Bruce Beutler
Bruce Alan Beutler is an American immunologist and geneticist. Together with Jules A. Hoffmann, he received one-half of the 2011 Nobel Prize in Physiology or Medicine, for "discoveries concerning the activation of innate immunity." Beutler discovered the long-elusive receptor for lipopolysaccharide. He did so by identifying spontaneous mutations in the gene coding for mouse Toll-like receptor 4 in two unrelated strains of LPS-refractory mice and proving they were responsible for that phenotype. Subsequently, and chiefly through the work of Shizuo Akira, other TLRs were shown to detect signature molecules of most infectious microbes, in each case triggering an innate immune response.
The other half of the Nobel Prize went to Ralph M. Steinman for "his discovery of the dendritic cell and its role in adaptive immunity."
Beutler is currently a Regental Professor and Director of the Center for the Genetics of Host Defense at the University of Texas Southwestern Medical Center in Dallas, Texas. In 2012, Beutler was appointed as an Honorary Professor in the School of Biochemistry and Immunology at Trinity College Dublin.
Early life and education
Born in Chicago, Illinois, to a Jewish family, Beutler lived in Southern California between the ages of 2 and 18. For most of this time, he lived in city of Arcadia, a northeastern suburb of Los Angeles in the San Gabriel Valley. During these years, he spent much time hiking in the San Gabriel Mountains, and in regional national parks, and was particularly fascinated by living things. These experiences impelled an intense interest in biological science. His introduction to experimental biology, acquired between the ages of 14 and 18, included work in the laboratory of his father, Ernest Beutler, then at the City of Hope Medical Center in Duarte, CA. There he learned to assay enzymes of red blood cells and became familiar with methods for protein isolation. He published his studies of an electrophoretic variant of glutathione peroxidase, as well as the inherent catalytic activity of inorganic selenite, at the age of 17.Beutler also worked in the City of Hope laboratory of Susumu Ohno, a geneticist known for his studies of evolution, genome structure, and sex differentiation in mammals. Ohno hypothesized that the major histocompatibility complex proteins served as anchorage sites for organogenesis-directing proteins. He further suggested that H-Y antigen, a minor histocompatibility protein encoded by a gene on the Y chromosome and absent in female mammals, was responsible for directing organogenesis of the indifferent gonad to form a testis. In studying H-Y antigen, Beutler became conversant with immunology and mouse genetics during the 1970s. While a college student at the University of California at San Diego, Beutler worked in the laboratory of Dan Lindsley, a Drosophila geneticist interested in spermatogenesis and spermiogenesis in the fruit fly. There, he learned to map phenotypes to chromosomal regions using visible phenotypic markers. He also worked in the laboratory of Abraham Braude, an expert in the biology of LPS.
Beutler received his secondary school education at Polytechnic School in Pasadena, California. A precocious student, he graduated from high school at the age of 16, enrolled in college at the University of California, San Diego, and graduated with a BA degree at the age of 18 in 1976. He then enrolled in medical school at the University of Chicago in 1977 and received his M.D. degree in 1981 at the age of 23. From 1981 to 1983 Beutler continued his medical training at the University of Texas Southwestern Medical Center in Dallas, Texas, as an intern in the Department of Internal Medicine, and as a resident in the Department of Neurology. However, he found clinical medicine less interesting than laboratory science, and decided to return to the laboratory.
Scientific contributions
Isolation of tumor necrosis factor and discovery of its inflammation-promoting effect
Beutler's focus on innate immunity began when he was a postdoctoral associate and later an assistant professor in the lab of Anthony Cerami at Rockefeller University. Drawing upon skills he had acquired earlier, he isolated mouse "cachectin" from the conditioned medium of LPS-activated mouse macrophages. Cachectin was hypothesized by Cerami to be a mediator of wasting in chronic disease. Its biological activity, the suppression of lipoprotein lipase synthesis in adipocytes, was thought to contribute to wasting, since lipoprotein lipase cleaves fatty acids from circulating triglycerides, allowing their uptake and re-esterification within fat cells. By sequential fractionation of LPS-activated macrophage medium, measuring cachectin activity at each step, Beutler purified cachectin to homogeneity. Determining its N-terminal sequence, he recognized it as mouse tumor necrosis factor, and showed that it had strong TNF activity; moreover that human TNF, isolated by a very different assay, had strong cachectin activity.Human TNF, isolated contemporaneously by other workers, had to that time been defined only by its ability to kill cancer cells. The discovery of a separate role for TNF as a catabolic switch was of considerable interest. Of still greater importance, Beutler demonstrated that TNF acted as a key mediator of endotoxin-induced shock. This he accomplished by raising an antibody against mouse TNF, which he used to neutralize TNF in living mice challenged with lipopolysaccharide. The often-lethal systemic inflammatory response to LPS was significantly mitigated by passive immunization against TNF. The discovery that TNF caused an acute systemic inflammatory disease presaged its causative role in numerous chronic inflammatory diseases. With J.-M. Dayer, Beutler demonstrated that purified TNF could cause inflammation-associated responses in cultured human synoviocytes: secretion of collagenase and prostaglandin E2. This was an early hint that TNF might be causally important in rheumatoid arthritis. Beutler also demonstrated the existence of TNF receptors on most cell types, and correctly inferred the presence of two types of TNF receptor distinguished by their affinities, later cloned and designated p55 and p75 TNF receptors to denote their approximate molecular weights. Before a sensitive immunoassay for TNF was feasible, Beutler used these receptors in a binding competition assay using radio-iodinated TNF as a tracer, which allowed him to precisely measure TNF in biological fluids.
Invention of TNF inhibitors
Beutler was recruited to a faculty position at UT Southwestern Medical Center and the Howard Hughes Medical Institute in 1986. Aware that TNF blockade might have clinical applications, he invented and patented recombinant molecules expressly designed to neutralize TNF in vivo. Fusing the binding portion of TNF receptor proteins to the heavy chain of an immunoglobulin molecule to force receptor dimerization, they produced chimeric reagents with surprisingly high affinity and specificity for both TNF and a closely related cytokine called lymphotoxin, low antigenicity, and excellent stability in vivo. The human p75 receptor chimeric protein was later used extensively as the drug Etanercept in the treatment of rheumatoid arthritis, Crohn's disease, psoriasis, and other forms of inflammation. Marketed by Amgen, Etanercept achieved more than $74B in sales.Discovery of the LPS receptor, and the role of TLRs in innate immune sensing
From the mid-1980s onward Beutler was interested in the mechanism by which LPS activates mammalian immune cells, sometimes leading to uncontrollable Gram negative septic shock, but also promoting the well-known adjuvant effect of LPS, and B cell mitogenesis and antibody production. A single, highly specific LPS receptor was presumed to exist as early as the 1960s, based on the fact that allelic mutations in two separate strains of mice, affecting a discrete genetic locus on chromosome 4 termed Lps, abolished LPS sensing. Although this receptor had been widely pursued, it remained elusive. Beutler reasoned that in finding the LPS receptor, insight might be gained into the first molecular events that transpire upon an encounter between the host and microbial invaders.Utilizing positional cloning in an effort that began in 1993 and lasted five years, Beutler, together with several postdoctoral associates including Alexander Poltorak, measured TNF production as a qualitative phenotypic endpoint of the LPS response. Analyzing more than 2,000 meioses, they confined the LPS receptor-encoding gene to a region of the genome encompassing approximately 5.8 million base pairs of DNA. Sequencing most of the interval, they identified a gene within which each of two LPS-refractory strains of mice had deleterious mutations. The gene, Tlr4, encoded a cell surface protein with cytoplasmic domain homology to the interleukin-1 receptor, and several other homologous genes that were scattered across the mouse genome. Beutler and his team thus proved that one of the mammalian Toll-like receptors, TLR4, acts as the membrane-spanning component of the mammalian LPS receptor complex. They also showed that while mouse TLR4 is activated by a tetra-acylated LPS-like molecule, human TLR4 is not, recapitulating the species specificity for LPS partial structures. It was deduced that direct contact between TLR4 and LPS is a prerequisite for cell activation. Later, an extracellular component of the LPS receptor complex, MD-2, was identified by R. Shimazu and colleagues. The structure of the complex, with and without LPS bound, was solved by Jie-Oh Lee and colleagues in 2009.
Jules Hoffmann and colleagues had earlier shown that the Drosophila Toll protein, originally known for its role in embryogenesis, was essential for the antimicrobial peptide response to fungal infection. However, no molecule derived from fungi actually became bound to Toll; rather, a proteolytic cascade led to the activation of an endogenous ligand, the protein Spätzle. This activated NF-κB within cells of the fat body, leading to antimicrobial peptide secretion.
Aware of this work, Charles Janeway and Ruslan Medzhitov overexpressed a modified version of human TLR4 and found it capable of activating the transcription factor NF-κB in mammalian cells. They speculated that TLR4 was a "pattern recognition receptor." However, they provided no evidence that TLR4 recognized any molecule of microbial origin. If a ligand did exist, it might have been endogenous. Indeed, numerous cell surface receptors, including the TGFβ receptor, B cell receptor, and T cell receptor activate NF-κB. In short, it was not clear what TLR4 recognized, nor what its function was. Separate publications, also based on transfection/overexpression studies, held that TLR2 rather than TLR4 was the LPS receptor.
The genetic evidence of Beutler and coworkers correctly identified TLR4 as the specific and non-redundant cell surface receptor for LPS, fully required for virtually all LPS activities. This suggested that other TLRs might also act as sensors of infection in mammals, each detecting other signature molecules made by microbes whether or not they were pathogens in the classical sense of the term. The other TLRs, like TLR4, do indeed initiate innate immune responses. By promoting inflammatory signaling, TLRs can also mediate pathologic effects including fever, systemic inflammation, and shock. Sterile inflammatory and autoimmune diseases such as systemic lupus erythematosus also elicit TLR signaling, and disruption of signaling from the nucleic acid sensing TLRs can favorably modify the disease phenotype.