Clues from mice could help explain why women face a higher risk of autoimmune disorders

Researchers have long known that women are more likely to develop autoimmune disorders than men, though they’ve struggled to fully understand why. Now a new study in mice suggests a key part of the answer may be that an RNA molecule that’s indispensable for female survival steers the body toward immune friendly fire.

A team led by Stanford scientists found that Xist, a molecule that teams up with proteins to keep female cells from activating a double (and deadly) dose of X chromosome genes, can trigger antibodies that latch onto complexes of the RNA and its protein partners.

Xist isn’t normally expressed in male cells, but to show that these complexes help drive autoimmunity, the authors created male mice that made Xist on demand. These animals were likely to show signs of autoimmune disease roughly as severe as in female mice.

It’s too early to tell for certain if the findings, published on Thursday in the journal Cell, will hold up in people. But the study’s authors found an encouraging hint. When they examined blood samples from healthy adults and patients with autoimmune diseases that mainly affect females, they found that the latter group was more likely to have antibodies that recognized Xist-protein complexes. That raises the possibility that the findings could be used to devise new and better ways to diagnose autoimmune diseases and to monitor whether treatments are working.

“This is like a completely different and novel explanation for female bias in immune disease,” said Howard Chang, a Stanford physician-scientist and the study’s senior author. “What our study really showed was that it’s not just the second X chromosome, it’s actually a very special RNA that comes from that second X chromosome, and just that RNA perhaps plays a major role.”

While many individual autoimmune disorders are rare, they’re together the third most common disease category after cancer and heart disease and affect about 8% of the population. The sex bias for these diseases is stark; four out of every five patients are female. In some cases, the imbalance is even more lopsided; 90 percent of lupus patients and 95% of those with Sjögren’s syndrome are female.

By comparison, males are generally more susceptible to infectious disease. Case in point: Men are more likely to die of Covid-19, while women have been more likely to develop long Covid, which experts say mimics aspects of autoimmune disorders.

Researchers have identified many potential explanations. Autoimmunity typically kicks in after puberty, suggesting that sex hormones play an important role, a theory supported by past research. The X chromosome itself is another potential culprit, as females carry two copies while males have one — well, most of the time. People with Klinefelter’s syndrome have two X’s and one Y, and, while they are biologically male, have an increased risk of autoimmunity compared to XY males.

That observation suggested to Chang and others that something on the X chromosome contributes to autoimmunity. That’s why his team zeroed in on Xist, an RNA molecule coded for by the X chromosome. Cells with two X’s can’t survive if both are active, as they’d produce a deadly double dose of proteins. Xist helps shut down one of the two X’s in each female cell by coating the chromosome in a way that looks a bit like a cloud of cotton candy spooled around a cone at a county fair.

If you’ve ever seen a calico cat, you’ve seen X inactivation. The gene that codes for fur pigment is on the X, and the orange and black patches on these cats reflect areas where one X chromosome was inactivated versus the other. And since X inactivation doesn’t happen in males, nearly all calico cats are female.

Chang’s lab had previously found that Xist associates with scores of proteins that help with X inactivation, and he’d noticed that these RNA-protein complexes resemble known targets of autoantibodies, antibodies often linked to autoimmunity that recognize the body’s own proteins rather than pieces of a pathogen. That led the team to wonder if Xist-protein complexes might play an important role in driving the sex bias seen in autoimmunity.

But the researchers wanted to separate the role of Xist from that of the rest of the X chromosome and from the effects of female sex hormones, so they went looking for clues in an unlikely place: male mice. The scientists genetically engineered male mice to carry Xist on one of their chromosomes and to produce the RNA molecule any time the animals were injected with doxycycline, an antibiotic. And they modified Xist so that it wouldn’t silence the chromosome but would simply cling to it.

The authors then tried to trigger lupus in the animals by injecting them with pristane, an oil known to induce inflammation and autoimmunity in mice. They found that most of the male mice producing Xist developed the same sort of multi-organ autoimmunity typically seen in females, but only if the mice came from a strain known to be susceptible to developing autoimmunity. Other experiments showed that switching on Xist in male mice caused their T cells to activate genes in ways that more closely resembled females.

To see if their findings matched what was happening in people, researchers analyzed blood samples from healthy donors and from patients with dermatomyositis, lupus, and systemic sclerosis. They found that autoimmune patients, about 86% of whom were female, had higher levels of autoantibodies against proteins associated with Xist than healthy donors. Some of these autoantibody responses were largely disease-specific while others were shared across all three conditions.

Chang says the findings suggest that, in women genetically predisposed to autoimmunity, a bit of inflammation or tissue damage might expose Xist-protein complexes that are usually cloistered in a cell’s nucleus, and that this exposure could trigger autoantibodies and increase their risk of developing disease. He adds that his team is now exploring whether they can use their findings to develop better ways to spot autoimmune disease and monitor treatment effectiveness, as a drop in autoantibodies to Xist-associated proteins might be an encouraging sign a therapy is working.

But it’s too early to say for sure that the autoantibodies the authors focused on are a key driver of autoimmunity, cautioned Montserrat Anguera, a researcher at the University of Pennsylvania who was not involved in the study. Anguera, who studies X inactivation and sex biases in autoimmunity, notes that it would be helpful to know if researchers find autoantibodies to Xist-linked proteins in autoimmune diseases that don’t have a sex bias. And she adds that Xist likely contributes to autoimmunity in many ways, citing recent evidence that the molecule itself can directly trigger inflammation.

“I don’t think the data is there yet to say that it’s the most important [factor] right now, because it’s sort of the first observation that this is possible,” she said of the Stanford team’s findings. “It just really highlights the fact that it’s not one pathway that involves the inactive X chromosome; there’s different ways that the inactive X can contribute to female bias in autoimmune disease.”