This year’s Nobel Prize in Medicine or Physiology was awarded to three renowned scientists, one of whom is the American Bruce Beutler who was instrumental in clarifying the structure of the mammalian Toll-like 4 (TLR4) receptor. TLR4 plays a key role in the ability of the human immune system to recognise and fight off bacterial intruders. The discovery of TLR4 triggered an explosion of research on the immunological defence of infections and on chronic and acute inflammatory disorders. Prof. Dr. med. Marina Freudenberg and Dr. Dr. h.c. Chris Galanos from the Freiburg-based Max Planck Institute (MPI) of Immunobiology and Epigenetics have played a crucial role in the investigation of TLR4 since the 1960s, and they also co-authored the key publication that led to the award of the Nobel Prize to Beutler. Galanos retired around nine years ago but his wife Marina Freudenberg still heads up a small group of researchers at the MPI in Freiburg. The two researchers still work together on TLR4 and in the search for answers to questions such as: what role does TLR4 play in contact allergies and other disorders? Why do some infected people suffer a life-threatening septic shock, while others don’t? These are just two of the many questions the researchers hope to answer.
Galanos began his research work at the MPI in 1964. Galanos’ doctoral student Marina Freudenberg, who he later married, joined his group in 1974 and still heads up a small group of researchers. Galanos developed LPS extraction and purification methods and identified a small phosphoglycolipid (known as lipid A) as the carrier of the immune activating abilities of LPS. The two researchers showed that very small amounts of LPS had positive effects on the human immune system because it trains the immune system and increases resistance to tumours and bacterial infections. The researchers also discovered that external influences can lead to a considerable increase in a mammal’s sensitivity to LPS with the result that even small quantities of LPS can trigger an immunological overreaction and potentially also septic shock. “Back in the 1970s, we were able to show that an organism’s sensitivity to LPS increases if it has previously been infected with certain bacteria, fungi, viruses and other microorganisms,” said Freudenberg. “We now believe that patients who have acute or chronic infections of Gram-negative bacteria, often with no disease symptoms, tend to have a more extreme reaction to subsequent infections.”
In order to develop causal therapies for the treatment of massive hyper-inflammatory reactions occurring as a result of bacterial lipopolysaccharides, researchers need to understand why and how immune cells react to LPS. Since the 1970s, immunobiologists around the world have been investigating the molecular structures on the surface of immune cells that recognise LPS and trigger the release of endogenous mediators. Freudenberg and Galanos are now two of the best-known international researchers in the field. Amongst other things, they have shown that the activation of macrophages by LPS is the main reason for the occurrence of septic shock. “With infectious diseases caused by Gram-negative bacteria, the macrophages of the human immune system come into contact with bacterial LPS and start to release substances such as TNF alpha, interleukins and prostaglandins,” said Galanos. “The macrophages are the starting point of a chain reaction that causes the immune system to overreact and potentially also trigger a life-threatening septic shock.”
After Galanos and Freudenberg showed that macrophages mediated the toxic activities of LPS and endogenous mediators, they went on to look for a receptor on the surface of macrophages that would recognise LPS. They jointly organised a conference held in Freiburg in 1990 at which Prof. Dr. Bruce Beutler from the Howard Hughes Medical Institute in Dallas/USA was invited to speak. “The three of us discussed different strategies we could use to identify the receptors we were looking for and we also talked about ways of working together,” said Freudenberg. “We firmly believed that such a project required an experienced molecular biologist.” Back then, there were two mouse strains whose immune system was known not to react to LPS and the researchers assumed that these mouse strains had defects in the structure that recognised LPS. “Virtually everybody was concentrating on the C3H/HeJ mouse strain,” recalls Freudenberg going on to add “but Chris and I deduced from a range of previous experiments that the C57BL/10ScCr mouse strain would be a more promising target.”Beutler and his colleagues started to look for the LPS receptor and the American and Freiburg researchers met frequently over the eight years that followed to exchange their findings and experiences. Although Beutler, along with many others, was concentrating on the LPS-resistant C3H/HeJ mouse strain, all results obtained with this strain were double-checked using the genetic material of the C57BL/10ScCr mouse strain from Freiburg. It was not until the late 1990s that Beutler and his partners from Freiburg discovered a gene that had a point mutation in C3H/HeJ but which was completely missing in the C57BL/10ScCr strain. This particular gene turned out be the elusive LPS receptor, which is now known as Toll-like receptor 4 (TLR4). When TLR4 binds, signals are activated that cause inflammation and, in the case of excessive doses of LPS, septic shock. It is one of the most important molecules used in infectious disease research as well as research into other diseases caused by disorders of the immune system.The researchers published the results of their joint study in the renowned journal “Science” in 1998 and continued their cooperative project by introducing an intact TLR4 gene into the LPS-resistant mice. This provided them with evidence that the mice were once again able to react to LPS, which was the proof that they had actually discovered the molecule they were looking for. The 2011 Nobel Prize committee awarded the prize to Beutler on the basis of the Science paper and the groundbreaking role it played in the development of new methods for preventing and treating disease. After receiving the award, Beutler sent an email to Freudenberg and Galanos acknowledging their contribution to the discovery of TLR4 as LPS receptor.
In close cooperation with Dr. Christian Termeer from the Department of Dermatology at the Freiburg University Medical Centre, the couple also succeeded in coming up with evidence that TLR4 is not only activated by LPS, but also by the human body’s own products. These products can lead to sterile inflammatory reactions and come about as a result of damage or “stress”. Recent research has shown that these products have a decisive influence on the development of many human diseases. One such disease is contact allergies, skin reactions with symptoms such as rash or blisters that result from exposure to allergens or irritants (e.g., nickel ions). “In humans, nickel directly activates TLR4. We have been able to prove this finding by introducing the human TLR4 variant into mice.”
Galanos and Freudenberg have since continued their research on clinically relevant aspects of TLR4 and LPS. Galanos retired nine years ago, but still works as guest researcher at the University of Freiburg. The small group of eight researchers very much depends on collaboration with other individuals and groups, including contact allergy specialists such as Prof. Dr. Stefan Martin from the Department of Dermatology at the Freiburg University Medical Centre. Freudenberg and Galanos are also working with another clinical research group led by Dr. Bernd Hildenbrand from the Freiburg Tumour Biology Center to find a way to use laboratory-engineered LPS derivates as adjuvants in cell-based applications. In addition, the researchers are looking into the causes of the LPS hypersensitivity of certain organisms and ways of treating it. “We are still in close contact with Bruce Beutler and were very pleased to hear the news that he has been awarded the Nobel Prize,” said Freudenberg.
Further information:Prof. Dr. med. Marina A. FreudenbergMPI of ImmunobiologyStübeweg 5179108 FreiburgTel.: +49 (0)761/ 5108 404Fax: +49 (0)761/ 5108 403E-mail: freudenberg(at)ie-freiburg.mpg.de