Everybody, it is my distinct pleasure to welcome our colleague and friend, Dr. Francisco Javier Cubero, to give the AASLD Hepatotoxicity Special Interest Group talk to us today. Javier is a full professor in the Department of Immunology, Ophthalmology and ENT at the Universidad Complutense de Madrid, School of Medicine, where he is interested in exploring signaling pathways, modulating endoplasmic reticulum and oxidant stress in the context of liver physiology and pathophysiology from acute DILI to chronic liver diseases such as mastoid, MASH, liver cancer and alcohol-related liver disease. Javier focused his master's and then his PhD thesis on inborn errors of metabolism in the liver. His postdoctoral experience began at Trinity College, Dublin in Anatomy and Physiology and in the Division of Liver Diseases at Mount Sinai School of Medicine, where he studied the pathophysiology of alcohol-related liver disease. He then moved to the University Hospital of Aachen, Germany, where he focused on intracellular cascades in animal models of MASLD and MASH. He's co-directed several editions of the Spanish Liver Society, translational meetings, EMBO meetings and the Young Investigators Seminar Edition. And as part of the Spanish Research Network in liver disease, he's a member of AEEH, EASL, A-S-L-D, I-S-H-S-R. During his career, he has received relevant honors, awards, basic award in medicine from the Royal Spanish Academy, most recently in 2020 through three, congratulations on that. The Iraq Best Publication Award, International Billiard Liver Scholar Award, German Liver Foundation Award, Ramon El-Kahal Fellowship and the M.Sc. Fulbright Fellowship in 2006. He is a leading expert in the field of drug-induced liver injury, a well-known investigator to all of us. He's published extensively and we've read his work. It is my pleasure to welcome you, Javier, to this ASLD talk on the interplay of ER stress and autophagy in APAP toxicity. Please take it away. Well, thank you very much for this kind introduction, Lily. It's for me a pleasure to be here presenting at the SIG Hepatotoxicity Group for the ASLD. So today I'm gonna show you some of the results that we haven't published. Some of them are already published, but I want to show you a little bit what we are doing now in the lab regarding ER stress and autophagy in APAP toxicity. So this is the agenda that I will discuss today. First, I will talk a little bit about the X-Binding Protein-1 which links ER stress and autophagy in APAP. Then I will address a little bit the role of XBP-1 in muscle D-MASH, because we have this consortium with muscle D-DILI. I will talk a little bit about the common target of several ER stress branches, which is Junk, as the Junk signaling pathway, cause or consequence in DILI. And finally, I will show you very recent data that we have gotten regarding the oxidative stress and pathways which modulate oxidative stress, such as NRF2, which is an emerging pathway, also really linked to ER stress because it's linked to the first branch. Just to start with the first point, I want to just give a little bit of an introduction of ER stress. So endoplasmic reticulum stress exerts several physiological functions, including protein folding, synthesis of fatty acid and esterols, detoxification of xenobiotics and the storage of intercellular calcium. So upon exposure to potential stressors, such as drugs, the ER initiates the unfolded protein response, the UPR, that I will be discussing during my talk today. APAP, as you already know, is just a significant public health concern and it accounts for over 55% of cases of acute liver failure in the UK, reaching almost 40% in the US. And the mechanistics of APAP hepatotoxicity are still not fully understood. So the clinical treatment, as you know, is very limited. The role of ER stress in APAP-induced hepatocellular injury is supported by a considerable amount of data. And more importantly, there have been some literature and some reports on the IRE1-alpha-XBP1 arm of the UPR response that plays a critical role in paracetamol-induced liver injury via the regulation of C3-1 activity. So as you can see here, there are the three branches of the ER stress. The first one is PERC. The second one is IRE1-alpha-XBP1. So this IRE1-alpha is an endonuclease which triggers the splicing of XBP1 into the splice form that goes to the nucleus and exerts specifically a function on lipid biogenesis as you will see every time we modulate XBP1, we will modulate metabolism. Also cytokine inflammation is very linked with inflammation and is very associated with a junk signaling pathway. The other branch that I will discuss today is PERC. PERC is linked to NRF2 and is associated with the antioxidant response via ATF4. So just to start with what we did with XBP1, we first check how is the UPR activation during acetaminophen-induced acute liver injury. We use HEPRGs with different concentrations of APAP and we use the normal C57 black 6 mice with different concentrations of APAP from zero to 500 milligrams per kilo. We kill them at different time points. We sacrifice the mice at different time points. And then we develop or we generated mice with the specific deletion of XBP1 in hepatocytes. This was done, we also challenged them with different concentrations of APAP. And at the end of the study, we use an inhibitor of IRA1-alpha to block the splicing of XBP1. We just collected serum transaminases, we performed Westerblot, RT-PCR, immunohistochemistry and immunofluorescence. So the first thing that we did was to check a database, well, not a database, a biobank of patients that we had access. These are patients from UK, from Newcastle. Helen Reeves provided us with these patients and we checked by immunostaining the splicing of XBP1. We observed that it was very linked to hepatocytes. So XBP1 expression increased during human acetaminophen DILI. And then in hepargese, what we check, it was that where we saw that the correlation between spliced and unspliced XBP1 was increasing along the increase in the concentration of APAP in hepargese and also in the mouse model. So from concentrations from 150 to 300 milligram per kilo, we already saw an increase in the splicing of XBP1. Basically with mice, we observed also this, the presence of XBP1 specifically in hepatocytes and the positive cells were significantly increased after 300, 400, 500 milligram kilo. So we ran some Westerblot to understand how was the UPR and the ER stress response in these mice. And as you can see, the phosphoeric one alpha and the splicing of XBP1 occurs already at 10, when a concentration of 10 APAP in hepargese and already a concentration of 150 milligram per kilo in the mouse model. This was nicely observed and we observed also very interestingly that CHOP was also activated at a concentration of 10 in hepargese and already at 300 milligram kilo in the mouse model. Moreover, we started to, we've generated these mice that I discussed before, the XBP1 delta hepa mice. These mice, we use DMSO and we use APAP to compare them. These mice are already characterized in basic conditions. They have hyperactivation of phosphoeric one alpha. And when you treat them with APAP, this hyperactivation is already observed also as well, but decreases in the mouse model. This is one of the two characteristics of these mice. The first is the activation of phosphoeric one alpha and the second one is CPT1 as I will show you later. They didn't have, we didn't see activation of VIP and the splicing was already gone in these mice compared to the control mice and CHOP1 also disappear. We also observed no expression of this, the branch of perg. In junk, considering junk, we also observed decreased phospho-junk in these mice. So the junk signaling pathway was also decreasing XBP1 delta hepa mice after APAP. I'm gonna show you some of the data that we obtained and the transaminases were significantly decreased, ALT and AST and ALDH in XBP1 delta hepa mice compared to the normal mice after APAP. The necrotic foci were significantly also decreased in XBP1 delta hepa compared to the fluxed mice. Less cell death was observed in tunnel. And this is the quantification that we saw in the necrotic area significantly decreased and the cell death in tunnel staining. CLIFCASPACE3 was, as expected, significantly decreased after APAP. And one of the characteristics that I told you is that CYP2A1 activity per se in this XBP1 delta hepa mice almost is blunted. And this is one of the characteristic of XBP1 delta hepa which gives them the survival towards APAP. So the advantage to have the benefit of being protected against APAP liver injury. We also check sonocludense 1, CETL1. And as you can see, CYP2A1 staining and 4-hydroxynonanol which is a marker of lipid peroxidation was also decreasing in this mice, XBP1 delta hepa after APAP. This is where we also perform OlredO. This is the CYP2A1 which was decreased, already decreased in the normal XBP1 delta hepa mice and no difference after the APAP between the flux and the delta hepa. And this is the lipid peroxidation, quantification of positive cells. So why we linked autophagy to XBP1? There were already some reports linking the UPR response with hepatic autophagy and the activation of, or the mediation of the activation, the splicing of XBP1 and the activation of elements which are linked to autophagy. This was one of the papers that we observed. The second one that it was very clear as you see in XBP1 on autophagy, the protection was linked to in endothelial cells. And this is one of the most famous papers regarding phosphoryl 1-altivation in APAP hepatotoxicity that I will come back later on during my talk. So we first did electron microscopy. We perform electron microscopy in XBP1 delta hepa and we already observed the presence of autophagosomes in the microscopical examination of these specimens. We observed increased ATG5 and other genes related to autophagy and decreased P62. When we run the proteins related to energy metabolism and autophagy, we observed increased phospho-INPK, decreased phospho-IKT and decreased P62 and increased LC3.28. Therefore, autophagy seemed characteristic of the protection exerted by what we observed in XBP1 delta hepa. One of the reviewers asked if we could, sorry, if we could perform these experiments in wild-type mice. So we thought to use an inhibitor of the splicing, an inhibitor of the phosphoryl-1-alpha. And we used this one, which is STF083010. It's a very well-known inhibitor of the phosphoryl-1 branch of the ER stress. And again, we observed decreased necrotic foci, very significant when we perform 300, 400, 500 milligrams per kilo in wild-type mice in combination with APAP. We did this before APAP. During, at the same time with APAP and post-APAP to see what was the correlation. This was one of the reviewers' concerns. We also observed decreased ALT, decreased AST and decreased LDH. And we performed again the Westerblot of autophagy and there were again decreased expression of ATG5, beckling, P62, AKT and increased LC3 puncture rate. Therefore, from this part, we can summarize that if we prevent the splicing of XPP1, we can prevent the activation of junk and the junk-related cell death because we prevent also CHOP activation. This we observed in XPP1 delta-HEPA mice and we also check it when we gave the inhibitor of the phosphorylated one endonuclease. So, but what happens in XPP1? Because there, as I told you at the beginning, XPP1 is linked to hepatic lipogenesis and well, all lipogenesis in general, no? So, there have been some reports relating XPP1 in NASH to NASH. As you can see here, XPP1, this is specifically macrophages, which you can see that it's decreased compared to the MCD diet normal flux mice. The NAFLD activity score decreased significantly. ALT, AST also in this study is shown is decreased and hepatic triglycerides was a significant increase. And the role of XPP1 was specifically linked to macrophages. Regarding this paper, we saw the differentiation from the group of Jacqueline Maja. Caroline, the words she showed, or she challenged the mice to fructose diet and she observed the same, no? That they had decreased junk activation and increased C. juna P38. So, we thought that I'm not gonna show you the first results because I'm just gonna tell you that with the Delta HEPA mice, we tried to see if the role of XPP1 in hepatocytes was important in NAFLD NASH or MASLD MASH. And we didn't see any differences. We did this several times, several rounds. And so far, we didn't see any difference at all. So, we check if the culprit of XPP1 role was the hepatic stellate cell. So, we discussed with Robert Schwaben from Columbia University and he provided us with the LRAD CRE mice. So, we created mice with a specific deletion of XPP1 in hepatic stellate cells. And we ran three different models. We ran MCD model, we ran CCL4, and we ran a model of NASH. So, FAT-NASH, which is a model developed by the laboratory of Scott Friedman, whose birthday is today, by the way, if you want to say hello to him. So, the MCD diet, just to mimic specifically MASLD MASH and fibrosis with CCL4. The first part of the results are regarding MCD diet. And as you can see, in contrast to APAP, XPP1 in hepatic stellate cells, they show increased damage. They have increased ALT levels, increased AST, and they had already increased lipid deposition that we measure with LRAD-O. This was very significant in mice with a specific deletion of XPP1 in hepatic stellate cells. And it was very obvious in this model after MCD diet. The number of tunnel-positive cells was extremely increased after MCD diet in XPP1 mice, knockout in stellate cells. And so was the CHI-67 as a marker of proliferation. So, we then decided to go for fibrosis. Fibrosis, we did typical two injections per week. During 12 weeks, and then we sacrificed the mice and we observed the same. We observed that these mice, they had increased liver fibrosis, increased ALT, the serous red staining and the deposits of serous red were significantly higher in XPP1 in mice knocked out in stellate cells. The same was we observed with alpha-spuma cell ducting, which is a marker of hepatic cell, hepatic stellate cell activation. And, and also the mRNA expression, the fold increase was a significant increase compared to the flux mice. And so was the, and we didn't see in this first round of mice, the differences in collagen 1A1. Cell death was also significantly increasing this model in mice with a stellate cell deletion of XPP1. And we also observed increased proliferation. So, compression proliferation was also significantly increased after CCL4. So when, then we, we check also for inflammation markers, such as TNF, interleukin-6 and TGF-beta that we have run by mRNA expression and also by ELISA. So then we, we thought what else we can do with, with XPP1 knockouts in stellate cells. And we decided to, to combine this new model of FAT-NASH that Scott developed. This model, after 20 weeks, is a very typical model of a patient with a big NASH and combined with obesity. So they have some metabolic syndrome changes. And at the end, if you extend the model, you will get hepatocellular carcinoma, but we haven't reached that stage yet. So we observe also very, very increased lipid deposition in knockout in stellate cells for XPP1 in this model. And as you can see, the quantification is significantly increased comparing the flux mice with mice with a stellate cell deletion of XPP1. The serous rate in this model is also very, very notorious because you can, you can, you can, you can see that already after, after these 20 weeks, the flux mice, they have F1, F2, but our model, it was a score much, much higher. The serous rate was significantly increased also in these mice and alpha-sbumacella actin was significantly increased in mRNA expression. So we can say that liver fibrosis and hepatic stellate cells were characteristic of mice with a stellate cell deletion of XPP1. Tunnel was explosive. We could see a lot of cell death in these, in these, in these livers. This was, this was all in, in the five mice that we, we have sacrificed so far. We have another group coming, coming soon. And, and these are a guy 67 staining the same. We have a high, very high compensatory proliferation to the cell death in XPP1 in stellate cells, knockout. The same here for, for inflammation markers, they were increased specifically interleukin-6 and no differences in TNF or TGF-beta. Therefore, we checked papers to correlate if there was any literature on XPP1 in hepatic stellate cells and in the role of, of, of XPP1 in stellate cells. And we observed that there was this, this nice paper published in scientific reports showing that the, the, the, the transcripts of XPP1 precede primary hepatic stellate cell activation in culture. And this already is observed after day two, the total XPP1 is very clearly overexpressed. And then the same for X, for the splice form of XPP1, and then other related markers, such as EDM, EDM1, or markers of hepatic stellate cell activation, like, like collagen 1A and ACTA2. Therefore, in collaboration with Roben Nogueiras from the, from Santiago, from CIMU Santiago, here in the north of Spain, we analyzed the transcripts of XPP1, if they were characteristic of human LX2 cells, no? So we, we check, we induce these cells with TGF-beta1 at different time points. I'm showing here 16 hours and 24 hours. So here on the, here, this panel is 16 hours and here is 24 hours. And we also check the collagen expression at this time to make sure that these, these hepatic stellate cells were active or activated. And, and we can see that there are different transcripts. So we, we observe ERA1 activation, spliced XPP1, total XPP1 as well, CHOP activation, and markers of oxidative stress, such as the KIP1, NRF2, HO1, the NQ01, the kinone, GLC, GCLC. This, I will talk a little bit now in detail about this. I want, I put them together so I can explain you a little bit about this pathway also in APAP. So I try to, to show you what, what we have gathered so far regarding XPP1 in APAP and muscle D, but we had already published a little bit about the junk. I'm not going to go into, into much into detail, but just to remind you that, that as Professor Kaplovich already showed, the J and K genes are very, very important in, in APAP-induced liver injury, but specifically J and K2 as we, as we also check in, in, in ibuprofen. And well, interestingly, we found that when we remove both, we observe more damage. This was a little bit strange because it was completely unexpected and we, we did it many times, but I can explain you a little bit what we, what we observed. So this is basically the MAP kinase pathway, which is activated by cytokines, T-cells, oxidative stress, irradiation, which activate MAP kinase, kinase, kinase, MAP3 kinase, which in turn activate MAP2 kinases. And at the end you have junk activation, which they are responsible for inflammation, cell death, specifically apoptosis proliferation. And they play a very important role in immunity as well as in cell survival. So I wanted to show you what, that all these experiments that will be performed on HEPRGs and the wild-type mice with APAP, we also observed activation of the junk signaling pathway already at, at a very low concentrations of APAP and on HEPRGs. And, and this was, we got them a little bit later with, I mean, a little bit with a high dose, like a 300 dose we needed to, to start activating junk. We also check phosphojunct1 and phosphojunct2 once these antibodies became available. There has been some, well, some reports such as the one I mentioned before, published in Journal of Experimental Medicine, showing that in the XBP1 deficient animals, they have, they have no junk activation. And this was what I showed you before. So this is just to confirm the data that we, that we show. Specifically, we, we check also at 24 hours because at early time points, we didn't see also any, any difference. So, this, this, these samples, we got them from, in Germany, and these are different samples from patients with, with different, sorry, with different etiologies of, of DILI, like paracetamol, fem-, procumon, and, and, and SAH, autoimmune hepatitis. And we observed in, in, in some of the liver explants that we, that we had, activation of the junk signaling pathway, very, very clear, as you can see. So, this was expected. And what the, what the pathologists told us is that the, the activation of junk was related to hepatocytes, not only to hepatocytes, but also to the infiltrating cells. So, we had a dual activation there, but it was clearly hyperactivated, both junk 1 and junk 2. What was very clear is that the junk in hepatocytes, junk 1, specifically, we didn't see any difference. And this was confirmed in the data already published on, on, with regard to APAP, whereas in liver fibrosis, junk 1 deletion is protective. Junk 1 delta-hepa in hepatocytes play no role, whereas when we remove both junk 1 and junk 2, we observe extensive necrosis, necrosis. So, we thought that junk 1, delta-hepa were sensitized towards APAP liver injury. We performed microarray. This was done in, in Wageningen, in, in, in the Netherlands. And, and the, the, both in, in the livers of junk, complete knockout mice and hepatocytes, and we compared them to wild-type hepatocytes and livers. These are the hepatocytes, and these are the livers. And we saw specifically genes related to cell proliferation, apoptosis, and inflammation. So, as you can see here, this complete junk deficient livers, they had more fibrosis, more serous red staining, and more collagen 1, this is immuno, immunostain, immunofluorescence, after CCL4, 28 days. We also check after CCL4, junk, so we did serous red quantification of, of all the samples. Collagen 1 mRNA expression was significantly increased compared to junk 2, but also we included all controls. We, we used junk 1, which were really down as, as published before, as I said, and junk 1 delta-hepa. Hydro, hydroxyproline was also highly, the content of hydro, hydroxyproline in these livers was significantly increased, and the expression of alpha smooth muscle actin was always characteristic of this complete junk deficient mice. We also observed after CCL, CCL4, after 20, 28 days, cell death and compensatory proliferation were significantly increased in, in junk deficient mice. So, these are the quantification of TANL, CHI-67 gas phase 3 activity with a kid, we didn't get any significance, but it was, there was a clear trend, and some Western blots that we ran with PCNA, cyclin D, et cetera. And we also observed some inflammation, as I said before, the junk activation was related to, not only to hepatocytes, but also to the infiltrating or immune cell compartment. So, interleukin 1 beta and TNF, for example, were significantly induced in this, in hepatocyte junk deficient animals. So, we isolated the hepatocytes, and we performed some functional studies to corroborate our findings. Already, we did different time points, but as you can see, these, these hepatocytes, they die after 12 hours, they die very quickly, very fast. And we, and we, when we used the inhibitor of the junk signaling pathway, SP6-00125, we could prevent somehow the cell death, but not totally. The same, sorry, the same for mitochondrial damage, we use mitosox here, and they had a very, very large mitochondria in damage, and we could prevent it both with, partially with, with the inhibitor of Junk 1 and Junk 2. These are the ROS positive cells that we observed, and this is what happens in the junk, the blue one here, junk deficient animals, they had, or hepatocytes, and this is when, when we block SP6, this inhibitor of, of, of junk, that we were also able to, to, to improve a little bit the damage. Tunnel cell death was, as I said, in these hepatocytes, most of them were dead after plating, specifically after 12 hours of plating, and we could reverse a little bit the damage using the SP6 inhibitor. This is the quantification as well, as I can say, as you can see here. And finally, what we observed is that these hepatocytes, when basically they, they were dying of, of increased necrosis, no? We also saw apoptosis, but Rekinase 1 and Rekinase 3 were significantly expressed in these, in these hepatocytes, and with the, with the use of SP6, we could see that the Rekinase or cell death was a little bit ameliorated. So, the reviewers asked to, to check other targets, and we did proteomics, and our proteomics analysis showed that these junk delta-hepa mice, they have decreased phospho-AMPK. So, we used the same mice, the MSO APAP and SP, APAP with combination of SP6, and all our results showed that specifically APAP activates protein kinase C alpha, which triggers junk activation and cell death. And in the proteomics analysis that, that I'm not showing here is that we, we have decreased JunD activation. This was very, very clear in the, in the junk delta-hepa mice. And, and this could be, could be prevented partially by using SP6, which restore a little bit AMPK as an energy of the cell, no? So, activating survival. Therefore, we could say that, that junk phosphorylation was prevented by phosphorylation of AMPK in this, in this model. Later, we, we check what, what was the purpose, what was the role, no, of junk in, in ibuprofen delay in humans. This was a medical doctor who performed his thesis, Miguel Zubek in, in, in Aachen. And, and we mentored both Christian Trauben and myself, his thesis. He, he first, this was one of the first surprising results, not that the, in ibuprofen delay biopsies that we had by, by chance, we observed that there was a very significant increase junk expression and, and, and activation. So, the, the, the staining was very, very clear. AST, so we, we, we use the same mice as we had, we had wild type, we have JunD delta-hepa, and as we didn't have at that time Jun2 mice specifically knockout in hepatocytes, we use an sRNA that blocked Jun2. This is a, to, to, to, to cut the story short, this sRNA was given to us, or was tested by a company, a very, a company, a collaborating company in, in Norway, in Norway, in Westphalia. And, and they supply us this sRNA, and it was very, very efficient. We first tested in hepatogens in different primary hepatocytes, and we could completely deplete the levels of the expression of Jun2. So, we, we used this Jun2 in normal wild type mice that had the same background as the Jun1 delta-hepa, and we observed that when we blocked Jun2, these mice, they show a very significant increase AST and ALT levels compared to the Jun1 delta-hepa and the wild type. What was the mechanism? The mechanism, we check PKC, protein kinase alpha phospho-AKT, which was significantly increased, and this was linked to the inhibition of Jun2. We observed decreased expression of phospho-ERK, which is linked, as you know, to cell survival, and the rekinases were specifically activated because we had increased phospho-AKT. Therefore, ibuprofen intoxication is highly dependent on Jun2, and Jun2 in hepatocytes protects against ibuprofen. So, when you depleted it, the, it can be a good, a good target. Finally, I want to show you a little bit the latest results that we have from the lab, and this is the last branch of the pathway that I haven't talked today, and it's PERC. PERC is also a link to NRF2. As I said, this pathway is really associated to the antioxidant response, and we are very, very, we are very interested in understanding NRF2 signaling pathway. As you know, NRF2 is kept in the cytoplasm by KIP1, and when it's activated, it goes into the nuclei and activates the antioxidant response elements, which are HO1, specifically, GLCL, and the kinones. So, I will show you some of the results linking regulation of linking XBP1 and NRF2. This is a paper published in 2018, and in this, and in this paper, they show that when you block XBP1 or mice with a total depletion of XBP1, they have no expression of NRF2. So, there is, there must be some kind of association, and as you can see, they have this knockout mice with tunica myosin, they have decreased NRF2 expression. Also, Nrf2 here is decreased. And these are the antioxidant response elements, NqO1, hemoxygenase-1, and GST. They are significantly depleting in XPP-1. So this is how you can modulate several branches of the ER at the same time by modulating lipid biogenesis and the antioxidant response. So we generated mice with Nrf2 overexpression. How we did this? We use, in a collaboration with Christoph Burtz from Aachen, he had this KIP-1 knockout mice. And we generated KIP-1 mice, specifically knockout in hepatocytes. And we treated them with a PAP at different time points. I'm gonna show you here now, six hours. And we use, we collected the liver, we collected serum, and we did a lot of experiments. The first one is to check really what is the expression of Nrf2. I'm sorry about the stain, it doesn't look here quite good. But we could see clearly that Nrf2 was significantly overexpressed during the doses of APAP. And specifically at 500 milligram per kilo, but also at 300, it's already starting to be significant. This, we run different experiments, but the best way to show Nrf2 activation is to isolate the nuclei and to check it by Westerblot. Because you need to see that the Nrf2 is not sequestered in the cytoplasm, but it goes into the nuclei. And we check also elements of the antioxidant response, such as NqO1, it was already significant after 150 milligram per kilo, and very significantly after 500. The same for GCLC, GST1 only at 500, we could see a difference compared with the MSO mice and hemoxygenase. So this already was telling us that Nrf2 might play a role in APAP daily. So we use APAP, the highest concentration, which is subtoxic at 500 milligram. And you can see here that we check the necrotic foci and they were enlarged compared to flux mice. Also tunnel staining was significantly increasing in this KIP1 delta HEPA mice compared to the flux mice. AST transaminases, the mice were dying some very, very fast. Also ALT, this is a mistake, sorry, it's a 1000 not to 4000. So it's 10 times, so it's one zero less and the same here. But we have the differences in AST, ALT and ALTH and the mice look quite bad. Therefore, what we did is to isolate the hepatocytes and to check what was going on with the hepatocytes. We did so far six hours, we have done 06, 12 and 24 with 10 millimolar because we didn't want to induce a huge injury in these hepatocytes. And as you can see at 24 hours is when the cells start to die. We did a very nicely phospho-Nrf2 staining it looks fantastic with this antibody. And you can really see that how the nuclei start to get activation after six hours and is complete in all after 24 hours. This was linked also with cell death and tunnel. So we can see that at 24 hours, they had a high cell death and high phospho-Nrf2 staining. What we also did was to isolate the nuclei and perform a Western blot for Nrf2. But interestingly, in a collaboration with the Institute of Physics in Prague with Oleg Lunov, Laura, who was my PhD student and now is at the University of Lausanne in Switzerland. She did, she used a KIPP knockdown to check Nrf2 activation. And in the confocal microscopy, they have a very, very nice microscopy there. And they observed, they saw that KIPP1 knockout when we blocked KIPP1 in hepatocytes these cells induce mitochondrial fission. So we advanced in understanding why was that probably the effect that we were seeing in KIPP1 delta-hepamines was linked to mitochondrial fission and DRP1 which is a marker of mitochondrial fission in these mice. This is what we are doing at the moment. I want to show you also some of the preliminary data that we have with sRNA of KIPP1 in cells, in hepargies. And this, as you can see, the treatment with, sorry. The treatment, I'm sorry about that. The treatment of these cells with APAP after 24 hours in combination with Nrf2 activation by blocking KIPP1, you can already see that the mitotracker signal is increasing significantly. These are nice data, but I will, the next time you invite me, I will give you more details because we are running out these experiments to finalize the study. Just to wrap up my talk, I just want to show you what happens in these mice with CCL4 and what happens is basically the same. We have activation of phosphor Nrf2 when we challenge KIPP1 delta-hepa. We also check all these antioxidant response elements like the, as I said, the quinone and NqO1 and it's significantly overexpressed. You can see here in the, this is the mRNA expression, GCLC as well, and GST, which is another marker of the antioxidant response after CCL4 in these mice was significantly induced. And this is how the livers look like. The necrotic foci, they are also, they were much larger than in flux mice and the cells were dying. And what we can see also here, these are the normal transaminases. This AST, ALT, and LDH were significantly higher in KIPP1 delta-hepa mice treated with CCL4 after 24, 28 days. This is just the inflammatory profile of these mice that they have more infiltrates of CD45, CD11B, and F4AT probably resident and non-resident macrophages in the liver. And this is how the serous rep looks like in these mice. They have significantly expression of deposits of collagen in KIPP1 delta-hepa mice after 28 days, more expression of alpha-semicellactin, and this is collagen 1A1 immunofluorescence. So therefore, these mice, these KIPP1 delta-hepa mice are more sensitive to both APAP and CCL4-induced liver injury, one acute and the other one more chronically, although we have tested CCL4 at very short times. So just I want to thank my laboratory. Hanhan did all the experiments regarding the XPP1 together. Laura, who started all the project with NRF2, Alejandro is now continuing these experiments, and Hui was the one performing the XPP1 delta-hepa mice with APAP. I want to thank all our collaborators and all the source of funding that we have now in the lab. Thank you very much for having us today. Wow, fantastic. Javier, what a tour de force. Sorry? What a tour de force. So much data. You showed us so much unpublished data. I have so many questions. You know, I barely have 45 minutes to talk. So all the talks are 15 minutes. So now I have to take advantage of so much time, you know? Yeah, usually the talks are like 20 minutes, right? Like, so you don't know what to show, but the beauty of your work is that everything ties in so well together. I'm going to start us off with questions. Please, everyone, turn on your camera or put in there just quickly that I have a question and then I will, let's see, Orian also has a question. So I'll go first and then I'll give it to Orian. My question to you is, you know, I've been in this field for a long time and I've switched to checkpoint inhibitors. So I don't know the most recent literature, but clarify for me the whole issue, for example, with CHOP, if you, with the three arms of the UPR, if you inhibit XBP1 or knock out XBP1, wouldn't CHOP still be activated by a PERC or another pathway? So this is a great question. There is a journal of hepatology on CHOP. I don't remember, it was like a few years ago, maybe two or three years. And when you block CHOP, it's also protective, you know? So what we saw is in normal conditions, when you splice XBP1, you have also induction of CHOP, but in XBP1 knockout mice, you don't have a junk activation and you also don't have CHOP expression. So it must be some interaction that we don't know between XBP1 and CHOP. Yeah, but I agree with you that it should be- It should be like you said, no? I was expecting to have a still CHOP in XBP1 knockout mice, but we don't have. Yeah, maybe XBP1 is like more important and more upstream than we realize. Also, it's very, I want to compliment you on, I have a hard time with phospho-IRE1. We tried it for a very long time. I want to compliment you on your blots. They look great. Aryan, go ahead. Hey, great talk. Lots of data. We're trying to keep up. The data on the NRF2 overexpression are kind of surprising because if you down-regulate, keep one, you should get more NRF2 activation, which really should be protective, isn't it? With things like APAP, I'm not so sure about CarbonTED, but especially with APAP, that's kind of been shown before. So I was wondering, you're sort of showing different. Your mice are more susceptible. So I was wondering what's going on. So I can give you some, well, this depends, highly depends on the time when you do the knockout and the experiment. So if you do the, so this depends on, if you do it at two, six hours, 12 hours, 24 hours, during the course of the injury, or let's say the challenge of APAP, the response is different. And there was a small paper, I think, in scientific reports showing with very few mice that they were protected somehow. However, after 24 hours, all the mice start to die because they have very strong NRF2 activation. So it might be that at a very short time, at a very short time points, like two, six, four hours, when you have the high peak of junk activation, it might be lethal and then replenish, and then again, it's lethal. So it has to have maybe some waves. So you think that delayed over activation of NRF2 is a bad idea because it does something to the regenerative process, maybe? Yeah, it is. There's some data on that. It is because there have been some publications showing exactly that, well, with this longterm NASH model that they become HTC when you block one of the parasites. Right. They are a little bit better. They don't get tumors, but they get a very strong NASH. Right. All right. I see what you mean. Okay. Interesting data. Now, we certainly need more experiments. That's why we have been doing this for a long time already with this project, and we are blocking them in different cell types to see what is the specific role of NRF2 activation and keep one in sequestering, no? Right. NRF2 in the cytoplasm. And, you know, Javier, actually, I was thinking about this and, you know, Partha just also messaged, I don't know if he wants to ask you himself as well. You know, when we give acetaminophen as well, we previously, I haven't worked on this again for years, we used to see, you know, CHOP-UP regulation. Okay. And GRP78. And I don't know what sort of a stress response that is, but I guess Harden is asking, do you know what exactly activates ER stress? Well, this is, what's the first, the chicken or the egg? I would say it's oxidative stress, in my opinion, because if you block oxidative stress, so if you give an NAC, you see a lot less XPP1 expression. So it might be that the oxidative stress is first coming and then ER stress, but it can also be linked to mitochondrial dysfunction. So there are cell compartments, which are very, very important here, no? This is very difficult to answer. Yeah. And then one other thing that I had a question about is, I'm trying to interpret the results of the experiment where you had a knockout JNK1 and 2, SP600125 was still protective. So in the hepatocytes. So the interpretation could be that either SP600125 have off-target effect, has off-target effects, which has been suggested, right? Because you controlled with the MSO in the other arm. Yeah. Oh, it's not the MSO. Or there's some effect of the non-parenchymal cell JNK on hepatocyte death, right? I don't know if you can like pull up that, pull up the imaging, but it was very clear that there's less cell death when you added. Yes, yes, absolutely, absolutely. Although in vitro, there's no, when you do it in vitro, there's no other cells. So what's going on? Yeah, but it's still, if you do it in vivo, you can see already that the non-parenchymal compartment plays a very significant role. So the JNK in the non-parenchymal compartment. So this, we cannot exclude it. In vitro, obviously it's clear with the experiments with primary hepatocytes, the off-target effect of SP was confirmed with the proteomic analysis that we ran. But what is that target? What do you think? How is it? Well, it was the guy who analyzed, we said in the paper, the guy who analyzed, we put a supplementary data, all the proteomic analysis, and it was a protein phosphatase 2C. So it's one of the protein that activates phospho-MPK, or it's supposed to interact with the conformational changes of PMPK. So this is what I can tell you so far, but for me, it was not clear because for that, you need another paper. Yeah, I mean, the off-target effects were very clear. However, I can tell you that after that, after doing this, running this gastroenterology paper with the junk and everything, then you can see that this junk 1, 2 knockout, they have a specific or a spontaneous phenotype. You know, they have increased cell death in the liver by itself, if you don't touch it. After 33 weeks, they start to develop these cysts. And these cysts are like, they have been reported several times by a group of Tonlude also. This is like Caroli's disease, you know, or Caroli's syndrome. So this might also influence a little bit the phenotype that we observed, although we did it with eight-week old mice that they were still okay. But after 33 weeks, you already see these cysts. And after 72 weeks, you have already the presence of cholangiocarcinoma sometimes, at least cholangioma. Yeah, that's what I was going to say. Caroli's is a disease of the bile duct plate. So it's very- Exactly. So you have hyperproliferation of the bile duct. I think the way you guys do your hepatocyte-specific knockouts, I remember that it was, you knock it out like alpha-fetoprotein or something. You knock out hepatocytes and cholangiocytes. It's not like a TBG CRE or a specific- No, no, no. We had both in our hand. We had the albumin CRE and alpha-fetoprotein CRE. So albumin is supposed to start expressing eight days postnatal. And alpha-fetoprotein, as you said, is both hepatocytes and cholangiocytes, yeah. Yeah, yeah. So that's why you see that. Interesting that you see cholangio. That J- Yeah. Because I don't know a lot about the role of JNK in, you know, like in PSC models or in, I'm sure it's been studied. Yes, yes, yes, yes. Biopaths as well? Yeah, yeah. I think Christian Traubens is still working on junk and PSC and primary biliary cirrhosis as well. With models of bile-like ligation. And I can tell you that one of the last papers was junk knockout animals in a model of PSC. They had the increased apalene receptor signaling. So apalene was supposed to be the link between, they also observed this in patients, you know. In patients- Did it make things worse there too? So did it- They have a very, they exacerbate all these hyperproliferation. So Zika-19 is all over the parenchyma, the liver parenchyma. This cholangiocyte proliferation. Yeah, yeah. Very, very interesting. Are there any other questions before we wrap up? It's 9.01. I think that's it. I think on behalf of everyone, I want to thank you for sharing all this unpublished data. We appreciate it. Thank you very much for this kind invitation. I'm very glad to see you very soon in person in San Diego. Absolutely. See you in San Diego. Okay. So have a nice day. For me, I have to wrap up almost the day. Yeah, yeah. Absolutely. Bye everyone. Okay. Bye, Jocelyn. Thank you.

hepatotoxicity

endoplasmic reticulum stress

autophagy

acetaminophen toxicity

XBP1

drug-induced liver injury

JNK signaling pathway

NRF2 signaling

liver fibrosis