When you think of the immune system, you probably think of white blood cells. Often portrayed as the soldiers of the body that defend against pathogens, white blood cells, also known as leukocytes, circulate the blood system and perform a variety of functions, including phagocytosis and the production of antibodies. 

However, when an initial wave of white blood cells fails to neutralize a pathogen, sometimes a granuloma forms. Simply put, granulomas are an aggregation of immune cells that form around the lesion, or area of infection. Think of them as a last-resort barricade that keeps the pathogen in one place, preventing it from spreading to other parts of the body.

The classic example of granuloma formation occurs in response to a bacterium called Mycobacterium tuberculosis, which causes inflammatory immune cells, primarily macrophages, to surround the sites of infection. Although researchers discovered granulomas centuries ago, advancements in biotechnology and immunobiology allowed scientists to make vast new discoveries about this phenomenon. 

Above: A diagram of a granuloma. Image courtesy of Andersson (2019).

The Miao Lab

One lab at Duke University that is at the forefront of such explorations is Dr. Edward Miao’s lab. Based in the Department of Integrative Immunology, the Miao Lab’s primary focus has been on the innate immune system, the first line of immune defense providing rapid but nonspecific response to microbes. In the past, the Miao lab has made extensive leaps in the field of innate immunology. For example, the Miao Lab was the first lab to demonstrate the existence and function of pyroptosis, a special form of lytic cell death that is regulated by a network of signaling factors and controls infections. 

Above: Dr. Edward Miao examines Chromobacterium violaceum, streaked onto a plate. Image courtesy of Duke University.

More recently, the Miao Lab has delved into the molecular mechanisms behind the formation, clearance, and regulation of granulomas in mouse models. To study granulomas, the lab employs Chromobacterium violaceum (Cv) as its bacterial model to investigate granuloma formation. Cv is an environmental pathogen that resides in soil and water sources and produces a vivid purple color when cultured on agar plates. Normally, the innate immune system completely clears Cve, but it can prove fatal in immunocompromised individuals. 

However, the Miao Lab discovered that while Cv can induce granuloma formation in the mouse liver, the innate immune system is ultimately capable of clearing the infection. This represents a significant distinction from its more dangerous counterpart, Mycobacterium tuberculosis, which sparks the formation of granulomas that do not clear bacteria. Furthermore, while other pathogens, like tuberculosis, are more dangerous and inefficient to work with (It can take months for infection to set in!), Cv is relatively harmless and has a much quicker turnaround time. Cv takes only a couple of days for the infection to set in, improving the efficiency of experiments. 

Above: Yu-Ming Li, a second-year PhD student in the Miao Lab. Image courtesy of the Duke University School of Medicine.

Yu-Ming Li’s Journey to a PhD in the Miao Lab

I had the chance to talk with Yu-Ming Li, a second-year PhD student in the Miao Lab studying the cell death response of hepatocytes, or liver cells, in mouse models infected with Cv.

Li’s scientific journey began as an undergrad student in Taiwan, where he later attended medical school and earned his MD. Although he studied medicine, Li always knew he wanted to pursue pure science in his career. At the time, Li was initially interested in cancer biology research and joined a lab that studied the cancer biology of glioblastoma multiforme, a deadly brain tumor. However, after about a year, he decided to explore other options. 

This led him to join a journal club hosted by another lab at his school that happened to study immunology. The papers presented were extremely advanced and beyond the scope of his knowledge—Li described the immunological terms and concepts as “a whole other language” to him. Despite this, Li persisted and continued participating to enhance his general scientific knowledge. One day, he asked the project investigator of that lab how he could better understand the research that was discussed. His PI’s response? Read immunology textbooks. 

Above: Medical Science Research Building III, the home of the Miao Lab. Image courtesy of Surface.

And so he did, marking the first step in his journey in immunology. After discovering his passion for immunology, he joined this PI’s lab and began basic research. Slowly, he transitioned from passive observation to active participation. After he graduated from medical school, Li applied for an immunology PhD, landing him at Duke and in the Miao Lab. 

Currently, Li is in his second year of his PhD program, one of the busiest years for PhD students. While the later years consist primarily of hard research, the first two years include a chaotic whirl of classes, rotation through labs, TAing for classes, and finally, the prelim—a qualifying exam conducted by the same committee of professors that will judge students’ final dissertation. 

Above: A box of liver histology slides in the Miao Lab. Image courtesy of Duke Health. 

From Histology to Literature: Li’s Project

Li’s observations in histology, the microscopic study of tissues and cell morphology, sparked his interest in hepatocyte necrosis. Normally, when a mouse is infected with a Cv, neutrophils crowd around the site of infection as early as six hours post-infection. Despite being the first responders and equipped with destructive reactive oxygen species, neutrophils fail to clear Cv, prompting the formation of a granuloma. 

Yet, when looking at both wild-type (WT) and neutrophil-deficient mice infected with Cv, he also noticed signs of coagulative necrosis in noninfected hepatocytes that surrounded the neutrophil swarm. In fact, the coagulative zone was so prominent that it formed a distinct zone around the neutrophils as soon as one day post-infection! A relatively old phenomenon that researchers discovered more than five decades ago, coagulative necrosis is a unique form of cell death where dying cells stick together. Li compared coagulative necrosis with a frying egg: the way the egg whites break apart and stick together is akin to how cells lyse and adhere together after death.

How did Li confirm that it was coagulative necrosis? He used the same tools that allowed him to make his initial observations: histology. Often sparked by strong stimuli like heat, coagulative necrosis is characterized by the denaturing of proteins. Using hematoxylin and eosin (H&E) staining, Li examined the tissue samples under a microscope. H&E staining selectively colors the nucleus a dark purple and other cellular structures, including the cytoplasm and motor proteins, a lighter pink. Li observed the aggregation of proteins within the cells, which appeared as a tangled network of dead hepatocytes. This mesh-like entanglement of necrotic cells is characteristic of coagulative necrosis, confirming the observations Li made through histology.

Above: An example of H&E staining with the acidic nucleus being colored a bluish purple and the basic cytoplasm being colored a lighter pink. Image courtesy of MedCell.

Most scientists would disregard this phenomenon, labeling it as a simple side effect of some sort of uncontrolled stimuli. Indeed, in the past, most immunologists were more intrigued by regulated cell death pathways, such as the well-known apoptosis, rather than the disordered process of necrosis observed here.

However, Li saw repeated instances of this coagulative necrosis in infected mice, regardless of the mouse’s genotype. Intrigued by how this coagulative necrosis could function as a host defense strategy against pathogens, Li hypothesized that this coagulative necrosis acted as sort of a “primitive” innate immune defense, boxing the pathogen to one area and controlling it to a single site. According to Li, this type of critical thinking, integrating inductive methods for data analysis and interpretation with deductive reasoning to formulate hypotheses, is essential for scientific training. Immunologists, in particular, must constantly think about host-pathogen interactions from different perspectives. 

Above: Examples of CFU plating in the Miao Lab. Image courtesy of Will Sun (Trinity ’27).

To test this hypothesis, Li will devise a method of inhibiting this coagulative necrosis by knocking out critical proteins and molecules in the pathway. If this inhibition is marked by increased Cv CFUs (colony forming units, a measurement of bacteria abundance) or altered host response to Cv, it could confirm that coagulative necrosis does indeed have a critical function in pathogen response. However, this process is much easier said than done, as the molecular pathway of the observed coagulative necrosis is almost entirely unknown. 

Yet, certain clues may hint at the mechanisms controlling coagulative necrosis. The lysing of cells, proliferation of cell ‘guts’ everywhere, and attraction of neutrophils via damage-associated pattern molecules closely resemble pyroptosis, a better-understood process. Thus, Li is exploring the possible relationship between the observed coagulative necrosis and pyroptosis. After completing an intensive literature review to identify the key proteins and molecules involved, Li plans to develop and administer inhibitors of these molecules in mouse models to test their roles in coagulative necrosis.

Above: A microscopic image of a cell undergoing pyroptosis, marked by the proliferation of caspase-1 (in red), the enzyme that sparks the process. Image courtesy of Antibodies Incorporated. 

Despite the project’s promise, Li faces significant uncertainty in this process. Although he hopes to successfully classify coagulative necrosis into a specific type of cell death, the observed coagulative necrosis may be an entirely new cell death pathway—or even worse, not a pathway at all and simply a random occurrence. According to Li, one pro of the project is that he has several interesting things to explore. A con? He still has so many things to explore—all from scratch. After all, building an entire molecular pathway purely from histological observations and literature is not easy!

The Implications of Li’s Project

Li’s project could have rippling implications for immunology in both academic and clinical settings. Similar to non-apoptosis necrosis, hepatocyte cell necrosis has not received significant attention from the immunology community. The most accepted school of thought is that hepatocyte necrosis is a negative reaction to some sort of cell insult. If Li can show that coagulative necrosis can actually be a beneficial mechanism to the host, and not just a random and natural response to stimuli, the finding could open new avenues for research into non-immune cell death. Essentially, this discovery may pave the way for greater exploration of new host response mechanisms, putting Li at the crux of a new hyper-specific niche within immunology. 

In terms of clinical applications, Li hopes that his project will have a large impact on the current understanding of liver health in humans. The liver performs a multitude of critical functions for humans—from regulating metabolism to conserving energy. However, livers are prone to ischemia reperfusion injury, an increase in cellular death and disorder when blood flow is restored to areas where they were previously absent. Both Li and Dr. Miao speculate that the hepatocyte death associated with ischemia reperfusion injuries may be linked to the coagulative necrosis observed in mouse models. 

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Above: A diagram of Ischemia in mice livers, seen by the lighter color in the injured liver that represents a loss of blood to those lobes. Image courtesy of Abe et al., 2008.

Thus, if Li can find a method of inhibiting the molecular pathway of coagulative necrosis in mice, doctors and caregivers could apply this method in clinical cases where hepatocyte necrosis is harmful in humans. For instance, during liver transplantations, the transplanted organ experiences significant ischemia reperfusion injuries. A drug developed to inhibit liver cell death following the same molecular mechanism would significantly reduce the impacts of ischemia reperfusion injuries and enhance the recovery of live transplantation surgeries. Although this is certainly an intriguing idea, it remains a very distant future for now.

Overall, Li’s project is still in its early stages; his hypothesis is driven solely by histological observation and requires further functional assays to prove the importance of this necrosis. Despite the huge uncertainty in the project, Li embraces the challenge. To him, PhD projects begin by “using imagination with a little bit of evidence to tell a comprehensive story.”

A Network of Scientists and Students: Mentorship in PhD Programs

Thankfully,  PhD students have a plethora of resources to turn to as they complete their projects. For Li, Duke’s interconnected science community has proven particularly useful. While Dr. Miao provides direct feedback on Li’s ideas, Li can easily turn to other faculty and students when he encounters an issue that is outside of Dr. Miao’s expertise. For any project, a single professor might only be familiar with about 10% of the project’s scope, requiring PhD students to leverage their network of professors and experts for guidance on specific techniques or ideas. For instance, Fernando Souza, another PhD student in the Miao Lab, recently gave a talk at a departmental seminar and received advice on how to utilize flow cytometry from another faculty member. Having attended multiple conferences, seminars, and talks, Li is skilled at communicating with other scientists and getting the help he needs—a fundamental skill for future PhD students.

Li encourages future PhD students and scientists to embrace uncertainty. To him, no matter how prepared a researcher might be, uncertainty is always intertwined with the essence of science. Even if an idea of interest is not 100% certain, Li urges young scientists to pursue it nonetheless. He also advises future students to begin exploring science as early as possible. Spending time in the lab, working on a graduation with distinction project, and even just talking to PIs about science as he did all help prime students to enter the tumultuous yet rewarding world of science.