NICHD grantees discover how placenta protects itself from virus infection

In this Research Conversation, NICHD's Dr. John Ilekis interviewed NICHD grantees Yoel Sadovsky and Dr. Carolyn Coyne about their discovery that cells of the placenta secrete tiny, balloon like structures called vesicles.  The vesicles are absorbed by other cells, and help to program the recipient cells to ward off infection by viruses.  The researchers hope to one day transfer this virus-fighting ability to other organs and tissues.

NICHD Research Conversations are audio interviews with NICHD scientists and grantees, on the latest NICHD-supported scientific findings. Listen to this Research Conversation at http://www.nichd.nih.gov/news/releases/Documents/NICHD_Research_Conversation_081313.mp3 (MP3 - 2 MB).

Mr. Robert Bock: Welcome to the National Institutes of Health. I'm Robert Bock, Press Officer for the NIH's Eunice Kennedy Shriver National Institute of Child Health and Human Development, the NICHD. Thank you for joining us for today's Research Conversation. Our host is John Ilekis of the NICHD's Pregnancy and Perinatology Branch. Today, Dr. Ilekis will interview Dr. Yoel Sadovsky of the Magee-Womens Research Institute and Foundation at the University of Pittsburgh, and Dr. Carolyn Coyne in the Department of Microbiology and Molecular Genetics at the University of Pittsburgh. They have published a recent paper in the Proceedings of the National Academy of Sciences on how the cells of the placenta resist infection by viruses. Dr. Ilekis?  

Dr. John Ilekis: Yeah, thanks, Bob. First, let me set the background. The placenta is an organ that connects the developing fetus to the wall of the mother's uterus and hooks up with the mother's blood supply. Essentially, the placenta is a lifeline that conveys oxygen, nutrients to the fetus, and removes waste. In fact, the fetus couldn't survive without it.

A number of disorders and conditions can occur in the placenta and jeopardize the health of the fetus, and often the mother. A big part of the NICHD's mission is to ensure that all children are born healthy, and for this reason we support research on the placenta, to learn how it develops under normal situations and the kinds of things that can go wrong. Information from these studies will be useful in preventing and treating conditions that affect the placenta and hence the well-being of the fetus.

I'm very proud today to welcome our guest, Yoel Sadovsky. Yoel studies the cells of the developing placenta and the molecular reactions that occur within them. He and other researchers in his lab concentrate on three main areas: how the placenta transports nutrients, how it adapts to injury and changing conditions, and the role of molecules called microRNAs that are in the placenta and play a role in the placenta's growth and development.

Next, I'd like to introduce Dr. Carolyn Coyne, who is a partner in the study. She studies how the host cell wards off invading viruses, and her main interest is research in protecting the fetus against microbial infection.

Without getting too bogged down in details, let me first explain that RNAs are molecules that carry out instructions of DNA, translating these instructions to make a protein. There are several kinds of RNAs. Dr. Sadovsky and his colleagues study a particular kind of RNA found in the placenta, and other cells as well, called microRNAs. As the name implies, microRNAs are much smaller than are other RNAs; however, they aren't involved in making protein. Rather, they function to regulate other kinds of RNAs.

Cells produce microRNAs to regulate their function. In addition, cells can also secrete microRNAs in tiny, balloon-like sacs called vesicles. Once these vesicles are secreted, they can latch onto other cells. After attaching to the surface of the cell, the vesicle injects its content into the cell. Once inside a receiving cell, the microRNAs can influence the functioning of a recipient cell as well.

Dr. Coyne and Dr. Sadovsky and the other members of the research team studied trophoblasts. Trophoblasts are the outside layer of cells of placenta, the placental cells that come directly in contact with the mother's uterus. They found that trophoblasts resist being infected by many different kinds of viruses. They traced this virus-fighting ability to a family of microRNAs within a trophoblast cell.

What's even more significant is that these virus-fighting microRNAs could be transferred to other types of cells, where they help the recipient cell ward off viruses also. Their discovery raises the intriguing possibility that this family of microRNAs might one day be harnessed to help cells in other parts of the body to fight off infection with viruses and thus be used as a potential new antiviral therapeutic drug.

Carolyn and Yoel, thanks so much for being here today and joining us. Yoel, I have a first question to you: Could you please tell how you conducted this study and how you figured out that all that activity was going on inside a trophoblast cell?

Dr. Yoel Sadovsky: Sure. John, thank you very much for this superb review. We know that viruses can affect many tissues in diverse types of organisms, and, in fact, almost every living organism can be susceptible to diverse effects by viral infections and its potential adverse impact on different cell types.

Pregnancy is a unique state because of, number one, the need of the mother to protect the developing embryo, the fetus, and, second, because of the potential of immune compromise when pregnant mothers, because of high level of hormones, may potentially be more susceptible to diverse types of infections, including viral infections.

So, Carolyn Coyne came to our lab with the simple question: If we take trophoblast cells―which, as you said, are the cells lining the placenta and are directly bathed with the maternal blood―if we take those cells and culture them in vitro, are they going to be more or less or equally susceptible to viral infections, compared to other cell types in the body, which we know are susceptible?

And, much to our surprise, when we did this experiment, Carolyn has found in her lab, using our cells, that trophoblast cells―the placental primary cells―are much more resistant to diverse type of viruses, compared to other cell types that we used. So, that was our initial observation. It was quite surprising. We infected cells in the same way in parallel experiments, but trophoblast cells, primary human trophoblast cells derived from term placentas, were much more resistant to viral cells, compared to other cell types. That question led us to try to figure out how this actually comes about: What might be the mechanism by which trophoblasts can protect themselves against viral cells?

And the first experiment we did to address this question was to take medium from the trophoblast cells, transfer them to cells that used to be susceptible―that were easy to infect with viral cells―and, much to our surprise, we found that nonplacental cells that used to be highly susceptible to viral infections now became resistant by exposure to medium that used to bathe trophoblast cells.

So, this is how we made our initial observations, led us to believe that trophoblast cells―specifically, primary trophoblast cells from term placenta―produce some kind of chemicals or mediators or molecules that can be transferred to other cells and confer viral resistance to recipient cells.

Dr. Ilekis: Very, very interesting. Yoel, I have another question to ask: So, how did you make the connection that this family of microRNAs or molecules had antiviral properties?

Dr. Sadovsky: We actually found it in a bit of an indirect way, John. When we transferred the condition medium to nonplacental cells, we realized that we may be transferring some kind of a vehicle or a shuttling container that may confer this resistance to the nonplacental cells. And we tried to use all kinds of chemical and mechanical means to try to disrupt that signal, and we found through a series of experiments that that signal is contained within small vesicles that cells produce. They are of nanometer size, so they're called nanovesicles, and in biological terms they're called exosomes. And these nanovesicles, these small, nanosized containers, are able to transmit that antiviral signal to the recipient cells.

When we interrogated the placental-specific types of those nanovesicles, we found out that one of the only things that they contain in a high amount, which is unique to trophoblast cells and not found in other cells that are capable of producing those vesicles, we found that there's a family of small RNA molecules that you alluded to before, called microRNAs. And there is this whole family of those microRNAs that are expressed only in trophoblast cells and cannot be found in other, nonplacental cell types.

This is a family of microRNAs called the chromosome 19 microRNA cluster, or abbreviated C19MC, because of that name. The gene that encodes those microRNAs is located in chromosome 19, the human genome. They are found only in primates and in the human genome, and not in any other lower organisms. And we found that, during pregnancy, if you compare the level of those―of microRNAs from that cluster to all other microRNAs, we found that those microRNAs are by far expressed the highest amount, compared to any other microRNA. And, also, they are highly packaged within those small nanovesicles, the exosomes that I alluded to.

So, to confirm that relationship between those microRNAs within the nanovesicles and the antiviral effect, what we did was, we took―isolated the exosomes only and transferred them to the nonplacental cells, and were able to confer the same antiviral effect by the transferring of the vesicles alone, not the entire medium volume, to the recipient cells.

And then we were also able to reproduce the effect specifically by transferring the microRNAs that come from this cluster, the chromosome 19 microRNA cluster, the C19MC. Those microRNAs, when introduced in isolation into the recipient cells, were able to confer the antiviral effect

Dr. Ilekis: Wow! Very interesting. Carolyn, I have a question for you: From your perspective as an infectious disease expert, and Yoel, based on your work on the placenta, specifically what are the microRNAs doing to help cells fight viruses? And, in addition, is this virus-fighting ability happening only within the placenta or does it benefit other types of cells as well?

Dr. Carolyn Coyne: I guess I'll start with your second question, which is, no, the mechanism by which we have found that these microRNAs work is actually not specific to the placenta and is a process that seems to also be enhanced in these nonplacental recipient cells when we expose them to these placental microRNAs, or these nanovesicles or exosomes, as Yoel just mentioned. And when we began sort of looking into the mechanisms by how these microRNAs could exert such potent antiviral effects, one of the things that I think struck us immediately was that these microRNAs are actually antiviral to really diverse viruses. And so these are DNA viruses, and RNA viruses and viruses that do not really share any sequence homology or any sort of shared characteristics between them.

And so, what that at least told me, as someone that works with viruses, is that it must be some sort of cellular-based mechanism, so that these microRNAs were not directly, you know, killing the viruses. They were somehow working on the host cell to perhaps alter the properties of these cells to make them more resistant.

And so, we looked at a number of kind of pathways that have been described for many years that are antiviral, and we actually found that the microRNAs were upregulating a pathway in cells that exists as a cell survival pathway. It's a process that's called autophagy, which is kind of a complex name, I suppose. But the process is quite simple in that autophagy exists to rid damaged organelles from a cell. And so, it's a pathway that normally exists within a cell to promote cell survival.

And what had been shown previously by other groups was that autophagy could also function as an antiviral pathway in some circumstances, whereby if a cell had enhanced level of autophagy, they may actually be able to resist viral infections a little more. And what we found was that these microRNAs actually very, very potently upregulate autophagy in these nonplacental recipient cells, and that the upregulation of this pathway was actually required for the antiviral effect. And if we blocked this pathway, we could actually now make the cells, again, susceptible to viral infections.

And I think what's really sort of fascinating about this is―then, the question sort of becomes, are the trophoblasts themselves also upregulated in this pathway? And we actually found that they were. So, if we just look at trophoblast cells, they actually exhibit a very, very high level of just basal autophagy, much higher than you normally see in other nonplacental cells. And we have some evidence, at least, that this upregulation in the trophoblast cells themselves is actually one of the mechanisms. They likely have many mechanisms to resist viral infection, but we feel that this is probably one mechanism that the trophoblast cells actually utilize to resist viral infections themselves.

Dr. Ilekis: Wow! Very interesting. So, Carolyn, let me ask you an intriguing question: What would be involved in harnessing this virus-fighting ability, using it to prevent viruses from infecting other kinds of organs and tissues?

Dr. Coyne: Well, you know, it's a question that Yoel and I have discussed often, sort of amongst―you know, we have coffee together once a week and discuss this project. And as we kind of were kind of working on this project, that sort of became the next step, is: How could we use this? It's a really fascinating phenomenon, and I think it tells us a lot of great information about the placenta and perhaps how the placenta has evolved to, not only itself resist infection, but perhaps confer this resistance to other cells.

And what I find really fascinating is that these microRNAs and, presumably, the microRNAs within these nanovesicles or exosomes, can actually be found circulating in the serum of pregnant women. So, women that are pregnant have these microRNAs circulating. And so that would at least indicate that the microRNAs themselves may not have any sort of off-target or potentially dangerous effects.

And so, we certainly thought about, you know, if we could engineer exosomes that resembled the exosomes from placental cells, we could certainly use those as delivery vehicles, to deliver either microRNAs or, potentially, even other forms of therapeutics to fight viral infections. And so that's something that we're definitely interested in and talk about often.

Dr. Ilekis: Well, it sounds like fantastic work, and I know both of you will move forward in elucidating even further. Yoel and Carolyn, I want to thank you so much for telling us about your important work. I should mention that in addition to NICHD, this research was also supported by the NIH's National Center for Advancing Translational Sciences.

Well, I guess that concludes our Research Conversation for today. I'd like to thank Dr. Sadovsky and Dr. Coyne for speaking with us, and I'd like to thank our listeners for joining us. Have a good day.

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About the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD): The NICHD sponsors research on development, before and after birth; maternal, child, and family health; reproductive biology and population issues; and medical rehabilitation. For more information, visit the Institute's website at http://www.nichd.nih.gov/.