Innate Immune System II: Primary Protective Barriers, Recognition & Phagocytosis

Learning Outcomes

• By the end of this topic you should be able to:

  • Identify and describe the primary protective barriers (anatomical, chemical, biological) and key soluble/cellular molecules of the innate immune system.

  • Explain how each barrier/molecule contributes to preventing pathogen access and/or eliminating pathogens once inside the host.

  • Discuss the molecular mechanisms of phagocytosis, distinguishing oxidative (ROS/RNS-mediated) from non-oxidative (lysosomal) attack.

Functions of the Innate Immune System

• Prevent pathogen entry

  • Achieved via primary barriers (skin, mucosa, chemical secretions, commensal flora).

• Recognise and respond rapidly to invaders

  • Uses germ-line encoded pattern-recognition receptors (PRRs) and soluble opsonins to detect pathogen-associated molecular patterns (PAMPs).

• Recruit immune cells to infection sites

  • Cytokines and chemokines drive inflammation and chemotaxis.

• Destroy pathogens/foreign material

  • Phagocytes, complement, and antimicrobial peptides (AMPs) provide effector functions.

• Bridge to adaptive immunity

  • Antigen-presenting cells (APCs) activate T and B lymphocytes.

Primary Protective Barriers

1. Anatomical (Physical) Barriers

• Skin

  • Epidermis: thin, tightly packed, mostly dead keratinised cells – strong mechanical shield.

  • Dermis: vascularised connective tissue with hair follicles, sebaceous & sweat glands.

  • Resident immune cells: macrophages, dendritic cells, mast cells patrol beneath.

  • Biochemical weapons of skin:

  • Low surface pH (≈ 5.5).

  • Fatty acids in sebum – membrane-disruptive to microbes.

  • AMPs e.g. psoriasin (potent vs. E.\ coli).

• Mucosal Epithelium (GI, respiratory, urogenital tracts)

  • Single-layer or pseudostratified epithelial cells, tight junctions.

  • Continuous mucus layer from goblet cells – traps microbes, prevents adhesion.

  • Ciliary escalator in airways propels trapped microbes outward.

2. Chemical Defences

• Saliva, tears, mucus contain:

  • Lysozyme – hydrolyses peptidoglycan, making Gram-positive walls permeable; also exposes Gram-negative membranes to other AMPs.

  • Defensins (α, β), cathelicidins, histatins – cationic peptides that insert into microbial membranes and form pores.

  • Secretory IgA – opsonises bacteria/viruses for clearance.

• Stomach acid \text{pH}\approx 2; vaginal acid \text{pH}\approx 4.5 – hostile to most pathogens.

• Bile acids (e.g. deoxycholic acid) damage bacterial membranes; effective vs. H.\ pylori.

• Pulmonary surfactant (6 lipids, 4 proteins) lowers alveolar surface tension; collectins within surfactant opsonise microbes and enhance macrophage uptake.

3. Biological Barrier – Commensal Microflora

• Competitive exclusion (“colonisation resistance”): commensals out-compete pathogens for nutrients & binding sites.

• Immune maturation: microbial stimuli drive development of gut-associated lymphoid tissue, pattern-shape innate & adaptive responses.

• Metabolic support: help digest polysaccharides, produce vitamins, short-chain fatty acids beneficial to host epithelium.

• Direct antimicrobial activity: bacteriocins, fatty acids inhibit pathogens (e.g. human breast-milk flora vs. S.\ aureus; bacterial fatty acids limit Candida\ albicans).

• Dysbiosis linked to inflammatory bowel disease and systemic disorders.

4. Overview Table (mechanical, chemical, microbiological)*

• Skin: keratin barrier; fatty acids, β-defensins; normal skin flora.

• Gut: peristalsis; low pH, enzymes (pepsin), α-defensins (cryptdins), RegIII lecticidins; rich microbiota.

• Lungs: mucociliary clearance; surfactant collectins, cathelicidin; resident alveolar flora sparse.

• Eye/Nose/Mouth: tears & nasal secretions with lysozyme; β-defensins; commensals of conjunctiva & oral cavity.

Innate Immune System Molecules (beyond barriers)

• Cells (see previous lecture): neutrophils, monocytes, macrophages, dendritic cells, NK cells, mast cells, eosinophils, basophils.

• Soluble factors:

  • Complement proteins (classical, lectin, alternative pathways).

  • Opsonins: C3b, C4b, C1q, MBL, C-reactive protein (CRP), pentraxins.

  • Cytokines/chemokines.

  • Interferons (type I, III).

  • Acute-phase proteins.

• Antimicrobial peptides (AMPs):

  • >800 identified; <60 aa, cationic, amphipathic.

  • Mechanisms: membrane disruption; interference with DNA/RNA/protein synthesis.

  • Seen in plants, invertebrates, vertebrates – evolutionarily conserved.

  • Pharmaceutical interest as alternatives for antibiotic-resistant bacteria (MacNair et al., 2024, Nature Reviews Microbiology 22:262–275).

• Major Histocompatibility Complex (MHC) I & II molecules – link innate detection with adaptive T-cell activation.

Recognition of Pathogens

Pathogen-Associated Molecular Patterns (PAMPs)

• Conserved microbial structures essential for survival, absent from host:

  • LPS (Gram-negative outer membrane), lipoteichoic acid (Gram-positive), peptidoglycan motifs, flagellin, unmethylated CpG DNA, double-stranded RNA, mannan.

Pattern-Recognition Receptors & Molecules (PRRs)

1. Soluble PR molecules

   • Mannose-Binding Lectin (MBL): trimeric collectin, binds terminal mannose/fucose/N-acetylglucosamine; activates lectin complement pathway; functions as opsonin.

   • C1q, CRP, serum amyloid P, pentraxins – bind microbial ligands → complement activation/opsonisation.

2. Phagocytic Receptors (cell-bound)

   • C-type lectin receptors (e.g. Dectin-1) – recognise fungal β-glucans.

   • Scavenger receptors (SR-A, MARCO) – bind anionic polymers & oxidised lipoproteins.

   • Complement receptors (CR1, CR3, CR4) – detect C3b/iC3b-coated microbes.

   • Fc receptors (e.g. FcγR) – bind antibody-coated targets (important bridge to adaptive immunity).

3. Signalling PRRs

   • Toll-like receptors (TLR1–13 in mammals): plasma membrane & endosomal; each TLR recognises distinct PAMPs (e.g. TLR4 → LPS; TLR3 → dsRNA) → MyD88/TRIF pathways → transcription of cytokines & type I IFNs.

   • C-type lectin signalling receptors (e.g. Dectin-1, Dectin-2).

   • NOD-like receptors (NLRs) – cytosolic, detect intracellular peptidoglycan fragments (NOD1/NOD2) or form inflammasomes (NLRP3) → IL-1β production.

Opsonisation – Concept Refresher

• Term from Greek "Opsōneîn" = "to prepare for eating".

• Opsonins (C3b, antibodies, MBL, CRP) coat microbes → bind specific receptors on phagocytes → greatly enhanced phagocytosis (≈ "molecular seasoning").

Phagocytosis Mechanisms

Key Phagocytic Cells

• Neutrophils (polymorphonuclear leukocytes – PMNs): rapid responders; abundant granules.

• Monocytes (blood) differentiate into tissue macrophages.

• Macrophages: resident in tissues (Kupffer cells liver, microglia CNS, alveolar macrophages lung, etc.).

• Dendritic cells: specialised for antigen presentation after uptake.

Step-wise Process

1. Recognition & attachment via PRRs or opsonin receptors (CRs, FcRs).

2. Engulfment: actin-driven membrane extension forms phagosome.

3. Phagosome maturation: sequential fusion with granules/lysosomes → phagolysosome.

4. Killing & degradation via oxidative and non-oxidative pathways.

5. Exocytosis of debris or antigen presentation on MHC II.

Oxidative (Respiratory) Burst

• Trigger: ligation of PRRs/opsonin receptors → activation of membrane-bound NADPH oxidase (NOX2) complex.

• Biochemistry:
  \text{NADPH} + 2\,O2 \;\xrightarrow{NOX2}\; \text{NADP}^+ + 2\,O2^{\bullet-} + H^+

• ROS generated:

  • Superoxide anion O_2^{\bullet-} (primary product).

  • Via superoxide dismutase (SOD) → hydrogen peroxide H2O2.

  • Myeloperoxidase (MPO) in neutrophil granules converts H2O2 + Cl^- \rightarrow OCl^- (hypochlorite, powerful microbicide).

  • Hydroxyl radicals \bullet OH via Fenton chemistry.

• Inducible nitric-oxide synthase (iNOS) produces RNS:
  L\text{-arginine} + O2 \xrightarrow{iNOS} L\text{-citrulline} + NO^{\bullet} → peroxynitrite ONOO^- when reacts with O2^{\bullet-}.

• Collectively, ROS/RNS damage proteins, lipids, DNA → pathogen death.

• "Respiratory burst" = transient ↑ O₂ consumption by phagocyte.

Non-Oxidative Killing

• Fusion with primary/secondary granules or lysosomes delivers:

  • Lysozyme – cleaves peptidoglycan.

  • Proteases (elastase, cathepsins) – degrade proteins.

  • AMPs (defensins, cathelicidin, azurocidin, BPI).

  • Acid hydrolases; acidic pH 3.5 \text{–} 4.0 further bacteriostatic.

  • Nutrient competitors: lactoferrin (binds Fe^{3+}), vitamin B₁₂-binding protein.

Neutrophil-Specific Details (Fig 3.5 schematic)

• Bacterial N-formyl-methionyl-leucyl-phenylalanine (fMLP) peptides activate G-protein signalling → Rac2 GTPase.

• Rac2 assembles cytosolic (p47, p67, p40) & membrane (gp91^phox, p22^phox) NOX2 subunits on the phagolysosome.

• Potassium influx acidifies lumen → releases cationic proteases from granule matrix for optimal activity.

Antimicrobial Mechanisms Summary (Table-style)

• Acidification: pH\approx 3.5!\text{–}!4.0 → bacteriostatic/cidal.

• Toxic oxygen products: O2^{\bullet-}, H2O2, ^1O2, \bullet OH, OCl^-.

• Toxic nitrogen products: NO^{\bullet}, ONOO^-.

• AMPs: α-defensins (HNP1-4), β-defensins (HBD4), cathelicidin (LL-37), azurocidin, BPI.

• Enzymes: lysozyme, acid hydrolases, elastase.

• Competitors: lactoferrin (iron), transcobalamin (vit B₁₂).

Signalling PRRs & Interferon Induction

• 13 mammalian TLRs (surface or endosomal).

• Ligand engagement → adaptor proteins (MyD88, TRIF) → NF-κB, IRF3/7 activation.

• Outcomes: transcription of pro-inflammatory cytokines (TNF-α, IL-6), chemokines (CXCL8/IL-8), and type I interferons (IFN-α/β) critical for antiviral state.

• C-type lectin receptor signalling (e.g. Dectin-1) activates Syk → CARD9 → IL-23/IL-17 axis (anti-fungal).

• NOD-like receptors can assemble inflammasomes (NLRP3) → caspase-1 mediated maturation of IL-1β & IL-18.

Definitions (Exam Focus)

• Antimicrobial Peptides (AMPs): short (<60 aa), cationic peptides produced by epithelial cells & leukocytes that disrupt microbial membranes and/or block vital biosynthetic pathways.

• Pathogen-Associated Molecular Patterns (PAMPs): conserved molecular motifs unique to microbes (e.g. LPS, flagellin, unmethylated CpG DNA) recognised by the innate immune system.

• Pattern-Recognition Receptors/Molecules (PRRs): germ-line encoded receptors (cellular or soluble) that bind PAMPs and initiate innate immune responses (e.g. TLRs, NLRs, MBL, CRP).

• Reactive Oxygen Species (ROS): chemically reactive oxygen-derived molecules such as O2^{\bullet-}, H2O2, ^1O2, \bullet OH generated during oxidative burst, capable of damaging microbial macromolecules.

• Oxidative (Respiratory) Burst: rapid increase in oxygen consumption by phagocytes leading to large-scale ROS production via NADPH oxidase during phagocytosis.

Clinical / Practical Connections

• Chronic Granulomatous Disease (CGD): NADPH oxidase defect → impaired oxidative burst → recurrent catalase-positive bacterial & fungal infections.

• Antibiotic resistance crisis fuels interest in synthetic or recombinant AMPs.

• Probiotics/prebiotics aim to restore beneficial microbiota and reinforce colonisation resistance.

• TLR agonists/antagonists under investigation as vaccine adjuvants and anti-inflammatory therapeutics.

Suggested Reading

• Kuby Immunology, 8th ed., Ch. 4 (Innate Immunity).

• Janeway’s Immunobiology, 9th ed., Ch. 1-3 (overview, innate mechanisms, phagocyte biology).

• Roitt’s Essential Immunology (relevant chapters on innate defence).

• Parham, The Immune System, 3rd ed.

• MacNair et al., “Alternative therapeutic strategies to treat antibiotic-resistant pathogens,” Nature Reviews Microbiology, 22:262\text{–}275\ (2024).

*Figure numbers referenced correspond to Janeway’s Immunobiology 8th edition; visuals not reproduced here.