Kim M. Olthoff, MD: Molecular Pathways of Injury and Repair in Liver Transplantation
As you know, the liver is one of the very few adult quiescent organs that has the ability to regenerate when something happens to it, as shown by Prometheus here who unluckily stole fire from the gods and therefore had his liver eaten out every night, but it was able to regenerate every day. Therefore it was eaten out every night again, so unfortunate for him, the liver was a regenerative organ.
The liver has the ability to go from a quiescent stage, then with some sort of stimulus, such as partial hepatectomy or injury, goes from the G0 phase into the S phase and into DNA synthesis and replication.
[ Slide 02 ] Transcriptional Control of Liver Regeneration
This is the proposed model of transcriptional regulation of liver regeneration that has been outlined by my collaborator and mentor, Becky Taub, where some sort of injury, such as partial hepatectomy, is performed and with some stimulus, and it's still not clear exactly what the initiating stimulus is, there is a release of constitutive cytokines. Most people feel it's from the Kupffer cell, but we'll address that later. There is induction of transcription factors within the hepatocyte, positive feedback causing more release of certain cytokines resulting in immediate early genes, glucose regulation as well as increase in other transcription factors and finally DNA synthesis.
Recovery after liver transplantation is something where I've tried to apply some of Becky Taub's work. There have been many elegant studies that have been performed looking at what causes injury at both the graft level, at the clinical level, at the cellular level and at the molecular level. Some of them have been in Tim Billiar's lab as well. But not too many people have looked at how the liver can recover and what cell and what molecular cascade of events causes the cell to recover and regenerate in response to the injury that has happened after liver transplantation and ischemia-reperfusion injury.
There's certain cytokines and growth factors that have been associated with ischemia-reperfusion injury in the liver and there is now some studies that have shown that there is activation of some transcription factors in the liver after ischemia-reperfusion injury. And what our lab is now focusing on is the role of the cell cycle and initiation of regeneration within the liver after injury induced by ischemia-reperfusion. So this morning I'd like to talk about two lines of investigation that my lab is looking at. One is the actual looking at the pathway that's involved in recovery of these cells after ischemia reperfusion in the transplant model and also looking at the cells that are actually contributing to the cytokines and growth factors that are inducing this pathway.
We initially focused on three transcription factors that have been known to have an important role in hepatocyte regeneration. STAT3 is one of them. The STATS have been shown to be very important in cellular proliferation and STAT3 in particular is very important in this role in moving from G1 to the S phase within the hepatocyte. It's induced by IL-6, which has been shown to be extremely important in liver regeneration.
NF-kappa B is another transcription factor, initially described in lymphocytes and important in immunology and inflammatory responses. It's also been shown to be induced immediately after partial hepatectomy within the liver. It's a TNF responsive transcription factor. And in response it then induces a multitude of inflammatory and other cytokines and growth factors that then have an effect on the rest of the cell cycle.
AP-1 is a transcription factor that includes the Jun and the Fos families, which dimerizes in response to IL-6 and TNF alpha, also shown to be very important in cellular proliferation.
So the aims that my lab first looked at was to decipher the pattern of initiation of molecular pathways involved in hepatocellular recovery following ischemia-reperfusion injury and to show that much of the recovery is dependent upon the initiation of these cytokine-responsive transcription factors and the cascade of events of the up-regulation of early genes following injury. We also wanted to determine the consequence of constitutively expressed and up-regulated cytokines on these pathways and the source of these cytokines.
We utilized a rat liver transplant model and initially our studies have been in a syngeneic combination in order to eliminate the added complexity of the immune response. We looked at three groups with minimal ischemia where there's little injury, moderate ischemia where there's more significant injury, and a 24-hour model where it's actually a nonsurvival model and none of those animals survived longer than a few hours.
We utilized several molecular biology techniques in order to look at the various pathways involved in recovery in our proposed model, looking at cytokines, transcription factors, immediate early genes and finally regeneration. So this is a RT-PCR of intragraft cytokines within transplanted liver grafts. This is the first group, which has very minimal ischemia, you can see there's induction of both IL-6 and TNF alpha, both having been shown to be very important in liver regeneration. They're up regulated in the group with longer ischemia time and very minimally up regulated within the sham animals.
And then to follow this is the up regulation of the transcription factors, STAT3 as you see, and AP-1 and NFkappa-B are all much higher... much more activated in the animals that have much longer ischemic injury, cold injury and following reperfusion. You can see that in the groups that have the 24 hour ischemia, there's an initial activation at about two hours of all three of these transcription factors, but by four hours, and this is about where the animal is dying, there's hardly any up regulation at all, so there's an initiation of the cascade, but the animal or the graft is unable to proceed and continue along the pathway of repair.
This is the immediate early genes that we looked at by Northern analysis. If you look at these four genes, the jun, the fos, the c-myc, the c-jun, these are four genes that have been associated with hepatocyte regeneration and with longer ischemia time you see increased up regulation of these immediate early genes. HPX or hemopexin, is an acute phase reactant and that's equally up regulated in both, so just the actual event of transplantation causes equal up regulation. But you see much more of an increase in the immediate early genes involved with regeneration in the animals that had longer ischemic time.
And finally the initiation of this pathway should result in DNA replication and recovery of the animal by replacing lost cells or injured cells. We looked at BrdU labeling of these grafts 48 hours after liver transplantation and we did see that there was a marked increase in both parenchymal and nonparenchymal cell labeling, showing increased DNA replication.
And these are the actual slides. You can see that with minimal ischemia, there's little injury, the cells look quite normal. There is some BrdU uptake, much more than the sham animals, but it's not a significant amount, whereas with the 12 hour ischemia where there's areas of necrosis and infarction within the liver, there's much more increase in BrdU upregulating almost equal to the amount you see with a 70 percent hepatectomy. And this was a surprise to many people. We haven't really seen that in this setting. Not too many people have looked at regeneration and recovery from ischemia-reperfusion.
This is just a sham animal showing that there's very little uptake. I wanted to show this slide just because - maybe it's hard to see from the audience - this is the 24-hour ischemia livers and obviously the longest we got out was four hours because the animals all died. But there was a significant amount of hepatocyte ballooning, showing significant injury to the cells.
So in conclusion to what we had looked at in the regeneration following the ischemia-reperfusion model, we did see that the inflammatory response induced by cold ischemia and reperfusion of liver grafts was associated with initiation of signal transduction and transcription of genes that were responsible for cellular repair and regeneration. And it appeared that the magnitude of the response was related to the extent of the injury to a certain point, after which the extent of the injury was too great so the cell could no longer respond to the amount of injury and proceed down the pathway to regeneration.
Well, as you know, liver graft recovery is never in isolation and it always has the added complexity of the immune response. There's an interrelationship between injury and the immune response. And that is something that we've been interested in looking at. Significant cold ischemia and reperfusion in the early time points after transplantation, and I mean within the first few hours, may significantly affect the alloimmune response by causing increased allogenicity in the graft itself. Likewise, an alloimmune response may also affect the ability of the organ to repair itself if there's a significant amount of injury. And many of the factors involved, the cytokines, the growth factors, are involved in both the alloimmune response and the cellular response.
We wanted to use IL-6 as a representative cytokine since it was well characterized in the regenerative models following partial hepatectomy, and we propose that the constitutively expressed intrahepatic IL-6 and other inflammatory cytokines that are released from functional Kupffer cells within the liver, and therefore repair and/or regeneration, is directly dependent upon an intact liver macrophage and hepatocyte axis. We wanted to show that there was a distinct relationship between the immune response as well as the inflammatory response and recovery. And even though most people believe it's the Kupffer cells that are involved, this hasn't been definitively proven. So we examined the interrelationship between the cells of the immune system and the pathways that were triggering hepatocyte regeneration.
So for this we utilized a knockout model. We used the IL-6 knockout mice. What we did with them, and it's pretty much well described in the little cartoon down here, the IL-6 knockout mice were lethally irradiated, a bone marrow transplant was then performed with wild-type bone marrow. So we had a knockout mouse but the bone marrow was replaced with wild-type bone marrow. This was allowed to engraft for six weeks and then at that point in time a 70 percent partial hepatectomy was performed. And then we were going to see if regeneration was possible. As a background to this, IL-6 knockout mice show very impaired regeneration. They're not able to regenerate, they show a lot of injury within the liver and they also don't show the usual STAT3 response that you do see with a wild-type mouse. In contrast, if we replaced a wild-type mouse with IL-6 knockout bone marrow, then that would prevent activation of STAT3 and preclude normal regeneration.
So we had to do two things first. First, we had to prove that we had completely replaced the bone marrow-derived cells in the recipient. And we did this by looking at splenocyte IL-6 ELISA in response and we also did a RT-PCR for the SLR locus of the Y chromosome, when we did a male to female bone marrow transplant. And then we wanted to obviously demonstrate a regenerative response within the remnant liver after partial hepatectomy. And we did this both by looking at RT-PCR for intrahepatic IL-6 as well as the STAT3 response, which is very IL-6 dependent, and finally DNA replication by BrdU immunohistology.
So this was first to prove that we had actually replaced the bone marrow and here we see in the wild-type male to knockout females we indeed did see the Y chromosome here. This is the wild-type to knockout male to female. Here's the knockout male to female where it also shows that the Y chromosome has been replaced and these are the controls, the wild-type female and the wild-type male DNA. I don't have the splenocyte data up here but that also showed marked increase in IL-6 production in the wild type to knockout animals.
Then we wanted to show that we actually did replace the IL-6 and that we were able to get an IL-6 response in the IL-6 knockout mouse by replacing the hematopoietic cells within the liver. So this is an intrahepatic IL-6 mRNA following partial hepatectomy pre and post hepatectomy within these animals that had the bone marrow replaced. And although this might be a little confusing, what you can see is these pre-(knockout to wild-type and wild-type to knockout) show very minimal IL-6 response, but the knockout to wild-type post partial hepatectomy also shows a very minimal response, whereas the wild-type to knockout post shows a very aggressive IL-6 response, showing that we were able to get the IL-6 response back once we replaced the bone marrow cells, the hematopoietic cells, within the liver.
Also this demonstrates that we have also had resurgence of the STAT3 activation following replacement of the IL-6 knockout mice with wild-type bone marrow. These are the mice that were IL-6 knockouts that received IL-6 positive or wild-type bone marrow. They have an aggressive STAT3 response whereas the mice who were the wild types and then received knockout bone marrow had a very minimal IL-6 response.
And finally, sort of the proof of the pudding, is in whether it actually induces regeneration. And we can see here that there's, although not as aggressive as we saw earlier in the ischemia-reperfusion models, there is much more BrdU uptake in the wild-type to knockout group as opposed to absolutely no BrdU uptake in the knockout to wild-type group.
So we concluded from these studies that activation of transcription factors depends upon the presence of constitutive and upregulated expression of inflammatory cytokines and that these cytokines appear to come from hematopoietic origin and therefore there's a close relationship between the cells of the immune system and the functional capacity of the hepatocyte to recover and regenerate.
Our future studies are going on to characterize these cells a little bit more by using more of the knockout mice that are available to us as well as the other mice that are deficient in various hematopoietic cells so that we can further characterize whether it truly is the macrophage or other cells that might be contributing to the release of these cytokines within the liver in order to initiate the recovery and regeneration within the injured liver.
I'd like to thank the people in my lab. Most of them are post-docs within the lab and several are surgeons who have been doing all the microsurgery for us for many years. Specifically I would like to thank Avi Shaked, who's always been a mentor for me, but he's also taught me the art of immunology. And also I wanted to mention the Taub lab. I know David Dunn had mentioned the importance of collaborators, Dr. Taub has a genetics laboratory and without her collaboration and help I don't think I would have ever gotten to do molecular biology. They've always been very tolerant of having a surgeon come to their very erudite laboratory meetings. They're rather quite amused by my very erratic schedule, but they've always been extremely supportive of the work.
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