Gene expression profiling of filaggrin-insufficient keratinocytes exposed to inflammatory mediators and allergens
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ABSTRACT:
PROVIDER: PRJNA839775 | ENA |
REPOSITORIES: ENA
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Project description:Loss-of-function mutations in the filaggrin gene are heavily associated with the incidence and severity of atopic dermatitis. Moreover, filaggrin insufficiency in epidermal keratinocytes can lead to persistent allergic inflammation and an associated immune response suggesting a role for this protein in regulation of immunity. Here, we investigate how filaggrin insufficiency affects the gene expression pattern in a filaggrin-insufficient (shFLG) keratinocyte cell line HaCaT in response to stimulation with various inflammatory mediators and allergens.
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Project description:Filaggrin (FLG) is a key structural protein expressed in epidermal keratinocytes. Mutations in the filaggrin gene (FLG) are strongly linked to atopic dermatitis (AD) and allergic inflammation occurring later in life at distant body locations in AD patients. Keratinocytes secrete exosomes, small, lipid-rich membrane vesicles to communicate with distant cells, including within the immune system. The FLG gene was silenced in the HaCaT keratinocyte cell line by shRNA delivered using a lentiviral vector. HaCaT cells transduced with scrambled shRNA were used as a control.
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Project description:The epidermis, outermost layer of the skin, forms a barrier and is involved in innate and adaptive immunity in an organism. Keratinocytes participate in all these three protective processes. However, a regulator of keratinocyte protective responses against external dangers and stresses remains elusive. We found that upregulation of the orphan gene 2610528A11Rik was a common factor in the skin of mice with several types of inflammation. In the human epidermis, peptide expression of G protein-coupled receptor 15 ligand (GPR15L), encoded by the human ortholog C10orf99, was highly induced in the lesional skin of patients with atopic dermatitis or psoriasis. C10orf99 gene transfection into normal human epidermal keratinocytes (NHEKs) induced the expression of inflammatory mediators and reduced the expression of barrier-related genes. Gene ontology analyses showed its association with translation, mitogen-activated protein kinase (MAPK), mitochondria, and lipid metabolism. Treatment with GPR15L reduced the expression levels of filaggrin and loricrin in human keratinocyte 3D cultures. Instead, their expression levels in mouse primary cultured keratinocytes did not show significant differences between the wild-type and 2610528A11Rik deficient keratinocytes. Lipopolysaccharide-induced expression of Il1b and Il6 was less in 2610528A11Rik deficient mouse keratinocytes than in wild-type, and imiquimod-induced psoriatic dermatitis was blunted in 2610528A11Rik deficient mice. Furthermore, repetitive subcutaneous injection of GPR15L in mouse ears induced skin inflammation in a dose-dependent manner. These results suggest that C10orf99/GPR15L is a primary inducible regulator that reduces the barrier formation and induces the inflammatory response of keratinocytes.
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Project description:We used microarray analysis to investigate if keratinocytes excert an immuno-inflammatory response towards streptococcal M1 protein. Keratinocytes respond to M1 protein by activating pathways to induce the production of inflammatory signaltransducers, AP-1 and NFkB transcription factor subunits, together with inflammatory mediators such as IL-8, CCL20 and IL-1 family proteins etc.
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Project description:In this study we aim to determine the role of IL-4/STAT6 in gene expression in human keratinocytes using RNA-sequencing approach. Human keratinocytes were cultured for 2 or 5 days with calcium chloride to induce terminal differentiation as determined by the expression of epidermal differentiation complex genes. The cells were then stimulated with IL-4 for 3 and 24 hours, or along the 5 days culture period. We observed that IL-4 inhibits fully differentiation of keratinocytes, induces genes involved with production of inflammatory mediators, and reduces the healing capacity of human keratinocytes. Moreover, STAT6 controlled important genes involved with calcium binding, inflammation and epidermis development. Human keratinocytes were differentiated with calcium chloride for 2 days and incubated with media alone or 20ng/ml of recombinant human IL-4 for 3 and 24 hours. Human keratinocytes were differentiated with calcium chloride for 5 days with or wihout recombinant human IL-4 (20ng/ml). Keratinocytes transfected with control or STAT6 siRNA were differentiated with calcium chloride for 2 days and then stimulated with recombinant huma IL-4 for 24 hours.
2015-12-15 | E-GEOD-59275 | biostudies-arrayexpress
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Project description:Shisha smoking is widely believed to be a hazard-free habit. Studies have reported shisha smoking to be associated with oral lesions as well ascarcinomas of the lung, esophagus, bladder, and pancreas. A better understanding of the underlying mechanism may contribute to the identification of biomarkers for targeted therapy and better prognosis of the disease. In this study, we have established a chronic model of shisha-exposed oral keratinocytes to study the effect of shisha smoking on oral cells. Normal oral keratinocytes (non-transformed and immortalised), OKF/TERT1, were chronically treated with shisha extract for 8 months. In vitro cellular assays as well as total proteomic analysis were performed using the OKF6/TERT1-Parental and shisha-exposed cells (OKF6/TERT1-Shisha) to understand the effect of shisha smoking on cellular transformation. Chronic exposure of OKF6/TERT1 cells with shisha resulted in a significant increase in cellular proliferation and cell invasion compared to the OKF6/TERT1-Parental cells. Quantitative proteomic analysis of OKF6/TERT1-Parental and OKF6/TERT1-Shisha cells resulted in the identification of more than 5,515 proteins. Of these 5,515 proteins, 82, 77, 106, 110 proteins were found to be differentially expressed (1.5-fold) in OKF6/TERT1 cells chronically treated with shisha extract for 2M, 4M, 6M and 8M, respectively when compared with OKF6/TERT1-Parental. Pathway and gene ontology-based analysis revealed dysregulation of interferon pathway, upregulation of proteins involved in cell growth and downregulation of immune processes. This study reveals that chronic treatment of normal oral keratinocytes with shisha leads to cellular transformation. Elucidation of dysregulated proteins in shisha smoke-exposed cells will provide insights into the effect of shisha smoking on oral cells and aid in identification of therapeutic targets.
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Project description:Hydrolyzed wheat proteins (HWPs) contained in cosmetics have occasionally caused immediate-type hypersensitivity following repeated skin exposure. Although the Cosmetic Ingredient Review Expert Panel concluded that <3,500 Da HWP is safe for use in cosmetics, it remains biologically unknown how allergenic HWPs evoke immediate-type allergy percutaneously. Keratinocyte-derived thymic stromal lymphopoietin (TSLP) induces type 2 immune responses, which play an essential role in the pathogenesis of immediate-type allergy. Previously, we demonstrated that protein allergens in cultured human keratinocytes strongly induced long-form TSLP (loTSLP) transcription. However loTSLP-regulating signaling by HWP is poorly understood. In this study, we performed global gene expression analysis by microarray to investigate how the allergenic HWP acts on epidermal keratinocytes and the induction of loTSLP.
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