 It's about now 37 years ago that I first came to the University of Chicago and I was sitting in this room just like all of you are now There were perhaps fewer of us. I never dreamed I'd be here dressing you all some generations of medical students later But I am grateful to the University of Chicago always will be just because it gave me the chance that it did And I've been trying to pay you back ever since I Want to talk about something that I think is just enormously important in Biology and medicine and that's the issue of how we recognize microbes. It's the old question of self non self discrimination and Where we studied the matter we applied the question to innate immunity the kind of immunity that you're born with as we all have The inheritability to recognize infection when it occurs but I'm going to be a bit historical and First tell you why I think the question is so very important and then I will Discuss the major landmarks in the evolution of our thinking about the question Of course infection has been known to physicians since antiquity and it's been known for its inflammatory and transmissible Character that was so even long before anyone had any inkling that there were such things as microbes at all Let alone that they were the cause of infectious disease and it's been a terrible source of attrition for our species In fact, if we look across the whole world even today or in this case, I think a few years ago this pie chart was drawn Infection kills about one quarter of all people on the planet and that actually is an enormous improvement because if we had gone back a couple hundred years and asked the same question It would almost certainly have been more than half of people born that would ultimately die of infection What you die of and how long you live Depends very much on when and where you live and I have some statistics that I think are really enlightening in this regard They came from a paper written by general on Casanova a review in which he simply looked at Actuarial statistics across the world and it may come as no surprise to you that in the United Kingdom in the year 2000 people lived on average quite long and full lives and median survival As you see here from the median survival line was somewhere close to 80 years of age in the same year in Mozambique things were very different and The median survival was something like 38 or 39 years of age really quite a shocking difference and in large part as you might imagine this would be attributable to infectious disease cause death the fact that there wasn't quite the same level of sanitation in Mozambique nor The use of antibiotics or immunization as there was in the United Kingdom in the same year More surprising still is that if you went back from the year 2000 mere 140 years to the city of Liverpool England in 1860 things were even much worse than in Mozambique Now this was the Victorian era in Britain the British Empire was at the peak of its power and Dominated the globe and yet the median survival of citizens in Liverpool was 10 years That was what you could expect when you were born Still more surprising maybe is the fact that if you went back in time just a little bit more to the city of Breslau in 1690 then things hadn't really changed very much Nor was the situation worse and neolithic times nor imperialistic times In fact people in the United Kingdom in England we could say we're living in the wild state in the year 1860 when it came to surviving the predation of microbes There's something else about infection that makes it very special as a source of mortality And that is that it tends to kill people at a very early age Really disproportionately so we're looking here at Infectious disease death and it all other causes of death in the developing world and we have to remember that all of the world was the developing world not very long ago and you can see that through the Maximum age of reproduction which we might artificially call 44 years of age If you add up the red columns, they're the dominant source of mortality and this tells us of course that this has been an enormous Selective pressure on our species throughout most of its history It's been such a selective pressure that ultimately if we go back even further and look at the whole history of multicellular Organisms that infection impaled the development of an elaborate immune system really two immune systems the innate and the adaptive immune system Many genes certainly have been co-opted from other functions to serve immunity in one way or another Or they've evolved de novo to do so and this means that ultimately there's a lot that can go wrong with immunity That many mutations can lead to immunodeficiency or autoimmunity The major causes of death in Victorian England though, they weren't really recognized in the way I'm portraying them in smallpox typhoid tuberculosis and cholera and Even before people became aware of microbes as a cause of infection There were empirical attempts to deal with infection as a problem I think all of you have probably seen this painting of Edward Jenner injecting James Phipps with some fluid from a blister On the hand of the milkmaid Sarah Nelms The idea then that was in popular law was that people who had had Cowpox couldn't develop smallpox and indeed this turned out to be quite an effective strategy in preventing smallpox More effective than variolation, which had a pretty high mortality associated with it Even in those days just as now people were suspicious of new technologies This is a painting also from those times from the anti-vaccine society There was a pervasive belief that this approach might lead to cows growing from appendages or from people's noses or mouths and We still have I would say the same type of resistance to immunization today But all of these approaches took place to that time in a kind of intellectual darkness Because no link had been made between infection and microbes. It's a second nature today That's even hard to imagine such a time and yet until there was such a link made There were simply vague ideas about how infection spread between individuals and that was the main substance of argumentation about infection in those days There were those who favored the model of my asthma and those who talked about contagion The my asthma model had to do with airborne spread of infection or some kind of flux that would affect people and on the other hand there were those who felt that no there had to be direct contact and The argument raged for a long time without anyone really realizing that it was completely missing the point It was not lost on people even from very ancient times that in many respects infections Resembled pitrefaction of organic material. So here you see a terrible looking lug wound in the upper panel And it isn't so unlike rotten meat or rotten vegetable material in its production of odors gases like hydrogen sulfide Or ammonia or captains and it was suspected that somehow when things putrified They developed a toxin of some kind and this came to be known as the putrid poison Obrecht von Holler and Francois Magendi began to systematically look for the putrid poison And they ground up meat that had been allowed to spoil or vegetables and injected animals with extracts of these Materials and as it would surprise none of us in fact that is very toxic There is something extremely toxic Associated with rotting organic material, but they could go no further at the time in characterizing just what that might be Others their successors did go quite a bit further as there were the rudiments of analytical chemistry and Ernst von Bergmann began purifying what he called sepsum Ted or Bill Walt also began purifying a putrid poison and Peter Panham who is very famous as an Epidemiologist for his studies of measles in the Faroe Islands Probably went the furthest of any of them and really did purify something even in the premicrobial age That was quite significant later on We have to remember that Peter Panham really straddled the microbial and premicrobial as and here He was writing about his experiences in 1874 when he did know about microbes, but he'd actually performed the work in 1856 when he didn't To be very brief about it He believed that the putrid poison as he called it was something that was heat stable That would differ from typical enzymes responsible for fermentation It was insoluble in alcohol, but soluble in water it wasn't a protein at all, although proteins could seemingly absorb it and 12 milligrams of his best preparations of this poison would be sufficient to kill a large dog Of course a huge leap was made with the identification of microbes as the causative agents of infectious disease And that we have to credit Pasteur and Koch for discovering And it was a student of Koch named Richard Pfeiffer who made the next step And he had the huge advantage of working with pure cultures of microbes rather than simply rotten material or pus As was the starting material for Peter Panham Richard Pfeiffer was a military surgeon who was billeted in Berlin He came to work with Koch and he was given a choice of working on tuberculosis or on cholera And he chose to study cholera And he was interested in a phenomenon that he observed Namely that he could heat and activate cholera vibrio Give them to a guinea pig and the guinea pig would sicken and die looking very much like an animal with a real infection Though he couldn't culture any live organisms from the predneal cavity afterward And so he coined the term endotoxin to discover what he saw He believed that a heat-stable toxin was associated with what he called the body substance of the bacteria and It was responsible for most of the pathologic effects of an infection We can look forward quite a few decades in a moment But I want to point out that while Richard Pfeiffer is a pretty obscure Microbiologist today by the end of his life. He had been nominated for the Nobel Prize in physiology or medicine 33 times Now why was that you why was he so celebrated? It was certainly the fact that hundreds of people died of endotoxin induced shock every day Which is the result of severe gram negative bacterial infection And he had isolated a principle that seemed to mediate most of these effects and named it endotoxin We know that endotoxic shock is a very severe systemic form of Inflammation and if you think about it all forms of inflammation were Ideologically obscure at that time no one knew of a spark that lit inflammation and here potentially was a single molecule that could do so Fulfur was a bit confused as to whether it was gram negative only or some gram-positive Bacteria that made endotoxin and today we know that endotoxin is strictly a product of gram negative bacteria It's an amphiphilic molecule that we've called lipo polysaccharide And it's the major structural component of the outer leaflet of the outer membrane of gram negative bacteria Which of course have two membranes Chemists looked at the molecule and they found that it was the lipid a moiety of endotoxin that was really toxic responsible for father's effect and Eventually lipid air molecules were synthesized entirely artificially by the 1980s and were shown to reproduce all of what LPS can do I'd like you to keep it in mind that some partial structures of LPS are In fact species specific in their action and here I refer to a molecule called lipid for a Which has four acyl chains rather than the six of the matured lipid a of an E. Coli or Salmonella Lipid for is agonistic when applied to mouse cells But a pure antagonist in human cells and that comes into the story later, so you should remember it in In my own career in medical school, of course, I became aware of endotoxin I had heard of it before but I began to see people with endotoxic shock and definitely took an interest in it and thought This was an important question as to how endotoxin worked But I never actually began working with endotoxin until quite by chance after my medical training I went to the Rockefeller University and There in the laboratory of Anthony Surami. There was an interest in the phenomenon of wasting in chronic disease This might seem to have nothing at all to do with endotoxic shock But it turned out to have quite a lot to do with it in a way The model that was set forth in those days was that it must be that products of trepenisomes Would act on cells of the immune system to stimulate the release of a hormone Which we called cackectin that would act on energy storage tissues of the host to suppress the uptake of fats from the plasma and prevent de novo fat synthesis and the reason this was Postulated was that in the cow I showed you a moment ago There might be only a few grams of the pathogen and there was no way to invoke a competitive mechanism To explain the wasting that was observed It was also suggested that maybe certain tumors would activate production of the same protein and furthermore that endotoxin might do so further that there might be Exogenous sources or rather we could say endogenous sources of the of the Protein that it might be made autonomously by tumors at times or by other tissues. I Looked at this model and as time went on I found that by far endotoxin would induce more of this activity Which we called cackectin than anything else in fact trepenisomes seemed to be a very feeble source of it If they could induce any at all and I set up an assay system in which Macrophages of the raw two six four point seven line were exposed to LPS Then after a period of time I harvested medium from the cells I applied it to 3t 3l 1 Preadipocytes and I would measure the suppression of lipoprotein lipase an enzyme needed for fat uptake This was suppressed almost to zero and was the perfect endpoint to follow in isolating this substance. I Did isolate the protein through? The fractionation procedure shown here and the main point I'd like to make is I found it was quite abundant it amounted to one or two percent of the Secretory product of macrophages within the first hour or so after they were exposed to LPS And this is how it appeared on a gel in those days It was a seven point five Kd band that I saw and at times at that time I believed it was a unique protein However, as it developed it turned out that mouse cacectin was strongly homologous to human tumor necrosis factor Factor known to be also induced by endotoxin from human cells that was measured by Looking at the ability of the protein to cause death of tumor cells in vitro or hemorrhagic necrosis of tumors in living animals This was extremely exciting because here I realized that one factor induced strongly by LPS Had these very different biological activities on the one hand it would act on Atical sites in the way I described on the other hand it would act on tumor cells And I began to wonder if this factor might be the central endogenous mediator of endotoxic shock I thought perhaps it mediates all of the effects of LPS including the lethal effect When I had sufficient amounts of the material to give to mice I found that yes TNF could kill mice about 20 micrograms was all it took and the mouse would die looking very much like an Endotoxin poisoned animal although in fact no endotoxin had been given But that wasn't really convincing enough I thought it would be necessary to see whether Anti-TNF passive immunization against TNF could block endotoxicity and it turned out that that too was the case There was a significant shift in the dose lethality curve if mice were immunized against TNF before being challenged with doses of LPS as time went on receptors for TNF were identified by David Wallach and David Goodell and These receptors were found to be widespread on many cell types and Wherever TNF was applied to cells it would cause inflammatory effects For example in fiber blasts it would cause the secretion of Collagenase and PGE2 and neutrophils it would cause adhesion to endothelial surfaces And it would modify the endothelial surface also to make it procoagulant and we could go through the entire list But suffice it to say this really was one of the central mediators of endotoxin effect, and it was highly inflammatory Probably for that reason I thought it would be a good thing to neutralize TNF for clinical effect And I didn't have confidence at the time that antibodies would do the job long-term at least in chronic inflammatory diseases Because I thought they would be immunogenic and that didn't really turn out to be as great a problem as I'd imagined But I decided to make a TNF inhibitor by fusing the ectodomain of the TNF receptors to IG heavy chains And indeed these molecules were stable non immunogenic for long periods of time in animals and Extremely specific and avid in their ability to bind and neutralize TNF So they did eventually seek clinical use and are still used today to treat rheumatoid arthritis and other inflammatory diseases after moving to Dallas in 1986 I began to study the regulation of TNF biosynthesis, but all the while I was aware I was really looking at endotoxin signaling as that was the inducer I was using to activate the TNF gene And I began to wonder more and more about the nature of the LPS receptor In fact, I became quite obsessed by this question because when I thought about it It wasn't clear at all how microbes were recognized within the first minutes to hours after inoculation And this was to me the greatest question that was outstanding in immunology It was really the central question of self-non-self discrimination Where LPS was concerned something was known about the problem It was shown by Yulevich and Wright in 1990 that a surface molecule called CD14 which had leucine-rich repeat domains was necessary for endotoxin responses But this molecule was anchored to the cell surface by a GPI tether It had no cytoplasmic domain and no one could understand how the signal got across the membrane We and others postulated that there must be a co-receptor of some kind that would transduce that signal But this was elusive and nobody knew the nature of the signal elicited by this co-receptor Where the TNF gene was known at least one thing had to happen in order to activate transcription Nuclear translocation of NF Kappa B had to occur There were four NF Kappa B binding motifs in the TNF promoter region and without NF Kappa B One couldn't get expression of TNF mRNA Even when the mRNA was expressed we knew that it was locked in an untranslatable form and today We would say probably it's sequestered in P bodies or stress granules and a second signal from the receptor Whatever it was was needed to activate translation of TNF mRNA and then allow production and processing of the protein the core question all came to revolve around the receptor and what it might be and We made many attempts to try to find the receptor using Conventional approaches I would say although all along there was a genetic route open to us In 1965 it had been noticed that the C3H HEJ substrain of C3H mice was refractory to LPS and these animals couldn't be killed by any dose of LPS that was given to them It appeared that there was a proximal defect because they didn't make a cytokine response There are macrophages ex vivo wouldn't respond to LPS either and furthermore The mutation that they seem to have was quite specific for LPS It didn't impair the response to nucleic acids for gel on or lipoproteins For example other components of microbes that were known to be inflammatory Even before the nature of the LPS receptor was known these mice were used to demonstrate that it was cells of hematopoietic origin That transmitted the lethal effect of LPS This was shown by reciprocal bone marrow transplantation experiments In which the donor type of the marrow would always predict what happened to the chimera Resistant marrow would give an irradiated rescued mouse a resistant phenotype and Susceptible marrow would cause susceptibility to LPS It had been known from the 1950s that LPS would help to drive an adaptive immune response In effect LPS was an adjuvant and this effect of LPS also was absent in C3 HHEJ mice So it was a very broad effect that the mutation seemed to have Paradoxically although HHEJ mice were resistant to LPS They were hyper susceptible to real gram negative infections and that said that if the mouse didn't recognize LPS It remained unaware of a gram negative infection until it was too late And it would be overwhelmed by the growth of the microbe before it could mount an immune response Only one or two salmonella typhimorium organisms would kill an HHEJ mouse where about 10,000 Was the MLD for C3 HHEN mice By the late 70s an entirely unrelated strain of mouse called C57 block 10 SCCR Was also found to be refractory to LPS and by breeding these two strains together It was demonstrated that Nalilic defect existed the same locus had been altered in both those strains Formal mapping of the mutation which became fixed in the HHEJ population sometime in the early 60s Was accomplished by Watson and Riblett who showed that the mutation was between two visible phenotypic markers Not one polysyndactyly on mouse chromosome 4, but they're the problem lay Because there were not sufficient molecular markers to do positional cloning in those days We thought we might shortcut the problem and we spent several years actually Cross-inunizing C3H HHEJ and HHEN mice hoping to find an antibody that would allow us to identify the protein that was missing in the HHEJ strain We also Attempted to rescue the phenotype with CDNA cloning taking RNA from sensitive C3H HHEN mice making libraries and transfecting C3H HHEJ macrophages and That was also an unsuccessful approach We looked proteomically to see if we could find a membrane protein that differed between the two strains and When all of these approaches had failed we turned to the genetic approach as a last resort Noting that by that time there were about 300 markers that would be informative in the mouse between various strains genome-wide My colleagues in undertaking this project were Betsy Layton Foremost Alexander Poltorak who really was a hero through the entire project Christoph von Uefel and Irina Smirnova We've started by crossing C3H HHEJ to the SWR strain and at that time We had only 11 informative markers covering mouse chromosome 4 for that particular cross Those are the ones shown in white circles The location of muc1 and polysyndactyly were not known with certainty and in fact they still aren't known today I guess the question is not of particular interest to anyone at the moment But we looked up and down the mouse chromosome and we set all of these 11 markers in order with respect to one another on 493 MayoC's the objective here of course is to know the location of the mutation to a point between closely spaced markers and then try to find the gene content of the area We were quite certain that the mutation was between this cluster of four markers and this single distal marker on chromosome 4 but those were still an enormous difference apart distance apart from each other Probably more than 16 million base pairs and this was much too large an interval to tackle so we went further up to 2093 MayoC's and finally arrived at a critical region that we could never shrink It was defined by markers B and 83.3, which we'd isolated DeNovo from Clones in the region and we thought that this was about 2.6 million base pairs of DNA In fact, it turned out probably to be about 5.8 million base pairs now that we know the sequence of the mouse genome Nonetheless, we decided we'd invested too much and we were going to go ahead and clone all of that DNA and try to Find whatever genes were present there We had to build an enormous back contact spanning this region And I looked at other positional cloning projects where there were only two or three backs And I was quite envious about them because this was certainly a long hard slog Of course, it was all terra incognita and when we began there were no genes that could be said to be in this interval And we spent a long time looking for genes having assembled the contact and for the most part For a period of about three years We did this chiefly by fragmenting the back sequencing them bi-directionally and blasting Against EST databases in order to find matches This went on from about 1995 through the summer of 1998 and in all that time we found only a collection of pseudo genes Which was most disappointing only of course we didn't know they were pseudo genes when we saw them about each and every one of These we could make up a very nice story about why it ought to be the LPS locusts but eventually we would find first of all that they probably weren't expressed one after another and we found that That there was no mutational difference to distinguish the sensitive and resistant strains Everything you see in yellow was sequenced essentially to completion in those things in pink to about 90 percent We were working from the center outward which was logical because the center was where the mutation ought to be and By the end of the summer of 98 I was getting extremely anxious because we had covered 90 percent of the contact And we had not found the mutation and I felt that either we Had to find the mutation soon or we were simply in the wrong area having made some kind of a mapping error Which was almost unthinkable Nonetheless that was the reality looking at us one evening in September of 1998 I was looking at the day's blast results in my study at about 9 30 at night And I saw a very strong hit with a new EST in the back that we'd begun to examine at that time called i-17 And within a few minutes I saw another very strong hit and these were much better matches than the pseudo genes had been before And I thought that this was almost certainly an authentic gene And as I looked at the nature of the gene and at the last list I got more and more excited and soon I was hyperventilating and I Called Alexander Polterac on the phone and breathlessly told him about this new gene in the contigue that was called TLR4 What was TLR4? TLR4 is depicted here Of course, we didn't know it looked exactly like that in those days But we did know that it had leucine rich repeats in its acto domain and that made a nice story because I told you CD14 had leucine rich repeats too and it made sense that by Proximity or by transfer the LPS might go just as I showed you from the co-receptor to the membrane spanning receptor and illicit a signal Also exciting was the fact that the cytoplasmic domain of TLR4 was quite similar to that of the aisle one receptor Which was known to activate NFKappa B and Which also was known for its inflammatory potential and this made a good story that this might be what we were after In fact, it had been shown at that time that artificial ligation of TLR4 as Illustrated by the work of Medjatov and Janeway would activate NFKappa B Though there was no inkling about what the ligand for the receptor might be There was another piece of evidence that had me excited as well a few years earlier Jules Hoffman whom I hadn't met to that point had shown that in fruit flies the namesake of the family told Which has presumably a similar structure was important in fly defense against fungal infection Now told never comes in contact with any component of fungi Nonetheless, the story seemed good to me that here a mutation in one member of the mammalian family Might lead to a situation where mice would be overgrown with gram-negative bacteria Just as flies could be overgrown by fungi If they lack toll all of this was hand-waving and it would mean nothing unless we found a mutation But we were confident enough to drop everything and look for a mutation and in due course we found that in the Third exon of the gene. There was a single amino acid transversion a single nucleotide transversion that led to an amino acid substitution of Conserved proline for a histidine which would be within the cytoplasmic domain of the receptor Of course people might have said and would have said at the time This is just happenstance these strains have been separated for 35 years And it was incumbent upon us to look at the other strain as well And they only found the TL4 wasn't expressed at all on northern blot as you can see here quite clearly and in the Formus of time we found that there was a deletion of 74k and this cut out all three axons of the gene So we had one earlier that was obviously destructive and the other that very likely was and that was enough for us to Conclude that this indeed was a part of the LPS receptor complex But the question remained and people debated it for some time afterward is this really the LPS receptor is there really direct contact between LPS and this molecule In the case of the free-flow remember toll. There is no contact between any Component of the fungus and the toll receptor instead the fungus will activate a proteolytic Cascade in the hemolymph that leads to the cleavage of an endogenous molecule called spetzla that acts as the ligand for toll To address the question of whether there was contact we made use of the fact that some partial structures of lipid a are Agonistic in humans in the mouse, but antagonistic in the human We set up a system in which he jay macrophages were Transpected with either the mouse or the human version of toll like receptor for remember these cells can't signal ordinarily the presence of LPS because of the mutation they have in the homozygous form and we asked them Will this one molecular difference Recreate the specificity that we see for responses That differ between human and mouse to the lipid a and lipid for a and what we found In fact was that the human toll like receptor for which support signaling by lipid a but not lipid for a where the mouse TLR for which support signaling by both Because that was the only molecular difference and it was responsible for determining whether there were four or six acyl chains We reasoned that this molecule must be in quite close proximity with the log and and in effect It was probably in contact with it These are the actual data and the key thing to look at is the response to lipid for a the mouse Responds and the human does not It wasn't known to us at the time But there was still another subunit in the receptor complex and that was discovered the following year by kensuki Miyaki This is the molecule called MD2, which is a small molecule with a deep hydrophobic pocket and it's a part of the complex and LPS or lipid a inserts into the pocket of MD2 and ultimately has contact with both the leucine rich repeats and with MD2 You can actually see a bit better how this looks from a ribbon diagram And maybe as it rotates around you can see that one of the acyl chains is directly in proximity With the backbone of TLR for and this is where all of the endotoxin response begins all of the complexity of endotoxic shock begins with this one molecule with this one complex and It's worth noting that in this complex in in the mouse There are probably only about 100 nanograms of the protein complex itself that mediates the lethal effect of lipopolysaccharide So there's enormous signal amplification after activation occurs One exciting thing that was obvious to us from the start was the fact that there are numerous tall like receptors We know there are 10 in humans and 12 in mice and it made sense to hypothesize that each one would recognize a different inflammatory molecule of microbial origin and Shizuo Kira in large part Established that that was true by knocking them out one after another Now that we can see the structures of these molecules We know that each of them engages a different molecule TLR5 is a receptor for flagellum double-stranded RNA signals by way of TLR3 Lipopeptides through the TLR2 complexes and so forth Collectively they detect almost any microbe we might ever encounter Another point that struck us right in the beginning was the very broad use of this molecule as a sensing mechanism across the tree of life I told you the story in the fly and in mammals. It is a fact that in humans meningococcal sepsis susceptibility seems to be influenced by mutations in TLR4 But what really surprised me in the beginning was that even plants rely on similar molecules to detect infection these are all Cytoplasmic molecules with a two-domain structure similar to the cytoplasmic domain of tall-like receptors Mutating any one of them creates a susceptibility state in plants mutating this one makes the flax plant susceptible to a fungal rust called merensualini But it wasn't clear that plants had surface receptors similar to TLRs at least not cleared us Until much later than it should have been This is a lady named Pamela Ronald who discovered that in fact there are surface receptors similar to tall-like receptors in Plants and she's not really because she's an absolutely outstanding scientist She did her work before the fly people or the mammalian people and she's also my second cousin as we realized after the fight She also took a positional cloning approach She's tailed with susceptible and resistant rice plants susceptible or resistant to the bacterial blight Xanthomonas or Aussie so you can see that these plants die when painted with the bacterium Whereas these survive and she positionally cloned the difference she found that susceptibility was Determined by the absence of a molecule called XA21 a leucine which repeats surface molecule Which had a non-ID kinase domain rather than a tear domain for signaling She went further than that and found the punitive ligand for XA21, which she called a X21 This is a sulfonated peptide that's processed and secreted by the plant through a series of Secretory apparatus proteins that are all listed here. These were all tracked down genetically by Pam Ronald So we have the story in the plant in vertebrates and also in insects And you can see that it seems to be a very similar picture overall From this point we wanted to know how it told like receptors signal And I'll talk about this more this afternoon about the whole subject of forward genetics and how we've Dedicated ourselves to finding things by an unbiased approach But the strategy we took was simply to make mutations at random in the mouse with a mutagen Enu track them all down as methods were getting to be much better and faster than they had been before and then ultimately Understand what was required for signaling this has the advantage of course There's no hypothesis and one can understand a complex system by making it fail repeatedly And we thought that this would be helpful to us in in our cost I'd argue too for all of you in medicine that Seems to me having done this for a while This is why medicine is so effective as a tool for studying Biologies because physicians look at failing systems and it's incumbent upon them to understand them And this is why we've come to know so much about how mammals operate Forward genetics of course produces a list of parts and One then has to set them together using various experiments, and that's what we do in biology all the time In practice what we do is to mutagenize mice with Enu We then breed them in breed them through two generations and we then screen G3 animals to look for phenovariants And this way of course you can see a lot of very peculiar looking mice and these animals each have stories to tell But therefore another time We focus mainly on immune phenotypes and those include phenotypes related to TLR signaling the antibody response control of viral infection and Also the prevention of excessive inflammation a Lot of details about TLR signaling are known from forward genetic Reverse genetic and biochemical studies, and I'll tell you just quite quickly how it all works We know first the system has to be set up And there are Chopper-owned proteins that escort or like receptors to where they need to be among these are prop 4 and GP 96 which are needed for some of the surface TLRs like TLRs for and the TLR to Complexes to get where they need to be At the same time unk 93 be a multi-span ER protein is needed for the nucleic acid Sensing TLRs to get to the endosome, and it's there in the acidified the acidified environment the endosome that they signal That's not enough of course There's a protein called slack 15a4 identified by a mutation called feeble That also is needed to condition the endosome for it to signal properly and feeble can only get where it needs to be If it's escorted there by another complex of proteins the AP3 complex And all of these were revealed by mutations in our in our lab We know of co-receptors like CD14 that are needed for signaling CD36 is another contributor to signaling by the TLR to complexes Signaling involves the recruitment of adapter proteins of which there are four and all But the one that's used by most TLRs except TLR 3 is called mighty 88 the others are include Mal or Tyrapp as it's alternately called and where TLR 4 signaling was concerned There is recruitment of Iraq 4 by a death domain interaction with mighty 88 That phosphorylates and activates Iraq one as well as rip and trough six a ubiquitin ligase is then brought to the complex It ubiquitinates itself as well as tack one and then later on also the icapa be kinase complex and tab two and tab three are proteins that hold the whole complex together as a kind of molecular glue The icapa be kinase complex activates other molecules including NF capa B by causing the degradation of icapa B and The transcription factors NF capa B AP1 and CREB orchestrate the inflammatory response by activating hundreds of genes including the TNF gene Which we take as the endpoint in this screen TNF has to be processed and mutations Associated with that were picked up one of them a mutation in Adam 17 Which clues the propeptide of TNF another in a worldwide like factor that gets Adam 17 where it needs to be Signaling can occur also by this alternative pathway involving trip another adapter protein That activates a separate trough family member and then different kinase is leading to the activation of IRF 3 Which triggers the interferon beta gene and that happens both from TLR 3 and TLR 4 TLR 4 can signal unconventionally to activate IRF 7 which activates the interferon alpha gene and IRF 7 and IRF 3 together Stimulate the interferon response the interferon signal through a stat dependent mechanism and they in turn activate hundreds of other genes This can be shown a little more formally with all of our mutations supporting the signaling pathway as I've shown you in Boxes and sometimes we had many alleles of these mutations I can make it a little more plain by getting rid of all those but what I really want to show you is that the structure of biologists have been at work, too and They have solved the structures of most of the components of these signaling pathways This was not our work, but it's very satisfying to look at and while some of the interactions are still fanciful, I like to think that we're approaching something on the order of the watch that I showed you earlier a last way to think about the whole scheme is as follows Everything I've shown you about the TLRs is in this gray area here And I've emphasized that tall like receptors in mammals are similar to tall in the fly and tall signals Ultimately to activate an NF Kappa B like analog which induces antimicrobial peptide synthesis It was later found that in the fruit fly. There's a second immune pathway called the IMD pathway and It also activates NF Kappa B a different NF Kappa B called relish and induces a different set Vanity microbial peptides this pathway detects gram-negative organisms in the fly this one gram positives and fungi The remarkable thing is that IMD signaling is for all the world very similar to TNF signaling You have a fad and trad connection you have a cast-base a dread connection the NF Kappa B's are there The tab molecules are there. So this really is the fly TNF signaling pathway What you can say is that in insects there are two quite separate sensing pathways in mammals There's really only one but it's hooked together With the other by TNF and if a tall like receptor signals everything will proceed from end to end in this pathway Because TNF is there and it was just rather lucky that I started to work on something that turned out to be TNF Or I would never have completed this story as I did Now we know the other signaling pathways now not discuss them at all for want of time But we know about the Riga like hella cases that activate similar transcription factors and detect cytoplasmic nucleic acids We know of the not like receptor family and in a way This is a facsimile of the TLR signaling pathways as well It's main function is to process and activate Interleukin one which signals by a tear domain mechanism as I mentioned But the TLRs are perhaps the most universal of all these pathways and animals don't survive very long if they don't have TLR signaling What does all this do for us? Well We might imagine that finally we can mitigate some inflammatory Diseases that seem to be sterile and not initiated by microbes at all We might also Be able to diagnose and treat certain immune deficiency diseases that are presently obscure We could imagine that we could design better vaccines than ever before with lower toxicity than before Molecular adjuvants could be used rather than these crude mixtures of things like alum that are mechanistically obscure The greatest question is what are the internal logans the endogenous logans I told you the microbial story, but what activates TNF production to access in a disease like rheumatoid arthritis Or in Crohn's disease or psoriasis In fact, we're beginning to have some understanding of those things The disease that may be the best understood at the moment would be systemic lupus erythematosis And in just the last one or two minutes, I'll close with our current understanding there Based on a lot of things that I haven't had time to talk about We believe that lupus involves first of all some inappropriate means of lysis of cells not clean apoptosis as should normally occur and it involves the release of ribonucleoproteins and Also deoxyribonucleoprotein complexes We all have B cells in the periphery that have specificity for such molecules they're not all eliminated centrally and It would be normal that these complexes would eventually find their way to B cells be internalized and then in the Acidified compartment of the endosome. They're likely to encounter the toll-like receptors Which in this case drive proliferation of B cell clones and where you had only a few B cells with pathologic Specificities you begin to have many of them and some of them will differentiate and become plasma cells They'll then make and secrete anti DNA and anti RNA protein complexes And these are likely then to cause Immune complex formation in the periphery and complement fixation They'll cause the manifestations of the disease and they'll also feed back to cause even further amplification of the pathologic clones You can see them that there would be a way to interrupt this cycle and at least a mice blocking TLR signaling has a rather pronounced effect on The course of lupus as it's modeled in that species It's too early to say whether it will work in humans as well But I like to think that we have found something that's important to many sterile inflammatory diseases I'll just close by showing you my current group in Dallas I just moved back to Dallas from Scripps where I was for 11 years and these people are capable of finding Mutations much faster than ever before That's what I'll describe This afternoon The fact is we've improved about a thousand fold in the speed at which we can find mutations And we're limited mainly by how fast we can generate phenotype. So I think the future is quite bright In terms of assembling all of the components of whatever machine you choose to study and with that all thank you very much