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Separating the Bad from the Good in Neuroimmunology
Q&A with Michael Dustin, Ph.D.
November 22, 2010
Michael Dustin, Ph.D.
Muriel G. and George W. Singer Professor of Molecular Immunology
New York University
Dana Foundation Grantee, 2007-2009
Michael Dustin, Ph.D., is one of the world’s foremost authorities on visualizing immune responses in the nervous system. In the 1990s, he defined the “immunological synapse” as the stable interaction between antigen-recognizing T cells and antigen-presenting cells.
With fellow NYU investigator Wenbaio Gan, Ph.D., and funding from the Dana Foundation, Dustin has been using new brain and spinal cord imaging techniques to study the behavior of T cells in real time, in mouse models of meningitis and multiple sclerosis. He recently spoke to writer Jim Schnabel.
What’s the main theme of your lab’s research these days?
Michael Dustin: In general, we’re trying to understand how the immune system interacts with the nervous system. We’re especially interested in determining the roles of different immune cells and their surface receptors in infections or autoimmune conditions that affect the nervous system.
You had a recent project, concerning a meningitis model, which resulted in a paper in Nature. What were you trying to do there?
My colleague, the neurobiologist Wenbiao Gan, had developed a technique known as transcranial microscopy. It involves thinning the skull of a mouse to create a sort of window. Through this window we can use a microscope to track fluorescently tagged cells in the brain or in the brain’s outer membranes, the meninges.
My lab members learned the technique from Wenbiao soon after I joined the Skirball Institute in 2001, and around that time we were urged to use it to look at lymphocytic choriomeningitis virus (LCMV) infection in the mouse, which is a model of human meningitis. It was then widely believed that LCMV meningitis is caused when cytotoxic T lymphocytes (CTLs, or CD8 T cells, or “killer T cells”) attack virus-infected cells in the meningeal membranes surrounding the brain. Our hypothesis was that we would be able to use this direct, into-the-brain imaging technique to see that happening.
You found that the CTLs weren’t acting as expected.
That’s right. We did the paper in collaboration with Dorian McGavern, Ph.D., who at the time was starting a lab at the Scripps Research Institute in California. He had been studying LCMV and the immune responses to it in transgenic mice, and had some perplexing results in mutant mice: key molecules used by the CTLs to kill were not required for the pathology in the disease. They didn’t know what was causing the pathology. So we set up the transcranial microscopy experiments to look at it.
Ideally these CTLs would target the virally infected cells very specifically and then kill them. We know this process is most efficient when a CTL latches onto an infected cell for five to ten minutes. But in the meningeal environment in this LCMV model, the CTLs rarely latched onto their targets for that long and instead stayed unusually mobile.
Why was that?
We aren’t entirely sure. These T cells are responsive to ‘stop’ signals and ‘go’ signals. Usually their recognition of viral antigens on the surface of an infected cell acts as a ‘stop’ signal and makes them stick to the cell and kill it. But it appears that something in the environment of the meninges is providing a competing ‘go’ signal that makes them more likely to keep moving.
The brain is an immune-privileged space, presumably so that brain cells, most of which can’t be replaced, are protected from lethal inflammation. The meninges are on the border between the brain and the rest of the body, so perhaps that inhibiting influence of the brain on the immune system is already being felt there, such that the ability of T cells to kill virally infected cells in the meninges is reduced.
Yet the immune response turned out to be worse, because the CTLs called up reinforcements.
We observed that they put out multiple immune-stimulating molecules. Apparently as a result, a very large number of white blood cells known as monocytes and neutrophils arrived in the meninges. It was only when we blocked these neutrophils and monocytes that we saw a significant delay in the course of the disease.
What does this suggest in terms of designing drugs or other interventions against this kind of process in human illnesses?
MD: Since neutrophils and monocytes turn out to be largely responsible for the fatal outcome, we now can think about ways to prevent or inhibit their entry into the meninges. One way would be to modify the behavior of the CTLs so that they are less likely to promote this invasion of monocytes and neutrophils, and instead perform the targeted killing they were meant to do.
For a more recent publication, in the Journal of Immunology, you did a different set of animal imaging experiments, using a mouse model of multiple sclerosis (MS).
Yes, these involved the induction of an autoimune condition in the mice known as Experimental Autoimmune Encephalitis, or EAE, which is routinely used as a model of MS. The imaging setup was similar to what we had used in the meningitis study, except that here we were trying to image fluorescently-tagged T cells in the spinal column.
How could you look at the spinal cord of a living mouse without damaging it?
You can use a microscope to peek at the spinal cord through gaps in the supporting spinal vertebrae, provided that you immobilize the mouse. We could have bought very fancy, very expensive stereotactic equipment to do that, but instead we followed the practice of Wenbiao Gan’s lab and made a device ourselves, using cheap items such as double-edged razor blades. The opening that each of these blades has, where it fits into a razor handle, turns out to be perfect for making a little clamp for the mouse spinal cord.
The only significant problem with our setup was that it proved to be impossible to immobilize the spinal cord in a healthy control mouse. Normally there is a small layer of fluid surrounding the spinal cord, probably to isolate it from shocks and to allow the cord to bend without hitting the surrounding vertebrae. In healthy mice the cord can still move a bit within that space. In mice with EAE, though, the cord swelled enough that it became immobile, so we were able to do the imaging.
What were you trying to see using this setup?
In EAE, helper T cells, also known as CD4 cells, migrate into the spine and–similarly to what’s seen in MS–attack the myelin sheaths of nerve fibers. We wanted to understand the function of one of the receptors, CXCR6, on these CD4 cells. So we genetically engineered mice that had a green fluorescent protein gene in place of one or both of their CXCR6 genes. These fluorescent proteins became beacons that allowed us to track the altered CD4 cells throughout the course of EAE–to see whether the partial or complete loss of their CXCR6 receptors made a difference to their behavior.
Did knocking out CXCR6 make a difference?
Well, it didn’t make a difference to the course of EAE, which wasn’t significantly delayed even in the mice that completely lacked the receptors. That wasn’t entirely a surprise, since these immune cell receptors often perform functions that are covered by other receptors too. The immune system has a lot of redundancy built into it, for obvious reasons.
We did find, however, that CXCR6 is necessary for an added pathological feature of MS that isn’t normally modeled in EAE mice, but that we were able to model. In some MS patients, T cell infiltration occurs not just in the spine and the white matter nerve bundles in the brain, but also in the nearby gray matter, the neurons.
One hypothesis is that this infiltration occurs due to ordinary tiny lesions in the brain because of head injuries or tiny strokes. The T cells are already in the vicinity and they migrate towards these injuries. Using our skull-window setup and a laser, we were able to model this cortical pathology by creating tiny lesions in the cortices of these EAE mice and observing the migration of nearby CD4 cells into these lesions. But the migration didn’t happen in the CD4 cells that lacked CXCR6, so we concluded that CXCR6 is necessary for this cortical migration. It isn’t sufficient, because there has to be a nearby injury to which this migration occurs, as well as a general state of inflammation.
But what you really want is something that stops MS earlier in the course.
Right, and in terms of targets we’re continually looking for an immune receptor or other factor that has a non-redundant function in MS, and isn’t just generally necessary for T-cells to move around and function. Certainly we’ll be using our new brain and spinal imaging techniques to test out our ideas.
To understand the challenge, consider the most effective MS drug to date, whose brand name is Tysabri. It blocks the VLA4 receptor, which T cells normally use to move around effectively, and it thus inhibits T cells from migrating into the spinal cord. For most MS patients that’s an effective, even life-changing therapy. But this impairment of T cell migration reduces the immune system’s ability to fight a rare viral infection called progressive multifocal leukoencephalopathy (PML). A number of patients taking Tysabri have come down with PML, and some have died.
So the big challenge here, as in other conditions involving autoimmune responses or excessive immune responses to infection, is to find a therapy that can specifically target the wayward immune response without impairing the immunity that a person needs.