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

Resumo

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.

Resumo

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.

Introdução

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 diseases^5,6,7,8. 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'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-γ 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-α, IL-6, IFN-β) 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-1α, oncostatin M(OSM), and TNF-α) produced by immune cell infiltration has been highlighted^12,13. Indeed, previous studies have shown that increased levels of IL-17A, IL-22, IL-1α, TNF-α, 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 psoriasis^{15,16,17,18,19,20}. 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 filaggrin^20,21,22, 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 KRT1/10^24,25 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.

Protocolo

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 PBS and add 2 mL of 0.25% trypsin-EDTA solution to the cells. Gently shake the culture dish to let the solution completely coat the monolayer of cells.
4. Incubate the cells at 37 °C in a humidified 5% CO₂ incubator for 5 min until the cells are visibly detached from the surface of the culture dish under phase-contrast microscopy.
   NOTE: The morphology of cells should appear round. If the cells are not well detached, incubate for an additional period of 5 min along with manual agitation.
5. Once the cells are detached, add 5 mL of DMEM plus 10% FBS to inactivate trypsin and collect the cell suspension in a 50 mL centrifuge tube using a pipette.
6. Centrifuge the tube containing cell suspension at 180 x g for 5 min. Decant the supernatant.
7. Add 10 mL of DMEM plus 10% FBS medium to the pellet and gently pipette the medium up and down to bring the cells into suspension.

2. Cell seeding in the 6-well plate

1. Seed the cells at a density of 2.0 x 10⁵ cells/well in 6-well culture plate.
2. Incubate the 6-well culture plate at 37 °C in a humidified 5% CO₂ incubator overnight before M5 combination stimulation to let the cells adhere to the plate.

3. M5 stimulation of HaCaT cells

1. Prepare M5 cytokine combination mix medium containing 10 ng/mL of recombinant IL-17A, IL-22, IL-1α, TNF-α, and oncostatin M protein in DMEM containing 2% FBS.
   NOTE: M5 combination consisted of recombination of IL-17A, IL-22, IL-1α, TNF-α, and oncostatin M. Synergistic action of M5 combination recapitulates some features of psoriasis, like upregulation of chemokines and antimicrobial peptides production³¹.
2. After overnight cell culture, remove the supernatant, and pipette 2 mL of M5 combination mix medium into each well.
3. Continue culturing the cells; cell lysates were collected at 24 h for mRNA quantification (step 4) or culture supernatant were collected at 48-72 h for the determination of cytokine levels by ELISA assay^{32,33,34,35,36,37} (step 6).

4. Harvest mRNA of M5 stimulated HaCaT cells

1. Aspirate the M5 combination mix medium and wash once with 1-2 mL of cold PBS.
2. Aspirate PBS and add 1 mL of commercially available guanidium Isothiocyanate solution directly to the culture dish to lyse the cells.
3. Pipette the lysate up and down several times to homogenize and transfer the cell lysate into a 1.5 mL microcentrifuge tube. Leave at room temperature for 5 min.
4. Add 200 µL of chloroform, shake the tube vigorously for about 20 s and incubate the sample for 5 min at room temperature.
5. Centrifuge at 12,000 x g for 10 min at 4 °C.
6. Transfer the aqueous phase to a fresh 1.5 mL microcentrifuge tube.
7. Add 500 µL of isopropanol to the aqueous phase and mix gently. Leave at room temperature for 5 min.
8. Centrifuge at 10,000 x g for 15 min at 4 °C.
9. Aspirate isopropanol and add 1 mL of 75% ethanol. Wash the pellet once.
10. Centrifuge at 7,500 x g for 5 min at 4 °C. Pour off the ethanol and let the pellets air-dry.
11. Add 30 µL DEPC treated water to the RNA pellet. Prepare for RT-PCR detection.

5. Analysis of mRNA expression by Real-Time PCR

1. Perform cDNA synthesis with 1 µg of total RNA using a commercially available kit following the manufacturer's instruction.
   NOTE: 1 µg of total RNA for SYBR Green RT-PCR assay.
2. Prepare the PCR reaction mixture. 12.5 µL of SYBR premix EX Taq II, 1µL of PCR Forward Primer (10 µM), 1 µL of PCR Reverse Primer (10 µM), 2 µg of cDNA, and 8.5 µL of dH₂O. The final volume is 25 µL per reaction.
   NOTE: List of primer sequences of genes used for RT-PCR analysis in this study is shown in Table 1.
3. Incubate in thermocycler. The cycling conditions included a denaturing step at 95 °C for 30 s, 40 cycles of 95 °C for 5 s, 60 °C for 30 s, 72°C for 20s and a melting curve analysis.
4. Analyze RT-PCR experiment data by calculating relative differences in gene expression from Ct (threshold cycle) values using 2^-ΔΔCTequations. Use β-actin as an internal control for RT-PCR relative quantitative gene expression standardization.

6. Harvesting cell culture supernatant for ELISA

1. Pipette each cell culture media into a 1.5 mL microcentrifuge tube.
2. Centrifuge at 1500 x g for 10 min at 4 °C.
3. Aliquot the supernatant and store at -80 °C immediately.

7. Analysis of cytokines expression by ELISA

1. Follow the manufacturer's instruction to prepare all reagents, working standard, and samples. A three-fold serial dilution of standard with sample diluent ranging from 2,000 to 2.74 pg/mL. Dilute samples 1:2 with sample diluent.
2. Add 100 µL of standard and sample per well. Cover wells with lid and seal the plate with paraffin film. Incubate for 2.5 h at room temperature.
3. After 2.5 h, discard the solution. Add 300 µL of wash buffer to each well of the plate for 3 min and aspirate. Repeat the process for four additional times. After the last wash, invert the plate and tap on absorbent paper to remove remaining liquid.
4. Add 100 µL of the detection antibody solution to each well. Incubate the plate on a shaker for 2 h at 37 °C.
   NOTE: Biotinylated detection antibody solution is prepared in each ELISA kits.
5. Aspirate and wash each well of plate five times using 300 µL of Wash buffer. After the last wash, invert the plate and tap on absorbent paper to remove the remaining liquid.
6. Add 100 µL of HRP-Streptavidin conjugate to each well. Incubate for 45 min at room temperature with gentle shaking.
7. Discard the solution by aspirating with a pipette after the incubation. Wash each well of plate five times using 300 µL of Wash buffer. After the last wash, invert the plate and tap on the absorbent paper to remove the remaining liquid.
8. Perform detection by adding 100 µL of TMB substrate to each well. Incubate for 30 min at 37 °C in the dark.
9. Add 50 µL of stop solution, when color develops, to each well. Gently tap the plate to ensure thorough mixing. Read at 450 nm immediately.

Resultados

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...

Discussão

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...

Materiais

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