Primary Keratinocytes Research Articles

A Tissue Engineered 3D Model of Cancer Cell Invasion for Human Head and Neck Squamous-Cell Carcinoma.

Head and neck squamous-cell carcinoma (HNSCC) is associated with aggressive local invasiveness, being a main reason for its poor prognosis. The exact mechanisms underlying the strong invasive abilities of HNSCC remain to be elucidated. Therefore, there is a need for in vitro models to study the interplay between cancer cells and normal adjacent tissue at the invasive tumor front. To generate oral mucosa tissue models (OMM), primary keratinocytes and fibroblasts from human oral mucosa were isolated and seeded onto a biological scaffold derived from porcine small intestinal submucosa with preserved mucosa. Thereafter, we tested different methods (single tumor cells, tumor cell spots, spheroids) to integrate the human cancer cell line FaDu to generate an invasive three-dimensional model of HNSCC. All models were subjected to morphological analysis by histology and immunohistochemistry. We successfully built OMM tissue models with high in vivo-in vitro correlation. The integration of FaDu cell spots and spheroids into the OMM failed. However, with the integration of single FaDu cells into the OMM, invasive tumor cell clusters developed. Between segments of regular epithelial differentiation of the OMM, these clusters showed a basal membrane penetration and lamina propria infiltration. Primary human fibroblasts and keratinocytes seeded onto a porcine carrier structure are suitable to build an OMM. The HNSCC model with integrated FaDu cells could enable subsequent investigations into cancer cell invasiveness.

LncRNA LINC00941 modulates MTA2/NuRD occupancy to suppress premature human epidermal differentiation.

Numerous long non-coding RNAs (lncRNAs) were shown to have a functional impact on cellular processes such as human epidermal homeostasis. However, the mechanism of action for many lncRNAs remains unclear to date. Here, we report that lncRNA LINC00941 regulates keratinocyte differentiation on an epigenetic level through association with the NuRD complex, one of the major chromatin remodelers in cells. We find that LINC00941 interacts with NuRD-associated MTA2 and CHD4 in human primary keratinocytes. LINC00941 perturbation changes MTA2/NuRD occupancy at bivalent chromatin domains in close proximity to transcriptional regulator genes, including the EGR3 gene coding for a transcription factor regulating epidermal differentiation. Notably, LINC00941 depletion resulted in reduced NuRD occupancy at the EGR3 gene locus, increased EGR3 expression in human primary keratinocytes, and increased abundance of EGR3-regulated epidermal differentiation genes in cells and human organotypic epidermal tissues. Our results therefore indicate a role of LINC00941/NuRD in repressing EGR3 expression in non-differentiated keratinocytes, consequentially preventing premature differentiation of human epidermal tissues.

Inhibition of RNase 7 by RNase inhibitor promotes inflammation and Staphylococcus aureus growth: Implications for atopic dermatitis.

The antimicrobial ribonuclease RNase 7 is abundantly expressed in the epidermis of lesional skin of atopic dermatitis (AD). Host RNase inhibitor (RI) binds to RNase 7 and blocks its ribonuclease activity. This study aimed to evaluate the impact of RNase 7-RI interactions on AD. Cultured human primary keratinocytes, with siRNA-mediated downregulation of RNase 7 and RI, were stimulated with the synthetic RNA polyinosinic-polycytidylic acid (poly I:C). Induction of proinflammatory mediators was analyzed by real-time PCR and ELISA. RI expression in AD non-lesional and lesional skin biopsies and healthy controls was analyzed by real-time PCR and immunostaining. RI protein release invivo on the AD skin surface was determined by western blot. Antimicrobial and ribonuclease assays were used to investigate the functional role of RI. RNase 7 inhibited the RNA-induced expression of proinflammatory mediators in keratinocytes. Accordingly, downregulation of RNase 7 in keratinocytes enhanced RNA-mediated induction of proinflammatory mediators, whereas downregulation of RI had the opposite effect. RI was released by damaged keratinocytes and epidermis. Invivo expression and release of RI on the skin surface were enhanced in lesional AD skin. Rinsing solution from the surface of lesional AD skin blocked the ribonuclease activity of RNase 7. The anti-Staphylococcus aureus activity of RNase 7 was abrogated by RI. Our data suggest a novel role of RI as a trigger factor of inflammation in AD by blocking the ribonuclease and antimicrobial activity of RNase 7, thereby enhancing RNA-mediated inflammation and S. aureus growth.

Potential functionality of Cutibacterium acnes extracellular vesicles in atopic dermatitis and acne vulgaris: A comparative proteomic analysis.

Cutibacterium acnes is a commensal bacterium residing in healthy skin and plays a critical role in maintaining skin homeostasis. C. acnes has been considered closely related to acne vulgaris, while recent studies suggest that C. acnes and its metabolites may have a protective role in atopic dermatitis (AD) by modulating the immune system and maintaining skin homeostasis. Extracellular vesicles (EVs) are small membranous vesicles secreted by bacteria that participate in bacteria-host interactions. This study first compared C. acnes EVs from AD lesions (AD-EVs), acne lesions (Acne-EVs), and healthy skin (NC-EVs), using Label-free quantitative LC-MS/MS and validated differently expressed proteins by parallel reaction monitoring (PRM). Then Normal Human Epidermal Keratinocytes (NHEK) and human primary keratinocytes (KC) were treated with C. acnes EVs isolated from different groups, and the expressions of inflammatory factors were measured by quantitative real-time PCR and Western blotting. Compared with the acne group, the AD group showed greater downregulation of proteins related to energy metabolism and carbon source utilization pathway. Differences in protein profile in AD and acne lesion-separated C. acnes EVs correspond to the abnormal sebum secretion pattern in both diseases. C. acnes EVs from different groups affected different expressions of Th1 and Th2 inflammatory factors and epidermal barrier markers in NHEK and KC, indicating different immunomodulatory potentials. This study observed distinct proteomic differences between AD-EVs and Acne-EVs, and provided insights into the functional differences of C. acnes EVs in AD and acne.

Single-nucleus transcriptomics uncovers a geroprotective role of YAP in primate gingival aging.

Aging has a profound impact on the gingiva and significantly increases its susceptibility to periodontitis, a worldwide prevalent inflammatory disease. However, a systematic characterization and comprehensive understanding of the regulatory mechanism underlying gingival aging is still lacking. Here, we systematically dissected the phenotypic characteristics of gingiva during aging in primates and constructed the first single-nucleus transcriptomic landscape of gingival aging, by which a panel of cell type-specific signatures were elucidated. Epithelial cells were identified as the most affected cell types by aging in the gingiva. Further analyses pinpointed the crucial role of YAP in epithelial self-renew and homeostasis, which declined during aging in epithelial cells, especially in basal cells. The decline of YAP activity during aging was confirmed in the human gingival tissues, and downregulation of YAP in human primary gingival keratinocytes recapitulated the major phenotypic defects observed in the aged primate gingiva while overexpression of YAP showed rejuvenation effects. Our work provides an in-depth understanding of gingival aging and serves as a rich resource for developing novel strategies to combat aging-associated gingival diseases, with the ultimate goal of advancing periodontal health and promoting healthy aging.

Differential Contributions of Interferon Classes to Host Inflammatory Responses and Restricting Virus Progeny Production.

Fundamental to mammalian intrinsic and innate immune defenses against pathogens is the production of Type I and Type II interferons, such as IFN-β and IFN-γ, respectively. The comparative effects of IFN classes on the cellular proteome, protein interactions, and virus restriction within cell types that differentially contribute to immune defenses are needed for understanding immune signaling. Here, a multilayered proteomic analysis, paired with biochemical and molecular virology assays, allows distinguishing host responses to IFN-β and IFN-γ and associated antiviral impacts during infection with several ubiquitous human viruses. In differentiated macrophage-like monocytic cells, we classified proteins upregulated by IFN-β, IFN-γ, or pro-inflammatory LPS. Using parallel reaction monitoring, we developed a proteotypic peptide library for shared and unique ISG signatures of each IFN class, enabling orthogonal confirmation of protein alterations. Thermal proximity coaggregation analysis identified the assembly and maintenance of IFN-induced protein interactions. Comparative proteomics and cytokine responses in macrophage-like monocytic cells and primary keratinocytes provided contextualization of their relative capacities to restrict virus production during infection with herpes simplex virus type-1, adenovirus, and human cytomegalovirus. Our findings demonstrate how IFN classes induce distinct ISG abundance and interaction profiles that drive antiviral defenses within cell types that differentially coordinate mammalian immune responses.

The cytotoxic and immunomodulatory mechanisms of photodynamic therapy evaluated in in vitro studies on cultures of selected skin cell lines

SignificanceThe aim of the study is to evaluate the cytotoxic and immunomodulatory effects of HYP-PDT in vitro studies on cultures of selected skin cell lines, as a possibility of using PDT for patients with atopic dermatitis and psoriasis. ApproachPrimary Dermal Fibroblast and Primary Epidermal Keratinocytes was used in the study. After incubation the cells were treated with hypericin, exposed to VIS visible light. Then cell viability was determined by the MTT assay and cytokines markings were done with Bio-Plex Pro™ Assay kit and with Bio-Plex Suspension Array System. ResultsThe experiment showed a significant cytotoxic effect of HYP-PDT and in sublethal doses, an immunomodulatory effect manifested in changes in the concentrations of selected cytokines ConclusionsThe obtaining results confirming the destructive effect of HYP- PDT on the selected, skin cells lines show a possibility of extending the indication for HYP-PDT, for patients with inflammatory skin diseases.

Antiviral and synergistic effects of photo-energy with acyclovir on herpes simplex virus type 1 infection

This experimental study aimed to evaluate the antiviral and synergistic effects of photoenergy irradiation on human herpes simplex virus type I (HSV-1) infection. We assessed viral replication, plaque formation, and relevant viral gene expression to examine the antiviral and synergistic effects of blue light (BL) with acyclovir treatment. Our results showed that daily BL (10 J/cm2) irradiation inhibited plaque-forming ability and decreased viral copy numbers in HSV-1-infected monkey kidney epithelial Vero cells and primary human oral keratinocyte (HOK) cells. Combined treatment with the antiviral agent acyclovir and BL irradiation increased anti-viral activity, reducing viral titers and copy numbers. In particular, accumulated BL irradiation suppressed characteristic viral genes including UL19 and US6, and viral DNA replication-essential genes including UL9, UL30, UL42, and UL52 in HOK cells. Our results suggest that BL irradiation has anti-viral and synergistic properties, making it a promising therapeutic candidate for suppressing viral infections in clinical trials.

Quantitative imaging and semiotic phenotyping of mitochondrial network morphology in live human cells.

The importance of mitochondria in tissue homeostasis, stress responses and human diseases, combined to their ability to transition between various structural and functional states, makes them excellent organelles for monitoring cell health. There is therefore a need for technologies to accurately analyze and quantify changes in mitochondrial organization in a variety of cells and cellular contexts. Here we present an innovative computerized method that enables accurate, multiscale, fast and cost-effective analysis of mitochondrial shape and network architecture from confocal fluorescence images by providing more than thirty features. In order to facilitate interpretation of the quantitative results, we introduced two innovations: the use of Kiviat-graphs (herein named MitoSpider plots) to present highly multidimensional data and visualization of the various mito-cellular configurations in the form of morphospace diagrams (called MitoSigils). We tested our fully automated image analysis tool on rich datasets gathered from live normal human skin cells cultured under basal conditions or exposed to specific stress including UVB irradiation and pesticide exposure. We demonstrated the ability of our proprietary software (named MitoTouch) to sensitively discriminate between control and stressed dermal fibroblasts, and between normal fibroblasts and other cell types (including cancer tissue-derived fibroblasts and primary keratinocytes), showing that our automated analysis captures subtle differences in morphology. Based on this novel algorithm, we report the identification of a protective natural ingredient that mitigates the deleterious impact of hydrogen peroxide (H2O2) on mitochondrial organization. Hence we conceived a novel wet-plus-dry pipeline combining cell cultures, quantitative imaging and semiotic analysis for exhaustive analysis of mitochondrial morphology in living adherent cells. Our tool has potential for broader applications in other research areas such as cell biology and medicine, high-throughput drug screening as well as predictive and environmental toxicology.

Loss of mutant p53 in HaCaT keratinocytes promotes cadmium-induced keratin 17 expression and cell death

BackgroundCadmium exposure induces dermatotoxicity and epidermal barrier disruption and leads to the development of various pathologies. HaCaT cells are immortalized human keratinocytes that are widely used as alternatives to primary human keratinocytes, particularly for evaluating cadmium toxicity. HaCaT cells bear two gain-of-function (GOF) mutations in the TP53 gene, which strongly affect p53 function. Mutant forms of p53 are known to correlate with increased resistance to various stimuli, including exposure to cytotoxic substances. In addition, keratin 17 (KRT17) was recently shown to be highly expressed in HaCaT cells in response to genotoxic stress. Moreover, p53 is a direct transcriptional repressor of KRT17. However, the impact of TP53 mutations in HaCaT cells on the regulation of cell death and keratin 17 expression is unclear. In this study, we aimed to evaluate the impact of p53 on the response to Cd-induced cytotoxicity. Methods and resultsEmploying the MTT assay and Annexin V/propidium iodide staining, we demonstrated that knockout of TP53 leads to a decrease in the sensitivity of HaCaT cells to the cytotoxic effects of cadmium. Specifically, HaCaT cells with TP53 knockout (TP53 KO HaCaT) exhibited cell death at a cadmium concentration of 10 μM or higher, whereas wild-type cells displayed cell death at a concentration of 30 μM. Furthermore, apoptotic cells were consistently detected in TP53 KO HaCaT cells upon exposure to low concentrations of cadmium (10 and 20 μM) but not in wild-type cells. Our findings also indicate that cadmium cytotoxicity is mediated by reactive oxygen species (ROS), which were significantly increased only in TP53 knockout cells treated with 30 μM cadmium. An examination of proteomic data revealed that TP53 knockout in HaCaT cells resulted in the upregulation of proteins involved in the regulation of apoptosis, redox systems, and DNA repair. Moreover, RT‒qPCR and immunoblotting showed that cadmium toxicity leads to dose-dependent induction of keratin 17 in p53-deficient cells but not in wild-type cells. ConclusionsThe connection between mutant p53 in HaCaT keratinocytes and increased resistance to cadmium toxicity was demonstrated for the first time. Proteomic profiling revealed that TP53 knockout in HaCaT cells led to the activation of apoptosis regulatory circuits, redox systems, and DNA repair. In addition, our data support the involvement of keratin 17 in the regulation of DNA repair and cell death. Apparently, the induction of keratin 17 is p53-independent but may be inhibited by mutant p53.

AMPK activation modulates IL-36-induced inflammatory responses by regulating IκBζ expression in the skin.

The interleukin (IL)-36 pathway is a critical player in the pathogenesis of pustular psoriasis. However, therapies targeting this pathway are limited or unaffordable (e.g. the anti-IL-36 receptor antibody). AMP-activated protein kinase (AMPK), a regulator of cellular energy and metabolism, is known to participate in inflammatory diseases. However, its role in IL-36-induced skin inflammation remains unclear. Therefore, we sought to investigate the role of AMPK signals in regulating IL-36-induced responses in the skin. IL-36-stimulated primary normal human epidermal keratinocytes (NHEKs) and IL-36-injected (intradermally) BALB/c mice served as the cell and animal models, respectively. Additionally, 5-aminoimidazole-4-carboxamide riboside (AICAR) and A769662 served as AMPK activators. AICAR and A769662 significantly suppressed the IL-36-induced IL-8 (CXCL8) and CCL20 production from NHEKs. IL-36-induced IκBζ protein expression was prominently reduced and IKK/IκBα phosphorylation was attenuated by AICAR and A769662. Conversely, AMPKα knockdown increased IκBζ protein expression and IKK/IκBα phosphorylation in IL-36-treated NHEKs. Furthermore, AICAR and A769662 enhanced IL-36-induced-IκBζ protein degradation via the proteasome-dependent but not the lysosome-dependent pathway. Pretreatment of NHEKs with IL-36 slightly suppressed the AICAR- and A769662-triggered phosphorylation of AMPK and acetyl-CoA carboxylase. In the mouse model, topical application of AICAR significantly reduced ear swelling, redness, epidermal thickening, neutrophil infiltration and inflammatory and antimicrobial peptide gene expression. AMPK activation suppresses IL-36-induced IL-8 and CCL20 release by regulating IκBζ expression in keratinocytes and reduces IL-36-induced skin inflammation in mice, suggesting that AMPK activation is a potential strategy for treating patients with IL-36-mediated inflammatory skin disorders.

Abstract 5899: Evaluation of Up284 as a selective proteasome inhibitor for prostate cancer therapy

Abstract Recent research into prostate cancer treatment has unveiled a promising contender known as Up284. In laboratory experiments, this compound has exhibited a remarkable preference for targeting prostate cancer cells as opposed to normal cells. Specifically, in vitro cell viability assays using MTT have shown that prostate cancer cells display high sensitivity to Up284 at low nanomolar IC50 values, while primary human prostate epithelial cells and mouse keratinocytes exhibit minimal sensitivity. This establishes a notable therapeutic window between cancerous and non-cancerous cells, with Up284 significantly inhibiting colony formation in PC3 cells. Additional functional assays have confirmed Up284's potential, including the 4UbFL Reporter Gene assay, which has revealed robust, dose-dependent proteasome inhibition. This effect is supported by the accumulation of polyubiquitinated proteins in various prostate cancer cell lines following Up284 treatment, suggesting a distinct mechanism of toxicity compared to bortezomib. Furthermore, Up284 has been found to induce apoptosis in PC3 cells, surpassing the efficacy of bortezomib. Competitive binding assays have shown that Up284 exhibits a stronger binding affinity to the proteasomal receptor RPN13 than the prototype inhibitor RA190B. This is further supported by a Cellular Thermo Stabilization Assay (CETSA), which indicates that Up284 stabilizes RPN13 in cells. A preliminary in vivo study utilizing the bone metastatic prostate cancer genetically engineered mouse model(BMPC GEMM) model for metastatic prostate adenocarcinoma has demonstrated that Up284 leads to significant tumor regression and increased levels of poly-ubiquitinated proteins in residual tumor tissues. Moreover, when Up284 is conjugated with an E3 ligase ligand, it successfully degrades RPN13, resulting in the death of PC3 cells. Up284 has also shown favorable pharmacokinetics/pharmacodynamics (PK/PD) profiles and safety in animal models. It is well-tolerated in mice and holds promise for oral administration, demonstrating a proteasome-dependent reporter protein stabilization effect superior to ixazomib. Importantly, Up284 has induced tumor regression and improved survival rates in various cancer models, including ovarian and breast cancers. In conclusion, Up284 emerges as a lead candidate for targeted prostate cancer therapy due to its high specificity, substantial therapeutic index, and potent anti-cancer activity both in vitro and in vivo. Further development and clinical investigation of Up284 are warranted to fully explore its therapeutic potential. Citation Format: Balasubramanyam Karanam, Ravi Anchoori, Yung Chang. Evaluation of Up284 as a selective proteasome inhibitor for prostate cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5899.

Abstract 7070: MEK-ERK signaling maintains epidermal proliferation through H2A.Z deposition

Abstract Recent studies have connected dysregulation of H2A.Z deposition to glioblastoma as well as lung, pancreatic, and metastatic breast cancers. The H2AZ1 and H2AZ2 isoforms, collectively known as H2A.Z, are histone variants that regulate gene expression, stem cell identity, and differentiation during development and in homeostatic tissue. Most tumors originate from epithelial tissues. We utilize skin epidermis, an accessible model of epithelial tissue, to understand the regulatory mechanisms controlling proliferation and differentiation. Primary human epidermal keratinocytes retain their abilities to proliferate, differentiate, and stratify in vitro. First, we observed that progenitor keratinocytes express high levels of H2A.Z mRNA and protein, and that H2A.Z levels significantly and progressively decrease during four days of calcium-induced differentiation. Using ChIP-sequencing, we find a significant, genome-wide decrease in H2A.Z chromatin occupancy in differentiated keratinocytes. Therefore, we hypothesized that H2A.Z is critical for progenitor function. Knock down of either H2AZ1 or H2AZ2 significantly impaired keratinocyte proliferation in holoclone assays. Furthermore, H2AZ1 or H2AZ2 knockdown inhibited epidermal stratification in organotypic culture. Canonically, two chromatin remodeling complexes, SRCAP and EP400, deposit H2A.Z into nucleosomes. While both SRCAP and EP400 knockdown diminish keratinocyte proliferation, only SRCAP knockdown decreases H2A.Z deposition in epidermal keratinocytes. Finally, we find that MEK and ERK signaling is required to sustain expression of SRCAP, H2AZ1, and H2AZ2. MEK or ERK inhibition is sufficient to significantly decrease H2A.Z occupancy genome-wide in progenitor keratinocytes. Citation Format: Stephenie Droll, Benny Zhang, Celia Xue, Maxwell Levine, Xiaomin Bao. MEK-ERK signaling maintains epidermal proliferation through H2A.Z deposition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7070.

Abstract 761: The relationship between ANGPTL4 and epidermal malignant transformation

Abstract High body mass index (BMI) is a risk factor for several types of cancers. Although epidemiology suggests obesity is negatively associated with non-melanoma skin cancer (NMSC), bariatric surgery has been shown to reduce the risk, suggesting a possible role of adipose tissue in the initiation and promotion of skin cancer. To gain mechanistic insight into how adipose tissue drives cutaneous carcinogenesis, we cultured non-tumorigenic mouse keratinocytes (JB6 P+ cells) with or without factors released from murine visceral adipose tissue (mFTF) and performed RNA-Sequencing. Published work demonstrated mFTF induces anchorage-independent cell growth, a surrogate marker for malignant transformation. Transcriptomic analysis demonstrated a significant induction of Angptl4 mRNA in JB6 P+ cells cultured with mFTF for 3 and 8 hours compared to the vehicle-treated cells. ANGPTL4 is an endogenous inhibitor of lipoprotein lipase that modulates free fatty acid delivery to adipose tissue and oxidative tissues such as muscle and liver. The C-terminal protein demonstrates tumor-associated activities such as angiogenesis, metastasis, protection against anoikis, and enhancement of cell survival. Therefore, we hypothesized that the induction of this gene promotes malignant transformation. RNA-seq data was validated in multiple cell models, including primary keratinocytes with human adipose tissue. Immunohistochemistry was used to assess ANGPTL4 expression in 37 actinic keratosis (pre-malignant) and cutaneous squamous cell carcinoma (cSCC) from patients with different BMIs (18.6 - 49.5). Notably, ANGPTL4 was only observed in the suprabasal epidermal layers in normal skin. For samples containing both lesional and perilesional skin, there was an increase in ANGPTL4 expression in the lesional epidermis relative to the normal perilesional skin. Furthermore, ANGPTL4 expression increased from transition to actinic keratosis to cSCC in situ, suggesting that ANGTL4 is induced during malignant transformation as observed in our pre-clinical models. Interestingly, once cSCC developed an invasive component, ANGTPL4 expression was lost in the invasive keratinocytes but persisted in the in situ component. There was no association between ANGPTL4 expression and BMI in lesional tissue. These data suggest that targeting ANTPLT4 may be a novel therapy for the prevention of skin cancer and that ANGPTL4-high patients or cancer patients with obesity may be a critical demographic for potential risk assessment or therapeutic intervention. Future studies will determine the impact of knocking out ANGTPL4 in our models of malignant transformation. Citation Format: Nat Ato Yawson, Jonathan D. Diedrich, Karen T. Liby, Jesse Veenstra, Jamie Jenna Bernard. The relationship between ANGPTL4 and epidermal malignant transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 761.

3D Approaches to Culturing Bovine Skin: Explant Culture versus Organotypic Skin Model

Introduction: Digital dermatitis (DD) in cattle appears with high prevalence; nevertheless, the knowledge on its pathogenesis is still limited. In this context, in vitro skin models represent a valuable tool to facilitate the study of DD. Methods: Two in vitro skin models were established using bovine distal limb skin: a skin explant model and an organotypic skin model. For the skin explant model, skin samples were cultured with an air-liquid interface for up to 7 days. Besides routine histopathological examination, readout parameters were Ki-67 and cleaved Caspase-3 stainings. For the organotypic model, primary keratinocytes were layered on top of a dermal equivalent containing mainly mitotically inactive fibroblasts and maintained for up to 21 days. At regular intervals (days 7, 14, and 21), cultured skin samples were taken for (immuno)histological analysis. Results: Both cultures could be maintained for the entire duration of the intended culture period. In the histopathological assessment, explant skin cultures showed ballooning degeneration of keratinocytes and segmental necrosis starting at day 5 of culturing. Initially, basal keratinocytes in the organotypic model differentiated as demonstrated by positive Keratin 14, Desmoglein-1, Loricrin, and Involucrin immunofluorescent stainings. Ki-67 was observed occasionally and suprabasally still after 21 days of culture. Conclusion: Both in vitro models proved dependable and constitute a viable option for replacing experiments on live animals, each with its own benefits. Whereas skin explants include all cell types available in vivo and can therefore reflect realistic cell-cell interactions and signaling pathways, the organotypic model offers a higher standardization and reproducibility. Depending on the focus of future studies, both models can be used for specific experimental purposes of bovine dermatological research in general or specialized questions concerning (infectious) claw diseases as, e.g., DD.

Differentiation of keratinocytes or exposure to type 2 cytokines diminishes S. aureus internalization.

Staphylococcus aureus is a leading cause of skin and soft tissue infections. Colonization by this bacterium is increased in individuals with chronic cutaneous diseases such as atopic dermatitis, psoriasis, and bullous pemphigoid. The greater abundance of S. aureus on the skin of subjects with atopic dermatitis in particular has been linked to recurrent cutaneous infections. The primary cell type of the epidermal layer of the skin is the keratinocyte, and it is thought that S. aureus internalized in keratinocytes associates with an increased incidence of skin infections. This study addresses whether keratinocyte differentiation and/or inflammation, two important characteristics altered in cutaneous diseases, influence bacterial internalization. To do this, S. aureus internalization was measured in immortalized and primary keratinocytes that were differentiated using high Ca2+-containing media and/or exposed to cytokines characteristic of atopic dermatitis (IL-4 and IL-13) or psoriasis (IL-17A and IL-22) skin. Our results indicate that S. aureus internalization is uniquely decreased upon keratinocyte differentiation, since this was not observed with another skin-resident bacterium, S. epidermidis. Additionally, treatment with IL-4 + IL-13 diminished bacterial internalization. We interpret this decrease as a mechanism of keratinocyte-based bacterial killing since a similar number of bacterial genomes were detected in cytokine-treated cells, but less viable internalized S. aureus was recovered. Finally, of the receptors reported for S. aureus binding/internalizing into keratinocytes, expression of the α5 component of the α5β1 integrin was in greatest accordance with the number of internalized bacteria in the context of keratinocyte differentiation.IMPORTANCEIndividuals with chronic cutaneous diseases demonstrate heightened susceptibility for severe and recurrent infections from Staphylococcus aureus. What drives this altered susceptibility remains poorly understood. Previous publications have detected S. aureus as deep as the dermal layer of skin in subjects with atopic dermatitis, suggesting that the cutaneous environment of this disease enables deeper bacterial infiltration than occurs in healthy individuals. This observation indicates that S. aureus has greater opportunity to interact with multiple skin cell types in individuals with chronic inflammatory skin diseases. Identifying the characteristics of the skin that influence bacterial internalization, a common method to establish reservoirs and evade the immune response, is critical for our understanding of S. aureus pathogenesis. The significance of this research is the novel identification of epidermal characteristics that influence S. aureus internalization. With this knowledge, methods can be developed to identify patient populations at greater risk for cutaneous infections.

MicroRNA-155 mediates multiple gene regulations pertinent to the role of human adipose-derived mesenchymal stem cells in skin regeneration.

Introduction: The role of Adipose-derived mesenchymal stem cells (AD-MSCs) in skin wound healing remains to be fully characterized. This study aims to evaluate the regenerative potential of autologous AD-MSCs in a non-healing porcine wound model, in addition to elucidate key miRNA-mediated epigenetic regulations that underlie the regenerative potential of AD-MSCs in wounds. Methods: The regenerative potential of autologous AD-MSCs was evaluated in porcine model using histopathology and spatial frequency domain imaging. Then, the correlations between miRNAs and proteins of AD-MSCs were evaluated using an integration analysis in primary human AD-MSCs in comparison to primary human keratinocytes. Transfection study of AD-MSCs was conducted to validate the bioinformatics data. Results: Autologous porcine AD-MSCs improved wound epithelialization and skin properties in comparison to control wounds. We identified 26 proteins upregulated in human AD-MSCs, including growth and angiogenic factors, chemokines and inflammatory cytokines. Pathway enrichment analysis highlighted cell signalling-associated pathways and immunomodulatory pathways. miRNA-target modelling revealed regulations related to genes encoding for 16 upregulated proteins. miR-155-5p was predicted to regulate Fibroblast growth factor 2 and 7, C-C motif chemokine ligand 2 and Vascular cell adhesion molecule 1. Transfecting human AD-MSCs cell line with anti-miR-155 showed transient gene silencing of the four proteins at 24h post-transfection. Discussion: This study proposes a positive miR-155-mediated gene regulation of key factors involved in wound healing. The study represents a promising approach for miRNA-based and cell-free regenerative treatment for difficult-to-heal wounds. The therapeutic potential of miR-155 and its identified targets should be further explored in-vivo.

Gene-edited cells: novel allogeneic gene/cell therapy for epidermolysis bullosa.

Epidermolysis bullosa (EB) is a group of rare genetic skin fragility disorders, which are hereditary. These disorders are associated with mutations in at least 16 genes that encode components of the epidermal adhesion complex. Currently, there are no effective treatments for this disorder. All current treatment approaches focus on topical treatments to prevent complications and infections. In recent years, significant progress has been achieved in the treatment of the severe genetic skin blistering condition known as EB through preclinical and clinical advancements. Promising developments have emerged in the areas of protein and cell therapies, such as allogeneic stem cell transplantation; in addition, RNA-based therapies and gene therapy approaches have also become a reality. Stem cells obtained from embryonic or adult tissues, including the skin, are undifferentiated cells with the ability to generate, maintain, and replace fully developed cells and tissues. Recent advancements in preclinical and clinical research have significantly enhanced stem cell therapy, presenting a promising treatment option for various diseases that are not effectively addressed by current medical treatments. Different types of stem cells such as primarily hematopoietic and mesenchymal, obtained from the patient or from a donor, have been utilized to treat severe forms of diseases, each with some beneficial effects. In addition, extensive research has shown that gene transfer methods targeting allogeneic and autologous epidermal stem cells to replace or correct the defective gene are promising. These methods can regenerate and restore the adhesion of primary keratinocytes in EB patients. The long-term treatment of skin lesions in a small number of patients has shown promising results through the transplantation of skin grafts produced from gene-corrected autologous epidermal stem cells. This article attempts to summarize the current situation, potential development prospects, and some of the challenges related to the cell therapy approach for EB treatment.

On- and off-target effects of paired CRISPR-Cas nickase in primary human cells

Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double-nickase-based strategies to generate staggered DNA double-strand breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-seq pipeline, dual CAST, to characterize chromosomal aberrations induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from patients with epidermolysis bullosa. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following paired-nickase editing. While the double-nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases, the chromosomal on-target aberrations were qualitatively different and included a high proportion of insertions. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects but still leave substantial chromosomal aberrations at on-target sites.

The POU-HD TFs impede the replication efficiency of several human papillomavirus genomes

Human papillomavirus (HPV) is a double-stranded DNA virus that infects cutaneous and mucosal epithelial cells. HPV replication initiates at the origin (ori), located within a noncoding region near the major early promoter. Only two viral proteins, E1 and E2, are essential for replication, with the host cell contributing other necessary factors. However, the role of host cell proteins in regulating HPV replication remains poorly understood. While several binding sites for cellular transcription factors (TFs), such as POU-HD proteins, have been mapped in the regulatory region, their functional importance is unclear. Some POU-HD TFs have been shown to influence replication in a system where E1 and E2 are provided exogenously. In this study, we investigated the impact of several POU-HD TFs on the replication of the HPV5, HPV11, and HPV18 genomes in U2OS cells and human primary keratinocytes. We demonstrated that OCT1, OCT6, BRN5A, and SKN1A are expressed in HPV host cells and that their overexpression inhibits HPV genome replication, whereas knocking down OCT1 had a positive effect. Using the replication-deficient HPV18-E1− genome, we demonstrated that OCT1-mediated inhibition of HPV replication involves modulation of HPV early promoters controlling E1 and E2 expression. Moreover, using Oct6 mutants deficient either in DNA binding or transcriptional regulation, we showed that the inhibition of HPV18 replication is solely dependent on Oct6’s DNA binding activity. Our study highlights the complex regulatory roles of POU-HD factors in the HPV replication.