T2: Innate Immunity

3 main components of innate immunity

1. barriers - physical & chemical

2. phagocytic & NK cell activities

3. molecules - complement system

1) barriers

1. physical barrier - what is it & found where

epithelial lining found in skin, GI tract, respiratory tract

barrier function of epidermis is mediated by what cells

corneocytes in stranum corneum (outermost layer of epidermis - skin surface layer)

integrity of physical barrier depends on what

integrity = how intact & functional

junction adhesion molecules & tight junction proteins

1) barriers

2. chemical barriers

   → 3 main components

1. biomolecules

2. skin pH

3. microbiome

2 types of biomolecules that act as chemical barriers are

1. antimicrobial peptides

2. biomolecules of skin

characteristic of antimicrobial peptides

amphipathic - hv 2 regions: hydrophillic & hydrophobic

function of antimicrobial peptides (2)

antimicrobial activity

cell activation in response to inflammation - influences host immune cells

antimicrobial peptides produced by

1. keratinocyte (mainly skin cells)/epithelial cells

2. skin-infiltrating leukocytes (eg. macrophages, PMNs (polymorphonuclear leukocytes))

AMPs produced by keratinocytes/epithelial cells

human beta defensins (HBD)

cathelicidin LL-37

mechanisms of AMPs

membrane-active AMPs

intracellular active AMPs

explain mech of membrane-active AMPs

1. positively charged region of AMP binds to negative charge microbial cell membrane

2. hydrophobic regions of AMP insert into lipid bilayer

3. membrane disruption → creates pores

4. increases leakage of cell contents

5. microbial death

explain mech of intracellular-active AMPs

AMPs enter bacteria by either direct penetration or endocytosis (thru vesicles) → interacts w/ target inside cells interfering with vital microbial processes eg. DNA replication, RNA transcription → growth inhibition/death of microbe

1) barrier

2. chemical barriers

2) biomolecules of skin

• main component & explain function

• main component = lipids

• converted into sphingosine & dihydrosphingosine (active form of lipids) → direct killing of pathogens & interference w/ microbial adherence & invasion

• antimicrobial activity against certain bacterial strains

2. chemical barriers

2) skin pH

function

• inhospitable environment for potential pathogens eg. staph aureus

• hospitable environment for commensal bacteria - normal non-harmful micro organisms that normally live inside body

2. chemical barriers

3) microbiome - what is it & function

• community of microorganisms on skin

• function:

  • prevents colonization of skin by pathogenic microbes

  • secretes their own antimicrobial agents which inhibit growth of pathogenic bacterial strains

antimicrobial agent that microbiome secretes

bacteriocins

major commensal microbes inhabitating skin

staphylococcus epidermis

propionibacterium acnes

specificity:

innate immunity recognises

common structures shared by classes of microbes or PAMPs (pathogen-associated molecular patterns)

specificity:

adaptive immunity recognises

unique structures on microbes - structural detail of microbial molecules or ANTIGENS

receptors in innate immunity (2)

• encoded by …

• diversity =

encoded in germline - fixed, inherited, same in everyone

limited diversity, are pattern recognition receptors

receptors in adaptive immunity (2)

• encoded by …

• diversity =

encoded by genes produced by somatic recombination of gene segments → created individually in each lymphocyte by gene rearrangement

greater diversity

is there discrimination of self & non-self in innate & adaptive immunity

yes, both types do not recognize healthy host cells

pattern recognition receptors - which type of immunity

innate

PRRs are expressed where (2)

surface of phagocytic vesicles & in cytosol of various cell types

toll-like receptors - located where

plasma membrance & endosomal membranes of dendritic cells, phagocytes, B cells, endothelial cells etc.

examples of TLR

TLRs 1-9

NOD-like receptors (NLRs) - located where

cytosol of phagocytes, epithelial cells etc.

which PRR does extracellular pathogen bind to when first entering host cell

TLR

which PRRs does intracellular pathogens bind to

NOD-like receptors (NLR), RIG-like receptors (RLRs), Cytosolic DNA sensors (CDS)

NLR recognize what

bacterial peptidoglycan

RLR recognize what

viral RNA

CDS recognize what

microbial DNA

TLR recognize what

extracellular pathogens & intracellular pathogens that wants to enter endosome

name 2 soluble pattern recognition molecules found in innate immune system

pentraxins, complement proteins

pentraxins are

C-reactive protein

pentraxin function

binds to phospholipids on microbes

which TLRs recognizes gram-positive bacteria (3)

TLR1:TLR2 (dimeric form, yuu gun pen 2 tua)

TLR2

TLR2:TLR6

which TLR recognizes bacterial peptidoglycan in gram-positive bacteria

TLR2

which TLR recognizes gram-negative bacteria

TLR4

which TLRs recognize nucleic acid on microbe

TLR3,7,8,9

Main Overview Mechanism of Innate Immunity - 2nd line of defence - phagocytic cells

PRRs + PAMPs → innate immune responses → 1) phagocytosis 2) inflammation 3) antiviral state

PRRs are expressed at surface of what cells - specify

innate immune cells - macrophage, dendritic cells, neutrophils

explain mech of phagocytosis

phagocytes (neutrophil & macrophage): PRRs on phagocytes bind w/ PAMP on microbe → phagocytes engulf & ingest microbes → microbes killed by action in phagolysosomes w/ microbicidal molecules

PRRs associated w/ phagocytosis (3)

C-type lectin receptors, scavenger receptors, opsonin receptors

microbicidal molecules than kill microbes in phagolysosomes (3)

ROS, nitric oxide (NO), proteolytic enzymes

2 examples of ROS

hydrogen peroxide & superoxide

proteolytic enzyme example

elastase

how does neutrophils kill microbes

1. ingestion & killing of microbes by activated phagocytes - engulfs microbe into phagosome → phagosome fuses with lysosome to form phagolysosome → killing occurs

2. extrudes their DNA & granules contents forming NETs

composition of NETs (neutrophil extracellular traps) (4)

DNA, histones, neutrophil granule proteins: elastase, MPO, cathepsin G

2) inflammatory response

give full inflammation mechanism

(starting from when microbe enters host)

1. injury/barrier break → microbe entry

2. microbes/injury activates sentinel cells (macrophages, dendritic cells, mast cells)

3. sentinel cells secrete inflammatory mediators (eg. cytokines)

4. vasodilation & increased vascular permeability → fluid & proteins (fibrinogen, complement proteins, antibodies) enter tissues/ site of infection (→ swelling)

5. complement, antibodies & antimicrobial proteins kill microbes

6. adhesion molecules & chemokines cause leukocyte migration into tissue

7. phagocytosis & killing of microbes

the main cells that produce cytokines

dendritic cells, macrophages

major pro-inflam cytokines & function

TNF-alpha, IL-1, IL-6

→ recruits neutrophils to kill microbes at infection site

which PRR leads to cascade of events that causes inflammation

TLR

explain mech of how cytosolic PRRs promote inflammation

nod-like receptor + PAMPs/DAMPs → forms inflammasome -> activates caspase-1 + produces inflam cytokines IL-1 & IL-18

possible consequence of inflammasome activation of capase-1

pyroptosis (pro-inflammatory programmed cell death)

what are potent inflammasome activators

crytalline substances

what disease does crystalline substances cause

monosodium urate crystal deposition → gout

explain mechanism of leukocyte recruitment to site of infection (5)

1. TLRs bind to microbe → release of pro-inflam cytokine: IL-1, IL-6, TNF-alpha by macrophage

2. endothelial cell expresses more ligand for neutorphil attachment → neutrophil attaches to endothelial cell via selectin ligand (low-affinity binding) → neutrophil rolling

3. other ligands w/ higher affinity (eg. integrin) also used → neutrophil attaches even more tightly to endothelial cell

4. neutrophil moves thru gaps in endothelial cell to site of infection

5. neutrophil removes microbes thru phagocytosis & NETs

local inflammation

• explain how endothelial cells are activated

1. endothelial cells are stimulated by TNF & IL-1 → express adhesion molecules → helps leukocytes stick to vessel walls

local inflammation

• explain how leukocytes are activated

• activated by TNF & IL-1 → causes leukocytes to release IL-1, IL-6, chemokines → attracts more immune cells

• amplifies & sustains inflammation

systemic consequences of inflammation

1. brain

• what cytokines

• what effect

TNF, IL-1, IL-6 → cause hypothalamus to produce prostaglandin → fever

systemic consequences of inflammation

2. liver

• what cytokines

• what effect

• TNF, IL-1, IL-6

• induces liver to produce acute-phase proteins - biomarkers of inflammation

systemic consequences of inflammation

3. bone marrow

• what cytokines

• what effect

• TNF, IL-1

• stimulates bone marrow to produce more leukocytes

systemic pathologic effects of inflammation

1. heart

   • what cytokine

   • what effect does too much of this cytokine cause

• TNF

• too much TNF → inhibits myocardial contraction → low CO → hypotension

systemic pathologic effects of inflammation

1. endothelial cells/blood vessel

   • what cytokine

   • what effect does too much of this cytokine cause

1. TNF

2. fluid leaks out of blood circulation → pleural effusion, dengue infection

systemic pathologic effects of inflammation

1. multiple tissues - eg. skeletal muscle

   • what cytokine

   • what effect does too much of this cytokine cause

• TNF

• insulin resistance - advantage of pro-inflam cytokine

3) antiviral state

• explain mechanism of induction of type 1 interferons by viruses

• hint: 2 pathways

1. virus enters host cell & contained in endosome

pathway 1:

2. PRRs: TLR 3,7,8,9 recognizes genetic material of virus (that is within endosome)

pathway 2:

2. virus exits endosome & enters cytoplasm

3. PRRs: MDA-5, RIG-1, DAI recognize virus in cytoplasm

4. virus binds to the specific PRRs in cytoplasm/endosome → produces type 1 interferons: IFN-alpha & IFN-beta

explain mechanism of what happens after virus-infected cell secretes type 1 IFN - how does type 1 IFN cause antiviral state in neighbouring cells

1. type 1 IFN secreted by virus-infected cell binds to IFN receptor on uninfected cell

2. IFNs induce expression of enzymes that block viral replication

3. leads to:

   • inhibition of viral protein synthesis

   • degradation of viral RNA

   • inhibition of viral gene expression & virion assembly (new virus particles being put tgt)

4. prevents viral infection in uninfected cell → neighbouring cell in antiviral state

what are NK cells

subpopulation of lymphocytes (10-15%in total blood)

functions of NK cells (2)

1. cytokine secretion

2. cytotoxicity - directly attacks target cell

CD56 bright → marker for which type of NK cell & how many percent

NK cells resp. for cytokine production, 10%

(these NK cells hv high expression of CD56 marker)

CD-56 dim → marker for which type of NK cell & how many percent

cytotoxic NK cells, 90%

(these NK cells hv low expression of CD56 marker)

2 types of NK cell receptors & function

activating - activates NK cell function

inhibiting - inhibits NK cell function

explain mechanism of cytokine secretion in NK cells

(hint: 2 cytokines secreted, cells: NK & macrophage)

macrophage releases IL-12 to tell NK cell that its unable to kill phagocytosed microbe → NK cell secretes IFN-gamma → helps macrophage produce toxic agent (eg. NO, ROS) → killing of phagocytosed microbe

explain mechanism of antibody-dependent cellular cytotoxicity (ADCC)

IgG antibody binds to surface antigen - antibody-coated cell → NK cell binds to antibody-coated cell → secrete perforin & granzyme → induce microbe apoptosis - killing of antibody-coated cell

apart from NK cell, which other cells have ADCC

none

NK cell receptor binds to what molecule on cells

MHC class 1

explain how NK cell function is inhibited in normal cells

MHC class 1 binds to inhibitory receptor on NK cell → inhibits NK cell function → normal cell not killed

explain how NK cells kill virus-infected cell

virus downregulates MHC class 1 on cell → nothing binds to inhibitory receptor on NK cell → only activating receptor is bound to ligand on infected cell → NK cell activated & kills infected cell

3) Molecules

Complement System

• what is it & produced where

a protein family produced by liver

state the 3 main pathways to activate complement system

1. classical pathway

2. alternative pathway

3. lectin pathway

complement system

• explain full mechanism of classical pathway of complement system + state the trigger is

(up until formation of C5 convertase)

trigger = antibody-antigen complex (IgM or IgG bound to microbe)

1. C1 complex binds to Fc region of antibody on microbe

2. this activates cleavage of C4 → C4b & C2 → C2a ——> forms C4b2a

3. C4b2a is the C3 convertase

4. C3 convertase cleaves C3 → C3a + C3b

5. C3b joins C4b2a → forms C5 convertase

complement system

• explain full mechanism of alternative pathway of complement system + state the trigger is

(up until formation of C5 convertase)

trigger = direct binding of complement protein C3b to microbial surfaces (no antibody required)

1. C3 recognizes lipopolysaccharide on microbe → hydrolyzes to C3b → attaches to microbial cell wall

2. with factor B & factor D → C3 convertase is formed (C3bBb)

3. C3 convertase cleaves C3b molecule → forms C5 convertase (C3bBbC3b)

complement system

• explain full mechanism of lectin pathway of complement system + state the trigger is

  (up until formation of C5 convertase)

trigger = recognition of microbial sugar patterns - mannose by mannose-binding lectin

1. mannose-binding lectin (MBL) binds mannose on microbes

2. activates MASP1 & MASP2 (serine proteases)

3. this cleaves C4 → C4b & C2 → C2a ——> forms C4b2a or C3 convertase

4. C3 convertase cleaves C3 → C3a + C3b

5. C3b added to C4b2a → forms C5 convertase (C4b2a3b)

complement system

• state the final common pathway (after C5 convertase formation - what C5a & C5b does)

1. C5 convertase cleaves C5 → C5a + C5b

2. C5a = strong inflammatory mediator

3. C5b = starts MAC assembly

   → C5b binds C6,C7, C8 & multiple C9 molecules (poly-C9) → forms MAC (membrane-attack complex) → forms pore in plasma membrane → lysis of pathogen

components of MAC

C5b, C6, C7, C8, poly-C9

state the 3 effector functions of complement

1. opsonization & phagocytosis

2. stimulation of inflammatory reactions

3. complement-mediated cytolysis

complement system - effector functions

1. explain the mechanism of opsonization & phagocytosis

C3b binds to microbe (opsonization) → recognition of bound C3b by phagocyte C3b receptor → phagocytosis of microbe

complement system - effector functions

1. explain the mechanism of stimulation of inflammatory mediators

the proteolytic products - C5a, C3a & C4a (but to lesser extent) stimulates leukocyte recruitment & activation → destruction of microbes by leukocytes

complement system - effector functions

1. explain the mechanism of complement-mediated cytolysis

activation of late components of complement (C5b, C6, C7, C8, C9) → formation of MAC → osmotic lysis of bacteria

what is a regulator of complement activation

CD59

CD59 function

blocks C9 binding → prevents formation of MAC

(controls complement activation → mai hai yur gern to prevent inflammation & tissue damage)