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 Adaptive immunity
•Conserved throughout evolution •Evolved 500 million yrs ago?
•In all multicellular organism •Unique to vertebrates
•Organisms possess a set •Infinite number of recognition
number molecules (antibodies)
of recognition molecules •Cells require priming
•Cells are immediately active •Provides memory of infection
•Has no memory
 Innate and adaptive immunity work together in humans
 In the absence of innate
immunity, infections cannot
be controlled. Need innate
immunity to initiate adaptive
immunity.

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

 In normal individuals,
infections are cleared by the
innate and adaptive
immune responses.
The discovery of the innate immune system

•Metchnikoff first described

role of phagocytes in
invertebrates (star fish)
How can our cells tell that something is foreign?

• Recognition of • Recognition of
exogenous microbial endogenous
products “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
Recognition of a:
MAMP/DAMP (danger signal)

By a :
PRM (pattern recognition molecule)

To induce:
Signal transduction
(changes in the cell)

Destruction of microbes
 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-positive Gram-negative
(Staphylococcus) (E. coli)

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
• Flagella

Flagella

- base unit making up flagella is called “flagellin”

* Protein
 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 intracellular
ROS

• 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 • Cytoplasmic

– TLR – 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 1988 1989 1991 1996 1997 1998 2009?

 Nicolas Gay: Toll and inner part of the
Human IL-1 receptor is similar
IL-1R Toll
• Searching for proteins
similar to Toll
• Shows cytoplasmic domain membrane
of Toll related to that of
cytoplasm
hIL-1R (now called a TIR
“TIR”
domain for Toll IL-1 Domain
Receptor)

Why does a protein involved in human

inflammation look like one involved in fly neural
tube development?

1985 1988 1989 1991 1996 1997 1998 2009?

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

1985 1988 1989 1991 1996 1997 1998 2009?

 Ruslan Medzhitov & Charles Janeway:
discovery of human Toll

• Argued that an ancient

immune defence system
based on the Toll signalling
might exist in humans

membrane
• Searched for human proteins
that totally resemble
Drosophila Toll hToll

• (also report similarity to plant

proteins)

1985 1988 1989 1991 1996 1997 1998 2009?

 Fernando Rock & Fernando Bazan:
identification of five human “TLRs”
• Identified 5 human Tolls,
which they called Toll like
receptors (TLRs)

• TLR4 same as Medzhitov’s

human Toll

1985 1988 1989 1991 1996 1997 1998 2010?

 Bruce Beutler: TLR4 Activated by LPS
• C57/Bl6 C3H/HeJ
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 LPS
cytoplasmic domain of TLR4

• TLR4 is the long-sought after signaling

receptor for LPS!

1985 1988 1989 1991 1996 1997 1998 2009?

Publications on TLRs sky-rocket!
Publications on Toll-like Receptors
1200

1000

TLR3/dsRNA
800

TLR5/Flagellin
600
TLR2/

TLR9/CpG DNA
lipoproteins
400
TLR4/LPS
Toll Toll-like
200 MyD88

1996 1997 1998 1999 2000 2001 2002 2003

• 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 MAMPs
Receptor MAMP Origin of MAMP
TLR1 Triacyl lipopetides (with TLR2) Bacteria and Mycobacteria

TLR2 Zymosan (with TLR6) Fungi

Lipotechoic acid Gram-positive bacteria
Lipoprotein/lipopeptides (with TLR1/6) Various pathogens
TLR3 Double-stranded RNA Viruses

TLR4 Lipopolysaccharide Gram-negative bacteria

TLR5 Flagellin Bacteria

TLR6 Zymosan (with TLR2) Fungi

Diacyl lipopetides (with TLR2) Mycoplasma
TLR7 Single-stranded RNA (ssRNA) Viruses
Imidazoquinoline Synthetic compounds
TLR8 Single-stranded RNA (ssRNA) Viruses
Imidazoquinoline 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 Receptors

ssRNA (mouse) and

Lipotechoic acid

Pili
ssRNA Profilin
(human)

TLR11
TLR8 (mouse only)
CD14
 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:
– NFκ B
– IRF (interferon regulatory factor)
– Inflammasome (we will hear about this one
with the NLRs)
• These are the major regulators of the
innate immune response
 NFκ B: 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 NFκ B
• How is the interaction between LPS and
TLR4 transmitted inside the cell?
LPS

(Adaptor)

(Transcriptio
factor)

Defense response
Signal Transduction to NFκ B

Pro-inflammatory products
 Activation of NFκ B
2. Signal transduction: Iκ B is
phosphorylated - “tagged” for
destruction

1. Held inactive in the

cytoplasm and “guarded”
by Iκ B

3. Iκ B is degraded

4. NFκ B moves
into the nucleus
and binds DNA
sequences

5. Effectors of the innate

immune response are produced
 NFκ B in action
NFκ B in nucleus

NFκ B in
cytoplasm

Unstimulated Stimulated (2 cells)

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

(Adaptor)

IKKε (1° Kinase)

(Transcription factor)

nucleus

Interferon-mediated defense response

 Cytoplasmic PRMs
• Nod-like receptors
• RigI-like receptors
 Pathogen recognition:
ouside-in versus inside-in

Extracellular: Intracellular:
MAMP
TLR
MAMP

NLR
RLR

Cellular
response Cellular
response
 Keeping peace with the intestine….
LPS bacteria

Mucosal epithelial cells are unresponsive

to extracellular MAMPs due to lack or low
TLR expression.
TLR
*Important for tolerance of the
epithelium to the normal microbial flora Macrophage
(Surveillance cells)
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

LPS Pathogen

Danger

Inflammation Bring in re-

enforcements
NLRs in action: invasive bacteria still induce
NFκ B in TLR-negative epithelial cells

Commensal-infected Shigella-infected

NFκ B in cytoplasm NFκ B in nucleus

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

NLR RLR

Iκ
B

IKK

NFκ B in the
nucleus
 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
NFκ B

(sensor) PG

Nod1

Rip2 (adaptor)
Iκ B

NFκ B (transcription factor)

nucleus

Pro-inflammatory products
 NLRPs detect danger and form the
“inflammasome”

DANGER

NLRP3
PYRIN NBD LRR LRR LRR LRR LRR

ASC PYRIN CARD

The inflammasome activates Caspase 1 into
an enzmatically active protein

NLRP3 Inflammasome LPS

NLRP3
Caspase-1
NOD PYRIN Pro-Caspase-
Leucine-rich 1
repeats ASC PYRIN CARD p10
CARD p20 p10
p20
CARD p20 p10
PYRIN CARD p10
NOD PYRIN CARD p20 p10
p20
CARD p20 p10
Active Caspase 1 cleaves pro-IL-1β into an active
cytokine

Caspase-1 Secreted
IL-1β
p10 from the
p20
cell

Pro-IL-
1β
RLRs - RIG-I-like receptors
 Alerting the cell:
RLRs sense nucleic acid (usually VIRAL) and
activate NFκ B & IFN
Viral nucleic acid

RIG-I Helicase CARD CARD

MAVS

NF-κ 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:
• NFκ B
• IRF
• Inflammasomes
• Initiation of the defense response
– Genes “turned on” and IL-1β initiate the defense response
 Next Lecture
• The consequence of NF-κ B, IRF and
IL-1β activation: the mediators of the
defense response