Bruce Beutler's research into the basis of innate immunity and his elucidation of the functional roles of many different proteins involved in the immune response caught our attention. Curious to know more, BioTechniques contacted him to find out about the ambition, character, and motivation that led to his success.The central answer
How did you first start studying immunity?
As a postdoctoral fellow and assistant professor at Rockefeller University, I purified a mouse protein that suppressed lipoprotein lipase activity in fat cells, thereby preventing these cells from taking in lipids. When I sequenced this protein, I found that the N-terminal portion was homologous to human tumor necrosis factor (TNF), which had been cloned contemporaneously. At the time, TNF was defined as a factor that could cause necrosis or death in certain tumor cell lines in vitro, and we showed that it was inflammatory as well.
It occurred to me that the effects of TNF, specifically suppression of lipoprotein lipase and hemorrhagic necrosis of tumors, were also known effects of lipopolysaccharide (LPS). So I tested whether this molecule could mediate the lethal effects of LPS in initiating an immune response. I began to passively immunize mice against TNF and found that they were strongly protected against challenge with LPS. How LPS caused inflammation became the central focus of my research after that, and I think my greatest achievement was to find the LPS locus.
What was it like to identify the LPS receptor locus and discover such a central component of the immune response?
There was a lot of speculation about the LPS receptor: some thought it was a member of the immunoglobulin family or CD18; others argued that it wasn't a surface receptor at all, rather a plasma molecule that bound LPS; still others suggested that it was a protease or an isozyme of protein kinase C. It was a very important question and many labs were working to find the answer. I was worried every day that I would open a copy of Nature or Science and find that somebody had finished the job.
During the years we were positionally cloning the LPS receptor locus, we had very little to show for our work. Many people criticized our approach and thought we would never find what we were after. But I knew that they were wrong. I knew if we kept continuously liquidating the critical region, we would eventually find it.
The biggest surprise of my career was to find that the LPS receptor locus was toll-like receptor 4 (TLR4). This was completely unexpected and surprising enough that I kept repeatedly looking at the sequence throughout the day to make sure the mutation was real. After spending five years looking for this and following many false leads in the process, it was hard to believe that we had really found it. We had a second strain of mice, unrelated to the one where we found the mutation; both were unresponsive to LPS. We repeatedly tried to PCR amplify the TLR4 locus in the second strain for verification but just couldn't do it; it was simply missing. We eventually found the limits of a deletion spanning the locus. All three exons of the gene were eliminated and no other genes were affected. This absolutely confirmed that TLR4 sensed LPS.
You won the Nobel Prize for this work. How did you respond to learning about the award?
Although I had won some predictive prizes, I wasn't confident that this would ever happen. I received an email at 2:30 in the morning informing me of the prize, but I was skeptical. I went to my laptop and tried to access the nobelprize.org site, but there was too much traffic. I then searched my name with Google and at first there was nothing. I was very concerned, but when I refreshed the browser, many listings of my name started showing up. Then my phone began to ring. It was a day of much joy, but also difficult to believe, as though I might just be dreaming.
What do you think is the most important open question in your discipline?
Since the discovery of the LPS receptor, many more immune sensors for microbes have been identified, giving us a clear idea of where inflammation begins following infection. Now I think it is important to find the endogenous equivalents of LPS that lead to autoimmune inflammatory diseases. In some cases, we are beginning to understand. For example, mouse models of systemic lupus erythematosus have provided evidence that inflammation depends on TLR7 signaling; for rheumatoid arthritis, TNF is pathogenically important. But what about other inflammatory diseases? And what is driving these diseases if you look upstream? Currently we are working to resolve these questions using pharmacogenomic and mutagenic approaches.