Journal of Investigative Dermatology

Acne vulgaris is a common skin disease that affects >85% of teenage young adults among which >8% develop severe lesions that leaves permanent scars. Genetic heritability studies of acne in twin cohorts have estimated that the heritability for acne is 80%. Previous genome-wide association studies (GWAS) have identified 50 genetic loci associated with increased risk of developing acne when compared to healthy individuals. However only a few studies have investigated genetic association with disease severity. GWAS of disease progression may provide a more effective approach to unveil potential disease modifying therapeutic targets. Here, we performed a multi-ethnic GWAS analysis to capture disease severity in acne patients by using individuals with normal acne as a control. Our cohort consists of a total of 2,956 participants, including 290 severe acne cases and 930 normal acne controls from FinnGen, and 522 cases and 1,214 controls from BioVU. We also performed mendelian randomization (MR), colocalization analyses and transcriptome-wide association study (TWAS) to identify putative causal genes. Lastly, we performed gene-set enrichment analysis using MAGMA to implicate biological pathways that drive disease severity in Acne. We identified two new loci associated with acne severity at the genome-wide significance level, six novel associated genes by MR, colocalization and TWAS analyses, including genes CDC7, SLC7A1, ADAM23, TTLL10, CDK20 and DNAJA4, and 5 novel pathways by MAGMA analyses. Our study suggests that the etiologies of acne susceptibility and severity have limited overlap, with only 26% of known acne risk loci presenting nominal association with acne severity and none of the novel severity associated genes reported as associated with acne risk in previous GWAS.

AbstractAtopic Dermatitis (AD) and psoriasis (PsO) are two frequent dermatologic conditions that may co-occur in a cluster of patients, yet current understanding of how these two conditions relate to one-another remains poorly understood. One way to better understand their relationship is through a process called phenotypic switching, where AD and PsO can turn into one another. We utilized a pharmacovigilance-based epidemiological approach to better understand this phenomenon. By generating adverse event-related disproportionality signals for various therapies and therapeutic classes used in AD and PsO, several potential mechanisms for the AD-PsO phenotypic switch were uncovered. This includes mechanisms involving TH2 and TH22 repolarization, TH17 and TH22 repolarization, and immune shifting between TH1, TH17, and TH2 cells. Clinically and immunologically related conditions were also analyzed to gain a clearer understanding of the specificity of the switch from PsO to an eczematous phenotype. Together, these findings provide mechanistic insight into the underpinnings behind the AD-PsO phenotypic switch through a novel approach, adding evidence to the fluid nature of immune phenotypes in common medical conditions. One Sentence SummaryThe atopic dermatitis-psoriasis phenotypic switch creates an overlap phenotype that is likely TH22-driven.

Psoriasis is a common chronic inflammatory skin disease characterized by disturbed interactions between infiltrating immune cells and keratinocytes. To enhance our understanding of the underlying molecular and cellular mechanisms driving psoriasis pathobiology, and to identify potential biomarkers for disease severity, we conducted RNA sequencing of skin biopsies from 75 patients with psoriasis vulgaris and 46 non-psoriatic controls. To increase the robustness of the results, we meta-analysed our data with four publicly available datasets, bringing the total number of samples to 534. By comparing lesional psoriatic (PP) to healthy control (NN) skin, we identified 2269 differentially expressed genes (DEGs) (|log2FC|>1.0, FDR <0.1), and 58 DEGs when comparing non-lesional psoriatic (PN) to NN skin. We also identified 54 DEGs associated with disease severity (PASI [&ge;]10 vs. PASI <10). Cellular deconvolution analysis showed that differentiated keratinocytes emerged as the most prominent cell type among the DEGs in PP/NN. Functional enrichment analysis in PN/NN revealed several IL-17 related pathways and confirmed a previously reported pre-inflammatory signature across all psoriatic skin. This study provides insights into the psoriasis transcriptome and identifies a severity-specific signature, which may serve as candidate for future studies aimed at identifying psoriasis biomarkers and predicting disease progression.

Atopic eczema (AE) is the most common inflammatory dermatosis, affecting up to 20% of children. Loss of function mutations in the Filaggrin (FLG) gene are the most strongly implicated genetic risk factor for AE, but little is known about the signalling pathways altered in response to loss of FLG. To explore the downstream effects of loss of FLG on the cellular environment, we combined RNAseq analysis of siRNA knockdown of normal human keratinocytes and analysis of tape strip (TS) samples from AE patients who were clinically phenotyped and genotyped for FLG mutation status. RNA-seq analysis revealed an increase in BMP signalling following FLG KD, which we validated in vivo using TS samples and biopsies. Recombinant BMP2 or BMP6 increased FLG and suprabasal keratin expression in vitro. Phosphoproteomic analysis identified 237 significantly differentially phosphorylated proteins following FLG KD. Kinase enrichment analysis identified downregulation of ERK1/2 and AKT1 signalling which was confirmed in AE biopsies. cFOS was downregulated following FLG KD and correlated with FLG expression in repository datasets. cFOS was downregulated following BMP6 treatment, implying that cFOS may be an important link between FLG and BMP signalling. Finally, proteomic analysis of TS samples identified altered desmosomal expression and phosphorylation following either loss of FLG or increased BMP signalling. Therefore, we have identified a SMAD1/Filaggrin/AKT axis as a potential therapeutic avenue in AE.

TGF-{beta} family ligands are key regulators of dendritic cell (DC) differentiation and activation. Epidermal Langerhans cells (LCs) require TGF- {beta} family signaling for their differentiation and canonical TGF-{beta}1 signaling secures a non-activated LC state. LCs reportedly control skin inflammation and are replenished from peripheral blood monocytes, which also give rise to pro-inflammatory monocyte-derived DCs (moDCs). Among all the miRNAs differentially expressed in LC vs moDCs, we observed miR-424 to be strongly induced during moDC differentiation from monocytes. We discovered that miR-424 is required for moDC differentiation from human and murine precursor cells in vitro and for inflammation-associated moDC in vivo. Mechanistically we found that low levels of miR-424 facilitate TGF-{beta}1-dependent LC differentiation at the expense of moDC differentiation. Loss of miR-424 in monocyte/DC precursors resulted in the induction of TGF-{beta} pathway. Therefore, miR-424 plays a decisive role in anti-inflammatory LC vs pro-inflammatory moDC differentiation from monocytes, and its repression allows TGF-{beta} ligands to promote LC differentiation. Short summaryMonocytes give rise to two distinct DC subsets in skin inflammation, exhibiting opposite roles in inflammation. This study identified miR-424(322) as a molecular switch controlling pro-inflammatory (moDC) vs anti-inflammatory LC subset differentiation by modulating TGF-{beta} signaling.

Surgical intervention is considered as the mainstream therapy for refractory vitiligo. In this study, we developed a modified autologous cultured epithelial grafting (ACEG) technique for the surgical treatment of vitiligo. A total of 726 patients with vitiligo treated with ACEG were enrolled from January 2015 to June 2019 in China. Patient characteristics, such as sex, age, clinical type, lesion sites, course of the disease, and disease stable period, were recorded. In 2118 skin lesions from 726 patients who received ACEG, total efficacy rate was 82.81% (1754/2118). However, the repigmentation rate of the ACEG was 64.87%, which was higher than that of conventional surgical interventions (52.69%). Patients with segmental vitiligo, skin lesions in the lower limbs, aged 18 years or below, and a stable period of over 3 years might have a good response to ACEG. Single-cell RNA sequencing was performed to observe different cell compositions in the skin before and after ACEG. The number of melanocytes increased by 50% after transplantation. In addition, there was a significant increase in hair follicle outer root sheath-derived keratinocytes in ACEG, and the numbers of these cells in the repigmentation sites 1 year after ACEG were still higher than those in the skin lesions. Therefore, ACEG is a promising therapeutic agent for refractory vitiligo. Age, clinical type, lesion site, and lesion stable period before surgery have significant impacts on repigmentation in ACEG. ACEG can increase the number of melanocytes and KRT6C+ keratinocytes in skin lesions, thereby restoring a skin microenvironment suitable for melanocyte survival. One sentence summaryAutologous cultured epithelial grafting (ACEG) technique is a promising therapy for refractory vitiligo.

ECM composition and organization are greatly altered during inflammation but it is still elusive if ECM dynamics may protect tissue stem cells against aberrant inflammation. Fibulin 7 (encoded by Fbln7) is part of the basement membrane ECM where epidermal stem cells (EpSCs) reside. It supports the long-term potential of fast-cycling EpSCs and moderates aging-related inflammatory markers in keratinocytes. Here, we assessed fibulin 7s role during imiquimod (IMQ)-induced inflammation in 1-year-old mouse dorsal skin. We found that loss of Fbln7 aggravates epidermal inflammation, marked by increased epidermal thickness, proliferation, and phosphorylation of JNK (c-Jun N-terminal kinase). Fast-cycling EpSCs labeled with Slc1a3-creER-TdTomato demonstrated that IMQ-induced proliferation in Fbln7 KO mice is contributed by cell divisions in the suprabasal layers, a hallmark of inflammatory epidermal responses. EpSC transcriptomes further reveal IMQ-modulated genes that are more substantially affected in Fbln7 KO mice, including IL-17 pathway-related genes known in psoriasis pathogenesis. Mechanistically, fibulin 7 directly binds to IL-17A and decreases IL-17A-mediated p38 MAPK activation. In public human psoriasis datasets, FBLN7 is reduced in lesional skin compared with non-lesional or normal skin, and it is significantly correlated with common psoriasis-associated genes. Altogether, fibulin 7 is potentially beneficial to protect against skin inflammation.

Vitiligo is an autoimmune disorder characterized by melanocyte destruction. We performed a rank-based meta-analysis of six independent transcriptomic studies (115 samples) spanning microarray, bulk and single-cell RNA-seq platforms to identify consensus signatures of lesional skin. Robust Rank Aggregation identified 114 differentially expressed genes (FDR < 0.05) with striking asymmetry: 108 downregulated versus 6 upregulated. Downregulated genes were dominated by melanocyte markers (MLANA, TYRP1, DCT, PMEL, KIT). Upregulated genes included interferon-stimulated genes (OAS1, OAS2, EPSTI1). Pathway-level meta-analysis confirmed uniform suppression of melanogenesis, while immune activation was heterogeneous across datasets. Single-cell data from three included studies confirmed melanocyte depletion. The 108 downregulated genes showed exclusive expression in melanocytes. These include neural genes (PLP1, GPM6B, NRXN3), consistent with melanocytes neural crest origin. We also identified candidate melanocyte markers such as CYB561A3 and QPCT with high melanocyte specificity and consistent downregulation in vitiligo. These findings reveal a robust melanocyte loss signature in vitiligo detectable across all platforms, and study-dependent immune activation possibly influenced by sampling method and disease characteristics.

Despite recent advances in understanding skin scarring, mechanisms triggering hypertrophic scar formation are still poorly understood. In the present study we performed single-cell sequencing of mature human hypertrophic scars and developing scars in mice. Compared to normal skin, we found significant differences in gene expression in most cell types present in scar tissue. Fibroblasts (FBs) showed the most prominent alterations in gene expression, displaying a distinct fibrotic signature. By comparing genes upregulated in murine FBs during scar development with genes highly expressed in mature human hypertrophic scars, we identified a group of serine proteases, tentatively involved in scar formation. Two of them, dipeptidyl-peptidase 4 (DPP4) and urokinase (PLAU), were further analyzed in functional assays, revealing a role in TGF{beta}1-mediated myofibroblast differentiation and over-production of components of the extracellular matrix (ECM) without interfering with the canonical TGF{beta}1-signaling pathway. In this study, we delineate the genetic landscape of hypertrophic scars and present new insights into mechanisms involved in hypertrophic scar formation. Our data suggest the use of serine protease inhibitors for the treatment of skin fibrosis.

Atopic dermatitis (AD) is a common skin disorder, characterized by impaired skin barrier function and cutaneous inflammation. The pathophysiology of AD is incompletely understood, and has considerable genetic contributions. To obtain a detailed molecular understanding of AD, we integrated the genomic, skin transcriptomic, and clinical measurements from 30 AD and 30 healthy control (HC) subjects. We found that the AD group had mild-to-moderate disease severity and only showed slightly increased genetic risk compared with HC. When comparing within the AD group, we found that the lesional skin of patients with increased genetic risk was characterized by a possible "self-protection" mechanism, including elevation of the anti-inflammatory cytokine IL-34, activation of fibroblasts, wound healing, and the complement system. We hypothesize that this mechanism may contribute to halting further progression of AD.

Serine/Arginine-rich splicing factor 3 (SRSF3) is one of 12 SRSFs that regulate gene expression via alternative splicing. SRSF3 is upregulated in cutaneous squamous cell carcinoma (cSCC) and several squamous cancer cell lines in relation to normal keratinocytes. We suppressed SRSF3 with a specific inhibitor, SFI003, and observed an increase in epidermal differentiation. Our data suggests that in cSCC, SRSF3 overexpression suppresses cellular differentiation to enable cancer progression. In a clinical setting, patients taking known SRSF3 inhibitors digoxin and amiodarone exhibited higher cSCC-free survival compared to a propensity score-matched cohort treated with beta blockers. Thus, SRSF3 upregulation may be a novel therapeutic target in cSCC that can improve patient prognoses.

Commensal bacteria are core players in wound healing whose function in the opposite pathophysiological process-scarring is presently unclear. Here, we document the association between bacteria and a specific skin fibrotic disease-keloid, which might offer a promising avenue for translational practice. ABSTRACTWound healing is an intensely studied topic involved in many relevant pathophysiological processes, including fibrosis. Despite the large interest in fibrosis, the network that related to commensal microbiota and skin fibrosis remain mysterious. Here, we pay attention to keloid, a classical yet intractable skin fibrotic disease to establish the association between commensal microbiota to scaring tissue. Our histological data reveal the presence of microbiota in the keloids. 16S rRNA sequencing characterize microbial composition and divergence between the pathological and normal skin tissue. Moreover, the data show elevation of interleukin-8 both in the circulation and keloid tissue, which elicited the collagen accumulation and migratory program of dermal fibroblasts via CXCR1/2 receptor. Our research provides insights into the pathology of human fibrotic diseases, advocating commensal bacteria and IL-8 signaling as useful targets in future interventions of recurrent keloid disease.

NLRP1 is an inflammasome sensor protein expressed in barrier tissues of humans. Its activation in response to microbes or cellular stress triggers a cascade of molecular events, leading up to IL1{beta}-driven inflammation and pyroptosis. Rare germline mutations of NLRP1 cause its persistent activation, resulting in autoinflammatory syndromes. Multiple self-healing palmoplantar carcinoma (MSPC) is one such syndrome, characterized by the appearance of recurrent keratoacanthomas (KAs) on the palms and soles. Here, we aimed to compare the subcellular localization of mutant NLRP1 in MSPC-associated lesions, to wild-type NLRP1 in non-MSPC KAs and in skin from healthy donors. Using mass spectrometry, immunohistochemistry and immunoelectron tomography, we found that NLRP1 localized to mast cell (MC) granules in all samples, a novel finding which implicates MCs in NLRP1-associated responses in human skin. Moreover, we found that MCs expressing the A66V pathogenic variant of NLRP1 overpopulated MSPC-KAs, infiltrated the epidermis and degranulated, a behavior not seen in other lesions from this study. The released granules had the highest NLRP1 protein content and also contained NLRP3 and IL1{beta}, indicating coexistence of inflammasome pathways within MCs. Taken together, our findings establish cutaneous MCs as a NLRP1 reservoir in health and disease, opening a new area of research in NLRP1-related syndromes.

Atopic dermatitis (AD) is a common skin disease with most of the health, social and economic impact driven by those with more severe disease. Determining the molecular pathways that influence severity is therefore crucial, offering opportunity to identify novel drug targets, as well as use in risk prediction tools. In this large-scale multi-omics study, we used complementary methods and datasets to identify molecular markers with robust evidence for involvement in AD severity. We undertook a case-only genome-wide association study meta-analysis (N=100,766) and subsequent transcriptome-wide association study (TWAS), differential expression meta-analysis in blood (N=340) and skin (N=185) as well as a differential protein abundance analysis in blood (N=75). A total of 440 genes/proteins showed evidence of association across all the analyses. Of these, four were significant in two or more analyses. For CEP85 (Pexpression=2.8x10-7; PTWAS=8.2x10-13), a gene not previously associated with AD, we also found strong evidence that the genetic variants affect CEP85 mRNA expression in monocytes. Functional in vitro follow-up showed that CEP85 over-expression in monocyte-derived macrophages can disrupt phagocytosis which we hypothesise may contribute to severity by impairing phagocytosis of S aureus. Together this work provides evidence of genetic risk and candidate molecular pathways to severe AD.

Impairment of desmosomal cell-cell adhesion leads to several life-threatening diseases such as the autoimmune skin blistering disorder pemphigus vulgaris (PV). Disease management strategies that stabilize intercellular adhesion, in addition to the existing immunosuppression therapies, may result in improved clinical outcomes. Previous findings showed that the serine protease inhibitor SERPINB5 promotes intercellular adhesion by binding to and regulating the localization of the desmosomal adapter molecule desmoplakin (DSP) at the plasma membrane. We here show that SERPINB5 overexpression prevents PV-IgG-mediated loss of cell-cell adhesion and the loss of DSP from the cell membrane. We mechanistically demonstrate that SERPINB5 loss deregulates TGF-{beta} signalling, a pathway known to destabilize DSP in keratinocytes. TGF-{beta} signalling was also activated in skin biopsies of PV patients and keratinocytes treated with PV autoantibodies, suggesting a contribution to disease. Inhibition of TGF-{beta} activation ameliorated PV-IgG-mediated loss of cell-cell adhesion, increased DSP membrane expression, and prevented PV-IgG-induced blister formation in a human ex-vivo skin model. Together, SERPINB5 modulates DSP and intercellular adhesion through the regulation of TGF-{beta} signalling. Further, TGF-{beta} signalling was identified as a potential target for pemphigus treatment.

Psoriasis is a chronic immune-mediated inflammatory skin disease characterized by excessive keratinocyte proliferation, immune cell infiltration and dysregulated inflammatory signaling. Despite the availability of biologic therapies targeting inflammatory cytokines, many patients experience incomplete responses or relapse, highlighting the need to better understand molecular regulators of cutaneous inflammation. CD109 is a glycosylphosphatidylinositol (GPI)-anchored protein previously identified by our lab as a co-receptor and negative regulator of Transforming Growth Factor-{beta} (TGF-{beta}) signaling that inhibits fibrotic responses. Emerging evidence suggests that CD109 also modulates immune and inflammatory pathways. In this study, we investigated whether epidermal CD109 overexpression influences cutaneous inflammatory responses. Transgenic (TG) mice overexpressing CD109 under the keratin-14 (K14) promoter were used to restrict transgene expression to the epidermis. TG and wild-type (WT) littermates were subjected to lipopolysaccharide (LPS)-induced skin inflammation. CD109 TG mice exhibited significantly reduced immune cell recruitment, including macrophages and neutrophils, along with decreased expression of the pro-inflammatory mediators IL-1 and MCP-1/CCL2 compared with WT mice. Transcriptomic analysis of primary keratinocytes revealed downregulation of multiple inflammatory signaling pathways in CD109-overexpressing cells, including TNF-/NF-{kappa}B, IL-2/STAT5, IFN-{gamma}, IFN-, and IL-6/JAK/STAT3 pathways. Together, these findings demonstrate that epidermal CD109 overexpression attenuates cutaneous inflammatory responses by suppressing key inflammatory signaling networks and limiting immune cell recruitment, suggesting that CD109 may represent an important regulator of inflammatory signaling in the skin and a potential target for inflammatory skin diseases such as psoriasis.

Vulvar diseases are a critical yet often neglected area of womens health, profoundly affecting patients quality of life and frequently resulting in long-term physical and psychological challenges. Lichen sclerosus (LS) is a chronic inflammatory skin disorder that predominantly affects the vulva, leading to severe itching, pain, scarring, and an increased risk of malignancy. Despite its profound impact on affected individuals, the molecular pathogenesis of vulvar LS (VLS) is not well understood, hindering the development of FDA-approved therapies. Here, we utilize single-cell and spatial transcriptomics to analyze lesional and non-lesional skin from VLS patients, as well as healthy control vulvar skin. Our findings demonstrate histologic, cellular, and molecular heterogeneities within VLS, yet highlight unifying molecular changes across keratinocytes, fibroblasts, immune cells, and melanocytes in lesional skin. They reveal cellular stress and damage in fibroblasts and keratinocytes, enhanced T cell activation and cytotoxicity, aberrant cell-cell signaling, and increased activation of the IFN, JAK/STAT, and p53 pathways in specific cell types. Using both monolayer and organotypic culture models, we also demonstrate that knockdown of select genes, which are downregulated in VLS lesional keratinocytes, partially recapitulates VLS-like stress-associated changes. Collectively, these data provide novel insights into the pathogenesis of VLS, identifying potential biomarkers and therapeutic targets for future research.

Cutaneous melanomas harboring a B-RafV600E mutation are treated with immune check point inhibitors or kinase inhibitor combination therapies relying on MAPK inhibitors (MAPKi) Dabrafenib and Trametinib (Curti and Faries, 2021). However, cells become resistant to treatments over the timespan of a few months. Resistance to MAPKi has been associated with adoption of an aggressive amoeboid phenotype characterized by elevated RhoA signaling, enhanced contractility and thick cortical filamentous actin (F-actin) structures (Kim et al., 2016; Misek et al., 2020). Targeting active RhoA through Rho-kinase (ROCK) inhibitors, either alone or in combination with immunotherapies, reverts MAPKi-resistance (Misek et al., 2020; Orgaz et al., 2020). Yet, the mechanisms for this behavior remain largely unknown. Given our recent findings of cytoskeletons role in cancer cell proliferation (Mohan et al., 2019), survival (Weems et al., 2023), and metabolism (Park et al., 2020), we explored possibilities by which RhoA-driven changes in cytoskeleton structure may confer resistance. We confirmed elevated activation of RhoA in a panel of MAPKi-resistant melanoma cell lines, leading to a marked increase in the presence of contractile F-actin bundles. Moreover, these cells had increased glucose uptake and glycolysis, a phenotype disrupted by pharmacological perturbation of ROCK. However, glycolysis was unaffected by disruption of F-actin bundles, indicating that glycolytic stimulation in MAPKi-resistant melanoma is independent of F-actin organization. Instead, our findings highlight a mechanism in which elevated RhoA signaling activates ROCK, leading to the activation of insulin receptor substrate 1 (IRS1) and P85 of the PI3K pathway, which promotes cell surface expression of GLUT1 and elevated glucose uptake. Application of ROCK inhibitor GSK269962A results in reduced glucose uptake and glycolysis, thus impeding cell proliferation. Our study adds a mechanism to the proposed use of ROCK inhibitors for long-term treatments on MAPKi-resistant melanomas.

Lichen Planopilaris (LPP) is a lymphocyte-mediated scarring alopecia characterized by progressive follicular destruction and fibrosis. In this clinical trial, patients with biopsy proven LPP were treated with deucravacitinib (an oral inhibitor of tyrosine kinase 2 (TYK2)) 6 mg BID for 24 weeks (NCT-06091956). Bulk and single-cell RNA sequencing was performed on paired pre- and post-treatment scalp biopsies from baseline and week 4. Patients (N=10) demonstrated improvements in PGA (88.9%, p=0.008), LPPAI (-2.3 points, SD 1.1, p=0.002) and Skindex-16 (-21.0 points, SD 22.1, p=0.014) scores at week 24. Bulk transcriptomic analysis of untreated LPP revealed upregulation of type I Interferon (IFN)-stimulated genes and pathways related to inflammation, immune activation, keratinization, and extracellular matrix remodelling, with downregulation of immune and inflammatory pathways following treatment. Single-cell RNA-seq of LPP was characterized by enrichment of CD8+GZMK+ T cells which showed downregulation of T-cell receptor signaling as well as antiviral pathways with treatment. Basal keratinocytes exhibited reduced cytokine and interferon signaling and decreased communication with NK cells following treatment. CCL19+ fibroblasts were prominent in untreated disease was attenuated after treatment, with downregulation of type I IFN signaling. Selective TYK2 inhibition with deucravacitinib effectively suppresses these inflammatory circuits in LPP and represents a promising therapeutic strategy.

The lipid composition of the epidermis plays a critical role in the skins barrier function, and defects in lipid synthesis or assembly can cause a spectrum of skin diseases, ranging from dry skin to severe ichthyoses. The aim of this study was to develop an in vitro model of human skin with tuneable lipid deficiency. Human N/TERT keratinocytes were engineered to express doxycycline-inducible short hairpin RNAs targeting ceramide synthase 3 (CerS3), which is essential for synthesis of ultra long chain ceramides and skin barrier function. We show that 3D human skin equivalents (HSEs) with induced knockdown of CerS3 display normal stratification and terminal differentiation but have reduced Nile Red staining for polar lipids. Further analysis of the lipidome by mass spectrometry confirmed a significant reduction in specific classes of ceramides and ceramide chain length in the CerS3 depleted HSEs. We also show that CerS3 knockdown is reversible upon removal of doxycycline and can be used to study recovery and repair of epidermal lipids. Together, these findings provide an overall strategy for genetically tuning the lipid composition within human skin models and establish a new in vitro model of ceramide deficiency.