March 22, 2024 | The single biggest risk factor for autoimmune disease is being biologically a female and many explanations have been proposed as to why that is, among them sex hormones and the X chromosome with its large number of immune-related genes. Scientists have more recently pointed to another culprit—a molecule called Xist (X-inactive specific transcript) involved in the generation of antibodies to a woman’s own tissues.
Xist is critical for the establishment of X chromosome inactivation, ensuring that females, like males, have one functional copy of the X chromosome in each body cell. Depending on the cell type, Xist in doing its job binds with different proteins to form ribonucleoprotein (RNP) complexes which, it is hypothesized can become visible to the immune system and misidentified as foreign and harmful, according to Diana Dou, Ph.D., a basic life research scientist at Stanford University. RNPs already exist that are clinical autoantigens for autoimmune disease, “so the Xist RNP may be eliciting a similar response from the immune system.” she says.
Dou was lead author on a study that published recently in Cell (DOI: 10.1016/j.cell.2023.12.037) showing that the mere existence of Xist RNP is enough to promote a higher background of autoimmune permissibility and severity. The research team also identified over two dozen new autoantigens that could serve as additional diagnostic markers and help build profiles for identifying patients at risk and the specific type of autoimmune disease some of them later develop to inform their treatment, she says.
Arriving at an early diagnosis is not always possible today because detection is based largely on symptoms that can be mixed, faint, or overlapping with other autoimmune diseases, Dou notes. The condition of patients often worsens before doctors can determine the cause.
“Every XX cell needs to have a mechanism to maintain gene dosage compensation between X and autosomes achieved through epigenetic silencing in which Xist is critically important,” explains Dou.
The level of gene activity produced by a single X chromosome is the normal “dosage” for a human and anything above that level would be consequential and, in some cases, lethal.
During X chromosome inactivation, an X chromosome is compacted to make a small, dense structure called a Barr body whose genes are mostly inactive, she says. Females, as well as males born with Klinefelter syndrome who have an extra copy of the X chromosome, will convert one X to a Barr body in each cell.
Given that women get lupus at a ratio of 9 to 1 relative to men (14 to 1 for XXY men), and for Sjogren’s syndrome the odds are 19 to 1, “something is going on with having two X chromosomes,” says Dou.
Conditions like these disrupt the body’s natural “immune tolerance balance” between self-protection and reactivity to outside pathogenic cells, she continues. In essence, the immune system overreacts and mistakenly attacks its own healthy cells.
As Dou views it, “the straw that breaks the camel’s back” with the development of autoimmune diseases is a collection of contributing factors. The immune system has many different checkpoints designed to prevent it from coming on too strong and things don’t necessarily go awry at only one of those regulatory pathways.
Dou and her team aren’t the only scientists looking at Xist, but they are the only group to show the particles being expressed is enough to contribute to “background autoimmunity,” as she puts it. Elsewhere, people are focusing on how Xist function is impaired, when its silencing activity is dysregulated, or what genes are escaping silencing from the Barr bodies in patients.
Among other hypotheses circulating in the scientific community is that abnormal phenotypes may be the result of imperfect (“leaky”) X chromosome inactivation from the Barr bodies, Dou shares. Many studies are also trying to parse out the genetic and environmental aspects of disease development.
A study published a few years ago strengthened evidence that Epstein-Barr virus (EBV) infection may play a causal role in the pathogenesis of multiple sclerosis (MS), an idea supported by the increased MS risk seen in people who have had infectious mononucleosis (Science, DOI: 10.1126/science.abj8222). EBV is the most common cause of mono.
While autoimmune diseases tend to run in families, studies in identical twins indicate they are not entirely genetic, says Dou. One might develop lupus while the other presents with scleroderma or another autoimmune disease. The odds of both twins developing scleroderma are less than 5%.
Xist is long non-coding RNA that recruits the proteins it needs to “mark the X it is going to silence and ... recruit the complex that is actually going to put down the silencing marks and create the Barr body,” says Dou, referring to a PRC2 complex that does not directly bind to Xist (Genes & Development, DOI: 10.1101/gad.337196.120) . The latest study focuses squarely on Xist minus all other considerations.
The research team used transgenic mice where the males were induced to express Xist on chromosome 11 in a “safe harbor” genomic region where it is non-silencing but still able to coat the chromosome and pull the usual protein partners together to form the RNP complex. The wild-type Xist in the females wasn’t disrupted, she says.
The wild-type Xist in the mice had a complex comprising 81 unique binding proteins, only three of which disappeared in the transgenic models, she notes. The three proteins binding to the A-repeat domain of Xist include SPEN, which is essential for silencing function.
To test their theory that the RNP complex was triggering autoimmune disease, researchers began by inserting the modified, non-silencing form of Xist into the male mice to set aside possible competing causes such as female hormonal background and differences in sex development, says Dou. In these susceptible mice, the males developed lupus-like autoimmunity at a rate approaching that of females and considerably more so than in the non-bioengineered males.
Interestingly, in both some of the female mice and susceptible male mice, the absence of autoimmunity showed that some kind of environmental tissue-damaging stress (in the study, injection of an irritant) is another requirement for an autoimmune disease to develop. This again suggests the necessity of an appropriate genetic background for autoimmunity to develop.
In the paper, the authors depicted a model of Xist RNP in autoimmune progression to show that epigenetic changes can influence if, how, and when autoimmune diseases manifest. Specifically, researchers established an order that is reflective of what is known in the field—that is, “chromatin remodeling precedes gene expression changes, followed by autoantibodies and then disease symptoms,” Dou says.
Since some people with autoantibodies never develop disease, she adds, multiple checkpoints along this order of events must be overridden for progression to autoimmune disease. As the study demonstrates, a genetic component and often an environmental component are prerequisites.
“Not all females get autoimmune disease, and not all males are spared from autoimmune disease,” Dou says. In women, increased risk for disease development appears to be these Xist particles present in every single cell in their body. When released, perhaps in response to cell death or damage, the immune system mounts a reaction—at least in people who are genetically predisposed to do so.
Tackling ‘Wrongful Recognition’
One of the current limitations in the autoimmune disease field is that diagnosis and treatment are largely symptom-based. In their study, Dou and her colleagues did a preliminary screen of different autoimmune disease agents in four patients with dermatomyositis, 64 with scleroderma, and 40 with lupus in search of those eliciting autoantibodies against the proteins complexing with Xist that weren’t present in the blood of healthy controls (16 individuals).
Given a larger sample size, Dou says she expects even more autoantigens would be identified for upgrading screening panels for autoimmune disease. “We can never really have enough diagnostic biomarkers because this will help us build profiles for not only identifying high risk but also at onset the type of autoimmune disease and, as it progresses, what kind of diverse outcomes patients might be trending towards and the stage of their disease... for the design of more specific therapies.”
Many screening panels have been developed based on cell lines derived from male patients, but today there is more consciousness of sex differences that are arising and a movement towards considering the importance of sex in preclinical studies, she says. This is reflected in the questions now routinely asked of researchers by the National Institutes of Health when grants are requested and by journals when study manuscripts are going through the review process.
Next up for Dou and her colleagues is to learn the cell types involved in the creation of the “wrongful recognition” of a perceived threat that progresses toward disease and overrides all the body’s immune checkpoints. “If we can figure out where the signal is coming from, and what is recognizing these particles, that would be great for understanding the pathways involved in this triggering... and also help us intervene at critical steps.”
As with scientific research in general, it will be a long, complex but worthwhile process, she says, noting that the latest study took over six years to produce not including previous work that went into developing the technique for visualizing the proteins binding to Xist. More than anything, says Dou, she wants the general population to know important work is being done and that the results will be impactful for society. In the U.S. alone, somewhere between 24 and 50 million people have an autoimmune disease.