INNATE IMMUNITY TBL STUDY GUIDE Comprehensive Review for Tomorrow's IRAT/TRAT

1. PATTERN RECOGNITION RECEPTORS (PRRs) & TOLL-LIKE RECEPTORS (TLRs)

What Are PAMPs and DAMPs?

• PAMPs (Pathogen-Associated Molecular Patterns)

  • Unique molecular structures found ONLY on pathogens, NOT on human cells.

  • Examples:

  • Bacterial cell wall components (e.g., LPS, peptidoglycan)

  • Viral RNA/DNA

  • Fungal cell wall components

  • Serve as "red flags" signaling to the immune system: "DANGER - INVADER PRESENT".

• DAMPs (Damage-Associated Molecular Patterns)

  • Molecules released by damaged or dying HOST cells.

  • Examples:

  • ATP

  • Uric acid crystals

  • Heat shock proteins

  • DNA from damaged cells

  • Signal: "Something is wrong with our own cells - investigate!"

Pattern Recognition Receptors (PRRs) - The Detectors

• PRRs are receptors on immune cells that recognize PAMPs and DAMPs.

• Analogy: Think of PRRs as security cameras placed in different locations to detect threats.

Location of Different PRRs

1. Cell Surface/Extracellular PRRs - Detect threats OUTSIDE the cell

   • TLR1, TLR2, TLR4, TLR5, TLR6

   • C-type lectin receptors (recognize carbohydrates on pathogens)

   • N-Formyl methionine receptor (recognizes bacterial proteins)

2. Endosomal PRRs - Detect threats INSIDE vesicles (after cell has ingested something)

   • TLR3, TLR7, TLR8, TLR9

   • These recognize viral nucleic acids

3. Cytoplasmic PRRs - Detect threats in the cell's cytoplasm

   • NOD-like receptors (NOD1, NOD2, NALP3/NLRP3)

   • RIG-I-like receptors (RIG-I, MDA5)

   • DAI (DNA sensor)

   • Cryopyrin

Detailed TLR Table - Memorize This

Why TLR Location Matters

• Surface TLRs (1,2,4,5,6): Recognize extracellular bacteria and fungi

• Endosomal TLRs (3,7,8,9): Recognize NUCLEIC ACIDS from viruses that have been internalized to prevent accidental recognition of our own DNA/RNA in cytoplasm

Other Important Cytoplasmic PRRs

TLR SIGNALLING PATHWAY - The Cascade

1. Step 1: TLR Recognizes PAMP/DAMP

   • TLR has leucine-rich repeats binding to the pathogen molecule

   • TLR has a TIR (Toll-IL-1 receptor) domain inside the cell

2. Step 2: Recruitment of Adaptor Proteins

   • MyD88 is the main adaptor protein

   • Adaptors bring in kinases and other signaling molecules

3. Step 3: Activation of Transcription Factors

   • Two main pathways branch from here:

     • PATHWAY A: NF-κB Activation

     • NF-κB normally sits in the cytoplasm, inactive

     • TLR signaling frees NF-κB to enter the nucleus

     • Turns on genes for:

       • Pro-inflammatory cytokines: IL-1, IL-6, TNF-α

       • Adhesion molecules: E-selectin, ICAM-1, VCAM-1

       • Costimulatory molecules: CD80, CD86

     • RESULT: Acute Inflammation

     • PATHWAY B: IRF (Interferon Regulatory Factor) Activation

     • IRFs are transcription factors that make interferons

     • Turns on genes for: Type I Interferons (IFN-α, IFN-β)

     • RESULT: Antiviral state, warns neighboring cells

     • Neighboring cells upregulate antiviral proteins; virus replication is blocked

CLINICAL CORRELATIONS - TLR Defects

Memory Tricks for TLRs

• TLR4 = LPS = Gram-negative sepsis

  • Think: "4 Letters: L-P-S"

• TLR5 = Flagellin = 5 letters in "Flagel"

• TLR9 = CpG DNA = "9 looks like G"

• TLR 3,7,8,9 are all ENDOSOMAL = recognize nucleic acids

• TLR2 is promiscuous = recognizes MANY things (peptidoglycan, LTA, zymosan)

2. THE INFLAMMASOME (NLRP3)

What Is an Inflammasome?

• An inflammasome is a large protein complex that assembles in the cytoplasm when danger is detected.

• Analogy: Consider it an "alarm system" that triggers inflammation.

The Three Components

1. SENSOR:

   • NLRP3 (NOD-like receptor family, pyrin domain containing 3)

     • Also called Cryopyrin

     • Detects danger signals in the cytoplasm

2. ADAPTOR:

   • ASC (Apoptosis-associated Speck-like protein containing a CARD)

     • Bridges the sensor and the effector

3. EFFECTOR:

   • Pro-caspase-1 (inactive enzyme)

     • When activated, converts to Caspase-1

What Activates NLRP3?

• The inflammasome can be triggered by many different danger signals:

  • Pathogen-Related Signals:

  • Bacterial products (e.g., peptidoglycan fragments)

  • Extracellular ATP (released from damaged cells)

  • Viral DNA in cytoplasm

  • Pore-forming toxins

  • Crystal/Particle Signals:

  • Uric acid crystals (associated with gout)

  • Cholesterol crystals (related to atherosclerosis)

  • Silica, asbestos, amyloid-β (related to Alzheimer's)

  • Cellular Stress Signals:

  1. K+ efflux (potassium exiting the cell)

  2. Reactive Oxygen Species (ROS)

  3. Lysosomal damage

The NLRP3 Inflammasome Pathway - Step by Step

1. PRIMING STEP (Signal 1):

   • TLR or other receptor detects PAMP

   • NF-κB is activated

   • Cell produces more pro-IL-1β (the inactive form)

   • Cell produces more NLRP3 protein

   • No action yet—just preparation

2. ACTIVATION STEP (Signal 2):

   • Danger signal appears (ATP, crystals, K+ efflux, ROS, viral DNA)

   • NLRP3 sensor detects it

   • NLRP3 together with ASC and Pro-caspase-1 assemble

   • Multiple pro-caspase-1 molecules activate each other to become Active Caspase-1

3. EFFECTOR STEP:

   • Active Caspase-1 cleaves Pro-IL-1β → Mature IL-1β

   • Active Caspase-1 cleaves Pro-IL-18 → Mature IL-18

   • IL-1β and IL-18 are SECRETED from the cell

   • RESULT: IL-1β → powerful pro-inflammatory cytokine

     • Causes FEVER (acts on hypothalamus)

     • Activates endothelial cells → adhesion molecules

     • Recruits more immune cells to the site of inflammation

   • IL-18 → activates NK cells and T cells

Why Two Signals?

• This two-signal system prevents accidental inflammation:

  • Signal 1 (TLR) = "There might be danger"

  • Signal 2 (NLRP3 activator) = "Confirmed danger!"

  • Need BOTH to trigger inflammation.

Pyroptosis - Inflammatory Cell Death

• When the inflammasome is strongly activated:

  • Caspase-1 cleaves Gasdermin D

  • Gasdermin D forms pores in the cell membrane

  • Cell swells and bursts → PYROPTOSIS

  • Releases all the inflammatory contents.

  • Different from apoptosis, which is quiet and controlled.

CLINICAL CORRELATIONS - Inflammasome Defects

• GAIN-OF-FUNCTION Mutations (Inflammasome too active):

  • Syndrome

  • Familial Mediterranean Fever (FMF)

    • Symptoms: Recurrent fever, peritonitis, arthritis

  • Muckle-Wells syndrome

    • Symptoms: Urticaria, deafness, amyloidosis

  • NOMID/CINCA

    • Symptoms: Neonatal onset, rash, CNS inflammation

  • Gout

    • Uric acid crystals → NLRP3 → inflammation

  • Type 2 Diabetes

    • Metabolic stress → NLRP3 → insulin resistance

  • Atherosclerosis

    • Cholesterol crystals → NLRP3 → plaque inflammation

• LOSS-OF-FUNCTION (Inflammasome doesn't work):

  • Increased susceptibility to infections

  • Can't mount proper inflammatory response

  • NOD-2 Mutations (Different but related):

    • Crohn's Disease (IBD)

    • Can't properly sense bacteria in gut

    • Blau syndrome

    • Granulomatous inflammation

Memory Tricks

• "NLRP3 needs 2 hits" - Two signals required

• "Crystals = Crisis" - Crystal deposition activates inflammasome (e.g., gout, atherosclerosis)

• "K+ OUT = Danger OUT" - Potassium efflux is a danger signal

3. NEUTROPHIL EXTRAVASATION (LEUKOCYTE RECRUITMENT)

The Big Picture

• When there's infection or injury in tissue, neutrophils need to leave the bloodstream and get to the site.

• This process is called extravasation or diapedesis, a multi-step process.

THE FIVE STEPS - In Detail

STEP 1: CAPTURE & ROLLING

• What Happens:

  • Neutrophils in blood are flowing rapidly and need to slow down to detect the problem.

• Molecules Involved:

  Endothelial Molecule	Neutrophil Molecule	Interaction Type
  P-selectin	PSGL-1 (P-selectin glycoprotein ligand-1)	Weak binding
  E-selectin	Sialyl-Lewis X (carbohydrate)	Weak binding
  GlyCAM-1, CD34 (on HEV in lymph nodes)	L-selectin	Weak binding

• The Mechanism:

  • Selectins are like "sticky fingers" that grab neutrophils as they pass by.

  • The bond is WEAK - it breaks easily, allowing a ROLLING motion along the vessel wall.

  • Rolling slows down the neutrophil from approximately 1000 μm/sec to 50 μm/sec.

• What Triggers Selectin Expression?

  • Macrophages in tissue detect bacteria and release IL-1 and TNF-α, which activate endothelial cells.

  • Endothelial cells express P-selectin (from storage) rapidly and E-selectin (newly made) slowly.

STEP 2: ACTIVATION

• What Happens:

  • While rolling, the neutrophil encounters chemotactic signals that instruct it to STOP HERE because this is the infection site.

• Chemotactic Signals (Chemoattractants):

  • IL-8 (also called CXCL8) - MOST IMPORTANT

  • C5a (complement fragment)

  • LTB4 (Leukotriene B4 - from arachidonic acid)

  • fMLP (N-formyl-methionine peptides - bacterial product)

  • Platelet Activating Factor (PAF)

• What These Signals Do:

  • Bind to G-protein coupled receptors on neutrophils, triggering inside-out signaling.

  • Integrins on neutrophils change shape from low-affinity to HIGH-AFFINITY.

  • Neutrophil is now activated and ready to adhere firmly.

STEP 3: ARREST (FIRM ADHESION)

• What Happens:

  • The activated neutrophil now STOPS rolling and sticks tightly to the endothelium.

• Molecules Involved:

  Endothelial Molecule	Neutrophil Molecule	Interaction Type
  ICAM-1 (Immunoglobulin superfamily)	LFA-1 (CD11a/CD18, αLβ2 integrin)	STRONG binding
  ICAM-1	Mac-1 (CD11b/CD18, αMβ2 integrin)	STRONG binding
  VCAM-1 (Immunoglobulin superfamily)	VLA-4 (α4β1 integrin)	STRONG binding

• Key Points:

  • ICAM-1 = Intercellular Adhesion Molecule-1

  • VCAM-1 = Vascular Cell Adhesion Molecule-1

  • LFA-1 = Lymphocyte Function-Associated Antigen 1

  • Mac-1 = Macrophage-1 Antigen (also called CR3, Complement Receptor 3)

  • VLA-4 = Very Late Antigen-4

• The Mechanism:

  • Integrins on the neutrophil are now in the high-affinity state (activated in Step 2).

  • They bind tightly to ICAM-1 and VCAM-1

  • Neutrophil STOPS moving → ARREST

  • Neutrophil flattens out on the endothelium

• What Upregulates ICAM-1 and VCAM-1?

  • TNF-α and IL-1 (from macrophages) activate endothelial cells, causing ICAM-1 and VCAM-1 expression over several hours.

STEP 4: TRANSMIGRATION (DIAPEDESIS)

• What Happens:

  • The neutrophil squeezes between endothelial cells and crosses the vessel wall into tissue.

• Molecule Involved:

  Molecule	Location	Function
  PECAM-1 (CD31)	Junctions between endothelial cells AND on neutrophils	Mediates migration through junctions

• The Mechanism:

  • Neutrophil extends pseudopodia (like little arms) searching for junctions between endothelial cells.

  • PECAM-1 on the neutrophil binds PECAM-1 on endothelial junction (homophilic binding).

  • Neutrophil squeezes through the junction and crosses the basement membrane using enzymes like collagenase.

  • Now the neutrophil is in the tissue!

• Other Molecules Involved:

  • JAM proteins (Junctional Adhesion Molecules)

  • CD99

  • Matrix metalloproteinases (MMPs) - digest basement membrane.

STEP 5: MIGRATION (CHEMOTAXIS)

• What Happens:

  • Once in tissue, neutrophil follows the gradient of chemoattractants to the exact site of infection.

• Chemotactic Signals in Tissue:

  • IL-8 - highest concentration near infection

  • C5a - generated at the site of complement activation

  • LTB4 - from inflammatory cells

  • Bacterial products (fMLP)

• The Mechanism:

  • Neutrophil has receptors for these molecules

  • Moves toward HIGHER concentrations (chemotaxis)

  • Eventually reaches bacteria to initiate PHAGOCYTOSIS!

SUMMARY TABLE - MEMORIZE THIS!

CLINICAL CORRELATIONS

• Leukocyte Adhesion Deficiency (LAD)

  • Defect: CD18 deficiency (β2 integrin subunit)

  • Affected molecules: LFA-1 and Mac-1 do NOT WORK

  • Result:

  • Neutrophils CAN'T arrest on endothelium

  • Clinical features include recurrent bacterial infections (skin, mucosal), delayed umbilical cord separation (classic sign), and high WBC count (leukocytosis) - neutrophils stuck in blood, cannot reach tissues.

  • Infections WITHOUT pus (neutrophils can't reach the sites).

• Selectin Deficiency (Rare)

  • SLeX (Sialyl Lewis X) deficiency, leading to inability of neutrophils to roll → inability to reach tissues.

  • Presenting symptoms are similar to LAD.

Memory Tricks

• "SELECTINS = Slow and Sloppy" - represent weak binding for rolling.

• "INTEGRINS = Iron Grip" - represent strong binding for arrest.

• "PECAM = Pass Through" - signifies transmigration process.

• Order: Rolling → Activation → Arrest → Transmigration → Migration

  • Think: "RAAT-M" or "Really Active Athletes Train More"

4. PHAGOCYTE FUNCTIONS (NEUTROPHILS vs MACROPHAGES)

Overview - The Professional Phagocytes

• Both neutrophils and macrophages are professional phagocytes; their main job is to eat and destroy pathogens.

NEUTROPHILS vs MACROPHAGES - Detailed Comparison

Detailed Neutrophil Features

• Granule Contents:

  • Azurophilic (Primary) Granules:

  • Myeloperoxidase (MPO) - produces HOCl (bleach!)

  • Defensins - perforate bacterial membranes

  • Lysozyme - decomposes bacterial cell walls

  • Elastase, cathepsin G - proteases

• Neutrophil Extracellular Traps (NETs):

  • When a neutrophil is dying, it can expel its DNA, forming a NET (like a spider web).

  • Traps bacteria so they cannot spread and kills them even after neutrophil death.

  • Clinical relevance: NETs can contribute to thrombosis and autoimmune diseases.

Detailed Macrophage Features

• Tissue Names for Macrophages (same cell, different locations):

  Location	Name
  Liver	Kupffer cells
  Lung	Alveolar macrophages
  Brain	Microglia
  Bone	Osteoclasts
  Kidney	Mesangial cells
  Skin	Langerhans cells (type of dendritic cell)
  Connective tissue	Histiocytes
  Peritoneum	Peritoneal macrophages

• Macrophage Activation States:

  • M1 (Classically Activated) - "Killer mode"

  • Triggered by: IFN-γ, LPS, TNF

  • Produces:

    • IL-12, TNF, NO (nitric oxide), ROS

  • Function: Kill intracellular pathogens, tumor cells

  • Memory Trick: "M1 = Mean"

  • M2 (Alternatively Activated) - "Healer mode"

  • Triggered by: IL-4, IL-13, IL-10

  • Produces:

    • TGF-β, IL-10, arginase

  • Function: Tissue repair, wound healing, parasite control

  • Memory Trick: "M2 = Mender"

PHAGOCYTOSIS - The Process (Both Cell Types)

STEP 1: RECOGNITION

• How do phagocytes find bacteria?

  • Pattern Recognition Receptors (PRRs):

  • TLRs: recognize PAMPs (e.g., LPS, flagellin)

  • Mannose receptor - recognizes mannose on bacterial surfaces

  • Scavenger receptors - bind modified lipids, dead cells

  • Complement receptors (CR1, CR3) - recognize C3b, iC3b

• What are Opsonins?

  • An "Opsonin" = "to prepare food for eating" (Greek origin)

  • Molecules that TAG bacteria to increase their appeal to phagocytes

  • C3b and IgG are the main opsonins (similar to putting ketchup on bacteria to make them more appetizing).

STEP 2: LIGAND BINDING & RECEPTOR CLUSTERING

• What Happens:

  • Phagocyte receptors bind to bacteria (or opsonins on bacteria).

  • Multiple receptors cluster together around the bacterium, sending strong signals into the cell.

• Mechanisms:

  • Zipper mechanism (FcγR): receptors zip up around the antibody-coated particle.

  • Trigger mechanism (complement receptors): one receptor triggers uptake.

STEP 3: ACTIVATION & SIGNALING

• Intracellular Signaling Cascades:

  • Receptor clustering activates tyrosine kinases.

  • PI3K (phosphoinositide 3-kinase) becomes activated, producing PIP3 at the membrane and recruiting cytoskeletal proteins.

  • Changes in the Cell:

  • Actin polymerization starts.

  • Membrane begins to extend around bacteria.

  • Energy (ATP) consumption occurs.

STEP 4: ENGULFMENT

• What Happens:

  • Membrane extends around the bacterium like arms forming pseudopodia (false feet).

  • Pseudopodia fuse together on the opposite side.

  • The bacterium is now trapped inside a membrane-bound vesicle called a PHAGOSOME ("eating body").

  • The phagosome still DOES NOT kill the bacterium yet.

STEP 5: PHAGOSOME MATURATION

• The phagosome undergoes changes:

  • Early phagosome (pH ~6.0-6.5)

  • Recruits Rab5 (small GTPase)

  • Intermediate phagosome

  • Rab5 → Rab7 switch; pH drops to ~5.5-6.0

  • Late phagosome

  • Acidic (pH ~5.0-5.5)

  • Acquires lysosomal markers

STEP 6: PHAGOSOME-LYSOSOME FUSION

• What Happens:

  • Lysosomes (organelles enriched with digestive enzymes) fuse with the phagosome, creating a PHAGOLYSOSOME.

  • The bacterium is now in serious trouble!

• What's in Lysosomes?

  • Acid hydrolases: work effectively at low pH.

  • Proteases: break down proteins.

  • Lipases: break down lipids.

  • Nucleases: break down DNA/RNA.

  • Lysozyme: breaks down the bacterial cell wall.

STEP 7: MICROBIAL KILLING

• Two main mechanisms:

  • A. OXYGEN-DEPENDENT KILLING (Respiratory Burst)

  • Key Enzyme: NADPH Oxidase

  • Multi-component enzyme complex activated during phagosome formation.

  • Located in phagolysosome membrane.

  • The Reaction:

    • NADPH oxidase transfers electrons from NADPH to O2, producing Superoxide anion (O2−).

    • Superoxide dismutase converts O2− → H2O2 (hydrogen peroxide).

    • Myeloperoxidase (MPO, in neutrophils) converts H2O2 → HOCl (hypochlorous acid = BLEACH).

    • Also produces hydroxyl radicals (•OH).

    • Result: These Reactive Oxygen Species (ROS) are TOXIC.

    • They damage bacterial DNA, proteins, and lipids, effectively killing most bacteria.

  • In Macrophages:

  • Produce Nitric Oxide (NO) via iNOS (inducible NO synthase).

    • NO + O2− → Peroxynitrite (ONOO−) - extremely toxic; kills intracellular bacteria (such as TB).

  • B. OXYGEN-INDEPENDENT KILLING

  • Even in the absence of oxygen, phagocytes can kill:

    • Mechanisms include:

    • Low pH (~4.5): many bacteria can't survive.

    • Lysozyme: cleaves peptidoglycan (bacterial cell wall).

    • Lactoferrin: chelates iron (bacteria require iron).

    • Defensins: small peptides that perforate bacterial membranes.

    • Proteases (e.g., cathepsins, elastase): digest bacterial proteins.

    • Phospholipase A2: destroys bacterial membranes.

STEP 8: BACTERIAL DESTRUCTION & DEGRADATION

• What Happens:

  • Bacterial components are broken down into small molecules - detailed mechanisms to be added here.