A gene network regulated by the transcription factor VGLL3 as a promoter of sex-biased autoimmune diseases.

Autoimmune diseases affect 7.5% of the US population, and they are among the leading causes of death and disability. A notable feature of many autoimmune diseases is their greater prevalence in females than in males, but the underlying mechanisms of this have remained unclear. Through the use of high-resolution global transcriptome analyses, we demonstrated a female-biased molecular signature associated with susceptibility to autoimmune disease and linked this to extensive sex-dependent co-expression networks. This signature was independent of biological age and sex-hormone regulation and was regulated by the transcription factor VGLL3, which also had a strong female-biased expression. On a genome-wide level, VGLL3-regulated genes had a strong association with multiple autoimmune diseases, including lupus, scleroderma and Sjögren's syndrome, and had a prominent transcriptomic overlap with inflammatory processes in cutaneous lupus. These results identified a VGLL3-regulated network as a previously unknown inflammatory pathway that promotes female-biased autoimmunity. They demonstrate the importance of studying immunological processes in females and males separately and suggest new avenues for therapeutic development.

Activation of the NLRP1 inflammasome in human keratinocytes by the dsDNA mimetic poly(dA:dT).

The accrual of cytosolic DNA leads to transcription of type I IFNs, proteolytic maturation of the IL-1 family of cytokines, and pyroptotic cell death. Caspase-1 cleaves pro-IL1ß to generate mature bioactive cytokine and gasdermin D which facilitates IL-1 release and pyroptotic cell death. Absent in melanoma-2 (AIM2) is a sensor of dsDNA leading to caspase-1 activation, although in human monocytes, cGAS-STING acting upstream of NLRP3 mediates the dsDNA-activated inflammasome response. In healthy human keratinocytes, AIM2 is not expressed yet caspase-1 is activated by the synthetic dsDNA mimetic poly(dA:dT). Here, we show that this response is not mediated by either AIM2 or the cGAS-STING-NLRP3 pathway and is instead dependent on NLRP1. Poly(dA:dT) is unique in its ability to activate NLRP1, as conventional linear dsDNAs fail to elicit NLRP1 activation. DsRNA was recently shown to activate NLRP1 and prior work has shown that poly(dA:dT) is transcribed into an RNA intermediate that stimulates the RNA sensor RIG-I. However, poly(dA:dT)-dependent RNA intermediates are insufficient to activate NLRP1. Instead, poly(dA:dT) results in oxidative nucleic acid damage and cellular stress, events which activate MAP3 kinases including ZAKα that converge on p38 to activate NLRP1. Collectively, this work defines a new activator of NLRP1, broadening our understanding of sensors that recognize poly(dA:dT) and advances the understanding of the immunostimulatory potential of this potent adjuvant.

SARS-CoV-2 ORF8 Mediates Signals in Macrophages and Monocytes through MyD88 Independently of the IL-17 Receptor.

SARS-CoV-2 has caused an estimated 7 million deaths worldwide to date. A secreted SARS-CoV-2 accessory protein, known as open reading frame 8 (ORF8), elicits inflammatory pulmonary cytokine responses and is associated with disease severity in COVID-19 patients. Recent reports proposed that ORF8 mediates downstream signals in macrophages and monocytes through the IL-17 receptor complex (IL-17RA, IL-17RC). However, generally IL-17 signals are found to be restricted to the nonhematopoietic compartment, thought to be due to rate-limiting expression of IL-17RC. Accordingly, we revisited the capacity of IL-17 and ORF8 to induce cytokine gene expression in mouse and human macrophages and monocytes. In SARS-CoV-2-infected human and mouse lungs, IL17RC mRNA was undetectable in monocyte/macrophage populations. In cultured mouse and human monocytes and macrophages, ORF8 but not IL-17 led to elevated expression of target cytokines. ORF8-induced signaling was fully preserved in the presence of anti-IL-17RA/RC neutralizing Abs and in Il17ra-/- cells. ORF8 signaling was also operative in Il1r1-/- bone marrow-derived macrophages. However, the TLR/IL-1R family adaptor MyD88, which is dispensable for IL-17R signaling, was required for ORF8 activity yet MyD88 is not required for IL-17 signaling. Thus, we conclude that ORF8 transduces inflammatory signaling in monocytes and macrophages via MyD88 independently of the IL-17R.

Pathophysiology of generalized pustular psoriasis.

Psoriasis is a chronic, immune-mediated skin disease that affects over 3% of adults in the United States. Psoriasis can present in several clinical forms. Of these, generalized pustular psoriasis is an acute, severe form, associated with increased morbidity and mortality. Unlike the more common plaque psoriasis, which is thought to feature dysregulation of the adaptive immune system, generalized pustular psoriasis reflects heightened autoinflammatory responses. Recent advances in genetic and immunological studies highlight a key role of the IL-36 immune axis in the pathogenesis of generalized pustular psoriasis. In this article, we review the psoriatic subtypes and discuss diagnostic criteria of generalized pustular psoriasis, discuss several newly identified genetic variants associated with pustular disease in the skin, and discuss how these mutations shed light on pustular disease mechanisms. Furthermore, we gather insights from recent transcriptomic studies that similarly implicate a pathogenic role of the IL-36 immune axis in generalized pustular psoriasis.

Retrospective pharmacogenetic study of psoriasis highlights the role of KLK7 in tumour necrosis factor signalling.

BACKGROUND: Multiple treatment options are available for the management of psoriasis, but clinical response varies among individual patients and no biomarkers are available to facilitate treatment selection for improved patient outcomes. OBJECTIVES: To utilize retrospective data to conduct a pharmacogenetic study to explore the potential genetic pathways associated with drug response in the treatment of psoriasis. METHODS: We conducted a retrospective pharmacogenetic study using self-evaluated treatment response from 1942 genotyped patients with psoriasis. We examined 6 502 658 genetic markers to model their associations with response to six treatment options using linear regression, adjusting for cohort variables and demographic features. We further utilized an integrative approach incorporating epigenomics, transcriptomics and a longitudinal clinical cohort to provide biological implications for the topmost signals associated with drug response. RESULTS: Two novel markers were revealed to be associated with treatment response: rs1991820 (P = 1.30 × 10-6) for anti-tumour necrosis factor (TNF) biologics; and rs62264137 (P = 2.94 × 10-6) for methotrexate, which was also associated with cutaneous mRNA expression levels of two known psoriasis-related genes KLK7 (P = 1.0 × 10-12) and CD200 (P = 5.4 × 10-6). We demonstrated that KLK7 expression was increased in the psoriatic epidermis, as shown by immunohistochemistry, as well as single-cell RNA sequencing, and its responsiveness to anti-TNF treatment was highlighted. By inhibiting the expression of KLK7, we further illustrated that keratinocytes have decreased proinflammatory responses to TNF. CONCLUSIONS: Our study implicates the genetic regulation of cytokine responses in predicting clinical drug response and supports the association between pharmacogenetic loci and anti-TNF response, as shown here for KLK7.

Cytokine responses in nonlesional psoriatic skin as clinical predictor to anti-TNF agents.

BACKGROUND: A major issue with the current management of psoriasis is our inability to predict treatment response. OBJECTIVE: Our aim was to evaluate the ability to use baseline molecular expression profiling to assess treatment outcome for patients with psoriasis. METHODS: We conducted a longitudinal study of 46 patients with chronic plaque psoriasis treated with anti-TNF agent etanercept, and molecular profiles were assessed in more than 200 RNA-seq samples. RESULTS: We demonstrated correlation between clinical response and molecular changes during the course of the treatment, particularly for genes responding to IL-17A/TNF in keratinocytes. Intriguingly, baseline gene expressions in nonlesional, but not lesional, skin were the best marker of treatment response at week 12. We identified USP18, a known regulator of IFN responses, as positively correlated with Psoriasis Area and Severity Index (PASI) improvement (P = 9.8 × 10-4) and demonstrate its role in regulating IFN/TNF responses in keratinocytes. Consistently, cytokine gene signatures enriched in baseline nonlesional skin expression profiles had strong correlations with PASI improvement. Using this information, we developed a statistical model for predicting PASI75 (ie, 75% of PASI improvement) at week 12, achieving area under the receiver-operating characteristic curve value of 0.75 and up to 80% accurate PASI75 prediction among the top predicted responders. CONCLUSIONS: Our results illustrate feasibility of assessing drug response in psoriasis using nonlesional skin and implicate involvement of IFN regulators in anti-TNF responses.

Hypersensitive IFN Responses in Lupus Keratinocytes Reveal Key Mechanistic Determinants in Cutaneous Lupus.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease in which 70% of patients experience disfiguring skin inflammation (grouped under the rubric of cutaneous lupus erythematosus [CLE]). There are limited treatment options for SLE and no Food and Drug Administration-approved therapies for CLE. Studies have revealed that IFNs are important mediators for SLE and CLE, but the mechanisms by which IFNs lead to disease are still poorly understood. We aimed to investigate how IFN responses in SLE keratinocytes contribute to development of CLE. A cohort of 72 RNA sequencing samples from 14 individuals (seven SLE and seven healthy controls) were analyzed to study the transcriptomic effects of type I and type II IFNs on SLE versus control keratinocytes. In-depth analysis of the IFN responses was conducted. Bioinformatics and functional assays were conducted to provide implications for the change of IFN response. A significant hypersensitive response to IFNs was identified in lupus keratinocytes, including genes (IFIH1, STAT1, and IRF7) encompassed in SLE susceptibility loci. Binding sites for the transcription factor PITX1 were enriched in genes that exhibit IFN-sensitive responses. PITX1 expression was increased in CLE lesions based on immunohistochemistry, and by using small interfering RNA knockdown, we illustrated that PITX1 was required for upregulation of IFN-regulated genes in vitro. SLE patients exhibit increased IFN signatures in their skin secondary to increased production and a robust, skewed IFN response that is regulated by PITX1. Targeting these exaggerated pathways may prove to be beneficial to prevent and treat hyperinflammatory responses in SLE skin.

Progression of acute-to-chronic atopic dermatitis is associated with quantitative rather than qualitative changes in cytokine responses.

BACKGROUND: Although multiple studies have assessed molecular changes in chronic atopic dermatitis (AD) lesions, little is known about the transition from acute to chronic disease stages, and the factors and mechanisms that shape chronic inflammatory activity. OBJECTIVES: We sought to assess the global transcriptome changes that characterize the progression from acute to chronic stages of AD. METHODS: We analyzed transcriptome changes in paired nonlesional skin, acute and chronic AD lesions from 11 patients and 38 healthy controls by RNA-sequencing, and conducted in vivo and histological assays to evaluate findings. RESULTS: Our data demonstrate that approximately 74% of the genes dysregulated in acute lesions remain or are further dysregulated in chronic lesions, whereas only 34% of the genes dysregulated in chronic lesions are altered already in the acute stage. Nonlesional AD skin exhibited enrichment of TNF, TH1, TH2, and TH17 response genes. Acute lesions showed marked dendritic-cell signatures and a prominent enrichment of TH1, TH2, and TH17 responses, along with increased IL-36 and thymic stromal lymphopoietin expression, which were further heightened in chronic lesions. In addition, genes involved in skin barrier repair, keratinocyte proliferation, wound healing, and negative regulation of T-cell activation showed a significant dysregulation in the chronic versus acute comparison. Furthermore, our data show progressive changes in vasculature and maturation of dendritic-cell subsets with chronicity, with FOXK1 acting as immune regulator. CONCLUSIONS: Our results show that the changes accompanying the transition from nonlesional to acute to chronic inflammation in AD are quantitative rather than qualitative, with chronic AD having heightened TH2, TH1, TH17, and IL36 responses and skin barrier repair mechanisms. These findings provide novel insights and highlight underappreciated pathways in AD pathogenesis that may be amenable to therapeutic targeting.

Photosensitivity and type I IFN responses in cutaneous lupus are driven by epidermal-derived interferon kappa.

OBJECTIVE: Skin inflammation and photosensitivity are common in patients with cutaneous lupus erythematosus (CLE) and systemic lupus erythematosus (SLE), yet little is known about the mechanisms that regulate these traits. Here we investigate the role of interferon kappa (IFN-κ) in regulation of type I interferon (IFN) and photosensitive responses and examine its dysregulation in lupus skin. METHODS: mRNA expression of type I IFN genes was analysed from microarray data of CLE lesions and healthy control skin. Similar expression in cultured primary keratinocytes, fibroblasts and endothelial cells was analysed via RNA-seq. IFNK knock-out (KO) keratinocytes were generated using CRISPR/Cas9. Keratinocytes stably overexpressing IFN-κ were created via G418 selection of transfected cells. IFN responses were assessed via phosphorylation of STAT1 and STAT2 and qRT-PCR for IFN-regulated genes. Ultraviolet B-mediated apoptosis was analysed via TUNEL staining. In vivo protein expression was assessed via immunofluorescent staining of normal and CLE lesional skin. RESULTS: IFNK is one of two type I IFNs significantly increased (1.5-fold change, false discovery rate (FDR) q<0.001) in lesional CLE skin. Gene ontology (GO) analysis showed that type I IFN responses were enriched (FDR=6.8×10-04) in keratinocytes not in fibroblast and endothelial cells, and this epithelial-derived IFN-κ is responsible for maintaining baseline type I IFN responses in healthy skin. Increased levels of IFN-κ, such as seen in SLE, amplify and accelerate responsiveness of epithelia to IFN-α and increase keratinocyte sensitivity to UV irradiation. Notably, KO of IFN-κ or inhibition of IFN signalling with baricitinib abrogates UVB-induced apoptosis. CONCLUSION: Collectively, our data identify IFN-κ as a critical IFN in CLE pathology via promotion of enhanced IFN responses and photosensitivity. IFN-κ is a potential novel target for UVB prophylaxis and CLE-directed therapy.

Six-transmembrane epithelial antigens of the prostate comprise a novel inflammatory nexus in patients with pustular skin disorders.

BACKGROUND: Pustular skin disorders are a category of difficult-to-treat and potentially life-threatening conditions that involve the appearance of neutrophil-rich pustules. The molecular basis of most pustular skin conditions has remained unknown. OBJECTIVE: We sought to investigate the molecular basis of 3 pustular skin disorders: generalized pustular psoriasis (GPP), palmoplantar pustulosis (PPP), and acute generalized exanthematous pustulosis (AGEP). METHODS: Microarray analyses were performed to profile genome-wide gene expression of skin biopsy specimens obtained from patients with GPP, PPP, or AGEP and healthy control subjects. Functional enrichment, gene network, and k-means clustering analyses were used to identify molecular pathways dysregulated in patients with these disorders. Immunohistochemistry and immunofluorescence were used to determine protein localization. Quantitative RT-PCR and ELISA were used to determine transcript and secreted cytokine levels. Small interfering RNA was used to decrease transcript levels. RESULTS: Molecules and pathways related to neutrophil chemotaxis emerged as common alterations in patients with GPP, PPP, and AGEP, which is consistent with the pustular phenotypes. Expression of two 6-transmembrane epithelial antigens of the prostate (STEAP) proteins, STEAP1 and STEAP4, was increased in patients' skin and colocalized with IL-36γ around neutrophilic pustules. STEAP1/4 expression clustered with and positively correlated with that of IL-1, the IL-36 family proteins, and CXCL1/8. STEAP4 expression was activated by cytokines and suppressed by inhibition of mitogen-activated protein kinase kinase 1/2, whereas STEAP1 expression appeared less prone to such dynamic regulation. Importantly, STEAP1/4 knockdown resulted in impaired induction of a broad spectrum of proinflammatory cytokines, including IL-1, IL-36, and the neutrophil chemotaxins CXCL1 and CXCL8. STEAP1/4 knockdown also reduced the ability of keratinocytes to induce neutrophil chemotaxis. CONCLUSION: Transcriptomic changes in 3 pustular skin disorders, GPP, PPP, and AGEP, converged on neutrophil chemotaxis and diapedesis and cytokines known to drive neutrophil-rich inflammatory processes, including IL-1 and members of the IL-36 family. STEAP1 and STEAP4 positively regulate the induction of proinflammatory neutrophil-activating cytokines.

HERC6 regulates STING activity in a sex-biased manner through modulation of LATS2/VGLL3 Hippo signaling.

Interferon (IFN) activity exhibits a gender bias in human skin, skewed toward females. We show that HERC6, an IFN-induced E3 ubiquitin ligase, is induced in human keratinocytes through the epidermal type I IFN; IFN-κ. HERC6 knockdown in human keratinocytes results in enhanced induction of interferon-stimulated genes (ISGs) upon treatment with a double-stranded (ds) DNA STING activator cGAMP but not in response to the RNA-sensing TLR3 agonist. Keratinocytes lacking HERC6 exhibit sustained STING-TBK1 signaling following cGAMP stimulation through modulation of LATS2 and TBK1 activity, unmasking more robust ISG responses in female keratinocytes. This enhanced female-biased immune response with loss of HERC6 depends on VGLL3, a regulator of type I IFN signature. These data identify HERC6 as a previously unrecognized negative regulator of ISG expression specific to dsDNA sensing and establish it as a regulator of female-biased immune responses through modulation of STING signaling.

A Critical Role for IFN-ß Signaling for IFN-κ Induction in Keratinocytes.

Background/Purpose: Cutaneous lupus erythematosus (CLE) affects up to 70% of patients with systemic lupus erythematosus (SLE), and type I interferons (IFNs) are important promoters of SLE and CLE. Our previous work identified IFN-kappa (IFN-κ), a keratinocyte-produced type I IFN, as upregulated in non-lesional and lesional lupus skin and as a critical regulator for enhanced UVB-mediated cell death in SLE keratinocytes. Importantly, the molecular mechanisms governing regulation of IFN-κ expression have been relatively unexplored. Thus, this study sought to identify critical regulators of IFN-κ and identified a novel role for IFN-beta (IFN-ß). Methods: Human N/TERT keratinocytes were treated with the RNA mimic poly (I:C) or 50 mJ/cm2 ultraviolet B (UVB), followed by mRNA expression quantification by RT-qPCR in the presence or absence neutralizing antibody to the type I IFN receptor (IFNAR). IFNB and STAT1 knockout (KO) keratinocytes were generated using CRISPR/Cas9. Results: Time courses of poly(I:C) and UVB treatment revealed a differential expression of IFNB, which was upregulated between 3-6 hours and IFNK, which was upregulated 24 hours after stimulation. Intriguingly, only IFNK expression was substantially abrogated by neutralizing antibodies to IFNAR, suggesting that IFNK upregulation required type I IFN signaling for induction. Indeed, deletion of IFNB abrogated IFNK expression. Further exploration confirmed a role for type I IFN-triggered STAT1 activation. Conclusion: Collectively, our work describes a novel mechanistic paradigm in keratinocytes in which initial IFN-κ induction in response to poly(I:C) and UVB is IFNß1-dependent, thus describing IFNK as both an IFN gene and an interferon-stimulated gene.

Multi-ancestry Genome-Wide Association Meta-Analysis Identifies Novel Loci in Atopic Dermatitis.

Atopic dermatitis (AD) is a highly heritable and common inflammatory skin condition affecting children and adults worldwide. Multi-ancestry approaches to AD genetic association studies are poised to boost power to detect genetic signal and identify ancestry-specific loci contributing to AD risk. Here, we present a multi-ancestry GWAS meta-analysis of twelve AD cohorts from five ancestral populations totaling 56,146 cases and 602,280 controls. We report 101 genomic loci associated with AD, including 15 loci that have not been previously associated with AD or eczema. Fine-mapping, QTL colocalization, and cell-type enrichment analyses identified genes and cell types implicated in AD pathophysiology. Functional analyses in keratinocytes provide evidence for genes that could play a role in AD through epidermal barrier function. Our study provides new insights into the etiology of AD by harnessing multiple genetic and functional approaches to unveil the mechanisms by which AD-associated variants impact genes and cell types. Disclosure Statement: BRG, MO, CH, KMS are employees of AbbVie. FT was an employee of AbbVie at the time of the study. JEG (University of Michigan) has received research support from AbbVie, Janssen, Almirall, Prometheus Biosciences/Merck, BMS/Celgene, Boehringer Ingelheim, Galderma, Eli Lilly, and advisor to Sanofi, Eli Lilly, Galderma, BMS, Boehringer Ingelheim. MKS, RU, MTP, QL, RW, JMK, LCT are employees of University of Michigan and have no funding to disclose. MEM, AHS, FDM, DW, JTG, HH are employees of the Children's Hospital of Philadelphia and no funding to disclose. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

Phototherapy Restores Deficient Type I IFN Production and Enhances Antitumor Responses in Mycosis Fungoides.

Transcriptional profiling demonstrated markedly reduced type I IFN gene expression in untreated mycosis fungoides (MF) skin lesions compared with that in healthy skin. Type I IFN expression in MF correlated with antigen-presenting cell-associated IRF5 before psoralen plus UVA therapy and epithelial ULBP2 after therapy, suggesting an enhancement of epithelial type I IFN. Immunostains confirmed reduced baseline type I IFN production in MF and increased levels after psoralen plus UVA treatment in responding patients. Effective tumor clearance was associated with increased type I IFN expression, enhanced recruitment of CD8+ T cells into skin lesions, and expression of genes associated with antigen-specific T-cell activation. IFNk, a keratinocyte-derived inducer of type I IFNs, was increased by psoralen plus UVA therapy and expression correlated with upregulation of other type I IFNs. In vitro, deletion of keratinocyte IFNk decreased baseline and UVA-induced expression of type I IFN and IFN response genes. In summary, we find a baseline deficit in type I IFN production in MF that is restored by psoralen plus UVA therapy and correlates with enhanced antitumor responses. This may explain why MF generally develops in sun-protected skin and suggests that drugs that increase epithelial type I IFNs, including topical MEK and EGFR inhibitors, may be effective therapies for MF.

Suppression of TCF4 promotes a ZC3H12A-mediated self-sustaining inflammatory feedback cycle involving IL-17RA/IL-17RE epidermal signaling.

IL-17C is an epithelial cell-derived proinflammatory cytokine whose transcriptional regulation remains unclear. Analysis of the IL17C promoter region identified TCF4 as putative regulator, and siRNA knockdown of TCF4 in human keratinocytes (KCs) increased IL17C. IL-17C stimulation of KCs (along with IL-17A and TNF-α stimulation) decreased TCF4 and increased NFKBIZ and ZC3H12A expression in an IL-17RA/RE-dependent manner, thus creating a feedback loop. ZC3H12A (MCPIP1/Regnase-1), a transcriptional immune-response regulator, also increased following TCF4 siRNA knockdown, and siRNA knockdown of ZC3H12A decreased NFKBIZ, IL1B, IL36G, CCL20, and CXCL1, revealing a proinflammatory role for ZC3H12A. Examination of lesional skin from the KC-Tie2 inflammatory dermatitis mouse model identified decreases in TCF4 protein concomitant with increases in IL-17C and Zc3h12a that reversed following the genetic elimination of Il17c, Il17ra, and Il17re and improvement in the skin phenotype. Conversely, interference with Tcf4 in KC-Tie2 mouse skin increased Il17c and exacerbated the inflammatory skin phenotype. Together, these findings identify a role for TCF4 in the negative regulation of IL-17C, which, alone and with TNF-α and IL-17A, feed back to decrease TCF4 in an IL-17RA/RE-dependent manner. This loop is further amplified by IL-17C-TCF4 autocrine regulation of ZC3H12A and IL-17C regulation of NFKBIZ to promote self-sustaining skin inflammation.

Single-cell sequencing reveals Hippo signaling as a driver of fibrosis in hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a chronic inflammatory disease characterized by abscesses, nodules, dissecting/draining tunnels, and extensive fibrosis. Here, we integrate single-cell RNA sequencing, spatial transcriptomics, and immunostaining to provide an unprecedented view of the pathogenesis of chronic HS, characterizing the main cellular players and defining their interactions. We found a striking layering of the chronic HS infiltrate and identified the contribution of 2 fibroblast subtypes (SFRP4+ and CXCL13+) in orchestrating this compartmentalized immune response. We further demonstrated the central role of the Hippo pathway in promoting extensive fibrosis in HS and provided preclinical evidence that the profibrotic fibroblast response in HS can be modulated through inhibition of this pathway. These data provide insights into key aspects of HS pathogenesis with broad therapeutic implications.

Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease.

Mutation of the ATP2A2 gene encoding sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2) was linked to Darier disease more than 2 decades ago; however, there remain no targeted therapies for this disorder causing recurrent skin blistering and infections. Since Atp2a2-knockout mice do not phenocopy its pathology, we established a human tissue model of Darier disease to elucidate its pathogenesis and identify potential therapies. Leveraging CRISPR/Cas9, we generated human keratinocytes lacking SERCA2, which replicated features of Darier disease, including weakened intercellular adhesion and defective differentiation in organotypic epidermis. To identify pathogenic drivers downstream of SERCA2 depletion, we performed RNA sequencing and proteomics analysis. SERCA2-deficient keratinocytes lacked desmosomal and cytoskeletal proteins required for epidermal integrity and exhibited excess MAPK signaling, which modulates keratinocyte adhesion and differentiation. Immunostaining patient biopsies substantiated these findings, with lesions showing keratin deficiency, cadherin mislocalization, and ERK hyperphosphorylation. Dampening ERK activity with MEK inhibitors rescued adhesive protein expression and restored keratinocyte sheet integrity despite SERCA2 depletion or chemical inhibition. In sum, coupling multiomic analysis with human organotypic epidermis as a preclinical model, we found that SERCA2 haploinsufficiency disrupts critical adhesive components in keratinocytes via ERK signaling and identified MEK inhibition as a treatment strategy for Darier disease.

Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease.

Mutation of the ATP2A2 gene encoding sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2) was linked to Darier disease more than two decades ago; however, there remain no targeted therapies for this disorder causing recurrent skin blistering and infections. Since Atp2a2 knockout mice do not phenocopy its pathology, we established a human tissue model of Darier disease to elucidate its pathogenesis and identify potential therapies. Leveraging CRISPR/Cas9, we generated human keratinocytes lacking SERCA2, which replicated features of Darier disease, including weakened intercellular adhesion and defective differentiation in organotypic epidermis. To identify pathogenic drivers downstream of SERCA2 depletion, we performed RNA sequencing and proteomic analysis. SERCA2-deficient keratinocytes lacked desmosomal and cytoskeletal proteins required for epidermal integrity and exhibited excess MAP kinase signaling, which modulates keratinocyte adhesion and differentiation. Immunostaining patient biopsies substantiated these findings with lesions showing keratin deficiency, cadherin mis-localization, and ERK hyper-phosphorylation. Dampening ERK activity with MEK inhibitors rescued adhesive protein expression and restored keratinocyte sheet integrity despite SERCA2 depletion or chemical inhibition. In sum, coupling multi-omic analysis with human organotypic epidermis as a pre-clinical model, we found that SERCA2 haploinsufficiency disrupts critical adhesive components in keratinocytes via ERK signaling and identified MEK inhibition as a treatment strategy for Darier disease.

Keratinocytes sense and eliminate CRISPR DNA through STING/IFN-κ activation and APOBEC3G induction.

CRISPR/Cas9 has been proposed as a treatment for genetically inherited skin disorders. Here we report that CRISPR transfection activates STING-dependent antiviral responses in keratinocytes, resulting in heightened endogenous interferon (IFN) responses through induction of IFN-κ, leading to decreased plasmid stability secondary to induction of the cytidine deaminase gene APOBEC3G. Notably, CRISPR-generated KO keratinocytes had permanent suppression of IFN-κ and IFN-stimulated gene (ISG) expression, secondary to hypermethylation of the IFNK promoter region by the DNA methyltransferase DNMT3B. JAK inhibition via baricitinib prior to CRISPR transfection increased transfection efficiency, prevented IFNK promoter hypermethylation, and restored normal IFN-κ activity and ISG responses. This work shows that CRISPR-mediated gene correction alters antiviral responses in keratinocytes, has implications for future gene therapies for inherited skin diseases using CRISPR technology, and suggests pharmacologic JAK inhibition as a tool for facilitating and attenuating inadvertent selection effects in CRISPR/Cas9 therapeutic approaches.