 here, so that's all that's going to happen. All right, Dr. Warner's here so we can start. I just want you to know I have to leave early. That's okay. Very early, like right away. Well, then you leave right away. No, nothing against that. Hey, that's okay. All right, so I think we'll get started here. And I just want to say that I think I can speak on behalf of all the residents, fellows, and faculty that have worked with Dr. Chris Conraddy, that we're very glad he came here to the Moran to join our residency program. He came to us from the MD-PhD program at the Dean McGee Eye Center in Oklahoma, and he's been the VA intern for the last three to four months. In his role there, Dr. Conraddy has been incredibly efficient coming in after his morning runs at 6.30 to churn through the paperwork, and even being so ahead that he can see retina clinic patients in addition to his regular clinic load. He apparently was also a model of efficiency during his PhD years, turning out 10 first-author papers in three short years. And we really look forward to hearing more about that PhD work. But before we hear from Dr. Conraddy, I just want to share a little story about how I found out that his nickname was Kamikaze. As part of the resident retreat, Renee, Chris, and I went on a little mountain biking trip to the Flying Dog Jeremy Ranch. There's a particularly steep downhill switchback, and Dr. Conraddy cut it a little bit too close and hit his front brake a little too hard. He started a head-first dive over the handlebars. I'm behind him envisioning really bad consequences for him and for me who took him down this trail. However, mid-air, Dr. Conraddy managed to convert this head-first over the handlebars momentum into a flying hurdle of his handlebars and land on his feet in a run, five feet down the trail, and managed to avoid the aspen on the other side. I would say that if reaction times during mountain biking crashes have anything to do with surgical skill, Dr. Conraddy will turn out to be a very excellent cataract surgeon. With that, we look forward to hearing about his PhD work. Thanks, Dr. Wong. Like Dr. Wong said, I will be talking about some of my PhD work. I've entitled this talk, The Great Recognition of HSV-1, a view from within the cornea. We'll talk much deeper about this topic, probably to the point that you guys are sick of hearing about innate immunity, but just to kind of give you a good context of that, we'll go through it. Then I first got my interest in the scientific world from actually kind of a different background. I was actually first introduced to the sciences in the botanical world, and I was fascinated with science from actually the botanical side. For those of you that know flowers or don't know flowers, this is a lily of the Nile, and I've transposed some different colors on top of that. I haven't genetically modified the flowers, which is what Eileen was thinking I'd done to this flower, but no, I've not genetically modified it. First, let me tell you to every story, there's the good guy and the bad guy. I want to introduce you to a topic that we all interact with almost on a daily basis is herpes simplex virus type 1. It's a highly prevalent DNA virus. It's neurotropic. 60 to 90% of the population is seropositive for the actual virus, meaning that they've been infected at some point and likely harbor the virus, whether they actually show symptomatic signs of that is very, very broad spectrum, and we'll talk a little bit about that as we go. The reason that we were interested in this topic obviously from the Dean McGee, I Institute perspective was that it's the leading cause of corneal blindness to an infectious etiology in the developed world. So basically we see it quite a bit and that was the reason that we became interested in it. And just a little foreshadowing to the rest of the talk that I'll give is that herpes simplex is shown to activate tolite receptor 2, 3, 7 and 9 and several other innate sensors and we'll go in excruciating detail over this topic. But that's just a little foreshadowing. Then the interesting thing that you all know about herpes simplex is that it has a wide array of symptoms, clinical symptoms. And so we have patients that come in that don't have any symptoms at all. Then in the context of HSV2, a very close relative of HSV1, you can actually get females that asymptomatically shed HSV2 into the vaginal vault, basically spreading the virus without even having symptoms of the virus itself. Then that can progress or not necessarily progress. But in other cases, you can get kind of a nuisance cold sore. Then as we progress in increasing morbidity and mortality, we can get corneal infections where you get the kind of pathenumonic dendritic staining of the cornea and that's what we'll be focusing on today. And then a little bit of my work touched on herpes encephalitis where you actually can get mortality and actually in 70% of those that survive, you'll get long-term neurological deficits. And so it's a pretty broad spectrum when it comes to clinical symptomology. And then to introduce to you the good guys, and this will be something that we focus most of this talk on, is the immune system. So the immune system comes in two flavors to dust off all those cobwebs from immune classes in medical school or in graduate school. There's two flavors of immunity. The first, the innate nonspecific arm that's driven by NK cells, macrophages, and actual host cells themselves. And it's based on these conserved pathogen-associated molecular patterns where you have a pathogen that has a conserved sequence and then these innate sensors respond to those conserved sequences. Then that's in contrast to the adaptive or more specific arm of the immune system that's driven by T and B cells. And this is a very, very basic form of immunology, but just to kind of give you a basic grasp of it for those of you that haven't really thought about immune system in a while. But we're gonna focus on this first half, or the innate immune system that's driven by this, by toll-like receptors, DNA and RNA sensors, and we'll go in more detail as we go. So what exactly are innate sensors? So in the most basic form, think of a watchtower on a castle. So they're literally surveying the surrounding tissue inside the cell itself, depending on the sensor itself, and so they're trying to activate the earliest immune response to invading pathogens. And so that's driven by toll-like receptors, DNA sensors, RNA sensors, so there's a whole broad spectrum of innate sensors. And as over the last probably 20 years, toll-like receptors were first hypothesized in the late 1980s, then identified actually in the mid-1990s. However, in the last three years, three or four years we'll say, DNA and RNA sensors have become, I guess, a center part of the innate immune system. And then they, like I said, they've activated the innate immune system, and they do that by inducing the production of chemokines, so basically molecules that attract infiltrating leukocytes to the site of infection. They also drive a pro-inflammatory state with pro-inflammatory cytokines, and they up-regulate cell surface receptors, and probably the key that we'll talk about today is they drive interferon production. And from the context of toll-like receptors, I need you to kind of focus on this, and I'll boil down the innate immune system to three points I want you to hold on to, and this is one of them, but we'll rehash that here in a second. But all toll-like receptors signal through one of two adapter proteins, either a Mighty 88 or TRF. So all signaling through the 12 or 13 toll-like receptors, depending on who you're talking to, signal through one of these two adapter proteins. And then just to kind of overwhelm you with actual innate immunity, so this is showing just RNA and DNA sensors and the pathways that they drive doesn't show the 12 or 13 toll-like receptors that have been identified as well. But you can see that it's a very complex process, basically to lead to the production of the interferon and driving interferon products. And so to kind of boil that down, to kind of, I guess, dumb that down to just HSV-related sensors, and so in the literature, you can dig through the literature and see that there are three toll-like receptors that actually drive innate immunity in some context to HSV, and that's toll-like receptor two, three, and nine. And then there are three DNA sensors that have been identified, DAI, DDX-41, IFI-16, and then there's a controversial role of an RNA sensor, or maybe it's actually two sensors, RIGI, MDA-5. That still shows that this is a pretty complex process, and what exactly is happening is hard to identify in a lot of tissues due to the complexity of the innate immune system. But just to boil that down a little bit further, so if we want to look at basically the most basic innate immune response, all innate immunity basically drives an NF-Kappa-B signaling cascade to drive pro-inflammatory cytokine production to drive a pro-inflammatory state, and then from the antiviral perspective, you have IRF, so interferon regulatory factor, protein is phosphorylated, dimerizes, nuclear translocates to drive interferon production, the most downstream signaling portions of these innate sensors. And so we'll be looking at a lot of those signaling cascades, but we'll talk more about them, and I'll continue to rehash that as we go. Then, at least from an antiviral state, interferon is then released once the innate sensor is activated and activates interferon production. Interferon is released from the cell basically binds in an autocrine and paracrine manner, so to the cell itself and then to the surrounding cells to this heterodimeric interferon receptor, that then activates what you all are probably most familiar with, the jack-stat signaling cascade. We're not going to talk much about that today, but basically to say that the jack-stat signaling cascade is important in driving basically interferon regulatory proteins to drive antiviral immunity to viruses. Then interferons themselves, so what exactly are interferons? They're antiviral cytokines, they're produced following these innate sensor activation, and they activate a couple of proteins that are important in antiviral immunity, so PKR, RNA cell, they induce apoptosis, increase MHC Class 1 presentation, and just to dust off the cobwebs of immunology, that's important in cytotoxic CD8 T-cell surveillance of whether a cell needs to be killed because it's infected or whether it's actually just host tissue that's being presented and a cytotoxic T-cell goes on its way. All these proteins and pathways are basically, I guess, activated to reduce the amount of virus or the ability of the virus to replicate. So if you kill the cell itself, so if you basically go on a kamikaze mission and basically destroy yourself, virus can't replicate in a dead cell. PKR and RNA cell both basically degrade all intracellular protein, so it's very difficult for a virus that hijacks intracellular proteins to replicate itself to do that when there are no proteins to replicate with. And then lastly, the sensitivity of MHC class I expression and this kind of shows the role of interferons bridging innate immunity with adaptive immunity. There's currently six subtypes of interferons known. Two of them we'll talk about today because they seem to be the predominant antiviral interferons. And like I said, I would blow this down to three key points that I need you to hold on to for the rest of the talks. I present some data. Something I've already basically hounded on and that's that all tolite receptors signal through either this TRF or Mighty 88 adapter protein. And don't worry if you forget some of these things, we'll hound on them a little bit more later. Then interferon production requires the nuclear translocation of these IRF subspecies, so these interferon regulatory factor proteins. And so if you don't get that nuclear translocation, you don't get interferon production. Then lastly, the activation of downstream interferon proteins results in increased cell death, decreased cell sensitivity to actual viral infection. So that's the key component of interferon. So then to take you through the literature up to date, what we knew going into this study, at least from tolite receptors in innate immunity in the cornea. And so it was very clear from literature that both bacterial and fungal infections require tolite receptor signals to clear the infection in the cornea. So if you knock out tolite receptors, you get an increased sensitivity to bacteria and fungus. Then it was widely assumed that interferon production was actually driven by tolite receptors in the cornea. And that was actually in part by a study that took human corneal epithelial cells and treated those with tolite receptor 3 agonists, and then they noted that interferon beta was actually produced. Then lastly, groups have gone through and taken corneal transplant, well, tissues that are of patients that are getting corneal transplants after HSV1 infection of the cornea, and noted that TLR mRNA is actually upregulated during HSV1 infections, suggesting that the virus, or at least the immune system, is upregulating its innate sensors to increase sensitivity to surveying the surrounding tissue. That would at least be the hypothesis from what they proposed. So then the two questions that kind of immediately popped up in our head was what drives the innate recognition of HSV1 in the cornea. Nobody had really nailed that down. And probably the more important question is why should we care? And so let me first target that second question. So for those of you that keep up with any sort of vaccine literature, there's no HSV1 or 2 vaccine on the market. Secondly, there have been multiple failed attempts most recently in 2012 where actually it showed an increased susceptibility to HSV infections. So not a great vaccine. Then there's emerging literature that would actually suggest, or actually not suggest, it's pretty firmly established at this point by multiple groups around the world that in order to get an efficacious vaccine, you have to activate the proper innate immune sensor. And so like I've already shown you, there's a dozen, and I didn't even show you all the innate immune sensors. There's a dozen of them. And so if you're making a vaccine with only a protein product but you actually need to activate the RNA sensor or the DNA sensor to create an efficacious vaccine, you're not going to get an efficacious vaccine. Then the other half of that, at least from the ophthalmology perspective, is that the therapeutic options and herpetic infections of the cornea are rather, let's say, I guess... You have two choices. You start antivirals, and then whether you start glucocorticoids or not later is kind of the question. So we'll talk about this as we go, but is there a more specific therapy possible? Because we all know the systemic... well, not necessarily systemic, but a lot of us have glucocorticoids, and then also I'll kind of elude on some of that as we go. And then let's return back to this question of what drives innate recognition of HSV1 and the cornea. And so to kind of introduce you to the first model, we basically used a mouse model in which we scarify corneas, topically inoculate HSV1 onto the cornea, and then we can look at specific time points compared to uninfected controls, and then we'll actually use global knockouts and proteins to look at the innate immune system in the cornea, at least in mice. Then we can do anything basically you could ever think of. We'll use plaque assays, basically where we grind up the corneas and then we can evaluate the amount of virus in the tissue by adding the tissue to Vero cells, and then HSV will actually form a plaque, and so we can actually count the amount of plaques in the tissue to give us a good logarithmic idea of how much virus is in the tissue. Then we can do all sorts of imaging, and we'll kind of walk through some of this data I actually won't show you. So then our first question became if interferon beta seems to be the predominant subtype produced during interferon infection, or at least when you treat cells with tolycperceptor-3 agonist, what is actually the predominant interferon subspecies when it comes to HSV1 infection? And so we looked at both interferon alpha and beta. They're predominant interferon or antiviral subtypes of the six interferons I suggested earlier. And so we looked at interferon beta. I don't show that data here due to time, but basically all interferon beta is restricted to the lumbus and infiltrating leukocytes. Then if we look at interferon alpha, you can see here in the middle panel, and this is kind of a busy panel, but I'll just kind of quickly highlight some of this, that if we look at HSV1 infection, so HSV infected cells are in red, blue is nuclei, so it's DAPI staining, and then interferon alpha is in green. We see that it seems to be interferon alpha that's actually produced by virally infected cells, and then also that it's the surrounding, as you see here with this yellow arrow, that cells that are uninfected will actually secrete the interferon alpha as well, and that's not a surprise since it binds in an autocrine and paracrine manner to basically reduce the ability of the virus to spread. So then at this point we're already scratching our heads thinking, okay, it's not interferon beta that seems to be driving the innate immune system in Acornia, but it seems to be interferon alpha. So then we took all the literature we had at the time and basically hypothesized what much of you would hypothesize at the time with the data that I've shown you, that the loss of toluic receptor signaling reduces HSV1 containment in Acornia due to loss of type 1 interferon production. And to first touch on that, we have, and I'll refer to it as our gold standard mouse, but it's a mouse that is deficient in one of the heterodimeric proteins of the interferon receptor, and so when you knock it out, you get no jack stat signaling and therefore you activate no antiviral pathways, at least when it comes to interferon. However, the mouse is still able to produce interferon, it just cannot respond to the signal. And so if we show you how susceptible these mice are to infection, this slide is very busy, but basically to boil it down to this. If you compare these knockout mice to wild type controls, you can clearly see that by three days post infection in almost any tissue you look at in a mouse's body, you have significantly more virus. So if you look in the brainstem, the trigeminal ganglia, the lymph node, the spleen, the thymus, then you can actually show that these mice, well, in these type 1 interferon receptor deficient mice that the virus is actually able to go by remake. These mice get extremely sick by day 5 or day 6 post infection. 90 to 100% of these mice are dead by day 5 post infection, likely from an encephalitis. So they're extremely susceptible to the virus and showing the extreme role of type 1 interferon in antiviral immunity. Then if we look at the complications of what happens when, at least from a pathological standpoint, of when we knockout type 1 interferon signaling an acornia, we can show you here on the left is a wild type control at 5 days post infection and then here start the image quality. It's not the best, but here on the right you can see that you get a ton of edema and actual stroma itself infiltrating neutrophils and I don't show the flow cytometry to confirm that, but we've confirmed that. Then you basically lose all epithelion of the actual CD118 mouse showing that these mice are extremely susceptible when it has pathological consequences on the cornea. Then we can basically conclude at this point that interferon is critical in host defense against the ocular HSC infection and we'll go down that road a little bit more. And then secondly that the loss of interferon signals results in rapid dissemination and death of a host. So these are absolutely critical signals. Then to get back to our hypothesis of whether toluic receptors are responsible for driving innate immunity we then took mice deficient in proteins and TRIF in Mighty 88, so the adapter proteins to all toluic receptors compared those to wild type controls and then our gold standard the type 100 interferon or CD118 deficient mouse. And we can see from this that the loss of those toluic receptors has no effect on viral containment. And then if we wanted to then that's at five days post infection and these are using those plaque assays to quantify the amount of virus in the cornea. If we then look at 24 hours post infection it appears that the role of type 100 interferons at least that we can appreciate from plaque assays is somewhere after 24 hours post infection. And so then at this point and this isn't actually a completely firm conclusion but the loss of toluic receptor signaling has no effect on HSV containment in the cornea when compared to CD118 mice five days post infection. And so your question probably or maybe you haven't really thought about this yet but quickly became apparent in my mind is what if a toluic receptor if you knock out that signal if another toluic receptor actually pick up the slack and actually recovers for missing toluic receptor. So if we're only knocking out one adapter protein there are other toluic receptors that can signal. And so I told you there's three toluic receptors that have all shown some role in HSV and aid immunity and so if we have others to cover for it if we're only knocking out one adapter protein then we may not be identifying the actual compensatory mechanism. So what we did is we actually back crossed toluic receptor adapter protein deficient mice into a double knockout and you see that when we knock out both toluic receptor adapter proteins it has no effect on viral containment in the cornea which would highly suggest that the loss of toluic receptors has no effect on the aid immunity in the cornea. And then just to kind of further solidify this I don't show the double knockout here but it has a basic finding that you see here you still see interferon production in all of these mice and like I said CD118 mice can still produce interferon they just can't respond to the signal because of the receptor. Then if we look down here and it's kind of small and I apologize for that but basically some of the interferon regulatory proteins we looked at a few of those by real-time PCR and they're activated in much the same way as wild type controls in our toluic receptor adapter protein deficient mice. And so basically we can conclude at that point that the loss of toluic receptors has no effect on downstream signaling at least when it comes to aid immunity. And I don't show this but the NF-Kappa B signaling cascade so this toluic receptor pathway or not toluic receptor pathway NF-Kappa B doesn't vary in that as well so it's NF-Kappa B and interferon production and it seemed to be completely fine even if you knock out toluic receptors. So then probably the biggest question that had us scratching our heads for quite some time is what is driving interferon production in NF-Kappa B signaling despite a loss in toluic receptors? So I've already told you that there are DNA sensors there's RNA sensors and probably any graduate student's worst nightmare is that you're going on a phishing experiment for an unknown protein. And so we decided to then look at what was known and then go with that. So I've already shown you that toluic receptor 2, 3, and 9 through the loss of toluic receptor adapter proteins seemed to have no effect in the cornea. So then that brought us to 4 sensors which seemed manageable unless it was an unknown sensor. And so we have, I'll first start with RIG-IMDA5 so that's an RNA sensor and in the literature if you dig back there's been a couple of reports in regards to HSV innate surveillance and they're not real strong reports in fact others have actually suggested that there's another sensor actually covering for the loss of RIG-IMDA5 and so it doesn't appear that they play a significant role in HSV surveillance. So then that brought us to the DNA sensors and so the first DNA sensor actually identified was DAI and I'm not going to tell you the actual full-length names of these proteins that are much easier just to give you the abbreviated because these are like four or five word-long proteins and it would just get very, very difficult to follow. So DAI had been shown that if you knock out DAI in macrophages you lose interferon beta production in regards to HSV infection. However if you take a mouse that's deficient in DAI has no effect on viral containment suggesting that DAI wasn't the innate sensor that we were looking for because we're looking for a sensor that makes the mouse exquisitely sensitive to the virus itself. Then DDX41 was actually a recently identified protein that had a similar role of DAI and been shown to play a role in macrophages but not much more than that. Then kind of the protein that we decided to hang our hats on at least from a research perspective was IFI-16. So I literally walked in so this paper was published in Nature Immunology as I'm trying to figure out the innate sensor responsible for HSV surveillance in Acornia. Paper was published in Nature Immunology the next morning I walk into my mentor's office and I say after six months of coming up and de-handed that I know what the innate sensor is and he was like Chris how can you be so confident in that? And it was due to the fact that this sensor had just been shown to drive an interferon response in regards to HSV infection in macrophages. So similar story that's how most DNA RNA sensors are identified in macrophages. However the other literature if you dig back to the late 1970s and early 1980s is that IFI-16 is actually expressed in prostate epithelium that when it is mutated prostate cancers now proliferate unabated and so that would actually suggest remember I told you interferons were critical in apoptosis and so it had been known for quite some time that if you lose interferon signaling and cancers that cancers proliferate and so we were looking at a protein that seemed to be regulating more than just interferon, beta production in macrophages in regards to HSV infection that it seemed to have some role in other tissues specifically in prostate epithelium and while the prostate is not the same as the cornea it at least was an epithelial tissue and so it was at least something that we could kind of grasp at that point. And so first we had to identify whether this protein was even expressed in corneal epithelium and so you can see here this is DAPI staining of the nuclei then P204 is the mouse homologue of IFI-16 and we have staining for that in red and you can clearly see that it's localized to both the nucleus and the cytoplasm and I didn't hound on this but this protein actually goes back and forth from the cytoplasm to the nucleus so it's no surprise that you see it in both places. Then to kind of further hang our hats on this we knew that the epithelial tissue was extremely sensitive to HSV infection and so this virus or this innate sensor was predominantly expressed in the corneal epithelium as you see here and let me use this instead as you see here in this 3D reconstruction of the corneal so this is the epithelial tissue here then the stroma here and then endothelium you can't really appreciate very well here but most of it's actually expressed in the epithelial tissue. Then we did something that we didn't actually think would work but that's a lot of science I guess so we took mice, we actually anesthetized them then transfected them with S-I-R-N-A to control and then also mice with P204 to knock down that protein to see if it had any effect on HSV surveillance because there was no global knockout of P204 IFI 16 and you can clearly see here that the loss of P204 results an increased susceptibility to HSV infection however you're probably thinking well that isn't near as drastic as what we saw earlier and so we first thought that maybe that was due to the fact that we were using a different model but probably the bigger thing was that when we look at the actual knockdown of how much knockdown we're actually getting we were getting anywhere from 40 to 50% knockdown of that protein of P204 so of IFI 16 in the corneal so that would probably mitigate a large portion of what we're seeing however we would then want it to look a little further and like I said IRF subspecies are translocated to the nucleus after activation of innate sensors and so if we look at the cytoplasmic portion of IRF3 which is the important IRF at least in regards to interferon alpha we can see that there's really no difference in the cytoplasmic portion of IRF3 however you can clearly see that when we knock out P204 with this 50% knockdown that you lose a good portion of the nuclear portion of IRF3 suggesting that we're losing the signal to drive interferon production with the loss of P204 then just to kind of further solidify that we then took mice, did the exact same thing transfected them, infected them and then did hole mounts and looked 48 hours post infection and you can see here that in our control you get interferon alpha production just like we saw previously however when we knock down P204 you lose that interferon alpha production in the cornea further solidifying the role of P204, IFI16 in interferon production in the cornea so then to take you back to this signaling cascade like I said we'd continue to return to this just to remind you, I have to remind myself as well IFI16 has an adapter protein STING STING then activates another protein that then activates another protein and there's probably five or six proteins that I don't show here needless to say it activates interferon regulatory factors so IRF nuclear translocation to drive interferon production so we did know that there was a STING deficient mouse out on the market so we actually contacted a group and wanted to look at what the loss of STING had in the cornea, so the downstream adapter protein of our IFI16 P204 so you can clearly see here that the loss of STING in CD118 there's no difference between their viral control at 48 hours post infection furthermore both of them are significantly elevated when it comes to the amount of virus you can recover from the cornea further solidifying the role of IFI16 P204 and viral surveillance of the cornea so then what probably any clinician then begins to ask is this just a mouse phenomenon so we wanted to kind of attest to that so we took an immortalized corneal epithelial cell line and wanted to first see if IFI16 was expressed in that cell line and you can see that there's a protein with a predicted size of IFI16 and then if we actually do confocal microscopy of these cells at least in this portion of the cell cycle most of the IFI16 protein is localized to the nucleus but it is expressed furthermore if we then look at IRF nuclear translocation so if we use control if we use SIRNA to knock down IFI16 and compare that to controls and we've quantified this but I'll show you the sexy images instead you get clear nuclear translocation as you can see here with purple staining where you have red and blue overlapping showing the nuclear portion of IRF however when you knock down IFI16 that protein is restricted to the cytoplasm showing that it's not activated and actually being nuclear translocated furthermore if you knock down IFI16 you have a significant rise the amount of recoverable virus from these cells then like any good Oklahoman we like to fish the picture up to this point in innate immunity had basically referred to sensors very specifically as sensors due to the fact that no one had actually shown that these innate sensors actually directly interact with the protein, DNA, or RNA that they're sensing or that they're just kind of prodding along, sensing the environment around and so what we did is we took these human corneal epithelial cells we transfected DNA very similar sequences to HSV into these human corneal epithelial cells within cross linked all proteins in the cells lice the cells then used IFI16 antibody to then pull down and then we could clearly show that when you have IFI16 you pull down DNA both single strand and DNA double strand DNA products that we transfected into the cell however when you don't have IFI16 or if you don't have DNA products you don't pull anything down suggesting that there is a direct actual sensing of IFI16 with DNA in the host cell whether that's actually cytoplasmic this would actually probably suggest that it's cytoplasmic because it's very difficult to get proteins into the nucleus or DNA into the actual nucleus this would suggest that it's a cytoplasmic interaction but it's still debatable at this point whether this interaction actually takes place in a cytoplasm where a virus would actually release its DNA products to then traffic into the nucleus or whether this is actually in the nucleus occurring so then the next question became does this happen with other pathogens are we just finding a nice phenomenon that just so happens to work that's not all that we've checked so far it didn't I'm just showing you the highlights there were a lot of negative data in this whole process and so if we look at an RNA virus vesicular stomatitis virus and I can clearly show you here with real-time PCR because it's not a plaque forming virus so this would be a way that we can quantify the amount of virus that when you lose our type 1 interferon signal you lose the ability to contain the virus so type 1 interferons are critical and the innate immune response to VSV however if you knock down P204 it does not have the same effect of losing P204 in regards to HSV infection suggesting that or I guess solidifying that this is a DNA sensor IFI 16 P204 and that at least at this point it's HSV specific but it's likely all DNA viruses then to kind of give you a model of what I've shown so far and some of this data I haven't shown you because just due to time constraints virus replicates epithelial tissues then activates P204 IFI 16 to then activate Sting and then probably a half dozen other proteins to then lead to the activation and dimerization and phosphorylation of IRF to then nuclear translocate to drive interferon alpha production and what I haven't shown is interferon alpha production while it drives PKR RNA cell apoptosis of cells and MHC class 1 expression we also show that it actually binds to the upstream promoter of a chemokine that's critical in driving inflammatory monocytes to the side of infection so while I'm showing you the major portion of innate immunity there is a bone marrow derived response while it is small it is significant it seems to be driven by inflammatory monocytes then I don't show the data on this but in the vaginal mucosa so in epithelial tissue it seems to be the same response so we actually knocked down IFI 16 P204 in mice and then infected with HSV2 and you get a significant rise in the amount of virus however we knew from literature that there's a group of children in France that are highly susceptible to recurrent herpetic encephalitis and that has been I guess remotely connected to a loss of tolite receptor 3 signaling and so we knew that when tolite receptors were first identified the person he actually didn't win the Nobel Prize and was kind of a controversial thing but a friend of his won the Nobel Prize for tolite receptors the guy that actually identified tolite receptors hypothesized that tolite receptors are omnipotent they actually drive all innate immunity and all cells and all tissues well it seems like a very strong statement and we were seeing something somewhat different and so we wanted to first test that and so if we look in the central nervous system and I'm only going to show briefly this just because I think it's interesting and the loss of type 1 interferon seems to be critical and you get HSV infection of appendable cells then you can further confirm that you lose the cilia of appendable cells you get some red blood cell hemorrhaging in those sites that you don't see in wild type controls and these mice get extremely swollen heads have neurological pathology that would be any neuropathologist dream but I'm not going to show any of that but to then go on to show that human appendable cells are actually extremely susceptible to the virus however it seems to be that if we take mouse brains and we actually take 200 micron sections of mouse brains and keep them alive on tissue culture and tissue culture we can show that if you lose the TRF adapter protein so the tolite receptor 3 adapter protein then when you lose that you lose innate immunity in the actual central nervous system then if you deplete macrophages with chloronate it's a very specific depletion that you actually lose that so it seems to be tolite receptor 3 in macrophages that's driving innate immunity in the central nervous system so to conclude the loss of p204-i5-16 results in enhanced viral susceptibility I've pretty clearly shown that then interferons play a crucial antiviral role in acornia as well as the entire host then the role of tolite receptor 3 in IFI-16 is tissue specific and that actually got us a lot of heat as we were trying to publish some of these papers as a Nobel Prize I guess non-winner but should have been winner was reviewing our papers and then lastly that p204-i5-16 drives recognition of HSV in acornia and it's I don't show well I didn't show as strong of data for the human side of that but I did show that in human corneal epithelium it seems to matter as well obviously you would have to do well you couldn't do those experiments then I'm going to leave you with a few questions just foods for thought and so first will inhibiting tolite receptors decrease complications of herpetic infections of acornia I don't show the data but groups have shown that tolite receptor 2 and even tolite receptor 9 seem to drive a very pro-inflammatory state so is it possible in acornia that when you have tolite receptor 2, 3, 9 IFI-16, p204 all being activated by HSV we clearly show that p204 is the antiviral innate sensor responsible for antiviral immunity but are these other sensors just driving a pro-inflammatory state so could you specifically target so instead of using glucocorticoids could you specifically target tolite receptors to decrease the pro-inflammatory state and hopefully the pathology that you see with herpetic infections of acornia that's anybody's guess at this point but that would be at least be you know something plausible with tolite receptor antagonists on the market at this point and then the second half of that question is the entire immune response a good thing and I've actually written on this subject before but in a central nervous system it's clear that the entire immune system if you add in the adaptive side of things that there's a pathological side of the immune system but then there's an important antiviral portion of the immune system patients they get long-term neurological pathology it's likely due not to the virus I mean the virus is activating the immune system but it's actually likely due to the immune system trying to clear the virus so you're actually getting that central nervous system pathology due to that and that's actually being tested in a huge clinical trial in Europe where they're using glucocorticoids and herpetic and cephalitis patients then the next question it's probably true but we haven't tested that so I can't say that it is but does p204-i5-16 drive an aid immunity to VSV so Zoster and so they're both alpha herpes viruses and so they're very very similar when it comes to structure of the viruses themselves and so it's likely that this is the same sensor for both viruses and so if you were to change therapy you would change it for both then lastly kind of the biggest question for thought is will HSV vaccination attempts to activate this innate sensor produce an efficacious vaccine and then furthermore could you actually design a vaccine that you could take into third world countries that's actually a topical eyedrop vaccine that would be much easier to take than a bunch of syringes only time would tell if you could actually do that and not induce pathology in the eye but it does seem plausible at this point my mentor is actually not necessarily testing well he is I guess testing a live attenuated virus very attenuated to see if he can actually activate this immune response then I'm only going to touch on one other thing because our lab has shown this quite clearly I've done a little bit of work on this but it was actually another graduate student in our lab that if you don't contain the virus early you get an increase in the amount of lymphatic vessels into the cornea and so from the pathological side early antiviral containment is crucial and interestingly this story actually goes back to HSV protein actually specifically drives VEGF-A production to drive lymphatic growth into the cornea and that's all been shown by this previous graduate student but it just hounds on the idea that you have to contain this virus early or you're going to have long-term complications and then here are my citations you can look those up if you'd like and then I have to give thanks to a lot of people and I boil it down to just a few people mainly from the people in my lab but let me just point out two people first my mentor Dr. Dan Carr at the D. McGee Institute and then a technician in the lab Min Zhang that was absolutely phenomenal let me do a lot of this work that I probably wouldn't have been able to do with my own hands and then with that said Dr. Wong asked me to put up some pictures of myself she actually wanted me as a kamikaze something I don't know when I was a kid I didn't have any of those and so this actually I guess my honeymoon with my wife were actually in Australia river rafting and here I am playing I guess a paddle guitar and my wife I guess right here is I guess about to bring the hammer down with somebody with her paddle so with that said any questions I can take at this point Dr. Harry so live attenuated so when you give live attenuated viruses if you actually look at almost every viral vaccine their live attenuated viruses the one example well there's two that come to mind that aren't so the flu vaccine you can actually get that intranasally or also polio and so I would actually argue that in the polio vaccine the live attenuated pill is actually better for you because it activates the proper innate system and actually now in the US for giving injections so it's not as efficacious and there's literature that actually shows that but it would probably be due to the fact that you're actually having that viral life cycle to activate the proper innate immune system and so if it is a YFI 16 P204 you would actually have to have DNA from the virus presented to this cytoplasmic nuclear sensor so the viral life cycle is absolutely critical and so in previous vaccine attempts with HSV they've done protein based vaccines and those like I said they make you actually more susceptible to the virus and so it's probably due to the fact that you're actually giving this actual viral life cycle to induce the immune system yes Dr. Wong they're coming around now we had to get to the point where we had to have someone pretty big in the field help us with a couple of experiments she was actually the one that identified YFI 16 P204 and so she's in a she works in a lab at UMass and so we had her do an experiment for us more is like hey can you check our work because reviewers are absolutely just slamming this idea and it was the same guy over and over again because you could tell from the writing in these reviews and so once she signed off on it and did this experiment for us it was like oh well if she signed off on it that must be true because this little Dean McGee lab in the middle of Oklahoma doesn't know what they're talking about but now that someone from UMass has used huge papers in the field now that she is actually signing off on it and it's done some of the work well I say she did some of the work she basically confirmed in her lab that what we were seeing was the same thing and so yeah from that they're finally catching on it's actually becoming more prominent in the field now so we'll see, time will tell yes well so P204 IFI16 hasn't been shown to directly activate those proteins but what it does is it activates interferon, interferon then binds to the heterodimeric interferon receptor then signals to the jack stat pathway to induce MHC class 1 expression keep in mind that MHC is always class 1 is always expressed on cells but it's actually upregulated in viral infections or anytime you have interferon production the virus actually has specific mechanisms to down regulate the MHC class 1 expression so it's a huge battle that I didn't describe here so it's a downstream effect of the sensor but it doesn't directly activate those two proteins or at least it hasn't been shown to this point which would be skipping a step in this long cascade so it's likely not directly driving those protein products or even apoptosis but it is having a downstream effect of that. Thanks, is that okay? So here is your goal down the road to some point to do cornea? I've thought about it but I'm kind of going back probably, I'll probably end up I'm pretty interested in uveitis from the innate side but honestly presentations I have a more basic science question of why I saved it so you mentioned that you believe that it was actually a DNA sensor and not an RNA sensor did I mishear it when it seemed like the IFI 16 was more with the DNA? P204 is the mouse homolog Oh that's right, that's right and that's a different name so same name and so all the names in innate literature there's a name like four names so then how did you then with RNA what was their respective thing oh rig I and that was the one there and then I noticed when you're showing the graphs