Nobel Prize Critiques: Bruce Beutler

(February 14th, 2012) The 2011 Nobel Prize for Medicine has been dogged by controversy. Both Jules Hoffmann and Bruce Beutler have been accused of unfairly promoting their own contributions to the discoveries. Here, Jeremy Garwood investigates the “case” of Bruce Beutler and rival claims.

The 2011 Nobel Prize for Medicine was controversial. In the first part, we looked at Bruno Lemaitre’s claim that Jules Hoffmann had been mistakenly credited with Lemaitre’s own groundbreaking discoveries on innate immunity in Drosophila, notably the role of the Toll protein (“A Nobel Not Immune from Error?” and “Borrowed Plumes”). In this second part, we will consider the other half of the Nobel Prize, awarded to Bruce Beutler for his discovery that a mammalian Toll-like receptor (TLR) was the cellular receptor for lipopolysaccharide (LPS), a highly conserved biochemical signature for gram negative bacteria that is probably the most immunogenic substance known to man.

Controversial Award – A better choice?

The first many of us were aware of doubts about Bruce Beutler came with a very public letter to Nature signed by 23 prominent immunologists: “Nobels: Toll pioneers deserve recognition” (10th Nov. 2011). Not that they directly criticised the choice of Beutler. Rather they stressed that the Nobel Committee “should have acknowledged the seminal contribution of immunologists Charles A. Janeway Jr. and Ruslan Medzhitov”.

However, Beutler was the only one of the 3 winners to have worked, like Janeway and Medzhitov, on mammalian Toll-like receptors (TLRs). Furthermore, the 1998 discovery for which Beutler received the Prize (Poltorak et al., Science 1998) came over a year after Medzhitov and Janeway had published their key discovery of a role for TLR in innate immunity (Medzhitov et al., Nature 1997). The implication was that the Nobel Prize committee had made a big error and chosen the wrong person. 

In fact, comments had begun appearing within days of the Nobel announcement. Science magazine (7th Oct. 2011) informed us that “research on TLRs has long been very competitive” and there were already “some grumblings” over who did not get chosen. “For example, Ruslan Medzhitov, a Russian immunologist at Yale University who worked on TLRs.”

They also interviewed an immunologist from Dublin, Luke O’Neill, who admitted to having prepared a confidential report in 2009 on the history of the field in order to help guide the Nobel panel’s prize deliberations. He said he had “struggled with it” since there were “at least five or six key people” who could have been honoured.

The Lancet (15th Oct. 2011) explained matters more directly: “Eyebrows have been raised in some quarters about the decision by the Nobel committee to overlook Medzhitov, and consequently the contribution of his mentor, the late Charles Janeway, to the elucidation of the mechanisms of innate immunity. The decision is given extra piquancy by the frosty relations between Medzhitov and Beutler, whom Medzhitov has in the past taken to task for undermining Janeway.” It was noted that Medzhitov had maintained a “stoney silence” on the issue: “Privately, however, he is said to be furious at what he sees as the success of a political campaign on the part of Beutler to sweep Janeway's legacy under the rug.”

A more complete explanation could be found in the science blog: “A bitter sweet Nobel – Beutler, Janeway, and the Dawn of Innate Immunity”. The author, Kevin, studies signaling networks downstream of Toll-like Receptors (TLRs). He began by saying that the 2011 Nobel should have been a happy occasion for his lab – it was awarded for early discoveries in his own field of innate immunity. But, unfortunately, “it was given to a scientist that many scientists I know feel is undeserving of the honor while at the same time sullying the legacy of my scientific great-grandfather.” (Kevin’s lab boss had been Medzhitov's postdoc, “putting him (and me) in Janeway's direct lineage.”)

“No love lost between Medzhitov and Beutler”

“For the past decade, Beutler has been publishing review articles playing up his own contribution and down-playing that of Janeway,” continues Kevin. “And the feud isn't restricted to the peer reviewed journals. The latest sortie in this rivalry occurred earlier this year, after Medzhitov gave a presentation to an audience of innate immunologists (including Beutler). At the end of his talk, Medzhitov announced that he wouldn't take questions, but wanted to comment on the fact that ‘someone’ had changed the Wikipedia entry on Charlie Janeway to read:

‘Janeway and coworkers did not actually demonstrate the existence of receptors for LPS or any other PAMPs. However, receptors for specific molecules made by microbes were eventually identified by other workers, notably [[Bruce Beutler]] , who discovered the LPS receptor [...]’

Medzhitov “didn't name names, but said ‘there's only one person that would do this, and we all know who.’ And I don't think he was referring to Sarah Palin”!

In order to better understand this dispute, it helps to look at the background to TLRs and innate immunity.

“The Immunologist’s Dirty Little Secret”.

Charles Janeway died of cancer in 2003, aged 60. His major contribution to immunology can be dated back to a paper he wrote in 1989. Unusually, it was a review of the field of immunology. Even more unusually, it was the introduction to a scientific meeting! The theme of 1989’s Cold Spring Harbor Symposium was ‘Immunological Recognition’.

In his 13 page introductory essay, “Approaching the asymptote? Evolution and revolution in immunology,” Janeway began by presenting an overview of what had been achieved in the field of immunology to date.

Major advances had been made in understanding the mechanisms of the adaptive immune response. Two populations of lymphocytes had been characterised: T and B. The B-cells made antibodies that could be released into the blood to fight infection. The T-cells, representing ‘cell-mediated immunity’, could recognise and kill infected host cells. Both are ‘adaptive’ because the receptors they use to recognise pathogens are generated at random by gene rearrangements – potentially, antibodies can be generated to recognise any molecule. Furthermore, the adaptive immune system has a ‘memory’ – it learns from infections and remembers, responding selectively to reinfection.

However, Janeway reminded his audience that there was also the less fashionable innate immune response. This is what we’re born with – it is a non-adaptive system that crudely recognises that an infection has occurred. Innate immune receptors are made by cells that are present at all times in tissues (rather than being recruited there during an infection like B-cells and T-cells). In 1989, it was widely believed that these cells were simply there to hold infections at bay while waiting for the B-cells and T-cells to show up and do ‘the real work’.

But, Janeway said immunologists had a “dirty little secret” that most of the field had chosen to ignore. In effect, the adaptive immune system cannot mount an immune response on its own. Injecting purified proteins into a rabbit or a mouse or a man will not produce an immune response, or activation of B- and T-cells: “In order to obtain readily detectable responses to these proteins, they must be incorporated into a remarkable mixture termed complete Freund’s adjuvant, heavily laced with killed Mycobacterium tuberculosis organisms or precipitated in alum and mixed with dead Bordetella pertussis organisms.”

“Why do we need adjuvants? To be quite honest, the answer is not known.” But Janeway then went on to to propose a kind of ‘grand unified theory’ for immunology that could bring together the innate and adaptive immune systems and show how they co-existed functionally.

In effect, there was already experimental evidence that activation of the adaptive immune system required two distinct signals. T-cells and B-cells received one signal through their main receptors, but they also required a second signal, or ‘co-stimulation.’ Adjuvants provided such a signal, and certain microbial products like LPS, a component of bacterial cell walls, had strong adjuvant activities.

Janeway's insight was that the second signal for T-cell and B-cell activation was linked to recognition of particular molecular patterns associated with infection.

“I contend that the immune system has evolved specificalty to recognize and respond to infectious microorganisms, and that this involves recognition not only of specific proteins, but also of certain characteristics or patterns common on infectious agents but absent from the host [...].” 

Janeway predicted that the costimulatory signal for lymphocyte activation was inducible on antigen-presenting cells, that include dendritic cells (whose discovery posthumously won Ralph Steinman the 2011 Nobel) and macrophages. Furthermore, he postulated that conserved microbial products induced this costimulatory signal, placing activation of adaptive immunity under the control of pathogen-sensing mechanisms.

Pathogen sensing, he thought, was mediated by a set of germline-encoded pattern recognition receptors that detect the distinct conserved products of microbial biosynthetic pathways (known as ‘pathogen-associated molecular patterns’ (PAMPs)). From an evolutionary standpoint, he reasoned, this innate immune recognition in vertebrates must be related to the immune systems of invertebrates that do not display adaptive immunity.

Evidence for Janeway’s model

Ruslan Medzhitov describes the search by Janeway’s lab for the pattern recognition receptors he had predicted (Medzhitov 2009 ImmunityApproaching the asymptote: 20 years later”). Originally from Uzbekistan, Medzhitov obtained his PhD from Moscow university then joined Janeway’s lab at Yale in 1994 as a post-doc (age 28). His project was to identify the receptors. Several receptors for innate immune recognition had been discovered by other labs, for example, CD14, a component of the LPS receptor complex (Wright et al. 1990 Science), but Janeway was looking specifically for cell-surface receptors expressed on macrophages and dendritic cells that could induce signaling pathways that controlled activation of the adaptive immune system.

“At that time, nothing was known about these receptors and what they were supposed to look like. Based on the assumption that such receptors should activate the NFkappaB signaling pathway, we identified a human homologue of the Drosophila Toll protein in early 1996. The Toll protein in Drosophila was known at the time to signal through the NFkappaB signaling pathway during embryonic development. However, in early 1996, Bruno Lemaitre and Jules Hoffmann discovered that the same receptor is also involved in the fly’s immune defense. This further reinforced our assumption that mammalian Toll may be involved in microbial recognition and activation of adaptive immunity.” 

(For further scientific details about the background to the discovery of Toll’s immune function in Drosophila, and how this fits in with the discoveries of Medzhitov and Beutler, see ‘The science behind Lemaitre’s discovery’). 

In 1997, Medzhitov and Janeway published their big paper: “A human homologue of the Drosophila Toll protein signals activation of adaptive immunity” (Nature, 388:394). They had cloned ‘human Toll’ (now known as TLR4). Like Drosophila Toll, TLR4 was a transmembrane protein with an extracellular domain consisting of a leucine-rich repeat (LRR) domain, and a cytoplasmic domain homologous to the cytoplasmic domain of the human interleukin (IL)-1 receptor. It was already known that both the Drosophila Toll and the human IL-1 receptor signal through the NFkappaB pathway. They now showed that a constitutively active mutant of human Toll could also induce activation of NFkappaB and the expression of inflammatory cytokines (IL1, IL6, IL8) and a costimulatory molecule required for the activation of T-cells.

Their subsequent research continued to define the TLR4 signaling pathway (e.g. they demonstrated that it employs an adaptor protein, MyD88, and induces activation of NF-kappaB via the kinase IRAK and the TRAF6 protein (Medzhitov Mol Cell. 1998).

TLRs as immune receptors

Medzhitov and Janeway still didn't know the natural activator of human Toll/TLR4, but based on their evidence and the known immune function of Drosophila Toll, they hypothesised that it was involved in the innate recognition of microbial products, and would lead to co-stimulation for the adaptive immune system.

Meanwhile it turned out that there were other members of the TLR family, and soon it was shown that TLRs could indeed respond directly to microbial products (e.g. Yang Nature 1998 395:284; Kirschning J Exp Med 1998 188:2091). This suggested that all mammalian TLRs might directly recognise different microbial ligands, unlike Drosophila Toll which is activated by binding of another Drosophila protein, Spatzle.

This is when three labs published strong evidence that TLR4 was the receptor for lipopolysaccharide (LPS). The Japanese group of Shizuo Akira had already been sufficiently convinced by the work on Drosophila and human Toll to generate a knockout mouse for TLR4. Based on several other results, notably the finding that TLR4 mapped to the LPS loci on both mouse and human chromosomes, they showed that the knockout mice no longer responded to LPS (Hoshino J Immunol 1999 162:3749).

Two other labs used a different genetics approach – Beutler’s (Poltorak, Science 1998 282:2085) and Danielle Malo’s (Qureshi, J Exp Med 1999 189:615). By positional cloning, they had mapped the mutation on a mouse strain that did not respond to LPS down to a region of chromosomal DNA small enough that they could sequence right through it and identify TLR4 as the most likely receptor.

These 3 papers were submitted and came out within weeks of each other. Yet, for the Nobel committee, it is only Beutler who has been recognised for this discovery.

Who is Bruce Beutler?

Bruce Beutler was born in Chicago in 1957. He became a medical doctor (MD) in 1981, but specialised in research. His early success came at Rockefeller University as a postdoctoral fellow working on inflammatory cytokines in Anthony Cerami’s lab. In the mid-1980s, he isolated mouse TNF (tumour necrosis factor) and showed its role in inflammation (Beutler et al. Nature 1985, Science 1985).

In 1986, he moved to the University of Texas Southwestern Medical Center in Dallas where he became an Assistant Investigator of the Howard Hughes Medical Institute (HHMI), the largest private funder of biomedical research in the US. It was here that he made his Nobel winning discovery that the mouse receptor for lipopolysaccharide corresponded to TLR4 (Medzhitov’s ‘human Toll’).

Origins of the Rivalry with Medzhitov?

Beutler has a different perspective on his discovery from many other immunologists. He does not agree that his finding that TLR4 was the probable LPS receptor may have been influenced by the work of Medzhitov and Janeway. In fact, in his 1998 Science paper, he seems to be at pains to avoid mentioning them at all.

The only mention: “TLR4 mRNA is reportedly expressed predominantly in lymphoid tissues (ref. 33 = Medzhitov).” ‘Reportedly’? He is using a word that suggests he doubts their result. But then he admits “Our own data are in agreement with this finding, but in addition (Fig. 4) suggest…”

Beutler explains that he realised that TLR4 was probably the LPS receptor because “the proinflammatory interleukin-1 (IL-1) receptor, like TLR4, is a member of the Toll receptor family.” No reference is given?  “Further, a human mutation causing coresistance to LPS and IL-1 (ref. 30) attests to the likelihood that the IL-1 and LPS use structurally related receptors.”

After he made his discovery, Beutler seems to have embarked upon an extensive publicity campaign to promote the idea that it was ‘the’ major result in innate immunity and that his was the only name that should be associated with it. Furthermore, often he either omits to mention rival labs or even suggests there are problems related to their work.

One need only look at his publication record (from his lab’s home page at UTSouthwestern) to see how his review articles came to dominate his activity:

The discovery was made in 1998. That year, Beutler published 4 articles, with the initial LPS/TLR4 observation published rapidly in mid-September, then with some functional data in Science in mid-December. In 1999, there were 3 research articles. But then Beutler began writing reviews about his discovery: 3 of them in 2000, 11 in 2001, 7 in 2002, 11 in 2003, 10 in 2004, 9 in 2005, etc. !

In these he wrote, for example, that “the general view of innate immunity sensing has been advanced in a dramatic fashion pursuant to the discovery that LPS and TLR4 are one and the same.” (Beutler J Endotoxin Res 2000, 6:269), and “The discovery that (the mammalian endotoxin receptor) is a member of the Toll-like receptor family has illuminated the sensing mechanism used in the innate immune system of vertebrates.” (Beutler Europ Cytokine Network 2000 11:143).

Promotional Lecture Tours

Beutler has also given many talks all over world. His cv lists 22 for 2002, 14 for 2003, 31 for 2004, 37 for 2005, 32 for 2006, 31 for 2007, 17 for 2008, 28 for 2009, 24 for 2010 etc. A typical example, taken from September/October 2005 has him travel from Norway to France to Germany, the UK, New York, California, China (twice), and on to Sweden.

You can even see one of his polished performances online: the Kenyoun Lecture et the NIH, 25/10/2007: ‘How we sense microbes.’

In it, we learn about another point of divergence between Beutler and scientists like Janeway and Medzhitov – apparently they even have different philosophies of science?

Seven minutes into his talk, Beutler explains how his own genetics method is different from the scientific method:

“The scientific method can be applied in all fields, in physics, chemistry and biology, to try to understand phenomenon and the approach is to make a hypothesis, do experiments, to exclude the hypotheses as rigourously as you can, then failing to reject them, build an inferential model of how the system works."

"In biology, we have a special approach, the genetic method, which is quite different and doesn’t depend on hypotheses at all, at least in the beginning. Geneticists instead make a phenotype, they track it down, they find its mutational cause and then they know at least one molecular component that’s required for the phenomenon to work. The great strength of this method is that it’s almost entirely unbiased."

“I’ve had people argue with me that we’re biased in our choice of screens and I would take issue with that. I would say that if you use a pure genetic approach, you’re letting the organism tell you how it works and don’t come with a preconceived notion and try to prove it, a trap which too many people fall into.”

But when is the ‘beginning’ for Beutler’s genetic approach? In his next slide, he shows us a picture of the C3H/HeJ mice, whose relative resistance to LPS was first noted in 1965 (Heppner and Weiss, J Bacteriol.). It was the final DNA sequencing of the LPS locus in this mouse strain that won Beutler the Nobel Prize in 2011. Are we to assume that the researchers who originally generated  and screened these mice were also operating in the absence of a scientific hypothesis? Apparently, Beutler is oblivious to the concept that someone had applied the ‘scientific method’ in order to generate and screen for these mutant mice in the first place. The original 1965 paper describes experimental observations and provides possible explanations in a fairly standard scientific fashion.

In September 2011, when he received the Shaw Prize, Beutler wrote: “In the early 1990s, at UT Southwestern, I first embraced a classical genetic approach to the study of innate immune responses. The genetic philosophy is one in which hypothesis is renounced in favour of a search for exceptions to the norm, induced by mutation. Using genetics to answer questions about mammalian immunity was an epiphany for me.”

By 10 minutes 20s into the talk, Beutler is describing what would become his Nobel Prize winning moment, when he identified TLR4 as the LPS receptor. He says TLR4 was, “at that time, known only for its structural similarity to Drosophila protein Toll.” Which is not exactly the case!!

At 15 minutes, he says that the two approaches that helped him for TLR4 were the Drosophila work on Toll and the work defining the IL-1 receptor. There is no mention of Janeway/Medzhitov or anybody else. However, he does thank S. Akira for the later knockouts from his lab affecting TLR4 partners, like MyD88, omitting to mention that Akira’s lab had also produced a TLR4 knockout mouse and shown it to be LPS sensitive at the same time as he was publishing his Science’98 paper.

Medzhitov’s style

You can even compare Beutler’s seminar style to his great rival, Ruslan Medzhitov. There is an NIH seminar online from 24/4/09: ‘Innate host defense’. Medzhitov is probably one of those that Beutler would criticise for their “preconceived notions” about how the organism works. He begins by presenting theories about how the innate immune system functions based on experimental evidence. He then explains how his own group’s research has progressed from this base. The seminar is much more focussed on ongoing experimental research results, and finishes with some pertinent questions about the nature of human allergy and allergen response.

Did Beutler deserve the Nobel for his discovery?

Which brings us back to Kevin’s comments on Beutler. For the Janeway/Medzhitov supporters: “This Nobel was awarded for the mechanisms triggering the activation of innate immunity and mediating the communication between innate and adaptive immunity. This was the paradigm suggested by Janeway in 1989 and validated by discoveries in the late 90's. Beutler just found a receptor that bound to LPS. Janeway and Medzhitov were the first to establish the link to adaptive immunity, and Medzhitov and others (not Beutler) followed his initial discovery by showing the direct links to T-cell activation, B-cell activation and establishing adaptive immune responses.”

However, Charles Janeway was no longer a candidate (he died in 2003). So, what about Medzhitov? “One could argue that Medzhitov shouldn't get it either, since he wasn't the principal investigator when his TLR paper was published. But his initial discovery, and his later work as an independent investigator firmly established the role of pattern recognition in the activation of adaptive immunity.”

Kevin’s conclusion: “Regardless of the arguments for or against Medzhitov, giving the reward to Beutler, especially in light of the efforts he took to discredit Janeway and his contribution is a bit of a slap in the face. When the history books are written about this era in immunology, I wonder if this Nobel will help Beutler in sweeping Janeway under the rug?”

Photo: Nobel Prize Foundation

Last Changes: 08.03.2012

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