Xue-Cui Wei, Yu-Jie Chen, Fang-Yuan Long, Shan-Shan Yu, Song Xu, Xu Chen,,＊
1 School of Public Health, Nanjing Medical University, Nanjing, Jiangsu 211166, China; 2 Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Hospital for Skin Diseases (Institute of Dermatology), Chinese Academy of Medical Sciences & Peking Union Medical College, Nanjing, Jiangsu 210042, China; 3 Key Laboratory of Basic and Translational Research on Immune-Mediated Skin Diseases, Hospital for Skin Diseases (Institute of Dermatology), Chinese Academy of Medical Sciences & Peking Union Medical College, Nanjing, Jiangsu 210042, China.
Abstract
Keywords: damage-associated molecular patterns, interleukin-1β, high-mobility group box 1, keratinocytes,inflammatory skin disorders
Inflammation is a complex bodily defense system that is activated in response to endogenous and exogenous stimuli, and it is the immune system’s fastest reaction to tissue damage.1The body initiates an inflammatory response2when exposed to internal or external stimuli such as pathogens, harmful stimuli (eg, chemicals), or physical damage (eg, ultraviolet B [UVB]3.Usually, inflammation is beneficial and is an automatic defense response of the body.However, inflammation can sometimes be harmful;in such cases, various inflammatory cytokines may damage the body’s tissues.Some researchers refer to inflammation induced by non-pathogens and their products as aseptic inflammation, in which the involved molecular patterns of immune stimulation are called damage-associated molecular patterns (DAMPs).4
DAMPs are molecules released in response to cellular stress or tissue injury.They are considered endogenous danger signals that regulate immune effects, such as intrinsic immunity, by interaction with pattern recognition receptors including NOD-like receptors (NLRs) and Toll-like receptors.5-6DAMPs can have different sources,including the extracellular matrix and various compartments or organelles within the cell.Intracellular DAMPs include high-mobility group box 1 (HMGB1), which can be secreted from the nucleus and autophagosomes;interleukin-1β (IL-1β), which originates from autophagosomes; and various DAMPs such as mitochondrial DNA,adenosine triphosphate, and transcription factor A from mitochondria and hyaluronic acid, versican, and fibrinogen from the extracellular matrix.5,7-11
The skin is an organ that is in direct contact with the external environment and has the roles of protection,excretion, body temperature regulation, and sensation of external stimuli.Its surface comprises epidermal cells, most of which are keratinocytes.Keratinocytes are the structural cells that make up the epidermis.The diversity of the biological functions of the epidermis is gradually being recognized.The epidermis can respond to numerous exogenous and endogenous stimuli; it is an important and active participant in the skin’s immune response as well as an important component of post-inflammatory tissue repair.12-13Keratinocytes play an important role in skin barrier function and are frequently exposed to adverse external factors.Research has shown that keratinocytes can secrete various DAMPs,14-16such as HMGB1, non-coding double-stranded RNA, and low-molecular-weight hyaluronan.
IL-1β is a considerable mediator of inflammatory reactions.It is involved in multiple cellular activities;for example, it is released from cells after stimulation by pathogen-associated molecular patterns or other DAMPs, and it is also released from necrotic cells.17-18HMGB1 is widely present in mammalian cells; it is an evolutionarily conserved nuclear protein and has been found to act as a key player in a variety of inflammatory responses.19-20An excessive inflammatory response can adversely affect the organism, and common cutaneous diseases resulting from excessive inflammatory responses include skin diseases caused by UV damage, contact dermatitis, and psoriasis.The present study was performed to explore and compare the transcription and secretion of HMGB1 and IL-1β in keratinocytes after 3 types of damage: UVB irradiation; tumor necrosis factor-α(TNF-α) and interferon-γ (IFN-γ) (TI) co-stimulation(simulation of T helper 1 cell inflammatory challenge21);and treatment with M5, a mixture of 5 proinflammatory cytokines (TNF-α, oncostatin M, IL-1α, IL-17A, and IL-22) as a psoriasiform modelin vitro.22
Antibodies
The antibodies used in this study were purchased from Cell Signaling Technology (Danvers, MA, USA).The primary antibodies were glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (D16H11) (#5174), HMGB1(D3E5) (#6893), histone deacetylase 1 (HDAC1) (10E2)(#5356), and cleaved IL-1β (Asp116) (D3A3Z) (#83186).The secondary antibody was anti-rabbit immunoglobulin G, horseradish peroxidase-linked antibody (#7074).
Cell culture
We used Dulbecco’s Modified Eagle Medium which contains 1% Antibiotic-Antimycotic and 10% fetal bovine serum to culture HaCaT cells (China Center for Type Culture Collection, Wuhan, China).In this study, Dulbecco’s Modified Eagle Medium, Antibiotic-Antimycotic, and fetal bovine serum were Gibco products purchased from Thermo Fisher Scientific (Waltham, MA, USA).
UVB irradiation
The cells were exposed to UVB at 50 mJ/cm2as previously described.23Before UVB irradiation, we first used an irradiator to determine the intensity of irradiation.After the irradiator display had become stable, the ratio of irradiation intensity and total dose (50 mJ/cm2) was used to determine the irradiation time.The control cells were subjected to the same operation as the irradiated cells with the exception of the irradiation.The cells were then incubated in an incubator until the corresponding experimental time point.
TI co-stimulation
The cells were stimulated with 20 ng/mL of TI (TNF-α[R&D Systems, Minneapolis, MN, USA] and IFN-γ[PeproTech, Cranbury, NJ, USA]) as previously described.21We cultured the cells in 6-well plates, and when the density was appropriate, the cells were cultured in medium with or without 20 ng/mL of TI until the indicated time points.
M5 treatment
The cells were treated with 10 ng/mL of M5 (TNF-α,oncostatin M, IL-1α, IL-17A, and IL-22; all from R&D Systems) as previously described.22We cultured the cells in 6-well plates.When the density was appropriate, the cells were incubated with serum-free medium for 24 hours and then stimulated with or without 10 ng/mL of M5 for the appropriate time.
Western blotting
The nuclear and cytoplasmic proteins of HaCaT cells were extracted by an NE-PER™ Nuclear and Cytoplasmic Extraction Reagent Kit (#78833; Thermo Fisher Scientific).And a bicinchoninic acid assay (BCA Assay Kit; Beyotime Biotechnology, Shanghai, China) was used to detect the protein concentrations in the samples.Protein samples were prepared in equal masses, and each protein sample was loaded into Smart PAGE™ Precast Protein Gel(4%-20%; Smart-Lifesciences Biotechnology, Changzhou,China).Secretory proteins were extracted from an equal volume of cell culture medium using methanol and chloroform, and the proteins were then dried using a freeze dryer to obtain secretory protein samples.The secreted proteins obtained were then dissolved with RIPA lysis buffer (P0013B, Beyotime Biotechnology), which contains a phosphatase inhibitor (PhosSTOP; Roche Applied Science, Basel, Switzerland) and a protease inhibitor cocktail (Roche Applied Science).Subsequent equal-volume preparation samples were loaded into preformed gels.After electrophoresis, the proteins were transferred to the polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA, USA), followed by EveryBlot Blocking Buffer (Bio-Rad Laboratories) blocking the PVDF membranes for 10 minutes.The PVDF membranes containing the target band were then incubated with the indicated primary antibodies at 4°C overnight.We washed the bands with 1× Tris-buffered saline with Tween®20(TBST) on the next day, then incubated the bands with the secondary antibodies for approximately 1 hour.We then washed the bands again and performed imaging of the indicated protein bands by chemiluminescence (Bio-Rad Laboratories).GAPDH and HDAC1 served as the loading controls in this study.We used ImageJ software to quantify the density of the protein bands of interest.The data were calculated by the following formula: ratio of the intensity of the target protein to the intensity of the corresponding loading control.
RNA extraction and quantitative reverse-transcription polymerase chain reaction analysis
Total RNA of HaCaT cells was extracted by a SteadyPure Universal RNA Extraction Kit.Subsequently the samples of RNA were reversely transcribed to complementary DNA by Evo M-MLV RT Premix for qPCR in accordance with the manufacturer’s instructions.Quantitative PCR was conducted on LightCycler®480 Instrument II (Roche Diagnostics) using a SYBR Green Premix Pro Taq HS qPCR Kit.The data are presented as fold differences by 2-ΔΔCTmethod, andGAPDHserved as the reference gene.Table 1 lists the primer sequences.The reagents used in this experiment were all purchased from Accurate Biotechnology (Hunan, China).
Table 1 Names and sequences of the 3 target genes.
Enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent assay (ELISA) kits were used to detect the secretion of IL-1β and HMGB1 in this study.The ELISA kits included Human IL-1β (#CHE0001;Beijing 4 A Biotech, Beijing, China), HMGB1 Detection Kit (#6010; Chondrex, Woodinville, WA, USA), and Human HMGB1 ELISA Kit (E-EL-H1554c; Elabscience Biotechnology, Wuhan, China).
Statistical analysis
All independent experiments were performed with at least 3 replications.Statistical analysis was performed with SPSS software.The data, which are shown as mean± standard deviation, were analyzed by the Student’sttest.ThePvalues of ＜0.05 were regarded as statistically significant.
mRNA and protein levels of IL-1β and HMGB1 in HaCaT cells exposed to UVB irradiation
After UVB irradiation, the results of quantitative reverse-transcription polymerase chain reaction (qRTPCR) assay showed that theIL-1βtranscription level was 1.887 ± 0.270 in UVB-challenged HaCaT cells and 0.966 ± 0.137 in the control at the 12th hour post-exposure.At the 24th hour after UVB irradiation, theIL-1βtranscription level was 5.201 ± 0.796 in UVB-challenged HaCaT cells and 1.003 ± 0.069 in the control (P＜ 0.001)(Fig.1A).The ELISA results showed that the IL-1β protein level was 19.818 ± 4.848 pg/mL in UVB-challenged HaCaT cells and 4.098 ± 0.826 pg/mL in the control at the 48th hour post-irradiation (P= 0.027) (Fig.1B).The above results showed that the transcription level ofIL-1βincreased at both the 12th and 24th hour after UVB irradiation and that the protein secretion increased at the 48th hour post-irradiation (P＜ 0.05).
Figure 1.Transcription and secretion of IL-1β in HaCaT cells after UVB irradiation.HaCaT cells were exposed or not exposed to 50 mJ/cm2 UVB.(A) qRT-PCR analysis of mRNA expression of IL-1β at the indicated time after UVB irradiation (n = 9).(B) ELISA was performed to assess the level of IL-1β in the culture medium at the 48th hour after UVB irradiation (n = 3).All data are presented as mean ± standard deviation.＊P ＜ 0.05, ＊＊＊P ＜ 0.001.ELISA: enzyme-linked immunosorbent assay; IL-1β: interleukin-1β; NS: nonsense; qRT-PCR: quantitative reverse-transcription polymerase chain reaction; CON: control; UVB: ultraviolet B; h: hour.
After UVB irradiation, the results of qRT-PCR assay showed that theHMGB1transcription level was 0.449 ± 0.212 in UVB-challenged HaCaT cells and 1.048 ± 0.077 in the control at the 12th hour post-exposure.At the 24th hour after UVB irradiation, theHMGB1transcription level was 0.306 ± 0.147 in UVB-challenged HaCaT cells and 1.009 ± 0.077 in the control (P＜ 0.001)(Fig.2A).The expression of HMGB1 in the nucleus and cytoplasm did not show significant differences between the 2 groups at the 24th hour post-irradiation.The quantitative results showed no statistically significant differences (P＞ 0.05) (nucleus:P= 0.436, cytoplasm:P= 0.361) (Fig.2B).Furthermore, we determined the protein secretion level of HMGB1 from HaCaT cells by ELISA at the 48th hour after UVB irradiation (Fig.2C).We found that the secretion level of HMGB1 was 876.630 ± 164.648 pg/mL in UVB-challenged HaCaT cells and 607.471 ± 248.643 pg/mL in the control at the 48th hour post-irradiation (P= 0.016).
mRNA and protein levels of IL-1β and HMGB1 in HaCaT cells after TI co-stimulation
Figure 2.Transcription levels and protein expression of HMGB1 in HaCaT cells after UVB irradiation.HaCaT cells were exposed or not exposed to 50 mJ/cm2 UVB.(A) The mRNA expression of HMGB1 was detected by qRT-PCR at the 12th and 24th hour after UVB exposure(n = 9).(B) At the 24th hour after UVB exposure, expression of HMGB1 in the cytoplasm and nucleus was determined by western blotting.GAPDH and HDAC1 served as the loading controls.Quantification of the protein bands of interest was shown (n = 3).(C) At the 48th hour after UVB exposure, secretion of HMGB1 was determined by ELISA.All data are presented as mean ± standard deviation (n = 9).＊P ＜ 0.05,＊＊＊P ＜ 0.001.ELISA: enzyme-linked immunosorbent assay; NS: nonsense; qRT-PCR: quantitative reverse-transcription polymerase chain reaction; CON: control; UVB: ultraviolet B; h: hour.
The qRT-PCR assay showed that theIL-1βtranscription level was 4.306 ± 0.477 in stimulated HaCaT cells and 1.002 ± 0.066 in the control after 12 hours of co-stimulation with 20 ng/mL TI and 9.655 ± 3.413 in stimulated HaCaT cells and 1.001 ± 0.051 in the control after 24 hours of co-stimulation (P＜ 0.001) (Fig.3A).The results of western blotting showed increased secretion of IL-1β protein after 24 and 48 hours of stimulation (Fig.3B).
The qRT-PCR assay showed that theHMGB1transcription level was 0.816 ± 0.048 in stimulated HaCaT cells and 1.002 ± 0.066 in the control after 12 hours of co-stimulation with TI and 0.625 ± 0.038 in stimulated HaCaT cells and 1.000 ± 0.014 in the control after 24 hours of co-stimulation (P＜ 0.001) (Fig.4A).The data showed that the transcription level ofHMGB1decreased at both 12 and 24 hours of stimulation (P＜ 0.05).There was no difference in HMGB1 expression found in the nucleus and cytoplasm after 24 hours of stimulation by western blotting.The quantitative results showed no statistically significant differences (P＞ 0.05) (nucleus:P=0.519, cytoplasm:P= 0.445) (Fig.4B).After 48 hours of stimulation, secretion of HMGB1 was increased in HaCaT cells (P＜ 0.05).The ELISA results showed that the HMGB1 protein level was 824.616 ± 86.190 pg/mL in treated HaCaT cells and 679.660 ± 77.066 pg/mL in control cells (P= 0.002) (Fig.4C).
mRNA and protein levels of IL-1β and HMGB1 in HaCaT cells after M5 treatment.
The results showed that theIL-1βtranscription level was 14.479 ± 5.311 in treated HaCaT cells and 1.003 ± 0.087 in the control after 12 hours of M5 treatment and 9.494 ± 2.345 in treated HaCaT cells and 1.002 ± 0.072 in the control group after 24 hours of M5 treatment (P＜0.001) (Fig.5A).The ELISA results showed that the IL-1β protein level was 79.503 ± 8.908 pg/mL in treated HaCaT cells and 22.774 ± 2.570 pg/mL in the control (P＜ 0.001)(Fig.5B).
Figure 3.Transcription and secretion of IL-1β in HaCaT cells after TI co-stimulation.HaCaT cells were stimulated with or without 20 ng/mL of TI for the indicated times.(A) The RNA levels of IL-1β were assessed by qRT-PCR (n = 9).(B) Secretion of IL-1β was measured by western blotting.All data are presented as mean ± standard deviation.＊＊＊P ＜ 0.001.IL-1β: interleukin-1β; NS: nonsense; qRT-PCR:quantitative reverse-transcription polymerase chain reaction; CON:control; TI: tumor necrosis factor - α and interferon - γ; h: hour.
To explore the changes in HMGB1 transcription and secretion levels in keratinocytes, we treated HaCaT cells with the same method.The qRT-PCR assay showed that theHMGB1transcription level was 0.705 ± 0.050 in treated HaCaT cells and 1.003 ± 0.076 in the control after 12 hours of M5 treatment and 0.605 ± 0.023 in treated HaCaT cells and 1.003 ± 0.084 in the control after 24 hours of M5 treatment (P＜ 0.001) (Fig.6A).The differences at the transcriptional level were statistically significant(P＜ 0.05).No changes were found in the cytoplasmic or nuclear HMGB1 after 24 hours of treatment.The quantitative results showed no statistically significant differences(nucleus:P= 0.961, cytoplasm:P= 0.550) (Fig.6B).After 48 hours of treatment, the ELISA results showed that the HMGB1 protein level was 0.481 ± 0.102 ng/mL in treated HaCaT cells and 0.604 ± 0.248 ng/mL in the control(P= 0.196) (Fig.6C).The differences were not statistically significant (P＞ 0.05).
Our study demonstrates that IL-1β and HMGB1 are closely involved in the responses of keratinocytes exposed to stimulation by inflammatory damage, including UVB irradiation, treatment by TNF-α and IFN-γ, and psoriasis-like stimulation by M5 treatment.IL-1β increased at either the transcriptional level or secretory level after these 3 types of challenge.After UVB exposure or stimulation with TNF-α and IFN-γ, the secretion of HMGB1 increased; however, the secretion of HMGB1 in keratinocytes exposed to M5 treatment was distinct from that in keratinocytes exposed to UVB treatment or TI co-stimulation.We speculate that this phenomenon might be caused by different responses of epidermal keratinocytes due to the regulation of specific signaling pathways in the distinct disease scenarios.Furthermore, these differences might lead to different contributions of keratinocytes to the pathogenesis and development of skin inflammation associated with UVB exposure, contact hypersensitivity,or psoriasis.In addition, the mRNA levels of HMGB1 decreased with all 3 types of stimulation.These findings indicate that IL-1β is a highly involved cytokine in the keratinocyte response to stress stimulation because both transcription and secretion are rapidly upregulated after stimulation.Interestingly, after UVB exposure or challenge by TNF-α and IFN-γ, keratinocytes suppressed the transcription ofHMGB1while increasing secretion of HMGB1.This suggests that keratinocytes might employ negative feedback in the regulation of HMGB1 signaling to avoid an excessive inflammatory response associated with HMGB1.
Figure 4.Transcription levels and protein expression of HMGB1 in HaCaT cells after TI co-stimulation.HaCaT cells were stimulated with or without 20 ng/mL of TI.(A) qRT-PCR analysis of HMGB1 transcription levels after TI co-stimulation for 12 and 24 hours (n = 9).(B) The expression of HMGB1 in the cytoplasm and nucleus was measured by western blotting at the indicated time after stimulation.GAPDH and HDAC1 served as the loading controls.Quantification of the protein bands of interest was shown (n = 3).(C) Secretion of HMGB1 was detected by ELISA after TI co-stimulation for 48 hours (n = 9).All data are presented as mean ± standard deviation.＊＊P ＜ 0.01, ＊＊＊P ＜ 0.001.ELISA:enzyme-linked immunosorbent assay; NS: nonsense; qRT-PCR: quantitative reverse-transcription polymerase chain reaction.
Figure 5.Transcription and secretion of IL-1β in HaCaT cells after treatment with M5.HaCaT cells were stimulated with or without 10 ng/mL of M5 for the indicated times.(A) The RNA levels of IL-1β were assessed by qRT-PCR (n = 9).(B) ELISA was performed to determine the IL-1β level in the culture medium (n = 3).All data are presented as mean ± standard deviation.＊＊＊P ＜ 0.001.ELISA: enzyme-linked immunosorbent assay; IL-1β: interleukin-1β; NS: nonsense; qRT-PCR: quantitative reverse-transcription polymerase chain reaction.
IL-1β is a potent proinflammatory cytokine that is critical in the host defense response to damage and infection.24It can be actively secreted by immune cells after endogenous or exogenous stimulation or passively released by necrotic cells.11One study suggested that IL-1β secreted by keratinocytes after exposure to UVB may be an upstream signal of the inflammatory response and may promote melanogenesis.25Tsujiet al.26found that metformin suppresses the secretion of IL-1β by damaging NLRP3 inflammasomes in keratinocytes, thereby suppressing skin inflammation in mice with imiquimod (IMQ)-induced psoriasiform dermatitis.Our study reconfirms that a rapid transcriptional and secretory response of IL-1β from keratinocytes occurs in all 3 types of stimulation mimicking common inflammatory environments of skin damage induced by UV exposure, contact hypersensitivity, and psoriasis.Our study further demonstrates that IL-1β is a critical cytokine for keratinocytes to perform immunomodulatory functions in inflammatory responses.
HMGB1 is a non-histone nuclear protein which is employed as a common signal in manipulating the immune responses to tissue injury and infection.27HMGB1 can translocate to the cytoplasm and also to the extracellular space, acting as a DAMP to promote inflammatory reactions in response to danger signals.28Parket al.29found that heme inhibits the release of HMGB1 from keratinocytes by activating adenosine monophosphate-activated protein kinase or heme oxygenase-1 and scavenging reactive oxygen species, thereby reducing skin damage caused by UVB exposure.Galbiatiet al.30reported that keratinocytes exposed to contact allergens such as paraphenylenediamine, 2,4-dinitrochlorobenzene(DNCB), and citral can secrete HMGB1, which induces the synthesis and secretion of IL-18 in keratinocytes,thereby promoting the development of contact dermatitis.Zhanget al.31showed that in a mouse model of IMQinduced psoriasis, the production and secretion of IL-18 in keratinocytes was promoted by HMGB1 secreted from keratinocytes, thereby promoting the development of psoriasis.Our study revealed that keratinocytes could restrain transcription ofHMGB1when the secretion of HMGB1 was induced in certain stimuli (eg, UVB exposure or stimulation by TNF-α and IFN-γ).We speculate that this is a crucial negative regulatory mechanism in keratinocytes to avoid excessive inflammatory reactions that could lead to severe tissue damage.
Figure 6.Transcription levels and protein expression of HMGB1 in HaCaT cells after treatment with M5.HaCaT cells were treated with or without 10 ng/mL of M5.(A) HaCaT cells were stimulated with M5 for the indicated times, and the transcription of HMGB1 was assayed by qRT-PCR (n = 9).(B) The expression of HMGB1 in the cytoplasm and nucleus was determined by western blotting in HaCaT cells treated with M5 for 24 hours.GAPDH and HDAC1 served as the loading controls.Quantification of the protein bands of interest was shown (n = 3).(C) HaCaT cells were treated with M5 for 48 hours, and ELISA was performed to analyze the HMGB1 levels in the culture medium (n = 9).All data are presented as mean ± standard deviation.＊＊＊P ＜ 0.001.ELISA: enzyme-linked immunosorbent assay; NS: nonsense; qRT-PCR:quantitative reverse-transcription polymerase chain reaction.
Studies have shown that keratinocytes actively participate in the inflammatory responses in skin tissue by secreting cytokines and DAMPs.Liuet al.32found that in keratinocytes exposed to UVB radiation, knockdown of heat shock protein 27 decreased cell viability and enhanced cell apoptosis.We previously found that in a mouse model of DNCB-induced contact hypersensitivity,deficiency or inhibition of MLKL-mediated necroptosis led to blocking of the activation and recruitment of inflammatory cells in contact hypersensitivity by reducing the release of HMGB1 in keratinocytes.33Liet al.34reported that cytokine thymic stromal lymphopoietin(TSLP) in mouse keratinocytes is involved in the initiation of an atopic dermatitis-like phenotype in mice.Wilsonet al.35found that TSLP from keratinocytes enhances inflammatory responses by activating neurons and immune cells.Fu and Hong36found that osthole treatment alleviates atopic dermatitis by directly suppressing the production of TSLP from keratinocytes.Suet al.37found that IL-17A in keratinocytes activated IL-23 expression in keratinocytes via IL-17RC and IL-17RD signaling to promote inflammatory responses in a mouse model of IMQ-induced psoriasis-like dermatitis.Thus,these studies suggest that keratinocytes, as important structural cells of skin tissue, not only are the key target tissues in damage caused by inflammatory diseases but also actively participate in the regulation of promoting inflammatory reactions through their autocrine function.Critically, in psoriasis, keratinocyte-derived secreted substances (including cytokines and DAMPs) were discovered to play a key role in amplifying the psoriatic inflammatory circuit.Therefore, clarifying the details of the regulatory mechanism by which keratinocytes produce and secrete key cytokines and DAMPs under inflammatory conditions is of great value for exploring new strategies to intervene in inflammatory skin diseases.The main limitation of this study is that the results were obtained using keratinocytes cultured in vitro.Further investigations should involve mice exposed to UV radiation, stimulation of contact allergens, or challenges mimicking psoriasis-like dermatitis.
In conclusion, IL-1β is a vital cytokine that has a critical immunomodulatory function in keratinocytes in the inflammatory response.This is supported by its elevated secretion in 3 common inflammatory microenvironments in the present study.In UVB irradiation or TNF-α and IFN-γ co-stimulation, the change in secretion of HMGB1 was consistent with that of IL-1β, whereas the transcription level of HMGB1 was restrained despite its increased secretion.
This work was supported by grants from the National Natural Science Foundation of China (81972952,81872531, 82173438, and 82273550), the Nanjing Incubation Program for National Clinical Research Center (2019060001), CAMS Innovation Fund for Medical Sciences (2021-1-I2M-059, 2017-I2M-1-017),Jiangsu Province Foundation (LGY2018095, WSW-016),PhD Programs Foundation of Ministry of Education of China (No.20131106120046).