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Fly Development Genes Lead to Immune Find

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Science  25 Sep 1998:
Vol. 281, Issue 5385, pp. 1942-1944
DOI: 10.1126/science.281.5385.1942

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Guided by fruit fly genetics, scientists are finding that the human innate immune system may be more specialized than they had thought

Like a lowly foot soldier toiling in the shadow of better equipped and better trained cavalry units, the innate immune system, the body's first line of defense against invading pathogens, has long been eclipsed by its partner, the adaptive immune system. In part, this relative lack of interest can be traced to immunologists' view of innate immunity as a sort of brute-force system that unleashes blunt, nonspecific weapons at any and every invader, keeping the foe at bay until the adaptive system with its highly specific weapons—antibodies and T cells—can take over. But now, aided by results from an unlikely source—the developmental control genes of the fruit fly—researchers are developing a new and more intriguing picture of the innate immune system.

Over the past few years, researchers have found that a family of proteins related to the Toll protein of fruit flies, which was first identified as a developmental protein, plays a key role in triggering innate defenses against bacterial and fungal invaders—not only in flies, but in organisms as divergent as tobacco plants and humans. Scientists are still sorting out the roles of the newly discovered proteins, but a few trends are emerging. There is strong evidence that the innate system not only provides a first line of defense, but also alerts the more specialized adaptive immune system to the presence of a dangerous microbe. And there are tantalizing clues that the innate system itself, instead of mounting a single generalized response as long thought, may have specific pathways to target particular pathogens.

Drug companies are especially interested in these findings, as they could eventually help scientists design more effective and safer vaccines and provide better treatments for chronic inflammatory and autoimmune diseases and severe microbial infections. Indeed, more than half a dozen pharmaceutical and biotechnology firms, plus dozens of academic labs, are doing research on the Toll pathway, and results are coming thick and fast. Five human Toll-like proteins have been published to date, molecular biologist Fernando Bazan of DNAX in Palo Alto has described at least five more at meetings, and there are likely more waiting in the wings. The field “is going to be dynamite” for the next few years, says molecular biologist Paul Godowski at Genentech Inc. in South San Francisco.

Thirteen years ago, when the first toll gene was identified, no one would have anticipated that the proteins would be playing such a “dynamite” role in immunology. The gene was discovered in a screen for mutations that interrupt the early stages of embryonic development in the fruit fly Drosophila melanogaster; the toll mutation disrupts proper formation of the insect's front and back. Developmental biologist Kathryn Anderson of the Sloan Kettering Institute in New York and her colleagues eventually showed that the toll gene makes a receptor protein that picks up developmental signals at the cell membrane and sends them to the nucleus. But as Anderson and other developmental biologists began identifying the various intracellular molecules that relay those signals, they found their work taking an unexpected direction—merging with work on the innate immune system.

In the late 1980s, researchers studying inflammation—one of the main weapons of innate immunity—began uncovering signaling pathways that converge on a protein called NF-κB, which turns on genes that make proteins that trigger inflammatory responses in other cells. They found that one of the cell surface receptors that passes its signal to NF-κB is the receptor for the protein interleukin-1 (IL-1), which among other things helps to induce fever.

As it happened, the developmental biologists found that one of the proteins they had identified in their signaling path, which goes by the name Dorsal, is structurally similar to NF-κB. Soon they also realized that the upstream proteins Toll and the IL-1 receptor had similarities, too. A few years later, researchers linked the Toll pathway to the immune system of insects when they found that Dorsal and a related protein, Dif, travel to the nucleus in response to infection. Even more remarkably, plant scientists found that proteins resembling Toll help plants fend off attacks from bacteria and fungi. And last summer, immunologist Charles Janeway of Yale University and his colleagues made the first link to human immunity. They identified the first human Toll protein, now called Toll-like receptor-4 (TLR4), and showed that it activates NF-κB, an indication that it may play a role in the innate immune system.

Indeed, just last week researchers reported the first direct evidence for such a role. Godowski, Austin Gurney, and their colleagues at Genentech reported in Nature that one of the human Toll-like receptors helps to alert immune cells to the presence of lipopolysaccharide, a component of certain bacterial cell walls, including Escherichia coli and Salmonella. Although scientists knew that lipopolysaccharide triggers an innate immune response that involves NF-κB, exactly how the cell detects its presence was a mystery. Toll had already become a leading suspect in the lipopolysaccharide reaction, however, because previous work in flies had shown that Toll activates Dorsal—the NF-κB relative—in response to fungal invasion.

To test if any of the human Toll-related receptors really do respond to lipopolysaccharide, the Genentech scientists exposed immune cells to purified lipopolysaccharide. In response, the RNA instructions for making Toll-like receptor-2 (TLR2) increased, indicating that more of the receptor was being made. What's more, the researchers found that the receptor could respond to lipopolysaccharide by increasing NF-κB activity. Molecular biologists Mike Rothe and Carsten Kirsching of Tularik, a biotech firm in South San Francisco, have reported very similar results at several meetings.

Other experiments, chiefly in the fruit fly, suggest that there are specialized Toll receptors that respond to different pathogens. For example, Toll is known to trigger the production of an antifungal peptide, drosomycin, in the fly. But molecular biologist Jules Hoffmann, of the Institute for Cellular and Molecular Biology in Strasbourg, France, has reported that Drosophila larvae lacking Toll could still turn on an antibacterial peptide called diptericin. Hoffmann and his colleagues suspect that a parallel path—perhaps through one of the four Toll-related proteins reported so far in flies—responds to bacteria, while the original Toll responds to fungal infections. They have shown that different proteins in the Toll pathway respond to fungal and bacterial infections, as do Anderson's recent unpublished experiments.

The human innate immune system may have similar specificity. Godowski found that TLR2 has varying sensitivity to lipopolysaccharide molecules from different kinds of bacteria—two strains of E. coli and one of Salmonella. Although that may be due to different preparation techniques, Godowski says it “raises an intriguing possibility that different Toll receptors may have the ability to recognize different pathogens.” What's more, in unpublished work Rothe and his colleagues have tested several other members of the human TLR family, and only TLR2 responded to lipopolysaccharide, Rothe says. Says Godowski: “There's going to be an incredible amount of interesting work that will come out of looking at these Toll receptors individually and perhaps in combination.”

Because so far the handful of Toll proteins with known functions seem to respond to one kind of pathogen, Anderson speculates that both humans and flies may have specific protein pathways for different invaders, although this isn't proven yet. Scientists hope that with a better understanding of how specific pathogens trigger the immune system, they might be able to selectively shut down certain proteins to treat inflammatory diseases.

Although researchers are still teasing out all the action of the various Toll proteins, they do know that Toll provides a link between the adaptive and innate immune systems. Naïve T cells—members of the adaptive immune system—that have not been exposed to antigens need two signals to become active. The first comes from the binding of an unfamiliar protein, or antigen, and the second comes from a protein called B7.1 and its relatives. And Janeway's work now links B7.1 to the innate immune system via the Toll pathway.

Janeway reported last summer that the active form of TLR4 increases production of B7.1. Immunologist Douglas Fearon of the Wellcome Trust in Cambridge, U.K., says that B7.1 may be a sort of red alert, released if the innate immune system, through its Toll-like receptors, has recognized an infectious invader.

While researchers pursue Toll proteins in hopes of medical applications, they are also thinking about what these new findings are telling us about evolution. Because the Toll immune proteins are similar across plants, flies, and mammals, most scientists think that the defense system arose before the divergence of plants and animals—perhaps at the dawn of multicellular life. Only later were the immune proteins co-opted by developmental systems. “You can't do anything as luxurious as making all sorts of fancy body parts without an immune response,” says molecular biologist Michael Levine of the University of California, Berkeley.

It may be that only flies have used Toll in developmental roles, however—to date, there is very little evidence that Toll relatives are important for mammal or plant development. “In all of our experiments doing knockouts in mice, we've never seen a developmental phenotype,” says David Baltimore of the California Institute of Technology in Pasadena, who helped characterize the NF-κB pathway.

Meanwhile, even as researchers continue to probe the functions of the Toll proteins, more of them continue to be uncovered. “We're not sure where this family ends,” says molecular biologist Michael Karin of the University of California, San Francisco. “It's a very exciting field.”

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