Seeing Targets Through the Eyes of the Innate Immune System
By Jason Socrates Bardi
Jane and Michael, both good parents, brought their two-year-old,
Mitch, to the emergency room when he came down with a fever
and a spotted rash and began vomiting in the middle of the
night. Had the child been able to communicate it, he would
have complained of nausea and a persistent stiff neck as well.
Still, the symptoms were enough to trigger warning bells
for the hospital staff, and the attending physician ordered
a blood culture prepared and the local health department notified.
The cultures returned with the expected results: the cultures
grew the bacterium Neisseria meningitidis.
The antibiotic rifampicin was given to the child, the parents,
ER doctors and nurses, and daycare workers who came into contact
with the child, but within a few days, several others were
sick as well.
This case depicts the beginnings of an outbreak of an N.
meningitidis infection, abstracted from a sample outbreak
scenario described in a public health conceptual data model
published by the Centers for Disease Control and Prevention.
Meningococcal sepsis and shock following poisoning with endotoxins—chemical
components of certain bacteria that are particularly harmful
to people—is a dangerous and potentially fatal condition
striking over 2,500 people a year in the United States. About
half of those who contract meningococcal sepsis are younger
than two, and the disease has an overall case fatality rate
of 12 percent.
"It's a very fast-moving, dramatic, and often fatal disease,"
says Immunology Professor Bruce Beutler, who is interested
in both meningococcal sepsis and, more broadly, in discovering
genes that serve innate immunity—the body's broad-based,
fast-acting defense against pathogens. "It has often been
suspected that there are genetic factors that determine who
gets the [severe form] of the disease and who does not," he
adds. "And meningococcal sepsis is just one rather rare form
of Gram-negative infection. In all, about 200,000 severe Gram-negative
infections occur in this country each year."
Beutler has studied a human gene used by the innate immune
system to help the immune system clear pathogens from the
body. This gene codes for a protein that resembles a receptor
called Toll, produced in flies. People with mutations in this
Toll-like gene have a higher probability of contracting meningococcal
sepsis.
Phenotype Follows Genotype—or Is It the Other Way
Around?
Toll is encoded in the genome of Drosophila melanogaster.
The protein is a plasma membrane receptor with one transmembrane
domain and a series of leucine-rich ectodomain repeats. In
the fly, Toll is important for both embryonic development,
during which it triggers dorsoventral patterning, and for
immune functions of the developed organism. In adult Drosophila,
the protein is an essential receptor molecule that defends
against fungal infections.
Mammals have a number of genes similar to Toll used in immune
defense. The interleukin–1 receptor, for instance, a
protein that initiates fever and inflammatory responses by
activating lymphocytes during an infection, has strong homology
to Toll in its cytoplasmic domain—as does the interleukin–18
receptor cytoplasmic domain.
There are also several mammalian proteins that have cytoplasmic
domain similarities to Toll and also have leucine-rich ectodomain
repeats, displaying gene homology to Drosophila Toll
over their entire coding regions. Ten of these have been identified
to date, including one essential gene in the innate immune
system called Toll receptor 4 (Tlr4), which is important
in endotoxin recognition.
"They are the eyes of the innate immune system,"says Beutler.
Tlr4 is a powerful pro-inflammatory receptor, responsible
for activating the immune system to attack invading gram-negative
bacteria like N. meningitidis. During a mammalian innate
immune response, Tlr4 recognizes endotoxins from the bacteria
and activates macrophages, which then ingest and destroy the
foreign pathogens.
The function of Tlr4 emerged from Beutler's research into
genetic defects that had arisen purely by chance in 1965 in
two different strains of mice. These mice were curiously unable
to sense endotoxin, or respond adequately if they became infected
by Gram-negative bacteria, and this odd phenotype was presumed
to result from a mutation that affected the endotoxin receptor.
This receptor was known, based on work carried out in the
early 1990s in the Ulevitch lab, to include the protein CD14
(Richard Ulevitch is the Immunology Department Chair at TSRI).
But while it was also known that at least one other subunit
of the receptor must exist, all attempts to identify this
protein were unsuccessful.
The genetic locus involved, termed Lps, was mapped
and cloned by Beutler's group while he was an Howard Hughes
Medical Institute investigator at the University of Texas
Southwestern Medical Center. The cloning took over five years,
and the efforts of Beutler, seven postdocs, four technicians,
and a string of summer students, because the solution required
a complicated process known as positional cloning.
Positional cloning entails the use of classical genetic mapping
methods to confine the location of the gene to a particular
area in the genome, extensive sequencing of the region in
question, and the performance of computer-aided searches through
databases to find homology between sequences in that region
and known genes. This is the process Beutler followed in his
Tlr4 work, and it is the process he continues to employ in
his studies on other systems today.
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Principal investigator Bruce Beutler's interest in meningococcal
sepsis is tied to his basic interest in an individual's innate
immune system—the body's broad-based, fast-acting defense
against pathogens.
“It
has often been suspected that there are genetic factors that
determine who gets the [severe form] of the disease and who
does not.”
—Bruce
Beutler
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