Lecture 122,123: Innate Immunity I and II

Innate IR

1st line:

• physical, chemical, biological
• skin, ear wax, tears, mucus

2nd line:

• inflammation, fever, complement, interferon, phagocytosis, NK cells

1st line physical barriers

Skin
Mucous membranes (trap organisms)
Respiratory track
Urinary Track (voiding)

Chemical Barriers

pH (skin= 5.5, stomach = 1-3, vagina = 4.5)
Microcidal molecules:

• lysozyme, defensins, RNAse, DNase, Salt

Inflammation

• response to tissue damage
• inflammatory mediators (histamine, C5a, TNFalpha)
• Coordinated by macrophages

Cardinal signs of inflammation

Heat, pain, redness, swelling and loss of function

What changes happen in inflammation?

• increased blood supply to area
• increase in capillary permeability
• increase in leukocyte chemotaxis

Acute vs chronic inflammation and order of cells activated?

fast clearance of Ag - acute

persistent Ag - chronic

Neutrophils - macrophages (resident: always there) - T helper cells - cytotoxic - B cells

Cytokines role in inflammation

Cytokines: TNF alpha, IL1, IFN gamma

• activate NF-kB (master switch of immune system)

TNF alpha:

• prothrombotic
• leukocyte adhesion and migration
• macrophage activation
• hematopoiesis and lymphocyte development

4 major plasma enzyme systems in Inflammation

1. Clotting system
2. Fibrinolytic system (plasmin)
3. Kinin system
4. Complement system

Plasmin vs Kinin system

Plasmin system (tissue repair and remodeling)

• dissolve fibrin
• activate MMP (remodeling of collagen)
• promote angiogenesis

Kinin system (bradykinin)

• venular dilation due to release of NO
• increased vascular permeability
• smooth muscle contraction

Mediators of Inflammation

Mast cells, basophils, and platelets release histamine and serotonin and pain

• IgE sensitizes mast cells and basophils
• C3a and 5a - trigger mast cell degranulation
• Leukotrienes and prostaglandins

PRR (pattern recognition receptor)

recognize PAMPS(pathogen associated molecular patterns: LPs, PG)

• coded by genome: identical in all individuals

recognize:

• secreted molecules (CRP, MBL)
• receptors present on cell surface and on endocytic vesicles (TLR)
• intra-cytoplasmic recognition molecules (NOD like receptors)

Action of PRRs

• trigger inflammation
• immobilize infectious microbe
• promote ingestion by phagocytes
• activate host cell secretion of antimicrobial substances

CRP

C reactive protein

• acute phase protein produced by hepatocytes in response to IL6
• member of pentraxin family of proteins
• opsonizes microbes (pneumococci)
• activates classical complement pathway
• binds to necrotic/apoptotic cells

TLRs:

found in most tissues

• activate phagocytes - APC
• activate NF-kB
• TLR3 deficiency susceptibility to HSV
• TLR 7 higher viral load and disease progression in HIV

TLR types

TLR1: gram - bacteria + mycobacteria
TLR 2: gram +, mycobacteria, fungi
TLR 3: dsDNA virus
TLR4: LPS gram -
TLR5: bacteria flagellin
TLR 6: mycobacteria
TLR7,8: ssRNa virus
TLR9: unmethylated CpG DNA: bacteria

Fever:

exogenous (LPS) or endogenous (IL1,6): pyrogens

Benefits:

• Inhibits multiplication of temperature- sensitive microorganisms
• impedes the nutrition of bacteria by reducing the availability of iron
• increases metabolism & stimulates immune reactions & naturally protective physiological processes

Interferon

Type 1: IFN alpha (Dendritic) and IFNB (fibroblasts)

• raise antimicrobial defenses in infected and non infected cells

Type 2: IFN gamma (adaptive IR):

• regulatory molecules, induce MHC

• class switching of abs

• induce chemokines on epithelial cells

MOA of interferons

RNA dependent protein kinase - blocks mRNA synthesis and activates apoptosis

Complement

= inactive, ancient

Activated:

• Classical (adaptive IR): latest pathway (Ag-Ab response)

• Alternative (innate IR): always active at low levels

• Mannan-binding lectin (MBL): innate IR (binds to mannose)

• all merge at C3a+b - C5-9: MAC complex

Functions of complement

• opsonization (C3b-4b): destroys capsulated bacteria

• lysis of bacteria and enveloped viruses (MAC)

• inflammation and chemotaxis (C3a+5a)

• priming of B cells: dev of Ab response

• C5b-9: Clearance of immune complexes and apoptotic cells

Complement C3b

C depleted and deficient mice have reduced Ab responses

• C opsonized particles are more active in triggering Ab production

Triggers of C3b:

• B-cell maturation & migration to lymphoid organs
• B-cell activation by binding to opsonized Ag
• DC select for correct B-cell

Classical vs Lectin vs Alternative pathway

Classical:

• Ab (first IGM then IgG) bound to antigen and requires Ca

Lectin: microbial carbs

• mannose and NAG

Alternative:

• constant state of low level activation
• requires Mg and factor B
• activated by surface molecules (LPS)

MAC function

embedded in membrane - makes holes - ions leak - lysis

Effect of complement deficiencies

susceptibility to encapsulated bacterial infections

Classical (C1, C2, C4): lupus like + bacterial (autoimmune)
MAC (C5-9) deficiency: Neisseria + PNH
Alternative: recurrent bacterial infections

Complement protection systems

• prevent consumption of components
• protect host from damage

Proteins in complement protection system

C4b: plasma
Factor H: plasma
CD55: DAF (decay accelerating)
CD46: MCP (membrane cofactor)
CD35: complement receptor

CD59:

• inhibit binding to C9
• broadly expressed
• Deficiency: PNH: paroxysmal nocturnal hemoglobinuria: urine black

NO MAC complex formed

Phagocytosis chemotaxis

tether - trigger - adhesion

• Once phagocyte binds to endothelial lining - leave to infection site - roll over and squeeze between squamous to get in
  • attracted to site of inflammation
• chemokines - adhesion to selectins in venules (epithelial cells)
  Integrins bind to CAMs (ICAM, VCAM) - sends to specific locations
  Both ligands and receptors vary resulting in selection of leukocytes entering tissues

Chemokines:

receptor/ligand vary in selection of leukocytes entering tissues

Defect:

• LAD syndrome (leukocyte adhesion deficiency): B integrins

Therapeutic:

• Abs against VLA4 (ligand for VCAM1)
• MS patients: limit migration of T cells into CNS: decrease attack of myelin

What receptors do HIV bind to to enter cell?

CCR5 + CXCR - chemokine receptors

Process of phagocytosis:

Attachment: TLR, CR,Fc
Recognition (don't eat me signal: CD47)
Engulfment (phagosome)
fusion of phagosome with lysosome
Digestion
Expulsion/Ag presentation

What receptors recognize intracellular pathogens?

Cytosolic receptors part of PRR

• NLR (NOD like receptors)

recognize Pg

• RLH - recognize dsDNA (retinoic acid gene I like helicase)

NLR form inflammasome which triggers inflammatory cell death (pyroptosis) of infected cells

NOD polymorphisms (increased risk)

atopic eczema
asthma
increased IgE
increased risk of Crohn's disease
increase in bacterial infections (reduced innate recognition → impaired immunity)

Types of phagocytes

PMNs: polymorphonuclear cells

• neutrophils (recruited by macrophages)
• eosinophils: pink, granules
• one nucleus with multiple shapes

MN:

• mononuclear cells
• monocytes
• Dendritic cells
• osteoclasts

Macrophage populations
bone marrow
liver
spleen
lymph nodes
lung
skin
bone

bone marrow: stromal
liver: Kuppfer
spleen: red pulp macrophages
lymph nodes: subscapular macrophages
lung: alveolar macrophages
skin: langerhan cells
bone: osteoclasts

Differences between neutrophils and Macrophages

Neutrophils:

• short lived
• acute response
• fast clearance
• stores wide range of polypeptides in intracellular granules
• produces oxygen radicals

Macrophages:

• long lived
• chronic inflammation
• less cidal
• recruit neutrophils
• requires activation by PRRs
• higher degree of specificity

Phagocyte cytokines

secrete IL1: pyrogen
TNF alpha: inflammation
IL8 and IL12: attract neutrophils and NK cells

How do pathogens evade phagocytosis?

capsule

• inhibit phagolysosome fusion (M tuberculosis)
• escape phagosome (L monocytogenes)

stimulate autophagy in macrophages (M tuberculosis, salmonella)

Macrophage types

Th2: IL4/IL13 and IL10 wound healing
T reg: IL10, TGFbeta: anti-inflammatory (steroids)
TH1: IFN gamma: TNF alpha, ILB: proinflammatory cell mediated immunity

NK cells

killer activation and killer inhibition receptors

KAR recognizes stress associated molecules (MICA, MICB)

NK cells use killer inhibition receptors (KIR) to assess MHC I

• not enough: kills target host cells

Viruses and malignant events depress MHC I

Chediak Higashi syndrome

defect in phagosome lysosome fusion

recurrent bacterial infex

Chronic granulomatous disease

defect in microbicidal enzymes (O2 radicals)

Recurrent fungal/bacterial infections

NADPH oxidase deficiency → impaired oxidative burst

Phagocyte disorders in Innate IR

• IFN gamma receptor deficiency: fail to activate macrophages → disseminated mycobacterial infections

• Leukocyte adhesion deficiency (LAD -1/2): Defective β2 integrins; neutrophils cannot migrate → life-threatening bacterial infections.

Complement Disorders

• Defect in alternative pathway and MBL lead to increase in susceptibility to infections

• defect in classical pathway not associated with increase in susceptibility to infections

• Deficiency in C1,2,4: inefficient clearance of immune complexes (increase in type III hypersensitivity)

• deficiency in C regulation (C1 esterase inhibitor: hereditary angioedema)

Which antibody is the most efficient activator of the classical pathway of complement?

A. IgM

B. IgG

C. IgA

D. IgE

E. IgD

A

Antibody	Ability to Activate Classical Complement	Why
IgM	✅ Most efficient	Pentameric structure → binds multiple C1q molecules easily upon antigen binding
IgG	✅ Efficient (especially IgG1, IgG3)	Monomeric → needs clustering; less efficient than IgM
IgA	❌ Does not activate classical pathway	Involved in mucosal immunity; does not bind complement well
IgE	❌ No complement activation	Functions in allergic responses; binds mast cells, not complement
IgD	❌ No known role in complement activation	Primarily a B-cell receptor

"GM makes Classic music"

• (Ig)G and M = Only IgG and IgM activate the Classical pathway

• But M is More powerful due to its pentameric structure

USMLE Mnemonic for TLRs

"TLR3 = TRIPLE-stranded (dsRNA) → Viruses"
"TLR4 = LPS = Gram-negative"
"TLR9 = 9 is upside-down 'P' for CpG DNA"

M1 macrophage phenotype fx

Inflammation (kill)

M2 macrophage phenotype fx

Interferon gamma (repair)

First Line

Prevent pathogen entry

Second Line

Eliminate pathogens that breach first line

Chemical Barrier - Lysozyme

Enzyme that breaks down bacterial peptidoglycan (cell wall)

Chemical Barrier - Defensins

Pore-forming antimicrobial peptides that disrupt microbial membranes

Chemical Barrier - Cathelicidins

Antimicrobial peptides that disrupt bacterial membranes

Chemical Barrier - RNases/DNases

Enzymes that degrade microbial RNA and DNA

KAR (Killer Activation Receptor) fx

Activates NK cell to kill

KIR (Killer Inhibitory Receptor) fx

Inhibits killing if MHC I is present

KAR (Killer Activation Receptor) recognizes

Stress molecules like MICA, MICB

KIR (Killer Inhibitory Receptor) recognizes

MHC class I molecules on healthy host cells

Healthy Cell: Normal MHC I levels bind KIR →

NK cell inhibited → No killing

Infected/Malignant Cell: Low/absent MHC I →

"no KIR signal → KAR engages stress ligands → NK cell kills the target; This is especially important because viruses and tumors often downregulate MHC I to evade CD8⁺ T cells — but this makes them vulnerable to NK cells."

MICA/MICB is recognized by

"KAR to signal cell stress (infection, transformation)"

NLR detects

NOD-like receptors

RLH detects

Retinoic acid-inducible gene I-like helicases

NLR pathogen

Peptidoglycan (PG) fragments - bacteria

RLH pathogen

Double-stranded RNA (dsRNA) - viruses

NLRs can assemble into a cytosolic complex called the inflammasome, which

"activates caspase-1, which:

- Converts pro-IL-1β to active IL-1β

- Triggers pyroptosis (inflammatory programmed cell death) to eliminate infected cells"

Mnemonic for Phagocytosis Steps:

"ARR-FEED" → Attach, Recognize, Receptor binding, Form phagosome, Engulf, Expel, (optional) Display antigen

Classical Pathway - Complement Activation

Ab (IgG/IgM) binding antigen (requires Ca²⁺)

Alternative Pathway - Complement Activation

Direct activation by pathogen surfaces (e.g., LPS) (requires Mg²⁺)

Lectin Pathway - Complement Activation

Mannose or NAG on microbes

CD59 inhibits binding of

C9

Blocking CD47 promotes anti-tumor immunity by

allowing phagocytes to engulf cancer cells

CD47 Fx

"Inhibits phagocytosis of self

Prevents clearance of healthy or early senescent cells

Expressed on many tumor cells"

Classical Pathway

Activated later; needs prior adaptive immune response

Lectin Pathway

Part of early innate response; pattern recognition-based

Alternative Pathway

First activated; always "on standby" for rapid response

C1, C2, C4 deficiencies →

impaired clearance → type III hypersensitivity