases, lactoferrin, transferrin and HCl.
• Defensins are human antimicrobial peptides (AMP) with both hydrophilic and hydrophobic regions.
• Defensins penetrate microbial membranes, disrupting their integrity and leading to lysis.
• They are constitutively secreted at mucosal surfaces to maintain levels of microbiota and can be induced by infection.
• Paneth cells in intestinal crypts are major producers of defensins in the intestines, while epithelial cells and neutrophils can also produce them.
• Pentraxins are cyclic proteins that circulate in the blood and lymph and bind to pathogen surfaces, serving as a target site for phagocyte attachment.
• Production in the liver is induced by IL-6 and locally in tissues by infiltrating immune cells upon innate receptor activation.
• Lysozyme is an enzyme found in saliva, tears, mucus, plasma, tissue fluids, and phagocytic granules which destroys bacterial cell walls by hydrolyzing the 1,4 beta linkages between NAG and NAM, increasing permeability and causing the bacteria to burst. Effective against gram-positive bacteria.
• Phospholipase A2 penetrates the bacterial cell wall and hydrolyzes the membrane phospholipids.
• Cathelicidins are proteins released by skin and mucosal epithelial cells and are cleaved into toxic peptides by bacteria.
• Lactic and fatty acids are found in perspiration and sebaceous secretions and operate to inhibit bacterial growth.
• Lactoferrin and transferrin, found in body secretions, plasma, and tissue fluid, sequester iron for use by human cells and inhibit bacterial use.
• Hydrochloric acid is found in gastric secretions and destroys the membranes of swallowed bacteria.
• Urea is antibacterial, anti-inflammatory, and promotes healing.

Microbiota

• Mammalian babies have no commensal microbes before birth.
• Aspiring pathogens must compete with the well-adapted commensals for nutrients and space
• A Westernized diet is thought to reduce microbiome diversity, while increased fiber intake is associated with increased diversity.
• Aging lowers microbiome diversity, as does a reduction in physical activity.
• Up to 80% of immune cells reside in the gut epithelium functioning as the first line of defense against harmful agents

Complement System

• Complement is a collection of >30 proteins constitutively made by the liver and present in the blood, lymph, and extracellular fluids.
• Complement is activated once a pathogen penetrates the epithelial barrier.
• Complement proteins coat the bacterial surface and extracellular viral particles to make them more easily phagocytosed, especially encapsulated bacteria who could otherwise resist phagocytosis.
• Complement proteins are produced as zymogens; upon activation, these proteases cleave proteins in a series.
• Complement activation results in microbial lysis, inflammation, and enhanced phagocytosis (opsonization).

Complement Component 3 (C3)

• C3 is released from the liver in an inactive form and then undergoes a spontaneous conformation change or is cleaved to expose the thioester bond.
• Nucleophilic attack can occur by water (H2O), amino (R-NH2) or hydroxyl (R-OH) groups of proteins/carbs on the pathogen surface.
• C3b binds to the pathogen proteins/carbs and is termed complement fixation.

Alternate Pathway of Complement

• C3 undergoes a spontaneous conformation change in the aqueous environment of the blood/tissues; the thioester bond attaches C3 to H2O → iC3.
• The amount of iC3 increases near the surface of the pathogen (but not attached to the pathogen).
• iC3 binds to Factor B and is then cleaved by Factor D, resulting in iC3Bb
• iC3Bb effectively cleaves C3 → C3a (released) + C3b (attaches to pathogen surface) - and is a C3 convertase
• Pathogen-bound C3b binds Factor B and promotes cleavage by Factor D.
• C3bBb is a potent C3 convertase that remains attached to the pathogen surface.
• Properdin (Factor P) enhances alternative C3 convertase activity by stabilizing the alternative C3 convertase complex (C3bBb) and prevents degradation by other proteases.
• Factor H decreases the number of alternative C3 convertase complexes -> Factor H binds to C3b together with Factor I resulting C3b is cleaved to iC3b, preventing any further C3 convertase formation

Complement Activation on Human Cells

• DAF (Decay-accelerating factor) and MCP (Membrane cofactor protein) are membrane proteins on the surface of human cells.
• DAF binds to C3b and dissociates Bb to inactivate the C3 convertase.
• MCP binds to C3b and recruits Factor I for the cleavage of C3b into iC3b.
• Factor H can also bind to sialic acid on human cells and recruit Factor I to cleave C3b-Streptococcus pyogenes and Staphylococcus aureus cover their cells with sialic acid to inactivate complement.
• C3b initiates the next function of complement activation, eventually being cleaved into C5a and C5b fragments

Membrane Attack Complex (MAC)

• C5b initiates formation of the Membrane Attack Complex (MAC) to directly lyse pathogens
• C5b binds to C6, forming a stable complex with a binding site for C7.
• C7 binds to C5bC6, exposing a hydrophobic region of C7 for initial attachment to the pathogen membrane.
• C8 binds to C5bC6C7, exposing a hydrophobic region of C8 that inserts into the pathogen membrane.
• C9 monomers assemble by binding to C8 and spanning the membrane, leading to osmotic lysis of the pathogen.

Blocking Alternative C5 Convertase Activity in Human Cells

• Protectin (CD59) prevents the recruitment of C9 monomers to the C5bC6C7C8 complex.
• Homologous restriction factor (HRF) operates the same way.
• S protein prevents C7 interaction with the cell membrane.
• Clusterin and Factor J operate the same way.
• Disruption of these mechanisms can lead to paroxysmal nocturnal hemoglobinuria, where red blood cells are lysed by complement

Inflammation

• During complement activation, two smaller fragments, C3a and C5a, are generated, which function to promote inflammation
• C3a and C5a result in:
  • Degranulation of mast cells and basophils in tissues
  • Increase vascular permeability by released products of mast cells and basophils
  • Vasodilation (by histamine)
  • Increase recruitment of neutrophils and monocytes from the blood to the site of inflammation
  • Increase complement receptors on phagocytes
  • Contraction of visceral smooth muscle
• Too much C3a and C5a can induce anaphylactic shock.

Macrophages

• First effector cells that a pathogen encounters upon invasion are the resident macrophages.
• Found in connective tissues, and linings of the Gl and respiratory tracts, brain, alveoli of the lungs and the liver and are long-lived phagocytic cells
• Macrophages express complement receptors that enhance phagocytosis

Blood Tests for Complement Deficiencies

• AH50:
  • Measures the activity of the alternative complement Pathway
  • High AH50 indicates:
    • A deficiency in one or more of the early components of the alternative complement pathway, which could increase susceptibility to certain infection
  • Low AH50 indicates:
    • Improved survival during critical illness
    • Fewer bloodstream infections
• CH50:
  • Measure total Classical complement activity
  • High CH50 indicates:
    • May indicate an inflammatory process in the body, (less common)
  • Low CH50 indicates:
    • Suggests a potential deficiency in the classical complement pathway, requiring further investigation to identify the specific complement component involved