Gender Bender

Men and women have a few crucial differences, but when it comes to MS, those that matter aren’t the obvious ones. This disease strikes three times as many women as men, and reasons for this imbalance have stumped scientists (Orton et al., 2006, and “Discriminatory Disease”). Researchers have now identified a protein that governs sex-specific responses in human T cells, providing new insight into women’s increased risk of MS.

The study, published 28 May in the Proceedings of the National Academy of Sciences, reports that sex matters when it comes to cytokines, signaling molecules that regulate the immune system. According to the work, human and mouse T helper (Th) cells, also called CD4+ T cells, make different amounts of key cytokines, depending on whether they come from a female or a male. 

After Th cells—which activate and direct other immune cells—encounter their first antigen, subsets of them specialize to follow distinct paths. Each type of Th cell secretes characteristic cytokines, which confer distinct physiological capabilities. For example, one class of specialized cells, called Th1 cells, is thought to play a central role in provoking the inflammation that underlies MS. Previous studies have found stronger Th1 responses in female compared to male mice, but the evidence for such a disparity in humans has been inconclusive. The current work not only demonstrates the phenomenon in human cells but also zeros in on a clue to the molecular basis for such a sex bias.

“This is a beautiful study in terms of doing human and mouse comparisons,” says Caroline Whitacre, an immunologist at Ohio State University in Columbus. “Using similar techniques in both species is really very rarely done. Because [the authors] did such a comprehensive analysis, I think it makes [the findings] much more believable.”

Shannon Dunn, an immunologist at the University of Toronto in Canada, and her colleagues isolated naïve CD4+ T cells—that is, ones that have not yet encountered an antigen—from humans and mice, and myelin-reactive CD4+ T cells from mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS and other T-cell-mediated autoimmune diseases (see “Animal Arsenal”). The CD4+ T cells were stimulated with two antibodies that jointly mimic the machinery that displays, or presents, antigens to T cells. Cells from females secreted larger quantities of the cytokine interferon γ, which typifies a Th1 response, and proliferated more vigorously than did cells from males. These findings reflect an especially robust immune reaction. Activated CD4+ T cells from males showed a different—and surprising—effect. A different cytokine, called interleukin (IL) 17, predominated in these cells, although this result reached statistical significance only in the mouse experiments. This cytokine reflects the activity of a T helper cell pathway called Th17. The finding was not predicted, as scientists have generally assumed that the immune system of male mice favors another T helper cell pathway called Th2. This study didn’t detect any Th2 at all, Dunn notes.

Data from mice with EAE provide hints about how these immune activation patterns might relate to MS. Previous studies showed that castration worsens the condition in male mice, implicating androgens in immune regulation; in the current work, castrated mice with EAE produced a more “feminized” pattern of cytokine production, suggesting that androgens are responsible for maintaining the seemingly protective cytokine profile normally observed in males.

To examine the mechanism underlying the sex-specific difference in cytokine profiles, Dunn and her colleagues turned to proteins called peroxisome proliferator-activated receptors (PPARs). These nuclear hormone receptors turn on genes involved in burning fat. Alongside their well-characterized role in regulating metabolism—the diabetes drug Avandia activates PPARγ, for instance—researchers have known for more than a decade that they also deliver potent anti-inflammatory effects. Indeed, Dunn began studying the proteins with an eye toward repurposing them to treat MS. Some evidence of sex-based differences in PPAR expression in liver and adipose tissue suggested that the molecules might respond to sex hormones. As a postdoc in Lawrence Steinman’s lab at Stanford School of Medicine, Dunn and her colleagues showed that the alpha form of PPAR in T cells protects against Th1-mediated autoimmunity in male but not female mice (Dunn et al., 2007).   

In the current work, Dunn’s team examined PPAR involvement more closely. The PPARα gene, the researchers found, is expressed equally poorly in naïve CD4+ T cells of both sexes, but upon stimulation of the cells, its expression surged more strongly in male cells than in female ones. Activity from the PPARγ gene, conversely, showed a weaker signal in males compared with females after stimulation.

Next, Dunn and her colleagues measured cytokine quantities after dampening PPARα activity using a technique called RNA interference, which blocks gene expression. In male mouse and human cells, this procedure increased amounts of interferon γ in CD4+ T cells. In female cells, however, it did not change the cytokine’s quantities. Conversely, boosting PPARα activity with compounds known to stimulate the protein slashed interferon γ quantities in males but not in females. PPARα thus seems to act as a brake on the Th1 immune response—but only in males—and additional experiments indicated that this regulation occurs through a complex mechanism that controls production of interferon γ.

Similar experiments with the gamma form of the molecule showed a complementary pattern in female cells: Curbing PPARγ gene expression revved up IL-17 production in females more intensely than in males, whereas boosting the protein’s activity turned down IL-17 production in females, but not in males.

The results suggest that these two forms of PPAR contribute significantly to the “maleness” or “femaleness” of the cytokine profile. The proteins “have key roles in modulating whether a T cell will become a Th1 or a Th17 cell,” Dunn says. “Alpha seems to be very specific in inhibiting the Th1 pathway, and gamma seems to be inhibiting the Th17 pathway.”

What’s still unexplained, Dunn says, is the male bias toward the Th17 pathway, which recent work has implicated in the pathogenesis of MS and other autoimmune-related disorders. “Th17 has been kind of the new poster child for autoimmunity,” she notes, but males are clearly protected from illness—given the relatively low incidence of MS in men—despite their apparently Th17-leaning immune systems. “We still don’t understand why,” she says. “We just wanted to put it out there” for the research community to consider.

The next step, Dunn says, is to determine whether the sex difference is present in cells taken from female and male MS patients. “If so, maybe the inflammation that develops in males versus females is slightly different, and we should be treating them differently,” she says.

Another focus of future studies will be to understand why female CD4+ cells proliferate more than male cells. “In mice it’s a really striking effect, and it may be the more important effect” in terms of how the disease manifests, Dunn says, adding that it’s not yet clear whether PPARs are involved in the process.

The mechanism identified in Dunn’s study could be interesting, says George Ebers, an MS geneticist at the University of Oxford, but it doesn’t explain a key element of the sex discrepancy in MS: the fact that the skew toward women has increased dramatically over just a few generations, and that it has done so in a geographically specific manner, with an increased bias at higher latitudes. “So right off the bat, before you even start, you know this has to be a gene-environment interaction,” Ebers says. His work suggests that environmental effects such as vitamin D levels interact with the so-called major histocompatibility complex genes, which are key elements of the human immune system; some of them have been identified as strong determinants of MS risk (see “Genetic Associations”). 

Dunn agrees that environmental factors are at play but points out that even Jean-Martin Charcot, who first described MS in the 1860s, found hints that the disease disproportionately afflicts women. The existence of an underlying bias, perhaps exacerbated by other factors, wouldn’t be surprising, she says, considering that sex differences have been well-documented in other fields of immunology, such as response to vaccinations. She notes that PPARs, too, “are well-poised to be candidates as potential sensors of these environmental risk factors”—perhaps through their involvement in regulating fat and metabolism, particularly as obesity rates rise.  

Dunn’s new finding is undoubtedly not the whole story in sex disparities in autoimmune disease, she says. Still, “I believe that we are elucidating some of the biology of the sex difference.”

Key open questions

• Will the sex difference identified in this study hold for human cells from MS patients?
• Why do male and female CD4+ cells proliferate to different extents in response to stimulation, and are PPARs involved?
• Which key properties of female cells are most important to autoimmune development: cytokine production, the ability to expand in vivo, the threshold of T cell activation, or something else?