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