Epidermal Keratinocytes Research Articles

920 Epidermal keratinocytes produce thymus and activation-regulated chemokine in patients with generalized pustular psoriasis

920 Epidermal keratinocytes produce thymus and activation-regulated chemokine in patients with generalized pustular psoriasis

761 Epidermal keratinocytes from psoriatic resolved skin keep disease residual transcriptomic and epigenomic profiles

761 Epidermal keratinocytes from psoriatic resolved skin keep disease residual transcriptomic and epigenomic profiles

731 Fluoxetine exerts anti-inflammatory effects on human epidermal keratinocytes, and suppresses endothelin release via inhibiting PI3K

731 Fluoxetine exerts anti-inflammatory effects on human epidermal keratinocytes, and suppresses endothelin release via inhibiting PI3K

Temperature-Responsive Hydrogel for Silver Sulfadiazine Drug Delivery: Optimized Design and In Vitro/In Vivo Evaluation.

Response surface methodology (RSM) was applied to optimise a temperature-responsive hydrogel formulation synthesised via the direct incorporation of biocellulose, which was extracted from oil palm empty fruit bunches (OPEFB) using the PF127 method. The optimised temperature-responsive hydrogel formulation was found to contain 3.000 w/v% biocellulose percentage and 19.047 w/v% PF127 percentage. The optimised temperature-responsive hydrogel provided excellent LCST near to the human body surface temperature, with high mechanical strength, drug release duration, and inhibition zone diameter against Staphylococcus aureus. Moreover, in vitro cytotoxicity testing against human epidermal keratinocyte (HaCaT) cells was conducted to evaluate the toxicity of the optimised formula. It was found that silver sulfadiazine (SSD)-loaded temperature-responsive hydrogel can be used as a safe replacement for the commercial SSD cream with no toxic effect on HaCaT cells. Last, but not least, in vivo (animal) dermal testing-both dermal sensitization and animal irritation-were conducted to evaluate the safety and biocompatibility of the optimised formula. No sensitization effects were detected on the skin applied with SSD-loaded temperature-responsive hydrogel indicating no irritant response for topical application. Therefore, the temperature-responsive hydrogel produced from OPEFB is ready for the next stage of commercialisation.

Anti-inflammatory effect and metabolic mechanism of BS012, a mixture of Asarum sieboldii, Platycodon grandiflorum, and Cinnamomum cassia extracts, on atopic dermatitis in vivo and in vitro

BackgroundAtopic dermatitis (AD) is a chronic, relapsing skin disease accompanied by itchy and dry skin. AD is caused by complex interactions between innate and adaptive immune response. AD treatment include glucocorticoids and immunosuppressants. However, long-term treatment can have serious side effects. Thus, an effective AD treatment with fewer side effects is required. Natural materials, including herbal medicines, have potential applications. PurposeThis study evaluated the in vivo and in vitro therapeutic effects of BS012, a mixture of Asarum sieboldii, Platycodon grandiflorum, and Cinnamomum cassia extracts, on AD and investigated the underlying metabolic mechanisms. MethodsThe anti-inflammatory effects of BS012 were assessed using a mouse model of AD induced by 1‑chloro-2,4-dinitrobenzene (DNCB) and in tumor necrosis factor-alpha/interferon-gamma (TNF-α/IFN-γ) stimulated normal human epidermal keratinocytes (NHEKs). In DNCB-induced mice, total dermatitis score, histopathological analysis, and immune cell factors were assessed to evaluate the anti-atopic activity. In TNF-α/IFN-γ-stimulated NHEKs, pro-inflammatory cytokines, chemokines, and related signaling pathways were investigated. Serum and intracellular metabolomics were performed to identify the metabolic mechanism underlying the therapeutic effects of BS012 treatment. ResultsIn DNCB-induced mice, BS012 showed potent anti-atopic activity, including reducing AD-like skin lesions and inhibiting the expression of Th2 cytokines and thymic stromal lymphopoietin. In TNF-α/IFN-γ-stimulated keratinocytes, BS012 dose-dependently inhibited the expression of pro-inflammatory cytokines and chemokines by blocking nuclear factor-kappa B and signal transducer and activator of transcription signaling pathways. Serum metabolic profiles of mice revealed significant changes in lipid metabolism related to inflammation in AD. Intracellular metabolome analysis revealed that BS012 treatment affected the metabolism associated with inflammation, skin barrier function, and lipid organization of the stratum corneum. ConclusionBS012 exerts anti-atopic activity by reducing the Th2-specific inflammatory response and improving skin barrier function in AD in vivo and in vitro. These effects are mainly related to the inhibition of inflammation and recovery of metabolic imbalance in lipid organization. BS012, a novel combination with strong activity in suppressing the Th2-immune response, could be a potential alternative for AD treatment. Furthermore, the metabolic mechanism in vivo and in vitro using a metabolomics approach will provide crucial information for the development of natural products for AD treatment.

Deciphering the mechanism of PSORI-CM02 in suppressing keratinocyte proliferation through the mTOR/HK2/glycolysis axis.

Hyperplasia of epidermal keratinocytes that depend on glycolysis is a new hallmark of psoriasis pathogenesis. Our previous studies demonstrated that PSORI-CM02 could halt the pathological progression of psoriasis by targeting inflammatory response and angiogenesis, but its effect(s) and mechanism(s) on proliferating keratinocytes remained unclear. In this study, we aim to identify components of PSORI-CM02 that are absorbed into the blood and to determine the effect(s) of PSORI-CM02 on keratinocyte proliferation and its molecular mechanism(s). We used the immortalized human epidermal keratinocyte cell line, HaCaT, as an in vitro model of proliferating keratinocytes and the imiquimod-induced psoriasis mouse (IMQ) as an in vivo model. Metabolite profiles of vehicle pharmaceutic serum (VPS), PSORI-CM02 pharmaceutic serum (PPS), and water extraction (PWE) were compared, and 23 components of PSORI-CM02 were identified that were absorbed into the blood of mice. Both PPS and PWE inhibited the proliferation of HaCaT cells and consequently reduced the expression of the proliferation marker ki67. Additionally, PPS and PWE reduced phosphorylation levels of mTOR pathway kinases. Seahorse experiments demonstrated that PPS significantly inhibited glycolysis, glycolytic capacity, and mitochondrial respiration, thus reducing ATP production in HaCaT cells. Upon treatments of PPS or PWE, hexokinase 2 (HK2) expression was significantly decreased, as observed from the set of glycolytic genes we screened. Finally, in the IMQ model, we observed that treatment with PSORI-CM02 or BPTES, an inhibitor of mTOR signaling, reduced hyperproliferation of epidermal keratinocytes, inhibited the expression of p-S6 and reduced the number of proliferating cell nuclear antigen (PCNA)-positive cells in lesioned skin. Taken together, we demonstrate that PSORI-CM02 has an anti-proliferative effect on psoriatic keratinocytes, at least in part, by inhibiting the mTOR/HK2/glycolysis axis.

Identification immune response genes in psoriasis after treatment with secukinumab

BackgroundSecukinumab is a fully human IgG1κ MoAb that selectively binds to IL-17A with high affinity, and it has been proven effective for the treatment of psoriasis. However, the immune response pathways and mechanisms during the treatment are still masked. Therefore, the current study was designed to investigate the potential immune response genes via bioinformatics approaches.MethodsGene expression data of severe plaque-type psoriasis was retrieved from the GEO database. Quantification of immune infiltration by ssGSEA and identification of differentially infiltrated immune cells were conducted to validate the treatment effect of secukinumab. After data processing, differentially expressed genes were identified between the treatment and untreated group. TC-seq was employed to analyze the trend of gene expression and clustering analysis. IL-17 therapeutic immune response genes were selected by taking the intersection of the genes inside the key cluster set and the MAD3-PSO geneset. Based on these therapeutic response genes, protein–protein interaction networks were built for key hub gene selection. These hub genes would work as potential immune response genes, and be validated via an external dataset.ResultsEnrichment scores calculated by ssGSEA illustrated that the immune infiltration level of T cells had a strong difference before and after medication, which validated the treatment effect of Secukinumab. 1525 genes that have significantly different expression patterns before and after treatment were extracted for further analysis, and the enrichment result shows that these genes have the function related to epidermal development, differentiation, and keratinocytes differentiation. After overlapping candidate genes with MAD3-PSO gene set, 695 genes were defined as anti-IL7A treatment immune response genes, which were mainly enriched in receptor signaling and IL-17 signaling pathways. Hub gene were pinpointed from the PPI network constructed by anti-IL7A treatment immune response genes, their expression pattern fits TC-seq gene expression pattern.ConclusionOur study revealed the potential anti-IL7A treatment immune response genes, and the central hub genes, which may act critical roles in Secukinumab, induced immune response. This would open up a novel and effective avenue for the treatment of psoriasis.

Abstract 5254: Topical inhibition of UV-induced PD-L1 expression and inflammatory signaling by the small molecule inhibitor BMS-202

Abstract Immune evasion is a molecular hallmark of cancer, and overexpression of the immune checkpoint protein PD-L1 (CD274) has been documented in several tumors including cutaneous squamous cell carcinoma (cSCC). Notably, while baseline levels of PD-L1 in normal human epidermis are low, acute solar-simulated light (SSL) exposure increases the expression of this protein in epidermal keratinocytes, an effect which can be replicated in cultured keratinocytes and in mouse models. While current immunotherapies targeting the PD-1/PD-L1 axis using monoclonal antibodies are providing therapeutic benefits in many cancers including cSCC, small molecule inhibitors are also being developed to target PD-L1 which may be amenable for topical formulation and therefore early intervention with reduced systemic exposure. We have recently tested one such molecule, BMS-202, in vitro and in vivo. BMS-202 application to keratinocytes in culture inhibits UV-induced PD-L1 RNA and protein expression. Notably, exposure to this inhibitor also reduced UV-induced stress signaling as measured by AP-1 luciferase assay in cultured keratinocytes. Topical application of BMS-202 to immunocompetent SKH-1 mouse skin resulted in significant inhibition of SSL-induced epidermal PD-L1 as determined by immunoblot analysis. NanoString nCounter Pathway™ transcriptomic analysis of full-thickness SKH-1 mouse skin shows strong inhibition of SSL-induced inflammatory responses, chemokine activity, innate immune response and NF-κB activation in BMS-202 treated samples compared to SSL-only controls. Our results indicate that early intervention against PD-L1 expression/activity could be a viable target for skin cancer photochemoprevention. Topical application of small molecule PD-L1 inhibitors such as BMS-202 may provide novel treatment options for populations at high risk for cSCC. Citation Format: Prajakta Vaishampayan, Jana Jandova, Yuchen (Ella) Ai, Viktoria Kirschnerova, Clara Curiel-Lewandrowski, Georg T. Wondrak, Sally E. Dickinson. Topical inhibition of UV-induced PD-L1 expression and inflammatory signaling by the small molecule inhibitor BMS-202. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5254.

Abstract 1218: Mercury(II)-induced abnormalities in processing of topoisomerase I-mediated DNA damage

Abstract Mercury (Hg) is a persistent environmental pollutant that has long been associated with neurotoxic and other noncancer health effects in exposed populations. Recent large epidemiological studies using different internal indices of Hg exposure have found significantly elevated risks for skin cancers in both men and women. To understand potential carcinogenic mechanisms for Hg(II), we examined its mutagenicity and impact on processing of different types of DNA damage. In agreement with its lack of DNA reactivity, we found that Hg(II) was not mutagenic at the mammalian Hprt locus and did not change DNA incorporation of rNMPs which are the most abundant endogenous nucleotide abnormalities in the genome. We next tested the impact of Hg(II) at nontoxic or only mildly toxic concentrations on sensitivity of cells to several genotoxicants producing specific classes of DNA damage. We found that even nontoxic concentrations of Hg(II) impaired cellular resistance to topoisomerase I-mediated DNA damage in human epidermal keratinocytes. Hg(II) had no significant effect on cytotoxicity of topoisomerase II-mediated DNA lesions and examination of numerous components of DNA damage response activated by topoisomerase II-targeting drugs showed no major differences between control and Hg(II)-exposed cells. In contrast, Hg(II)-treated keratinocytes displayed a distorted DNA damage response to topoisomerase I-produced DNA damage, manifested in a misbalanced activation of ATM and ATR targets. Consistent with their lower survival, Hg(II)-treated cells accumulated higher levels of toxic DNA double-strand breaks (DSBs). Further analyses indicated that Hg(II) caused impartment of DNA repair of replication-associated DSBs. Thus, although Hg(II) is not directly genotoxic, it can increase genomic instability by interfering with accurate processing of endogenous DNA damage produced by topoisomerase I. Citation Format: Giorgiana Madalina Ursu, Anna M. Cyran, Anatoly Zhitkovich. Mercury(II)-induced abnormalities in processing of topoisomerase I-mediated DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1218.

Abstract 4723: DNA G-quadruplex facilitates MYC enhancer-promotor interaction

Abstract Background: G-quadruplexes (G4) are nucleic acid secondary structures formed in guanine-rich regions of DNA or RNA. The biological significance of G4 has been attributed to the regulation of gene expression, and more recently, to the stabilization and generation of enhancer-promotor interaction. Since G4 are enriched in the promoter region of the proto-oncogene MYC, we sought to investigate the role of G4 in stabilizing the enhancer-promoter loop using the locus 8q24 (hg38) 127,700,000-128,500,000 that contains the MYC gene as a model. Methods: Two different cell lines were used, the spontaneously immortalized, non-oncogenic human epidermal keratinocyte (HaCaT), in which the enrichment of G4 in the MYC promoter was demonstrated, and the primary normal human epidermal keratinocyte (nHEK) that is depleted of G4 in MYC promoter. Both cell lines were exposed to the G4 selective stabilizer 360-A drug and the half-maximal inhibitory concentration (IC50) could be calculated. Gene expression was accessed by RT-qPCR and DNA methylation by Methylation-Sensitive High Resolution Melting (MS-HRM). Chromatin Conformation Capture (3C-qPCR) technique was performed interrogating the MYC gene (chr8:127,733,798-127,744,729) and the putative enhancer in the lncRNA PVT1 gene (chr8:127,914,509-127,922,393) to compare the frequency of promoter-enhancer interaction between the two cell lines. Results: The HaCaT cells showed interaction between the MYC promoter and the distal region. The nHEK cells that are not enriched for G4 showed no promoter interaction with the distal region. The use of 360-A in IC50 did not change the results. The gene expression analysis of the MYC showed higher expression in HaCaT cells compared to nHEK. There was no difference in DNA methylation in the MYC promoter between the two cell lines. Conclusions: The data suggest an important role of G4 for the interaction of the MYC promoter and downstream enhancer. Additional analyzes of other genomic regions enriched for G4 should be performed to confirm the mechanism. Citation Format: Dieila Giomo De Lima, Gustavo Narvaes Guimarães, Bianca Caroline Bianco, Ana Paula De Souza, Aline Cristiane Planello. DNA G-quadruplex facilitates MYC enhancer-promotor interaction. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4723.

Abstract 3211: OQL025, a topical cream for the prevention of epidermal growth factor receptor inhibitor-induced skin rash

Abstract Background: Epidermal growth factor (EGF) and its receptor (EGFR) are expressed by several cell types including epidermal keratinocytes. EGF facilitates skin healing by promoting DNA synthesis and cell proliferation. However, driver mutations in EGFR can cause uncontrolled cell growth and cancers. Thus, EGFR has become a therapeutic target for tumors, especially those with EGFR mutations. EGFR inhibitors (EGFRIs) are widely used to treat various tumor types (e.g., non-small cell lung & colorectal cancers). EGFRIs cause inflammatory skin reactions (e.g., acneiform rash) generally within the first 3 weeks of therapy. We propose a novel topically applied cream (OQL025) containing a prodrug, OQRC184 that releases tofacitinib, a Janus Kinase (JAK) inhibitor, as a prophylaxis for EGFRi-induced skin rash. Methods: OQRC184 is designed to enhance lipophilicity/solubility and when topically applied releases tofacitinib into cutaneous tissue. OQRC184 & tofacitinib potencies were characterized for JAK (JAK1, JAK2, JAK3, TyK2) in a cell-free isolated kinase assay. Pharmacokinetics (PK) were studied in Bama minipigs (n=15) with shaved dorsal skin (~10 mg/cm2) that were randomly assigned to 3 groups of OQRC184 @ 0.15, 0.3, 0.5 mg/cm2. Blood was collected (1 mL in EDTA-K2) at pre-dose & 0.5, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, and 168 h post-dose. Plasma concentrations of OQRC184 and tofacitinib (LLOQ of 0.01 ng/mL range 0.01 to 10 ng/mL) were determined with LC-MS/MS. Prevention of EGFRI-induced rash was studied with topically applied 3% or 6% OQRC184 or vehicle cream (CRM) in an SD rat EGFRI-induced rash model (oral afatinib 30 mg/kg (AF)), QD vs. vehicle (water (VW)). The four groups were VW+CRM; AF+CRM; AF+3%OQRC184; AF+6% OQRC184. Results: OQRC184 had low inhibitory activity compared to tofacitinib against JAK1, JAK2, JAK3, and TyK2 with IC50 ratios of OQRC184 to tofacitinib for JAK1 (3610:1), JAK2 (4435:1), JAK3 (3814:1) and TyK2 (>385:1). After a single topical administration, OQRC184 plasma concentrations were below the LLOQ in 42~52% of samples, and <10-fold of LLOQ in 83%~88% of samples. Tofacitinib plasma concentrations were below the LLOQ in 67~87% of samples and <10-fold of LLOQ in 90%~97% of samples. All rats treated with AF developed a rash by day 5. The incidence of rash grade ≥ 2 was lower in 3% and 6% OQRC184 groups vs. controls. Prophylactic treatment with 6% OQRC184 had almost no rash ≥ grade 2. The mean ±SE rash grade in the 3% and 6% OQRC184 prophylactic treatment groups was significantly lower than in the AF+CRM group (3% (0.53±0.23), 6% (0.30±0.16) vs. AF+CRM (1.03±0.44 (p<0.05)). Conclusion: OQRC184, a prodrug designed for releasing tofacitinib into dermal layers, lacks inhibitory potential for a family of JAK kinases. OQL025 topical cream containing OQRC184 showed minimal systemic exposure for either OQRC184 or tofacitinib and effectively prevented/reduced EGFRI-induced rash in preclinical models. Citation Format: Shilan Liu, Wenqin Zeng, Wenxi Li, Qing You, Robert Claude Tyler, Jie Luo, Hong Tang, Shiyi Zhang. OQL025, a topical cream for the prevention of epidermal growth factor receptor inhibitor-induced skin rash [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3211.

Cell cycle dysregulation on prenatal and postnatal arsenic exposure in skin of Wistar rat neonates

This study explores the effects of prenatal and postnatal (until weaning period) arsenic exposure given via pregnant females on Wistar rat neonates. Pregnant female rats were divided in four groups – control, low dose, moderate dose and high dose groups of sodium arsenite exposure during gestation and weaning period. Half of the neonates were sacrificed at day 1 of birth and other half at day 21 of birth. Cell cycle analysis in epidermal keratinocytes using flowcytometer revealed that there was a consistent increase in number of cells in G2/M phase from 0.04% in control group to 0.88%, 1.59% and 2.77% in low, moderate and high dose groups respectively for neonates sacrificed at day-1. Whereas, the increase in number of cells with increasing doses in G2/M phase of neonates sacrificed at day-21 was from 3.44% to 5.1%, 6.82%, and 9.17%. At postnatal day 21, mRNA expression of Cyclin A and B1, p53, Caspases 3, 7 and 9, and Bax were found to be up-regulated. Whereas that of Cyclin E, CDK 1 and 2 and Bcl2 were down regulated consistently in skin tissues of arsenic exposed groups.

Antibacterial efficacy of Citrus hystrix (makrut lime) essential oil against clinical multidrug-resistant methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates

The increasing incidence of methicillin-resistant S. aureus is a major public health concern. Recently, the performance of Citrus hystrix essential oil (CHEO) has been shown to contain broad-spectrum antibacterial activity. Therefore, this study aims to determine the antibacterial activity of CHEO alone and in combination with gentamicin against panels of clinical isolates of methicillin-susceptible S. aureus (MSSA, n = 45) and methicillin-resistant S. aureus (MRSA, n = 40). Antibiotic susceptibility testing revealed multidrug-resistant (MDR) patterns among 3 MSSA isolates and 39 MRSA isolates, indicating that the clinical MRSA isolates were associated with MDR (p < 0.05). For the drug resistant isolates, resistance was observed toward most antibiotics, except for chloramphenicol, trimethoprim-sulfamethoxazole, linezolid, and vancomycin. Antibacterial screening by disk diffusion demonstrated that CHEO alone had certain antibacterial activity toward all MSSA isolates (IZD: 16.0 ± 4.7 mm) and MRSA isolates (IZD: 16.5 ± 4.2 mm) (p > 0.05). The MIC values of CHEO are 18.3 ± 6.1 mg/mL in MSSA isolates and 17.9 ± 6.9 mg/mL in MRSA isolates (p > 0.05). The antibacterial activity of CHEO demonstrated the bactericidal effect with MIC index 1.0–1.4. Time-killing kinetics revealed that CHEO at 1 × MIC completely killed MSSA and MRSA within 12 h. Moreover, the checkerboard titration demonstrated the synergistic and additive interactions of CHEO with gentamicin with FIC index 0.012–0.625. CHEO against human epidermal keratinocyte; HaCaT cell line demonstrated the IC50 value at 2.15 mg/mL. The use of CHEO as an alternative antibacterial agent would reduce the emergence of resistant bacteria, especially MDR MRSA.

Designing a robust biocompatible porous polymeric membrane using Laponite and graphene oxide for versatile and selective adsorption of water contaminants

For reclaiming contaminated water, nanoparticle-incorporated porous polymeric membranes provide the requisite functionality for efficient and selective adsorption of toxic molecules. To this end, a simple yet innovative material selection strategy was adopted to prepare a robust and high-performance membrane system by the phase-inversion method using thermoplastic polyurethane (TPU), Laponite, graphene oxide (GO) and a hybrid derived from their aqueous “co-dispersion”, viz., Laponite/GO (LGO). The addition of 12 wt% LGO substantially improved the hydrophilicity, porosity, pure water flux (PWF), and mechanical properties of TPU membranes. Notably, the above membrane showed a striking 20-fold increase in PWF (from ∼27 to ∼652 Lm-2h−1), a 43-fold increase in elastic modulus, a 10-fold increase in tensile strength and a 3-fold increase in toughness and displayed synergistic improvement in mechanical properties. At 12 wt% LGO, the rejection efficiency of > 98%, >95%, and ∼86%, and the adsorption capacity of ∼47 mg/g, ∼49.3 mg/g and ∼16.8 mg/g for methylene blue (MB), crystal violet (CV) and rhodamine B (RhB) dyes, respectively were observed. The dye adsorption efficiency was comparable with that of poly(vinylidene fluoride) (PVDF)-LGO membranes. Owing to the anionic functional groups in LGO, the nanocomposite membranes displayed excellent selectivity of adsorption. The dye adsorption by the nanofiller-TPU membranes showed a pseudo-2nd-order kinetic behavior, indicating chemisorption; while the pristine TPU showed marginal physisorption of dyes. The LGO-filled membranes also showed efficient adsorption of Pb2+ and As5+ ions from water, with efficiency increasing with LGO content, showing > 90% efficiency with 15 wt% LGO. The nanocomposite membranes showed no cytotoxicity effect and supported the proliferation of human epidermal keratinocytes (HaCaT) cells. On these accounts, the LGO-TPU membranes promise the development of high-performance and cost-effective filtration membranes.

Translation-dependent skin hyperplasia is promoted by type 1/17 inflammation in psoriasis

BackgroundPsoriasis vulgaris (PV) is a chronic skin inflammatory disease and characterized by aberrant epidermal hyperplasia. The molecule eukaryotic initiation factor (eIF) 4E controls translation initiation of certain protein synthesis and determines cell cycle or differentiation fate. ObjectiveTo determine the role of eIF4E in keratinocytes abnormal differentiation in the context of psoriasis. MethodsThe expression of eIF4E in psoriatic skin lesions and normal skin from human subjects was examined by western blot and immunohistochemistry. In a murine model of psoriasis-like dermatitis that is induced by topical imiquimod, 4EGI-1 was used to inhibit eIF4E activities. To measure murine skin eIF4E and keratinocytes differentiation, immunofluorescence and western blot assays were conducted. Normal human epidermal keratinocytes (NHEK) were isolated, cultured, and stimulated with cytokines including TNF-α, IFN-γ, and IL-17A, respectively. Immunofluorescence and western blot were performed to test eIF4E and effect of 4EGI-1 in a co-culture system. ResultsCompared with healthy controls, skin lesions from patients with PV exhibited a higher expression of eIF4E, which was positively correlated with the epidermal thickness. This expression pattern of eIF4E was replicated by the imiquimod-induced murine model. Skin hyperplasia and eIF4E activities in the murine model were attenuated by the administration of 4EGI-1. Both IFN-γ and IL-17A, rather than TNF-α, are sufficient to induce NHEK abnormal differentiation. This effect can be disrupted by 4EGI-1. ConclusioneIF4E plays a crucial role in keratinocytes abnormal differentiation driven by type 1/17 inflammation in the context of psoriasis. The initiation of abnormal translation provides an alternative treatment target for psoriasis.

Ligand Activation of the Aryl Hydrocarbon Receptor Upregulates Epidermal Uridine Diphosphate Glucose Ceramide Glucosyltransferase and Glucosylceramides

Ligand activation of the aryl hydrocarbon receptor (AHR) accelerates keratinocyte differentiation and the formation of the epidermal permeability barrier. Several classes of lipids, including ceramides, are critical to the epidermal permeability barrier. In normal human epidermal keratinocytes, the AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin, increased RNA levels of ceramide metabolism and transport genes: uridine diphosphate glucose ceramide glucosyltransferase (UGCG), ABCA12, GBA1, and SMPD1. Levels ofabundant skin ceramides were also increased by 2,3,7,8-tetrachlorodibenzo-p-dioxin. These included themetabolites synthesized by UGCG, glucosylceramides, and acyl glucosylceramides. Chromatin immunoprecipitation-sequence analysis and luciferase reporter assays identified UGCG as a direct AHR target. The AHR antagonist, GNF351, inhibited the 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated RNA and transcriptional increases. Tapinarof, an AHR ligand approved for the treatment of psoriasis, increased UGCG RNA, protein, and its lipid metabolites hexosylceramides as well as increased the RNA expression of ABCA12, GBA1, and SMPD1. In Ahr-null mice, Ugcg RNA and hexosylceramides were lower than those in the wild type. These results indicate that the AHR regulates the expression of UGCG, a ceramide-metabolizing enzyme required for ceramide trafficking, keratinocyte differentiation, and epidermal permeability barrier formation.

The significance of M1‐polarized CD163+ macrophages in acute graft‐versus‐host disease (GVHD): Possible mechanisms of GVHD in the development of skin lesions

AbstractObjectivesGraft‐versus‐host disease (GVHD) is an important complication of bone marrow transplantation. Recent reports suggest the significance of T‐cell subsets (Th1, Th17, and cytotoxic CD8+ T cells) as well as CD163+ macrophages in the development of cutaneous GVHD. CD163+ macrophages produce various chemokines to establish the immunological microenvironment following stimulation by stromal factors in lesional skin. Thus, the purpose of this study is to determine the main source of IFN‐inducible chemokines in the lesional skin of GVHD.MethodsWe employed immunohistochemical (IHC) staining for CD163 as well as interferon (IFN)‐inducible chemokines (CXCL9, CXCL10, CXCL11) to determine if the main source of IFN‐inducible chemokines in the lesional skin of GVHD was CD163+ macrophages. Moreover, we investigated the possible cytokine profiles of lesional skin in GVHD by evaluating phospho‐signal transducer and activator of transcription (pSTAT) expression in epidermal keratinocytes.ResultsImmunohistochemical staining of serial sections for CD163 revealed that CXCL9‐expressing cells, CXCL10‐expressing cells, and CXCL11‐expressing cells were detected in adjacent to CD163+ TAMs in the dermis. In contrast, there were no CCL17‐expressing cells or CCL22‐expressing cells in the dermis. The nuclei of epidermal keratinocytes in GVHD expressed pSTAT1, pSTAT3, and pSTAT5B.ConclusionsThe chemokine expression patterns on CD163+ macrophages matched the expected phosphorylation pattern of epidermal STATs. Our present study suggested that CD163 + macrophages may be a therapeutic target in GVHD.

Chronic activation of Toll-like receptor 2 induces an ichthyotic skin phenotype.

Ichthyosis defines a group of chronic conditions that manifest phenotypically as a thick layer of scales, often affecting the entire skin. While the gene mutations that lead to ichthyosis are well documented, the actual signalling mechanisms that lead to scaling are poorly characterized; however, recent publications suggest that common mechanisms are active in ichthyotic tissue and in analogous models of ichthyosis. To determine common mechanisms of hyperkeratosis that may be easily targeted with small-molecule inhibitors. We combined gene expression analysis of gene-specific short hairpin RNA (shRNA) knockdowns in rat epidermal keratinocytes (REKs) of two genes mutated in autosomal recessive congenital ichthyosis (ARCI), Tgm1 and Alox12b, and proteomic analysis of skin scale from patients with ARCI, as well as RNA sequencing data from rat epidermal keratinocytes treated with the Toll-like receptor 2 (TLR2) agonist Pam3CSK4. We identified common activation of the TLR2 pathway. Exogenous TLR2 activation led to increased expression of important cornified envelope genes and, in organotypic culture, caused hyperkeratosis. Conversely, blockade of TLR2 signalling in keratinocytes from patients with ichthyosis and our shRNA models reduced the expression of keratin 1, a structural protein overexpressed in ichthyosis scale. A time course of TLR2 activation in REKs revealed that although there was rapid initial activation of innate immune pathways, this was rapidly superseded by widespread upregulation of epidermal differentiation-related proteins. Both nuclear factor kappa B phosphorylation and GATA3 upregulation was associated with this switch, and GATA3 overexpression was sufficient to increase keratin 1 expression. Taken together, these data define a dual role for TLR2 activation during epidermal barrier repair that may be a useful therapeutic modality in treating diseases of epidermal barrier dysfunction.

The Effects of Mucopolysaccharide Polysulfate on Steroid-Induced Tight Junction Barrier Dysfunction in Human Epidermal Keratinocytes and a 3D Skin Model

Introduction: The long-term use of topical corticosteroids (TCS) is associated with side effects such as skin atrophy and barrier deterioration. Moisturizers, such as mucopolysaccharide polysulfate (MPS), have been reported to prevent relapses in atopic dermatitis (AD) when used in combination with TCS. However, the mechanisms underlying the positive effects of MPS in combination with TCS in AD are poorly understood. In the present study, we investigated the effects of MPS in combination with clobetasol 17-propionate (CP) on tight junction (TJ) barrier function in human epidermal keratinocytes (HEKa) and 3D skin models. Methods: The expression of claudin-1, which is crucial for TJ barrier function in keratinocytes, and transepithelial electrical resistance (TEER) was measured in CP-treated human keratinocytes incubated with and without MPS. A TJ permeability assay, using Sulfo-NHS-Biotin as a tracer, was also conducted in a 3D skin model. Results: CP reduced claudin-1 expression and TEER in human keratinocytes, whereas MPS inhibited these CP-induced effects. Moreover, MPS inhibited the increase in CP-induced TJ permeability in a 3D skin model. Conclusion: The present study demonstrated that MPS improved TJ barrier impairment induced by CP. The improvement of TJ barrier function may partially be responsible for the delayed relapse of AD induced by the combination of MPS and TCS.

Effect of Hypertrophic Scar Fibroblast-Derived Exosomes on Keratinocytes of Normal Human Skin

Epidermal keratinocytes are highly activated, hyper-proliferated, and abnormally differentiated in the post-burn hypertrophic scar (HTS); however, the effects of scar fibroblasts (SFs) on keratinocytes through cell-cell interaction in HTS remain unknown. Here, we investigated the effects of HTSF-derived exosomes on the proliferation and differentiation of normal human keratinocytes (NHKs) compared with normal fibroblasts (NFs) and their possible mechanism to provide a reference for clinical intervention of HTS. Fibroblasts were isolated and cultured from HTS and normal skin. Both HTSF-exosomes and NF-exosomes were extracted via a column-based method from the cell culture supernatant. NHKs were treated for 24 or 48 h with 100 μg/mL of cell-derived exosomes. The expression of proliferation markers (Ki-67 and keratin 14), activation markers (keratins 6, 16, and 17), differentiation markers (keratins 1 and 10), apoptosis factors (Bax, Bcl2, caspase 14, and ASK1), proliferation/differentiation regulators (p21 and p27), and epithelial-mesenchymal transition (EMT) markers (E-cadherin, N-cadherin, and vimentin) was investigated. Compared with NF-exosomes, HTSF-exosomes altered the molecular pattern of proliferation, activation, differentiation, and apoptosis, proliferation/differentiation regulators of NHKs, and EMT markers differently. In conclusion, our findings indicate that HTSF-derived exosomes may play a role in the epidermal pathological development of HTS.