This work was supported by the National Natural Science Foundation of China [81703132, 31271483, 81472650, 81673061, 81573050, 31872739, and 81601462]

Perform steps 1 to 3 under sterile condition. All the culture medium contained 0.1 mg/mL penicillin and streptomycin.
1. Cell preparation
<ol>
	<li>Seed 1 x 106 HaCaT cells in 10 mL of Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in 100 mm cell culture dish. Incubate the culture dish at 37 &#176;C in a humidified 5% CO2 incubator for 2 days.</li>
	<li>When the culture reaches around 80% confluency, carefully remove the mediu...

Described herein is a method using five cytokines combination (IL-17A, IL-22, IL-1&#945;, TNF-&#945;, 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...

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 change with differentiation process: the stratum basal, stratum spinosum, stratum granulosum, stratum lucidum (found on palms and soles) and stratum corneum described here from the inner to outer surface4. Change in epidermis differentiation leads to a disturbed skin barrier, which is important for the pathogenesis of skin inflammatory diseases5,6,7,8. Keratinocytes play a vital role in maintaining an intact epidermal barrier to prevent water loss and against environmental triggers such as UVB exposure, allergens, and pathogens9. Healthy individuals show a balance between basal cells proliferation and stratum corneum desquamation, while multiple skin diseases including psoriasis, are characterized by an imbalances of this complex mechanism10.
In addition to forming barrier function, keratinocytes are also a critical component of skin&#39;s immune system. In the immunopathogeneses of psoriasis, activation of skin-resident Type 1 helper T cells (Th1) and Type 17 helper T cells (Th17), leads to increased production of IFN-&#947; and IL-17A, respectively. These cytokines induce increased synthesis of chemokines (CCL20, CXCL1/2/8/9/10/11), antimicrobial peptides (BD2, LL37, S100A7/8/9/12), and other inflammatory factors (TNF-&#945;, IL-6, IFN-&#946;) in keratinocytes, leading to the recruitment of more Th1, Th17, and neutrophils into the skin, further amplifying the IL-17/IL23 axis11. The crosstalk between keratinocytes and immune cells is responsible for the induction and maintenance of psoriasis11.
Complex cytokine networks have been described in psoriasis, and the central role of pro-inflammatory cytokines (such as IL-23, IL-22, IL-17, IL-1&#945;, oncostatin M(OSM), and TNF-&#945;) produced by immune cell infiltration has been highlighted12,13. Indeed, previous studies have shown that increased levels of IL-17A, IL-22, IL-1&#945;, TNF-&#945;, and OSM induced a profile of psoriasiform on normal human epidermal keratinocytes in vitro14.
Immortal keratinocyte cell lines (HaCaT) that are more easily obtained and cultured than primary keratinocytes with better reproducibility, have been widely used for the study of psoriasis15,16,17,18,19,20. Different from human papillomavirus16 E6/E7 transformed HEK001 and KerTr cells, HaCaT cell line is capable of expressing differentiation gene products, including Keratin1 (KRT1), Keratin10 (KRT10), Loricrin, and filaggrin20,21,22, thereby providing a promising tool similar to primary keratinocytes to study the regulation of keratinization and proinflammatory.
KRT5/14 is the major type I-type II keratin pair expressed in proliferative basal keratinocytes, whereas differentiated keratinocytes in the suprabasal layers downregulate KRT5/14 and express KRT1/10 as the major keratin pair23. Upon comparison of psoriasis lesion with healthy skin, the changes in keratin expression included decreases in KRT1/1024,25 and increases in KRT5/14 in the psoriatic epidermis26, characterized by hyperproliferation and parakeratosis27. Loricrin is a terminally differentiating structural protein comprising more than 70% of the cornified envelope, contributes to the protective barrier function of the stratum corneum28, down regulated in the skin of psoriasis patients29. Filaggrin is expressed at the final stages of keratinocyte differentiation and is involved in the aggregation of a scaffold-like cornified envelope30, decreased expression in psoriasis lesion skin29.
Overall, our goal was to generate inflammatory keratinocytes model in HaCaT cells using a cytokine combination that will be able to synergistically recapitulate some characteristics of psoriasis skin lesions, including initiating an immune response, keratinocytes proliferation and differentiation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM&#8212;Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10100139C</td>
			<td></td>
		</tr>
		<tr>
			<td>HaCaT cells</td>
			<td>China Center for Type CultureCollection</td>
			<td>GDC0106</td>
			<td>Less than 15 generations</td>
		</tr>
		<tr>
			<td>Human IL-1&#946; ELISA Kit</td>
			<td>Beyotime</td>
			<td>PI305</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-6 ELISA Kit</td>
			<td>Beyotime</td>
			<td>PI330</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-8 ELISA Kit</td>
			<td>Beyotime</td>
			<td>PI640</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-1 alpha Human</td>
			<td>Prospec</td>
			<td>CYT-253</td>
			<td>Recombinant protein</td>
		</tr>
		<tr>
			<td>IL-17 Human</td>
			<td>Prospec</td>
			<td>CYT-250</td>
			<td>Recombinant protein</td>
		</tr>
		<tr>
			<td>IL-22 Human</td>
			<td>Prospec</td>
			<td>CYT-328</td>
			<td>Recombinant protein</td>
		</tr>
		<tr>
			<td>OSM Human</td>
			<td>Prospec</td>
			<td>CYT-231</td>
			<td>Recombinant protein</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010049</td>
			<td>pH 7.4</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140163</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimeScrip RT reagent Kit</td>
			<td>TAKARA</td>
			<td>RR047A</td>
			<td></td>
		</tr>
		<tr>
			<td>TB Green Premix Ex Taq</td>
			<td>TAKARA</td>
			<td>RR420A</td>
			<td></td>
		</tr>
		<tr>
			<td>TNF alpha Human</td>
			<td>Prospec</td>
			<td>CYT-223</td>
			<td>Recombinant protein</td>
		</tr>
		<tr>
			<td>TRIzo Reagent</td>
			<td>Invitrogen</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Gibco</td>
			<td>25200072</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishing transcription profile of psoriasiform cutaneous in vitro using hacat cells stimulated with combination of cytokines

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-1&#945;, TNF-&#945;, 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-1&#946;, 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.

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...

Read this Scientific Article Protocol about Establishing Transcription Profile of Psoriasiform Cutaneous In Vitro using HaCaT Cells Stimulated with Combination of Cytokines at JoVE.com

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

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-1&#945;, TNF-&#945;, OSM) on HaCaT cell line.

Psoriasis is a common chronic inflammatory skin disease mediated by innate and adaptive immune systems, characterized by abnormal proliferation and ...

establishing-transcription-profile-psoriasiform-cutaneous-vitro-using

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

Abstract