Welcome to the sixth installment of “Dissertation Diaries.” We will be highlighting Machlan Sawden, a fifth-year Ph.D. student in the Shruti Sharma Lab at the Tufts Graduate School of Biomedical Sciences.
Sawden obtained his bachelor’s degree in biology at the School of Arts and Sciences at Tufts. Here, he became interested in aging research and learned how to design experiments after taking Professor Mitch McVey’s class, Biology of Aging, in his junior year. The summer before his senior year, he worked as an intern at Pfizer to investigate the interactions between macrophages and endothelial cells, which sparked his interest in immunology research.
Sawden knew he wanted to stay in the Northeast region, and applied to the Tufts Graduate School of Biomedical Sciences as one of five options. Despite not being invited to the first round of interviews, he was eventually brought into a second round of calls, and accepted his spot on the program in 2020.
The Shruti Sharma Lab focuses on the role of different innate immune cells and pathways in aging and how the immune system maintains these pathways. Specifically, Sawden’s research hones in on macrophages in the liver — known as Kupffer cells — and how these cells are affected by the aging process.
Macrophages act as the body’s security system, cleaning up dead cells and recycling their components to ensure that their remains do not cause pathogenic inflammation. Kupffer cells live in the blood vessels of the liver and filter the blood as it circulates throughout the body to catch any pathogens in the body.
Losing Kupffer cell function can greatly exacerbate liver disease because the cells are no longer there to return the body to a state of homeostasis. One such disease is metabolic dysfunction-associated steatohepatitis, in which fat accumulates in the liver, causing chronic inflammation and fibrosis, or constant scarring that can lead to organ failure. This disorder affects almost 6% of U.S. adults. Without Kupffer cell function, the disease’s induction phase is much more likely to cause bad tissue damage.
Sawden collaborated with his previous Pfizer labmate to study human liver tissue samples, staining them via immunofluorescence — a technique to locate specific components within the cell — and discovering that Kupffer cell count declines with age due to evolutionary conserved loss. In addition, they used a published data set of single-cell RNA sequencing from 2024 that studied various cells, including Kupffer cells, in the human liver from a variety of donors in different ages. Through the data, he discovered that, as Kupffer cells decline in number, they upregulate genes that are related to pyroptosis, a type of cell death.
Normal cell death occurs through a process called apoptosis, in which cells are broken down into all their cellular components and packaged into vesicles that are easily eaten by cells like macrophages. Pyroptosis is an inflammatory cell death often used to protect against intracellular infection by pathogens. But this pathway is activated in the wrong circumstances during aging.
“In aging research, stuff that helps you survive when you’re young doesn’t necessarily help you survive when you’re older,” Sawden said. “The same molecule or the same type of process could be helpful in one context, but pathogenic in another. Pyroptosis, for a decade or two, has been identified as one of those things that gets aberrantly activated with aging.” For younger adults, pyroptosis is important for fighting infection. In the context of aging, however, it can produce many inflammatory molecules and lead to tissue damage, and it is often associated with liver diseases.
Sawden then studied reporter mouse models — mice genetically engineered with the reporter gene that allows researchers to visualize biological processes inside — to watch the activation of pyroptosis in liver tissue sections. He discovered an increase in pyroptosis in Kupffer cells as the mice get older, and theorized that this is likely due to the damaged products that accumulate in some macrophages — specifically those that don’t do a perfect job breaking cells down. These products, in addition to other currently unknown factors, are potential triggers for pyroptotic death.
Sawden used several mouse lines that had gene knockouts for different steps along the pyroptosis pathway. For example, one cell line removed the enzyme that cleaves and activates the inflammatory cytokines that are associated with pyroptosis; another removed the molecule MRP3, which begins the entire pyroptosis cascade. Using flow cytometry to break down the tissue samples into singular cells for analysis and immunofluorescence, he discovered the Kupffer cells were only partially protected from the age-associated loss.
After five years of his research, Sawden noted that the hardest part of his Ph.D. is the independence of it: “The point of the Ph.D. is to independently ask scientific questions and design experiments. It’s the unknown of it. You have to ask questions that other people haven’t asked before. There’s not really a clear roadmap for the next step.”
Nevertheless, Sawden still hopes to continue discovering targets for therapeutics in an industry setting. “I’m curious [about] normal biology, but I also want to do research that has an impact on human health,” he said.
For any aspiring Ph.D. students, Sawden advised students to know what they want to get out of the program. “You have to have very good reasons to want to do one. Know what you want to do, and pick the right situation. I don’t think it makes sense to rush into one if it’s not a situation where you think you can succeed.”
