# Lecture 18: Innate Immunity

## Microbiology

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## Learning Objectives

By the end of this lecture, students will be able to:

1. Describe the physical, chemical, and biological barriers that constitute the first line of defense
2. Explain the role of pattern recognition receptors (PRRs) in detecting microbial invaders
3. Distinguish between PAMPs and DAMPs and provide examples of each
4. Describe the cellular components of innate immunity and their functions
5. Explain the complement system, its activation pathways, and effector functions
6. Describe the inflammatory response and the role of cytokines
7. Explain how innate immunity interfaces with adaptive immunity

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## Lecture Content

### I. Overview of Innate Immunity

The innate immune system provides **immediate, non-specific defense** against pathogens. It is present from birth and does not require prior exposure to a pathogen. In the classical view, innate immunity lacks immunological memory, though the emerging concept of "trained immunity" has begun to modify this understanding. The innate immune system comprises physical and chemical barriers (the first line of defense), cellular defenses including phagocytes, natural killer cells, and innate lymphoid cells, humoral factors such as complement, cytokines, and acute phase proteins, and the inflammatory response. It responds within **minutes to hours** of encountering a pathogen. Innate immunity is evolutionarily ancient and present in virtually all multicellular organisms.

### II. First Line of Defense: Physical and Chemical Barriers

#### A. Skin

The intact epidermis consists of stratified squamous epithelium with a keratinized outer layer that forms a formidable physical barrier to microbial invasion. Low moisture and an acidic pH of approximately 5.5 inhibit microbial growth. Sebaceous gland secretions contain fatty acids such as oleic acid with antimicrobial activity. The skin produces antimicrobial peptides, including **defensins** (both alpha and beta forms) and **cathelicidin (LL-37)**, which directly kill or inhibit microorganisms. The normal skin microbiota also contributes to defense by competing with potential pathogens for resources, a process known as colonization resistance.

#### B. Mucous Membranes

Mucous membranes line the respiratory, gastrointestinal, and urogenital tracts, providing a barrier that combines physical and chemical defenses. **Mucus**, a viscous glycoprotein layer composed of mucins, traps microbes and prevents their access to epithelial cells. The **mucociliary escalator** in the respiratory tract consists of ciliated epithelial cells that continuously move mucus-trapped particles upward toward the pharynx for expulsion. Several soluble factors enhance mucosal defense: **lysozyme**, found in tears, saliva, nasal secretions, and mucus, cleaves the beta-1,4-glycosidic bond between NAM and NAG in peptidoglycan. **Lactoferrin** sequesters iron from bacteria, limiting their growth. **Secretory IgA**, produced by plasma cells in the mucosa, agglutinates microbes and prevents their attachment, bridging the innate and adaptive immune systems.

#### C. Other Chemical Defenses

Additional chemical defenses protect specific body sites. **Gastric acid** at pH 1.5--3.5 destroys most ingested microbes. **Bile salts** exert a detergent-like action that disrupts microbial membranes in the small intestine. The flow of **urine** mechanically flushes the urinary tract. **Vaginal pH** of approximately 3.8--4.5, maintained by lactic acid from *Lactobacillus* species, inhibits the growth of many potential pathogens.

<image>A multi-panel figure of the body's physical and chemical barriers. Panel A: Cross-section of skin showing the keratinized epidermis, sebaceous glands secreting fatty acids, and antimicrobial peptides (defensins, cathelicidin) in the dermis; resident microbiota on the surface. Panel B: Cross-section of respiratory epithelium showing goblet cells producing mucus, ciliated cells forming the mucociliary escalator, and trapped bacteria being swept upward. Panel C: Cross-section of the stomach lining showing gastric pits secreting HCl (pH 1.5-3.5) and dead bacteria in the lumen. Panel D: Table summarizing chemical defenses at each body site: skin (fatty acids, defensins), eyes (lysozyme, lactoferrin), mouth (lysozyme, histatins), stomach (HCl, pepsin), intestine (bile salts, defensins, IgA), urogenital (urine flow, vaginal pH).</image>

### III. Pattern Recognition: PAMPs, DAMPs, and PRRs

#### A. Pathogen-Associated Molecular Patterns (PAMPs)

**PAMPs** are conserved molecular structures found in broad classes of microbes but absent from host cells, allowing the innate immune system to distinguish self from non-self. Important examples include **lipopolysaccharide (LPS)** from the Gram-negative outer membrane (detected by TLR4), **lipoteichoic acid (LTA)** and **peptidoglycan** from the Gram-positive cell wall (detected by TLR2), **flagellin** from bacterial flagella (detected by TLR5), **dsRNA** produced as a replication intermediate of many viruses (detected by TLR3, RIG-I, and MDA5), **CpG DNA** containing unmethylated CpG dinucleotides common in bacterial and viral DNA (detected by TLR9), **beta-glucan** from the fungal cell wall (detected by Dectin-1), and **mannose-rich glycoproteins** on microbial surfaces (detected by the mannose receptor and mannose-binding lectin).

#### B. Damage-Associated Molecular Patterns (DAMPs)

**DAMPs** are endogenous molecules released by damaged or dying host cells. Examples include HMGB1, ATP, uric acid, heat shock proteins, and mitochondrial DNA. DAMPs signal tissue damage and can trigger sterile inflammation even in the absence of infection.

#### C. Pattern Recognition Receptors (PRRs)

The innate immune system deploys several families of pattern recognition receptors to detect PAMPs and DAMPs. **Toll-like receptors (TLRs)**, of which 10 have been identified in humans (TLR1--10), are divided into cell surface receptors (TLR1, 2, 4, 5, 6) that detect extracellular PAMPs and endosomal receptors (TLR3, 7, 8, 9) that detect nucleic acids after phagocytosis. TLR signaling proceeds through the MyD88-dependent pathway, leading to NF-kB activation and production of pro-inflammatory cytokines, and through the TRIF-dependent pathway, leading to the production of type I interferons.

**NOD-like receptors (NLRs)** are cytoplasmic sensors. NOD1 and NOD2 detect peptidoglycan fragments (meso-DAP and MDP, respectively). The **NLRP3 inflammasome**, a multiprotein complex comprising the NLRP3 sensor, the ASC adaptor, and caspase-1, cleaves pro-IL-1-beta and pro-IL-18 into their active forms and can also trigger pyroptosis, an inflammatory form of cell death mediated by gasdermin D.

**RIG-I-like receptors (RLRs)** are cytoplasmic sensors of viral RNA. RIG-I detects short dsRNA with a 5'-triphosphate modification, while MDA5 detects long dsRNA. These receptors signal through MAVS to activate IRF3/7 and induce type I interferons (IFN-alpha and IFN-beta). **C-type lectin receptors (CLRs)**, including Dectin-1 and the mannose receptor, recognize carbohydrate structures on fungi and bacteria. The **cGAS-STING pathway** detects cytoplasmic dsDNA of viral or bacterial origin, producing cyclic GMP-AMP that activates STING and ultimately leads to type I interferon production.

<image>A comprehensive diagram of pattern recognition receptors and their ligands. Panel A: A cell showing TLR locations -- TLR4 on the cell surface binding LPS (with MD-2 and CD14 co-receptors), TLR2/TLR1 heterodimer binding lipopeptides, TLR5 binding flagellin; endosomal TLR3 binding dsRNA, TLR7/8 binding ssRNA, TLR9 binding CpG DNA. Signaling arrows from surface TLRs through MyD88 to NF-kB (pro-inflammatory cytokines: TNF-alpha, IL-1, IL-6) and from endosomal TLRs through TRIF to IRF3/7 (type I interferons). Panel B: Cytoplasmic sensors -- NOD1/NOD2 detecting peptidoglycan fragments; NLRP3 inflammasome assembly (NLRP3 + ASC + pro-caspase-1) leading to IL-1-beta and IL-18 release and pyroptosis. Panel C: RIG-I detecting 5'-triphosphate dsRNA, signaling through MAVS to produce interferons. Panel D: cGAS detecting cytoplasmic DNA, producing cGAMP that activates STING.</image>

### IV. Cellular Components of Innate Immunity

#### A. Phagocytes

**Neutrophils** (polymorphonuclear leukocytes, PMNs) are the most abundant white blood cells, comprising 60--70% of circulating leukocytes. They are the first responders to sites of infection, arriving within minutes, but are short-lived, surviving only about five days in circulation. Neutrophils kill microbes through phagocytosis, the production of reactive oxygen species (ROS) during the respiratory burst, reactive nitrogen species, and degranulation, which releases antimicrobial enzymes including lysozyme, defensins, and elastase. Neutrophils can also deploy **neutrophil extracellular traps (NETs)** -- web-like structures composed of DNA, histones, and antimicrobial proteins that trap and kill extracellular bacteria.

**Macrophages** derive from blood monocytes that migrate into tissues and differentiate. They are known by tissue-specific names: Kupffer cells in the liver, alveolar macrophages in the lungs, microglia in the central nervous system, and osteoclasts in bone. Macrophages perform phagocytosis, present antigens to T cells (linking innate to adaptive immunity), produce cytokines, and participate in tissue repair. Activated macrophages polarize into two functional states: M1 (classically activated) macrophages are pro-inflammatory and microbicidal, while M2 (alternatively activated) macrophages are anti-inflammatory and promote tissue repair.

**Dendritic cells (DCs)** are professional antigen-presenting cells that serve as sentinel cells in tissues -- in the skin, they are called Langerhans cells. Upon capturing antigens at an infection site, dendritic cells migrate to lymph nodes, where they present processed antigen on MHC II molecules to T cells. Dendritic cells are thus the critical bridge between innate and adaptive immunity.

#### B. Phagocytosis -- Step by Step

Phagocytosis proceeds through a coordinated sequence of events. **Chemotaxis** draws the phagocyte toward the microbe along a chemical gradient established by complement fragments (C5a), bacterial products, and chemokines. **Recognition and attachment** involve binding of the microbe by PRRs and opsonin receptors (Fc receptors and complement receptors). **Engulfment** occurs as pseudopods extend around the microbe, internalizing it into a **phagosome**. **Phagolysosome formation** follows as the phagosome fuses with a lysosome. **Killing and digestion** within the phagolysosome employ reactive oxygen species (superoxide, hydrogen peroxide, hypochlorous acid generated by myeloperoxidase), reactive nitrogen intermediates (nitric oxide), defensins, lysozyme, and an acidic pH of approximately 4.5. Finally, **antigen presentation** involves displaying processed peptides on MHC molecules for recognition by T cells.

#### C. Natural Killer (NK) Cells

**Natural killer cells** are large granular lymphocytes of the innate immune system that kill virus-infected cells and tumor cells without prior sensitization. They operate according to the **"missing self" hypothesis**: NK cells detect the downregulation or absence of MHC class I molecules, which viruses and tumors frequently downregulate to evade cytotoxic T cells. Activating receptors such as NKG2D recognize stress-induced ligands on abnormal cells, while inhibitory receptors called KIRs recognize self-MHC I and prevent the killing of normal cells. When activating signals outweigh inhibitory signals, NK cells kill their targets by releasing **perforin** (a pore-forming protein) and **granzymes** (serine proteases that induce apoptosis). NK cells also mediate **antibody-dependent cellular cytotoxicity (ADCC)** through their Fc receptors (CD16).

### V. The Complement System

The complement system comprises more than 30 plasma proteins, mainly produced by the liver, that enhance ("complement") immune defense. These proteins circulate as inactive zymogens and are activated through sequential proteolytic cleavage.

#### A. Activation Pathways

Three pathways converge on the generation of C3 convertase. The **classical pathway** is initiated when C1q binds to antibody-antigen complexes (IgG or IgM) on the microbial surface, leading to activation of C1r/C1s, cleavage of C4 and C2, and formation of the C3 convertase C4b2a. The **lectin pathway** begins when mannose-binding lectin (MBL) or ficolins bind mannose-rich microbial surfaces, activating MASP-1/MASP-2 to generate the same C4b2a C3 convertase. The **alternative pathway** is initiated by spontaneous low-level hydrolysis of C3 (the "tick-over" mechanism): C3b deposits on microbial surfaces and, together with Factor B and Factor D, generates the C3 convertase C3bBb, which feeds into an amplification loop. All three pathways converge on the cleavage of C3 into **C3a** (an anaphylatoxin) and **C3b** (an opsonin).

#### B. Effector Functions

Complement effector functions fall into three categories. **Opsonization** occurs when C3b coats microbes, enhancing their phagocytosis through complement receptors (CR1) on phagocytes. **Inflammation** is promoted by the anaphylatoxins C3a and C5a, which recruit and activate phagocytes, promote mast cell degranulation, and increase vascular permeability. The **membrane attack complex (MAC)**, formed by C5b, C6, C7, C8, and poly-C9, assembles a transmembrane pore that causes osmotic lysis of Gram-negative bacteria and enveloped viruses. Complement also aids in the **clearance of immune complexes**: C3b and C4b bound to immune complexes engage CR1 on erythrocytes, which transport the complexes to the liver and spleen for removal.

#### C. Regulation of Complement

Tight regulation prevents complement-mediated damage to host cells. **C1 inhibitor (C1-INH)** inhibits C1r/C1s and MASPs; its deficiency causes hereditary angioedema. **Factor H and Factor I** inactivate C3b on host cell surfaces, exploiting the fact that host cells have regulators while microbes lack them. **CD59 (protectin)** prevents MAC assembly on host cells, and **decay-accelerating factor (DAF/CD55)** dissociates C3/C5 convertases on host cells.

<image>A three-pathway diagram of the complement system. Left: Classical pathway -- antibody (IgG/IgM) bound to bacterial surface, C1q binding to Fc regions, sequential activation of C1r, C1s, C4, C2, forming C4b2a (C3 convertase). Center: Lectin pathway -- MBL binding to mannose residues on the bacterial surface, MASP-1/2 activation, leading to the same C4b2a convertase. Right: Alternative pathway -- spontaneous C3 hydrolysis, C3b depositing on the microbial surface, Factor B and Factor D generating C3bBb convertase. All three arrows converge at C3 cleavage into C3a and C3b. Below the convergence: three branches showing effector functions -- (1) C3b coating bacteria with phagocyte engulfing (opsonization), (2) C3a and C5a causing mast cell degranulation and neutrophil chemotaxis (inflammation), (3) C5b-C9 forming the MAC pore in a bacterial membrane (lysis).</image>

### VI. The Inflammatory Response

Inflammation is a localized tissue response to infection or injury, characterized by the cardinal signs: **rubor** (redness), **calor** (heat), **tumor** (swelling), **dolor** (pain), and **functio laesa** (loss of function).

#### A. Acute Inflammation

Acute inflammation begins when tissue-resident macrophages and mast cells detect PAMPs via PRRs. This triggers the release of **pro-inflammatory cytokines and chemokines**: **TNF-alpha** activates endothelial cells, induces adhesion molecule expression, and promotes fever. **IL-1-beta** contributes to fever, the acute phase response, and T cell co-stimulation. **IL-6** induces acute phase protein production in the liver, fever, and B cell differentiation. **IL-8 (CXCL8)** is a potent neutrophil chemotactic factor. **IL-12** activates NK cells and promotes Th1 differentiation.

These signals produce **vascular changes**: vasodilation driven by histamine and prostaglandins increases blood flow, causing redness and heat. Increased vascular permeability leads to plasma leakage into tissues, producing swelling (edema). Adhesion molecules (selectins, ICAM-1, VCAM-1) are upregulated on the endothelium, facilitating **leukocyte recruitment (extravasation)** through the sequential steps of margination, rolling (mediated by selectins), firm adhesion (mediated by integrins binding ICAMs), diapedesis (transmigration through the endothelium), and chemotaxis to the infection site. Neutrophils dominate the early cellular infiltrate (first 6--24 hours), with macrophages predominating later.

#### B. Fever

Fever is a systemic response to infection mediated by endogenous pyrogens, including IL-1, IL-6, TNF-alpha, and prostaglandin E2. These pyrogens act on the hypothalamus to raise the thermoregulatory set point. Moderate fever enhances immune function by increasing phagocytosis and lymphocyte activity while reducing microbial replication. However, excessive fever (above 40.5 degrees C) can be harmful, causing protein denaturation and seizures.

#### C. Acute Phase Response

In response to IL-6, the liver produces acute phase proteins that bolster the immune response. **C-reactive protein (CRP)** acts as an opsonin, binds phosphorylcholine on bacteria, and activates the classical complement pathway. **Mannose-binding lectin (MBL)** activates the lectin complement pathway. **Ferritin** sequesters iron from potential pathogens. **Fibrinogen** promotes clot formation to wall off infections. The **erythrocyte sedimentation rate (ESR)** and **CRP** are widely used clinical markers of inflammation.

### VII. Interferons and Antiviral Defense

**Type I interferons (IFN-alpha and IFN-beta)** are produced by virus-infected cells upon detection of viral nucleic acids through RIG-I, MDA5, TLR3/7/8/9, and the cGAS-STING pathway. They bind to the IFNAR receptor on neighboring cells, triggering JAK-STAT signaling and the induction of hundreds of **interferon-stimulated genes (ISGs)**. The products of ISGs include **PKR**, which phosphorylates eIF2-alpha to shut down protein synthesis; **OAS/RNase L**, which degrades viral RNA; **Mx proteins**, which block viral replication; and **APOBEC3**, which deaminates viral DNA. Type I interferons also enhance NK cell activity, upregulate MHC I expression, and promote adaptive immune responses.

**Type II interferon (IFN-gamma)**, produced mainly by NK cells and T cells, is a potent macrophage activator. **Type III interferons (IFN-lambda)** provide mucosal antiviral defense with functions similar to type I interferons but are more tissue-restricted.

### VIII. Trained Immunity

The emerging concept of **trained immunity** holds that innate immune cells -- monocytes, macrophages, and NK cells -- can exhibit enhanced responsiveness upon re-exposure to certain stimuli. This enhanced state is mediated by **epigenetic reprogramming** through histone modifications and metabolic rewiring, rather than by the genetic rearrangement that underlies adaptive memory. BCG vaccination, for example, induces trained immunity that provides non-specific protection against unrelated infections. While trained immunity is not antigen-specific and is less durable than adaptive memory, it challenges the strict dichotomy traditionally drawn between innate and adaptive immunity.