This Q&A accompanies the Editorial entitled Highlight on Dr. Rhonda Voskuhl: a pioneer in the exploration of sex differences in the immune response, published in Biology of Sex Differences' Collection: Sex Differences in Autoimmune Disease.
I (Dunn) was fortunate to interview Dr. Rhonda Voskuhl, a clinician scientist, who treats people with multiple sclerosis (MS), an autoimmune disease that targets myelin in the brain and spinal cord and affects women more than men. Dr. Voskuhl had also made important discoveries about sex differences in the immune system (see the accompanying commentary in Biology of Sex Differences). Her work has also paved the way for hormone therapies in multiple sclerosis. What struck me in the interview was (1) her passion and enthusiasm for research (it was infectious!), (2) her ability to see complex problems with clarity, and (3) how she always keeps her patients at the forefront of her research, even during the preclinical stage. Since Dr. Voskuhl is a pioneer in the sex-difference field, I first asked her about how she became interested in MS and sex differences in MS.
SD: Can you tell me a bit about your training history and how you got started in MS research?
RV: I wanted to be a neurologist, and when I started my residency, the question became, what sub-field in neurology? I fell in love with research after doing a research rotation in a lab at University of Texas Southwestern. One area that intrigued me is: Why does the immune system attack itself? It seemed so odd. This interest in autoimmunity and neurology naturally led to an interest and specialty in MS.
SD: What about sex differences? How did you get started researching this topic?
RV: When I was doing my fellowship, researchers were conducting clinical trials in MS, and there was always a 60:40 or 70:30 split of women to men enrolling in these trials. This naturally led to the question - why women? Since I am studying biology, I needed to study sex differences. I started by running experiments in the animal model of MS, comparing male and female mice, to see what was different between the sexes.
When, you find a sex difference, it is pretty simple, it has to be either sex hormones or sex chromosomes. I have spent my career studying both of these influences on neuroimmune mechanisms.
SD: Your research was amongst the first to report that female mice were more prone to develop MS-like disease compared to males. You were using a particular inbred strain of mice called "SJL" as a model system to study these sex differences. This strain of mouse, in contrast to more commonly used mouse strains such as C57BL6/J, shows a female bias in autoimmunity similar to that seen in humans. What makes the SJL mouse a good model to study sex differences.
RV: In simple terms, the genetic background of the SJL makes the mouse more pro-inflammatory. Because of this, the immunostimulatory effect of having two copies of the X chromosome is more easily seen. Females of this strain are more prone to develop autoimmune diseases. Remember, the key in research is to use a model system that best mimics the process you are trying to understand in humans. In this respect, the SJL is a good model for studying the female bias in autoimmunity.
SD: I was struck by the fact that, before your work, there were very few papers on the topic of sex differences in MS and MS models. Then, all of a sudden, there appeared to be a burst of research in this area. After your first paper, others including Dr. Halina Offner's group at University of Oregon and Dr. Stephen Stohlman's group at University of Southern California, were also reporting on sex differences in the immune system using the SJL mouse. Was there an inciting event that spurred a burst of research in sex differences in the late 1990s.
RV: It is interesting you mention this. These things sprang up independently. I think that more than one person can have a good idea. The world evolves to a point where there is sufficient information in the literature to prompt people to ask the same question independently. It wasn't competitive, and it wasn't collaborative until we became aware of each other's findings as we started to publish and present on our results at conferences.
SD: You, together with Dr. Art Arnold, were the first to apply the four-core genotype mouse model to study the influence of the sex chromosomes on the immune system. For our readers who may not be familiar with the model, this is a genetic model where the gene on the Y chromosome that specifies the testes to develop is missing and instead is expressed on an autosome. When this male mouse is bred with normal XX females, you can generate XX and XY females that have male or female gonads. How did this partnership with Dr. Arnold start?
RV: I had started examining the influence of sex hormones, since it was easier, but I always knew that I had to also look at the sex chromosomes. This was more challenging. When you remove the gonads in an adult or pre-pubertal mouse, and you see a sex difference in your phenotype, it doesn't necessarily mean it is due to sex chromosomes. This is because there are also developmental surges in gonadal hormones in early life that can influence the immune and nervous system.
When I first came to UCLA, Dr. Art Arnold spoke to me and told me about the four-core genotype model. I said, I have to do that! The four-core genotype model is the only model where you can sort out the influence of the sex chromosomes from gonads, since mice that have a difference in sex chromosomes can have the same gonads (either ovaries or testes), so you can examine one variable at a time. Using these mice, we found that there was a clear immunostimulatory effect of XX chromosome complement on the immune system.
SD: What is it about the X chromosome that is stimulatory for the female?
RV: There was a paper that I had read a while back that reported on the genes that were differentially expressed in females and males. What struck me is that there were many of them. That got me thinking that it isn't just one gene, but a host of genes that are differentially regulated between the sexes - not all of these had hormone-responsive elements. We did our own gene profiling studies in female and male immune cells to see what genes were present at a higher gene dosage in females on the X chromosome that could escape from X-inactivation. This led to us to KDM6A, a histone-modifying enzyme. KDM6a (and Kdm6a in mice) fit the model, since it could modulate the expression of many genes through epigenetic changes. So we knocked out the gene in mice, first in T cells, and then in microglia. This led to the discovery that it is immunostimulatory in females in both of these immune cell populations.
I really think that the reason why females are getting MS and why their disease is worsening after menopause is due to this greater expression of Kdm6a. It is pro-inflammatory, and in premenopausal women, this effect is countered by the anti-inflammatory effects of the female hormones. However, after menopause, when a woman loses these hormones, this balance between sex chromosome complement (XX) and sex hormones (estrogen) is lost. Men are different and have different genetic and hormone balances at play. These differences are why we have to tailor therapies differently in men and women.
SD: When you started at UCLA, you also were experimenting with the effects of sex hormones in the MS models. You showed that estrogens and androgens in the animal models were improving the MS-like disease in mice. Though estradiol is kind of the "flagship" estrogen that everyone knows about (the one that fluctuates with the menstrual cycle), you decided to work with estriol. What was your reasoning for studying estriol?
RV: When I started to dissect the mechanisms underlying sex differences, I was doing simple experiments, that is, taking the hormones away by ovariectomizing female mice and then adding hormones back. When you add the hormones back, you have to think about factors such as the type of hormone (e.g., progesterone or estrogen), the timing, and the dose. In these considerations, I was already thinking about the patient. I didn't want to be treating a mouse with a hormone that would not eventually be safe to use in people. Regarding estrogens, the Women's Health Initiative findings were already out there, and there was, and still is, the concern about treating women with estradiol because of the effects of the hormone in increasing breast cancer risk. We knew that estradiol was acting mainly through estrogen receptor-alpha. I started to read about different estrogens and became very interested in the chemistry of estriol, which is an estrogen that goes up during pregnancy, and primarily hits another estrogen receptor, called estrogen receptor-beta, which is not linked to breast cancer. Therefore, estriol seemed to be a safer choice. I also knew that women with MS generally do well when they are pregnant. So, I went all in on estriol and tested it in the MS model and found that it was extremely effective.
SD: I was also amazed at how quickly you were able to translate these basic research findings in an animal model of MS into clinical trials - many researchers take decades to make that leap. Was it easy getting a clinical trial going to test estriol and androgens in people with MS?
RV: It wasn't easy, but as a clinician, I could do the trials, and I had a lot of support from my Chair. When he saw the results of estriol in the MS models, he said, "you need to put this into people". So I learned how to run a pilot Phase 2a, single-arm crossover trial and then after getting promising results, went on to design a larger phase 2b, placebo-controlled trial. We are now seeking funding to run the larger Phase 3 trial in MS, as well as another phase 2 trial for older MS women who are menopausal.
SD: We are at a point where we have a lot of therapies that are effective at treating relapses (i.e., the inflammatory phase of MS), but are lacking treatments that can inhibit the neurodegenerative phase of this disease. Are there any plans to treat patients in the progressive phase with estriol?
RV: We found that estriol does two things, it is anti-inflammatory and can decrease the appearance of new lesions. The other thing that we saw is that it can improve processing speed, slow regional brain atrophy and lower serum markers of neuronal damage. We also showed that hitting estrogen-receptor-beta with agonists can promote remyelination. Putting this all together, estriol appears to be a potent neuroprotective agent and may promote brain repair mechanisms. Though progressive MS may be an indication, the next goal is to try to target women before they enter the progressive phase of disease.
When it comes to MS, progression seems to be age-dependent. When you hit 50, the risk of becoming progressive increases. This is also the age at which the majority of MS patients (which are primarily women) experience menopause. I would like to do a trial where women are put on estriol before menopause to see if we can prevent them from entering this stage of the disease.
With pregnancy, your immune system shifts away from cell-mediated immunity, and at the same time, the hormone environment is neuroprotective. Likely, estriol was evolutionarily selected for to help the baby: prevent the fetus from being rejected, but at the same time preserve the brain if there were any pregnancy-related complications. I believe that these same properties could benefit women with MS and healthy women during aging. We are also examining estriol treatment in women throughout the country to see if we can prevent or improve some of the brain symptoms that occur with menopause, such as loss of working memory and verbal memory.
SD: That is really cool. Estriol therapy is designed for women and this will be a sex-specific therapy. How much has sex been considered in the design of clinical trials testing other treatments for MS?
RV: When you design a clinical trial, there are a number of approaches. One approach, which is the one that was first applied, is to enroll anyone you can get and ask whether the treatment works. This is a flawed design, since if only one sex responds to a treatment and the other doesn't, you lose power and have a strong chance of missing a real effect of your treatment. For an optimal trial, you need to have the basic science done ahead of time to guide the best outcome measures, and to know who may best benefit from the therapy to ensure that you are not diluting-out the effect of your treatment. This has to be the future of MS therapy.
SD: Are there any other examples of sex-specific therapies in the pipeline for MS?
RV: We recently reported on the effect of the X-encoded gene Kdm6a in microglia in Science Translational Medicine. As I said before, Kdm6a was pro-inflammatory only in microglia in females. In this paper, we used a known drug, Metformin to treat males and females. This drug is not only a metabolic regulator, but is also an inhibitor of Kdm6a. We found that it only worked in the females that had the greater Kdm6a activity. If we proceed to a trial, enrolling males would likely dilute out the beneficial effects in females, thus losing power to show a significant difference of treatment.
SD: From a clinician perspective, you have been treating patients for several decades from a time when there was little to offer your patients to present day where there is an arsenal of immune-modifying treatments. However, we still don't have much that can help people in the progressive phase of disease. Do you feel hope for the future that we will find something to help these patients?
RV: There is a tremendous amount of hope. The best predictor of the future is what has happened in the past. We now have 20 treatments for MS. Scientists have done a great job with the immune system and found many ways to target it to inhibit MS attacks. There are many people who are not in wheelchairs because of these treatments. For the next phase, we need to use a two-pronged approach. We need to continue to inhibit the immune system as we have been, but also target the brain--keep the microglia and astrocytes calm, and protect the neurons. We also need to optimize treatments and run clinical trials to find out which treatments are best for a male or for a female. We also need to enact these treatments early in the disease, in the critical window, when we can prevent CNS damage. By giving a person an anti-inflammatory agent, coupled with a neuroprotective agent, we will fix MS. It is that simple. We are almost there!
SD: It is so great to hear your optimism about the future for MS patients!
One last question for you: if you were advising a young post-doctoral fellow or graduate student starting in research today, what advice would you give them?
RV: I would advise them to go into a room all by themselves or go for a walk in the woods and ask "What is the biggest question in the field that they are studying in?" You have to know the literature inside and out to know what is going on. You do not want to study something that someone else is already doing, since your studies will only be incremental.
You have to ask a big question? For me, I started with "Why are females more susceptible to MS". Then, "Why is pregnancy so helpful?" and then "Why is aging a factor in MS progression?".
I also use a "Bedside to bench to bedside" approach, which is different then the "bench to bedside" approach we always hear about. The difference is that I always start with the patient and ask what I can do that will help them. For PhD students who are not clinicians, I would still advise to ask the biggest clinical questions. If they don't know what they are, they can partner with clinicians to find out.
SD: Thanks so much Rhonda for your time. It was a great pleasure to speak with you!