Tolllike Receptors TLRs and LPS sensing in Innate Defense

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Toll-like Receptors (TLRs) and LPS sensing in
Innate Defense MLS 475 Sept. 28, 2007
Dr. James Stafford Z508 Department of Biological
Sciences 492-9258 (stafford_at_ualberta.ca)
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Learning Objectives

• Define PAMPs and PRRs and give an example of
  each.
• Know the basic structure of Toll-like receptors
  (TLRs).
• Identify one ligand for each of TLR2, TLR4, and
  TLR9 and know the source of each of these
  ligands.
• Know the basic structure of endotoxin (i.e.
  lipopolysaccharide) and be able to identify the
  active portion of this molecule.
• Describe how to measure macrophage responses to
  endotoxin.
• Understand the TLR4-mediated LPS recognition
  pathway.
• Describe the two major pathways of TLR-mediated
  intracellular signaling and the outcomes of these
  different signaling pathways.

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Toll-Like Receptors (TLRs) (also known and as
Pattern Recognition Receptors PRRs) Total of 13
TLRs have been identified in mammals Human (TLRs
1-10) Mouse (TLRs 1-9, 11-13) In general TLRs
recognize constituents of microbial cell walls or
pathogen-specific nucleic acids that are
essential to the integrity, function or
replication of microbes / viruses that cannot
readily be modified. Pattern Associate Molecular
Patterns (PAMPs)
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Toll-Like Receptors (TLRs) Basic Architecture
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TLR4 not so basic Architecture
ED TM CYT
LRR
Modified from Kim HM et al., 2007 Cell
130906-917
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Toll-Like Receptors (TLRs) (Beutler B et al.,
2003 Journal of Leuk Biol 74479-485)
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ENDOTOXIN
-Structural component of the outer leaflet of the
outer membrane of Gram negative
bacteria -Contains the O-antigenic polysaccharide
determinant. -Presence of polysaccharide and
lipid components. -Also termed Lipopolysaccharide
(LPS). -The lipid component or Lipid A
(glycophospholipid) is responsible for the
biological activities of LPS (toxicity / fever)
LPS (endotoxin)
Outer membrane
Peptidoglycan
Inner membrane
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LPS (endotoxin)
Outer membrane
Peptidoglycan
Inner membrane
The Basic Architecture of
LPS Structure of Lipid A
LPS
the toxic center of LPS
Lipid A is a glycophospholipid with phosphorylated
D-glucosamines
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Other Bacterial Poisons
Peptidoglycan
Lipopeptides
Lipoteichoic Acid
Unmethylated DNA Oligomers
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ENDOTOXIN Biological Properties
Biological Effects (i.e. fever, toxic shock)
Low doses
Interaction with a high affinity/specificity
receptor (LPS-sensing receptor) Linked to a
signal Amplification system Endotoxin Receptor
?? Hyporesponsive mice CH3/HeJ provided
the genetic basis for identifying the endotoxin
receptor.
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ENDOTOXIN Biological Properties
Detected by Immune Cells Myeloid cells -
MACROPHAGES
-If the LPS dose is small -Responses is
LOCALIZED and BENEFICIAL -If the LPS dose is
large -Response is SYSTEMIC and LETHAL (Septic
Shock) Generally non-immune cells and many
animals are LPS-insensitive (i.e. birds,
fish). Why have a potentially lethal
LPS-detecting system?
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ENDOTOXIN The Poison-Protection Dichotomy
-What is the purpose of sensing LPS? -Timely
detection of a small number of infectious
organisms and the mobilization of an immune
response to contain / eliminate them. -What
happens if there is no LPS-sensing? -C3H/HeJ
mice have increased bacterial infections
causing increased mortality (i.e. infection
death). -Therefore, LPS-sensing can be risky
but VITAL for the immediate or INNATE Immune
Response towards pathogens.
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TNF Mediates LPS Responses
Tumor Necrosis Factor. -Is a cytokine involved
in systemic inflammation. -Mainly secreted by
macrophages. -Mouse TNF was originally purified
from the conditioned medium of LPS-stimulated
Møs (1985). TNF causes a range of effects
similar to LPS. -Diarrhoea, shock, and
death. Possibility that TNF was the endogenous
mediator of LPS toxicity. Antibody-mediated
blockage of TNF caused a significant reduction in
LPS toxicity.
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Endogenous Mediator of LPS Biological Activities
Induction of Signaling Cascades NF?? Activation
TNF production
BIOLOGICAL EFFECTS
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LBP and CD14 the first Endotoxin Receptors
LPS-Binding Protein (LBP) -Plasma protein
produced in the Liver. -Delivers LPS from the
serum to macrophages. -Enhances the sensitivity
of macrophages for LPS. CD14 -GPI-linked
membrane protein (macrophages). -Also exists in
a soluble form recruited to endothelial
cells. -Works with LBP to bring LPS to the cell
surface. No link between LBP/CD14 and
intracellular signaling. However, LPS detection
by immune cells results in an intracellular
signaling response and production of TNF.
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LBP and CD14 the first Endotoxin Receptors
Macrophages
Endothelial
cells
Biological Activities
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Defective LPS signaling in C3H/HeJ and C57/10ScCr
mice mutations in the Tlr4 gene (Poltorak A. et
al., 1998 Science 2822085-2088)
C3H/HeJ and C57/10ScCr Mice -Hyporeseponsive to
LPS / Susceptible to G- negative bacterial
infections.
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Macrophage Responses to LPS
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MD-2, a Molecule that Confers Lipopolysaccharide
Responsiveness on Toll-like Receptor 4 (Shimazu.
et al., 1999 Journal Exp Med 1891777-1782)
It Appears that TLR4 does not act
alone. -Transfection of TLR4 does not confer LPS
responsiveness on a TLR4-deficient cell
line. -Suggest the requirement of an additional
molecule. -MD-2 identified as the TLR4
co-receptor for LPS. -Physically associated with
TLR4 on the cell surface. -Confers LPS
responsiveness to LPS on TLR4 cells.
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MD-2, a Molecule that Confers Lipopolysaccharide
Responsiveness on Toll-like Receptor 4 (Shimazu.
et al., 1999 Journal Exp Med 1891777-1782)
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MD-2, a Molecule that Confers Lipopolysaccharide
Responsiveness on Toll-like Receptor 4 (Shimazu.
et al., 1999 Journal Exp Med 1891777-1782)
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(No Transcript)
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(Beutler B et al., 2003 Journal of Leuk Biol
74479-485)
Earlier Signaling Events
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Earlier Signaling Events
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TLR Signaling The Basics

• 1. Different signaling cascades
• are initiated by different TLRs.
• 2. Some TLRs can initiate
• multiple signaling cascades.
• 3. Dependent on the adaptor
• molecules that are recruited to
• the TIR of each receptor.
• 4. Initiates / controls an
• appropriate immune response
• (i.e. anti-viral vs. anti-bacterial).

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MyD88-Dependent Signaling (most TLRs, IL-1 and
IL-18 Rc)
1. MyD88 is recruited to the TIR domain of TLR
following interaction with ligand (i.e. CpG
DNA). 2. MyD88 (myeloid differentiation factor)
is an adaptor molecule that also has a TIR
domain. 3. MyD88 recruits two related protein
kinases (IRAK 4 and IRAK-1) and the adaptor
TRAF6 to its own TIR domain IRAK IL-1
receptor associated kinase TRAF TNF receptor
associated factor 6 4. The IRAK-1 / TRAF6
complex then dissociates leading to the
activation of a series of Mitogen activated
protein (MAP) kinases (Erk, JNK, p38). 5. Also
activates the I-?? kinase (IKK) complex,
which then activates the transcription factor
NF-? leading to the up-regulation of
pro-inflammatory cytokines including TNF and
IL-1 (antibacterial). 6. MyD88 may also directly
interact with the transcription factor IRF-5
(Interferon Regulatory Factor), which is then
activated by phosphorylation and recruited to
the nucleus
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MyD88-Independent Signaling (TLR3 and TLR4)
1. TRIF is recruited to the TIR domain of TLR
following interaction with ligand (i.e.
dsRNA). 2. TRIF-TIR-domain containing adaptor
inducing interferon-?. 3. TRIF primarily
signals via TRAF3 and the protein kinases
TBK-1 and IKK? 4. This can result in the
phosphorylation of IRF-3 resulting in its
translocation to the nucleus 5. TRIF also
stimulates the TRAF6 pathway as seen with the
MyD88 pathway. 6. In contrast to the MyD88
pathway, TRIF directly interacts with TRAF6
(no IRAK used as mediators). 7. Leads to the
production of anti-viral factors such as type
I interferons.
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TLR4 can signal using both pathways
TRAM TRIF-related adaptor molecule TIRAP
Toll-interleukin 1 receptor (TIR)
domain-containing protein TLR4 signaling
induces the transcription of both
proinflammatory cytokines and IFN-? that are
required for anti-bacterial and anti-viral immune
responses, respectively.
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Cooperation of Toll-like receptor signals in
innate immune defense
Trinchieri and Sher Nature Reviews Immunology 7,
179190 (March 2007) doi10.1038/nri2038