Filaggrin Research Articles

A modified protocol for studying filaggrin degradation using a reconstructed human epidermis model under low and high humidity.

Filaggrin (FLG) is an essential protein that plays a vital role in maintaining skin barrier function and moisture levels, allowing the skin to adapt to dry environments. However, the precise temporal dynamics of FLG metabolism in the human epidermis remain poorly understood, and suitable tools to study these time-dependent effects are currently lacking. To investigate the molecular mechanisms and time course of FLG metabolism and skin barrier function under high- and low-humidity conditions, utilizing a reconstructed epidermis model. EpiSkin specimens cultured under humid or dry conditions for varying durations (2-48 h) were compared by assessing FLG degradation and skin barrier formation using immunofluorescence staining and western blotting. Under conditions of low humidity, the proteolysis of FLG in EpiSkin increased between 4 and 12 h and was accompanied by elevated levels of cysteine-aspartic protease (caspase)-14. The expression of peptidyl arginine deiminase 1 and calpain 1 also increased at 4 h. However, after 24 h, the expression of these three FLG-degrading proteins significantly decreased. Conversely, the levels of pyrrolidone-5-carboxylic acid and urocanic acid initially decreased at 2 h and then increased between 12 and 24 h. Additionally, the expression of skin barrier proteins, such as FLG, transglutaminase 5, loricrin and zonula occludens-1, decreased starting from 12 h. Notably, epidermal cell viability and activity were also inhibited. We propose a reliable and ethical model to study the temporal dynamics of FLG metabolism and its role in skin barrier function. Using a commercially reconstructed epidermis to mimic dry skin formation obviates the need for animal and human testing.

Expression of hornerin in skin lesions of atopic dermatitis and skin diseases.

We have previously identified the filaggrin (FLG)-like protein, hornerin (HRNR). Recently, there have been several reports regarding the relationship between HRNR and atopic dermatitis (AD). In the present study, we examined HRNR expression in the skin lesions of seven unrelated patients with AD to clarify the role of HRNR in the pathogenesis of AD. HRNR was detected in chronic AD lesions (n = 4), whereas no HRNR signals were observed in acute AD lesions (n = 3). HRNR was detected in the cytokeratin 6-expressing epidermis, and Ki67-positive keratinocytes were more abundant in the HRNR-positive epidermis. These findings suggest that HRNR may be associated with epidermal hyperproliferation in AD lesions. Next, we examined HRNR expression in skin diseases associated with hyperkeratosis. HRNR signals were irregularly observed in different cells from those expressing FLG in epidermolytic ichthyosis and actinic keratosis. Therefore, HRNR may play a unique role in the molecular process of cornification.

Novel roles of bovine milk-derived exosomes in skin antiaging.

Exosomes are small vesicles released from cells and are found in various mammalian biological fluids, such as bovine milk, which has been employed in skincare for many years, apart from its dairy applications. In addition, exosomes have been recognized as vehicles for intercellular communication. In this study, we aimed to investigate the novel effects of bovine milk-derived exosomes (MK-Exo) on antiaging in human skin. Initially, MK-Exo were co-cultured with keratinocytes and fibroblasts; subsequent analysis involved qPCR and western blotting to assess induced gene expression. Subsequently, MK-Exo were topically applied to the facial skin of 31 female volunteers twice daily for 28 days. The functions were evaluated after conducting safety assessments invivo. Purified MK-Exo demonstrated the ability to be taken up directly by keratinocytes and fibroblasts invitro, resulting in the upregulation of natural factors associated with skin moisturization, including filaggrin (FLG), aquaporin 3 (AQP3), and CD44 in keratinocytes, as well as hyaluronidase (HAS2) in fibroblasts. Concurrently, MK-Exo promoted fibroblast cell migration and restored the expression of type I and III collagen (Col I and Col III) following exposure to ultraviolet radiation. Furthermore, phototoxicity, photoallergy, repeated skin irritation, skin allergy, and patch tests confirmed the safety of MK-Exo for skin application. Finally, we elucidated the roles of MK-Exo in preserving moisture and reducing wrinkles in humans. Our findings unveil the novel contributions of MK-Exo to human skin aging, presenting a new avenue in the field of skincare.

Генетические факторы риска развития профессиональных контактных дерматитов

Occupational contact dermatitis is an important current occupational health problem with serious economic and social consequences. Among possible risk factors for this disease, researchers pay attention to genetic predisposition. Identification of polymorphisms associated with the occupational pathology will allow specialists to establish risk groups, carry out timely preventive measures, and adjust treatment, guided by a personalized medicine approach. The purpose of this review was to summarize the results of studying genetic risk factors for occupational contact dermatitis. Three researchers did an independent search in the PubMed, Google Scholar, eLibrary, and CyberLeninka databases and further analysis of scientific literature on genetic predisposition to occupational dermatitis published in 1990 to 2023. Of 88 papers analyzed, 32 articles were included in this review. We established that genetic risk factors for occupational contact dermatitis were usually studied in metallurgical workers with a focus on potential candidate genes among skin barrier function-related genes, pro-inflammatory and anti-inflammatory genes, and xenobiotic metabolism and biotransformation genes. The most compelling evidence for the use of genetic polymorphisms as risk factors for occupational contact dermatitis has been demonstrated for the filaggrin (FLG) gene, which is involved in maintaining the skin barrier, and tumor necrosis factor alpha (TNF-α), which is involved in protecting the body and cells from inflammation and apoptosis. However, the data available are insufficient to use genetic polymorphisms as risk factors for occupational skin diseases. Further studies that take into account the mechanism of interaction of different genes during the development of occupational contact dermatitis are required.

Genetic risk factors for occupational contact dermatitis

Occupational contact dermatitis is an important current occupational health problem with serious economic and social consequences. Among possible risk factors for this disease, researchers pay attention to genetic predisposition. Identification of polymorphisms associated with the occupational pathology will allow specialists to establish risk groups, carry out timely preventive measures, and adjust treatment, guided by a personalized medicine approach. The purpose of this review was to summarize the results of studying genetic risk factors for occupational contact dermatitis. Three researchers did an independent search in the PubMed, Google Scholar, eLibrary, and CyberLeninka databases and further analysis of scientific literature on genetic predisposition to occupational dermatitis published in 1990 to 2023. Of 88 papers analyzed, 32 articles were included in this review. We established that genetic risk factors for occupational contact dermatitis were usually studied in metallurgical workers with a focus on potential candidate genes among skin barrier function-related genes, pro-inflammatory and anti-inflammatory genes, and xenobiotic metabolism and biotransformation genes. The most compelling evidence for the use of genetic polymorphisms as risk factors for occupational contact dermatitis has been demonstrated for the filaggrin (FLG) gene, which is involved in maintaining the skin barrier, and tumor necrosis factor alpha (TNF-α), which is involved in protecting the body and cells from inflammation and apoptosis. However, the data available are insufficient to use genetic polymorphisms as risk factors for occupational skin diseases. Further studies that take into account the mechanism of interaction of different genes during the development of occupational contact dermatitis are required.

Helicobacter pylori infection attenuates 2,4-dinitrochlorobenzene-induced atopic dermatitis-like skin lesions in C57/BL6 mice

BackgroundAlthough numerous studies have suggested a negative correlation between Helicobacter pylori (H. pylori) infection and allergies, there has been limited research on the relationship between H. pylori infections and atopic dermatitis (AD). The present study aimed to investigate the effects of H. pylori infection in an AD mouse model and identify potential mechanisms related to type 2 immunity, skin barrier defects, and pruritus.MethodsA model of AD-like symptoms was established with 2,4-dinitrochlorobenzene (DNCB) after infection of the gastric cavity with H. pylori. Analysis of the expression of key inflammatory cytokines and serum levels of immunoglobulin E (IgE) was based on enzyme-linked immunosorbent assay (ELISA). The expression of filaggrin (FLG) and loricrin (LOR) were analyzed by immunohistochemistry staining. The evaluation of STAT1, STAT3, phosphorylated STAT1 (phospho-STAT1), and phosphorylated STAT3 (phospho-STAT1) expression levels in skin lesions was performed using western blot.ResultsThe present study showed that the H. pylori-positive AD group (HP+AD+) exhibited milder skin lesions, including erythema, erosion, swelling, and scaling, than the H. pylori-negative AD group (HP−AD+). Additionally, HP+AD+ displayed lower levels of IgE in serum, and downregulated expression of interleukins 4 and 31 (IL-4 and IL-31) in serum. Furthermore, HP+AD+ demonstrated higher expression of filaggrin and loricrin than HP−AD+. Notably, H. pylori significantly reduced the amount of phosphorylated STAT1 and STAT3.ConclusionHelicobacter pylori infection negatively regulates the inflammatory response by affecting inflammatory factors in the immune response, and repairs the defective epidermal barrier function. In addition, H. pylori infection may reduce IL-31, thereby alleviating pruritus. These effects may be associated with the inhibition of JAK–STAT signaling activation.

Development of monoorganic and polyorganic phenotypes of bronchial asthma in children: the role of combined single-nucleotide variants

Background. Bronchial asthma (BA) in children is on one of the leading places in the morbidity and mortality structure among other allergic and atopic diseases. It can be developed in the form of a monoorganic phenotype (MOPh) or a polyorganic phenotype (POPh) with other nosologies of atopic march (AM): atopic dermatitis (AD) and allergic rhinitis/rhinoconjunctivitis (AR/ARC). This process is genetically determined, with single-nucleotide variants (SNV) of filaggrin (FLG), thymic stromal lymphopoietin (TSLP) and orsomucoid-like protein 3 ­(ORMDL3) genes playing a major role. The purpose of this study was to reveal the impact of rs_7927894 FLG, rs_11466749 TSLP and rs_7216389 ORMDL3 SNV genotype combinations in the deve­lopment of MOPh and POPh of atopic BA in children. Materials and me­thods. One hundred and twenty-one children of the main group and 105 controls took part in the study. The criteria for inclusion into the main group were: age from 3 to 18 years, clinically established and laboratory confirmed diagnoses of MOPh BA, POPh BA + AR/ARC and AD + AR/ARC + BA. The criteria for inclusion in the control group were: age from 3 to 18 years, exclu­ded diagnoses of BA, BA + AR/ARC and AD + AR/ARC + BA. All children underwent swabbing of the oral mucosa and real-time polymerase chain reaction with the obtained material to detect variants of rs_7927894 FLG, rs_11466749 TSLP and rs_7216389 ORMDL3 genotype combinations. The results were processed ­using the following statistical tools: logistic regression analysis with determination of odds ratio (OR) with 95% confidence interval (95% CI), receiver operating characteristic (ROC) analysis with determination of the area under the ROC curve (AUC), sensitivity (Se), specificity (Sp), Pearson’s correlation coefficient (r), Fisher’s ­exact test, Student’s t-test. The significance value was set at p < 0.05, trend to reliability — at p = 0.0–0.1. Results. The structure of the significantly most frequent genotypes in the cohorts of the main group was as follows: C/T rs_7927894 FLG + C/T rs_7216389 ­ORMDL3 — BA = 8.7 %; C/T rs_7927894 FLG + T/T rs_7216389 ORMDL3: BA = 21.7 %, BA + AR/ARC = 18.1 %, AD + AR/ARC + BA = 15.4 %; C/T rs_7927894 FLG + A/A rs_11466749 TSLP: BA + AR/ARC = 31.9 %, AD + AR/ARC + BA = 42.3 %. Next, indicators of the genotypic combinations impact on the risk of BA phenotypes development related to the control group are provided. MOPh BA: C/T rs_7927894 FLG + T/T rs_7216389 ­ORMDL3: r = 0.299, OR = 9.44 (95% CI 2.07–43.03), AUC = 0.594 (0.507–0.682), Se/Sp = 21.7/97.1 % (p < 0.001). POPh BA + AR/ARC: C/T rs_7927894 FLG + A/A rs_11466749 TSLP: r = 0.136, OR = 1.88 (95% CI 0.94–3.74), AUC = 0.560 (0.493–0.626), Se/Sp 31.9/80.0 % (p = 0.071); C/T rs_7927894 FLG + T/T rs_7216389 ORMDL3: r = 0.260, OR = 7.49 (95% CI 2.05–27.37), AUC = 0.576 (0.528–0.624), Se/Sp = 18.1/97.1 % (p < 0.001). POPh AD + AR/ARC + BA: C/T rs_7927894 FLG + A/A rs_11466749 TSLP: r = 0.207, OR = 2.93 (95% CI 1.18–7.31), AUC 0.612 (0.507–0.716), Se/Sp = 42.3/80.0 % (р < 0.05); C/T rs_7927894 FLG + C/T rs_7216389 ORMDL3: r = 0.173, OR = 2.50 (95% CI 0.99–6.30), AUC = 0.592 (0.489–0.695), Se/Sp = 38.5/80.0 % (p < 0.05); C/T rs_7927894 FLG + T/T rs_7216389 ORMDL3: r = 0.222, OR = 6.18 (95% CI 1.29–29.6), AUC = 0.563 (0.490–0.635), Se/Sp = 15.4/97.1 % (p < 0.01). The ratio of associations and risks for developing the phenotypes rela­ted to each other: BA + AR/ARC related to BA: C/T rs_7927894 FLG + C/T rs_7216389 ORMDL3: r = 0.171, OR = 3.50 (95% CI 0.75–16.41), AUC = 0.582 (0.504–0.659), Se/Sp = 25.0/91.3 % (p = 0.095); AD + AR/ARC + BA related to BA: C/T rs_7927894 FLG + C/T rs_7216389 ­ORMDL3: r = 0.345, OR = 6.56 (95% CI 1.26–34.23), AUC = 0.649 (0.537–0.761), Se/Sp = 38.5/91.3 % (р < 0.05); C/T rs_7927894 FLG + A/A rs_11466749 TSLP: r = 0.270, OR = 3.48 (95% CI 0.92–13.17), AUC = 0.625 (0.500–0.750), Se/Sp 42.3/82.6 % (p = 0.059). Conclusions. MOPh BA has a significant association and an increased risk of development with the SNV genotype combination C/T rs_7927894 FLG + T/T rs_7216389 ORMDL3. POPh BA + AR/ARC has significant associations and increased risks of development with the following SNV genotype combinations: C/T rs_7927894 FLG + A/A rs_11466749 TSLP and C/T rs_7927894 FLG + T/T rs_7216389 ORMDL3. POPh AD + AR/ARC + BA has the most associations and increased risks of development within the following SNV genotype combinations: C/T rs_7927894 FLG + A/A rs_11466749 TSLP, C/T rs_7927894 FLG + C/T rs_7216389 ORMDL3, C/T rs_7927894 FLG + T/T rs_7216389 ORMDL3.

Association between skin barrier development and early-onset atopic dermatitis: A longitudinal birth cohort study

A diagnosis of atopic dermatitis (AD) is common during infancy; however, it is unclear whether differential skin barrier development defines this period and signals disease onset in predisposed individuals. We sought to study (NCT03143504) and assess the feasibility of remote skin testing from birth to monitor skin barrier maturation and model association with an AD diagnosis by age 12 months. Biophysical testing and infrared spectroscopy were conducted at the maternity ward and family home. Tape stripping collected samples for desquamatory protease and natural moisturizing factor analysis. The 4 common European filaggrin risk alleles were screened. A total of 128 infants completed the study, with 20% developing mild disease. Significant changes in permeability barrier function, desquamatory protease activity, and molecular composition assessed spectroscopically were observed longitudinally, but only subtle evidence of differential skin barrier development was noted between infant subgroups. Common filaggrin risk alleles were strongly associated with early-onset disease and conferred a significant reduction in natural moisturizing factor and water content by age 4 weeks. Accounting for a family history of atopy, these parameters alongside a greater lipid/protein ratio and reduced chymotrypsin-like activity at birth were associated with AD. Measured in ambient conditions, transepidermal water loss did not signal disease risk at any stage. Skin barrier dysfunction lacked an acquired modality but was considered proportional to cohort severity and suggests that a portfolio of tests used in a community setting has the potential to improve current AD risk evaluations from birth.

Зв’язок розвитку фенотипів бронхіальної астми в дітей з однонуклеотидними варіантами генів філагрину, тимічного стромального лімфопоетину та орсомукоїд-1-подібного білка 3

Bronchial asthma (BA) composes the most severe form of atopic march (AM) in children, which also includes atopic dermatitis (AD),allergic rhinitis and rhino-conjunctivitis (AR/ARC). AM can clinically manifest as mono-organ phenotypes of AD, AR/AR and BA or poly-organ phenotypes of AD+AR/ARC, BA+AR/ARC, AD+AR/ARC+BA. Single nucleotide variants (SNVs) of genes: rs_7927894 of filaggrin (FLG), rs_11466749 of thymic stromal lymphopoietin (TSLP) and rs_7216389 of orsomucoid-1-like protein 3 (ORMDL3) are important factors in the development of AM in children. The aim was to study the role of different variants of rs_7927894 FLG, rs_11466749 TSLP and rs_7216389 ORMDL3 genotypes in the risks of AM - BA, BA+AR/ARC and AD+AR/ARC+BA phenotypes` development. Materials and methods. 121 children of the main group and 105 of the control group aged from 3 to 18 years were involved in the study. Cohorts of the main group: BA - 23 children, BA+AR/ARC - 72 children, AD+AR/ARC+BA - 26 children. The control group consisted of 105 children without AM nosoloies. All children were genotyped by polymerase chain reaction in real time for SNVs C/T, C/C T/T rs_7927894 FLG, A/A, A/G, G/G rs_11466749 TSLP and C/C, C/T, T/T rs_7216389 ORMDL3. For the statistical processing of the obtained results, the methods of variational statistics were applied with the statistical significance threshold at the value of p<0.05, trend to significance - p=0.05-0.1. Results. AD+AR/ARC+BA correlated to BA+AR/ARC: A/G rs_11466749 - reduces the risk, r=0.204, odds ratio (OR)=0.28 (0.08-1.0, p<0.05); T/T rs_7216389 - reduces the risk, r=0.172, OR=0.40 (0.13-1.17, p=0.09). AD+AR/ARC+BA correlated to BA: A/A rs_11466749 – elevates the risk, r=0.259, OR=3.06 (0.9-10.4, p=0.07), A/G rs_11466749 - reduces the risk, r=0.320, OR=0.20 (0.05-0.88, p<0.05). Conclusions. SNV C/T rs_7927894 FLG, A/A rs_11466749 TSLP and T/T rs_7216389 ORMDL3 are associated and elevate the risk of BA+AR/ARC phenotype by 1.87, 1.84 and 3.34 fold, and C/C rs_7216389 ORMDL3 and A/G rs_11466749 TSLP reduce it to 0.42 and 0.56 times. SNV C/T rs_7927894 FLG and A/A rs_11466749 TSLP are associated and increase the risk of developing the full AM AD+AR/ARC+BA phenotype by 3.34 and 3.27fold, and C/C rs_7927894 FLG and A/G rs_11466749 TSLP – are associated and reduce it by 0.45 and 0.15 fold. SNV A/A rs_11466749 TSLP is associated with an elevated risk of developing AD+AR/ARC+BA correlated to BA up to 3.06 fold, and SNV A/G rs_11466749 TSLP is associated and reduces it by 0.20 fold. SNV A/G rs_11466749 TSLP and T/T rs_7216389 ORMDL3 are associated and reduce the risk of AD+AR/ARC+BA phenotype development correlated to BA+AR/ARC by 0.28 and 0.40 fold, respectively. The research was carried out in accordance with the principles of the Helsinki Declaration. The study protocol was approved by the Local Ethics Committee of the institution mentioned in the work. The informed consent of the parents and the children was obtained for the conducting the study. No conflict of interests was declared by the author.

Multifunctional Biological Properties and Topical Film Forming Spray Base on Auricularia polytricha as a Natural Polysaccharide Containing Brown Agaricus bisporus Extract for Skin Hydration

Mushrooms are edible fungi containing valuable nutrients. They provide attractive bio-active properties, which have confirmed anti-oxidants, anti-aging, and anti-inflammatory properties. Mushrooms possess abundant natural polymers affecting skin hydration and acting as moisturizers supporting skin barrier function. In this study, cloud ear mushroom (Auricularia polytricha) water extract (CW) was produced as a natural polymer to evaluate a new film-forming spray (FFS) containing CW to increase skin hydration and protect transepidermal water loss. CW contained polysaccharides as 748.2 ± 0.02 mg glucose/g extract. CW significantly inhibited the secretion of IL-6 and TNF-α and enhanced skin hydration by increasing aquaporin-3 (AQP3) and filaggrin (FLG) in HaCaT cells. The FFS was formulated using CW, sodium polystyrene sulfonate, and glycerin. The selected formulation contained brown Agaricus bisporus (BE-FFS) evaluated physical appearance, spray angle, spray pattern, and in vitro skin permeation. The BE-FFS has a transparent thin film with suitable occlusive properties, drying time, and physical appearance. Afterward, in vitro skin permeation and human hydration property studies presented the long-lasting effects and provided safety and hydration potential after 4 weeks of use. Overall, all results indicate that the BE-FFS is a natural film-forming spray for skin hydration improvement.

Dendrobium officinale Extract Fermented with Lactobacillus plantarum GT-17F Enhances the Protection of UV-Mediated Photoaging.

Long-term and extensive exposure to UV irradiation can cause sunburn, photoaging, or skin cancer. Various studies have shown that Dendrobium officinale extract has a certain protective effect on skin-related diseases. Lactobacillus plantarum is a probiotic that has been reported to be used for co-fermentation with various plants to enhance the activity of extracts. This article discusses the effectiveness of fermentation of Dendrobium officinale extract with Lactobacillus plantarum GT-17F on protection against UV-mediated photoaging. The study found that fermented extract of Dendrobium officinale (FDO) has a stronger antioxidant effect, especially in free radical scavenging. Pretreatment with FDO enables human skin fibroblast (HSF) cells and reconstruction skin models (EpiSkin and T-Skin) to resist UV-mediated degradation of type I collagen and type III collagen, repair epidermal barrier function, and reduce the damage of barrier-related proteins, such as filaggrin (FLG) and loricrin (LOR). Those findings provide a basis for further studies to evaluate the effectiveness of fermented Dendrobium officinale in preventing UV-mediated damage and photoaging in humans.

The Protective Effects of Moisturizer Containing Potentilla anserina Extract in the Topical Treatment of Skin Damage Caused by Masks.

The use of face masks during the COVID-19 pandemic resulted in significant societal changes, particularly for individuals with sensitive skin. To address this issue, the researchers explored traditional medicine and identified Potentilla anserina extract as a potential solution due to its anti-inflammatory and moisturizing effects. This research investigated how this extract influences skin hydration, barrier function, and itching. The findings revealed that the extract had a hydrating effect by elevating Aquaporin-3 (AQP3) expression. Additionally, the study demonstrated that the extract improved skin barrier function, with Filaggrin (FLG) expression being approximately three times higher (p < 0.001) in the Potentilla-anserina-extract-treated group compared to the control group and the genes associated with itching being reduced. In this process, we researched and developed HPβCD (hydroxypropyl-β-cyclodextrin)-Liposome containing Potentilla anserina extract, gradually and sustainably releasing the active components of the Potentilla anserina extract. During four weeks of clinical trials involving individuals wearing masks for over 6 h a day, a moisturizer containing Potentilla anserina extract demonstrated a notable reduction in skin redness. Hemoglobin values (A.U.), which serve as indicators of skin redness, showed decreases of 5.06% and 6.74% in the test area inside the mask after 2 and 4 weeks, respectively, compared to the baseline measurements. Additionally, the moisturizer containing Potentilla anserina extract notably decreased Trans Epidermal Water Loss (TEWL), with reductions of 5.23% and 9.13% observed in the test area inside the mask after 2 and 4 weeks, respectively. The moisturizer, especially in the test area treated with the extract-containing moisturizer, significantly enhanced skin hydration compared to the control group. The Corneometer values (A.U) exhibited notable increases of 11.51% and 15.14% in the test area inside the mask after 2 and 4 weeks, respectively. These discoveries emphasize the potential of Potentilla anserina extract and its utility in tackling skin issues caused by mask wearing, including enhancing moisture, fortifying the skin's barrier, and alleviating itching. These results indicate that moisturizers incorporating specific ingredients provide greater benefits compared to conventional moisturizers.

Anti-aging Effect of Allium pseudojaponicum in UVA-irradiated Human Epidermal Keratinocytes.

This study was conducted to investigate the anti-aging effects of Allium pseudojaponicum extract on normal human epidermal keratinocytes (NHEKs). The effects were examined by analyzing gene expressions related to skin hydration using quantitative real-time polymerase chain reaction (qRT-PCR), hyaluronic acid (HA) production using HA-ELISA, cell viability using a cell viability assay, and a phospho-kinase array. Allium pseudojaponicum extract increased the gene expression levels of AQP3/HAS2 and HA protein production in NHEKs while decreasing the overexpressed mRNA levels of KRT1, 10 and FLG genes, known as differentiated keratinocyte markers in NHEKs. Additionally, A. pseudojaponicum extract reduced the phosphorylation of CHK2 and p53 proteins, which are related to cell cycle or epidermal differentiation. This study demonstrated the anti-aging effects of A. pseudojaponicum extract, which could potentially be used as a functional ingredient for skin hydration and anti-aging products.

Bacillus subtilis Fermentation Augments the Anti-Inflammatory and Skin Moisture Improvement Activities of Tetragonia tetragonoides through the Upregulation of Antioxidant Components

This study demonstrates that the fermentation of Tetragonia tetragonioides (T. tetragonioides) by Bacillus subtilis (B. subtilis) subsp. spizizenii enhances its antioxidant, anti-inflammatory, and skin-moisturizing activities. Fermented T. tetragonioides extracts (FTEs) showed a significant increase (p &lt; 0.05) of approximately 1.3 to 3.07 times in their total polyphenol content (TPC), total flavonoid content (TFC), and vanillic acid content compared to pre-fermentation T. tetragonioides extracts (TEs). Additionally, the 2,2-diphenyl-1-picrylhydrazyl (DPPH)- and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical-scavenging activities were significantly higher (p &lt; 0.05) in FTEs than in TEs, showing an increase of about 1.25 to 1.64 times. The anti-inflammatory effects, evaluated through the inhibition of nitric oxide (NO) in RAW 264.7 cells stimulated by lipopolysaccharide (LPS), revealed that FTEs exhibited significant (p &lt; 0.05) NO inhibition activity at less than half the concentration of TEs. It is particularly noteworthy that the FTE at 200 μg/mL significantly suppressed the expression of the cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and tumor necrosis factor-alpha (TNF-α) proteins. In HaCaT cells, FTEs substantially (p &lt; 0.001) increased the mRNA expression of filaggrin (FLG), hyaluronan synthase (HAS)-1, and HAS-3, indicating improved skin protection and moisturization. In conclusion, this study confirms that T. tetragonioides’ antioxidant, anti-inflammatory, and skin-moisturizing activities are enhanced by B. subtilis fermentation, suggesting the potential of FTEs as a cosmeceutical ingredient.

Identification of potential hub genes associated with atopic dermatitis-like recombinant human epidermal model using integrated transcriptomic and proteomic analysis.

Atopic dermatitis (AD) is a severe inflammatory skin disorder, characterized by elevated levels of proinflammatory cytokines that fuel a vicious cycle of inflammation. While inflammatory recombinant human epidermal (RHE) models relevant to AD have been established, comprehensive understanding remains limited. To illuminate changes and identify potential hub genes involved in AD-related inflammation, RHE models, stimulated by an inflammatory cocktail including polyinosinic-polycytidylic acid, tumor necrosis factor alpha (TNF-α), interleukin 4 (IL-4) and interleukin 13 (IL-13), were constructed and examined using tandem mass tags-proteomic coupled with RNA-seq transcriptomic analyses. Principal component analysis (PCA), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway functional enrichment were employed for the analysis of related genes and proteins. Protein-protein interaction networks helped identify hub genes, which were further confirmed by qPCR and western blot. We observed high expression of thymic stromal lymphopoietin in the inflammatory RHE. Our study identified 2369 differentially expressed genes and 880 differentially expressed proteins in the cocktail-induced group versus the normal control group. A total of 248 overlapping symbols were enriched in various biological processes and signaling pathways, including cornification envelope, cell-cell junction, calcium ion binding, extracellular matrix receptor, terpenoid backbone biosynthesis, and peroxisome proliferator-activated receptors signaling pathway, among others. Among the 248 overlapping symbols, CytoHubba identified 10 hub molecules, namely signal transducer and activator of transcription 3 (STAT3), integrin subunit beta 1 (ITGB1), filaggrin (FLG), involucrin (IVL), DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 (DDX58), small proline rich protein 1B (SPRR1B), interferon induced with helicase C domain 1 (IFIH1), desmoglein 1 (DSG1), collagen type XVII alpha 1 chain (COL17A1), and integrin subunit alpha 6 (ITGA6), based on the degree. These integrated results offer valuable insights into the molecular mechanisms of AD and present potential tools for screening cosmetic formulations intended for the treatment of AD.

P102 Severe atopic eczema is associated with reduced acne vulgaris in Bangladeshi children and young adults: a retrospective analysis from a cross-sectional study

Abstract Atopic eczema (AE) is the most common inflammatory dermatosis, affecting up to 20% of children. Loss of function (LoF) mutations in FLG are the most strongly implicated genetic risk factor for eczema. To understand the effect of FLG on the pathobiology of AE, we knocked down FLG in primary keratinocytes using short hairpin RNA and performed phosphoproteomic analysis, which showed downregulation of the AKT signalling pathway, confirmed using Western blotting to phospho-AKT and immunostaining of biopsies from patients with AE. Given that AKT signalling is increased in acne vulgaris, we hypothesized that loss of FLG may protect patients with AE from acne. The Tower Hamlets Eczema Assessment (THEA) study is a cross-sectional study that aims to phenotype and genotype 1000 Bangladeshi children and young adults with AE. So far, 620 participants have undergone genotyping, 37% of whom have LoF mutations in FLG. We selected patients from this cohort who were older than 12 years of age. In total, 204 patients (99 FLG wild type, 86 heterozygotes, 19 compound heterozygotes) were included. We identified a diagnosis of acne or previous prescription of topical acne therapies or tetracycline antibiotics from the patients’ general practice e-health records. We stratified the patients by genotype, Eczema Area and Severity Index (EASI) score, Patient Oriented Eczema Measure (POEM) score, transepidermal water loss (TEWL) and palmar hyperlinearity groups. The χ2-test was used to determine differences between groups. A lower proportion of patients with AE with FLG mutations had acne (27% wild type vs. 19% FLG mutations), which was more pronounced, but not statistically significant, in the compound heterozygote group (16%). We saw a nonsignificant reduction in acne in patients who had the top 25% of TEWL measurements, those with the most severe form of hyperlinearity and those with higher POEM scores. Patients with severe AE, measured as an EASI score &amp;gt; 21, had a more reduced incidence of acne than those with lower EASI scores (9% vs. 26%; P = 0.04). Overall, this suggests that patients with more severe AE are protected from developing acne. There was insufficient evidence to establish if this was mediated by a reduction in filaggrin (FLG) and AKT signalling. This may be because the groups were too small to determine this. Given that all patients with severe AE have reduced FLG expression regardless of genotype, it is possible that this is mediated through FLG–AKT signalling.

Excess filaggrin in keratinocytes is removed by extracellular vesicles to prevent premature death and this mechanism can be hijacked by Staphylococcus aureus in a TLR2-dependent fashion.

Filaggrin (FLG) protein is indispensable for multiple aspects of the epidermal barrier function but its accumulation in a monomeric filaggrin form may initiate premature keratinocytes death; it is unclear how filaggrin levels are controlled before the formation of storing keratohyalin granules. Here we show that keratinocyte-secreted small extracellular vesicles (sEVs) may contain filaggrin-related cargo providing a route of eliminating excess filaggrin from keratinocytes; blocking of sEV release has cytotoxic effects on those cells. Filaggrin-containing sEVs are found in plasma in both healthy individuals and atopic dermatitis patients. Staphylococcus aureus (S. aureus) enhances packaging and secretion of filaggrin-relevant products within the sEVs for enhanced export via a TLR2-mediated mechanism which is also linked to the ubiquitination process. This filaggrin removal system, preventing premature keratinocyte death and epidermal barrier dysfunction, is exploited by S. aureus which promotes filaggrin elimination from the skin that could help safeguard bacterial growth.

The Role of EAAT4 in Epidermal Differentiation and Calcium Homeostasis During Aging

The Role of EAAT4 in Epidermal Differentiation and Calcium Homeostasis During Aging

PDE4 inhibition by difamilast regulates filaggrin and loricrin expression via keratinocyte proline-rich protein in human keratinocytes

BackgroundDifamilast, a topical phosphodiesterase 4 (PDE4) inhibitor, has been shown to be effective for treating atopic dermatitis (AD), but the molecular mechanism involved is unclear. Since skin barrier dysfunction including reduced expression of filaggrin (FLG) and loricrin (LOR) contributes to AD development, difamilast treatment may be able to improve this dysfunction. PDE4 inhibition increases transcriptional activity of cAMP-responsive element binding protein (CREB). Therefore, we hypothesized that difamilast may affect FLG and LOR expression via CREB in human keratinocytes. ObjectiveTo elucidate the mechanism by which difamilast regulates FLG and LOR expression via CREB in human keratinocytes. MethodsWe analyzed normal human epidermal keratinocytes (NHEKs) treated with difamilast. ResultsWe observed increases of intracellular cAMP levels and CREB phosphorylation in difamilast (5 μM)-treated NHEKs. Next, we found that difamilast treatment increased mRNA and protein levels of FLG and LOR in NHEKs. Since reduced expression of keratinocyte proline-rich protein (KPRP) is reported to be involved in skin barrier dysfunction in AD, we examined KPRP expression in difamilast-treated NHEKs. We found that difamilast treatment increased mRNA and protein levels of KPRP in NHEKs. Furthermore, KPRP knockdown using siRNA transfection abolished the upregulation of FLG and LOR in difamilast-treated NHEKs. Finally, CREB knockdown canceled the upregulation of FLG, LOR, and KPRP in difamilast-treated NHEKs, indicating that PDE4 inhibition by difamilast treatment positively regulates FLG and LOR expression via the CREB-KPRP axis in NHEKs. ConclusionThese findings may provide further guidance for therapeutic strategies in the treatment of AD using difamilast.

Human epidermis organotypic cultures, a reproducible system recapitulating the epidermis in vitro.

The translatability of research is highly dependent on models that recapitulate human tissues and organs. Here, we describe a procedure for the generation of human epidermis organotypic cultures (HEOCs) from primary keratinocytes isolated from foreskin and adult skin as well as from an immortalized keratinocyte cell line (KerTr). We tested several media conditions to develop a defined HEOC growing and expansion media. We characterized the HEOCs and show that in optimal culture conditions they express the proliferation marker Ki67, the basement membrane protein collagen 17 (col17) and the epidermal differentiation markers keratin 15 (K15), keratin 14 (K14), keratin 5 (K5), keratin 10 (K10), keratin 1 (K1), transglutaminase 1 (TGM1), transglutaminase 3 (TGM3) and filaggrin (FLG). Thus, they recapitulate the human epidermis and are stratified from the basal layer to the stratum corneum. These HEOC can be generated reproducibly on a large scale, making it an invaluable model for screening therapeutic compounds and also for the study of pathologies affecting the epidermis.