Journal of Investigative Dermatology

Sulfite oxidase deficiency is a rare inborn error in metabolism leading to early childhood death due to rapidly progressing neurodegeneration. A new mouse model of sulfite oxidase deficiency carrying a homozygous deletion in the Suox gene resembles the human pathology in terms of neonatal death and elevation of sulfite and thiosulfate in plasma and urine, respectively. Homozygous Suox-/- mice are initially born healthy, display growth retardation starting at postnatal day 4 and die in average at day 9.6. Here we report that Suox-/- mice develop dry and scaly skin early postnatally, showing that sulfite oxidase is essential to maintain a functional skin barrier after birth. At postnatal day 5 Suox-/- mice develop altered epidermal morphology and dysregulated early and late keratinocyte differentiation accompanied by increased stress response. We propose a sulfite-induced cleavage of disulfide bonds in key epidermal proteins essential for a functional barrier.

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.

Chronic spontaneous urticaria (CSU) is one of the most intractable human-specific skin diseases. However, as no experimental animal model exists, the mechanism underlying disease pathogenesis in vivo remains unclear, making the establishment of a curative treatment challenging. Here, using a novel approach combining mathematical modeling, in vitro experiments and clinical data analysis, we show that the pathological state of CSU patients can be inferred by geometric features of the skin eruptions. Based on our hierarchical mathematical modelling and the analysis of 105 CSU patient eruption pattern geometries, analyzed by six dermatologists, we demonstrate that the eruption patterns can be classified into five categories, each with distinct histamine, basophils, mast cells and coagulation factors network signatures. Furthermore, our network analysis revealed that tissue factor degradation/activation likely determines boundary/area pattern, and that the state of spontaneous histamine release from mast cells may contribute to divergence of the boundary pattern. Thus, our study not only demonstrates that pathological states of diseases can be defined by geometric features but will also facilitate more accurate decision-making to manage CSU in the clinical setting.

Psoriasis is a complex chronic inflammatory skin disease with unclear molecular mechanisms. Here, we identify Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) as a novel accelerator of psoriasis development. Both genetic ablation of SHP2 in macrophages and pharmacological inhibition of SHP2 prevents the development of psoriasis-like skin inflammation in an imiquimod-induced murine model of psoriasis. Mechanistically, SHP2 promotes the trafficking of Toll-like receptor 7 (TLR7) from Golgi to endosome through its interaction with and dephosphorylation of TLR7 at Tyr1024, which promotes the ubiquitination of TLR7 and psoriasis-like skin inflammation. Importantly, SHP2 allosteric inhibitor SHP099 reduces the expression of pro-inflammatory cytokines in peripheral blood mononuclear cells from human patients with psoriasis. Collectively, our findings identify SHP2 as a novel regulator of psoriasis and suggest that SHP2 inhibition may be a promising therapeutic approach for psoriatic patients.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease characterized by recurrent painful abscesses and tunnels in flexural sites. The mechanisms driving HS pathogenesis, particularly the roles of keratinocytes and fibroblasts in the HS inflammatory ecosystem, remain poorly understood. To characterise the cellular and molecular landscape of HS, we analyzed lesional skin from severe HS patients using single-cell RNA-sequencing and spatial transcriptomics to identify key keratinocyte states and cellular interactions, with a focus on fibroblast-keratinocyte crosstalk. Our study identifies a novel migratory S100+ pathogenic keratinocyte state and highlights critical interactions between COL6A5+ fibroblasts in HS lesion formation. We also detect tertiary lymphoid organ (TLO)-like structures enriched with activated B and plasma cells interacting with APOD+ fibroblasts, implicating their role in HS chronic inflammation. We show that fibroblast interactions with HS keratinocytes and specific immune cells, such as Langerhans cells, are key drivers of HS pathogenesis, and that two main fibroblast niches are present within the HS microenvironment. Targeting these cellular networks may offer new therapeutic strategies for HS management, and highlight the potential limitations of targeting individual pathways in isolation, when treating pathology present in HS tissue.

AbstractPsoriasis is a multifactorial disorder mediated by IL-17-producing T cells, involving immune cells and skin-constituting cells. Semaphorin 4A (Sema4A), an immune semaphorin, is known to take part in T helper type 1/17 differentiation and activation. However, Sema4A is also crucial for maintaining peripheral tissue homeostasis and its involvement in skin remains unknown. Here, we revealed that while Sema4A expression was pronounced in psoriatic blood lymphocytes and monocytes, it was downregulated in the keratinocytes of both psoriatic lesions and non-lesions compared to controls. Imiquimod application induced more severe dermatitis in Sema4A knockout (KO) mice compared to wild-type (WT) mice. The naive skin of Sema4AKO mice showed increased T cell infiltration and IL-17A expression along with thicker epidermis and distinct cytokeratin expression compared to WT mice, which are hallmarks of psoriatic non-lesions. Analysis of bone marrow chimeric mice suggested that Sema4A expression in keratinocytes plays a regulatory role in imiquimod-induced dermatitis. The epidermis of psoriatic non-lesion and Sema4AKO mice demonstrated mTOR complex 1 upregulation, and the application of mTOR inhibitors reversed the skewed expression of cytokeratins in Sema4AKO mice. Conclusively, Sema4A- mediated signaling cascades can be triggers for psoriasis and targets in the treatment and prevention of psoriasis.

Store-operated Ca2+ entry (SOCE) is a mechanism by which STIM1/2 detect calcium depletion in the endoplasmic reticulum (ER) and activate ORAI1/TRPC channels on the cell membrane to induce calcium influx into the cytoplasm. This study aimed to investigate SOCEs role in the skin epidermis in vivo, using female mice with epithelial tissue-specific Stim1 and Stim2 knockout (Stim1/2 cKO) and control mice (Stim1/2fl/fl). Keratinocytes from Stim1/2 cKO mice exhibited reduced Stim1 and Stim2 gene expression levels and impaired SOCE function compared with the controls. Histological analysis revealed hyperkeratosis in the Stim1/2 cKO tissues; however, no significant changes were observed in the proliferation or migration of keratinocytes or wound healing of the back skin. RNA-seq analysis indicated altered keratinization and cell-to-cell adhesion in the Stim1/2 cKO mice. Additionally, transepidermal water loss (TEWL) was increased significantly, and a biotinylated reagent diffused from the subcutaneous area into the granular layer, signifying compromised barrier function in the Stim1/2 cKO mice. The Stim1/2 cKO mice exhibited alterations in desmoglein 1 (Dsg1) and elevated levels of Kallikrein-related peptidase (Klk) 6 and Klk7, leading to increased trypsin- and chymotrypsin-like serine protease activities, respectively. These results suggest that SOCE dysfunction leads to hyperkeratosis and impaired epidermal barrier function via Klk activation without affecting skin cell proliferation in vivo. These novel findings improve understanding of the molecular mechanisms underlying calcium signaling in the epidermis barrier and suggest potential avenues for investigating related skin pathologies linked to calcium homeostasis, such as Hailey-Hailey disease (HHD) and Darier disease (DD).

Differences in prevalence of inflammatory skin diseases including atopic dermatitis and psoriasis in African American (AA) versus White Non-Hispanic (WNH) population are well recognized. However, the underlying mechanisms are largely unknown. We previously observed significant differences in healthy AA skin transcriptome with differentially expressed genes (DEG) enriched for inflammation and cornification processes. Here we analyzed proteome in skin biopsies from healthy AA and WNH volunteers using Olink(R) Explore Inflammation 384 biomarker panel. Among proteins with higher expression in AA skin were IRAK1, IL1A, IL4, IL22RA1. IL1A binding to IL1R1 receptor is known to result in recruitment of adapter molecules such as IRAK1, and activation of downstream NF-{kappa}B and MAPK signaling. We confirmed NF-{kappa}B and ERK1/2 activation in AA skin by Western blot analysis of their phosphorylation at specific activating sites. Importantly, we observed similar differences between AA and WNH neonatal foreskin and between AA and WNH 3D skin organoids. Further analysis of DEG promoters by Gene Transcription Regulation Database (GTRD) pointed to NF-{kappa}B and AP1 as key transcription factors involved in AA DEG regulation. Overall, proinflammatory signaling in healthy AA skin starting early in childhood may contribute to the increased risk of certain inflammatory skin diseases within the AA population.

BackgroundMast cells (MCs) are tissue-resident immune cells that are classified into two subsets in mice: connective tissue-type MCs (CTMCs) and mucosal type MCs (MMCs). Although both CTMCs and MMCs can be induced from bone marrow (BM)-derived hematopoietic stem cells (HSCs) in vitro, recent research on MC ontogeny has revealed that MMCs are maintained with a supply of BM-derived HSCs, while CTMCs are maintained locally by self-proliferation in steady state in vivo. However, how CTMCs, such as skin MCs, are maintained in an inflammatory state such as atopic dermatitis (AD) remains to be fully elucidated. Methods: MC903-induced AD model was used to identify BM-derived MCs in the skin. The infiltration and proliferation of MCs were evaluated by flow cytometry using CD45.1 BM-chimera mice and parabiosis. BM-derived MCs in AD-like skin were compared to resident MCs (rMCs) in gene expressions by RNA sequence analysis. The fate of BM-derived MCs in AD-like skin was investigated for expressions of CTMC markers and responses to compound 48/80. Results: In AD-like skin, significant increase of both rMCs and BM-derived MCs was observed. BM-derived MCs were derived from circulating MC progenitors (MCps) and were distinguished from rMCs by integrin{beta}7 expression, which was gradually downregulated in the skin. RNA sequence analysis showed that integrin{beta}7+ MCs in the skin shared characteristics of both MMC and CTMC. Integrin{beta}7+ MCs proliferated in situ and acquired the CTMC phenotypes in AD-like skin. Conclusions: Skin MCs are maintained in AD-like skin by both local proliferation of rMCs and infiltration/proliferation of BM-derived MCs, which differentiate toward CTMC in the skin.

Hidradenitis Suppurativa (HS) is a proinflammatory painful skin disorder. This chronic disease is often associated with aggressive squamous cell carcinoma (SCC). The molecular pathogenesis of this disease progression remains undefined. The translation initiation factor eIF4E/4G/4A1 complex is overexpressed in a variety of human malignancies. In this study, we found that the expression of eIF4E/4G/4A1 as well as phosphorylated eIF4E were upregulated in HS skin. In the global transcription profiles derived from two public database, we were able to enrich 734 eIF4F-related genes. GSEA pathway enrichment analysis further demonstrated that RAS/MEK/ERK oncogene signaling pathway associated with inflammation signaling were significantly activated in HS lesion. The increase expression of eIF4 protein components was associated with enhanced eIF4E translation targets Cyclin D1 and c-Myc. Confocal fluorescence microscopy analysis further revealed that Cyclin D1 and c-Myc specifically co-localized in nuclei of certain cells in HS epithelium. We also found that many of the PCNA positive hyperproliferative cells were also positive for c-Myc expression. These data demonstrate that 5-cap{square}dependent translation is a potential pathway underlying the SCC pathogenesis in chronic HS lesions. Furthermore, being a druggable target, inhibition of eIF4F may block lesion-associated lethal SCCs in HS patients.

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.

Fibroproliferative diseases such as systemic sclerosis are complex conditions characterized by chronic skin inflammation and progressive fibrosis, with fibroblast activation as a central feature. While Transforming Growth Factor Beta (TGF-{beta}) signaling is a well-established driver of fibrosis in SSc, inflammatory pathways such as Nuclear Factor Kappa B (NF-{kappa}B) also contribute substantially to disease morbidity. We previously identified CD109 as a TGF-{beta} co-receptor and negative regulator of fibrotic signaling; however, its role in inflammatory signaling remains unknown. Here, we investigate the function of CD109 in regulating inflammatory signaling in skin fibroblasts. We show that, CD109 co-localizes and associates with Toll-like receptors (TLR2, TLR4) and tumor necrosis factor receptors (TNFRI, TNFRII), and that loss of CD109 enhances TNF--induced NF-{kappa}B activation and reprograms cytokine production in human dermal fibroblasts. Furthermore, both global and fibroblast-specific CD109 knockout mice exhibit increased immune cell infiltration and skin inflammation. In parallel, single-cell transcriptomic analyses across a pan-disease fibroblast atlas show that CD109 expression is preferentially maintained in structural and homeostatic fibroblast subtypes, whereas immune-interacting fibroblast subsets consistently display decreased CD109 levels. Pathway-level analyses of fibroblast pseudobulk samples reveal altered activity of canonical inflammatory pathways in SSc compared to healthy skin. Together, these findings identify CD109 as a fibroblast-intrinsic negative regulator of inflammatory signaling and suggest a broader role for CD109 in modulating inflammatory responses in systemic sclerosis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/736423v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@be9e08org.highwire.dtl.DTLVardef@794173org.highwire.dtl.DTLVardef@b81eb5org.highwire.dtl.DTLVardef@1e811f5_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract: CD109 Restrains Fibroblast-Driven Inflammation by Modulating NF-{kappa}B Signaling. Generated using FigureLabs.ai and edited using Adobe Photoshop. C_FIG

Hidradenitis suppurativa (HS) is a severe chronic inflammatory skin disease affecting human apocrine sweat gland-bearing skin regions. One unique feature of HS is the development of keratinized sinus tracts that grow extensively deep in the dermis and are highly immunogenic. Here, we demonstrated that the stem cell fate infidelity exists in the HS sinus tracts, which exhibit features of both surface epidermis and appendages. Using single cell transcriptome analyses, we finely dissected different compartments of the HS epithelium and identified their respective changes in cytokine expression during disease progression and the critical interactions with the immune cells. Together, our work provides advanced understanding of the pathological epidermal remodeling and important implications for HS therapeutics.

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.

The skin is a multifunctional organ, forming a barrier between the external and internal environment, thereby functioning as an initial safeguard against foreign and harmful environmental and biological factors. The barrier function is governed by the epidermis, a stratified epithelium composed of an outermost cornified envelope, several differentiated layers, and an innermost basal layer, anchored to the basement membrane (BM). Below the BM lies the dermis, which is composed of an extracellular matrix (ECM), formed by the dermal fibroblasts and dermal adipose tissue. The skin is subject to constant cellular turnover (terminal differentiation) that maintains the homeostatic state of the skin. Recently, autophagy has been implicated in epidermal differentiation and in preserving homeostasis in the skin. The AKT/mTORC1 pathway is a key regulator for epidermal development and differentiation, and conditions (e.g., psoriasis) are linked to the hyperactivation of these key autophagy regulators. A further process known as LC3-associated phagocytosis (LAP) uses some but not all components of autophagy. The Atg16l1 E230 mouse model (E230), deficient for LAP, has been widely used to study the effects of LAP-deficiency and autophagy in tissue homeostasis. Here, the E230 model was used to study the relationship between skin homeostasis and LAP and to determine whether LAP-deficient mice (WD) display a cutaneous skin phenotype. Histological analysis of male 1-year-old Wild Type (WT) and WD mice skin revealed morphological differences in dorsal and tail skin. qPCR analysis of key keratins showed no differences (p > 0.05) when compared between genotypes. This was confirmed by western blotting studies. In addition, the expression of general proliferation markers (Akt & ERK2) showed a small reduction (p < 0.05) in the WD mice skin. General skin barrier formation was assessed by dye permeation assays, which demonstrated full and proper formation of the skin barrier at E18.5 in both WT and WD. Notable dermal thinning in the WD mice skin (p < 0.0001), led us to examine the biomechanical properties to dismiss any abnormalities relating to biomechanical functionality. Results indicate that WT and WD mouse skin show identical biomechanical properties at these ages (p < 0.05 and p < 0.01, respectively). In summary, the noted differences in the dermal and epidermal histology of WD skin were shown to be functionally insignificant in terms of epidermal permeability and dermal biomechanics.

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.

Wound repair requires the coordination of multiple cell types including immune cells and tissue resident cells to coordinate healing and return of tissue function. Diabetic foot ulceration is a type of chronic wound that impacts over 4 million patients in the US and over 7 million worldwide (Edmonds et al., 2021). Yet, the cellular and molecular mechanisms that go awry in these wounds are not fully understood. Here, by profiling chronic foot ulcers from non-diabetic (NDFUs) and diabetic (DFUs) patients using single-cell RNA sequencing, we find that DFUs display transcription changes that implicate reduced keratinocyte differentiation, altered fibroblast function and lineages, and defects in macrophage metabolism, inflammation, and ECM production compared to NDFUs. Furthermore, analysis of cellular interactions reveals major alterations in several signaling pathways that are altered in DFUs. These data provide a view of the mechanisms by which diabetes alters healing of foot ulcers and may provide therapeutic avenues for DFU treatments.

Loss of cell polarity and tissue disorganization occurs in majority of epithelial cancers. Studies in simple model organisms identified molecular mechanisms responsible for the establishment and maintenance of cellular polarity, which play a pivotal role in establishing proper tissue architecture. The exact role of these cell polarity pathways in mammalian cancer is not completely understood. Here we analyzed the mammalian orthologs of drosophila apical-basal polarity gene lethal giant larvae (lgl), which regulates asymmetric stem cell division and functions as a tumor suppressor in flies. There are two mammalian orthologs of lgl (Llgl1 and Llgl2). To determine the role of the entire lgl signaling pathway in mammals we generated mice with ablation of both Llgl1 and Llgl2 in skin epidermis using K14-Cre (Llgl1/2-/- cKO mice). Surprisingly, we found that ablation of Llgl1/2 genes does not impact epidermal polarity in adult mice. However, old Llgl1/2 cKO mice present with focal skin lesions which are missing epidermal layer and ripe with inflammation. To determine the role of lgl signaling pathway in cancer we generated Trp53-/-/Llgl1/2-/- cKO and Trp53-/+/Llgl1/2-/- cKO mice. Loss of Llgl1/2 promoted squamous cell carcinoma (SCC) development in Trp53-/- cKO and caused SCC in Trp53-/+ cKO mice, while no cancer was observed in Trp53-/+ cKO controls. Mechanistically, we show that ablation of Llgl1/2 causes activation of aPKC and upregulation of NF-kB signaling pathway, which may be necessary for SCC in Trp53-/+/Llgl1/2-/- cKO mice. We conclude that Lgl signaling pathway functions as a tumor suppressor in mammalian skin epidermis.

A delayed-type hypersensitivity (DTH) response induced by the intradermal inoculation of leishmanin antigens is used to detect prior exposure to the protozoan parasite Leishmania. Leishmanin antigen preparations are an important tool in disease surveillance studies in endemic areas. Commercial scale leishmanin antigens are being developed for wider deployment in assessing vaccine efficacy and latent infections in the field. Previous studies with leishmanin induced DTH response were limited to analysis of PBMCs. To investigate the mediators of DTH response, we performed spatial transcriptomic analysis of the DTH skin biopsies obtained from a Leishmania endemic foci. Compared to healthy skin biopsy, macrophages and T cells, and IL-16 and TNF that attract CD4+ T cells, were elevated in Langerhans cells in the DTH biopsies. Both IFN-{gamma} and its receptors were similarly elevated in the DTH biopsies. Chemokines CCL2, CCL5, CCL8 and CCL19, and the corresponding receptors were elevated in DTH biopsies, with CCL19-CCR7 as the most salient interaction in our Cellchat analysis. These data reveal biomarkers of DTH response following leishmanin inoculation and enable appropriate formulation and reintroduction of leishmanin skin test antigens for disease surveillance.

miR-378a-3p has been reported to be upregulated in the lesional skin of patients with atopic dermatitis (AD); however, its function in AD remains unclear. Here, we demonstrate that miR-378a-3p expression is induced by IL-4 and live Staphylococcus aureus (S. aureus) in normal human epidermal keratinocytes (NHEKs) cultured in proliferative conditions or in a 3D epidermal culture model. Transcriptomic profiling and gene set enrichment analysis of miR-378a-3p-transfected NHEKs revealed positive enrichment of inflammatory response pathways alongside downregulation of genes associated with epidermal development. More specifically, miR-378a-3p enhanced expression of multiple NF-{kappa}B-dependent inflammatory mediators, accompanied by increased phosphorylation of p65, indicating activation of canonical NF-{kappa}B pathway. Notably, miR-378a-3p concomitantly reduced the expression of several NF-{kappa}B family members and upstream adaptor molecules, supporting a model in which miR-378a-3p promotes canonical NF-{kappa}B activity through coordinated modulation of multiple components within the NF-{kappa}B regulatory network. In NHEKs exposed to live S. aureus, miR-378a-3p significantly increased the secretion of IL-1{beta}, IL-1Ra, and IL-8, indicating that miR-378a-3p may amplify innate immune responses triggered by S. aureus colonization in AD. Collectively, these findings identify miR-378a-3p as a positive regulator of keratinocyte inflammatory responses that may contribute to AD exacerbation, particularly in the context of S. aureus colonization.