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Seeing Targets Through the Eyes of the Innate Immune SystemJane 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. "We're attempting to create mutations that destroy innate immunity and in this way, to identify all of the genes involved in innate immunity," Beutler says, "or at least as many as we can." The mutagenesis involves attacking genomes with ethylnitrosourea and looking for defective innate immune function phenotypes. Treatment with ethylnitrosourea creates about 10,000 to 30,000 randomly distributed mutations in the germline, mostly A–T or A–G substitutions. About 1.6 percent of these mutations fall within coding regions, and about three quarters of these result in an amino acid change. Thus in every separate mutant genome, there are between 100 and 300 mutations that create a coding change. By examining several thousand such genomes, one can get very deep coverage of the entire genetic content of a species—hundreds of thousands of mutations are induced in all, with at least one mutation in almost every gene and several mutations in some of the larger genes. Then, Beutler says, one simply looks for phenotypes that are of interest. In his case, these include instances of immunodeficiency. "Then if we find an individual that is immunocompromised," says Beutler, "we can go back and positionally clone the critical gene that we have hit." Though this may sound routine, there are many more failures than successes, and the competition can be fierce, especially when the mapping and sequencing steps take years, as they did in the case of Tlr4. Beutler described the long battle in a historical narrative recently published in the Journal of Endotoxin Research. Of the race to clone Lps, he wrote:
Bringing it Home to TSRIAfter the successful cloning effort, many new questions related to endotoxin signaling immediately presented themselves, and Beutler continues to study this protein and its relatives, hopeful that a full understanding of the events that occur in signaling may emerge. By making chimeras of human Tlr-4 and mouse Tlr-4 proteins, he and his coworkers hope to pinpoint the part of the protein that actually "touches" the endotoxin molecule, leading to cell activation. Other follow-up work has already panned out. A standard gene searching program called Basic Local Alignment Search Tool (BLAST) has revealed three novel Toll-like receptors in mammals, which were then independently cloned in 2000 by the Beutler and Ulevitch laboratories. These molecules were dubbed TLRs 7, 8, and 9. TLR10—which may prove to be the "last" TLR—was cloned shortly thereafter in the Ulevitch lab. The binding specificity of most of the TLRs remains unknown. "And," Beutler says, "everybody is interested to learn about the structure of these proteins." Beutler calls the process of positional cloning 'addictive.' "The methods used in hunting genes are compelling, and I will continue to use them whenever possible," says Beutler. "The power of a mutation to disclose function is tremendous, and provided that one begins with a clear phenotype, the genes that are ultimately identified will most certainly be relevant to the biological question that one has in mind." There is something in biology referred to as the phenotype gap—the discrepancy between the 30,000 to 40,000 genes we believe are present in the human genome and the mere total of 5,000 distinguishable traits that have been identified through studies of inherited diseases and knockout mutations produced by gene targeting. The inference to be made is that the critical function of most genes remains unknown. This applies in the field of immunology as in many other fields. The precise question of which genes serve innate immunity is one of several lines of inquiry Beutler brought with him when he came to TSRI last year. He is also interested in discovering why the placenta is not rejected during pregnancy as any "normal" tissue allograft would be. Since half of a fetus's genetic material hails from its father, the fetus is sufficiently non-self to cause an immune reaction. But in most cases, it does not. Beutler is trying to create a model in which semi-allogeneic pregnancy (in which the fetus is derived from genetically different parents) is not tolerated, but syngeneic pregnancy (in which the fetus is genetically identical to both parents) is tolerated. "We are asking how it happened that placenta could arise in evolution, although when it did, there was already a very good adaptive immune system," Beutler says. For Beutler, coming to TSRI was a definite homecoming. Raised in southern California and very familiar with San Diego from having attended the University of California at San Diego, he also joins his father, Ernest Beutler, who is chair of the Department of Molecular and Experimental Medicine. "I love it here," he says. "The scientific environment at Scripps is superb, my colleagues here are wonderfully supportive, and not least, I always wanted to come back to California, having missed it ever since I left 25 years ago." Beutler got his start in science while working in his father's laboratory as a teenager. "My father worked on red cell enzymes at the time, and my first project involved assaying glutathione peroxidase activity in human erythrocytes," says Beutler. "He was a fine teacher, who gave me an in-depth introduction to science at a young age." When asked his opinion, Ernest Beutler smiled widely and nodded in approval. "I'm glad he's here," he said.
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“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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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.