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MESSAGE IN A BOTTLE: How Cells Talk When They Are Sick

By Jon Kaletka

Human cells infected with Listeria (green) that grows inside of our cells

(Dr. Melinda Frame/Jon Kaletka)

There are countless apps to keep in touch with friends and family throughout the world. But have you ever wondered how your body's trillions of individual cells talk to each other? That's what I study to improve the diagnosis and treatment of bacterial infections.

An emerging field of cellular communication is the way tiny particles secreted by cells carry messages to other cells. Similar to how we use mail to stay in touch, these particles – called extracellular vesicles - deliver the messages to the other cells that change how these cells function. These vesicles are very small, ranging about 0.1-1 micrometer in size. For comparison, the cells in our body, such as the white blood cells of our immune system which we can only see with a microscope, are about 10 micrometers in size.

Extracellular vesicles are not just found with healthy cells…a wide range of diseases utilize their messaging abilities as well. For example, vesicles from tumors prevent the response to diseased cells, allowing the cancer to grow and spread. Extracellular vesicles provide a new way of understanding various diseases, could lead to different ways of treating them.

I study the role of extracellular vesicles during infection caused by Listeria. If this bacterial pathogen sounds familiar, you likely saw it in the news about recall of contaminated food because of it.

The large objects are extracellular vesicles, which carry messages between cells in our body (Dr. Alicia Withrow/Jon Kaletka)

An important part about Listeria infections is that it affects different portions of the human population differently. Specifically, pregnant women are much more likely to be infected. In fact, this is why pregnant women are recommended to avoid eating soft cheeses and deli meats, foods that are more commonly contaminated with Listeria. Often times Listeria-infected mothers show at worst mild symptoms, but the infection can be devasting on the developing fetus, as it often results in spontaneous abortions, birth defects, and stillbirths.

Listeria can also live inside healthy cells, even ones that normally kill bacteria called immune cells. Think of an immune cell as a jailhouse that contains the bad guys. When the criminal escapes custody, he starts destroying the jailhouse, making it useless. Then he moves onto other buildings, leading to chaos across the city.

This is how Listeria attacks a body – by invading, destroying, and spreading through cells. Listeria travels cell-to-cell, spreading throughout the mother’s body to invade and attack the placenta, which facilitates the interactions between the mother and the fetus. Our cells need to organize a proper response to combat this invasion, and I predict that one way to do that is to use extracellular vesicles.

To study this infection, I use a model of a type of placental cells, called trophoblasts, that are isolated from mice and can be easily grown and manipulated in the lab. I am able to efficiently research the interactions between Listeria invaders and the placenta because of these trophoblast cells.

The first step in understanding how Listeria infection changes the production of extracellular vesicles is getting the vesicles from the trophoblasts. The cells are grown in a special liquid solution that keeps them alive, and they spew the vesicles into the media where I can collect them. But because extracellular vesicles are so small, they can be very difficult to isolate from everything else the cells make. After the cells have been infected with Listeria, I collect the liquid that the cells live in (which contains all of the extracellular vesicles) and use a process called ultracentrifugation to isolate the vesicles. This just means the liquid is spun really, really fast so the vesicles pack together into a concentrated grouping that can be collected. Just how fast do they spin? The ultracentrifuge spins the samples at over 23,000 rotations per minute. For comparison, the motor in your car spins around at about 2,000 rpm. Spinning the samples at such high speeds creates a force equal to 100,000 times that of gravity, concentrating extracellular vesicles for collection.

With these vesicles, the first question I had was “Does Listeria change the amount of vesicles produced by the trophoblasts?” Again, since extracellular vesicles are so small, counting them can be difficult. I used a technology called nanoparticle tracking analysis, which basically works by shooting lasers to take images and videos of the tiny particles, that can use to count to count the number of vesicles.

I found that Listeria reduces the number of vesicles infected cells release. A possible reason for this is that Listeria actively targets and stops the pathways that are generate and release the vesicles, which I’ll talk more about in a little bit.

I was next interested in how extracellular vesicles from infected trophoblasts activate an immune response. For this, I used an immune cell type called macrophages, which are one of the front-line cells of the immune system that first fight against bacteria invaders. Although Listeria is well adopted to invade into these cells, certain activation signals can prime macrophages so that they are ready to attack and destroy the bacteria and infected cells. I believe that extracellular vesicles can also be used to ready the defense against the infection.

To determine if extracellular vesicles activate macrophages, I treated the cells with the vesicles from infected trophoblasts. I then measured the production of a protein called TNF-alpha, which macrophages make when they are ready to fight infections. I found that treating macrophages with vesicles from infected cells stimulated production of TNF-alpha, signaling that the vesicles can activate immune cells.

These results suggest that cells use extracellular vesicles to activate an immune response to fight the infection. Remember when I said that Listeria could be preventing the vesicle release? This could be a strategy the bacteria use to stave off the body’s defense, by preventing the activation of these immune cells.

It’s like the escaped criminal cutting off communication going out of the building and preventing calls for help, delaying this response will help the criminal stay free and cause more damage.

My next challenges are to find out what exactly is being carried by the vesicles and fully understanding how cells respond to the messages.

Bacterial infections have long been a burden in the world, responsible for thousands of deaths every year. I believe that research into extracellular vesicles can lead to new ways to treat these pathogens, or even to develop vaccines against them, preventing the infections from occurring in the first place.

JON KALETKA is a third year PhD student at Michigan State in the department of Microbiology and Molecular Genetics working with Dr. Jonathan Hardy.  He is originally from Mount Horeb, Wisconsin and received a B.S. in microbiology from University of Wisconsin-Madison. He is broadly interested in host-pathogens interactions.

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