Lukas Flippo, Photo Editor

In order to better understand macrophages — one of the first responders in our immune system — Associate Professor of Biomedical Engineering Kathryn Miller-Jensen and her lab decided to observe the mechanism of macrophage response at the level of the individual cell.

Macrophages respond to stimuli by following one of two pathways called M1 and M2, explained Ilana Kelsey, a postdoctoral associate of the Miller-Jensen lab who worked on the study. But she said that researchers have long thought that macrophages do not cleanly follow either the M1 or the M2 pathway. Although how exactly the mechanism works is still unknown, the Miller-Jensen lab’s research confirms the wide variety of macrophage responses to different stimuli — which could potentially guide future research on immune system responses.

Using single-cell RNA sequencing technology, the Miller-Jensen lab was able to tackle this question and study individual macrophage cells as they responded to cues in their environment. According to Kelsey, the Miller-Jensen lab exposed macrophage cells to conflicting signals — some that promote M1 responses and others that promote M2 responses — and found that the macrophage responses were highly varied. They measured this variability by studying the differences in the RNA transcribed by the various macrophage cells and the proteins they produced.

“This M1 versus M2 is not as clean as we like to make it out to be in the lab,” Miller-Jensen said. “There’s actually a lot of mixed activities going on. Sometimes the same macrophages seem to be both attacking and resolving at the same time.”

 In the M1 pathway, macrophages release signals causing inflammation and coordinating an immune system attack of the disease. The M2 pathway, on the other hand, is characterized by anti-inflammatory processes and tissue rebuilding. 

Scientists have determined that certain cues trigger M1 inflammatory responses in macrophages while others trigger M2 tissue rebuilding responses, according to Kelsey. She explained that these cues are usually tested individually in artificial lab settings, but in real life situations, macrophages are often faced with conflicting signals.

“In a variety of disease states — cancer is the one that’s sort of most famous right now — macrophages should recognize that there is a threat, so you would expect them to become pro-inflammatory,” Kelsey said. “But what happens in the tumor microenvironment is that the tumors are sending out signals that sort of trick or co-opt macrophages into looking more tissue rebuilding instead of tissue damaging.”

According to Miller-Jensen, scientists have studied macrophages for a long time, but always at the population level, combining millions of cells to be able to measure results. Thanks to technological advances within the past five to six years, however, scientists are now able to study how individual macrophages respond to different cues in their environments, and they are able to observe the variability of responses in the cells.

What the Miller-Jensen lab found was a wide variety of macrophage responses to this co-stimulation, which fell on a spectrum ranging from distinctly M1 responses to distinctly M2 responses, according to Kelsey. The co-stimulated macrophages fell into three different groups along this spectrum. A small percentage of the co-stimulated macrophages distinctly resembled only M1 or only M2 macrophages. Kelsey explained that the third and largest group included macrophages whose transcribed RNA was different enough from that of both the M1 and M2 stimulated groups of macrophage to be considered an entirely separate group and fell somewhere in the middle of the spectrum.

“The reason that we find this quite interesting is that all of these cells … should be genetically identical, and they’re all seeing the exact same signal, but somehow, between seeing the exact same signal and being genetically identical, we’re getting this variability,” Kelsey said. “What we really found was that every cell was a little different.”

But when they examined the proteins produced and secreted by the macrophages, as opposed to the RNA transcribed, they found that for certain protein secretions, the macrophages only produced either the ones associated with the M1 pathway or those associated with the M2 pathway and not both, according to Miller-Jensen. 

She said that these protein-making processes appear to be mutually inhibitive.

“Sometimes they actually are making choices for certain activities and for other activities they’re not, they’re just responding in a spectrum,” Miller-Jensen said. “And I guess what we want to know is why and how, but I’m not sure yet.”

The researchers of this study identified transcription factors that may cause macrophages to choose between either producing certain M1 versus M2 protein secretions when exposed to conflicting signals. 

Isaiah Yim GRD ’25, a graduate student in the Miller-Jensen lab, is starting new research that will look at KLF4, a transcription factor that regulates protein production. According to Yim, this transcription factor has been identified as a possible source of the variability seen in macrophage responses.

“Biological interactions are inherently just two proteins kind of hitting each other randomly, and so at some level there’s going to be noise,” Miller-Jensen said. “There’s no way a cell and molecules and proteins can respond without some sort of noise or variability. Maybe nature has figured out how to use that variability to its advantage.”

By better understanding how exactly a macrophage responds to cues in its environment, the researchers hope that future scientists can learn how to target these processes to encourage a more effective immune response. Kelsey gave the example of tumor-associated macrophages. She said that through a clearer understanding of their responses, researchers might learn how to prompt macrophages in the tumor microenvironment to be more pro-inflammatory and fight off the tumor.

Miller-Jensen, along with Professor of Dermatology, Pathology and Immunobiology Marcus Bosenberg, received a $2.8 million grant from the National Cancer Institute earlier this year to investigate how to target immune cells for cancer treatments.

Emilia Oliva | emilia.oliva@yale.edu