Taking Toll of Toll-Like Receptors

Courtesy of Alan Aderem BAR-CODED BACTERIA: Combined activation of Toll-Like Receptors (TLRs) produces different specific responses. In this example, Microbe 1 activates TLRs 4 and 5, and is therefore likely to be a Gram-negative, flagellated bacterium. Microbe 2 activates TLR 5 and TLR 2/6, and is therefore likely to be Gram-positive and flagellated. Because of slightly different specificities, Microbe 3 would elicit a different response. Twas a head-scratcher: Twelve years ago, researchers found that a receptor, known for its role in a fruit fly dorsoventral patterning called Toll, was related to an important mammalian immune receptor called interleukin-1 (IL-1), a pro-inflammatory cytokine.1 "How could this be?" recalls biochemistry professor Luke O'Neill, Trinity College, Dublin. "We have IL-1, which is inflammatory, and Toll, which is developmental.... Why would they be so similar?" But then it was shown in the adult fly that Toll has a role in antifungal defense.2 Since 1996, numerous researchers have investigated Toll-like receptors (TLRs) in mammals, which are involved in innate immunity, the body's first response to infection. Innate immunity is inborn; acquired immunity, which involves such downstream immune system components as dendritic cells and antigens, is obtained through lifelong contact with pathogens. Data derived from the Science Watch/Hot Papers database and the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. L. Alexopoulou et al., "Recognition of double-stranded RNA and activation of NF-KB by Toll-like receptor 3," Nature, 413:732-8, 2001. (Cited in 150 papers) F. Hayashi et al., "The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5," Nature, 410:1099-103, 2001. (Cited in 205 papers) K.A. Fitzgerald et al., "Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction," Nature, 413:78-83, 2001. (Cited in 111 papers) T. Horng et al., "TIRAP: an adapter molecule in the Toll signaling pathway," Nat Immun, 2:835-41, 2001. (Cited in 114 papers) Four papers in 2001 added more pieces to the ever-evolving, complex TLR story.3-6 These researchers began detailing a clearer and more specialized picture of innate immunity than anyone expected, offering new insights into the links between innate immunity and the adaptive immune response. The independent groups that wrote these Hot Papers found more specializations for TLRs, recognizing such exogenous particles as viral double-stranded RNA,3 bacterial flagellin,4 and that different receptors activate separate immune response pathways within the cell.5,6 As scientists began finding human TLRs, which are even more homologous to fly Tolls than IL-1, Bruce Beutler of The Scripps Research Institute, La Jolla, Calif. and others demonstrated that mammalian TLR 4 recognizes lipopolysaccharide (LPS), a bacterial cell-wall component responsible for septic shock and tens of thousands of deaths annually.7,8 "It was clear then that Tolls in humans were sensing pathogens," says O'Neill. Within a few years, other work would solidify connections: Viruses activate TLR 3, para-sites activate TLR 2, and bacteria activate TLRs 2, 4, 6, and 9. "It becomes clear that the job of these Tolls is to sense microbial products and trigger innate immunity," he adds. Courtesy of Lena Alexopoulou Lena Alexopoulou THE TOLLS MOUNT The published number of mammalian Toll-like receptors is now up to 10, "but there's suspicion of at least three more ... almost every month one more is coming up," quips Lena Alexopoulou, a postdoctoral associate of immunologist Richard A. Flavell, Yale University and a Howard Hughes Medical Institute investigator. Alexopoulou's research shows that TLR 3 recognizes double-stranded RNA, a molecular pattern associated with viral infection.3 "Before our paper, the pathogens that were believed to be recognized by Toll receptors were bacteria and fungi," says Flavell. "Nothing previous suggested the recognition of viruses [by TLRs]." After the cell recognizes a virus, cytokine genes are switched on, which mediate the inflammatory response that is part of innate immunity. The type-1 interferons, (IFNa and IFNB ), are antiviral agents specifically made in response to TLR 3. Also, TLR 3 activation causes dendritic cell differentiation and maturation, thereby initiating the adaptive immune response. This association suggests that activation of TLR 3 may enhance antiviral responses and that antagonists could reduce the immuno-pathology that stems from too much TLR 3 activation. Also in 2001, Alan Aderem, director of the Institute for Systems Biology in Seattle, found that bacterial flagellin is the agonist for TLR 5.4 Earlier in 1999, the Aderem lab reported that TLR 2 is recruited to macrophage phagosomes, where it samples the contents and identifies the pathogen contained within it.9 In 2000 they reported that different TLRs combine to increase the repertoire of recognition by these germ-line receptors.10 Courtesy of Alan Aderem Alan Aderem The flagellin paper stands out for several reasons, Aderem says. First, flagellin is a good adjuvant, and can stimulate the adaptive immune system. Understanding how flagellin activates TLR 5 could lead to the development of better vaccines. "This is being investigated as we speak," says Aderem. Second, TLR 5, if mutated, might predispose individuals to infectious diseases. "We recently found that people who have mutations in Toll 5 are more susceptible to Legionnaire's disease, which is caused by a Gram-negative flagellated bacteria," explains Aderem. Third, this paper speaks to the mechanism by which different TLRs signal different outcomes. "Toll receptors are able to read a bar code on the surface of the bacterium," says Aderem. TLR 5 and TLR 4 are activated if the encountered bacterium is a Gram-negative flagellate like Legionella. "Toll receptors supply information to the immune cell about the precise nature of the pathogen, and the immune system has to respond in a measured way," he says. "If the response isn't highly controlled, inflammatory disease could be the result." MAL CONTENTS O'Neill's group researches signal transduction and TLR function at the molecular level, and began studying TLRs through interleukin-1. "People are interested in it because it seems to be a key factor in arthritis. But no one knew how IL-1 worked," says O'Neill. In piecing together the TLRs' molecular interactions downstream, O'Neill's lab found that a new adapter protein, dubbed Mal for MyD88-adapter-like, was specific for TLR 4 signaling.5 Nearly all TLRs use MyD88 in their signal transduction. But here's the mystery: If MyD88 is knocked out from mice and TLR 4 still works, then MyD88 isn't essential for TLR 4 signaling. So O'Neill and colleagues began searching for more adapter proteins and found Mal, showing that Mal uses TLR 4 and not IL-1, suggesting specificity. Another group from Yale, headed by Rusian Medzhitov, also found Mal and named it TIRAP.6 They showed that their adapter protein required TLR 4, but not TLR 9, again emphasizing specificity in signaling. In resting cells, a Toll-like receptor is embedded in the membrane, its cytosolic portion dangling below the membrane into the cell's innards. Add an agonist such as LPS to cells, and MyD88, which is a cytosolic protein, is mobilized and interacts with the receptor's cytosolic face. This acts as a scaffold for other proteins to be brought into play such as the kinase IRAK 4, which begins the signaling process. Before this paper, researchers thought that all TLRs signal in the same way because TLRs have similar cytosolic domains, explains O'Neill. But the Mal discovery shows that signaling requires specificity. In other words, TLR 4 can turn on a set of genes that other TLRs can't and Mal is likely to be part of regulating that specificity. INNATE-ADAPTIVE CONNECTION "In the old days people thought that the most evolved part of the immune system was adaptive immunity," says O'Neill. "What's becoming clearer is that Tolls themselves can give rise to specific and complex pathways. Innate immunity was always thought of as something global and quite crude, but now it might be more refined. If the immune system encounters bacteria, a bank of genes gets turned on. If it encounters a virus, a separate bank gets turned on. And each bank is tailor-made to fight the bacteria or virus. This tailoring of the immune system is probably being driven by Tolls through the recruitment of specific adapters for each Toll." The TLRs were the missing puzzle pieces regarding the interface between innate and adaptive immune system, says Aderem. "We and others have been looking for this for 20 years. Once that last piece was in place, the field took off and our papers were cited over and over again." Karen Young Kreeger (kykreeger@aol.com) is a freelance writer in Media, Pa. References 1 N.J. Gay, F.J. Keith, "Drosophila Toll and IL-1 receptor," Nature, 351:355-6, 1991. 2. B. Lemaitre et al., "The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults," Cell, 86:973-83, 1996. 3. L. Alexopoulou et al., "Recognition of double-stranded RNA and activation of NF-kB by Toll-like receptor 3," Nature, 413:732-8, 2001. (Cited in 150 papers) 4. F. Hayashi et al., "The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5," Nature, 410:1099-103, 2001. (Cited in 205 papers) 5. K.A. Fitzgerald et al., "Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction," Nature, 413:78-83, 2001. (Cited in 111 papers) 6. T. Horng et al., "TIRAP: an adapter molecule in the Toll signaling pathway," Nat Immun, 2:835-41, 2001. (Cited in 114 papers) 7. A Poltorak et al., "Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice," Science, 282:2085-8, 1998. 8. K Hoshino et al., "Cutting edge: Toll-like receptor 4 (TLR 4)-deficient mice are hyporesponsive to lipopolysaccharide: Evidence for TLR4 as the LPS gene product," J Immun, 162:3749-52, 1999. 9. D.M. Underhill et al., "The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens," Nature, 401:811-5, 1999. 10. A. Ozinsky et al., "The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between Toll-like receptors," Proc Natl Acad Sci, 97:13766-71, 2000. function sendData() { document.frm.pathName.value = location.pathname; result = false if (document.frm.score[0].checked) result = true; if (document.frm.score[1].checked) result = true; if (document.frm.score[2].checked) result = true; if (document.frm.score[3].checked) result = true; if (document.frm.score[4].checked) result = true; if (!result) alert("Please select") return result; } .myradio{background-color : #94bee5} Please indicate on a 1 - 5 scale how strongly you would recommend this article to your colleagues? Not recommended 1 2 3 4 5 Highly recommended Please register your vote

Taking Toll of Toll-Like Receptors

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