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LPS-induced TLR4-mediated Cellular Activation
Abstract
Sepsis, the tenth leading cause of death in the United States, is a
whole-body inflammatory response to infection. Sepsis leads to
septic shock, a condition with a 30-40% mortality rate caused by
multiple organ failure and development of hypotension. Lack of
understanding of the pathophysiology of sepsis limits successful
treatment options. Gram-negative bacteria are a major cause of
sepsis. The outer membrane of Gram-negative bacteria contains
lipopolysaccharide (LPS).
LPS is recognized by a receptor
complex expressed by certain immune cells that includes the
transmembrane glycoprotein, Toll-like receptor 4 (TLR4), and
myeloid differentiation factor-2 (MD-2).
Over-stimulation of
immune cells by LPS through TLR4/MD-2 results in sepsis. TLR4mediated activation of immune cells is also responsible for allergic
contact dermatitis due to nickel, a common and less fatal condition
than sepsis.
The crystal structure of TLR4-MD-2-LPS has
elucidated residues involved in LPS binding to TLR4/MD-2 and in
TLR4 dimerization, which are essential events involved in immune
cell activation and induction of sepsis. A better understanding of
interactions between TLR4, LPS and MD-2 will help create better
drugs to disrupt the interactions. The Laconia SMART (Students
Modeling A Research Topic) Team used 3-D printing technology to
model the TLR4 dimer in collaboration with MSOE.
1. Gram negative bacteria such as E. coli are lysed by cells of
the innate immune system resulting in the release of LPS
from the outer membrane.
2. Free LPS attaches to LPS binding protein (LBP).
3. The LBP-LPS complex binds to a CD14 molecule on the
plasma membrane of an innate immune cell and transfers
LPS to a TLR-4/MD-2 molecule.
4. The binding of LPS to TLR4/MD2 facilitates the dimerization
of TLR4 molecules leading to intracellular signal transduction
resulting in the transcription and translation of cytokines.
Cytokines recruit and activate other immune cells.
Over-production of cytokines results in sepsis.
A
Nickel Allergy
B
B
Acinetobacter baumannii. Digital image. University of
Rochester Medical Center. University of Rochester, 27 Jan.
2009. Web. <http://www.urmc.rochester.edu/>. (ref. 7)
Introduction
Medshitov, R., Nature Reviews Immunology, 2001;1:135-145
• Nickel contact dermatitis is initiated by the binding of
inorganic nickel to His 456 and 458 (green in B, left) on
adjacent TLR-4 molecules resulting in their dimerization
and signaling.
Sepsis is an overwhelming, whole body inflammatory response to an
infection which if left unresolved progresses to severe sepsis, a state
of inflammation and organ failure.
The Clinical Burden of Sepsis
Common: There are 500,000
750,000 cases per year in US1,2
•
Cases of Sepsis in the United States
-
•
•
•
Expensive: The estimated annual
cost of treatment in the US is
estimated at $17 billion2
Fatal: 30%-40% of patients with
severe sepsis will die1
Severe sepsis is among the
leading causes of death in
non-coronary intensive care units2.
•
Martin, G., et al., N Engl J Med, 2003;348:1546-54 (ref. 1)
Sepsis is the 10th leading cause of death in United States3
Gram-negative bacteria (A, above), like E. coli, contain LPS in their outer membrane (red).
LPS is a lipopolysaccharide that contains a chain of polysaccharides attached to two
phosphorylated glucosamines that are connected to six lipid chains.
The extracellular domain of TLR4 (purple/violet) is always in a complex with MD2
(charcoal/silver) on the cell surface (B, above).
The binding of LPS causes two TLR-4/MD-2 complexes to interact to form a dimer (C, below).
Hydrogen bonding occurs between the R2-OH on LPS and Gln 436 (Royal Blue) on TLR-4.
The R2 on LPS is involved in hydrophobic interactions with the amino acids Phe 440, Leu
444, and Phe 463 (Royal Blue) on TLR-4. There are also charge interactions between the
amino acids Lys 388, Arg 264, Lys 341, and Lys 362 (Sky Blue) on TLR-4 and the phosphates
on LPS.
The LPS-bound TLR4/MD-2 dimer is stabilized by interactions between the TLR4 component
of one complex and the MD-2 component of the other complex. Thus, Phe 440, Leu 444, and
Phe 463 (Royal Blue) of TLR4 participate in hydrophobic interactions with MD-2 amino acids
Phe 126, Ile 124, Leu 87, Met 85, and Val 82. In addition, hydrogen bonding occurs between
TLR-4 amino acids Glu 439, Ser 416, and Asn 417 (Royal Blue) and MD-2 amino acids Gly
123, Lys 125, and Arg 90.6
About 50% of sepsis cases are caused by Gram-negative bacteria1
• According to UW Health nickel allergy affects 10% of the
U.S. population.
Conclusion
•
Currently, the number of cases of sepsis in the United
States is on the rise, and there are no effective
treatments due to a lack of understanding of the
pathophysiology of the disease.
•
Without a cure for sepsis, it will continue to take an
increasing toll on the human population, both
financially and in terms of human lives.
•
Understanding the structures and interactions
between TLR4/MD-2 complex and LPS could lead to
therapeutic advances.
C
Recognition of Foreign Pathogens by the Innate Immune System
LPS
Pathogen-Associated Molecular Patterns (PAMP)
• Lipopolysaccharide (LPS), which is a component of Gramnegative bacteria is an example of a PAMP
• Constitutive - always expressed
• Evolutionarily conserved - present on all pathogens of that
type
• Foreign - expressed on microbes but are absent from the host
Sources
1. Martin, G., et al., N Engl J Med, 2003;348:1546-54
2. Angus, DC., et al., Crit Care Med 2001;29:1303-10.
3. Xu J et al., National Vital Statistics Reports, Vol. 58, May 20,
2010
4. Rothenberg, M., Nature Immunology, 2010; 11:781-782
5. Medzhitov, R., Nature Reviews Immunology, 2001;1:135-145
6. Park, B.S., et al., Nature, 2009;458:1191-1196
7. Acinetobacter baumannii. Digital image. University of
Rochester Medical Center. University of Rochester, 27 Jan.
2009. Web. <http://www.urmc.rochester.edu/>.
Park, B.S., et al., Nature, 2009;458:1191-1196 (ref. 6)
Toll-like Receptors
Medshitov, R., Nature Reviews Immunology, 2001;1:135-145 (ref. 5)
Pattern Recognition Receptors (PRR)
• Receptors on the surfaces of
host cells that bind to PAMPs
• Toll-like receptor 4 (TLR4) is an
example of a PRR
Based on the 3fxi.pdb
SMART Teams are supported by the National Institutes of Health (NIH)- National Center
for Research Resources-Science Education Partnership Award (NCRR-SEPA), and an
NIH CTSA Award to the Medical College of Wisconsin.