My research has been focused on the innate immune system for the past 25 years. It grew out of my interest in the cell biology of macrophages. Our early work focused on the effects of bacterial lipopolysaccharide, LPS, on macrophage physiology. One of our discoveries was LPS-priming; the capacity of LPS to establish a state of hyper-responsiveness in macrophages. We had been searching for a long time for the LPS receptor, so working on the TLRs was a logical extension.

“...we felt it important to demonstrate that perturbations in the newly discovered innate immune mechanisms lead to disease in humans.”

Both Richard Ulevitch and I had been successful in analyzing the innate immune response to LPS, and the editors of Nature solicited the review. Richard had made a number of seminal discoveries, including the demonstration that CD14 and LPS-binding protein were required for LPS recognition and the identification of various components of the LPS-signaling pathway, to name a few. We had demonstrated that TLRs discriminate between different types of bugs; our initial observation was that TLR4 mediated recognition of gram-negative bacteria whereas TLR2 mediated recognition of gram-positive bacteria (Underhill DM, et al., "The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens," Nature 401: 811-5, 1999). Bacteria and other microorganisms are eaten by macrophages, a process called phagocytosis. We demonstrated that TLRs are recruited to the phagosome where they sample the content and determine the nature of the pathogen. This information permits a measured, controlled response. This is necessary to prevent inflammatory disease (Underhill DM, et al., "The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens," Nature 401: 811-5, 1999).

Once we wrote the review, everyone cited the review, not the primary paper. That’s typical. The primary paper was still cited 590 times, though, which is not too bad. A great many reviews are usually written in a rapidly growing field, and often important papers are cited secondarily.

We explored various TLR agonists and the TLR signaling pathway. I think that the observation that different TLRs interact with each other to extend their repertoire of recognition was important. There are a great many TLR agonists and a restricted number of TLRs. We showed in that TLR2 could pair with either TLR1 or TLR6 and that this heterodimerization dictated the specificity of the receptor complex (Ozinsky et al., "The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between Toll-like receptors," PNAS 97: 13766-71, 2000). This paper has been cited more than 600 times.

Another important discovery was the observation that TLR5 detects bacterial flagellin (Hayashi F, et al., "The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5," Nature 410: 1099-103, 2001).

Dr. Alan Aderem's most-cited paper with 972 cites to date: Aderem A and Ulevitch RJ, "Toll-like receptors in the induction of the innate immune response," Nature 406(6797): 782-7, 17 August 2000. 972 cites. Source: Essential Science Indicators.

It came out of left field. Till then all known TLR agonists were lipids or glycolipids, and a protein represented a different class of molecule. The paper also demonstrated how the innate immune system can phenotype bacteria. Purified TLR agonists are never seen in nature, it’s always a cocktail. Thus, if a bacterium activates TLR4 and TLR5, the cell is able to compute that it has been exposed to a gram-negative flagellated bug. That level of specificity in the innate immune system was also surprising.

Well, the field has clearly matured significantly as a result of the concerted efforts of a great many laboratories. As for our work, we have focused on the immunogenetics of the innate immune system in humans, the NOD-like proteins (which are in effect cytosolic TLRs), various aspects of the host response to flagellin, and a systems biology based analysis of the TLR pathway.

With respect to immunogenetics, we felt it important to demonstrate that perturbations in the newly discovered innate immune mechanisms lead to disease in humans. We demonstrated that mutations in TLR1, TLR2, TLR3, TLR4, TLR5, TLR9, and TIRAP, amongst others, cause susceptibility or resistance to various infectious diseases in humans. For example, a mutation in TLR5 makes you more susceptible to Legionnaire’s disease; a mutation in TLR2 makes you more susceptible to tuberculosis; a mutation in TLR4 makes you more resistant to leprosy; a mutation in TLR9 leads to rapid progression to full blown AIDS.

We have also focused on the NOD-like receptors, or NLRs, which are the intracellular counterparts of the TLRs. These detectors recognize components of intracellular bacteria and viruses. One NLR that we have been interested in is called Ipaf; it detects flagellin in the cytosol. So extracellular flagellin is sensed by TLR5 and cytosolic flagellin is detected by Ipaf; this dual detection system leads to tighter regulation of the inflammatory response flagellin.

Flagellin has remained an important interest of our laboratory. We have done a lot of structural work and also focused on the molecular mechanism by which a variety of bacteria elude this detection system. Flagellin promises to be a very important adjuvant and we have therefore become quite interested in its use in vaccination.

Our major focus is to understand complexity in the innate immune system. Most of the signaling pathways crosstalk with each other and are regulated at multiple levels. Many intracellular and intercellular feedback loops exist. For example, activation of TLR4 leads to the rapid production of TNF and a delayed production of interleukin-1; both of these cytokines stimulate the TLR pathway. And this is only the tip of the iceberg. I think that these questions can only be resolved using the tools of systems biology. That is, use wet bench techniques to make global measurements and perturbations of the system, use computational tools to analyze the data and predict and simulate the behavior of the system as a whole, and finally experimentally validate and extend the predictions. This is an interdisciplinary enterprise and requires teams of investigators working together. We have published one paper so far (Gilchrist M, et al., "Systems biology approaches identify ATF3 as a negative regulator of Toll-like receptor 4," Nature 441:173-8, 2006).

We are also very interested in vaccines and adjuvancy and participate in a number of large scale vaccine initiatives. We have continued to work on the immunogenetics, and the NLR programs which I mentioned previously. We have also focused quite a bit of attention on developing microfluidic devices to do single-cell analysis.

Not really. The discovery of the TLRs provided molecular insight into innate immune recognition. I had no doubt that it was going to open a floodgate of studies as investigators revisited more than 100 years of research into innate immunity.

I think we now understand many of the guiding principles. For that reason I think research in the field is going to slow down somewhat. Important unanswered questions include:

Commensals are symbiotic bacteria that live inside of us and are critical for life. Why do these bacteria not provoke a robust inflammatory response? A related question is the issue of locale. For example, there are bacteria that are normal commensals of the vagina but become pathogenic if they migrate to the uterus.

I think we’re going to find a number of endogenous activators of TLRs, and find that they have a role in inflammatory disease. There are already hints of this. We have shown that a point mutation in TLR5 is associated with systemic lupus erythematosis. We do not yet understand what this means but it would be interesting if an endogenous agonist of TLR5 had a role.

I also think that there will be an enormous focus on adjuvancy, particularly in the generation of cell-mediated vaccines. Work from a number of groups has demonstrated the promise of flagellin (TLR5) and CpG (TLR9).

It is very important to obtain structural information about TLRs and their agonists. I say agonists and not ligands since we don’t even know whether the agonists are bound directly. Personally, I think that structural studies by Ian Wilson’s group demonstrate this clearly, and put the question to rest, at least for TLR3. Structural information is also very important for the development of therapeutics that modulate the TLR pathway.

Once we decided to focus on complexity in the immune system we had to find a way to do it. This ultimately led to Lee Hood, Ruedi Aebersold (see also), and me co-founding the Institute for Systems Biology. It has been very challenging to establish a culture in which investigators from a large number of disciplines can interact creatively. Another challenge has been the development and implementation of technology and computational tools to enable the measurement and analysis of large data sets. The science of systems biology is still in its infancy and although we have overcome a number of hurdles many challenges lie ahead. It has also been critical to raise sufficient funds to feed the beast; that has been phenomenally time consuming and challenging.

I would first pinch myself to make certain that I am awake. Then I would do a comprehensive systems-level analysis of the immune response to HIV and try to understand precisely how the virus undermines the host response. With this information in hand I would develop a vaccine. You did say and ideal world with unlimited funding!

It is hard to say. There are things that are already within reach of the scientific community; these include the ability to define correlates of immunity to infection; a critical milestone in developing vaccines. We also have the ability to evaluate adjuvants and to develop better ones. We are also within reach of sequencing personal genomes. Ultimately, comprehensive systems level analysis should lead to predictive, preventive, and personalized medicine.

Alan Aderem, Ph.D.
Institute for Systems Biology
Seattle, WA, USA