Establishing Transcription Profile of Psoriasiform Cutaneous In Vitro using HaCaT Cells Stimulated with Combination of Cytokines

Summary

This paper presents a method of establishing an in vitro psoriasiform cutaneous inflammatory model at the transcription level using a combination of five cytokines (IL-17A, IL-22, IL-1α, TNF-α, OSM) on HaCaT cell line.

Abstract

Psoriasis is a common chronic inflammatory skin disease mediated by innate and adaptive immune systems, characterized by abnormal proliferation and differentiation of epidermal keratinocytes and infiltration of inflammatory cells. Skin-specific keratinocytes are key participants in innate immunity, responding to immune cells and environmental stimulation, thereby serving an important role in the immunopathogenesis of psoriasis. Here, we present a method for inducing psoriasiform keratinocytes inflammation at transcription level with HaCaT cell line using five proinflammatory cytokines combination (M5 combination), including IL-17A, IL-22, IL-1α, TNF-α, and oncostatin M. Results demonstrate that M5 combination induced HaCaT cells showed increased levels of antimicrobial peptides (BD2, S100A7, S100A8, and S100A9), chemokines, and cytokines (CXCL1, CXCL2, CXCL8, CCL20, IL-1β, IL-6 and, IL-18). The mRNA levels of keratinocytes differentiation markers (Keratin1, Keratin10, Filaggrin, and Loricrin) were down regulated, which was consistent with the transcriptome data derived from psoriasis-like keratinocytes. The method described here, therefore, establishes an in vitro psoriasiform cutaneous inflammation at transcription level and contributes to the research for molecular pathogenesis of psoriasis.

Introduction

Psoriasis is a common non-contagious chronic inflammatory skin disease triggered by a dysregulated immune response, affecting the keratinocytes that predominantly form the epidermis¹, characterized by abnormally rapid multiplication of keratinocytes with hyperkeratosis and parakeratosis. Psoriasis affects about 3% of the world-wild population². Disease burden is further increased by several comorbidities, including cardiovascular diseases and metabolic syndrome caused by the syndrome³.

Epidermis is composed of five layers of keratinocytes and undergo morphological chang....

Protocol

Perform steps 1 to 3 under sterile condition. All the culture medium contained 0.1 mg/mL penicillin and streptomycin.

1. Cell preparation

1. Seed 1 x 10⁶ HaCaT cells in 10 mL of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in 100 mm cell culture dish. Incubate the culture dish at 37 °C in a humidified 5% CO₂ incubator for 2 days.
2. When the culture reaches around 80% confluency, carefully remove the medium from the culture dish and wash the cells with 5 mL of 1x phosphate buffered saline (PBS). Gently rock the culture dish manually.
3. Remove PB....

Results

M5 combination stimulation induced inflammatory response of HaCaT cells.
HaCaT cells were stimulated with or without M5 cytokines combination for 24 h. mRNA expression of psoriasis-related genes, which are involved in the regulation of the immune and inflammatory chemokines and antimicrobial peptides, were evaluated. Neutrophil chemokines CXCL1³⁸, CXCL2³⁹, CXCL8⁴⁰, and T.......

Discussion

Described herein is a method using five cytokines combination (IL-17A, IL-22, IL-1α, TNF-α, OSM) into HaCaT cell line to establish an in vitro psoriasiform cutaneous inflammation profile at transcription level. This protocol can be adapted for the study on the mechanism of genes in the pathogenesis of psoriasis as well as the screening of therapeutic drugs for psoriasis. Recent reports have shown that overexpression of IL-17A and IL22 producing CD8 T cells in lesional skin suggests their involvement in the path.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM—Dulbecco's Modified Eagle Medium	Gibco	11965092
Fetal Bovine Serum	Gibco	10100139C
HaCaT cells	China Center for Type CultureCollection	GDC0106	Less than 15 generations
Human IL-1β ELISA Kit	Beyotime	PI305
Human IL-6 ELISA Kit	Beyotime	PI330
Human IL-8 ELISA Kit	Beyotime	PI640
IL-1 alpha Human	Prospec	CYT-253	Recombinant protein
IL-17 Human	Prospec	CYT-250	Recombinant protein
IL-22 Human	Prospec	CYT-328	Recombinant protein
OSM Human	Prospec	CYT-231	Recombinant protein
PBS	Gibco	10010049	pH 7.4
Penicillin-Streptomycin	Gibco	15140163
PrimeScrip RT reagent Kit	TAKARA	RR047A
TB Green Premix Ex Taq	TAKARA	RR420A
TNF alpha Human	Prospec	CYT-223	Recombinant protein
TRIzo Reagent	Invitrogen	15596018
Trypsin-EDTA (0.25%), phenol red	Gibco	25200072