Systemic sclerosis (SSc) is a debilitating autoimmune disease that preferentially afflicts women. The molecular origins of this female bias are unclear. A new study of plasmacytoid dendritic cells from SSc patients by Du et al. (https://doi.org/10.1084/jem.20231809) suggests the X chromosome may play a key role.
Plasmacytoid dendritic cells (pDCs) from women with systemic sclerosis (SSc), a heavily female-biased autoimmune disease, exhibit impaired maintenance of X-chromosome inactivation (XCIm), increased TLR7 expression from both the active (Xa) and inactive X chromosome (Xi), and exaggerated type 1 interferon responses, suggesting that faulty XCIm may promote systemic autoimmune disease in a female-biased fashion.
Many systemic autoimmune diseases, including SSc, systemic lupus erythematosus (SLE), and Sjögren’s disease, preferentially afflict females, though the biological basis of this female bias remains unknown (Jiwrajka and Anguera, 2022). Although these diseases have diverse clinical and serologic manifestations, they all exhibit a type 1 “interferon (IFN)-stimulated gene” (ISG) transcriptional signature in the circulation and/or affected tissues (Muskardin and Niewold, 2018), suggesting that the mechanisms underlying the shared female sex bias of these diseases could also contribute to this shared immunopathology involving aberrant type 1 IFN production and signaling. pDCs are a predominant source of type 1 IFNs that have been implicated in the pathogenesis of SSc (Kafaja et al., 2018; Kioon et al., 2018; Ross et al., 2021). pDCs express high levels of pattern recognition receptors that promote type 1 IFN responses, including TLR7, and uniquely in SSc, TLR8 (Kioon et al., 2018). Importantly, both TLR7 and TLR8 are X-linked, which encourages consideration regarding how their expression is appropriately regulated in females (XX) compared with males (XY).
The balance of X-linked gene dosage between XX and XY individuals requires XCI, a mechanism that epigenetically silences supernumerary X chromosomes in all female somatic cells. As many X-linked genes have important immune functions (TLR7, TLR8, FOXP3, CD40LG, CXCR3, etc.), impaired XCI maintenance (XCIm) in immune cells has been hypothesized to contribute to female-biased autoimmunity through the partial reactivation of immunomodulatory genes from the Xi (Forsyth et al., 2024; Jiwrajka and Anguera, 2022). However, no prior studies have investigated the fidelity of XCIm in pDCs isolated from women with female-biased autoimmune diseases. Similarly, although pDCs from patients with SSc ectopically express TLR8, it is unknown whether this expression originates from the Xa, the Xi, or both. More broadly, given their low abundance in circulation, it has been challenging to study the transcriptional heterogeneity of pDCs in health and disease.
Now, Du et al. help clarify these important unknowns through an in-depth examination of pDCs and the X chromosomes in SSc (Du et al., 2025). Using a pDC enrichment strategy followed by single-cell RNA sequencing, they define the transcriptional heterogeneity of pDCs isolated from healthy females and females with diffuse SSc. They identify subclusters of pDCs expressing high levels of ISGs and show that these ISGhigh subclusters are enriched in SSc. Given these findings, the authors investigate the fidelity of XCIm as a mechanism contributing to type 1 IFN production by pDCs. They find that some pDCs isolated from females with SSc exhibit expression of TLR7 and TLR8 from both the Xa and Xi, suggesting that impaired XCIm may contribute to female-biased type 1 IFN production in SSc. Notably, the authors also show that type 1 IFNs may impair the localization of XIST RNA, a component of the XCIm machinery, to the Xi in pDCs. These findings suggest that there may be a positive feedback loop for type 1 IFN production and signaling that is uniquely possible in XX individuals with SSc. The authors also posit that the increased expression of TLR7 may be due to the looping of this genomic locus outside of the Xi’s nuclear territory, where it may be more transcriptionally accessible. While this claim warrants additional investigation, it raises interesting considerations of the potential contributions of chromosome architecture to SSc pathogenesis. Thus, this work provides evidence that impaired XCIm, by derepressing Xi-linked TLR7 and TLR8 expression, contributes to aberrant type 1 IFN signaling in pDCs from patients with SSc, providing new insights into SSc’s female sex bias and its chronic type 1 ISG signature (see figure).
Impaired XCIm in pDCs from females with SSc promotes type 1 IFN responses. pDCs from patients with SSc exhibit greater expression of ISGs, ectopic TLR8 expression, and increased biallelic TLR7 expression due to impaired XCIm. The resulting type 1 IFN production may further destabilize the XCI machinery resulting in a feed-forward loop of type 1 IFN signaling that is uniquely possible in XX individuals.
This work is especially timely and offers considerations for the molecular basis of female bias in other systemic autoimmune diseases beyond SSc. While several recent studies have focused on the immunologic contributions of the XCI machinery upon its release from dying cells in females (Crawford et al., 2023; Dou et al., 2024), the present study builds upon a complementary body of work investigating the epigenetic contributions of the XCI machinery in maintaining a transcriptionally silent Xi in females (Wang et al., 2016; Souyris et al., 2018; Syrett et al., 2019a; Huret et al., 2024; Lovell et al., 2024, Preprint).
How does XIST RNA impact type 1 IFN?
Du et al. find that while SSc pDCs were enriched for cell clusters with high ISG expression, they generally exhibited lower XIST RNA expression (Du et al., 2025). In another recent study, Crawford et al. found that XIST RNA fragments can stimulate primary human pDCs in vitro to produce type 1 IFNs in a TLR7-dependent manner. Accordingly, they describe a positive correlation between ISG expression and XIST RNA levels in human peripheral blood mononuclear cells (PBMCs) from females with SLE (Crawford et al., 2023). These seemingly contradictory findings highlight the dichotomous yet complementary roles that XIST RNA may play in the genesis of female-biased autoimmunity—either as a proinflammatory mediator or an epigenetic regulator of XCI—and suggest that the relative contributions of these roles may vary across cell types, patients, and diseases. Although historically considered dispensable for XCIm, XIST expression is indeed important for maintaining silencing of the Xi, particularly in immune cells (Huret et al., 2024; Lovell et al., 2024, Preprint). These studies demonstrate that either reduced or complete loss of Xist expression in specific female immune cells can either spontaneously promote or exacerbate features of systemic autoimmunity in female mice through the aberrant expression of Xi-linked Tlr7-pathway members. Importantly, the effects of decreased Xist expression appear to be most apparent within a subset of X-linked genes expressed within well-defined immune cell populations that may exhibit a particular dependence on those genes. Thus, future investigations exploring the relationship between XIST RNA expression and XCIm in autoimmune disease should consider focusing on relatively homogenous immune cell subsets. Notably, in some cases, existing single-cell approaches may be inadequate to study rare cell subsets, underscoring the need for alternative approaches including cellular enrichment strategies like those described here.
How does type 1 IFN impact XIST RNA?
Another intriguing outcome of this study pertains to the impact of type 1 IFNs on XIST RNA. Despite the positive correlation between ISG expression and XIST RNA levels in PBMCs from females with SLE, Crawford et al. show that in vitro stimulation of SLE PBMCs with type 1 IFN does not affect XIST expression, indicating that XIST RNA is not an ISG, but instead a driver of type 1 IFN production (Crawford et al., 2023). Intriguingly, although Du et al. do not quantify XIST RNA levels in healthy human pDCs stimulated in vitro with type 1 IFN, they show that in vitro stimulation of pDCs with type 1 IFN results in the dispersal of XIST RNA particles beyond the Xi territory and increased biallelic TLR7 expression (see figure). These observations suggest a model where extracellular XIST RNA fragments stimulate pDC-mediated production of type 1 IFNs, which reciprocally destabilize the XCIm machinery, perhaps by modulating the expression of XIST RNA binding proteins which tether XIST to the Xi, as has been previously suggested (Syrett et al., 2019a; Pyfrom et al., 2021; Jiwrajka et al., 2023). Future work is necessary to determine how IFN signaling impacts XIST RNA’s localization to the Xi.
Where in the world is XIST RNA?
Regarding XIST RNA localization patterns, it is fascinating that, unlike most female somatic cells, pDCs from either healthy female donors or patients with SSc generally appear to lack XIST RNA clouds. These observations are consistent with prior observations in murine pDCs and several additional mouse and human immune cells (Wang et al., 2016; Syrett et al., 2019b) and prompt intriguing questions related to the fidelity of XCIm and both the identity and breadth of genes expressed from the Xi in female pDCs. Is the absence of cytologically visible XIST RNA associated with increased biallelic expression of additional proinflammatory genes, perhaps exclusively in the setting of disease? What epigenetic factors are reinforcing XCIm in these cells, and how are these factors modified in disease? Answering these questions could help identify novel mechanisms contributing to female-biased autoimmunity in SSc and beyond.
Du et al.’s study advances our understanding of the mechanisms contributing to the female bias of SSc. Ongoing work in this field could eventually lead to clinically translatable insights and potentially enable sex-specific prognostic biomarker discovery and treatment selection. For example, the recently opened phase 3 trial of anifrolumab in SSc would provide an opportunity to perform an exploratory analysis of sex-specific therapeutic effects resulting from type 1 IFN receptor blockade. Though the strong female sex bias of systemic autoimmune diseases presents a challenge in designing clinical trials that are adequately powered to identify sex-specific therapeutic effects, such efforts may enable us to reach a point in the era of precision medicine where we consider sex not just as a biological variable that informs our research, but as a clinically actionable variable that informs our medical decision making.
Acknowledgments
Author contributions: N. Jiwrajka: Conceptualization, Funding acquisition, Visualization, Writing - original draft, Writing - review & editing, M.C. Anguera: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing - review & editing.
References
Author notes
Disclosures: The authors declare no competing interests exist.