Notes on Innate Immunity: The First Lines of Defense (Chapter 2)

ANATOMIC BARRIERS AND INITIAL CHEMICAL DEFENSES

• Infectious diseases are caused by diverse living agents that replicate in their hosts. Pathogens include organisms that can be found in interstitial spaces, blood, lymph (intracellular and extracellular) and on epithelial surfaces.
• First barrier: Epithelial surfaces of the body provide the initial defense against infection.
• Infectious agents must overcome innate host defenses to establish a focus of infection.
• Epithelial cells and phagocytes produce several kinds of antimicrobial proteins.
• The barrier is composed of:
  • Skin, gut, lungs, eyes/nose/oral cavity
  • Mechanical defenses (tight junctions, cilia-driven flow, mucus movement)
  • Chemical defenses (low pH, fatty acids, enzymes)
  • Normal microbiota that compete with pathogens
• Protective immunity involves multiple effectors including Complement, Phagocytosis, Antibodies, NK cells, and T-cell/NK-cell dependent macrophage activation.
• Major antimicrobial components produced by epithelia and phagocytes include:
  • Enzymes: Lysozyme, secretory phospholipase A2 (sPLA2)
  • Antimicrobial peptides (AMPs): Defensins, Cathelicidins, Histatins, RegIII family (e.g., RegIIIα)
  • Lectins: RegIIIα among others
• Defenses are supplemented by normal microbiota which help prevent colonization by pathogens.

Epithelial barriers in detail (2-2 to 2-3)

• Epithelial surfaces provide the first barrier against infection:
  • Skin: Epidermis with tight junctions; stratum corneum forms a watertight lipid layer; lamellar bodies contribute to antimicrobial defense.
  • Gut epithelium: Goblet cells secrete mucus; Paneth cells produce α-defensins; crypts host RegIII peptides; microfolds aid antigen sampling; Various immune cells reside in underlying tissue.
  • Lungs: Bronchial ciliated epithelium; mucus-producing glands; cilia move mucus out of the airways.
  • Eyes/nose/oral cavity: Tears and saliva contain lysozyme; nasal passages have ciliated epithelia; pulmonary surfactant contributes to antimicrobial activity in the lungs.
• Antimicrobial components present across epithelia include:
  • Defensins: Short cationic peptides, amphipathic, disrupt microbial membranes; activated by proteolysis; α-defensins and β-defensins produced by different cell types.
  • Cathelicidins: Include human LL-37 in some epithelia; contribute to antimicrobial activity.
  • Histatins: Salivary antifungal peptides.
  • RegIII lectins: Carbohydrate-binding lectins with antimicrobial activity in the gut.
• Normal microbiota forms a protective background by occupying niches and producing antimicrobial substances that inhibit pathogen overgrowth.

Infectious agents must overcome innate defenses (2-3 to 2-4)

• Pathogens adhere to epithelium and may penetrate the epithelium, leading to local infection.
• Local infection can progress to local tissue involvement; phagocytes (especially in lung), wound healing processes, and antimicrobial proteins/peptides participate in defense.
• Innate immune responses include: Complement activation, cytokine and chemokine production, phagocyte recruitment, NK cell activation, and macrophage activation.
• Dendritic cells and macrophages sample antigen and can migrate to lymph nodes to initiate adaptive immunity.
• Blood clotting helps limit spread of infection.
• Clearance of infection typically requires adaptive immune components (antibody production, T-cell responses) but is supported by innate responses such as macrophage activation and cytotoxic mechanisms.

Antimicrobial proteins produced by epithelia and phagocytes (2-4)

• Antimicrobial enzymes:
  • Lysozyme: Found in tears and saliva; cleaves peptidoglycan.
  • Secretory phospholipase A2 (sPLA2): Found in secretions.
• Defensins:
  • Short cationic peptides; amphipathic; disrupt microbial membranes; active against bacteria, fungi, and enveloped viruses; secreted by keratinocytes, phagocytes, and mucosal epithelia; activated proteolytically.
  • Classes include α-defensins and β-defensins.
• Cathelicidins and histatins contribute to antimicrobial activity in mucosal surfaces.
• Lectins (e.g., RegIII family) contribute to bacterial targeting in mucosal surfaces.

The complement system and innate immunity (2-5 to 2-8)

• Core concept: The complement system recognizes features of microbial surfaces and marks them for destruction by coating them with C3b.
• Pathways of activation:
  • Lectin pathway: Soluble receptors recognize microbial surfaces to activate the complement cascade.
  • Classical pathway: Initiated by activation of the C1 complex; homologous to the lectin pathway.
  • Alternative pathway: An amplification loop for C3b formation; accelerated by properdin in the presence of pathogens.
• Activation is largely surface-restricted to the site of initiation.
• The complement system is ancient evolutionarily and involved in early host defense.
• The three pathways converge on common downstream events:
  • Formation of C3 convertases that cleave C3 into C3a and C3b and deposit C3b on pathogen surfaces.
  • Generation of C5 convertases that cleave C5 into C5a and C5b, leading to formation of the membrane-attack complex (MAC).
  • Activation products C3a and C5a promote local inflammation.
• Ingestion of complement-tagged pathogens by phagocytes is mediated by receptors for bound complement proteins (e.g., CR1/CD35).
• The small fragments of complement proteins (e.g., C3a, C5a, C4a) act as mediators of inflammation, increasing vascular permeability and promoting leukocyte recruitment.
• The terminal complement proteins polymerize to form pores in membranes (MAC) that can kill certain pathogens.
• Complement regulatory proteins protect host tissues from unintended activation and destruction and regulate all three activation pathways.
• Pathogens have evolved proteins that inhibit complement activation (various mechanisms listed below).

Stages and outputs of complement activation (2-5 to 2-6, 2-8)

• Stages of complement action (Fig. 2.13):
  • Pattern recognition trigger: Recognition of microbial surfaces.
  • Protease cascade amplification/C3 convertase formation: Propagates rapid amplification of C3 cleavage.
  • Inflammation: Release of inflammatory mediators (C3a, C5a) that recruit and activate leukocytes.
  • Phagocytosis: Opsonization with C3b and recognition by complement receptors on phagocytes.
  • Membrane attack: Formation of MAC leading to lysis of susceptible pathogens.
• Opsonization and phagocytosis:
  • Surface-bound C3b increases phagocytosis via receptors such as CR1.
  • C5a can activate macrophages to enhance phagocytosis via CR1.
• Local inflammation role:
  • C3a, C4a, C5a stimulate vascular permeability and adhesion molecule expression, promoting leukocyte recruitment.
• The MAC (C5b-9) creates pores in pathogen membranes, leading to lysis.

Complement components and convertases (2-6 to 2-8; Fig. 2.14, 2.16, 2.20-2.22)

• Functional protein classes in the complement system include:
  • Recognition and surface binding: C1q, MBL, ficolins; bind to antibodies and microbial surfaces.
  • Activating enzymes (convertases): C1r, C1s, MASP-1, MASP-2, MASP-3; the enzymes that cleave and activate downstream components.
  • Surface-binding proteins and opsonins: C4b, C3b; act as opsonins and form part of the convertases.
  • Anaphylatoxins: C4a, C3a, C5a; mediators of inflammation.
  • Membrane-attack proteins: C6, C7, C8, C9; form MAC.
  • Complement receptors: CR1 (CD35), CR2 (CD21), CR3, CR4, CRIg; mediate interactions with immune cells.
  • Regulators: CD59, DAF (CD55), MCP (CD46), C4BP, CR1, iC3b, Factor H; control activation and protect host cells.
• The C3 convertases (central catalytic complexes):
  • Classical and Lectin pathways: $C4b2a$
  • Alternative pathway: $C3bBb$
• The C5 convertases (accelerated by C3b deposition):
  • $C4b2a3b$ and $C3b2Bb$
• C3b deposition on pathogen surfaces marks for phagocytosis and contributes to C5 convertase formation.
• The anaphylatoxins promote inflammation by acting on blood vessels and immune cells: $C3a, C4a, C5a$.
• The membrane-attack complex (MAC) forms a pore in the target membrane: $C5b6789_{ ext{MAC}}$, enabling lysis of susceptible pathogens.
• Regulation of the convertases occurs at several steps (examples):
  • C1 inhibitor (C1INH) dissociates C1r and C1s from the active C1 complex.
  • DAF/CD55, C4BP, MCP/CD46 displace C2a from C4b2a; solubly cleaved forms become inactive (C4d, C4c).
  • iC3b formation and CR1/H roles in regulating C3b cleavage.
  • CD59 prevents final assembly of MAC at the C8–C9 stage.
• The thioester bond in C3 is critical for covalent deposition on surfaces:
  • In the C3 molecule, the thioester within the TED (thioester-containing domain) bond is protected before cleavage.
  • Cleavage by C3 convertase exposes a reactive thioester that can react with a nucleophile on a pathogen surface to form a covalent bond, producing C3b on the pathogen surface.
  • This enables stable opsonization and further convertase activity.
• Representative notation (for memory):
  • Classical/lectin C3 convertase: $C4b2a$
  • Alternative C3 convertase: $C3bBb$
  • C5 convertases: $C4b2a3b, C3b2Bb$
  • MAC: $C5b6789$
• The lectin pathway specifics (Fig. 2.20):
  • Mannose-binding lectin (MBL) and ficolins recognize microbial surface carbohydrates.
  • Activated MASPs (MASP-1, MASP-2, MASP-3) cleave C4 and C2 to form C4b2a (C3 convertase).
  • C4b2a cleaves C3 into C3a and C3b; C3b binds surface or convertase; one C4b2a can cleave up to ~1000 C3 molecules.
  • Formation of C4b2a3b leads to C5 convertase activity and MAC formation.
• The classical pathway specifics (Fig. 2.21-2.22):
  • C1 complex (C1q, C1r, C1s) binds pathogen surface directly or to antibody bound to surface.
  • Activation propagates via C1r and C1s; C4 and C2 are cleaved to form C4b2a (C3 convertase) and C2a (part of C3 convertase) with inflammatory peptide mediators C4a and C3a.
  • C4b2a forms C3 convertase; C3b acts as opsonin; C3b also initiates amplification via the alternative pathway.
  • C5 is targeted by the C5 convertase to generate C5a and C5b; C5b initiates MAC assembly.
• The alternative pathway specifics (Fig. 2.29, 2.31):
  • C3 undergoes spontaneous hydrolysis to C3(H2O), initiating deposition of C3 convertase on microbial surfaces.
  • Factor B binds to C3b; Factor D cleaves B to Bb forming C3bBb (initial C3 convertase).
  • Properdin stabilizes C3bBb, enhancing amplification on pathogens.
• Localization of activity:
  • Activation is largely restricted to the surface where initiated, limiting host damage.

Regulation and pathogen evasion (2-10, 2-16 to 2-17)

• Regulators maintain balance and protect host from complement-mediated injury:
  • C1INH blocks C1r/C1s activation in the classical pathway.
  • DAF (CD55) and CR1 (CD35) promote dissociation of C2a from C4b2a and C3b degradation, respectively.
  • C4BP and MCP (CD46) act as cofactors for Factor I to cleave C4b and C3b, generating inactive fragments (C4d, C4c; iC3b).
  • Factor H regulates the alternative pathway by restricting C3b deposition on host surfaces.
  • CD59 inhibits terminal MAC assembly at the C8–C9 stage on host cells.
• Microbial evasion strategies (Table/figures 2.37-2.38):
  • Surface and secreted proteins mimic host regulators or recruit them to surfaces to inhibit C3b deposition or convertase activity.
  • Examples of bacterial, fungal, and viral strategies include:
  • Neisseria meningitidis: factor H binding protein (fHbp) binds Factor H to inactivate C3b on its surface.
  • Borrelia burgdorferi: outer surface protein E (OspE) binds Factor H to inactivate bound C3b.
  • Streptococcus pneumoniae: PspC binds Factor H; C4BP can be recruited to inactivate C3b.
  • Staphylococcus aureus: Spa (protein A) binds immunoglobulins via Fc region, hindering C1 activation; SCIN inhibits C3 convertase activity; SAK cleaves immunoglobulins.
  • Immunoglobulin binding and cleavage by staphylococcal proteins interferes with C1 activation and downstream complement activity.

Summary of functional outcomes

• Complement tagging (C3b) promotes opsonization and phagocytosis via complement receptors (e.g., CR1/CD35).
• Anaphylatoxins (C3a, C4a, C5a) promote inflammation: increased vascular permeability, chemotaxis of macrophages and PMNs, and enhanced microbicidal activity.
• The MAC (C5b-9) can lyse susceptible pathogens by pore formation in membranes.
• Regulation ensures host protection and minimizes bystander damage; pathogens counter-regulate to enhance survival.

Connections to broader concepts (integrated view)

• Innate barriers provide immediate defense, buying time for the adaptive immune response to develop (early innate 0-96 h; adaptive response typically by >96 h).
• Dendritic cells bridge innate and adaptive immunity by transporting antigens to lymph nodes, enabling clonal expansion and differentiation of B and T cells.
• Complement acts as a central amplifier of innate defense, interfacing with phagocytes, antibodies, and inflammatory mediators to coordinate pathogen clearance.
• Real-world relevance: Defects in complement components or regulators can predispose individuals to infections or inflammatory diseases; understanding evasion mechanisms informs vaccine and therapeutic design.

Key numerical references and concrete details (LaTeX-formatted)

• C3 convertases (classical/lectin): $C4b2a$
• C3 convertases (alternative): $C3bBb$
• C5 convertases: $C4b2a3b$ and $C3b2Bb$
• Membrane-attack complex: $C5b6789_{ ext{MAC}}$
• One molecule of C4b2a can cleave up to approximately $10^3$ molecules of C3: $ext{C4b2a} o 3 ext{C3}$ (approx. 1000 cleavages per convertase)
• MAC pore diameter: about $10 ext{--}15 ext{ nm}$ (schematic representation in figures)
• Reactive thioester in C3 (TED) enables covalent attachment to pathogen surfaces; after cleavage, C3b is covalently bound via the TED to the pathogen surface.

Important terms for quick recall

• Pathogen-associated molecular patterns (PAMPs)
• Pattern recognition receptors (PRRs)
• Opsonization
• Anaphylatoxins: $C3a, C4a, C5a$
• Membrane-attack complex (MAC): $C5b6789_{ ext{MAC}}$
• Regulatory proteins: C1INH, DAF (CD55), MCP (CD46), CR1, CR2, Factor H, C4BP, CD59
• Pathogen evasion proteins and mechanisms (e.g., fHbp, PspC, SCIN, Spa, SAK)

Connections to prior and future topics

• Builds on general principles of innate immunity, pattern recognition, and host-pathogen interactions.
• Sets stage for adaptive immune activation, including antigen processing/presentation and clonal selection, which will be covered in later chapters.
• Highlights mechanisms of inflammation and tissue damage control, providing context for inflammatory and autoimmune conditions.