Imm 250 lecture 2 modified

Once the barrier is breached how are pathogens detected and dealt with?

Innate immune detection (this lecture)

Cellular and humoral innate factors (next week)

Innate immunity

• Innate denotes a property of some thing or action which is essential and specific to that thing or action, and which is wholly independent of any other object, action or consequence. Also known as “inborn”.

Innate immunity

• Represents THE immune system in most multicellular organisms (from plants, sea urchins, flies, …)

Innate versus adaptive immunity

Innate immunity•Conserved throughout evolution•In all multicellular organism•Organisms possess a set

number of recognition molecules•Cells are immediately active•Has no memory

Adaptive immunity•Evolved 500 million yrs ago?•Unique to vertebrates•Infinite number of recognition molecules (antibodies)•Cells require priming•Provides memory of infection

Innate and adaptive immunity work together in humans

In normal individuals, infections are cleared by the innate and adaptive immune responses.

In the absence of adaptive immunity, infection are first controlled by innate immunity but cannot be cleared.

In the absence of innate immunity, infections cannot be controlled. Need innate immunity to initiate adaptive immunity.

•Metchnikoff first described role of phagocytes in invertebrates (star fish)

The discovery of the innate immune system

How can our cells tell that something is foreign?

• Recognition of exogenous microbial products

• Recognition of endogenous “danger signals”

Dr. Charles Janeway Dr. Polly Matzinger

Turns out they are both right!

• Innate immunity depends upon the recognition of:

• MAMPs (microbial-associated molecular patterns)– “Signatures” of microbial infection - pieces of microbes

• DAMPs (danger-associated molecular patterns)- Danger signals released from dead or dying cells

Overview of innate immunity

MAMP/DAMPMAMP/DAMP  

PRMPRM   (pattern recognition molecule)(pattern recognition molecule)

Destruction of microbesDestruction of microbes

(changes in the cell)(changes in the cell)

Recognition of a:Recognition of a:

By a :By a :

To induce:To induce:

(danger signal)(danger signal)

Signal transductionSignal transduction  

What are MAMPs?

- Also known as PAMPs (pathogen-associated molecular patterns)

- Represent structural components of microorganisms unable to be modified

But first, a little more details about bacteria….

Bacteria can be differentiated into 2 groups based on the “Gram-stain”

Gram-positive Gram-negative

Bacteria can be differentiated into 2 groups based on the “Gram-stain”

Gram-negative(E. coli)

Gram-positive(Staphylococcus)

The basis for this stain is due to the cell walls of these bacteria

Gram-positive cell wall

Staphylococcus

Gram-negative cell wall

MAMPs include these bacterial cell wall products

• Lipotechoic acid (Gram +)

• LPS (Gram -; also called “endotoxin”)

• Peptidoglycan (both; also the target of some antibiotics)

* Lipidated carbohydrates and peptide-carbohydrates

And also “extensions”

• Flagella

* Protein

Flagella

Flagella

- base unit making up flagella is called “flagellin”

MAMPs also include

• Genetic material of bacteria and viruses

– DNA: CpG sequences (cytosine and guanine separated by a phosphate, “p”)

• Quite unique to microbes• These sequences are suppressed and/or modified in

mammalian genetic material

– RNA: double or single stranded• genetic material often associated with viruses

* Nucleic acid

MAMPs associated with fungus

• Zymosan - carbohydrate from the cell wall of yeast

• Beta-glucan - carbohydrate from the cell wall of other fungi

* carbohydrates

What are DAMPs?

• High intracellular levels of reactive oxygen species (ROS) – Produced by cells that are “blocked” in

phagocytic process (and probably destined to die)

• Release of potassium (K+)– Released by cells with damaged

membranes

K+ efflux

• K+ is kept at high concentrations INSIDE the cell

• Leakage means something is wrong - cell is dying

K+K+

K+

K+

K+

K+

K+Danger!

What are ROS?

• Highly toxic oxygen-derived molecules

• Generated by “oxidative burst” - use of oxygen by cells of the innate immune system to form ROS in order to kill microbes

Phagocytosis

ROS

*We will look at this in more detail in next week’s lecture

ROS as a DAMP• “Frustrated” phagocytosis leads to high

amounts of intracellularROS

• Triggers: asbestos (lung disease), uric acid crystals (gout), aluminium (vaccine adjuvant), amyloid (accumulates in Alzheimer’s disease)

MAMPs and DAMPs are the “sensees”, what are the sensors?

• PRM - pattern recognition molecules– Soluble PRMs

• Collectins (eg: surfactant, mannose-binding lectin), Ficolins

– Cell signaling PRMS• Toll-like receptors (TLR), Nod-like receptors

(NLR)

Collectins

• Assemble into multi-meric structures

• Bind microbial carbohydrates

• Link to the complement system for destruction of microbe (see next week’s lecture)

Destruction of microbe through complement pathway

Cell-signaling PRMs

• Membrane-associated

– TLR

• Cytoplasmic

– NLR

Membrane

NLRs

TLRs and NLRs

• History of the discovery of these PRMs

• The MAMPs/DAMPs they recognize

• How they tell the cell that they have recognized a MAMP/DAMP– Signal transduction and activation of gene

expression

Christiane Nuesslein-Volhard: discovery of Drosophila Toll

• Identified a protein she called “Toll” meaning “COOL” in German

• Helps the Drosophila embryo to differentiate its top from its bottom

(She won the Nobel prize for Medicine

for her work on Drosophila development in 1995)

1985 1991 2009?1996 1997 19981988 1989

Nicolas Gay: Toll and inner part of the Human IL-1 receptor is similar

1985 1991 2009?1996 1997 19981988 1989

• Searching for proteins similar to Toll

• Shows cytoplasmic domain of Toll related to that of

hIL-1R (now called a TIR domain for Toll IL-1 Receptor)

Why does a protein involved in human inflammation look like one involved in fly neural

tube development?

“TIR” Domain

TollIL-1R

membrane

cytoplasm

Bruno Lemaitre and Jules Hoffmann: Drosophila use Toll to defend from infection

with fungus

1985 1991 2009?1996 1997 19981988 1989

• Infected Toll-deficient adult flies with Aspergillus fumigatus

• All flies died after 2-3 days

• Flies use Toll to defend from fungi

• Thus, in Drosophila, Toll seems to be involved in embryonic development and adult immunity

Ruslan Medzhitov & Charles Janeway: discovery of human Toll

1985 1991 2009?1996 1997 19981988 1989

• Argued that an ancient immune defence system based on the Toll signalling might exist in humans

• Searched for human proteins that totally resemble Drosophila Toll

• (also report similarity to plant proteins)

hToll

membrane

Fernando Rock & Fernando Bazan: identification of five human “TLRs”

1985 1991 2010?1996 1997 19981988 1989

• Identified 5 human Tolls, which they called Toll like receptors (TLRs)

• TLR4 same as Medzhitov’s human Toll

Bruce Beutler: TLR4 Activated by LPS

1985 1991 2009?1996 1997 19981988 1989

• Normal mice die after being injected with LPS

• A strain of mice, C3H/HeJ, were known since the 1960s to have defective responses to LPS and survive

• Beutler’s group identifies a mutation in the C3H/HeJ mice affecting the cytoplasmic domain of TLR4

• TLR4 is the long-sought after signaling receptor for LPS!

C3H/HeJC57/Bl6

LPS

Publications on TLRs sky-rocket!

0

200

400

600

800

1000

1200

1996 1997 1998 1999 2000 2001 2002 2003

Toll Toll-likeTLR4/LPS

TLR2/lipoproteins

TLR3/dsRNA

Pub

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on

Tol

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TL

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MyD

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TL

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/Fla

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• More than 10 TLRs are identified in mammals• Much work goes into identifying what MAMP is recognized by which TLR

Toll-Like Receptors and their MAMPsToll-Like Receptors and their MAMPs

Receptor MAMP Origin of MAMP

TLR1 Triacyl lipopetides (with TLR2) Bacteria and Mycobacteria

TLR2 Zymosan (with TLR6)

Lipotechoic acid

Lipoprotein/lipopeptides (with TLR1/6)

Fungi

Gram-positive bacteria

Various pathogens

TLR3 Double-stranded RNA Viruses

TLR4 Lipopolysaccharide Gram-negative bacteria

TLR5 Flagellin Bacteria

TLR6 Zymosan (with TLR2)

Diacyl lipopetides (with TLR2)

Fungi

Mycoplasma

TLR7 Single-stranded RNA (ssRNA)

Imidazoquinoline

Viruses

Synthetic compounds

TLR8 Single-stranded RNA (ssRNA)

Imidazoquinoline

Viruses

Synthetic compounds

TLR9 CpG-containing DNA Bacteria, Malaria and Viruses

TLR10 Not determined Not determined

TLR11 (only in mice) Profilin-like molecule Toxoplasma gondii

Toll-Like ReceptorsToll-Like Receptors

CD14

ssRNA (mouse) and

TLR11 (mouse only)

PiliProfilin

TLR8

ssRNA (human)

Lipotechoic acid

Interaction of LPS with TLR4

1. LPS is transferred to LPS binding protein (LBP)

2. LPS is transferred to CD14

3. CD14 “presents” LPS to MD-2 bound to TLR4

4. Conformational change of TLR4 “transduces” the signal to the cell interior

5. Cell initiates defense “program”

Consequences of PRM activation:

How is the defense program initiated?

• Signal transduction culminates in the activation of:– NFB– IRF (interferon regulatory factor)– Inflammasome (we will hear about this one

with the NLRs)

• These are the major regulators of the innate immune response

NFB: Master regulator of innate immune defense

• Transcription factor - a protein that binds to specific sequences of DNA and thereby controls the transfer (or transcription) of genetic information from DNA to RNA and ultimately proteins

• “Turn on” gene expression of the mediators of the defense response (cytokines etc.)

Simplified overview of signal transduction

MAMP interaction with PRM

Conformational change

Adaptor interaction

Transcription factor activation

Gene expression

Signal Transduction to NFB

• How is the interaction between LPS and TLR4 transmitted inside the cell?

LPS

(Adaptor)

(Transcriptionfactor)

Defense response

Signal Transduction to NFB

Pro-inflammatory products

Activation of NFB

1. Held inactive in the cytoplasm and “guarded” by IB

4. NFB moves into the nucleus and binds DNA sequences

3. IB is degraded

2. Signal transduction: IB is phosphorylated - “tagged” for destruction

5. Effectors of the innate immune response are produced

NFB in action

Unstimulated Stimulated (2 cells)

NFB in nucleus

NFB in cytoplasm

TLR-dependent activation of IRF

• IRF is another transcription factor but is specific for genes encoding interferons (we will talk about these next lecture)

• Interferons are cytokines that help our cells defend themselves against infection

Signal Transduction to IRF

IKK (1° Kinase)

Interferon-mediated defense response

(Adaptor)

nucleus

(Transcription factor)

Cytoplasmic PRMs

• Nod-like receptors

• RigI-like receptors

Pathogen recognition:Pathogen recognition:ouside-in versus inside-inouside-in versus inside-in

MAMPMAMP

NLR

Intracellular:

TLRMAMPMAMP

Extracellular:

Cellular response Cellular

response

RLR

Keeping peace with the intestine….

LPSLPS

MacrophagMacrophagee

TLRTLR

(Surveillance cells)

Mucosal epithelial cells are unresponsive to extracellular MAMPs due to lack or low TLR expression.

*Important for tolerance of the epithelium to the normal microbial flora

bacteriabacteria

But epithelial cells play a sentinel role - they can signal that something is wrong when infected with a pathogen

Epithelial cells can detect pathogens despite the lack of TLRs: role of NLRs and RLRs

Commensal microbes

Bring in re-enforcements

Inflammation

PathogenPathogen

Danger

LPS

NLRs in action: invasive bacteria still induce NFB in TLR-negative epithelial cells

Commensal-infected

NFB in cytoplasm

Shigella-infected

NFB in nucleus

Cells have an intracellular mode of microbial recognition as a back-up

IKK

Commensal microbePathogen

IB

NFB in the nucleus

NLR RLR

Nod-like receptors - cytosolic sensors of bacteria and danger

NLRP

NLRs cytoplasmic sensors of MAMPs AND DAMPs

• Nod proteins - MAMPs– Eg: Nod1 and Nod2 detect fragments of

peptidoglycan

• NLRPs - DAMPs– K+ and ROS are triggers

Alerting the cell:Nods sense bacteria products and activate

NFB

Rip2

Pro-inflammatory products

Nod1

IB

NFBnucleus

PG

(adaptor)

(transcription factor)

(sensor)

NLRP3NBD LRR LRR LRRLRR LRR

NLRPs detect danger and form the “inflammasome”

PYRIN

ASC CARDPYRIN

DANGER

LPS

The inflammasome activates Caspase 1 into an enzmatically active protein

NOD PYRIN

NOD PYRIN

NLRP3

Leucine-rich repeats

Pro-Caspase-1

NLRP3 Inflammasome

PYRIN CARD

PYRIN CARD

ASC

CARD p20 p10

CARD p20 p10p20

p10

Caspase-1

CARD p20 p10

CARD p20 p10p20

p10

Active Caspase 1 cleaves pro-IL-1 into an active cytokine

p20

p10

Caspase-1

Pro-IL-1

IL-1 Secreted from the

cell

RLRs - RIG-I-like receptorsRLRs - RIG-I-like receptors

Alerting the cell:RLRs sense nucleic acid (usually VIRAL) and

activate NFB & IFN

Alerting the cell:RLRs sense nucleic acid (usually VIRAL) and

activate NFB & IFN

Helicase CARD CARD

Viral nucleic acid

RIG-IMAVS

NF-B IFN

Lecture summary• Activation of the innate immune response depends on the recognition of

MAMPs and DAMPs• PRMs of the innate immune system detect MAMPs and DAMPs

– Soluble (eg. collectins)– Cell signaling

• Membrane-associated: TLRS• Cytoplasmic: NLRs RLRs

• Signal transduction- conformational change, binding of adaptors

• Activation of regulators– Signal transduction culminates in:

• NFB• IRF• Inflammasomes

• Initiation of the defense response– Genes “turned on” and IL-1 initiate the defense response

Next Lecture

• The consequence of NF-B, IRF and IL-1activation: the mediators of the defense response