The statistics have been clear and consistent for decades: autoimmune conditions are at least three times more common among women than men. Some studies suggest it’s more like a factor of four. For specific diseases like Lupus, the disparity is 9-fold. For Sjogren’s syndrome, it’s 19 to 1.
This appears to be an objective biological phenomenon. It’s not a reflection of gender-based differences in seeking medical care, or of misdiagnosis—though there’s plenty of that. Neither is it strictly hormone-related.
Hormonal factors do play some role in autoimmune disorders. But the hormonal differences between biologically female and male individuals in no way fully account for the marked disparity of autoimmune diseases.
In humans, and in rodent models, the simple presence of the double X chromosome is far more predictive of autoimmune risk than any measure of hormone status.
A Vexing Question
What accounts for such a marked gender disparity? It’s a question that has vexed researchers, clinicians, and patients for a long time.
An international research team headed by two dermatologists at Stanford University believe they have an answer, or at least a strong clue.
They contend that Xist—a specific form of long non-coding RNA produced by biologically female mammals—is the decisive factor.
Xist has a very particular function: it squelches gene expression on one of the two X chromosomes in each cell, thereby preventing a lethal doubling of proteins coded by genes within the X chromosome. This X chromosome inactivation occurs during early embryogenesis.
Through a process that appears to be random, each cell in the embryonic female body “decides” which of its two X chromosomes it will inactivate. The one selected for silencing begins to produce Xist, which then binds to and coats the chromosome, thus inhibiting transcription of its genes.
This “decision” is preserved over the lifetime of the individual, through many cycles of cell division, ensuring that all of the cells in the body are only transcribing one set of X chromosome genes into actual proteins.
X Inactivation & Autoimmunity
In humans, Xist is a 19 kb long non-coding (lnc) RNA chain. The length of the chain differs slightly in other mammals, but the function is the same. Biologically male animals have the gene for Xist on their X chromosomes, but under normal circumstances it is never expressed. Production of Xist only occurs when two X chromosomes are a matched pair inside a cell.
So, what does this have to do with autoimmune disease?
Once Xist binds to the X chromosome that will be silenced, a wide range of other types of proteins bind onto Xist, forming ribonucleoprotein (RNP) complexes. It turns out that many of these are autoantigenic.
“Here, we show that the Xist ribonucleoprotein (RNP) complex comprising numerous autoantigenic components is an important driver of sex-biased autoimmunity,” writes lead author Diana Dou, PhD, in the introduction to the Stanford group’s recent landmark report, published in the journal, Cell.
Dr. Dou is a researcher in the lab of Stanford dermatologist and geneticist, Howard Y. Chang, MD, PhD. Nearly a decade ago, Chang, Dou, and their colleagues identified nearly 100 proteins that can bind to and accumulate around Xist. A more detailed bibliomic analysis showed that 30 of these Xist RNPs had been previously identified as targets for autoantibodies associated with human autoimmune diseases including Rheumatoid Arthritis, Systemic Lupus Erythematosus, Multiple Sclerosis, Myositis, and Sjogren’s syndrome.
This was not entirely surprising. The authors note that autoantibodies are often directed toward nuclear RNA binding proteins. “The nature and titer of such autoantibodies define the type and severity of autoimmune diseases in clinical practice.”
But the fact that these particular autoantibodies targeted a specific type of RNA complex that binds to the X chromosome, and is normally produced only by biologically female mammals, opened an entirely new line of inquiry about the etiology of autoimmune disease.
“Human patients with autoimmune diseases displayed significant autoantibodies to multiple components of XIST RNP. Thus, a sex-specific lncRNA scaffolds ubiquitous RNP components to drive sex-biased immunity,” they wrote.
This hypothesis was bolstered by the fact that in men with Klinefelter syndrome—a rare genetic condition defined by having one Y and two X chromosomes—the risk of many autoimmune disorders is comparable to that of women. Men with Klinefelter’s are phenotypically male. Though their testosterone levels are usually low, they do show typically male hormone patterns. It seems that in this population, the predisposition to autoimmunity is connected to the presence of that second X chromosome.
Dr. Chang, who is a clinician as well as a researcher, said his driving interest in this question is a desire to help his patients. “As a practicing physician, I see a lot of lupus and scleroderma patients, because those autoimmune disorders manifest in skin,” he said in a Stanford University press release following publication of the new study last February. “The great majority of these patients are women.”
Xist Increases Autoimmunity
To prove that Xist can generate autoimmune responses, the Stanford team assembled an interdisciplinary, international consortium of researchers who, together, undertook some very sophisticated bioengineering.
First, they developed transgenic strains of mice in which the males produced Xist in a similar manner as the females. The particular Xist gene which they inserted into the mouse genome is inducible by external chemical means. In this case, the chemical trigger was doxycycline, which the investigators could give to the mice in drinking water. This gave the them the possibility of turning on or shutting off Xist production in the males. Once induced, Xist forms similar RNP complexes in the males as seen in female mice.
The investigators then injected the test mice with pristane, an irritant that can induce a lupus-like autoimmune reaction in susceptible mice. In this way they could test whether male mice that produced Xist were more likely than normal males to show the autoimmune response.
Indeed, they were. The male mice with activated Xist were far more likely to show autoimmune reactions following pristane stimulation than the non-Xist males. The autoimmune reactions occurred at a rate comparable to that of females similarly stimulated with pristane.
“Increased disease severity and elevated autoreactive lymphocyte pathway signatures were observed in the mouse models of pristane-induced SLE,” the authors report. “Expression of Xist RNPs in male mice is sufficient to increase disease severity and change the expression and epigenomic profiles of both the B cell and T cell effectors of SLE pathogenesis.”
Chang and colleagues note that the presence of Xist alone does not induce autoimmunity; the Lupus-like syndrome still needed to be stimulated by the pristane injection. But the presence of activated Xist in genetically male mice that would not ordinarily express Xist did increase the likelihood and severity of the autoimmune reaction once the pristane stimulus was applied.
The findings give creedence to the core hypothesis that production of Xist, and the subsequent RNP complexes it forms, predisposes an organism to autoimmune reactions.
New Human Disease Markers
On its own, this mouse experiment could be dismissed as a very elaborate science fair project with limited clinical significance. But the Stanford team went a step further: based on the antigens they identified in the mice, they designed an antigen array to test serum obtained from humans with autoimmune diseases, which allowed them to assess reactivity to specific Xist RNP complexes.
Using this array, they were able to compare serum from patients with Dermatomyositis, Scleroderma, or SLE, with samples obtained from healthy donors who did not have autoimmune diagnoses.
They found that the autoimmune patients were significantly more reactive to 79 of the antigens tested than the healthy control subjects. Of those 79 antigens, 53 were associated with Xist RNPs. Nine of the protein complexes tested were antigenic in all three of the disease categories. Further 28 of the 53 Xist-associated antigens had not previously been described in association with specific autoimmune diseases.
“These results show that multiple proteins from the Xist RNPs are novel autoantigens in patients with DM, SSc, and SLE.” This suggests that some of them could eventually become potentially new markers for detecting autoimmune conditions.
The Stanford research goes a long way in making the case that Xist and its related protein complexes, play a role in the etiology of autoimmune disorders, though it’ll take more work to definitively prove it.
Nature vs Nurture?
The findings are provocative in that they suggest that the strong gender disparity in incidence of autoimmune disorders could be gene-linked, and therefore independent of diet, lifestyle, or environmental factors.
In an interview shortly after publication of the study, Dr. Chang stated: “This research says that the risk for autoimmune disease comes from your genes. It’s not something you did wrong. Sometimes people are like, “Did I eat something wrong? Did I do something wrong?” No. It’s not something you can control.”
That runs somewhat counter to core principles of functional, holistic, and naturopathic medicine which put autoimmunity in the context of modifiable factors like microbiome dysregulation, intestinal impermeability, food allergies, and exposure to things like mold and environmental toxins.
Understandably, the paper generated considerable interest within the functional medicine community.
Terry Wahls, MD, a professor of medicine at the University of Iowa, who reversed her own Multiple Sclerosis via nutritional and lifestyle changes, and has taught MS patients to do the same, described the Stanford study as “Super interesting.”
Though the clinical implications are not entirely clear, Wahls says the paper underscores the broad problem of “not having enough women in studies. A blind spot was created,” she told Holistic Primary Care.
Joel Evans, MD, a functional medicine practitioner specializing in women’s health, says the Xist research prompted a lot of conversation among his peers and colleagues. Though it clearly suggests that predisposition to autoimmunity is X-linked, this is definitely not a clear-cut nature versus nurture dyad. The predisposition may indeed be genetic, but exogenous factors still play a big role in determining whether or not someone will actually manifest an autoimmune disease, Dr. Evans says.
Indeed, while autoimmune conditions are more common in women than men, the majority of women do not have autoimmune diseases despite having an XX genotype and producing Xist RNA. Further, some men do have autoimmune conditions, though they do not produce Xist. In fact, one autoimmune disease—Type 1 diabetes—has a higher incidence in males versus females.
Studies of identical twins show that the development of autoimmune disorders is not always identical in twin pairs, despite the shared genome.
The discovery that Xist-linked protein complexes can function as autoantigens is compelling. It adds a new and very interesting piece to the autoimmune puzzle. But it is not the entire picture.
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