Primary Normal Human Epidermal Keratinocytes Research Articles

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.

Comprehensive Physicochemical Characterization, In Vitro Membrane Permeation, and In Vitro Human Skin Cell Culture of a Novel TOPK Inhibitor, HI-TOPK-032.

Nonmelanoma skin cancers (NMSC) are the most common skin cancers, and about 5.4 million people are diagnosed each year in the United States. A newly developed T-lymphokine-activated killer cell-originated protein kinase (TOPK) inhibitor, HI-TOPK-032, is effective in suppressing colon cancer cell growth, inducing the apoptosis of colon cancer cells and ultraviolet (UV) light-induced squamous cell carcinoma (SCC). This study aimed to investigate the physicochemical properties, permeation behavior, and cytotoxicity potential of HI-TOPK-032 prior to the development of a suitable topical formulation for targeted skin drug delivery. Techniques such as scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) spectroscopy, differential scanning calorimetry (DSC), hot-stage microscopy (HSM), X-ray powder diffraction (XRPD), Karl Fisher (KF) coulometric titration, Raman spectrometry, confocal Raman microscopy (CRM), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), and Fourier transform infrared microscopy were used to characterize HI-TOPK-032. The dose effect of HI-TOPK-032 on in vitro cell viability was evaluated using a 2D cell culture of the human skin keratinocyte cell line (HaCaT) and primary normal human epidermal keratinocytes (NHEKs). Transepithelial electrical resistance (TEER) at the air-liquid interface as a function of dose and time was measured on the HaCAT human skin cell line. The membrane permeation behavior of HI-TOPK-032 was tested using the Strat-M® synthetic biomimetic membrane with an in vitro Franz cell diffusion system. The physicochemical evaluation results confirmed the amorphous nature of the drug and the homogeneity of the sample with all characteristic chemical peaks. The in vitro cell viability assay results confirmed 100% cell viability up to 10 µM of HI-TOPK-032. Further, a rapid, specific, precise, and validated reverse phase-high performance liquid chromatography (RP-HPLC) method for the quantitative estimation of HI-TOPK-032 was developed. This is the first systematic and comprehensive characterization of HI-TOPK-032 and a report of these findings.

Cytoprotective, Antiproliferative, and Anti-Oxidant Potential of the Hydroethanolic Extract of Fridericia chica Leaves on Human Cancer Cell Lines Exposed to α- and β-Zearalenol.

Fridericia chica (Bignoniaceae) is a Colombian Caribbean plant with numerous health benefits, including properties such as wound healing, immune system stimulation, and antioxidant capacity, among others. Mycotoxins alpha-zearalenol (α-ZEL) and beta-zearalenol (β-ZEL) are phase I metabolites of zearalenone, a natural product involved in endocrine disruption and cell proliferation processes. This study aimed to investigate the cytotoxic potential of the hydroethanolic extract of F. chica leaves (HEFc) and determine their protective effects against proliferation induced by α-ZEL and β-ZEL on human hepatoma HepG2, lung cancer Calu-1, and primary normal human epidermal keratinocytes, neonatal (HEKn). The cytotoxicity of HEFc was measured in a range from 4 to 1000 µg/mL and from 0.4 to 100 μM for both α-ZEL and β-ZEL. Cell production of intracellular ROS was monitored using the H2-DCFDA probe. The cells exposed to HEFc presented IC50 of 128, 249, and 602 µg/mL for the HepG2, Calu-1, and HEKn cells, respectively. A greater selectivity was seen in HepG2 cells [selectivity index (SI) = 3.5] than in Calu-1 cells (SI = 2.4). Cells treated with mycotoxins remained viable during the first day, and cell proliferation increased at low tested concentrations (0.4-6.3 µM) in all three cell lines. However, after 48 h treatment, cells exposed to 50 and 100 µM of α-ZEL and β-ZEL displayed decreased viability. HEFc at 16 µg/mL was able to give some protection against cytotoxicity induced by high concentrations of β-ZEL in HepG2, reducing also cell proliferation elicited at low levels of α-ZEL and β-ZEL. ROS production was not observed in cells treated with this HEFc concentration; however, it prevented ROS formation induced by treatment with 50 µM α-ZEL or β-ZEL. In summary, HEFc isolated from plants grown in northern Colombia displayed promising results against cell proliferation and oxidative stress caused by mycotoxins.

CCN1 upregulates IL-36 via AKT/NF-κB and ERK/CEBP β-mediated signaling pathways in psoriasis-like models.

Psoriasis is a chronic skin disorder characterized by epidermal keratinocyte hyperproliferation and inflammatory infiltration. CCN1 (also termed CYR61 or cysteine-rich angiogenic inducer 61) is an extracellular matrix-associated protein that is involved in multiple physiological functions. In psoriasis, we recently demonstrated that the overexpression of CCN1 promoted keratinocyte proliferation and activation. Furthermore, CCN1 was highly expressed in psoriatic skin lesions from psoriasis vulgaris patients. Here, we dissect the underlying molecular mechanism in imiquimod (IMQ) and interleukin (IL)-23-induced psoriasis-like models. Our results demonstrate that CCN1 can significantly upregulate IL-36 production in the murine skin of IMQ and IL-23-induced psoriasis-like models. Injection of CCN1-neutralizing antibody improved epidermal acanthosis and significantly reduced IL-36 production in vivo. These results suggest that CCN1 can be a critical upstream pro-inflammatory factor in psoriasis. In primary normal human epidermal keratinocytes, we demonstrated that CCN1 can selectively induced the production of IL-36α and IL-36γ through the activation of the protein kinase B (AKT)/nuclear factor kappa light chain enhancer of activated B cells (NF-κB) and extracellular-regulated kinase (ERK)/CCAAT/enhancer binding protein β (CEBPβ) signaling pathways via integrin receptor α6β1 in vitro. Our results suggest that targeting CCN1 can be a potential therapeutic strategy for psoriasis.

847 Modeling basal cell carcinoma in 3D organotypic raft cultures

Basal cell carcinoma (BCC) is the most-commonly diagnosed cancer worldwide with limited availability for non-surgical treatments. The Hedgehog signaling cascade drives tumorigenesis, but efforts to develop effective chemotherapeutics have been thwarted by chemoresistant tumor recurrence. A contributing factor to this is an overall lack of human-based preclinical models. We endeavored to develop 3D organotypic raft models that mimic characteristics of human BCC by combining primary normal human epidermal keratinocytes (NHEK) with established murine BCC cells.

Probing the effects of sweet cherry (Prunus avium L.) extract on 2D and 3D human skin models

Natural products are not only positioned in the heart of traditional medicine but also in modern medicine as many current drugs are coming from natural sources. Apart from the field of medicine and therapeutics, natural products are broadly used in other industrial fields such as nutrition, skincare products and nanotechnology. The aim of this study was to assess the effects of sweet cherry (Prunus avium L.) fruit extract from the Greek native cultivar 'Vasiliadi', on the human 2D and 3D in vitro models in order to investigate its potential impact on skin. We focused on 2D culture of primary normal human epidermal keratinocytes (NHEK) that were treated with sweet cherry fruit extract. In the first place, we targeted fruit extract potential cytotoxicity by determining ATP intracellular levels. Furthermore, we assessed its potential skin irritability by using 3D skin model. To better understand the bioactivity of sweet cherry fruit. extract, we used qPCR to study the expression of various genes that are implicated in the skin functions. Our experiments showed that sweet cherry fruit extract is non-toxic in 2D keratinocytes culture as well as non-irritant in 3D skin model. Our results revealed that the extract mediated important pathways for the optimum epidermis function such as cell proliferation, immune and inflammatory response. The sweet cherry fruit extracts possesses significant activity in epidermis function without any potential of cytotoxicity or skin irritability, which makes it a rather promising active agent for skincare.

Spleen tyrosine kinase regulates keratinocyte inflammasome activation and skin inflammation induced by UVB irradiation

UVB can induce inflammatory responses contributing to diverse skin damage. UVB-triggered inflammasome activation of human keratinocytes underlies UVB-induced skin sunburn reaction. Pleiotropic functions of spleen tyrosine kinase (Syk) have rendered it as a potential therapeutic target. In immunocytes, Syk modulates immunoreceptor signaling and NLRP3 inflammasome activation. In skin, Syk mediates EGFR signaling, regulates keratinocyte differentiation and is involved in inflammatory disorders. However, roles of Syk in UVB-induced inflammasome activation in keratinocytes remain elusive. We investigated roles of keratinocyte Syk in UVB-triggered photo-responses. Primary normal human epidermal keratinocytes (NHEKs) isolated from skin were used. Syk knockdown or Syk inhibitor R406 was applied to investigate functions of keratinocyte Syk in UVB photobiology. The possible in vivo role of Syk was evaluated by checking UVB-induced skin damage in R406-treated mice. UVB was able to induce Syk phosphorylation in NHEKs that could be regulated by reactive oxygen species (ROS) generation and EGFR. Syk knockdown or Syk inhibitor (R406) treatment reduced UVB-triggered apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) crosslinking, procaspase-1 cleavage, active IL-1β formation, and gasdermin D activation, indicating roles of Syk in UVB-triggered inflammasome activation in keratinocytes. UVB-induced production of IL-8, TNF-α, ROS, and phosphorylation of JNK and p38 were attenuated after Syk knockdown or inhibition. R406 ameliorated UVB-induced mouse skin damage, including erythema and transepidermal water loss (TEWL). Thus, Syk participated in UVB-induced inflammasome activation and inflammatory response in vitro and in vivo, suggesting potential photo-protective effects of Syk inhibition in UVB-induced skin inflammation.

MiR-181a Promotes Multiple Protumorigenic Functions by Targeting TGFβR3

Cutaneous squamous cell carcinoma (cSCC) comprises 15‒20% of all skin cancers and has a well-defined progression sequence from precancerous actinic keratosis to invasive cSCC. To identify targets for chemoprevention, we previously reported a cross-species analysis to identify the transcriptional drivers of cSCC development and identified miR-181a as a potential oncomiR. We show that the upregulation of miR-181a promotes multiple protumorigenic properties by targeting an understudied component of TGFβ signaling, TGFβR3. miR-181a and TGFβR3 are upregulated and downregulated, respectively, in cSCC. miR-181a overexpression (OE) and TGFβR3 knockdown (KD) significantly suppresses UV-induced apoptosis in HaCaT cells and in primary normal human epidermal keratinocytes. In addition, OE of miR-181a or KD of TGFβR3 by short hairpin RNA enhances anchorage-independent survival. miR-181a OE or TGFβR3 KD enhances cellular migration and invasion and upregulation of epithelial‒mesenchymal transition markers. Luciferase reporter assays demonstrate that miR-181a directly targets the 3'-untranslated region of TGFβR3. miR-181a upregulates phosphorylated SMAD3 levels after TGFβ2 administration and results in elevated SNAIL and SLUG expression. Finally, we confirm invivo that miR-181a inhibition compromises tumor growth. Importantly, these phenotypes can be reversed with TGFβR3 OE or KD in the context of miR-181a OE or KD, respectively, further highlighting the physiologic relevance of this regulation in cSCC.

110 Metabolites of the skin commensal bacterium S. hominis enhance tight junction barrier integrity in primary human keratinocytes

Studies have shown that skin microbiota play a significant role in the health of the host and may play a role in disease. The epidermis of the skin is a two-way barrier, protecting the entire body from external insults and water loss. Tight junctions (TJs) are critical to this barrier. In this study, we investigated whether metabolites produced by human skin microbiota could impact TJ function in primary normal human epidermal keratinocytes (NHEKs). Skin swabs were collected from 4 body sites of 5 healthy participants and commensal bacteria were cultured in 6 different agars.

UV-Induced Reduction of ACVR1C Decreases SREBP1 and ACC Expression by the Suppression of SMAD2 Phosphorylation in Normal Human Epidermal Keratinocytes.

Activin A receptor type 1C (ACVR1C), a type I transforming growth factor-β (TGF-β) receptor, has been implicated in sensitive skin and psoriasis and is involved in the regulation of metabolic homeostasis as well as cell proliferation and differentiation. In this study, we identified a novel role of ACVR1C in the ultraviolet (UV)-irradiation-induced reduction of epidermal lipogenesis in human skin. UV irradiation decreased ACVR1C expression and epidermal triglyceride (TG) synthesis in human skin in vivo and in primary normal human epidermal keratinocytes (NHEK) in vitro. Lipogenic genes, including genes encoding acetyl-CoA carboxylase (ACC) and sterol regulatory element binding protein-1 (SREBP1), were significantly downregulated in UV-irradiated NHEK. ACVR1C knockdown by shRNA resulted in greater decreases in SREBP1 and ACC in response to UV irradiation. Conversely, the overexpression of ACVR1C attenuated the UV-induced decreases in SREBP1 and ACC. Further mechanistic study revealed that SMAD2 phosphorylation mediated the ACVR1C-induced lipogenic gene modulation. Taken together, a decrease in ACVR1C may cause UV-induced reductions in SREBP1 and ACC as well as epidermal TG synthesis via the suppression of SMAD2 phosphorylation. ACVR1C may be a target for preventing or treating UV-induced disruptions in lipid metabolism and associated skin disorders.

Citrus sudachi Peel Extract Suppresses Cell Proliferation and Promotes the Differentiation of Keratinocytes through Inhibition of the EGFR-ERK Signaling Pathway.

Citrus sudachi is a well-known fruit in Tokushima Prefecture, Japan, and its peels are rich in phytochemicals, including phenolic compounds. Although it is expected that the extract of the C. sudachi peel elicits various beneficial physiological activities, the effect on the skin has not been investigated. In this study, we report that the aqueous extract from the peel of C. sudachi suppresses cell proliferation of the immortalized human keratinocyte cell line, HaCaT, and primary normal human epidermal keratinocytes. The extract of C. sudachi peel suppressed epidermal growth factor (EGF)-induced EGF receptor activation and tumor necrosis factor (TNF)-α-induced extracellular regulated kinase (ERK) 1/2 activation, which suggests that the extract exerts its inhibitory effect through inhibition of both the EGF receptor (EGFR) and its downstream molecules. Additionally, the extract of C. sudachi peel potentiated calcium-induced keratinocyte differentiation. These results suggest that the extract of C. sudachi peel may have beneficial effects against skin diseases that are characterized by hyperproliferation of epidermal keratinocytes, such as those seen in psoriasis and in cutaneous squamous cell carcinoma.

251 Skin epidermal keratinocyte differentiation-associated processes regulate homeostatic antiviral protein expression

Epidermal keratinocytes produce antimicrobial peptides and proteins, including antiviral proteins (AVPs) as innate defense against various pathogens. We made the novel observation that distinct genes encoding innate AVPs are transcriptionally activated upon calcium-induced epidermal differentiation of primary normal human epidermal keratinocytes (NHEK). The AVPs include, but are not limited to, members of oligoadenylate synthetase (OAS) family, myxovirus resistance proteins (MX), interferon-induced proteins with tetratricopeptide repeats (IFIT), interferon-induced transmembrane protein 1 (IFITM1), interferon stimulated gene 15 (ISG15), and guanylate-binding proteins (GBP) (p < 0.05).

Role of Epidermal TRP Channels in the Development of Pruritogenic Signals

TRP ion channels are non-specific cationic channels with 6 transmembrane domains, most of them are permeable to Ca 2+ ions. Primarily, the multimodal thermosensitive members of the TRP family, TRPV1-4, TRPA1, and TRPM8, are proven to play role in skin sensory functions. In addition to sensory neurons in the skin, some of our previous work has also described the presence of sensory TRP channels on a number of non-neural cells and has been implicated in the physiological and pathological biological processes of keratinocytes and skin appendages (eg sebaceous gland, hair follicles). In this project, we investigate the role of keratinocytes and their heat-sensitive TRP channels in the development of itch and related pruriceptive signaling. Primary normal human epidermal keratinocytes (NHEK) isolated from surgical specimens and We studied the expression (RNASeq, Q-PCR, ELISA) and function (intracellular Ca2+ measurements) of TRP channels and other molecules involved in pruritic signaling in normal human epidermal keratinocytes (NHEKs). Expression of several sensory TRP channels was detected on NHEKs, among which TRPV3 and TRPV4 wee expressed at highest level. Their functionality was confirmed by Ca2+ measurements. In addition to TRP channels, keratinocytes also expressed a number of receptors and mediators that may play a role in the development of pruritus or pruritus-related dermatological disorders (eg, proteinase-activated receptor 2, TLR3, histamine H1 receptor, etc.). Subsequently, we investigated the role of TRP channels in the signaling pathways related to these potentially pruritogenic molecules in NHEKs. According to our results, activation of TLR3 upregulated TRPV3 and enhanced subsequent Ca2+ signals, which may contribute to other TLR3 evoked responses. Based on our results, it appears that keratinocytes may play a role in the transduction of certain itch-inducing signals. Supported by: NKFIH 120187; GINOP-2.3.2-15-2016-00050;ÚNKP-19-4-DE-285; ÚNKP-19-3-I-DE-140; János Bolyai Research Scholarship (TIB)

Intrinsic Differences between the Open Chromatin Regions of Oral and Epidermal Keratinocytes.

To investigate and characterise the differences between the open chromatin regions of oral and epidermal keratinocytes. Human immortalised oral epithelial cell lines (HIOECs) were used as the standard model for oral keratinocytes, and primary normal human epidermal keratinocytes (NHEKs) were chosen as the model for epidermal keratinocytes. Assay for transposase accessible chromatin using sequencing (ATAC-seq) and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) were used to evaluate the dynamic changes in open chromatin regions and active enhancers during oral keratinocyte differentiation. In silico prediction and dual-luciferase assays were used to evaluate the enriched motifs and maintain enhancer activity in specific enriched HIOECs. Integration and comparison of HIOEC ATAC-seq with NHEK ATAC-seq were used to identify oral keratinocyte-enriched open chromatin regions along with key motifs governing differential enhancer activity. The genomic regulatory elements and GWAS overlap algorithm was used to compare the annotation rate of HIOEC-overlapped craniofacial enhancers with other craniofacial enhancers for orofacial cleft-associated variants. During the differentiation of HIOECs, 14933 open chromatin regions became more accessible. Grainyhead-like (GRHL) and Krüppel-like factor (KLF) motifs were overrepresented in maintaining HIOEC-specific activity. Compared with NHEKs, 16161 open chromatin regions were uniquely accessible in HIOECs. Within these regions, the C/EBP motif governed HIOEC-specific enhancer regulating SOX2 and PITX2, which enhanced oral keratinocyte wound healing. When intersected with human craniofacial super-enhancers, open chromatin regions in HIOECS can better annotate the common variants associated with orofacial cleft. The intrinsic differences between the open chromatin regions of human oral and epidermal keratinocytes are directly maintained by a set of transcription factors.

Effect of Vitamin D3on nonmelanoma skin cancer cells: A comparative in vitro study

Background: The use of Vitamin D3, as an alternative drug, combined with common therapeutic strategies to treat nonmelanoma skin cancers, has recently attracted attention. However, in vitro data on Vitamin D3action on different tumor cell lines compared to healthy cells are lacking. Aims and Objectives: In this context, the present study aimed to investigate the potential role of Vitamin D3's ability as an antitumor treatment. Materials and Methods: Cell growth, cell viability, and apoptosis as well as cell cycle distribution were comparatively assessed in a squamous cell carcinoma (SCC) cell line, a basal cell carcinoma (BCC) cell line, and healthy primary normal human epidermal keratinocytes (NHEK) in response to various Vitamin D3concentrations. Results: Tumor and healthy cells clearly responded differently to Vitamin D3application with regard to metabolic activity and apoptosis. The application of Vitamin D3reduced the metabolic activity of the BCC and SCC cancer cell lines (and not NHEK) and induced cell cycle arrest. Furthermore, Vitamin D3-mediated increased apoptosis was observed in tumor cells but not in healthy primary keratinocytes. Conclusions: Our findings indicate an antiproliferative and proapoptotic Vitamin D3-dependent effect on skin cancer cells in vitro, highlighting Vitamin D3as a potential and beneficial alternative drug for further studies with respect to possible clinical strategies to treat nonmelanoma skin cancers.

Hypotonic stress response of human keratinocytes involves LRRC8A as component of volume-regulated anion channels.

The barrier function of the human epidermis is constantly challenged by environmental osmotic fluctuations. Hypotonic stress triggers cell swelling, which is counteracted by a compensatory mechanism called regulatory volume decrease (RVD) involving volume-regulated anion channels (VRACs). Recently, it was discovered that VRACs are composed of LRRC8 heteromers and that LRRC8A functions as the essential VRAC subunit in various mammalian cell types; however, the molecular identity of VRACs in the human epidermis remains to be determined. Here, we investigated the expression of LRRC8A and its role in hypotonic stress response of human keratinocytes. Immunohistological staining showed that LRRC8A is preferentially localized in basal and suprabasal epidermal layers. RNA sequencing revealed that LRRC8A is the most abundant subunit within the LRRC8 gene family in HaCaT cells as well as in primary normal human epidermal keratinocytes (NHEKs). To determine the contribution of LRRC8A to hypotonic stress response, we generated HaCaT- and NHEK-LRRC8A knockout cells by using CRISPR-Cas9. I- influx assays using halide-sensitive YFP showed that LRRC8A is crucially important for mediating VRAC activity in HaCaTs and NHEKs. Moreover, cell volume measurements using calcein-AM dye further revealed that LRRC8A also substantially contributes to RVD. In summary, our study provides new insights into hypotonic stress response and suggests an important role of LRRC8A as VRAC component in human keratinocytes.

Effects of a ceramide-containing water-in-oil ointment on skin barrier function and allergen penetration in an IL-31 treated 3D model of the disrupted skin barrier.

Atopic dermatitis (AD) is a chronically relapsing, pruritic inflammation of the skin with dryness and disturbed skin barrier function. Recently, we established that IL-31 treatment of human 3D skin models resulted in a disrupted skin barrier phenotype resembling AD. In this model, we found that IL-31 interferes with the differentiation of keratinocytes and inhibits the expression of terminal differentiation markers. In the present study, we investigated the effects of a ceramide-containing water-in-oil skin care ointment on the physical skin barrier structure and function in disrupted skin barrier models, generated either by using primary normal human epidermal keratinocytes (NHEK) or HaCaT cells. We observed that the physical skin barrier of the models recovered after daily topical treatment with the ceramide-containing ointment. Topical application of the ointment prevented downregulation of filaggrin and disorganization of other differentiation markers, such as keratin 10 and β4-integrin, as demonstrated by immunohistological analysis. The expression of Ki67 was also upregulated in response to the ointment. Furthermore, functional studies revealed that local application of the ointment diminished the increased uptake of fluorescently labelled recombinant allergens of timothy grass (phl p1) in our model. In conclusion, our data revealed that topical application of a ceramide-containing skin care ointment reduced IL-31 induced impairments of the physical skin barrier and skin barrier function in an in vitro model of the disrupted skin barrier. This standardized model can be utilized in the future to monitor ex vivo effects of various topical therapies on skin morphology, physiology, and gene expression.

1136 Galectin-3 regulates UVB-induced inflammation in skin

Galectin-3, a β-galactoside binding lectin, can be detected in diverse types of cells, including epidermal keratinocytes. Regulatory roles of galectin-3 in the pathogenesis of various malignant and inflammatory skin disorders have been reported, but the known roles of galectin-3 in UVB-induced inflammation in skin are still limited and the underlying molecular mechanisms remain elusive. In this study, we aimed to evaluate the regulatory roles of galectin-3 in UVB-induced inflammatory responses in skin by using primary normal human epidermal keratinocytes (NHEKs) and galectin-3 knockout mice as the models. Our results demonstrated knockdown of galectin-3 in NHEKs attenuated the UVB-induced production of active IL-. UVB-induced ASC cross-linking and active caspase-1 formation were reduced but LDH release was not affected after galectin-3 knockdown. In addition, after galectin-3 knockdown, UVB-induced IL-8, TNF-α and COX-2 expression in NHEKs were also down-regulated. Furthermore, UVB-induced production of reactive oxygen species in NHEKs was also reduced after galectin-3 knockdown. In the animal study, UVB-induced skin inflammation and damage were less severe in galectin-3 knockout mice than in wild type mice grossly and histologically. When measuring the physiological parameters, UVB-induced transepidermal water loss and skin erythema index were also reduced in galectin-3 knockout mice compared with wild type mice. In conclusion, current findings indicate that galectin-3 plays a crucial regulatory role in UVB-induced inflammation, suggesting a potential way to prevent UVB-induced skin damage by targeting galectin-3 in skin.

Effect of microRNA-143 on interleukin-13-induced expression of kallikrein 7 in human epidermal keratinocytes

Objective To evaluate the effect of microRNA-143 (miR-143) on interleukin (IL) -13-induced expression of kallikrein 7 (KLK7) in primary normal human epidermal keratinocytes (NHEKs) . Methods Some NHEKs at exponential growth phase were divided into 4 groups to be treated with recombinant human IL-13 at different concentrations of 0, 2, 10 and 50 μg/L respectively for 24 hours, and some NHEKs were treated with 50 μg/L IL-13 for 0, 6, 12, 24 and 48 hours separately. After the treatment, NHEKs were collected, and total RNA was extracted. Real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of KLK7. Some other NHEKs were divided into another 4 groups: NHEK group (blank control group) receiving no treatment, IL-13 group treated with 50 μg/L IL-13, miR-NC group transfected with miRNA mimics negative control followed by the treatment with 50 μg/L IL-13, and miR-143 group transfected with miR-143 mimics followed by the treatment with 50 μg/L IL-13. After 24-hour treatment with IL-13, real-time fluorescence-based quantitative PCR and Western blot analysis were conducted to determine the mRNA and protein expression of KLK7 respectively in the above groups. Results After 24-hour treatment with IL-13 at concentrations of 0, 2, 10 and 50 μg/L, the mRNA expression of KLK7 in NHEKs was 1.00 ± 0.12, 0.89 ± 0.04, 1.15 ± 0.09 and 1.70 ± 0.10 respectively, and significantly increased along with the increase of IL-13 concentrations (F= 92.48, P < 0.05) . After 0-, 6-, 12-, 24- and 48-hour treatment with 50 μg/L IL-13, the mRNA expression of KLK7 in NHEKs was 1.00 ± 0.05, 1.05 ± 0.12, 1.71 ± 0.20, 1.97 ± 0.19 and 2.48 ± 0.13 respectively, and significantly increased over time (F= 206.44, P < 0.05) . Compared with the miR-NC group, the miR-143 group showed significantly decreased mRNA and protein expression of KLK7 (t= 6.76, 4.23 respectively, both P < 0.05) . Conclusion In NHEKs, IL-13 can up-regulate the expression of KLK7, likely by the regulation of miR-143. Key words: Dermatitis, atopic; Keratinocytes; Interleukin-13; Kallikrein 7; MicroRNA-143

Comparative study on antimicrobial activity and biocompatibility of N-selective chitosan derivatives

N-selective derivatives of chitosan (CH) namely, N-methyl chitosan (NMC), trimethyl chitosan (TMC), diethyl methyl chitosan (DEMC) and carboxymethyl chitosan (CMC) were synthesized to study the effects of alkylation on antimicrobial property and biocompatibility. The derivatives were characterized by FT-IR confirming O-free alkyl substitution. XRD studies showed reduction in crystallinity and zeta potential measurement proved the increase in positive charges upon alkylation. Antimicrobial activity was observed to be in the order of DEMC<CMC<CH<NMC<TMC. MTT assay revealed that the modified chitosans were non-cytotoxic to primary normal human epidermal keratinocytes. Chitosan derivatives were tested using EpiDerm™ Skin Irritation Test as per OECD TG 439 and were categorized as non-irritants. Zebrafish embryo viability test was used to study the toxicity of the modified chitosans and LC50 for the samples in zebrafish embryo ranged from 20 to 50μg/mL. Results showed that amongst the alkyl derivatives, O-methyl free N,N,N–trimethyl chitosan (TMC) is the potential candidate material with enhanced antimicrobial property and biocompatibility. Herein, we report the first results of using chitosan or its derivatives in the EpiDerm in vitro skin irritation test. The four derivatives of chitosan are compared on a single platform for their physicochemical, biological and biocompatible properties.