Among the many health disparities characterizing the COVID-19 pandemic, one that’s received particular attention is the difference in outcomes between men and women. As early as February last year, researchers observed that, although men and women were contracting COVID-19 at similar rates, men seemed far more likely to die from the disease.
Evidence of the gap has continued to emerge as COVID-19 datasets have expanded. For instance, data aggregated by The Sex, Gender and COVID-19 Project indicate that, although statistics vary substantially among countries around the world, men with the disease are around 20 percent more likely to be hospitalized than women. Once hospitalized, men are more likely to require intensive care, and once there, they’re more likely to die.
One person unsurprised by these differences is Sabra Klein, a biologist at Johns Hopkins Bloomberg School of Public Health. Klein has been studying immune responses to viral infections for more than two decades, and says that COVID-19 is helping to shine a spotlight on an important truth in infectious disease biology: that viruses and other pathogens don’t equally affect women and men (for the purposes of this article, people with two X chromosomes and those with one X and one Y, respectively). And at least some of that difference can’t simply be explained by differences in exposure or risk-related behavior between women and men—it comes down to biology.
Once a controversial idea, the concept of sex differences in immune function has taken hold in the research community over the last few years. Retrospective analyses of data from the 2003 SARS and 2013–2014 MERS coronavirus outbreaks, for instance, have revealed that, among detected cases, men had a higher risk of death than women. And several other viruses such as hepatitis C are known to cause more serious infections in men. On the other hand, autoimmune conditions such as lupus, multiple sclerosis, and rheumatoid arthritis—which in many cases are thought to arise from the overactivation of antiviral immune pathways—are far more prevalent in women, leading Klein and other researchers to theorize that scientists might be observing two sides of the same coin.
Men with COVID-19 are around 20 percent more likely to be hospitalized than women. Once hospitalized, men are more likely to require intensive care, and once there, they’re more likely to die.
With the pandemic helping to draw attention to the influence of sex on disease outcome, Klein and others have been digging into the mechanisms underlying differences in immune responses between XY males and XX females. Both in humans and in other mammals, their findings reveal fundamental distinctions at the genetic, cellular, and organismal level that Klein says could influence how scientists approach the study, treatment, and prevention of human infectious diseases. "SARS-CoV-2 has just changed the discussion immensely. My hope is that moving forward, post-pandemic, this starts to spread and we begin to see these considerations more broadly in immunology and infectious diseases."
Documenting sex differences in immune responses
Perhaps the most consistent result that researchers have found as they study functional variation in the mammalian immune system is that females tend to mount "stronger" immune responses to viral infections than males do, says Marcus Altfeld, an immunologist at the Heinrich Pette Institute in Hamburg. This difference is particularly evident in the cells involved in the innate immune system, which tend to respond faster in females to stimulation by viruses that binds to those cells’ receptors and, once stimulated, launch a greater production of antiviral signaling molecules. "This is really the first step in the immune response against a virus," Altfeld explains.
His group has focused on toll-like receptor 7 (TLR7), a protein involved in detecting and responding to single-stranded RNA in the cell cytoplasm—an early warning sign that a virus has invaded the cell. As early as 2009, Altfeld, then at Harvard Medical School, and colleagues observed that certain TLR7-bearing immune cells known as plasmacytoid dendritic cells from women produced significantly more interferons in response to stimulation with bits of HIV RNA than did cells from men.
Other researchers have found that neutrophils, the most abundant type of white blood cell in the mammalian immune system, appear to be more responsive to interferon signaling if they’re sampled from women. Specifically, female cells show stronger upregulation of multiple genes involved in immune defense, according to a study published last year by Mariana Kaplan and colleagues at the National Institute of Arthritis and Musculoskeletal and Skin Diseases.
Research in the past few years has pointed to sex differences in the development of adaptive immunity, too. Klein, who uses influenza vaccines to experimentally study mice’s responses to immune stimulation, has found that female animals tend to generate higher neutralizing antibody responses. T cells, which are typically associated with adaptive immune responses but can play a role in innate immunity as well, also show sex-specific characteristics. For example, multiple studies point to higher numbers of helper T cells, which promote the activation of large swathes of the adaptive immune system, in female children and adults than in males.
Klein says that the stronger immune responses illustrated by these and similar studies are likely a double-edged sword from a health perspective. On the one hand, females’ heightened immune activation could help limit the amount of virus in the body—an effect observed in people with HIV, for example, as women tend to have much lower viral loads than men do in the first few years following infection. Additionally, females seem to generate better protective antibody responses than do males following vaccination against influenza, yellow fever, dengue, and several other viruses. On the other hand, that same biology likely also predisposes females to diseases stemming from overactive immune responses, Klein notes. "The downside of this robust immunity is that 80 percent of all autoimmune disease patients are women," she says. "We are so much more likely to have autoimmune diseases; we are significantly more likely to have multiple autoimmune diseases."
Women are also at greater risk for disease progression in some infections driven by immune system interactions, such as HIV infection, which progresses to AIDS faster in women than it does in men. Altfeld’s group has reported clinical data showing that women with HIV have higher T cell activation—a key predictor of disease progression—than do males with the same viral load, and concluded in their 2009 study that females’ stronger TLR7 signaling likely accounted for part of this difference.
Although many details of SARS-CoV-2 infection are yet to be described, several studies have already hinted that discrepancies in immune system function may be responsible for some of the sex differences in COVID-19 disease outcome, including the fact that men appear to be more at risk of death. Yale University’s Akiko Iwasaki and colleagues recently demonstrated that female patients admitted to the hospital with COVID-19 had higher numbers of activated T cells than male patients did, for example, reinforcing the idea that women generate a stronger immune response to SARS-CoV-2 and may be better protected from severe disease, Iwasaki tells The Scientist.
Meanwhile, early studies of long COVID, a loosely defined condition involving symptoms lasting weeks or months after infection, already indicate a female bias. Although the condition is far from being understood, some researchers have speculated that the disease might be mediated by T cells and could involve attack against self-antigens—molecular markers on a person’s own cells—just like an autoimmune condition.
Do SARS-CoV-2 Immune Responses Differ Between Sexes?
During the past year, many medical studies have reported that men are more likely than women to die from COVID-19, the disease caused by SARS-CoV-2. Although disease statistics vary substantially between countries and there are a number of socioeconomic and behavioral factors likely involved, there may be underlying biological mechanisms that also contribute to this imbalance.
Researchers who study sex differences in immunity have discovered several ways in which viral responses differ between people with two X chromosomes and people with one X and one Y (female and male, respectively, for the purposes of this article). Although the mechanisms of SARS-CoV-2 infection are not yet fully understood, it’s possible that some of these sex differences could help explain differences in how infection with the virus affects men and women.
SARS-CoV-2 uses the cell membrane proteins ACE2 and TMPRSS2 to enter human cells. Previous research has suggested that ACE2 expression may be downregulated by estrogen, while TMPRSS2 appears to be upregulated by male hormones, or androgens, in some tissues.
Viral genetic material inside cells can be detected with the help of TLR7, a protein encoded by an X-chromosome gene that escapes normal X inactivation. People with two X chromosomes produce more TLR7 than people with one do. Consistent with this idea, research on other viruses has indicated that TLR7-producing immune cells called plasmacytoid dendritic cells (pDCs) with two X chromosomes tend to release higher quantities of antiviral proteins called interferons than do cells with one X upon infection. In addition, the activity of pDCs and other immune cells may be upregulated by female sex hormones such as estrogen.
Innate immune cells such as neutrophils show higher activation in females than in males in response to some viral infections. These cells can be regulated by sex hormones and may have a more mature phenotype—one that is better prepared to respond to antiviral pathways—in females compared to males. Other immune cells, such as natural killer cells and macrophages, also show differential gene expression depending on sex, perhaps helping to explain the stronger immune responses often seen in females.
Females have higher numbers and/or higher activity of some types of T cells, which can help trigger adaptive immune responses to viral infections. Studies have also shown that antibody production tends to be lower in males than in females for a number of viral infections and vaccines, although data on sex differences in antibody responses to SARS-CoV-2 are inconsistent. The gene expression and development of cells involved in these responses is likely influenced by sex hormones such as estrogens and androgens.
As researchers make progress in defining the differences between male and female immune responses, they’re also trying to address a parallel question concerning how—and why—those differences arise in the first place. Typically, scientists have attributed biological differences between the sexes to variation in the concentrations of sex hormones—predominantly testosterone and other androgens in males, and estrogen and progesterone in females. This is almost certainly an important source of differences in the immune system, says Klein, noting that "every immune cell in your body has receptors" for these hormones.
In support of this idea, several in vitro studies have found that treatment with estrogen can reduce human immune cells’ susceptibility to infection by viruses such as HIV. Kaplan’s 2020 study of neutrophils also addressed the causes of differences in immune activation, finding that, compared to male immune cells, female neutrophils had a more mature phenotype that was better prepared to respond to interferon stimulation. The team showed that they were able to push neutrophils from men toward that female phenotype by treating the cells with estradiol, the most prevalent type of estrogen.
Further hints of hormones’ role come from observations that women’s risk for certain diseases changes after menopause, when estrogen and progesterone levels typically decline, says Klein. She reported in a recent paper, for example, that postmenopausal women’s immune response to the influenza vaccine is weaker than that of premenopausal women. Parallel experiments her group carried out in hormone-treated mice indicated a role for estrogen, too, with estradiol increasing the magnitude of female animals’ antibody responses to vaccination.
There are hints of a similar effect with SARS-CoV-2 infection. A preprint posted on medRxiv last year, for example, suggested that postmenopausal women may be at a greater risk of being diagnosed with COVID-19 than premenopausal women of a similar age, and may experience more serious symptoms. The same study reported that women under 45 years of age who were taking estrogen-containing contraceptive pills were less likely to be hospitalized than those who weren’t on such pills, and posited that estrogen may help protect women against the disease.
The picture that’s emerging is one of a suite of biological mechanisms that provide females with stronger immune responses to viruses at the cost of a higher risk of autoimmune conditions, often later in life.
Klein emphasizes that hormone-driven differences don’t necessarily come just from differences in hormone concentrations circulating in the blood; they could arise from different concentrations of sex-hormone receptors on male and female cells. She adds that there’s some evidence that levels of estrogen receptors decline in women after menopause, although, due to the difficulty of measuring receptor levels, "it’s not been well-studied enough."
Despite a likely important role for hormones, some consistent differences between males and females—different ratios of certain T cells and cytokines, for example—are seen in prepubescent boys and girls, suggesting that there are also nonhormonal drivers of divergence in immune system function. Indeed, recent research has provided evidence that at least some differences are mediated by genetics, and more specifically, by the sex chromosomes.
Because biological females have two X chromosomes, one X is typically silenced, or inactivated, early on during development, preventing an overdose of the proteins coded by X-linked genes. But in 2018, Jean-Charles Guery, the director of the center of pathophysiology at INSERM in Toulouse, and colleagues reported that the TLR7 gene, which is on the X chromosome, somehow escapes inactivation in females and in people with Klinefelter syndrome, who have two Xs and one Y chromosome.
The consequence of this escape, the team showed, was that a substantial proportion of immune cells containing two X chromosomes had higher levels of TLR7 protein and were far more responsive to TLR7-stimulating ligands than were cells with a single X. Guery says that the findings could help explain observational data indicating that XX females and people with Klinefelter syndrome are far more susceptible than XY males to certain autoimmune conditions such as lupus.
He adds that researchers have shown that a handful of other immune-related genes on the X chromosome escape inactivation, although the phenotypic effect of these escapes isn’t yet clear. "What we’re working on now is precisely to develop experimental models to provide direct links between the X chromosome–inactivation escape of some genes and the development of better responses of viruses or the development of autoimmune diseases," Guery says.
Researchers who spoke to The Scientist also noted early research on other mechanisms involved in shaping male and female immune responses, including the regulation of certain genes on the Y chromosome, or even sex-specific biases in the composition of the microbiome. The picture that’s emerging, says Klein, is one of a suite of biological mechanisms that provide females with stronger immune responses to viruses at the cost of a higher risk of autoimmune conditions, often later in life—a trade-off likely driven by natural selection.
"Many of us [in the field] hypothesize that this is tied to reproductive success," says Klein. "One of the things a mother can give her baby during pregnancy is antibodies, some of the immunity that we have. This is likely involved as a mechanism of protection of young, when they’re most vulnerable to severe outcomes."
Sex differences’ implications for disease treatment and prevention
Although research on COVID-19 has helped draw attention to research on sex differences in immunity, it’s also thrown some curveballs at the field. A preprint that was widely reported in the media last year suggested that sex differences in antibody responses might be less clear cut in SARS-CoV-2 infection than they appear to be for some other viruses. Blood tests of more than 300 people with mild disease indicated that men may actually produce more antibodies than women in response to the virus.
Although preliminary, these kinds of results are "causing me to question my own dogma," Klein says. "We’re seeing that those patients that have the most severe disease, including men and older-age adults, have greater antibody responses. Prior to this I would have told you, hands down: men have lower antibody responses . . . and older age is associated with lower antibody responses," she says. Without further data, "it’s a chicken-egg question," Klein adds. "Is it that being male and being older, you’re not controlling the infection so there’s more [viral] antigen stimulating the cells to drive that response, or is that [response] why you’re sick?"
Indeed, researchers are only just beginning to understand how age and other factors interact with sex to influence immune biology. Iwasaki’s study, for example, found a complex relationship between sex, age, and T cell activation in COVID-19 patients, with older male patients seeming to have a higher likelihood of worse disease and poorer T cell activation, but female patients showing no such correlation with age. Klein and her Johns Hopkins colleague Andrea Cox are now running one arm of a multicenter study coordinated by the National Cancer Institute that will try to tease apart some of the biological effects of factors such as age and sex on COVID-19, as well as to understand how other sex-related factors such as behavior and preexisting health conditions contribute to a person’s risk of severe illness following SARS-CoV-2 infection.
Work is continuing on the effect of biological sex on immune responses to other diseases, too, including bacterial infections and cancer, and on the success of treatments for those conditions. Klein coauthored a review last year, for example, arguing that variation in the health outcomes in cancer patients receiving immunotherapies such as checkpoint inhibitors—an approach that seems to have been more successful in men than in women to date—may partly stem from sex differences in immune responses.
Properly understanding such differences will be a critical step toward the holy grail of precision medicine—an individualized approach that has so far failed to make inroads into treatments for most infectious diseases, let alone vaccination or other prevention strategies, says Altfeld. "Everyone is speaking about personalized medicine, having personalized approaches for every individual," he says. "I think having a step before there, maybe stratifying the population into male and female, and optimizing treatment strategies there, is an important step."
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