# Escape From X Chromosome Inactivation as a Driver of Plasmacytoid DC Heterogeneity in Health and Disease

Franck Barrat, Jean-Charles Guéry

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## To cite this version:

Franck Barrat, Jean-Charles Guéry. Escape From X Chromosome Inactivation as a Driver of Plasma-cytoid DC Heterogeneity in Health and Disease. Immunological Reviews, In press, 336 (1), pp.e70074. 10.1111/imr.70074. hal-05364668

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Submitted on 14 Nov 2025

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Distributed under a Creative Commons Attribution 4.0 International License
Escape from X-chromosome inactivation as a driver of
plasmacytoid DC heterogeneity in health and disease.

Franck J. Barrat¹,² and Jean-Charles Guéry³

1: Inflammation and Autoimmunity Program and David Z. Rosensweig Genomics Research Center, HSS Research Institute, Hospital for Special Surgery, New York, NY, USA

2: Department of Microbiology and Immunology, Weill Cornell Medicine, New York, NY, USA

3: Institut Toulousain des Maladies Infectieuses et Inflammatoires (INFINITY), Université de Toulouse, INSERM, CNRS, UPS, Toulouse, France.

Correspondence : [barratf@hss.edu](mailto:barratf@hss.edu) or [jean-charles.guery@inserm.fr](mailto:jean-charles.guery@inserm.fr)
**Summary**

Decades of experimental work has helped define heterogeneity of the various cell types that compose the immune system. The different cell types arise from distinct hematopoietic stem and progenitor cells in a coordinated fashion during ontogeny, providing a set of diverse cells that contribute to host defence. Cells can also differentiate into different subsets in response to the cytokine and tissue environment, creating a level of cellular heterogeneity that helps direct the nature and magnitude of the immune response.

Here we are discussing a variation whereby cellular heterogeneity arises due to the expression of X-linked immune genes that escape X-chromosome inactivation, giving an advantage to a subset of cells more prone to respond to stimulation by external (pathogens) but also internal signals (i.e., mechanosensing). Interestingly, these inflammatory subsets are much more likely to be differentially enriched in patients with autoimmunity or inflammatory diseases which are well known to be predominant in females. We are using plasmacytoid dendritic cells (pDCs) as a model cell type, as these cells are a rare but critical subset of innate immune cells, with a rapid and massive capacity to produce type I IFNs (IFN-I) upon sensing of nucleic acids from pathogens, but also from the self, and these cells have been linked to the pathogenesis of many autoimmune diseases.

**Key words:** Autoimmunity, plasmacytoid DCs, X chromosome inactivation, TLRs
**Introduction**

Plasmacytoid dendritic cells represent less than 1% of peripheral blood mononuclear cells (PBMCs) in humans ¹⁻³. The most defining feature of pDCs is their capacity to produce high amounts of IFN-I upon sensing of nucleic acids from pathogens, but also from the self ⁴.

Activated human pDCs also secrete TNF and IL-6, and upregulate co-stimulatory molecules (CD80, CD86, CD40), allowing them to present antigen to T cells and bridge innate sensing with adaptive immune activation ¹,². Human pDCs are derived from the bone marrow and require Flt3 ligand (Flt3L) for development ². They are characterized by high expression of CD123 (IL-3Rα) and specifically express BDCA-2 (CLEC4C), BDCA-4 (Neuropilin-1) and ILT7 as well as other markers such as HLA-DR, CD68, ILT3 or CD4 and many chemokine receptors. However, human pDCs lack lineage markers (CD3, CD13, CD14, CD16, CD11b, CD11c, CD19, CD56) ¹⁻³,⁵. At steady state, pDCs circulate in the blood and can migrate into lymphoid tissues and sites of infection via chemokine receptors such as CXCR3 and CCR7. In the absence of infection, they remain in a quiescent state with minimal basal IFN expression and limited surface MHC-II or co-stimulatory molecules. The hallmark of pDC function is their unique ability to rapidly produce type I and type III IFNs following detection of nucleic acids ⁶⁻¹⁰. In humans, this sensing is mediated primarily by the ssRNA-sensing TLR7 and DNA-sensing TLR9, which are constitutively found in endosomal compartments of the cells.

Upon internalization of viral or endogenous nucleic acids —either directly or via immune complexes—pDCs traffic these ligands to endosomal compartments where TLR engagement occurs. Ligand binding triggers a cascade via MyD88, an adaptor protein that recruits IRAK1, IRAK4, and TRAF6⁴, ultimately activating the master transcription factor IRF7 ¹¹,¹².

Importantly, human pDCs express high basal levels of IRF7, enabling them to bypass the need for IFN-I priming required in other immune cells ¹¹,¹². This rapid IRF7 activation results in the transcription of hundreds of interferon-stimulated genes (ISGs), including all IFN-α subtypes, IFN-β, regulatory cytokines and chemokines and anti-viral molecules. The IFN-I output of activated pDCs vastly exceeds that of other immune cells and can constitute the majority of IFN in circulation during acute viral infection ¹⁻³,⁸,¹³. pDCs can also produce IFN-I indirectly by sensing DNA released from neutrophils infected by bacteria such as mycobacterium tuberculosis ¹⁴,¹⁵. The IFN-I produced by pDCs bind to the IFN-α/β receptor (IFNAR) expressed on nearly all nucleated cells which initiates JAK1 and TYK2 activation, leading to phosphorylation of STAT1 and STAT2, which dimerize and form a complex with IRF9 known as ISGF3 and this cascade leads to further induction of transcription factors that can induce ISGs. This complex drives the transcription of ISGs in various cells in circulation
and in tissue and in addition to antiviral defence, IFN-I is also enhance dendritic cell maturation, promote MHC expression, and support the activation of NK cells, CD8+ T cells, and B cells. Because of the adjuvant properties of pDCs, their activation is tightly regulated to balance immunity with self-tolerance. However, in inflammatory conditions, particularly in autoimmune settings, pDCs can become potent drivers of immune activation ³.

Indeed, a hallmark of many systemic autoimmune diseases is the presence of an IFN-I signature characterized by upregulation of ISGs in blood and affected tissues ¹⁶. Although the source of IFN-I likely depends on the disease, disease status and impacted tissue, it appears that pDCs contribute to this excess of IFN-I in autoimmune patients where we and others have shown that pDCs are chronically activated with changes in their metabolic landscape. Of note, TLR7 and TLR8 are key sensors of ssRNA and self-DNA/RNA released from apoptotic or necrotic cells, immune complexes containing nucleic acids, and mitochondrial DNA that can activate pDCs in autoimmune patients, as clearly illustrated in patients with systemic lupus erythematosus (SLE) or systemic sclerosis (SSc).

SSc is a complex autoimmune connective tissue disease characterized by vasculopathy, immune dysregulation, and progressive fibrosis of the skin and internal organs. Although SSc shares features with other systemic autoimmune diseases, it is distinguished by its intense fibrotic manifestations and high morbidity. pDCs have emerged as key amplifiers of both immune activation and tissue damage as these cells accumulate in fibrotic skin where they are found in perivascular regions and fibrotic dermis but also in lungs of patients with interstitial lung diseases (ILD).

In SSc, pDCs exhibit a chronically activated phenotype, spontaneously secrete IFN-I and CXCL4, infiltrate fibrotic tissue and are impacted at the metabolic level ¹⁷⁻²⁰. As discussed below, subsets of pDCs are enriched in patients with SSc due to the dysregulation of TLR7 and TLR8 expression and function¹⁸,²⁰. In tissue, we recently demonstrated that pDCs are highly sensitive to the biophysical properties of their environment²¹. It is likely that substrate stiffness and spatial confinement of pDCs in fibrotic tissue may be guided by chemokines that also contribute to their activation. We and others have shown that CXCL4, a key pro-fibrotic molecule, as well as other chemokines can complex and form nanoparticles with DNA, increasing its stability and uptake by pDCs and leading to superinduction of TLR-mediated IFN production, creating a feed-forward loop of inflammation and fibrosis²²,²³. In a murine model of SSc, depletion of pDCs significantly reduced inflammation and fibrosis, confirming the central role of the pDC-IFN axis in disease pathogenesis²⁰. Importantly, this study
highlighted TLR8 as a critical sensor in pDCs, suggesting that both TLR7 and TLR8 contribute to disease-promoting IFN responses.

It is well known that SSc and many other autoimmune diseases are more prevalent in women ²⁴⁻²⁷. As both TLR7 and TLR8 are encoded on the X chromosome, and with the observation that gain of function mutants of human TLR7 can induce autoimmunity ²⁸⁻³¹, we will discuss pDC heterogeneity in patients with SSc, whether escape from X chromosome inactivation (XCI) accounts for the dysregulation of TLR7 and TLR8 expression and chronic activation of pDCs in patients, whether changes in the metabolic landscape impact the epigenetic regulators of XCI in patients with SSc, and the functional consequences for the behaviour of these cells once they infiltrate the tissues.

**Relevance of X chromosome associated genetic risk to the female predominance in autoimmunity.**

Autoimmune diseases disproportionately affect females, often with a female-to-male ratio as high as 9:1 in some conditions like SLE or SSc ²⁵,²⁶. While hormonal and environmental factors contribute to this disparity, mounting evidence highlights the X chromosome complement and its unique regulatory mechanisms as critical genetic risk factors. This is supported by the observation that males with Klinefelter syndrome (XXY) have a similar risk of developing female-biased autoimmune diseases compared to females (XX) ³²⁻³⁴. Thus, having multiple X chromosomes enhances the susceptibility to develop female-biased autoimmunity. This observation strongly supports the idea that having two X chromosomes, regardless of hormonal background, contributes to autoimmunity risk. Indeed, in the four-core genotype (FCG) mouse model, XX mice have higher susceptibility to autoimmune diseases than XY mice, even when gonadal sex is controlled ³⁵,³⁶. These findings suggest that it is the number of X chromosomes and their gene dosage effects that are mainly responsible for the female bias in autoimmune disease, although additive effects of sex hormones cannot be ruled out as reported for pDCs ³⁷.

To equalize gene expression between XX females and XY males, one X chromosome in female cells is transcriptionally silenced in a process known as X chromosome inactivation (XCI) ³⁸. This process occurs early during embryogenesis and is maintained throughout life. XCI is orchestrated by a complex interplay of epigenetic modifications, including DNA methylation, histone modifications, and the action of the long non-coding RNA *XIST* (reviewed in ref ³⁹). In humans, XCI is established at an early stage of female embryogenesis and is then inherited through somatic cell divisions over life. However, XCI is not complete
and 15-23% of X-linked human genes escape XCI and are thus expressed from both the active (Xa) and inactive (Xi) X chromosomes, in certain cells, tissues or individuals<sup>40,41</sup>. Many immune-related genes are carried by the X chromosome. As such, potential defects in XCI could lead to altered expression of these X-linked genes and promote autoimmunity<sup>42</sup>.

Since XCI occurs randomly in early embryonic cells, females are natural mosaics of two cell populations: one expressing maternal X alleles and the other expressing paternal (Figure 1A). This mosaicism can provide resilience against X-linked deleterious mutations but also introduces heterogeneity in immune cell repertoires. The functional contribution of X-linked dosage to autoimmune phenotypes has been supported by mouse studies, as male BXSB-Yaa mice carrying two copies of *Tlr7* spontaneously develop features of an SLE-like syndrome<sup>29,43,44</sup>. Likewise, human TLR8 overexpression in a mouse induces the spontaneous development of systemic arthritis<sup>30</sup>. Duplication of *Cd40lg/CD40LG* in mice and humans is sufficient to induce autoimmunity<sup>45,46</sup>. Thus, biallelic expression of such immune regulatory genes may endow some female immune cells with enhanced functional properties. Indeed, human B cells with bi-allelic expression of *TLR7* class switch more frequently and exhibit enhanced capacity to differentiate into plasmablasts upon stimulation through TLR7 compared to B cells with monoallelic expression of *TLR7*<sup>47-49</sup>. Likewise, female pDCs with bi-allelic expression of *TLR7* expressed more *TLR7* transcript compared to monoallelic cells<sup>50,51</sup>. This gene dosage imbalance in XX individuals may lower the activation threshold of autoreactive B cells or innate immune cells and tip the balance toward loss of self-tolerance resulting in female bias in autoimmunity.

## Sex differences in pDC innate function in health: sex effects are everywhere.

Among immune cell subsets, pDCs exhibit robust sex bias in their function, most specifically their capacity to produce IFN-I (IFN-α/β) in response to stimulation by virus-associated ssRNA or their endosomal TLR ligands, particularly TLR7<sup>52</sup>. This sex-bias was first established in 2006 by Berghofer and colleagues who first reported that pDCs from healthy women were able to release larger amounts of IFN-I in response to stimulation by TLR7-specific ligands, as compared to pDCs from men<sup>53</sup>. Subsequent reports confirmed this observation and established that this sex bias was due to an increased frequency of pDCs capable to secrete IFN-α, rather than a sex bias in IFN-α production on a per cell-basis<sup>54-56</sup>. Further, supplementation of post-menopausal women with 17β-estradiol (E2) was found to substantially enhance IFN-α production by blood pDCs not only in response to TLR7 stimulation, but also to TLR9 ligands, including natural ligands such as self-nucleic acid-
containing immune complexes present in the sera of SLE patients <sup>55</sup>. Cell-intrinsic estrogen
receptor (ER) α activation by E2 was required to upregulate pDC innate functions both in
vivo and in vitro <sup>37,55,57,58</sup>. Estrogen-signaling was subsequently found to enhance IRF5
expression in both mouse and human pDCs through ERα-signaling <sup>57</sup>. Indeed, it has been
recently reported that pDCs from mice deficient for the IRF5 adaptor molecule Tasl failed to
produce IFN-α in response to TLR7 and TLR9 stimulation <sup>59</sup>. The positive impact of
estrogens on pDC innate function has been corroborated in transgender individuals who
undergo gender-affirming hormone (GAH) treatment focused on testosterone. A longitudinal
follow-up study in transgender-male, XX females undergoing masculinizing testosterone
treatment, reported changes in pDC-derived IFN-I production that gradually declines together
with estrogen levels <sup>60</sup>. These data suggested that testosterone administration modulated
intrinsic pDC functioning rather than cell type frequencies, probably through the indirect
down-regulation of blood estrogen levels, rather than through androgen receptor signaling
within pDCs <sup>60,61</sup>.

However, besides estrogens, X-linked genetic factors independently contribute to the
strong sex bias in the TLR7-driven response of pDCs <sup>37,55</sup>. In a humanized mouse model, the
frequency of IFN-α- and TNF-producing human pDCs in response to TLR7 ligands was
greater in female than in male host mice <sup>37</sup>. Unexpectedly, functional differences were also
observed depending on the sex of the human stem-cell donors <sup>37</sup>. Female (XX) pDCs
responded better to TLR7 stimulation compared to male (XY) cells, whatever the hormonal
back-ground of the host mice <sup>37</sup>. These data provided the first evidence for a critical role of X
chromosome dosage on the functional competence of female pDCs and suggested that both X
chromosome complement and sex hormone estrogens may act in concert to confer elevated
production of IFN-I by pDCs in response to TLR7 signaling <sup>37</sup>. This genetic effect may also
explain the persistence of the female superiority in the TLR7-driven IFN-α response of pDCs
across ages, including post-menopausal and elderly women <sup>62</sup>.

The *TLR7* gene is on the X chromosome, and it was shown that this gene can escape from XCI in immune cells <sup>47,48,50</sup>. *TLR7* belongs to the category of facultative escapees as expression from the Xi was not observed in all cells, but in a distinct subset ranging from 10-40% of immune cells, including B cells, pDCs and monocytes <sup>48</sup>. Actually, *TLR7* bi-allelic cells have been observed in all female subjects tested <sup>47,50</sup>, including HIV-1-infected women under anti-retroviral therapy (ART) <sup>51</sup>. In this latter study, the frequency of pDCs with bi-allelic expression of TLR7 was similar between uninfected control and HIV/ART females, despite enhanced functional differences in the TLR7-driven pDC responses in HIV/ART
women⁵¹. Consistent with the increased cytokine responses by pDCs from HIV/ART females, a significant upregulation of *TLR7* gene expression was observed in pDCs, including in mono-allelic cells where *TLR7* was expressed almost exclusively from the Xa. These results provided evidence for an increased TLR7-driven pDC responsiveness associated with enhanced transcriptional activity of the *TLR7* locus on both X chromosomes, in pDCs from HIV/ART females. This also suggested that enhanced *TLR7* mRNA levels in pDCs at single-cell resolution could promote enhanced responsiveness to TLR7 ligands, by inducing the transcriptional activity or bi-allelic expression of the *TLR7* gene or a combination of both ⁴⁷,⁵⁰,⁵¹. Indeed, single-cell resolution analysis of *TLR7* allelic expression in female pDCs revealed that *TLR7*-biallelic pDCs not only expressed higher levels of *TLR7* mRNA but also higher levels of transcripts coding for all IFN-α subtypes and IFN-β at steady state⁵⁰. The mechanism responsible for the congruent expression of basal levels of IFN-I transcripts in pDCs biallelic for *TLR7* is currently unknown. Whether it is a consequence of a continuous low-grade tonic TLR7/MYD88 signaling triggered by endogenous ligands in *TLR7* bi-allelic pDCs remains to be investigated. TLR7 protein is expressed at higher levels in female immune cells ⁴⁷,⁵⁶, and the TLR7-driven response of human pDCs is TLR7 protein dosage-sensitive, particularly in female cells⁵⁶. Indeed, the rs179008 c.32A/T which is a SNP that introduces a substitution of a leucine for a glutamine (p.Gln11Leu) in the leader sequence of TLR7, controls protein dosage at the translational level through the mRNA sequence itself, rather than through signal peptide function⁵⁶. Thus, the minor allele rs179008 c.32T impaired IFN-I production by pDCs in response to TLR7 ligands, but only in women, including c.32A/T heterozygous ones⁵⁶. Thus, the sex bias in pDC function mirrored the rs179008 genotype-dependent drop in TLR7 protein expression in female leukocytes, consistent with the notion that TLR7 dosage in pDCs determines IFN-I secretion potential ³⁷,⁵⁰. It is therefore likely that the heterogeneity of female pDCs, associated to the facultative escape of *TLR7* from XCI, may translate into the functional discrimination between mono-allelic and biallelic pDCs⁵⁰. Functional heterogeneity in steady state pDCs is supported by numerous observations ⁶³⁻⁶⁵; although sex was not considered as a critical biological variable in those studies. Constitutive production of low levels of IFN-I in pDCs has been reported to act in an autocrine/paracrine manner to drive high level production of IFN-I by pDCs⁶⁶. Thus, pDCs with high level expression of *TLR7* mRNA due to the cell-autonomous action of *TLR7* XCI escape ⁴⁷,⁵⁰, could belong to a group of early responder pDCs with superior ability to produce IFN-I⁶⁴. We propose that this “early responder” group of pDCs could be more
prominently represented in females thanks to the genetic dosage effects linked to XCI escape of genes contributing to TLR-signaling pathways (Figure 2).

**X chromosome linked mechanisms regulating TLR-signaling in pDCs.**

Little is known regarding the causal relationship between XCI escape of *TLR7* alone or in combination with other genes, and pDC functional heterogeneity contributing to the well-established sexual dimorphism in IFN-α secretion in response to TLR7 ⁵³⁻⁵⁵,⁶²,⁶⁷. The X chromosome encodes for approximately 1000 genes among which 150 genes have been directly or indirectly linked to immunity²⁵. We will focus in this chapter on the X-linked genes contributing to TLR signaling (Figure 2), besides TLR7/8, and recapitulate the reported evidence regarding their possible contribution to pDC functional heterogeneity in females.

*The BTK-IRAK1-IKBKG axis*

Several X-linked factors could contribute to the enhanced TLR7-mediated production of IFN-I by pDCs from women through non-exclusive mechanisms²⁴. Among them, the escape from XCI of genes directly or indirectly involved in TLR-signaling can be considered. Based on expression analysis in rodent/human hybrid fibroblast cell lines that retain a human inactive X, it has been shown that up to 15% of X-linked genes display some degree of escape from XCI. This includes immune-related genes such as *BTK*, *IKBKG* (NEMO) and *IRAK1* ⁴⁰, which are critical components of the endosomal TLR7/9-signaling pathways (Figure 2). This has been directly confirmed by analyzing allelic expression in female pDCs at single-cell resolution, at least for *BTK*⁵⁰, which we previously reported to regulate TLR9 rather than TLR7 signaling in human pDCs⁶⁸. In this study, a single-cell RT-PCR approach to tag allelic expression of *TLR7* using SNP markers has been extended to other X-linked loci (e.g., *RPS6KA3*, *CYBB*, *BTK*, *IL13RA1*)⁵⁰. Simultaneous examination of up to 3 different loci within the same cells showed a heterogenous profile of XCI escape at single-cell resolution, suggesting that coordinated XCI escape at multiple loci was a rare event in female human pDCs⁵⁰.

*EXITS genes including DDX3X.*

Unbiased analysis of genome sequencing data from over 4100 tumors representing 21 tumor types revealed that six of 783 non-pseudoautosomal region (PAR) X-chromosome genes—*ATRX*, *CNKSR2*, *DDX3X*, *KDM5C*, *KDM6A*, and *MAGEC3*—contained loss-of-function mutations more frequently in males than in females, whereas similar bias was never detected in 18,055 autosomal and PAR genes. These genes were called EXITS ("escape from
X-inactivation tumor suppressor” (EXITS) genes), based on the assumption that by
expressing spare tumor suppressor EXITS genes from the Xi, females would be better
protected from cancer incidence⁶⁹. Among these EXITS genes, *DDX3X*, *KDM6A* and
*KDM5C* are recognized escapee genes with female-biased expression across multiple tissues
⁴⁰,⁴¹. Similar observations have been made in the mouse, where enrichment in RNA
polymerase II and DNAse I hypersensitivity sites were reported for *Ddx3x* and *Kdm6a*⁷⁰.
Among these constitutive escapee genes, *Ddx3x*, *Kdm6a*, *Eif2s3x* and *Kdm5c* represent genes
that possess a conserved Y-linked paralog with a similar function⁷¹,⁷². These X/Y genes play
important roles in the regulation of transcription and translation and are highly dosage-
sensitive, which could explain why they consistently escape XCI in all tissues examined so
fare. DDX3X has multiple functions and has been reported to promote IFN-I induction
through its direct association with antiviral signaling pathways⁷³⁻⁷⁵. Whereas the contribution
of *KDM6A* and *KDM5C* has not been examined in pDCs, a recent study reported upregulated
expression of *DDX3X* in female pDCs compared to males⁷⁶. DDX3X was found to positively
regulate both TLR7- and TLR9-driven IFN-α production in female pDCs⁷⁶. DDX3X partially
translocated to the nucleus together with IRF7 facilitating the transcription of IFN-α and IL-6,
suggesting that because this gene constitutively escapes XCI and is dosage sensitive, it may
contribute to the sex-bias in pDC responsiveness by enhancing TLR-signaling in female cells
⁷⁶. It may also contribute to the functional heterogeneity of human pDCs, as a minority of
cells have been shown to produce IFN-I upon TLR stimulation in droplet-based microfluidic
systems⁶³⁻⁶⁵.

TASL: *the endosomal TLR7/8/9 adaptor mediating the IRF5-dependent pathways.*

CXorf21/TASL is a strong candidate gene in SLE, as a genetic polymorphism in the Xp21.2 region in the exon encoding the *CXorf21* gene has been strongly associated with SLE in European populations, and *CXorf21* mRNA expression levels in peripheral blood cells are indicators of SLE flares ⁷⁷,⁷⁸. The function of the *CXorf21* gene has been recently characterized and this gene renamed as “TLR adapter interacting with the SLC15A4 on the lysosome” (TASL) ⁷⁹. TASL interacts with the endolysosomal transporter SLC15A4 to activate the IRF5 pathway following the engagement of endosomal TLR receptors (TLR7, 8 and 9). TASL shares functional homology with the adapter proteins STING, MAVS and TRIF and appears to be a central “hub” in the activation of the TLR-IRF5 pathway ⁷⁹. Recently, a TASL paralogue encoded by Gm6377 (TASL2), has been identified and found preferentially expressed in cDCs, whereas TASL was prominently expressed in pDCs and B cells.
Accordingly, TASL deficiency impairs IRF5 activation and downstream cytokine production in mouse pDCs including IFN-I production in vitro and in vivo⁵⁹. In pDCs the endosomal TLR7/9-induced responses were mostly abrogated in TASLKO mice, whereas residual IRF5 activity was observed due to the expression of TASL2 in cDCs⁵⁹. CXorf21/TASL, which is an interferon inducible gene, escapes XCI in immune cells⁸⁰. Indeed, the mouse ortholog Tasl gene has been recently reported to escape XCI in monocytes/macrophages and B cells⁸¹. Whether TASL/TASL2 escape XCI in human pDCs is currently unknown. Interestingly, the TLR7/9-TASL-IRF5 pathway in human pDCs could be subjected to both X-linked dosage effects but also estrogen-mediated upregulation of IRF5 through cell-intrinsic ERα-signaling in human and mouse pDCs as mentioned earlier⁵⁷. Thus, the cumulative action of estrogen and XCI escape mechanisms may independently contribute to explain the robust female predominance in TLR7-driven production of IFN-I by pDCs in women, which can be observed not only during the reproductive years⁵³⁻⁵⁵, but also in the absence of sex hormones, in prepubescent⁶⁷ or elderly subjects⁶².

## Heterogeneity in pDC populations in patients with autoimmunity.

Because it has been well described that patients with SSc chronically express ISGs in PBMCs but also in tissues such as the skin, ³,¹⁶,⁸²⁻⁹² and since polymorphisms in transcription factors that regulates ISG expression have been associated with SSc ⁹¹, IFN-I is regarded as a driver of SSc pathogenesis. Hence, a large phase 3 trial of the IFNAR blocker Anifrolumab is currently being evaluated in patients with SSc ⁹³. Although the source of the IFN-I likely involves multiple cell types and this cocktail of IFN-producing cells may differ between tissues, we and others have identified that pDCs play a role in the overall IFN-I response in SSc patients ¹⁷,¹⁸,²⁰,⁹⁴, and these cells are chronically activated in patients and promote fibrosis ¹⁸,²⁰,⁹⁵. Evidence of their chronic activation is supported by their ability to chronically secrete IFN-α and CXCL4 or express ISGs ¹⁷,¹⁸,²⁰, infiltrate tissues ¹⁸,²⁰,²³,⁹⁴,⁹⁵ or being metabolically dysregulated ¹⁹,⁹⁶. This last observation may be critical to our understanding of the way pDCs contribute to disease in SSc, as we have shown that metabolism and in particular the proper function of the TCA cycle is associated with their ability to produce IFN-I ¹⁹,²¹. Moreover, it was also reported that pDCs from SSc patients with anti-topoisomerase I Ab (ATA+) exhibited a more activated or exhausted profile, compared to pDCs from anti-centromere Ab (ACA+) patients ⁹⁶. Although this study was performed on a limited number of patients with SSc, including some males, pDCs from limited cutaneous SSc (lcSSc) were more reactive to TLR9-stimulation than pDCs from diffuse cutaneous SSc (dcSSc) ⁹⁶. Although TLR7-driven
responses were not tested in this study, these data indicate that accumulation of higher TLR9 responder pDCs is inversely correlated with disease activity. Further, the number of skin-infiltrating pDCs was shown to be different in patients with limited versus diffuse SSc⁹⁵

Taken altogether, these data provide evidence for functional heterogeneity in pDCs in SSc which could contribute to disease pathogenesis. At the single cell level, the heterogeneity of pDCs in autoimmune patients has been suggested⁹⁵,⁹⁷, but the scarcity of pDCs in the blood and tissue has made such studies challenging. We used a different approach where pDCs were enriched before sequencing which led us to identify different subclusters of pDCs, even in healthy donors¹⁸. Indeed, the scRNA-seq analysis revealed enrichment of pDC subclusters with high ISG expression in patients with SSc (ref¹⁸ and **Figure 2**). RNA fluorescence in situ hybridization (RNA-FISH) performed on primary pDCs from HDs and SSc patients showed impaired maintenance of XCI at the TLR7/8 locus in the disease state (ref¹⁸ and **Figure 2**). In SSc pDCs, both TLR7 and TLR8 were more likely to escape XCI, correlating with altered TLR7 protein expression profiles. Combined RNA/DNA FISH analysis further indicated that the TLR7/8 locus tends to localize outside of the inactive X (Xi) chromosome territory when TLR7 is expressed, implying that expression from the Xi is associated with chromatin remodeling and higher-order loop formation (ref¹⁸ and **Figure 2**).

Additionally, expression of key regulators of XCI, including the long non-coding RNA XIST and its binding partner the transcriptional repressor SPEN, were significantly reduced in SSc pDCs compared to healthy controls¹⁸. This pattern is consistent with previous findings in SSc monocytes, where aberrant ISG expression was linked to dysregulation of chromatin modifiers, including reduced SPEN levels⁹⁸. Importantly, downregulation of SPEN was also observed in IFN-I-stimulated healthy monocytes, suggesting that IFN-I signaling may suppress SPEN expression in immune cells¹⁸. A key unresolved question is whether the enrichment in ISGhigh pDC subclusters observed in patients with SSc is a consequence of enhanced TLR7 and/or TLR8 signaling. Alternatively, epigenetic imprinting in pDC progenitors could favor the emergence of such ISGhigh subclusters. Our data clearly support a model in which IFN-I signaling promotes reactivation of genes on the Xi through SPEN/XIST axis disruption, and that XCI escape at the TLR7/8 locus becomes a key driver of persistent pDC activation in female SSc patients (**Figure 4**).

As discussed earlier, the accumulation of lower TLR9 responding pDCs in severe dsSSc is intriguing and suggests that such phenotype could be attributed to an enhanced proportion of pDCs with strong ISG signature, such as the pDC clusters we identified in our study¹⁸. Indeed, two independent studies have shown that pre-sensitization of pDCs with IFN-I is
characterized by a down-regulated TLR9-driven IFN-α production, contrasting with their dramatic enhanced capacity to rapidly produce IFN-α in response to TLR7-ligands ⁹⁹,¹⁰⁰.

It is also likely that other factors such as CXCL4, which is elevated in patients with SSc ¹⁷,²²,¹⁰¹ and regulates TLR signaling in pDCs ²⁰,²²,²³, macrophages ¹⁰² and B cells ¹⁰³, and/or other chemokines, some of which are induced by IFN-I ²³, may exacerbate the accumulation of ISGhigh pDCs in patients. Further studies are therefore warranted to investigate whether pDC heterogeneity correlates with the biallelic expression of TLR7 and/or TLR8 and also whether such cells are preferentially infiltrating tissues such as fibrotic skin or lungs. This said, our data suggest that the escape from XCI of the *TLR7/8* locus could be associated with the female predominance in SSc by lowering the TLR-driven activation threshold of pDCs. Whether dysregulated XCI maintenance also occurs in pDCs from SLE patients will warrant further investigation. In favor of such hypothesis, a recent analysis of XIST expression in PBMCs from cis-female adolescents with childhood-onset SLE (cSLE) revealed a negative correlation between XIST expression and IFN-I scores as well as *TLR7* gene expression ¹⁰⁴. This observation is supportive of a role for altered XCI maintenance in controlling the pathogenic mechanisms associated with cSLE in females.

## Chromatin re-organization of the TLR7/8 locus on the Xi drives pDC functional heterogeneity

The long non-coding RNA, *XIST/Xist* is a central effector of the X-inactivation process and coats the future *Xi* *in cis*. Gene expression silencing is subsequently induced by the recruitment to the *Xi* of the polycomb complexes PRC1 and PRC2 that are responsible for deposition of repressive epigenetic marks and chromatin locking (Figure 1C). The acquisition of these repressive epigenetic marks is involved in the stable maintenance of the inactive state of *Xi* ¹⁰⁵. Although *XIST/Xist* is necessary for the initiation of the XCI process, it is dispensable to maintain the *Xi* status ¹⁰⁶,¹⁰⁷. In general, in somatic cells such as fibroblasts, *XIST/Xist* is localized on the *Xi* in the form of a "cloud" visualized by RNA-FISH, suggesting that XCI is strongly maintained in this cell type. Dynamic regulation of *XIST/Xist* has been reported in lymphoid cells both in humans and in mice ¹⁰⁸,¹⁰⁹. Whereas common lymphoid progenitors (CLP) and common myeloid progenitors (CMP) present an inactive territory covered by *Xist* and H3K27me3 repressive marks, this is not the case of mature lymphoid cells ¹⁰⁸,¹¹⁰ and including the majority of mouse and human pDCs at steady state ¹⁸,¹¹¹. Although *Xist* is not visible on the *Xi* by RNA-FISH, it is however continuously transcribed and localizes diffusely in the nucleus. Remarkably, following the activation of mature B cells
in vitro by TLR ligands (LPS (TLR4) or CpG (TLR9), Xist as well as the repressive marks relocate to the Xi according to a dynamic process involving the transcription factor YY1 ¹⁰⁸. By contrast, in murine pDCs Xist RNA localization to the Xi and enrichment of H3K27me3 repressive marks were lost suggesting that the chromatin of the Xi may be prone to reactivation of some X-linked genes ¹¹¹. We made a similar observation in ex vivo purified human pDCs where robust XIST clouds were relatively scarce in both healthy donors and women with SSc ¹⁸. To gain further insights into the link between XIST repositioning to the Xi and TLR7 biallelic expression, we designed an in vitro system to promote XIST cloud formation to the Xi in female human pDCs. Upon in vitro culture with IL-3, we observed a major XIST cloud relocalization to the Xi in >60-70% of cells ¹⁸ (Figure 4). Despite this, strong TLR7 primary transcripts were detected in primary pDCs from both X chromosomes allowing us to examine the 3-D conformation of the TLR7/8 genomic region relative to the whole Xa and Xi chromosome territories using combined RNA/DNA FISH analyses. In monoallelic cells, as one would expect, the TLR7/8 locus was in close proximity to the two X domains, with no significant differences in the Euclidean distance between the TLR7/8 genomic region and the Xa and the Xi. In striking contrast, in cells where TLR7 escapes XCI, we observed that the TLR7/8 genomic region was localized outside of the XIST domain at ≥1 µm of the whole Xi territory (Figure 2). Such a chromatin looping of the TLR7/8 locus was specific to the Xi and not observed for the Xa. This Xi-specific 3D chromatin organization makes the TLR7/8 genomic region readily accessible to the transcription machinery ¹⁸. This observation, which is in agreement with previous observations for other facultative escapees ¹¹², provides a mechanistic model to explain why some pDCs are bi-allelic not only for TLR7, but also for TLR8 ¹⁸. Indeed, TLR8 escapes XCI in monocytes and CD4 T cells in women and Klinefelter syndrome men ¹¹³, and we provided evidence that pDCs even from healthy donors expressed primary transcripts of TLR8, although with lower frequencies compared to TLR7 ¹⁸,²⁰. Because of the higher proportion of TLR7 bi-allelic cells in SSc pDCs and the finding of chromatin organization on the Xi in biallelic cells, the data suggest that the chance for TLR8 expression from the Xi could also be upregulated, thereby contributing to the enhanced ectopic expression of TLR8 in SSc pDCs ²⁰. This enhanced transcriptional activity of the TLR7/8 locus was associated with enhanced expression of the full-length form of TLR7 protein in SSc pDCs and the capacity of SSc pDCs to produce a wide range of IFN-I and pro-inflammatory cytokines and chemokines in response to TLR8 ligands, demonstrating that these pDCs are competent in IFN-I induction upon stimulation of all endosomal TLR pathways, including TLR7/8/9. This observation is in agreement with compelling evidence
that pDCs from SSc patients chronically secrete IFN-I<sup>17,19,20,22</sup>. Likewise, enhanced TLR7-
signaling and increased production of IFN-α by pDCs has been reported to be positively
correlated with disease activity in SLE<sup>99,100,114</sup>, and pDCs are also less responsive to
corticosteroid treatment in these patients<sup>115</sup>. IFN-I exposure of pDCs from 2 to 24 hours also
boosted their capacity to produce IFN-α upon TLR7 stimulation and was associated with
increased TLR7 protein localization to late endosome and lysosome-related organelles<sup>99,100</sup>.
Whether IFN-I causes up-regulation of TLR7/8-dependent IFN-I production in SSc pDCs
through accumulation of full-length TLR7 protein in these organelles will deserve further
investigation.

Together, we posit that increased *TLR7/8* expression and responsiveness in SSc pDCs
could be related to the enhanced proportions of cells in which the *TLR7/8* locus escapes XCI.
Whether the looping of this genomic region outside the Xi territories is a general feature of
*TLR7* bi-allelic pDC in SSc will warrant additional investigation, however, this model would
propose that changes in chromosome architecture at the Xi is an essential contributor to pDC
functional heterogeneity not only in healthy individuals but also in SSc patients.

Feed-forward loop of IFN-I in promoting dysregulated XCI maintenance.

Although *Xist* has been considered dispensable for the maintenance of XCI in differentiated cells, recent evidence indicates that *XIST/Xist* expression level is indeed important for maintaining silencing in immune cells <sup>81,116,117</sup>. Using a genetic model where XCI is dysregulated due to *Ftx*-deficiency, a cis-regulatory controller of *Xist* expression, we observed sustained reduction in *Xist* expression by 50% on average in adult female immune cells. This was associated with enhanced reactivation of X-linked genes, including *Tlr7* and *Tasl* in macrophages and B cells, and spontaneous systemic B cell autoimmunity <sup>81</sup>. Although the impact of *Ftx*-deficiency on pDC innate function was not tested in this study, we reported enhanced TLR7-driven inflammatory responses in *Ftx<sup>KO</sup>* macrophages, whereas TLR4 and TLR9-signaling were unaffected <sup>81</sup>. Altogether, this study established that full-blown *Xist* expression is indeed important for maintaining silencing of the Xi in immune cells, and that altering this pathway is sufficient to lead to spontaneous lupus-like disease in female mice <sup>81</sup>.

In humans, *XIST* deletion in primary B cells promoted enhanced *TLR7* bi-allelic expression and the development of CD11c+ atypical memory B cells <sup>116</sup>. *TASL* was also identified among the XIST-dependent gene repertoire upregulated upon XIST deletion, suggesting that *XIST* is critical for Xi silencing of many X-linked immune response genes <sup>116</sup>. Interestingly, re-expression of *XIST* in XIST-CRISPRi B cells re-silenced the Xi
demonstrating the highly dynamic nature and reversibility of this process<sup>116</sup>. Thus, in human B cells XIST was continuously required to maintain the silencing status of the Xi, particularly for XIST-dependent facultative escapee genes. As for XCI initiation, this process required the recruitment of the transcriptional repressor SPEN, which positively regulates HDAC3 activity and mediates H3K27 deacetylation <sup>116,118</sup> (see **Figure 1**). Although these processes have never been investigated in pDCs, the influence of XIST/Xist levels on facultative escapees has been provided in other tissues via direct SPEN-mediated gene silencing, suggesting that this XIST-SPEN-mediated mechanism of XCI maintenance could be well conserved across mammals <sup>119</sup>. Indeed, we observed reduced XIST expression in SSc pDCs at single-cell resolution, together with reduced SPEN expression that inversely correlated with ISG signatures in pDCs <sup>18</sup>. Interestingly, SPEN, together with other histone modifying enzymes and readers, has been reported to be altered in SSc monocytes associated with increased H3K27 acetylation <sup>98</sup>.

Further, SPEN expression was negatively regulated by IFN-I in monocytes and its downregulation was already present in patients with early SSc, suggesting that aberrant expression of these epigenetic modifiers may contribute to disease onset <sup>98</sup>. Our results in SSc pDCs are in agreement with this observation in monocytes and suggest that the enrichment of pDCs with high ISG expression could be a consequence of enhanced IFN-I production due to dysregulated XCI maintenance and exacerbated TLR-signalling (**Figure 4**). As suggested <sup>120</sup>, chronic production of IFN-I may further enhance bi-allelic expression of *TLR7* and ectopic expression of *TLR8*, as well as other Xist-dependent genes, through SPEN down-regulation and inhibition of *XIST* localization to the Xi. Indeed, by quantifying the effect of IFN-I-signalling in *XIST* cloud formation in female pDCs, we observed reduction of robust *XIST* clouds on the Xi, associated with a concomitant dispersal of the *XIST* RNA particles beyond the Xi territory, together with enhanced TLR7 biallelic expression (**Figure 4**). These findings suggested that *XIST* clouds can be dynamically regulated by IFN-I in female pDCs, resulting in enhanced proportion of *TLR7* biallelic cells <sup>18</sup>. In our recent study, we also measured the type of *XIST* clouds between HD and SSc pDCs and segregated nuclei according to their Type I-II versus Type III-IV *XIST* cloud profiles <sup>18</sup>. We observed slightly higher frequencies of robust *XIST* clouds of Type I-II in pDCs in SSc patients as compared to HDs. By looking at the relative proportion of biallelic cells in pDCs lacking robust *XIST* clouds, we found upregulated frequencies of *TLR7* bi-allelic cells only in SSc pDCs, compared to HDs (**Figure 5**). This observation indicates that *XIST* mislocalization could indeed influence *TLR7* biallelic frequency in SSc pDCs (**Figure 5**). This observation is also in agreement with our hypothesis
that *XIST* cloud formation on the Xi can be dynamically down-regulated by IFN-I in female pDCs, resulting in enhanced proportion of *TLR7* biallelic cells (**Figure 4**) <sup>18</sup>.

Although future work is necessary to understand the underlying mechanisms by which IFN-I signalling inhibits *XIST* RNA localization to the Xi, IFN-I could modulate the expression and/or function of XIST cofactors which are important for tethering *XIST* to the Xi as suggested <sup>108,121-123</sup>. To our knowledge, signalling pathways (environmental cues) that control *XIST* RNA mislocalization to the Xi in immune cells have not been described yet, and IFN-I could represent such a pathway. By altering the silencing status of the Xi, thereby promoting expression of XIST-dependent immune response genes, IFN-I signalling could represent a critical contributor to the functional diversity of pDCs in XX individuals during chronic inflammation (**Figure 4**). Moreover, this pathway may antagonize the positive action of T cell-derived IL-3 or GM-CSF on *XIST* localization to the Xi. IL3/GM-CSF are indeed likely to be produced in large amounts in the inflammatory tissue microenvironment. In the absence of IFN-I such cytokines, by directly acting on locally recruited pDCs, may not only facilitate their survival/maintenance in tissues, but could unexpectedly promote in female pDCs XIST cloud re-localisation to the Xi, thereby enhancing XCI maintenance in female cells. On the other hand, the presence of IFN-I may counteract this process maintaining XIST away from the Xi, thereby destabilizing the XCI machinery. Thus, dysregulated XCI maintenance could be a specific feature of rheumatic diseases with sustained production of IFN-I acting as a feed-forward loop to maintain XCI escape of facultative escapees (**Figure 4**). Whether this also occurs in females suffering from interferonopathies or monogenic lupus will deserve further investigation <sup>124</sup>. IFN-I receptor blockade, which is in clinical trials in SSc <sup>93</sup>, could therefore be particularly well suited for XX individuals which are overrepresented in patients with systemic autoimmune diseases. Indeed, Anifrolumab, a fully antagonist mAb targeting human IFN-α/β receptor, has shown promising results in the treatment of SLE <sup>125-127</sup>. Whether disease control occurs through inhibition of IFN-I pathway involvement in immune related pathways or through additional mechanisms involving re-establishment of XCI maintenance, specifically in XX individuals, will warrant further investigation. It is also worth noting that IFN-III could also have a role in these processes. It is known that pDCs can produce copious amounts of IFN-III <sup>6-10,23</sup>, however a recent study showed that human pDC are the only DC subsets that respond directly to IFN-III as these cells express high levels of IFNLR1 and IL10RB, the two chains forming functional IFN-III receptor <sup>128</sup>. Similarly to IFN-I, IFN-III-signaling activates STAT1/2 and ISGF3, and IFN-III stimulated pDC upregulated many ISG including TLR7 and TLR7-signaling pathway
associated genes, suggesting that IFN-III may act like IFN-I to dysregulate XCI maintenance
by inducing the dispersal of *XIST* RNA beyond the Xi territory, thereby increasing *TLR7*
biallelic expression. We could speculate that IFN-I and IFN-III may act sequentially to
increase TLR7/8 responsiveness in pDCs, with IFN-I signaling in the lymphoid organs and
the blood and then IFN-III signaling in the inflamed tissues such as the skin <sup>129</sup>. This
hypothesis warrants further investigation.

A link between cellular metabolism and epigenetic changes, a connection with escape
from XCI?

We have reported that ER stress has a significant impact on pDC biology. Specifically, we
showed that activation of the unfolded protein response (UPR) can suppress the production of
IFN-I by human pDCs upon TLR7 or TLR9 stimulation. There, ER stress led to the activation
of the IRE1α-XBP1 axis which reprogrammed cellular metabolism by diverting glycolysis
toward serine biosynthesis. This metabolic shift resulted in reduced levels of pyruvate, a
central metabolite that fuels the TCA cycle and subsequently leads to decreased production
and overall levels of ATP <sup>19</sup>. In parallel, we identified that mechanosensing can similarly
suppress pDC activation and IFN-I production, and that this occurs through the induction of
the transcription factor NRF2. We observed that NRF2 activation induced the pentose
phosphate pathway (PPP), another major branch of glucose metabolism. While the PPP plays
critical roles in redox balance and nucleotide biosynthesis, its upregulation again diverted
glucose-derived intermediates away from pyruvate production. As a result, pyruvate levels
were blunted leading to reduced ATP generation, hence limiting the activation of pDCs <sup>21</sup>. In
both cases, the inhibition of pDC response was linked not only to the induction of specific
stress pathways, but also from the rewiring of the cellular metabolic response which had a key
impact on the TCA cycle that is a central element of cellular metabolism. This suggests that
pDC function is intimately regulated by glycolysis and that interference with this metabolic
pathway appears to play a pivotal role in regulating the functions of pDCs, including their
ability to produce IFN-I. We observed that the interference by stress pathways on pDC's
response was dysregulated in patients with autoimmunity, including SLE and SSc, and that
pro-fibrotic cytokines and chemokines, such as CXCL4 and IFN-I, could disrupt the
metabolic response to stress. These disruptions may contribute to the persistent activation and
dysfunctional immune responses by pDCs in patients.

As discussed above, we and others have observed heterogeneity in pDC populations in
patients with SLE and SSc with the enrichment of subsets with high levels of expression of
ISGs which were associated with escape from XCI of the immune receptors TLR7&TLR8<sup>18,95,97,130</sup>. We also observed changes in expression levels of key epigenetic regulators in the ISG<sup>high</sup> pDC subsets. Hence, it is tempting to speculate that changes in the cellular metabolism of pDCs may be involved in epigenetic changes in the cells that are likely to regulate XCI maintenance, or lack of, in pDCs of patients with SSc. Although escape from XCI is multifactorial, it is normally maintained by the function of the long non-coding RNA *Xist*, which recruits chromatin-modifying enzymes to silence one of the two X chromosomes in female cells (**Figure 1**) <sup>39,118</sup>. Disruption of this silencing mechanism is often associated with altered activity of histone-modifying enzymes and chromatin remodelers. As many of these enzymes can be regulated by metabolites, this suggests a potential link between cellular metabolic state and epigenetic integrity.

Many metabolic intermediates generated through glycolysis and during the TCA cycle pathways can regulate key enzymes that mark histones, suggesting that these can control gene regulation. This includes Acetyl-CoA that is required for the proper function of histone acetyltransferases (HATs), which are known epigenetic regulators by mediating histone acetylation and thus promoting gene transcription, but also α-ketoglutarate (α-KG) and NAD+ which act as co-factors of histone demethylases which remove repressive methylation marks. Hence, given this interaction between metabolites and histone modifying enzymes, it is possible that changes in cellular metabolism present in cells of patients could alter the chromatin landscape and potentially modulate XCI escape. This is illustrated in tumor cells where disruption in metabolic landscape is well known and escape from XCI is frequent <sup>69</sup>. Although these examples do not provide a direct and causal link between the metabolic landscape and disruption in XCI maintenance, the impact of metabolites on epigenetic enzymes suggests that changes in cellular metabolism of pDCs of patients with autoimmune disease may explain some of the increase in XCI escape and the enrichment of chronically activated cells in patients. Future studies are needed to explore how changes in metabolic flux and in particular in glycolysis under stress, might contribute to epigenetic instability in cells such as pDCs. A deeper understanding of these interactions could open new therapeutic strategies to interfere with the aberrant chronic activation of pDC function in patients with autoimmune diseases.

**Functional consequences of increased pDC activation in tissues**

The observations that, in patients with autoimmunity, pDCs are chronically activated with the presence of subclusters that express ISGs and with the increased proportion of cells with
biallelic expression of *TLR7*, raise the question as how these features impact the functional response of pDCs within tissues. We used the skin as model system as (i) pDCs are well known to contribute to skin inflammation both in preclinical models<sup>23,94,131-133</sup> and in patients<sup>134-138</sup>, (ii) these cells are absent in healthy skin but quickly infiltrate the tissue upon injury<sup>20,95,131,132</sup> providing an ideal system to study recently skin-infiltrating cells upon injury and (iii) the IFN-I they produce is known to induce lesions in the skin<sup>125-127</sup>. The presence of activated pDCs in the skin of patients with SSc or SLE suggested that the environment of the skin and in particular its stiffness would be sensed by pDCs as a danger signal and would trigger their activation. Indeed, mechanosensing can influence cellular response<sup>139-145</sup>. However, we observed that increased stiffness negatively impacted the ability by pDCs to produce IFN-I in response to TLR stimulation<sup>21</sup>. This was due to the induction of a stress response that reshaped the metabolic landscape of pDCs with reduced function of the TCA cycle and reduced ATP levels<sup>21</sup>. Although the signal induced by mechanosensing activated NRF2, other stressors that induce the UPR similarly led to metabolic changes in pDCs associated with reduced ATP levels and IFN-I response<sup>19,21</sup>. These data suggest that hostile environments can negatively impact the ability of pDCs to respond to TLR7 and TLR9 signaling, and we posit that this is a critical mechanism to resolve pDC activation following their sensing of nucleic acids. Indeed, these cells can induce robust IFN-I response by sensing nucleic acids, both self and non-self, and processes are needed to terminate their response.
We posit that following tissue injury or during an inflammatory response that pDCs are quickly recruited to the site of insult where they sense nucleic acids presented by cationic peptides which allow the trafficking of DNA/RNA to their endosome<sup>22,23,146,147</sup>. This leads to the activation of pDCs and the secretion of IFN-I which activates other immune cells present in tissue but also induces the expression of chemokines that promote the recruitment and infiltration of immune cells. This is likely an important function of pDCs in their wound healing function (**Figure 6**) <sup>131</sup>. However, this inflammatory response has consequences on the skin with the activation of macrophages and differentiation of myofibroblasts which remodel the tissue microenvironment with scarring provoking a significant increase in stiffness, which we propose then induces a stress response in pDCs which terminate their response (**Figure 6**).

In patients, the presence in the skin of chemokines like CXCL4 or CXCL10, which can act as nucleic acid delivery vectors<sup>20,23,148</sup>, can super-induce TLR7&9-induced inflammation. Further, we observed that disruption in XCI and escape of TLR7 in pDCs of patients with SSc leads to heterogeneity in pDCs circulating in the blood of patients, including cells that already
express ISGs and that are more prone to respond to nucleic acid signaling <sup>18</sup>. Combined, these
two elements likely overcome the normal resolution of the pDC activation by high stiffness,
leading to lesions or more fibrosis (Figure 6).

**Concluding remarks**

Since the seminal observation of the female preeminence of IFN-α production by TLR7-
stimulated pDCs <sup>53</sup>, it is now well accepted that human pDCs' innate function is positively
controlled by female sex-specific biological factors. Besides the influence of sex hormone
estrogens which could enhance both TLR9 and TLR7-mediated responses, probably through
the upregulation of IRF5 during reproductive ages <sup>55,57</sup>, the involvement of sex chromosome
dependent effects has subsequently emerged as a major trigger <sup>37</sup>. The contribution of gene-
dosage effects linked to X chromosome complement is likely to explain the enhanced capacity
of female pDCs to respond to the X-linked TLRs in autoimmune conditions, such as SSc <sup>18,20</sup>,
but also in healthy subjects across ages <sup>37,62</sup>. Besides IFN-I-mediated TLR overexpression due
to dysregulated XCI maintenance <sup>18,120 149</sup>, other X-linked mechanisms are likely contributors
to this robust sex bias, including genes constitutively escaping XCI, for which a role in the
sex-based differences in pDC innate function has started to emerge <sup>76</sup>. Chronic pDC activation
in disease conditions like SSc is likely sustained through several additional mechanisms
involving dysregulated ER stress and metabolic responses <sup>19</sup>, as well as the non-canonical role
of chemokines like CXCL4 as NA delivery vectors to super-induce TLR driven inflammation
<sup>23</sup>. All these pathways may contribute to skin fibrosis or inflammation as observed in SSc and
SLE <sup>92</sup>. Indeed, targeting pDCs has shown promise in clinical trials for cutaneous SLE <sup>136-138</sup>.
Whereas these latter pathways are likely to occur to a similar extent in both males and
females, how they are influenced by sex-specific factors is currently unknown. On the other
hand, the X-linked genetic risk of autoimmunity is extremely strong in humans and many
systemic autoimmune diseases preferentially afflict individuals with two XX chromosomes,
including females and KS males <sup>25</sup>. Interestingly, we recently reported that dysregulated XCI
maintenance in a genetic mouse model led to enhanced reactivation of X-linked genes and
female-specific spontaneous systemic B cell autoimmunity, suggesting a critical role of the Xi
and *XIST/Xist*-dependent mechanism of silencing in the emergence of female-biased
autoimmune diseases <sup>81,149</sup>. Understanding the molecular pathways involved in the
dysregulation of XCI maintenance will provide new insights into the intimate mechanisms of
female-specific immunity and may lead to the development of sex-specific biomarkers to
guide the diagnosis and management of autoimmune diseases in women, as well as sex-
specific therapeutic approaches aimed at reestablishing XCI maintenance in females.
## ACKNOWLEDGMENTS

We thank Dr. Peggy Crow for the critical reading of the manuscript and the numerous suggestions. This work was supported by grants from the Scleroderma Research Foundation (F.J.B.), the Scleroderma Foundation (F.J.B.), the Agence Nationale de la Recherche (ANR-20-CE15-0014-01; ANR-23-CE15-0002-01) (J.C.G.), FOREUM Foundation (2020-041) (J.C.G.), Fondation pour la Recherche Médicale (EQU202403018065) (J.C.G.), the Inspire Program from Région Occitanie/Pyrénées-Méditerranée (#1901175) (J.C.G.) and Novartis DREAMER (2023) (J.C.G.).

## DECLARATION OF INTERESTS:

F.J.B. is a co-founder and Scientific Advisor of IpiNovyx and EpiStemyx, two startup biotech companies. Otherwise, the authors have no other conflicts.
**Figure legends**

**Figure 1. Molecular mechanisms of X-chromosome inactivation. (A)** In female mammals, one of the two X chromosomes is randomly inactivated to equalize the dosage of gene expression between sexes. This process known as X chromosome inactivation (XCI) results in cellular mosaicism, where approximately half of the cells in a tissue expresses the maternal X chromosome and the other half the paternal one <sup>38</sup>. (B) XCI is initiated by the expression of the long noncoding RNA XIST, which spreads in cis along the future inactive X chromosome (Xi). (C) XIST functions through its interactions with the Xi and numerous interacting proteins. *Xist* RNA is essential for the initial recruitment, in a direct or indirect manner, of the factors involved in the lock-in of the Xi repressive state. The first step of XCI involves the interaction of XIST with the transcriptional repressor SPEN which activates pre-bound histone deacetylase 3 (HDAC3). XIST and SPEN are essential for the earliest steps of gene silencing on the Xi, but recent works indicate that they are also critical for XCI maintenance (see text). The subsequent recruitment of the Polycomb repressive complexes (PRC1) and PRC2 (step 2), as well as DNA methyltransferases (step 3) trigger the deposition of repressive histone marks (H2AK119ub and H3K27me3 respectively) and CpG island DNA methylation, respectively (Figure adapted from <sup>39</sup>).

**Figure 2: Candidate X chromosome-linked genes implicated in TLR-signaling in pDCs.**

(A) The X chromosome encodes around 1000 genes, among which 50 genes are involved in immune cell functions <sup>150</sup>, including genes with important function in TLR-signaling: *TLR7*, *TLR8*, *CXorf21* the IRF5 adaptor interacting with SLC15A4 on the lysosome (*TASL*), *BTK*, *IRAK1*, *NEMO/IKBKG*. *TLR7*, *TLR8*, *CXorf21/TASL* have been reported to escape XCI in a facultative manner in immune cells <sup>47,50,51,81,113,116</sup>.

**Figure 3. Impaired XCI maintenance at the *TLR7/8* locus is associated with pDC heterogeneity and enhanced ISG signature.**

SSc is an autoimmune disease that has a strong female predominance. Both the X-linked TLR7 and TLR8 can induce IFN-I by pDCs which can promote fibrosis. Using RNA FISH approaches, we recently reported that both *TLR7* and *TLR8* escape XCI at higher frequency in pDCs of SSc patients compared to healthy donor (HD). This was associated with enhanced ISG signature and TLR8-driven IFN responses in SSc pDCs <sup>18</sup>. Furthermore, the expression levels of *XIST* and the transcriptional repressor *SPEN* were reduced in SSc pDCs. These data reveal the heterogeneity of pDCs in SSc and suggest that altered XCI at the *TLR7/8* locus may contribute to the chronic activation of pDC in female SSc patients <sup>18</sup>.
**Figure 4.** Type I IFN-signaling and the dynamic of XCI maintenance in female SSc pDCs.

(A) Unlike most somatic cells, immune cells, including human pDCs, lack robust XIST cloud formation on the Xi. However in vitro stimulation of pDCs with cytokines (IL-3 or GM-CSF) leads to the relocalization of XIST RNA foci at the Xi in 70% of nuclei ¹⁸.

Combined RNA/DNA FISH analysis revealed that the *TLR7/8* locus is preferentially located outside of the Xi territory when *TLR7* is expressed, suggesting that higher-order loop formation is linked to *TLR7/8*-expression from the Xi in female pDCs (see Figure 2).

Incubation of IL3-pDCs with IFN-I resulted in the dispersal of *XIST* RNA particles beyond the Xi territory and upregulated the proportion of nuclei with bi-allelic expression of *TLR7*.

(B) The current hypothesis is that IFN-I signaling by inhibiting *SPEN/XIST* expression and preventing *XIST* localization to the Xi may impair XCI maintenance (XCIIm) in SSc pDCs, resulting in a feed-forward loop of TLR-driven IFN-I production, sustaining ISG signature, TLR7/8-expression and -signaling in individuals with two X chromosomes (adapted from ¹²⁰).

**Figure 5.** Higher frequency of TLR7 biallelic cells in SSc pDCs lacking robust XIST cloud I-II profiles.

Graph representing the relative frequencies of *TLR7* biallelic nuclei in control HD and SSc female pDCs among all nuclei counted (all) and only in nuclei lacking the I and II XIST patterns (XIST neg) as indicated. Statistical differences were analyzed using an ANOVA test corrected for multiple comparisons (q values are shown).

**Figure 6.** Inflammatory circuit leading to skin lesions and/or fibrosis in patients with autoimmunity.

Schematic representation of how the skin microenvironment is being remodelled by pDCs and the IFN-I that they produce and how this allows the resolution of the inflammation and wound healing. Also described is the hypothesis as to why such resolution does not occur in the skin of patients with SLE or SSc, including the presence of inflammatory mediators such as CXCL4 in the skin that can super-induce IFN-I from pDCs, but also of subsets of pDCs enriched in high ISG due to defects in XCI that supersede the inhibitory signals in pDCs, leading to sustained expression of IFN-I and skin lesions.
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<figure><img src="image_4.png" /><figcaption>Figure 1</figcaption></figure>

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<figure><img src="image_6.png" /><figcaption>Figure 2</figcaption></figure>
**TLR7-monoallelic cells**

<figure><img src="image_7.png" /></figure>

**TLR7-biallelic cells**

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Figure 4.</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>Figure 5.</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>Figure 6.</figcaption></figure>