Art by Miriam Kopyto.
Scientists, like English teachers, always ask “What?” then “Why?” First observe a pattern—of metaphors in a novel, of a phenomenon in nature—then investigate the reason for it. Months into the coronavirus pandemic, the majority of published research was still answering “what” questions: What age is at greatest risk of hospitalization? What sex is most likely to recover? But when it came to why these differences were observed—and how to use that information to develop better treatments—the Iwasaki lab at the Yale School of Medicine was uniquely poised to find answers. Their latest research revealed sex differences in the immune response that might explain differences in COVID-19 disease progression.
The Iwasaki lab, headed by Akiko Iwasaki, focuses on innate immunity against viruses and how it generates adaptive immunity. They work on projects ranging from the role of autophagy in innate viral recognition to the effect of temperature on the common cold virus. Their research has also provided them experience with several high biosafety level viruses. This experience with more dangerous viruses prepared them well for this COVID-19 study.
When the COVID-19 pandemic hit, Albert Ko from the Yale School of Public Health spearheaded the launch of the COVID-19 biorepository study framework called Yale IMPACT (Implementing Medical and Public health Action against Coronavirus (Connecticut, CT)) , allowing researchers like Iwasaki to start collecting patient samples from Yale New Haven Hospital to participate in COVID-19 research. This allowed researchers to analyze patients’ immune responses from day one and throughout disease progression to identify different signatures associated with the immune response against SARS-CoV-2.
A Tale of Two Immune Responses
For their “immunophenotyping” study of sex differences in COVID-19, researchers in the Iwasaki lab zoomed in on the characteristics of an individual’s immune response to SARS-CoV-2 infection. Specifically, the researchers compared four immune response markers in male in female patients: viral concentrations in nasopharyngeal swabs and saliva, anti-SARS-CoV-2 antibodies and cytokines (immune signaling molecules) in the blood plasma, as well as the relative amounts of different kinds of immune cells.
As the blood samples started to come in, lab members went into hyperdrive. According to first author Takehiro Takahashi, this study was an intense collaborative effort of immunophenotyping experiments, data collection, and extensive data analyses. “Blood came into the lab usually in the afternoon, then processing and preparation took around six hours, then after that we ran it in the [flow cytometry] machine, so the entire workflow often went past midnight every day,” Takahashi said. To identify and analyze blood cells, researchers use fluorescent antibodies—molecules that bind their specific targets like unique magnets. To quantify cell types, they use a flow cytometer machine to separate cells based on shape, size, or other distinguishing characteristics.
The study was broken down into two comparisons. Volunteers from among healthcare workers were the healthy control group, and a group of non-ICU patients who had not received any immunomodulatory drugs were labeled Cohort A. The first, baseline analysis of patients’ initial immune response compared male and female healthcare workers in the control group to male and female patients from Cohort A at the first sampling time point. The second comparison was a longitudinal analysis—comparing the immunophenotypes of male and female patients across multiple time points of disease progression.
The longitudinal analysis included patients who were further along in the disease, including some in the ICU or receiving immunomodulatory treatments. For this analysis, epidemiologists mathematically corrected for variables other than sex, such as age, BMI, and treatment status. Still, there are limits to these statistics. The sample size was relatively small, and options were limited for the healthy control group. “Of course, there are no seventy-year-old healthcare workers, so we just had to mathematically adjust,” Takahashi said.
From the mosaic of immunophenotype analyses across time in the two cohorts, two different immune response patterns began to emerge. At an early stage of infection (represented by Cohort A), male patients seemed to have a stronger innate immune response than females, indicated by higher levels of innate immune cells and the signaling molecules they secrete, such as interleukin-8. In contrast, female patients had a stronger adaptive immune response, indicated by higher levels of activated cytotoxic T cells, which are activated by the presence of a virus to recognize and kill infected cells. These two patterns carried over into later stages of COVID-19, as lower levels of T cells (adaptive immunity) were correlated with a worse disease progression in males, while higher levels of innate immune molecules were correlated with worse disease progression in females.
While the researchers urge caution extrapolating too far, their results align with previous findings that a higher innate immune response is associated with worse outcomes for COVID-19 patients. These findings could be used to study sex-differentiated treatments: male patients might benefit more from boosting the T-cell response to the SARS-CoV-2 virus, while female patients might benefit more from tamping down the innate immune response. Takahashi hopes that this research can impact future COVID therapies for both male and female patients.
Looking forward, the Iwasaki lab wants to look into the effects of COVID-19 on different organ systems. For example, autopsies of COVID-19 deaths have revealed evidence of infection in the brain. In addition, Iwasaki’s lab plans to investigate underlying immunological mechanisms of why some patients are facing long-haul syndrome—severe long-term consequences of COVID-19.
While these studies are exciting for the Iwasaki lab, Takahashi mentioned the intense need to disseminate results of our COVID-19 research rapidly, adding pressure to publish quickly. The lab published the sex differences study as a preprint, meaning it has not yet gone through the time-consuming process of peer review. Nevertheless, there are benefits to this way of sharing research. “Preprint is important because publication takes two or three months or even longer…but in this kind of pandemic you have to share information with people quickly and discuss more openly; and the pandemic has changed how the preprint papers are perceived,” Takahashi said.
Sex Differences on the Macro Scale
Sex differences, however, are not limited to immune responses and disease progression. While gender barriers have always existed in academia, they have become especially prevalent during the coronavirus pandemic, drastically affecting women’s careers. For example, women with children or those who need to take care of loved ones cannot risk going back to work. While Iwasaki is fortunate enough to be able to work from home, she recognizes the issues that many other women—including trainees in her lab—are facing. Seeing these barriers drives her further to contribute as a woman in science. “We’re seeing less and less of women being able to contribute scientifically because of all these other obligations…I have a double sense of duty…not just [to be] doing science, but communicating science,” Iwasaki said.
Even prior to the pandemic, the lack of women in STEM has always been a major issue, and one that an issue Iwasaki is passionate about. Iwasaki was born and raised in Iga, Japan. With her parents as role models—her father was a physicist and her mother fought for women’s rights in the workplace—she decided from a young age to pursue science. Inspired by her immunology professor at the University of Toronto, where she majored in biochemistry and minored in physics, Dr. Iwasaki got her Ph.D. in immunology. She spent two years as a postdoctoral fellow at the NIH, where she studied the roles of dendritic cells.
Iwasaki went on to do groundbreaking research after joining Yale’s Department of Immunology. She developed the ERVMap—a tool used to map endogenous retroviruses (ERVs) in the genome—and a two-stage vaccination strategy called prime and pull, which focuses on enhancing T cell response. Taking after her mother, she is a fierce advocate for women and minorities in the sciences. At the undergraduate and graduate levels, more than 50% of STEM students are women. However, attrition really begins in graduate school all the way through to professor levels. While more serious issues like sexual harassment and sexist comments must be addressed, more covert discrimination actually plays the biggest role in women being pushed out of science.
Reflecting on her own experiences, Iwasaki says that women are often left out of discussions, decisions, and other opportunities. When their voices are being overshadowed, it’s important that they have a support group they can rely on so that they can be heard. Iwasaki mentions that tackling these issues requires a fundamental restructuring of meetings and decisions to incorporate more women, and more awareness needs to be spread about the lack of representation so more people can recognize the discrimination and stand up for their female colleagues. These are things she implements into her own work, always making sure there is more than one woman in every meeting and raising awareness of issues facing women so that her male colleagues can promote female voices. On a more practical note, Iwasaki also says that since women tend to stay home and take care of children, having accessible childcare is also needed to better support women.
Looking back on a career filled with plenty of challenges, Iwasaki says there’s nothing she would change about her choices and her experiences. “Obviously I’ve made a lot of mistakes in my career and you learn from that, but I don’t regret anything,” Iwasaki said. “Finding out what’s going on in the COVID patients and being able to hopefully inform future therapy and potentially vaccines, that’s what motivates me.” While her lab’s study of COVID, her many other contributions to the world of immunology, and her work encouraging and empowering women in STEM have helped shape the world of science, Iwasaki’s research has also shaped her.
About the Authors:
Catherine Zheng is a sophomore BME major in Pauli Murray College. In addition to writing for YSM, she’s involved in research and other organizations on campus, and loves going out to eat with friends. Raquel Sequeira is a senior MB&B major in Timothy Dwight College. In addition to writing for YSM, she loves to play soccer and sing in the Glee Club.
The authors would like to thank Professor Akiko Iwasaki and Takehiro Takahashi for sharing their time and enthusiasm about their work.
Takahashi, T. et al. (2020). “Sex differences in immune responses that underlie COVID-19 disease outcomes.” Nature. https://doi.org/10.1038/s41586-020-2700-3.
Viegas, Jennifer. (2018) “Profile of Akiko Iwasaki.” Proceedings of the National Academy of Sciences of the United States of America. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294888/