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At the Intersection of Discovery and Technology: Understanding Inflammation in the SLAS Zone

Daniel Irimia, M.D., Ph.D., is an engineer at heart. However, he took a slight detour en route to his current role as associate professor and deputy director of the BioMEMS Resource Center at the Center for Engineering in Medicine, Massachusetts General Hospital (Boston, MA) after first training as a medical doctor in his native Romania. Now combining an engineer’s mind with a physician’s knowledge, he and his colleagues have designed a large-scale microparticle-cluster array to replicate human neutrophil swarming outside the body, which allows the study of signaling pathways and mediators during swarming.

“When I was in high school I liked math and physics and fixing things. I went into medicine thinking if I can fix man-made things, I should be able to fix diseases, too,” says Irimia. But he soon discovered that clinicians approach problems differently than engineers. “Doctors tend to decide what treatment to give based on past experience whereas engineers make quantitative measurements and use them to make decisions based on engineering or physics principles.”

After practicing medicine for a few years, Irimia returned to his roots. While earning his Ph.D. in bioengineering at the University of Illinois (Chicago), he was exposed to new ideas and technologies he never knew existed, like microfluidics. As a postdoc, he began working with immunology researchers and realized that clinicians not only solve problems differently than engineers, but they also view neutrophils differently than researchers. “If you talk to a clinician,” he says “they’ll tell you neutrophils are one of the most important cells in the body. They are the first cells to respond to pathogens; without them you would have uncontrollable infections. That’s why doctors are always measuring neutrophil counts. Yet, even though neutrophils make up 60 percent of white blood cells, a typical immunology textbook devotes maybe 5 percent of its pages to neutrophils.” He asked himself why there is such a discrepancy between how clinicians and researchers think about the same cell and its role in the body. The answer, he concluded, came down to available technology.

Advancing Technology

Irimia points out that over the past 40 years, the development of tools such as flow cytometry, mass spectrometry and ELISA have helped immunologists to understand the role of immune cells in disease. “Unfortunately,” he says, “the development of tools to study how immune cells function didn’t follow the same accelerated path. For example, the transwell assay, still the most commonly used assay for studying cell migration, was developed in the 1960s.”

Understanding cell migration is important not only for understanding how neutrophils fight infection but also for learning how they contribute to inflammation. Neutrophils are able to suppress large pathogens and prevent infection through a phenomenon called swarming, a process that involves communication between cells to coordinate cell migration to the site of tissue damage. But when swarming continues uncontrolled, it can lead to inflammatory conditions, including sepsis. In 2013, Lammermann et al. published a study in Nature where they used mice models to identify leukotriene B4 (LTB4) as a key communication signal in neutrophil swarming, suggesting it may be a potential target for anti-inflammatory therapies. This prompted Irimia to start thinking that the more we understand about neutrophil swarming, the better we can treat inflammatory diseases.

Until recently, the only way to study neutrophil swarming was to do in vivo imaging in experimental animal models. But, as Irimia explains, this is very limiting because it’s low-throughput and doesn’t provide enough detail about how cell behavior changes in different circumstances. That’s when he combined his medical background with his engineering training and introduced newer technology to solve the problem. Irimia presented this research at SLAS2018 (now available on demand), highlighting the advantages this novel array has over traditional animal models.

High-Throughput

With this array, researchers can print thousands of microparticle clusters on slices in a single dish to recreate a pathogen or injury, triggering thousands of neutrophil swarms that are all growing simultaneously. In this way, they can observe thousands of swarms at once as opposed to looking at one mouse and getting one data point.

More Accurate Model

Assuming that findings from mouse models can be used to predict human neutrophil swarming poses a potential problem because there are known differences in the migration and chemokine responses between human and mouse neutrophils. The new array overcomes this hurdle by using human cells, offering a more complex and realistic picture of what happens in the natural setting.

Reproducible Assays

In the study Irimia presented at SLAS2018, and also published in Nature Biomedical Engineering, neutrophil swarms from the same donor are compared in three separate assays and show very similar results each time, validating the reproducibility of the assay.

Comprehensive

Irimia believes this array touches on functions of neutrophils not being tested by other assays. “One of the key features of the microscale array is that we can synchronize thousands of neutrophil swarms,” he says. “Since they are all growing in the same dynamic, we can measure signaling molecules released at different stages of swarming and even compare particle interaction of swarming, non-swarming and inactive neutrophils simultaneously. We can measure the space between targets that trigger swarming and analyze how neutrophils arrive at the site of an injury, how they move and even how fast they move.”

Current Applications

In addition to looking at neutrophils from healthy individuals to understand the normal swarming process, Irimia and his group also are looking at neutrophils from trauma patients. They have observed that the neutrophil activity in healthy individuals is very similar from one person to the next. “It’s like temperature,” Irimia says. “All healthy people have a body temperature around 37 °C. Based on what we know about neutrophils in healthy individuals, we would expect them to behave the same way across the board.” But what they’ve found is that in some trauma patients, neutrophil swarming changes following an accident or trauma, while in other patients it doesn’t.

With the help of functional assays, Irimia and his team are able to monitor patients and how their neutrophils change during disease. “If we see a patient with normal neutrophil swarming after trauma, we have confidence they’ll be able to fight off infections like a healthy individual would. If, on the other hand, we see a patient with dysfunctional swarming, we know this person has a higher risk of developing infections.”

So how can this knowledge help the high-risk trauma patient? Irimia acknowledges that at this time there isn’t much we can do differently other than start antibiotics sooner. “The question,” he says, “is what stops swarming in the first place. If we know that, then in the future we may be able to develop drugs to help restore swarming functionality and give these patients the same level of protection as another individual.”

To find out what stops swarming, researchers in Irimia’s lab have collaborated with Dr. Charles Serhan at Brigham and Women’s Hospital (Boston, MA). Using mass spectrometry, they look at lipids in suspension above neutrophils and are able to verify that LTB4 is released and peaks at about one hour. Interestingly, lipoxin A4 (LXA4), a mediator in the inflammatory response, begins to increase at one hour, just when neutrophils are reaching their plateau. According to Irimia, “At least in terms of timing, this seems to be a potential limiting factor for swarming.” With the microparticle assay, they are able to validate their hypothesis that LXA4 is a stop signal for swarming. When LXA4 is added to neutrophils at the beginning of swarming, swarms stay small. Irimia clarifies that LXA4 doesn’t inhibit neutrophil activity. The neutrophils still come and engage the target, but the number of neutrophils is smaller so the swarm stays the size of the target. Without LXA4, normal swarming occurs.

What’s Next?

Irimia is excited about the potential for using the microparticle array to identify new targets for drug therapy which would stimulate or restrain swarming, similar to the endogenous LTB4 and LXA4 but finely tuned to various therapeutic needs. But he also envisions taking this new knowledge and leveraging neutrophils to deal with pathogens directly. For instance, he thinks that if a patient has a staph infection, we should be able to modulate neutrophils in a way that gives them an edge over the staph, instead of relying exclusively on antibiotics. He thinks it could be a way to circumvent antibiotic resistance.

He also sees potential for the array in drug lead optimization. As mentioned in his presentation, several new anti-inflammatory agents have been tested in clinical trials in recent years, some of which decrease the body’s ability to fight fungal infections. “With functional assays,” he says, “researchers can test their compound on human neutrophils to see its impact on swarming and predict whether or not the drug is likely to put patients at increased risk of opportunistic infections.”

Irimia hopes that this new array will serve as a platform for building other, more complex assays or what he calls “inflammation-on-a-chip” to study what happens beyond swarming. “Swarming is only one aspect,” he says. “It sets the stage for subsequent events in the inflammation chain involving monocytes, lymphocytes and dendritic cells. I think we need models of chronic inflammation in a dish that we can play with and are specific to certain diseases like arthritis or inflammatory bowel disease. For example, what happens when we have both neutrophils and monocytes in the same well? It’s something that doesn’t exist today so we have to build on what we have now.”

He knows it won’t happen overnight. As with any new discovery, it may take science some time to seize on the process of neutrophil swarming. “There are still more fundamental things we could understand about swarming, but if we find a niche where we can do something with what we currently understand, that will accelerate the process significantly.”

Building Bridges

Communicating these new ideas may be the key to finding that niche. That’s one of the reasons Irimia chose to present his work at SLAS2018. He says, “Unlike other, more academic conferences where people talk about ideas, SLAS brings people together from research who want to take action and make things happen.”

For his part, Irimia is also working to bring people together. “I don’t regret not practicing medicine. I work with physicians all the time. They do the medicine and I do the engineering. We understand the same language and we can have meaningful communication. It’s a great position to be in, bridging the medicine and engineering fields. I think that’s what’s needed to move things forward.”


Interested in presenting your work at SLAS2019? Submit an abstract now!


 

June 14, 2018