IL-36β Promotes Inflammatory Activity and Inhibits Differentiation of Keratinocytes In Vitro

Wenming Wang, Chao Wu, Xiaoling Yu, Hongzhong Jin＊

1Department of Dermatology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China

2Department of Dermatology, Dermatology Hospital of Southern Medical University,Guangdong Provincial Dermatology Hospital, Guangdong 510080, China

Key words: interleukin-36β; psoriasis; keratinocytes; inflammatory activity; differentiation

Objective Psoriasis is an immune-mediated inflammatory disease. Despite advances in the study of its pathogenesis, the exact development mechanism of psoriasis remains to be fully elucidated. Hyperproliferative epidermis plays a crucial role in psoriasis. This study aimed to investigate the effects of interleukin-36β (IL-36β)on keratinocyte dysfunction in vitro.Methods Human keratinocyte cell lines, HaCaT cells, were treated with 0 (control), 50 or 100 ng/ml IL-36β respectively for 24 h. Cell viability was determined with a cell counting kit-8 assay. Flow cytometry was used to assess the effects of IL-36β on apoptosis and cell cycle distribution. Expressions of the differentiation markers, such as keratin 10 and involucrin, were evaluated by quantitative real-time polymerase chain reaction (RT-qPCR). Expressions of the inflammatory cytokines, IL-1β and IL-6 were tested by ELISA.Results CCK8 assay showed the survival rate had no significant difference between the control and treated group (P ＞ 0.05). Flow cytometry analysis showed cell cycle arrest at S phase in the IL-36β-treated groups compared with the control group (P ＜ 0.05). RT-qPCR verified the decreased mRNA expressions of keratin 10 and involucrin in the IL-36β-treated groups compared with the negative control (P ＜ 0.01). ELISA showed 100 ng/ml IL-36β enhanced levels of IL-1β and IL-6 in culture supernatants of HaCaT cells compared with the negative control (P ＜ 0.05).Conclusion Taken together, these findings suggest that IL-36β could induce cell cycle arrest at S phase, inhibit keratin 10 and involucrin expressions and promote inflammatory activity in HaCaT cell lines.

AS a chronic, inflammatory skin disorder,psoriasis is caused by genetic predisposition and can be affected by environmental factors.[1,2]Histologically, it is characterized by excessive growth and aberrant differentiation of keratinocytes.[3]Secreted products of psoriatic keratinocytes influence immune activation, and thereby activated immune cells alter keratinocyte responses. Thus, crosstalk between keratinocytes and immune cells shapes and maintains the inflammatory status.[4-6]The interleukin-1 (IL-1) family consisting of 11 cytokines, plays important roles in immune and inflammatory processes.[7,8]It has showed that the IL-1 family plays an important role in the pathogenesis of psoriasis.[9]IL-36, including IL-36α, IL-36βand IL-36γisoforms, is a relatively newly characterized member of the IL-1 cytokine family.[10-12]Recent studies have demonstrated that IL-36 can promote myeloid cell infiltration, activation and inflammatory activity in skin.[10,13,14]In addition, IL-36 was found to be overexpressed in human psoriatic damaged skin and mouse models of psoriasis.[14-16]IL-36 expression is correlated with the upregulation of cytokines involved in the pathogenesis of psoriasis, such as IL-17A, IL-22, tumor necrosis factor-α, IL-23, and interferon-γ.[17,18]

Previous studies showed that IL-36βcan induce inflammatory gene expression in mature adipocytes[19]and exert pro-inflammatory effects in primary human joint cells.[20]The expression of IL-36βwas significantly increased in plaque psoriasis skin compared with healthy controls.[15]However, the association between changes of IL-36βexpression and the inflammatory process of psoriasis is still not fully elucidated. The goal of our present study was to address the effects of IL-36βon keratinocytes.

MATERIALS AND METHODS

Cell culture and grouping

HaCaT cells are an immortalized, human keratinocyte line. HaCaT cells, provided by China Infrastructure of Cell Line Resources, Beijing, China, were grown in minimum essential medium/Earle’s balanced salt solution (HyClone, Logan, UT, USA) supplemented with 10% inactivated fetal bovine serum (VACCA Biologics,Green Bay, WI, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (HyClone, Logan, UT, USA), in a humidified atmosphere of 5% CO2at 37°C.

HaCaT cells were grown overnight without treatment in 96-well plates (1×104cells/well). Then the cells were divided into the control, 50 and 100 ng/ml IL-36βgroups (3 wells in each group) and treated with 0, 50 or 100 ng/ml IL-36β(R&D Systems, Minneapolis,MN, USA) respectively for 24 h for further analysis.

Cell viability assay

Cell viability was assessed using the Cell Counting Kit-8 (CCK8) assay (Dojindo Laboratories, Kumamoto,Japan) according to the manufacturer’s instructions.The absorbance was measured at 450 nm to determine the cell viability with Thermo Scientific Varioskan Flash (Thermo Fisher Scientific, Waltham, MA, USA).Survival rate was calculated from optical density (OD)measurements according to the formula: Survival rate(%) = (ODtreated- ODblank)/(ODcontrol- ODblank) × 100%.

Flow cytometry analysis

After 24 h incubation, IL-36β-treated or -untreated HaCaT cells (Control) were harvested and stained with Annexin-V-fluorescein isothiocyanate (FITC) and propidium iodide (PI; KeyGEN BioTECH, Nanjing, China) to analyze apoptosis by flow cytometry, according to the manufacturer’s protocol. IL-36β-treated or -untreated HaCaT cells were incubated with PI after fixation with 70% ethanol overnight at 4°C to analyze cell cycle. The cells were then analyzed on an Accuri C6 cytometer (BD Biosciences, San Jose, CA, USA).The cell cycle distribution and apoptosis were analyzed using the ModFit program version 3.1 (Verity Software House, Inc., Topsham, ME, USA).

Quantitative real-time reverse transcriptionpolymerase chain reaction (RT-qPCR)

Total RNA was isolated from cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Total RNA (500 ng) from each sample was reverse-transcribed using Goscript?Reverse Transcription System (Thermo Fisher Scientifc,Waltham, MA, USA). PCR amplification was performed on an Applied Biosystems 7500 Real-Time PCR System(Applied Biosystems, Foster City, CA, USA) using Fast-Start Universal SYBR Green (Roche, Pleasanton, CA,USA). The PCR primers used were as follows: forward primer, 5′-TCCTCCAGTCAATACCCATCAG-3′ and reverse primer, 5′-CAGCAGTCATGTGCTTTTCCT-3′ for involucrin;forward primer, 5′-GAGCAAGGAACTGACTACAG-3′ and reverse primer, 5′-CTCGGTTTCAGCTCGAATCT-3′ for keratin 10; forward primer, 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ and reverse primer, 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′ for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH was used as internal control. The relative quantification of mRNA expression was calculated using the formula 2-ΔΔCt.

Enzyme-linked immunosorbent assay (ELISA)

After stimulation for 24 h, cell culture supernatants were collected and stored at -80°C until analysis. The proteins concentrations of IL-1βand IL-6 in the supernatants were measured using ELISA kits (NeoBioScience Technology Company, Shenzhen, China) according to the manufacturer’s instructions.

Statistical analysis

Data were analyzed using SPSS 21.0 for Windows (SPSS,Chicago, IL, USA). Numerical data were presented as the mean ± standard error of mean (SEM). One-way analysis of variance with post hoc least significant difference (LSD) was carried out to determine the statistical significance among groups.P-values less than 0.05 were considered to indicate a significant difference.

RESULTS

Effects of IL-36β on cell proliferation

CCK8 assay showed the survival rate had no significant difference between the 50 ng/ml group and the Control group (0.850 ± 0.077vs. 0.971 ± 0.017,F= 1.232,P= 0.228), between the 100 ng/ml group and the Control group (0.847 ± 0.077vs. 0.971 ± 0.017,F= 1.232,P= 0.218).

Effects of IL-36β on apoptosis and cell cycle

Flow cytometry (Figure 1) revealed the apoptosis rates had no significant difference between the 50 ng/ml IL-36βgroup and the Control group (11.700% ±0.608%vs. 12.767% ± 0.636%,F= 1.405,P= 0.291),between the 100 ng/ml IL-36βgroup and the Control group (11.267% ± 0.706%vs. 12.767% ± 0.636%,F= 1.405,P= 0.154).

As illustrated in Figure 2, flow cytometry showed in the IL-36β-treated groups, the percent of G0/G1 phase cells was significantly lower than that in the Control group (control group 47.350%±2.019%, 50 ng/ml group 40.097% ± 1.541%,F= 5.508,P= 0.020;100 ng/ml group 41.653%±1.221%,F= 5.508,P= 0.048); the retardation of cell cycle at S phase was significantly higher compared with the controls(control group 35.383%±1.575%; 50 ng/ml group 44.203% ± 1.052%,F= 7.899,P= 0.009; 100 ng/ml group 41.850%±2.082%,F= 7.899,P= 0.031).

Effects of IL-36β on expression of keratin 10 and involucrin mRNA

RT-qPCR showed the expression of keratin 10 mRNA was significantly lower in the 50 ng/ml group (0.404 ±0.022,F= 34.687,P= 0.000) and the 100 ng/ml group (0.430 ± 0.093,F= 34.687,P= 0.000) than that in the Control group (1.125 ± 0.072). The expression of involucrin mRNA decreased significantly in the 50 ng/ml group (0.391 ± 0.051,F= 30.047,P= 0.001) and the 100 ng/ml group (0.383 ± 0.013,F= 30.047,P= 0.001)vs. the Control group (0.902 ±0.078).

Effects of IL-36β on inflammatory cytokines

Figure 1. Representative dot-plots of flow cytometric analysis of HaCaT cells treated with IL-36β (0, 50 or 100 ng/ml) for 24 h.

Figure 2. Effect of IL-36β on HaCaT cell cycle.

ELISA revealed IL-1βin culture supernatants after treatment with 100 ng/ml IL-36β(6.360 ± 0.458 pg/ml)was significantly higher than that of the control group(4.443 ± 0.301 pg/ml,F= 12.935,P= 0.007), but that in culture supernatants of HaCaT cells treated with 50 ng/ml IL-36βwas slightly lower than that of the control group (4.153 ± 0.183 pg/ml,F= 12.935,P= 0.561).In addition, there was significantly more IL-6 in culture supernatants of the 50 ng/ml group (81.283 ±4.538 pg/ml,F= 9.284,P= 0.006) and the 100 ng/ml group (62.037 ± 17.130 pg/ml,F= 9.284,P= 0.027)vs. the control group (19.317 ± 3.320 pg/ml).

DISCUSSION

Here, we report that IL-36βtreatment of HaCaT keratinocytes induced the expression of pro-inflammatory cytokines, IL-1βand IL-6, which are also known to be upregulated in psoriasis patients.[21,22]In addition,IL-36βwas able to inhibit differentiation of HaCaT keratinocytes, suggesting that IL-36βmay be responsible for the aberrant differentiation and excessive growth seen in psoriatic lesions. Therefore, we suggest that IL-36βmay be a new molecular target for psoriasis.

Previous studies have shown that IL-36βplays a role in the pathogenesis of obesity,[19]spondylitis ankylopoetica,[23]rheumatoid arthritis[20]and psoriatic arthritis.[24,25]In consistent with our study, D'Ermeet al.[26]have demonstrated that IL-36βcan promote the expression of many pro-inflammatory genes in primary keratinocytes, such as IL-17C,β-defensin-2, S100A9, tumor necrosis factorα(TNF-α),and granulocyte colony-stimulating factor (G-CSF). In this study, we treated HaCaT keratinocytes with IL-36βand found that it upregulated the levels of the inflammatory factors, IL-1βand IL-6, indicating a pro-inflammatory role of IL-36βin these cells. However, the function of IL-36βin the inflammatory process of psoriasis need more studies to clarfy.

Skin consists of three layers, the epidermis,dermis and hypodermis.[27]The epidermis is mainly composed of keratinocytes, and normal keratinocyte differentiation is important for the formation of an intact epidermal barrier.[28-30]Previous studies have demonstrated that aberrant keratinocyte differentiation is involved in the pathogenesis of psoriasis.[31,32]Involucrin is a late marker of keratinocyte differentiation and keratin 10 is a terminal marker of keratinocyte differentiation.[33,34]In this study, we found that the added IL-36βnegatively regulated keratin 10 and involucrin mRNA expressions of HaCaT keratinocytes,which indicates a role of IL-36βin the perturbation of keratinocyte differentiation.

Accelerated epithelial cell proliferation is a crucial element in psoriasis and is thought to be related to a speeding up of the cell cycle or a shortening of the cell division time (19 days in normal epidermisversus37.5 h in psoriatic epidermis).[35,36]Previous studies also showed that the ability of psoriatic keratinocytes to resist apoptosis may play a crucial role in the pathogenesis of psoriasis.[37,38]In this study, we found that IL-36βhad no obvious influence on the proliferation or apoptosis of HaCaT keratinocytes. However, our work demonstrated that IL-36βcan promote the transition of HaCaT keratinocytes from G1 into S phase. Our study indicates that IL-36βmay enhance the proliferation ability of HaCaT keratinocytesin vitro.

Taken together, our study indicates that IL-36βmay be involved in skin inflammation. The limitation of the present study is that HaCaT cells cannot fully represent the pathogenesis of psoriasisin vivo. Further studies should be conducted regarding the role of IL-36βin patients with psoriasis.

Conflicts of interest statement

All authors declare no conflicts of interest.