MicroRNA‐181b negatively regulates the proliferation of human epidermal keratinocytes in psoriasis through targeting TLR4

Our study aims to explore the role of microRNA‐181b (miR‐181b) and TLR in the regulation of cell proliferation of human epidermal keratinocytes (HEKs) in psoriasis. Twenty‐eight patients diagnosed with psoriasis vulgaris were selected as a case group with their lesional and non‐lesional skin tissues collected. A control group consisted of 20 patients who underwent plastic surgery with their healthy skin tissues collected. Real‐time quantitative fluorescence polymerase chain reaction (RT‐qPCR), in situ hybridization and immunohistochemistry were used to detect the expressions of miR‐181b and TLR4 in HEKs of healthy skin, psoriatic lesional skin and non‐lesional skin respectively. The 3′ untranslated region (3′UTR) of TLR4 combined with miR‐181b was verified by a dual‐luciferase reporter assay. Western blotting and bromodeoxyuridine were applied for corresponding detection of TLR4 expression and cell mitosis. The expression of miR‐181b in HEKs of psoriatic lesional skin was less than healthy skin and psoriatic non‐lesional skin. In psoriatic lesional and non‐lesional skin, TLR4‐positive cell rates and the number of positive cells per square millimetre were higher than healthy skin. The dual‐luciferase reporter assay verified that miR‐181b targets TLR4. HEKs transfected with miR‐181b mimics had decreased expression of TLR4, along with the decrease of mitotic indexes and Brdu labelling indexes. However, HEKs transfected with miR‐181b inhibitors showed increased TLR4 expression, mitotic indexes and Brdu labelling indexes. HEKs transfected with both miR‐181b inhibitors and siTLR4 had decreased mitotic indexes and Brdu labelling indexes. These results indicate that miR‐181b can negatively regulate the proliferation of HEKs in psoriasis by targeting TLR4.     

The role of LncRNA MALAT-1 and MiRNA-9 in Psoriasis

BackgroundPsoriasis is a chronic skin disorder manifested by recurrent episodes of scaly, red, itchy skin patches that occur within apparently normal skin.ObjectivesThis study was performed to detect the expression of serum and tissue (lesion and non-lesion) LncRNA MALAT-1 and MiRNA-9 that might be used as biomarkers for psoriasis.MethodsBlood samples were obtained from 60 psoriasis patients and 40 controls, as well as 4 mm punch biopsy from lesional and non lesional skin of psoriatic patient and normal skin of healthy controls. Expression of LncRNA MALAT-1 and miRNNA-9 in serum and tissues was detected by real time qRT-PCR.Resultsa statistically significant increase in the expression of MALAT-1 in lesional and non-lesional skin and serum of psoriatic patients in comparison to controls were detected. Moreover, there was statistically significant increase in serum MiRNA-9 in patients in comparison to controls, while its tissue level was significantly lower in patients.ConclusionThis study highlights the dysregulation of LncRNA MALAT-1 and miRNA-9 in psoriasis. Elevated expression of MALAT-1 in lesional skin of psoriatic patients compared to non-lesional skin may possibly contribute to the development of psoriatic plaques.     

Allergic Contact Dermatitis in Psoriasis Patients: Typical,Delayed, and Non-Interacting

Psoriasis is characterized by an apoptosis-resistant and metabolic active epidermis, while a hallmark for allergic contact dermatitis (ACD) is T cell-induced keratinocyte apoptosis. Here, we induced ACD reactions in psoriasis patients sensitized to nickel (n = 14) to investigate underlying mechanisms of psoriasis and ACD simultaneously. All patients developed a clinically and histologically typical dermatitis upon nickel challenge even in close proximity to pre-existing psoriasis plaques. However, the ACD reaction was delayed as compared to non-psoriatic patients, with a maximum intensity after 7 days. Whole genome expression analysis revealed alterations in numerous pathways related to metabolism and proliferation in non-involved skin of psoriasis patients as compared to non-psoriatic individuals, indicating that even in clinically non-involved skin of psoriasis patients molecular events opposing contact dermatitis may occur. Immunohistochemical comparison of ACD reactions as well as in vitro secretion analysis of lesional T cells showed a higher Th17 and neutrophilic migration as well as epidermal proliferation in psoriasis, while ACD reactions were dominated by cytotoxic CD8+ T cells and a Th2 signature. Based on these findings, we hypothesized an ACD reaction directly on top of a pre-existing psoriasis plaque might influence the clinical course of psoriasis. We observed a strong clinical inflammation with a mixed psoriasis and eczema phenotype in histology. Surprisingly, the initial psoriasis plaque was unaltered after self-limitation of the ACD reaction. We conclude that sensitized psoriasis patients develop a typical, but delayed ACD reaction which might be relevant for patch test evaluation in clinical practice. Psoriasis and ACD are driven by distinct and independent immune mechanisms.     

Suppression of Molecular Inflammatory Pathways by Toll-Like Receptor 7, 8, and 9 Antagonists in a Model of IL-23-Induced Skin Inflammation

Psoriasis is a complex inflammatory disease resulting from the activation of T helper (Th) 1 and Th17 cells. Recent evidence suggests that abnormal activation of Toll-like receptors (TLRs) 7, 8 and 9 contributes to the initiation and maintenance of psoriasis. We have evaluated the effects of TLR antagonists on the gene expression profile in an IL-23-induced skin inflammation model in mice. Psoriasis-like skin lesions were induced in C57BL/6 mice by intradermal injection of IL-23 in the dorsum. Two TLR antagonists were compared: IMO-3100, an antagonist of TLRs 7 and 9, and IMO-8400, an antagonist of TLRs 7, 8 and 9, both of which previously have been shown to reduce epidermal hyperplasia in this model. Skin gene expression profiles of IL-23-induced inflammation were compared with or without TLR antagonist treatment. IL-23 injection resulted in alteration of 5100 gene probes (fold change ≥ 2, FDR < 0.05) including IL-17 pathways that are up-regulated in psoriasis vulgaris. Targeting TLRs 7, 8 and 9 with IMO-8400 resulted in modulation of more than 2300 mRNAs while targeting TLRs 7 and 9 with IMO-3100 resulted in modulation of more than 1900 mRNAs. Both agents strongly decreased IL-17A expression (>12-fold reduction), normalized IL-17 induced genes such as beta-defensin and CXCL1, and normalized aberrant expression of keratin 16 (indicating epidermal hyperplasia). These results suggest that IL-23-driven inflammation in mouse skin may be dependent on signaling mediated by TLRs 7, 8, and 9 and that these receptors represent novel therapeutic targets in psoriasis vulgaris and other diseases with similar pathophysiology.     

ZNF750 Is Expressed in Differentiated Keratinocytes and Regulates Epidermal Late Differentiation Genes

Disrupted skin barrier due to altered keratinocyte differentiation is common in pathologic conditions such as atopic dermatitis, ichthyosis and psoriasis. However, the molecular cascades governing keratinocyte terminal differentiation are poorly understood. We have previously demonstrated that a dominant mutation in ZNF750 leads to a clinical phenotype reminiscent of psoriasis and seborrheic dermatitis. Here we show that ZNF750 is a nuclear protein bearing a functional C-terminal nuclear localization signal. ZNF750 was specifically expressed in the epidermal suprabasal layers and its expression was augmented during differentiation, both in human skin and in-vitro, peaking in the granular layer. Silencing of ZNF750 in Ca2+-induced HaCaT keratinocytes led to morphologically apparent arrest in the progression of late differentiation, as well as diminished apoptosis and sustained proliferation. ZNF750 knockdown cells presented with markedly reduced expression of epidermal late differentiation markers, including gene subsets of epidermal differentiation complex and skin barrier formation such as FLG, LOR, SPINK5, ALOX12B and DSG1, known to be mutated in various human skin diseases. Furthermore, overexpression of ZNF750 in undifferentiated cells induced terminal differentiation genes. Thus, ZNF750 is a regulator of keratinocyte terminal differentiation and with its downstream targets can serve in future elucidation of therapeutics for common diseases of skin barrier.     

Co-localization of COX-2, CYP4F8, and mPGES-1 in epidermis with prominent expression of CYP4F8 mRNA in psoriatic lesions

Cyclooxygenase-2 (COX-2), cytochrome P450 4F8 (CYP4F8), and microsomal PGE synthase-1 (mPGES-1) form PGE and 19-hydroxy-PGE in human seminal vesicles. We have examined COX-2, CYP4F8, and mPGES-1 in normal skin and in psoriasis. All three enzymes were detected in epidermis by immunofluorescence and co-localized in the suprabasal cell layers. In lesional psoriasis the enzymes were also co-localized in the basal cell layers. Real-time RT-PCR analysis suggested that CYP4F8 mRNA was induced 15-fold in lesional compared to non-lesional epidermis. mRNA of all enzymes were present in cultured HEK and HaCaT cells, but the prominent induction of CYP4F8 mRNA in psoriasis could not be mimicked by treatment of these keratinocytes with a mixture of inflammatory cytokines or with phorbol 12-myristate-13-acetate. The function of CYP4F8 in epidermis might be related to lipid oxidation and keratinocyte proliferation.     

Localization of immuno-analogues of erythrocyte protein 4.1 and spectrin in epidermis of psoriasis vulgaris

The presence and localization of immuno-analogues of human erythrocyte protein 4.1 and spectrin were examined in the epidermis of psoriasis vulgaris. Immunoblot analysis with antibodies against human erythrocyte protein 4.1 revealed that psoriatic epidermis contains a 4.1-like protein of 80 kDa, and also minor immunoreactive polypeptides, including a 45-kDa polypeptide. The 45-kDa band was not detected in non-lesional epidermis. Lesional epidermis of psoriasis contains spectrin-like proteins of 240 kDa. Analysis with immunofluorescence microscopy revealed that 4.1-like proteins were detected mainly in the cytoplasm of the suprabasal cells in lesional epidermis and in the peripheral cytoplasm of the basal cells in non-lesional epidermis. On the other hand, spectrin-like proteins were localized to the peripheral cytoplasm of basal keratinocytes in both lesional and non-lesional psoriatic epidermis. The present results indicate that proteins related to protein 4.1 and spectrin are consistently detected within epidermal cells of psoriasis, a chronic skin disease characterized by epidermal hyperplasia; the expression and distribution of protein 4.1 in lesional epidermis of psoriasis differs from that in non-lesional epidermis. These membrane skeletal proteins may be of significance in the hyperproliferative epidermis of psoriasis.     

Toll-Like Receptor 4 Expression in the Epithelium of Inflammatory Periapical Lesions.: An immunohistochemical study

Toll-like receptors (TLR) are essential for the innate immune response against invading pathogens and have been described in immunocompetent cells of areas affected by periapical disease. Besides initiating the inflammatory response, they also directly regulate epithelial cell proliferation and survival in a variety of settings. This study evaluates the in situ expression of TLR4 in periapical granulomas (PG) and radicular cysts, focusing on the epithelial compartment.Twenty-one periapical cysts (PC) and 10 PG were analyzed; 7 dentigerous non-inflamed follicular cyst (DC) served as control. TLR4 expression was assessed by immunohistochemistry. TLR4 immunoreaction products were detected in the epithelium of all specimens, with a higher percentage of immunostained cells in PG. Although TLR4 overexpression was detected in both PG and PC, there were differences that seemed to be related to the nature of the lesion, since in PG all epithelial cells of strands, islands and trabeculae were strongly immunoreactive for TLR4, whereas in PC only some areas of the basal and suprabasal epithelial layers were immunostained. This staining pattern is consistent with the action of TLR4: in PG it could promote formation of epithelial cell rests of Malassez and in epithelial strands and islands the enhancement of cell survival, proliferation and migration, whereas in PC TLR4 could protect the lining epithelium from extensive apoptosis. These findings go some way towards answering the intriguing question of why many epithelial strands or islands in PG and the lining epithelium of apical cysts regress after non-surgical endodontic therapy, and suggest that TLR4 plays a key role in the pathobiology of the inflammatory process related to periapical disease.Key words: TLR4, periapical inflammatory granulomas, radicular cysts     

K14、K19在寻常型银屑病发病中的作用机制研究

目的:研究细胞角蛋白K14、K19在寻常型银屑病(PV)发病中的作用机制。方法:选择从2012年1月到2016年12月在西安交通大学第二附属医院皮肤科就诊的PV患者60例纳入本次研究。将PV患者在皮损区及未受累皮肤区分别取1份皮肤,制成存档蜡块,分别记为PV皮损组和PV未受累皮肤组各60例。另选同期在我院接受外伤手术治疗的健康皮肤60例作为对照组,分析K14、K19在PV中的阳性表达率以及表达水平,分析K14及K19在PV皮损区阳性表达的相关性。结果:各组的K14及K19阳性表达率以及表达水平比较差异有统计学意义(P0.05)。PV皮损组和PV未受累皮肤组的K14阳性表达率及表达水平明显高于对照组,K19阳性表达率及表达水平明显低于对照组,差异有统计学意义(P0.05)。PV皮损组的K14阳性表达率及表达水平明显高于PV未受累皮肤组,K19阳性表达率及表达水平明显低于PV未受累皮肤组,差异有统计学意义(P0.05)。根据Spearman法分析相关性发现,K14及K19在PV皮损区阳性表达中呈负相关(r=-0.871,P=0.000)。结论:K14及K19均在PV发病过程中出现异常表达,其中K14阳性表达较强,而K19阳性表达较弱,二者在PV皮损区阳性表达中呈负相关。临床上可考虑监测K14及K19的阳性表达水平,从而更好地辅助临床诊断及病情的预估,值得临床医师关注与重视。     

E6/E7 expression of human papillomavirus type 20 (HPV-20) and HPV-27 influences proliferation and differentiation of the skin in UV-irradiated SKH-hr1 transgenic mice

The functional role of UV irradiation, in combination with the E6 and E7 proteins of the cutaneous human papillomavirus (HPV) types in the malignant conversion of benign papillomatous lesions, has not been elucidated. Transgenic SKH-hr1 hairless mice expressing HPV-20 and HPV-27 E6 and E7 proteins in the suprabasal compartment were generated and exposed to chronic UV irradiation. Histological and immunohistochemical examination of skin samples revealed enhanced proliferation of the epidermal layers and papilloma formation in both transgenic strains in comparison to what was observed with nontransgenic mice. Squamous cell carcinoma developed in the HPV-20 E6/E7 transgenic line as well as in the HPV-27 E6/E7 transgenic line. Several weeks after cessation of UV-B exposure, enhanced proliferation, as measured by BrdU incorporation, was maintained only in HPV-20 transgenic skin. Keratin 6 expression was increased in the transgenic mice throughout all cell layers. Expression of the differentiation markers involucrin and loricrin was reduced and disturbed. p63alpha expression was differentially regulated with high levels of cytoplasmic expression in clusters of cells in the granular layer of the skin in the transgenic lines 20 weeks after cessation of UV-B exposure, in contrast to uninterrupted staining in the nontransgenic lines. p53 was expressed in clusters of cells in nontransgenic and HPV-27 transgenic mice, in contrast to an even distribution in a higher number of cells in HPV-20 transgenic animals.     

Keratinocyte TLR2 and TLR7 contribute to chronic itch through pruritic cytokines and chemokines in mice

Although neuronal Toll-like receptors (TLRs) (e.g., TLR2, TLR3, and TLR7) have been implicated in itch sensation, the roles of keratinocyte TLRs in chronic itch are elusive. Herein, we evaluated the roles of keratinocyte TLR2 and TLR7 in chronic itch under dry skin and psoriasis conditions, which was induced by either acetone-ether-water treatment or 5% imiquimod cream in mice, respectively. We found that TLR2 and TLR7 signaling were significantly upregulated in dry skin and psoriatic skin in mice. Chronic itch and epidermal hyperplasia induced by dry skin or psoriasis were comparably reduced in TLR2 and TLR7 knockout mice. In the dry skin model, the enhanced messenger RNA (mRNA) expression levels of pruritic CXCL1/2, IL-31, IL-33, ST2, IL-6, IL-17A, TNF-α, and IFN-γ were inhibited in TLR2^−/− mice, while CXCL2, IL-31, and IL-6 were inhibited in TLR7^−/− mice. In psoriasis model, the enhanced mRNA expression levels of pruritic CXCL1/2, IL-31, IL-33, ST2, IL-6, and TNF-α were inhibited in TLR2^−/− mice, while CXCL1/2, IL-31, IL-33, ST2, IL-6, IL-17A, and TNF-α were inhibited in TLR7^−/− mice. Incubation with Staphylococcus aureus (S. aureus) peptidoglycan (PGN-SA) (a TLR2 agonist), imiquimod (a TLR7 agonist), and miR142-3p (a putative TLR7 agonist) were sufficient to upregulate the expression of pruritic cytokines or chemokines in cultured keratinocyte HaCaT cells. Finally, pharmacological blockade of C-X-C Motif Chemokine Receptor 1/2 and high mobility group box protein 1 dose-dependently attenuated acute and chronic itch in mice. Together, these results indicate that keratinocyte TLR2 and TLR7 signaling pathways are distinctly involved in the pathogenesis of chronic itch.     

Regulation of Involucrin in Psoriatic Epidermal Keratinocytes: The Roles of ERK1/2 and GSK-3β

Psoriasis, a common inflammatory skin disease, is characterized by epidermal hyperplasia, abnormal differentiation, angiogenesis, immune activation, and inflammation. Involucrin is an early terminal differentiation marker of epidermal keratinocytes. In this study, we determined the immunolocalization of involucrin in psoriatic lesions and normal skin of individuals without psoriasis by means of immunofluorescence (IF) assay. Furthermore, the regulation of involucrin by interleukin (IL)-13, IL-17A, endothelin (ET)-1, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ was investigated by Western blot. Extracellular regulate protein kinases 1/2 (ERK1/2) and glycogen syntheses kinase-3β (GSK-3β) inhibitors were also included to define the roles of these signals in the production of involucrin in both psoriatic and normal keratinocytes. In psoriatic lesional skin, involucrin was detected in the stratum spinosum, but not in the basal or the cornified layer. In normal skin, involucrin was restricted to the granular layer and the upper stratum spinosum. IL-13, IL-17A, ET-1, TNF-α, and IFN-γ up-regulate expression of involucrin in both psoriatic and normal keratinocytes. However, this effect was abolished by ERK1/2 and GSK-3β inhibitors. In conclusion, involucrin is up-regulated in psoriatic keratinocytes. IL-13, IL-17A, ET-1, TNF-α, and IFN-γ could increase involucrin protein levels in psoriatic and normal keratinocytes. The ERK1/2 and GSK-3β signaling pathways may play positive roles in regulating epidermal differentiation as observed in psoriasis.     

Dolichos biflorus agglutinin (DBA) reveals a similar basal cell differentiation in normal and psoriatic epidermis

Summary Binding of N-acetyl galactosamine (GalNAc)-specific Dolichos biflorus agglutinin (DBA) conjugates to frozen sections of normal epidermis and of psoriatic uninvolved and lesional skin was studied in fluorescence microscopy. The DBA conjugates bound only to single basal cell layer in normal and uninvolved psoriatic epidermis from patients with different blood group status. In the lesional area of psoriatic skin a similar reaction with a single basal cell layer was revealed. Other lectin-conjugates applied, presenting also GalNAc specificity, reacted with most cell layers of normal and both uninvolved and lesional psoriatic epidermis and gave an attenuated reaction with the middle epidermal layers. The results show that the basal cell characteristics are confined only to the cells along the basal membrane also in psoriatic epidermis, although cells in three lowest layers may be able to proliferate.     

PSORS2 is due to mutations in CARD14

Psoriasis is a common, immune-mediated genetic disorder of the skin and is associated with arthritis in approximately 30% of cases. Previously, we localized PSORS2 (psoriasis susceptibility locus 2) to chromosomal region 17q25.3-qter after a genome-wide linkage scan in a family of European ancestry with multiple cases of psoriasis and psoriatic arthritis. Linkage to PSORS2 was also observed in a Taiwanese family with multiple psoriasis-affected members. In caspase recruitment domain family, member 14 (CARD14), we identified unique gain-of-function mutations that segregated with psoriasis by using genomic capture and DNA sequencing. The mutations c.349G>A (p.Gly117Ser) (in the family of European descent) and c.349+5G>A (in the Taiwanese family) altered splicing between CARD14 exons 3 and 4. A de novo CARD14 mutation, c.413A>C (p.Glu138Ala), was detected in a child with sporadic, early-onset, generalized pustular psoriasis. CARD14 activates nuclear factor kappa B (NF-kB), and compared with wild-type CARD14, the p.Gly117Ser and p.Glu138Ala substitutions were shown to lead to enhanced NF-kB activation and upregulation of a subset of psoriasis-associated genes in keratinocytes. These genes included chemokine (C-C motif) ligand 20 (CCL20) and interleukin 8 (IL8). CARD14 is localized mainly in the basal and suprabasal layers of healthy skin epidermis, whereas in lesional psoriatic skin, it is reduced in the basal layer and more diffusely upregulated in the suprabasal layers of the epidermis. We propose that, after a triggering event that can include epidermal injury, rare gain-of-function mutations in CARD14 initiate a process that includes inflammatory cell recruitment by keratinocytes. This perpetuates a vicious cycle of epidermal inflammation and regeneration, a cycle which is the hallmark of psoriasis.