In the present study, following treatment of primary keratinocytes with IL-17, reduced mRNA and protein levels of FLG and IVL were observed

In the present study, following treatment of primary keratinocytes with IL-17, reduced mRNA and protein levels of FLG and IVL were observed. resulted in reduced manifestation levels of FLG and IVL in the mRNA and protein levels. In addition, the gene manifestation levels of FLG and IVL were significantly reduced in the HaCaT cells by IL-4. Treatment with the mitogen-activated protein kinase (MAPK) inhibitors, SB203580 and PD98059, significantly inhibited the effects of IL-17 within the gene and protein manifestation levels of FLG and IVL. Finally, the protein levels of phosphorylated ERK and P38 were significantly increased following IL-17 activation. Taken together, the results revealed that IL-17 reduced the expression of FLG and IVL in HaCaT cells, and this effect involved the P38/ERK MAPK signaling pathways. strong class=”kwd-title” Keywords: atopic dermatitis, filaggrin, involucrin, interleukin-17, skin barrier Introduction Atopic dermatitis (AD) is usually a chronic, relapsing inflammatory skin disease, which affects ~10C20% and 1C3% of children and adults, respectively, in Western populations (1). Impaired epidermal barrier and 1,2,3,4,5,6-Hexabromocyclohexane immune function defects are common in patients with AD (2,3). AD is also characterized by a T helper type 2 (Th2) dominance, mediated 1,2,3,4,5,6-Hexabromocyclohexane by pro-Th2 cytokines, thymic stromal lymphopoietin and interleukin (IL)-33, which polarize dendritic cells and promote Th2 responses (4). CD4+ T cells are the main mediators of cellular immunity and are found in the cell infiltrate of the skin of patients with AD (5). Th17 cells, a distinct lineage of CD4+ helper T cells, are important in the host defense against extracellular fungal and bacterial pathogens, 1,2,3,4,5,6-Hexabromocyclohexane and the pathogenesis of inflammatory and autoimmune disorders (6). IL-17, also known as IL-17A, is the main effector cytokine of Th17 cells and regulates the functions of multiple cell types (7), including the activation of keratinocytes to produce cytokines, chemokines and vascular endothelial growth factor (6). Another important component in Goat monoclonal antibody to Goat antiMouse IgG HRP. AD is skin integrity (8,9). Of notice, skin barrier dysfunction in patients with AD is usually associated with abnormal protein expression of filaggrin (FLG), loricrin (LOR) and involucrin (IVL), which result in skin impermeability by cross-linking (10,11). FLG is usually a major structural protein in the stratum corneum of the epidermis, with reduced levels altering the shape of skin corneocytes (12). LOR comprises 80% of the total protein mass in the cornified layer (13), whereas IVL functions as a scaffold to other cross-linked proteins (14). Patients with AD with an acquired defect in the expression of FLG exhibit an atopic inflammatory response (15). Therefore, it is hypothesized that FLG and IVL can be regulated by AD-associated cytokines, including IL-17, as the expression of IL-17 is usually enhanced in acute lesions in AD skin, compared with that in normal skin, with increased numbers of Th17 cells in the peripheral blood in acute AD (16). IL-17 activates mitogen-activated protein kinases (MAPKs), and the P38/extracellular signal-regulated kinase (ERK) MAPK signaling pathways are involved in the pathogenesis of inflammatory skin diseases, including psoriasis (17). The present study aimed to examine the effects of IL-17 around the expression of FLG and IVL in human HaCaT keratinocytes, and investigate the regulatory mechanism. Materials and methods Cell culture The HaCaT cells (JennioBioech Co., Ltd., Guangzhou, China), a human keratinocyte cell collection, were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml of penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.), at 37C in a humid environment made up of 5% CO2. To examine the effects of cytokines around the expression of FLG and IVL, the keratinocytes were differentiated for 5 days by treatment with CaCl2 at 1.3 mmol/l. Cells seeded at 1105 cells/ml were allowed to grow to 70C80% confluence and were stimulated with medium made up of IL-4 (100 1,2,3,4,5,6-Hexabromocyclohexane ng/ml) or different concentrations of IL-17 (50, 100 and 200 ng/ml) for 24 h at 37C. IL-4 and IL-17 were purchased from PeproTech, Inc. (Rocky Hill, USA). Following treatment, the cells were harvested for protein extraction. Cells in passages 2C5 were utilized for all experiments. Treatment with MAPK inhibitors The MAPK inhibitors directed against P38 (SB203580; 1,2,3,4,5,6-Hexabromocyclohexane 5 M), ERK (PD98059; 20 M), or c-Jun N-terminal kinase (SP600125, 1 M), respectively, were added to the media for treatment of the HaCaT cells 1 h prior to the addition of IL-17 (100 ng/ml) at 37C. The cells were cultured for 24 h prior to harvest for mRNA and protein extraction. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis for quantitation of mRNA expression Total RNA was extracted from your HaCaT cells using the RNeasy Mini kit (Qiagen, Inc., Valencia, CA, USA), according to the manufacturer’s protocol. cDNA.