Almost Invisible to the Immune Response

R. Mark Wooten, Ph.D.
Professor, Department of Medical Microbiology and Immunology
University of Toledo College of Medicine
Dana Foundation Grantee: 2007-2010

According to the Centers for Disease Control and Prevention (CDC), more than 30,000 official cases of Lyme disease, an inflammatory disease caused when the spirochetal bacterium Borrelia burgdorferi is transmitted to humans through common ticks, are reported to the agency each year. However, more detailed calculations suggest the true number of cases is closer to 300,000, which is cause for great concern. Those who become infected can develop a host of long-term effects, including complications with the skin, heart, and joints. The disease also may result in debilitating neurological symptoms, including cognitive and memory impairment, headaches, neuropathic pain, facial palsies, encephalitis, and seizures. Mark Wooten, a microbiologist who studies how B. burgdorferi interacts with the mammalian immune system, says that it is an odd bacterium in that it is “almost invisible to the immune response.” His laboratory focuses on understanding how this particular bacterium seems to hide in plain sight after infection—and how certain immune system cells and mediators may actually facilitate its doing so, allowing it to elicit damage to the host tissues and result in the disease’s many symptoms.

What first interested you in studying Lyme disease?

I grew up on a farm in rural Arkansas surrounded by all sorts of animals and nature. While I’ve always had a love of science, I wasn’t quite sure where I fit. In school, I first gravitated towards zoology and chemistry, but eventually discovered microbiology and immunology, which was love at first sight. As time went on, I understood my passion was to investigate the immune response to infectious diseases. Because I grew up in an area with a lot of ticks, I found Lyme disease, which was then a relatively new discovery, to be really interesting.

Tell me more about why it’s so interesting to study.

Lyme is caused by a very weird kind of bacterium called B. burgdorferi, which is a spirochete. This means that instead of being a little round circle or rectangular shape like more typical bacteria, spirochetes have a unique long and “snake-like” spiral shape, which allows them to move and behave different from most bacteria within a host.

The other thing about this bacterium is that it is an obligate parasite, which means that it can only survive inside particular hosts, like ticks and certain vertebrate animals. While we can grow these bacteria in a test tube, this is an artificial system and they do not actually “act” in the same way in a test tube as they do in their natural environment—meaning the host’s tissues. After performing a lot of these test tube studies, we confirmed that they really did not accurately reflect what these supremely evolved pathogens do within their natural hosts to result in the damage they do. In fact, you cannot acquire this disease by eating the bacteria, or eating infected animals, or any other way other than being transmitted through the skin via ticks or needle-inoculation.

We realized that the only way we can really understand how the bacteria do what they do is by looking at it within an animal, which makes it more difficult to work with. Our lab has developed methods to better assess its behavior using laser confocal microscopy, which allows us to directly observe these bacteria within the intact skin of an anaesthetized live mouse in real time. That allows us to observe what they do within mouse skin tissues; how they move, how they disseminate to distant tissues, and how they interact with different immune cell populations that respond to infection.

And what are they doing in real time?

To infect an animal, B. burgdorferi has developed very unique methods, many of which we still don’t understand. But those methods allow it to become almost invisible to the host animal’s immune system. Its host animals are completely immunocompetent, meaning they have a perfectly normal immune response where the immune cells become activated soon after detecting the bacteria and go to the site of infection. Within a few days’ time, they will also develop antibodies and do all of the things that would normally clear an infection. But these bacteria are not cleared and continue to persist out in the extracellular spaces where they should be exposed to these immune mediators. Somehow the bacterium is adapting in the host to become nearly invisible to the immune response—and that’s what allows it to persist for so long.

That’s the big question. Why, after producing what appears to be a perfectly good and functional immune response, can’t the host clear these bacteria? When we watch B. burgdorferi in real-time using our microscopy method, we can see them running happily around between the skin cells for years without any seeming repercussions from the immune response. Why is it able to do that?

Tell me more about the challenges involved with developing your microscopy technique.

I did not invent two-photon laser scanning microscopy, of course—the field of immunology has been using it to look at immune cell populations for some time. That was the jumping off point for me to start using it to look at B. burgdorferi. But there were a few big hurdles we had to get over.

First, we had to engineer B. burgdorferi to be fluorescent. And that’s easier said than done. It isn’t like E. coli or other bacteria that are easier to manipulate genetically, where researchers can do a similar thing in a week or so. B. burgdorferi is very difficult to manipulate genetically for many reasons. Eventually, my collaborator, M.D. Motaleb at East Carolina University, did develop ways to do it. But once we got the fluorescent tag into the bacteria, we learned that these bacteria did not like the promoter we were using. It appeared to make too much fluorescence, which made it easier to see, but also corresponded to the bacteria being killed prematurely in the host. So we had to go back to the drawing board to try to generate the fluorescence using a more “natural” gene promoter that didn’t harm the bacteria.

The second obstacle we faced is that we didn’t have a real concept of how fast these bacteria can move inside tissue. They are really fast. We knew they were spirochetes—and these type of bacteria have multiple motors possessing flagella housed inside the cellular membrane rather than projecting outside the cell like most motile bacteria. What this means is that, instead of acting like a propeller–as seen in most bacteria–they instead contort the entire body of the spirochete and basically turn it into a sort of corkscrew.

Thus, when you put B. burgdorferi in liquid media, they just kind of flail around and don’t really go anywhere. That’s because they are meant to live and move in the dense tissues of the body, like within the skin. When we put B. burgdorferi into the skin, they turned into those corkscrews, and they started moving like crazy. When we measured them, they moved around 100 times faster than any other immune cell type that we measured traveling through the skin tissue. And, unfortunately, something moving that fast is really hard to image. With an immune cell, you can take an entire z-stack of images with a confocal microscope to get a three-dimensional image of the cells in a tissue. And you can take one of those every three to five minutes, stitching them together to make a nice video that shows you how immune cells move. But this bacterium moves too fast. You try to get the z-stack images and combine them, but you end up with just one big bacterial blur, while the slower immune cells look fine. We’ve had to work out the best way to measure B. burgdorferi—and how to image it and still get accurate measurements of how fast it moves. This is an obstacle we are still dealing with. It takes a lot of time and patience.

How may the inflammatory response in the skin tell us more about how B. burgdorferi leads to its terrible neurological symptoms. Do we know?

We don’t fully know. What we do know is that this bacterium has the ability to get into pretty much any tissue in the human body using its corkscrew motility. It seems to lack some inflammatory molecules on the surface of its body that the immune system normally associates with disease. For example, many bacteria have lipopolysaccharides on them. Our bodies have evolved to where, when the immune system detects that component, which is so unique to most bacteria, the immune cells will react violently and do everything they can to try to get rid of it. These spirochetes do not have those lipopolysaccharides. Our cells also directly recognize the flagella made by bacteria. However, these spirochetes house their flagella inside their cellular membranes, so our cells do not detect that signal.

B. burgdorferi does make a variety of different lipoproteins—and which ones it makes seem to depend on what host it is inhabiting. It makes certain types of lipoproteins in the tick, others in the mouse or rabbit, still others in the human. It can very quickly change these proteins on the surface and it’s likely they do so to better adapt to its host. And our immune systems will eventually recognize those lipoproteins and mount an immune response. In fact, the inflammation you see in Lyme disease is largely because our bodies are recognizing the lipoproteins.

You know that hallmark bull’s-eye rash that people often get when they are bitten by an infected tick? If you go in and look, what you see are the body’s immune cells kind of chasing after the spirochetes ahead of them. And it’s believed that the response to the lipoproteins is what is setting this inflammation off. As you can imagine, no matter what part of the body the bacterium goes into, there will be some kind of immune response if it is able to persist long enough. It’s that background inflammation that is actually the disease.

But why do some people get neurological symptoms when others do not? New work suggests that the way your body responds during the development of Lyme disease depends on your genetic make-up. We know, from looking at different strains of mice, that there are different strains of animals that can have exactly the same number of bacteria persisting in their tissues but get completely different severity of disease. While all humans basically have the same genes, the slight differences we see across different genomes may determine whether someone gets mild or severe disease. Since the disease is really our body’s inflammatory response to the bacteria’s presence, what kind of disease you get is also going to depend on where the bacteria are when the body mounts that response. If it’s in your heart, you can have cardiac problems or a heart arrhythmia. If you have it in your joints, you can get arthritis. And if it is in your brain, you’ll get foggy memory, shooting pains, or other problems.

Lyme is a very complex disease, because the bacteria want to be invisible. They want to get along so they can survive. And Lyme disease is our immune response refusing to ignore these bacteria—and trying to fight them off with an inflammatory response. And since it is the body’s own response, it leaves us with a big problem. Often Lyme disease isn’t diagnosed right away. It may not be diagnosed until you see some of the more advanced severe symptoms. You may be having this health issue—but are you going to connect it with a tick bite you probably don’t remember even getting from 2-5 months ago and then tell your doctor about that? Probably not. So better diagnosis and diagnostics are a big public health issue. This is because if you wait until the severe symptoms have developed, even if you clear the bacteria with antibiotics, a lot of damage has already been done. Then the next question becomes how do we heal people who have suffered neurologic, or other damage, once we cure the infection? Unfortunately, many of these tissues do not heal quickly or may not heal completely at all.

Some researchers are putting great stock in new gene-editing techniques such as CRISPR, and genetically engineered animals to help stop the spread of Lyme disease. What are your thoughts?

CRISPR technology is providing an exciting new universe of potential uses to treat disease at the genome level. The genetically engineered mice proposal put forward by the MIT Media Lab could potentially have the desired effect. However, there are several problems that are immediately apparent, as well as many that we do not yet have the experience to figure out. Their proposal depends on their ability to find the genes that prevent some mice from transmitting the Lyme bacterium. If this were done, a more direct and safer approach would be to just target those bacterial proteins that interact with the identified mouse gene products as a vaccine, which should have the same final outcome of making vaccinated humans (as well as pets) resistant to being infected without releasing genetically-altered mice into the wild. Personally, I do not think this would be an appropriate use of the CRISPR technology, but I do commend the researchers for thinking outside of the box.

How does your work fit in with current efforts to treat Lyme disease?

In the case of Lyme disease, there appears to be a dysregulated immune response. For most bacterial infections, the host first makes a pro-inflammatory response to clear the microbes—but then you subsequently need to generate an anti-inflammatory response so that your body does not damage itself after clearing the infection. Interestingly, our work has shown that this response is somewhat flipped. At the same time as the pro-inflammatory response is occurring, the host also makes a very large anti-inflammatory response that appears to shut down the beneficial antimicrobial responses prematurely, which helps the bacteria escape clearance and set up a persistent infection. That may be one way in which the bacteria become invisible. We’ve seen a lot of interesting things as we’ve looked at the immune cells and how they respond to B. burgdorferi. Things don’t work the way they are supposed to work.

If we can figure out how the bacterium is eliciting this dysregulated response, that could be a potential target for treatment. We might be able to restore the normal immune response so the immune system just takes care of the bacteria. If it’s a persistent infection, there may be a drug that you could use with antibiotics to help relieve the inflammatory response long enough to clear the disease. There may be something in there that could help point to a potential vaccine.

Another thing I study is the motility and chemotaxis, or the signals these bacteria receive that tell them how and where to move. When we saw how fast they can move—and the fact that they constantly move in the tissue—we realized that this was probably an essential part of how they evade the immune response. By taking apart different aspects of their motility and chemotaxis, we’ve realized that if you can mutate some of these molecules, you end up with bacteria that can’t move at all or only move in one direction. Subsequently, this allows the immune system to eradicate them completely, and quite quickly, too. That gives us two potential therapies: one, to find a way to target those motility molecules to subsequently paralyze the bacteria so they can be cleared by your own immune system. Or two, to generate motility-mutant bacteria that can persist in the host for a few days, providing the bacteria just enough time to express the virulent molecules that are essential for it to persist in the host. Such a mutant might represent an ideal vaccine—the bacteria persist only long enough to express those molecules that the host can generate antibodies against, and might lead to development of life-long immunity.

We’re now learning that inflammation plays an important role in many neuropsychiatric and neurodegenerative diseases. What can the intersection of immunology and neuroscience offer us in terms of preventing, diagnosing, and treating these diseases?

Diseases caused by bacteria and microbes come in two general forms. Either the microbe makes a toxin that causes bad things to happen or our own immune response—that inflammatory response—goes out of control and causes damage. More and more, we understand that anything we can learn about why inflammation occurs, and how it may occur inappropriately, have broad implications for a large number of diseases.

In the case of Lyme disease, my postdoctoral mentor, Janis Weis at the University of Utah, has identified many different host genes that seem to be responsible for severe versus mild disease. Many of these genes had never been associated with arthritis or neurological conditions before. But when they looked more closely, they learned that these genes were also linked to other inflammatory diseases, such as rheumatoid arthritis.

Thus, we may initially think some of these things are unique to Lyme disease—or even to inflammation in general—but we may learn that they are also critical to a number of other inflammatory diseases. Anything that helps us gain a better understanding of dysregulated inflammation can only help the medical field overall.