Multiple Sclerosis Discovery -- Episode 10 with Dr. Richard Ransohoff

[intro music]   Host – Dan Keller  Hello, and welcome to Episode Ten of Multiple Sclerosis Discovery, the podcast of the MS Discovery Forum. I’m your host, Dan Keller.   This week’s podcast features an interview with Dr. Richard Ransohoff about his group’s latest research. But to begin, here is a brief summary of some of the latest developments on the MS Discovery Forum at msdiscovery.org.   We reported on two research articles on B cells published in “Science Translational Medicine". The authors suggested B cells are activated in the peripheral lymph nodes before migrating to the CNS in patients with MS. It appears that after activation the B cells travel back and forth between the CNS and the lymph nodes. The findings have implications for monoclonal antibodies rituximab and ocrelizumab, which reduce peripheral B cell numbers, as well as natalizumab, which prevents lymphocyte migration across the blood-brain barrier.    Together, MSDF and our non-profit publisher, the Accelerated Cure Project – ACP – are committed to speeding the pathway toward a cure for MS. Among the news and resources we provide is a list of more than two dozen tissue repositories, including ACPs. Visit our website and click on the “tissue repositories” button under the “research resources” tab to browse through repositories from all over the globe.   Another part of our goal in working faster toward a cure is to provide a place where researchers can share their experiences and expertise with one another and also debate controversial issues in MS research. We encourage researchers and clinicians from all disciplines to log onto our forum and discuss their latest research, techniques, and discoveries. We also encourage you to help keep us up-to-date on your latest work by e-mailing us directly. Send information you’d like to share on job postings, meetings, funding opportunities, or other news to [email protected].   [transition music]   Now to the interview. Dr. Richard Ransohoff is the director of the Neuroinflammation Research Center at the Cleveland Clinic and a member of the MSDF scientific advisory board. His research group focuses on chemokines and chemokine receptors. He met with our senior science writer, Carol Morton, to discuss his latest findings.   Interviewee – Richard Ransohoff Glad to be here with you today, Carol. The paper that we just published is the result of at least five year's of work. The fundamental question that we tried to answer in this paper I guess if we take a step back and say one looks at an active MS lesion, and one sees a tremendous infiltrate of macrophages, which are clearly involved in the removal of myelin, which is the fundamental problem in MS. And because of the fact that these macrophages have two sources – meaning that some of them come from microglia that are resident within the brain and are macrophage like; and others are differentiated from blood monocytes that have infiltrated the tissue – it's worth wondering whether all the macrophages that look identical by their size and appearance and tissue staining are also functionally identical. And in the case of an MS like disease, by functionally identical, what we mean is are some of these cells that live in the tissue or in the lesion are some of them good guys and others are bad guys, or are they all uniformly bad guys? The reason it's worth asking that question – beyond simple curiosity – is that if you can figure out who are the good guys and the bad guys the treatment approaches become much clearer to you. So for example, if the infiltrating monocytes are the bad guys, then it's possible to apply certain kinds of medications simply to keep them out of the lesion, keep them in the bloodstream. And we already have medicines that we use in MS that take that sort of general approach. So medicines like natalizumab, which blocks cells from leaving the bloodstream and going into tissue, is one. A medicine like fingolimod, which locks up some cells in the lymph nodes and also may stabilize the blood-brain barrier, is another. And a medicine like alemtuzumab that simply removes a large fraction of the cells in the bloodstream that could participate in the tissue damage is a third. So we do know how to treat the blood cells in the way that I'm describing. Most of that work is focused on lymphocytes, not monocytes. But it could certainly be refocused. On the other hand, if the microglia are the bad guys, then the approach becomes completely different, and it's important then to figure out what sort of molecular cues are the microglia responding to and how can we block that part of the MS disease process.    Interviewer – Carol Morton Because the microglia already are in the brain. So the existing medicines have to do with peripheral immune cells that are coming into the brain during disease, right?   Dr. Ransohoff Correct. It would be important, then, to get the medicine in the brain. And when you take that approach, you have to really be very careful that the medicine doesn't have adverse effects on other brain cells. So we started from position that there were no existing markers to identify these cells in the tissue. And we were lucky enough to find out that recently developed genetic models, which tagged different myeloid cells with different fluorochromes could be used to discriminate between the monocyte derived macrophages and the microglia derived macrophages right at the onset of EAE. It turned out that we could first ask which of the two cell types contained myelin debris at the onset of EAE. That seemed like the simplest way to go after our question because we really wanted to know which cell was damaging and taking up myelin. And it turned out that both cell types had myelin in them, and so that really didn't answer the question for us. And if we gave it another moment's thought, we realized that some cells could be damaging myelin, and other cells could be removing debris. And we know that debris removal is part of the repair process. So we had to think of another way to address the problem. And through a relatively long process of developing different techniques, we figured out a way to adapt the data from studying conventional fluorescence confocal microscopy images of the cells and the tissue so that we could identify the same cells in EM sections. Because we had to go to the EM level really to answer the question that we were going after. And we used a new instrument at the Cleveland Clinic, which is one of only a few of this type of instrument at the time around the world, and it allows you to construct a 3D EM picture of certain tissue elements. And so, we set about doing that, and we identified the monocytes in the tissue and the microglia in the tissue using that approach, and we also pointed out how these two cell types were interacting with what you call the axoglial unit, which is the myelin wrap around the axons in the white matter of the spinal cord of the mouse. And we did quite a few samples from quite a few mice. And the answer to our question was very clear. Just to make it simple, the monocytes were the bad guys – they were attacking myelin and removing it from axons even when the axons appeared perfectly healthy and normal, and the myelin appeared normal where it wasn't being pulled off by the monocytes. And the microglia seemed a little bit later to begin to remove the debris. And the answer was pretty unambiguous; we never found microglia attacking myelin directly. And there was a very satisfying resolution to something that had been bothering us for a long time. We also got extra bits of information beyond what we were looking for. We found that the monocytes seemed to attack the axoglial unit not at random but at only selected places, and it seemed as though they focused primarily on the nodes of Ranvier. And it seemed as though that complicated structure was particularly vulnerable to attack. We think that that's because these structures – being as complicated and delicate as they are – become somewhat disrupted in the inflammatory environment, and the monocytes recognize something and take out after it. So we're now very intent on figuring out what the molecular signals are that attract the monocytes. We think that'll be important to know. At the same time, we thought it was important to figure out whether the gene expression in the monocytes and microglia reflected the different behaviors that we saw. And we found that the gene expression profiles in the monocytes and microglia were extremely different. The monocyte profile was almost exactly what you would predict if you watched their bad behavior and that was one of increasing many, many genes associated with inflammatory signaling, production of inflammatory mediators, and most importantly phagocytosis. The microglia, by contrast, showed a lot of downregulation of genes that are expressed in microglia from the healthy brain. So the microglia seemed to be repressed from doing things that they do in the healthy brain, and their metabolism seemed to be in a sense shutoff. They showed an inhibition of the ability to express genes by making RNA to maintain cytoplasmic and cytoskeletal organization to carryout phagocytosis and to make inflammatory mediators. So the microglia seem very repressed by comparison to microglia from the healthy brain. And so, our followup plans for the microglial side of things is to understand whether that phenotype persists for very long because the microglia need to be quite active in the process of repair and make a contribution to that process. And if their metabolism is sort of slowed down and stunted, they may have troubles to do that. So we believe the way they ask they question is to do not only additional gene expression studies but epigenetic studies, as well. And the epigenetic question we're asking is whether DNA methylation, which will turn off gene expression quite efficiently and in a long-lasting way, is changing as the microglia go from healthy brain to EAE onset and finally to recovery. We have some strong preliminary data that the gene expression decreases that we've already seen are associated with DNA methylation changes so they may be quite long-lasting. And we want to follow it through until the mice begin to recover and remyelinate to see whether the microglia recover their ability to contribute to repair. The reason we think that question is crucially important is because if this is characteristic of what happens in multiple sclerosis there may be ways to intervene such that the amount of damage is limited – we already do that pretty well – but also so the amount of repair is maximized. And I'm not certain that we understand how to do that as well as we would like.    MSDF You're calling them both macrophages. Is that a certain state of activation?   Dr. Ransohoff So when I say macrophage, it's a kind of a shorthand term for a relatively activated form of a mononuclear phagocyte in a tissue doesn't tell you where it came from – doesn't tell you if it came from a monocyte or a tissue macrophage or some other source – it just tells you what its lineage is, it's a mononuclear phagocyte, and that it's in a tissue and is displaying signs of activation.    MSDF I understand that the difference in the identity between the microglia and the macrophage has been a long-standing problem, and it sounds like you leapfrogged ahead of that to look at the functional difference. So is your study the last word on that? Is there other evidence that reinforces it, or I don't want to say contradicts but…   Dr. Ransohoff Are there other ways of looking at it?   MSDF  …or unresolved issues that it raises, like new questions that it raises. And I'm curious about the implications for thinking about that in mice and then the implications in people with MS.   Dr. Ransohoff It's always important to be mindful of the limitations of one's research approach. So we try to do that. The approach that we took had a bunch of technical reasons. Our fundamental question was are monocytes and microglia different in EAE tissues? In order to ask that and in order to ask it in a sort of a meaningful and balanced way, we chose the moment of onset of the EAE. And the reason was, first of all, that we thought the most important elements of damage to the myelin were taking place at that moment. So we wanted to know how did the disease get started. And secondly, because the preliminary work that we did showed that the monocytes and microglial cells were present in those lesions absolutely gathered together in a small space, densely intermixed with each other, and in equal numbers. So we weren't biasing the case against one cell or the other; there were equal numbers of both, and they were in the same time and place. So that's a good rationale. At a technical level, the labeling that we were using is most efficient at that moment. And so, there were lots of reasons to do it that way. That tells you a good deal about what's going on at that moment; it doesn't tell you what the fate of the monocyte derived cells is. And it doesn't tell you very much about the function of the microglia. The reason that the microglial function is a open question and an important one is because two groups – one about 10 years ago, Frank Heppner and Adriano Aguzzi and others; and then more recently Marco Prinz and his colleagues – published very strong evidence that microglia play some undefined, important role in EAE. Prinz's work is, I think, the most direct and convincing. He used a way to delete a particular inflammatory gene from microglia, and he showed that in mice where the microglia lack this inflammatory – it's a major contributor to inflammatory signaling, called TAC1 – those mice are almost resistant to EAE. And so, if you put his work together with ours, you really have a fascinating, open question. And that is okay, if the microglia at the onset of EAE look very repressed and nonfunctional, but the microglial inflammatory response is necessary for EAE to occur, then it suggests that before the onset – at some early time point – there is something the microglia are doing that makes the tissue able to support the EAE process. And so, we're going to go after that; we want to understand that to some extent. And it also, as I said, it leaves open what are the microglia doing in the process of repair, which we think is very important. So I think it is always the case that new bits of research open up new questions. We hope that we've opened up good and answerable questions.    MSDF  Great. Well, thank you. This is…I appreciate you taking the time to do this. So is there anything else that I should be asking about this study or more broadly that you wanted to emphasize…add or emphasize?   Dr. Ransohoff The only thing I want to add is that this is a particularly exciting time for this type of research. It's a time when all the work we've done over the years in MS begins to pay off not just for people with MS but also for people with other types of brain disease. I think that the people who have studied MS over the years have a unique contribution to make to understanding diseases that we worry about like Alzheimer’s disease. And vice versa people that study Alzheimer’s disease and other sorts of degenerations can make a contribution understanding MS. So it's a kind of coming together of these two different types of research.   MSDF Can you elaborate a little bit more? Is that because more of the basic science is understood, and people can apply it in these different disease contexts?    Dr. Ransohoff I think it's a combination of things. One is that people with a background studying MS have spent their lives working on inflammation, and a good deal has been learned. The thing that makes that now applicable to other conditions is that it is becoming clear that every time there's any sort of injury to the brain there will be a reaction. And elements of that reaction will resemble what you call inflammation, and that will lead to either repair process or further damage depending on the nature of the disease and other factors that we don't know about. Those of us who have worked on MS can make a contribution to that research enterprise by sharing what we know about inflammation. As I say, it's a time which is exciting, and I think we'll be productive.   MSDF  You're so good at explaining all of this. Thank you so much.   Dr. Ransohoff Oh, thank you.    [transition music]   Thank you for listening to Episode Ten of Multiple Sclerosis Discovery. This podcast was produced by the MS Discovery Forum, MSDF, the premier source of independent news and information on MS research. MSDF’s executive editor is Robert Finn. Msdiscovery.org is part of the non-profit Accelerated Cure Project for Multiple Sclerosis. Robert McBurney is our President and CEO, and Hollie Schmidt is vice president of scientific operations.    Msdiscovery.org aims to focus attention on what is known and not yet known about the causes of MS and related conditions, their pathological mechanisms, and potential ways to intervene. By communicating this information in a way that builds bridges among different disciplines, we hope to open new routes toward significant clinical advances.   We’re interested in your opinions. Please join the discussion on one of our online forums or send comments, criticisms, and suggestions to [email protected].   [outro music]