IL‑37/IL‑1R8 blocks keratinocyte acantholysis via suppressing ADAM17/EGFR

Introduction

Pemphigus vulgaris (PV) is a life threatening autoimmune blistering disease characterized by loss of cell-cell adhesion of keratinocytes (acantholysis) and the formation of non-healing suprabasal intraepidermal blisters (1,2). Its annual incidence ranges from 0.098-5 patients per 100,000 individuals (3). Although the use of corticosteroids and immunosuppressive agents has markedly improved clinical outcomes in patients with PV, prolonged administration of these drugs increases the risk of serious adverse events, with infections being particularly notable (4). Growing evidence indicates that autoantibody-induced keratinocyte acantholysis involves multiple mechanisms. Autoantibodies binding to PV antigens not only directly disrupt desmosomal adhesive function but also activate intracellular kinase signaling pathways downstream of ligated antigens. These include epidermal growth factor receptor (EGFR), Src, mammalian target of rapamycin (mTOR), p38 mitogen-activated protein kinases (MAPK), as well as elevated intracellular calcium and activation of cell death cascades, leading to the dissociation of keratinocyte and formation of acantholysis (5). Therefore, uncovering the mechanisms by which autoantibodies trigger acantholysis and cell death is crucial for the development of targeted therapeutics.

Recently, increasing evidence has demonstrated that interleukins play significant roles and represent promising therapeutic targets in skin-related diseases (6,7). Our previous study found that interleukin-37 (IL-37) inhibits keratinocyte dissociation and desmoglein-3 (Dsg3) endocytosis through upregulating caveolin-1 and inhibiting signal transducer and activator of transcription 3 (STAT3) pathways (8). IL-37 is predominantly expressed in circulating monocytes, macrophages, dendritic cells, tonsillar B cells, and plasma cells, as well as in epithelial cells of the skin and gut in response to inflammation (9). In psoriasis, IL-37 expression has been reported to be decreased (10); however, findings in atopic dermatitis remain inconsistent (9,11,12).

As a member of interleukin-1 superfamily, IL-37 functions as a natural suppressor of inflammatory and immune responses (13,14). It modulates both innate and acquired immunity by maintaining the cytokine balance away from excessive inflammation (15,16). Notably, IL-37 binds to the IL-18Rα and recruits the co-receptor IL-1R8 to form a tripartite complex, triggering a signaling cascade that involves mTOR, MAPK and NF-kB pathways (17). Johnston et al (18) identified synergistic interactions between IL-1 and EGFR, a pivotal regulator of epithelial homeostasis, in enhancing keratinocyte antimicrobial defense mechanisms. Furthermore, it has been demonstrated that PV-IgG induces EGFR activation, and clinical use of EGFR inhibitors such as lapatinib prevents keratinocyte blister formation (19). These findings led to the hypothesis that IL-37 may regulate EGFR-mediated keratinocyte acantholysis and cell death.

The present study stimulated HaCaT keratinocytes with anti-Dsg3 antibody to establish an in vitro PV model and co-cultured these cells with IL-37 to investigate its role and underlying mechanisms in PV pathogenesis. The results revealed that IL-37 treatment markedly reduced keratinocyte dissociation and apoptosis in anti-Dsg3 antibodies-treated-keratinocytes through the IL-1R8/TNF-alpha-converting enzyme (TACE/ADAM-17)/EGFR pathway. Collectively, these findings identified IL-37 as a pivotal regulator of EGFR activation and keratinocyte dissociation in PV, highlighting its potential as a therapeutic target.

Materials and methods

Collection of serum and skin tissues

Between June 2019 and June 2020, serum and skin tissues were collected from 15 patients with PV (8 males and 7 females; mean age 46.00±12.63 years, range 21-70) undergoing treatment in the Department of Dermatology in People's Hospital of Xinjiang Uygur Autonomous Region. Healthy control serum (15 samples, 7 males and 8 females; mean age 45.20±12.03 years, range 25-63) and skin tissues (5 samples) were collected from participants undergoing skin plastic surgery at the same hospital. The present study was approved by the Ethics Committee of the People's Hospital of Xinjiang Uygur Autonomous Region (approval no. 2019030616). The procedures followed were in accordance with the Helsinki Declaration of 1975, as revised in 1983.

Cell culture

HaCaT cells were obtained from Cell Bank of the Chinese Academy of Sciences (cat. no. SCSP-5091). The cell line has tested negative for mycoplasma, bacteria and fungi and has been verified by STR profiling. HaCaT cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc. Massachusetts, USA.) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 37°C and 5% CO2 incubator.

CCK-8 assay

HaCaT cells (2×103 cell/well) were cultured in 96-well plate and treated with different concentration of IL-37 recombinant protein (0, 25, 50, 100, and 200 ng/ml). Then, 10 μl of CCK-8 (Beyotime Biotechnology) were added and cultured for 1 h, the absorbance were detected at 450 nm.

Construction of PV-cell model

To establish the in vitro model of antibody-induced acantholysis, HaCaT cells placed in 6-well plates were treated with mouse monoclonal anti-Dsg3 antibody AK23 (cat. no. MA5-51877; Thermo Fisher Scientific, Inc.) at a dilution of 1:2,000 or 1:1,000 for 24 h at 37°C. Subsequently, the cells were treated with IL-37 recombinant protein (isoform b; 100 ng/ml; cat. no. 10155-HNAE; Sino Biological, Inc.) for 24 h at 37°C.

Cell transfection and treatment

To knockdown the expression of IL-1R8, IL-18Rα and ADAM17, HaCaT cells were transfected with 50 nM of IL-1R8-, IL-18Rα- and ADAM17-specific small interference RNA (si-RNA; Shanghai GenePharma Co., Ltd.) using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h at 37°C according to the manufacturer's instructions. The knockdown efficiency was detected by western blot assay. Subsequently, HaCaT cells were treated with AK23 for 24 h and 100 ng/ml of IL-37 recombinant protein for another 24 h. The sequences of siRNAs were as follows: si-IL-1R8 #1: 5'-GCAAGUUCGUGAACUUCAUCC-3', and 5'-AUGAAGUUCACGAACUUGCGG-3'; si-IL-1R8 #2: 5'-GCGUCGCUCCUCUGCUCAACA-3', and 5'-UUGAGCAGAGGAGCGACGCCG'; si-IL-1R8 #3: 5'-GGGAGAGUUUGCUCCCAAAGG-3', and 5'-UUUGGGAGCAAACUCUCCCCU-3'; si-IL-18R #1: 5'-GAGUGAGAUUGUCAGUGUAAG-3' and 5'-UACACUGACAAUCUCACUCUU-3'; si-IL-18R #2: 5'-GAGUCUUAAUCUUCAGAAACA-3' and 5'-UUUCUGAAGAUUAAGACUCGG-3'; si-IL-18R #3: 5'-GAGAAGAGAUGAAACAUUAAC-3' and 5'-UAAUGUUUCAUCUCUUCUCGU-3'; si-ADAM17 #1: 5'-GAUUUGACCUACAAAUCAAUC-3' and 5'-UUGAUUUGUAGGUCAAAUCUA-3'; si-ADAM17 #2: 5'-AGAGAUCUACAGACUUCAACA-3' and 5'-UUGAAGUCUGUAGAUCUCUUU-3'; si-ADAM17 #3: 5'-GAAUCGUGUUGACAGCAAAGA-3' and 5'-UUUGCUGUCAACACGAUUCUG-3'; si-NC: 5'-UUCUCCGAACGUGUCACGUTT-3' and 5'-ACGUGACACGUUCGGAGAATT-3'.

For EGFR inhibition assay, HaCaT cells were pre-treated with 1 μM of EGFR inhibitor AG1478 (MedChemExpress) at 37°C for 30 min followed by treatment with AK23 for 24 h at 37°C.

Enzyme-linked immunosorbent assay (ELISA)

The levels of IL-37 (cat. no. PI652), IL-6 (cat. no. PI325), IL-10 (cat. no. PI536), and IL-17 (cat. no. PI550) in the serum or cellular supernatant were measured using ELISA kits (Beyotime Biotechnology) according to the manufacturer's instructions. Briefly, the serum (50 μl sample analysis buffer and 50 μl serum/well) or cellular supernatant (100 μl/well) was added and cultured for 120 min at room temperature. Then, biotinylated antibody (100 μl/well) was added and cultured for 60 min at room temperature. Following this, streptavidin (100 μl/well) labeled with horseradish peroxidase was added and incubated at room temperature for 20 min away from light. Then, a color-developing agent (TMB solution; 100 μl/well) was added, and the reaction wells were sealed using a sealing plate film (white), and incubated at room temperature for 15 min away from light. Finally, a termination solution (50 μl/well) was added and the absorbance was measure at 450 nm immediately after mixing.

Immunohistochemical staining

Skin tissues were fixed with 4% paraformaldehyde for 15 min at room temperature, and then permeabilized with 0.5% Triton X-100 for 20 min at room temperature. After blocking with normal goat serum in PBS containing 0.1% Tween for 30 min at room temperature, the tissue sections were incubated with IL-37 antibody (cat. no. ab278499; Abcam) overnight at 4°C. The next day, the skin tissues were incubated with biotin-labeled goat anti-rabbit IgG (H+L) antibody (Thermo Fisher Scientific, Inc.; 1:200) for 15 min at room temperature. They were then counterstained with hematoxylin for 5 min at room temperature. Finally, images of the skin tissue sections were captured using a light microscope (Leica DM4000 B LED; Leica Microsystems GmbH).

Dsg3 immunocytofluorescence

HaCaT keratinocytes cultured with or without mouse anti-Dsg3 antibody (1:1,000 dilution) were fixed with 4% paraformaldehyde, incubated with 0.1% Triton X-100 for 10 min at room temperature and then blocked with 5% goat serum for 1 h at room temperature. Subsequently, mouse anti-Dsg3 antibodies (1:100 dilution; cat. no. NBP1-78984; Novus Biologicals; Bio-Techne) were added and incubated at 4°C. The next day, Alexa Fluor 594-labelled goat anti-rabbit IgG (cat. no. 33112ES60; Shanghai Yeasen Biotechnology Co., Ltd.) was added and incubated for another 2 h at 37°C in the dark. The nuclei were stained with DAPI at room temperature for 5 min (1:5,000 dilution; Beyotime Biotechnology). Images of fluorescence staining were captured using a fluorescence microscope.

Reverse transcription-quantitative (RT-q) PCR

Cells (1×106 cell) were collected and lysed with TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), after which their RNA was extracted. RNA (1 μg) was reverse transcribed into the cDNA using the PrimeScript RT Master Mix kit (Takara Biotechnology Co., Ltd.) following the manufacturer's instructions. The mRNA levels of these samples were then detected using SYBR® Premix Ex Taq II (Qiagen China Co., Ltd.) according to the manufacturer's instructions. The reactions program was as follows: 95°C for 5 min, followed by 40 cycles at 95°C for 5 sec and 60°C for 34 sec and finally extended at 72°C for 10 min. Gene expression levels were quantified using the 2−ΔΔCq method (20), with β-actin serving as the endogenous reference gene. The primers used were: IL-37: Forward: 5'-GACCAGGATCACAAAGTACTGG-3', Reverse: 5'-GAGCTCAAGGATGAGGCTAATG-3'; IL-10: Forward: 5'-GCTGGAGGACTTTAAGGGTTAC-3', Reverse: 5'-GATGTCTGGGTCTTGGTTCTC-3'; IL-6: Forward: 5'-CACTCACCTCTTCAGAACGAAT-3', Reverse: 5'-GCTGCTTTCACACATGTTACTC-3'; IL-17: Forward: 5'-GAAATCCAGGATGCCCAAATTC-3', Reverse: 5'-GAGGTGGATCGGTTGTAGTAATC-3'; β-actin: Forward: 5'-AGCCTTCCTTCCTGGGCAT-3', Reverse: 5'-TGATCTTCATTGTGCTGGGTG-3'. All experiments were performed in three independent biological replicates.

Western blotting

All the cell samples from all the groups and the skin tissue samples from patients with PV were lysed with RIPA buffer (cat. no. P0013C; Beyotime Biotechnology,) for 10 min at 4°C. Protein concentration was determined using the Bradford Protein Concentration Assay Kit (Beyotime Biotechnology). Total protein (20 μg) were electrophoresed on 6, 8, 10 or 12% SDS-PAGE gel and subsequently transferred to a PVDF membrane. The membranes were then blocked using a blocking buffer (Beyotime Biotechnology) for 1 h at room temperature. Following this, the membranes were incubated with the following specific primary antibodies overnight at 4°C: Rabbit anti-IL-37 monoclonal antibody (cat. no. ab278499; Abcam), rabbit anti-Bax monoclonal antibody (cat. no. ab32503; Abcam), rabbit anti-Bcl-2 polyclonal antibody (cat. no. ab59348; Abcam), rabbit anti-caspase 3 antibody (cat. no. 9662; Cell Signaling Technology, Inc.), rabbit anti-cleaved caspase3 antibody (cat. no. 9661; Cell Signaling Technology, Inc.), mouse anti-IL-1R8 antibody (cat. no. sc-271864; Santa Cruz Biotechnology, Inc.), mouse anti-IL-18Rα antibody (cat. no. PA5-115404; Thermo Fisher Scientific, Inc.), rabbit anti-EGFR phospho Y1173 antibody (cat. no. ab5652; Abcam), rabbit anti-EGFR antibody (cat. no. ab52894; Abcam), rabbit anti-ADAM17 polyclonal antibody (cat. no. ab39162; Abcam), anti-phosphorylated (p-) ERK1/2 antibody (cat. no. ab201015; Abcam), anti-ERK1/2 antibody (cat. no. ab184699; Abcam), anti-p-AKT (Ser473) antibody (cat. no. 9271; Cell Signaling Technology, Inc.), anti-AKT antibody (cat. no. 4691; Cell Signaling Technology, Inc.), anti-p-STAT3 antibody (cat. no. ab76315; Abcam), and anti-STAT3 antibody (cat. no. ab68153; Abcam). Then, the appropriate HRP-conjugated goat anti-rabbit IgG or goat anti-mouse antibodies were added and incubated for 1 h at room temperature. Finally, positive bands were visualized using an imaging system (Bio-Rad Laboratories, Inc.). Densitometric analysis was performed using ImageJ software (version 1.53; National Institutes of Health).

Dispase-based dissociation assay

HaCaT keratinocytes were cultivated on 24-well plates at a density of 1×106 per well. The cells were then stimulated with or without anti-Dsg3 antibody in the presence or absence of IL-37 and/or EGFR activator NSC228155 for 15 min at 37°C. After two washes using phosphate-buffered saline (PBS), each well was treated with 2 U/ml of Dispase II (MilliporeSigma) and incubated at 37°C for 30 min. Dispase II was then gently removed, and 1 ml of PBS was added. The cells were then pipetted up and down 10 times using a 1 ml electric pipette at a constant speed to disrupt the integrity of the monolayer cells and dissociate the cells. Finally, cell debris was stained with 0.01% crystal violet (MilliporeSigma) at room temperature for 1 min and the number of fragments was measured using an inverted microscope. The experiment was performed three times.

Mitochondrial membrane potential

The PV-cell model was treated with IL-37 recombinant protein for 24 h, after which it was washed twice with PBS. The cells were then stained with 1.0 mM of JC-1 (cat. no. C2005; Beyotime Biotechnology) at 37°C for 10 min. The fluorescence intensity of the cells was detected using a fluorescence microscope.

Flow cytometry

All the groups of cells were washed with PBS for three times and then double-stained with annexin V-FITC and propidium iodide at 37°C for 20 min. The apoptosis of each sample group was analyzed using a fluorescence-activated cell sorter (FACS; CytoFLEX; Beckman Coulter, Inc.). Data analysis was conducted using FlowJo software (version 10.8.1; Becton, Dickinson & Company, Oregon, USA). Apoptotic rates were calculated as the sum of the percentage of early and late apoptotic cells.

Co-immunoprecipitation assays

Co-immunoprecipitation assays were conducted according to the manufacturer's instructions (cat. no. 88804, Thermo Fisher Scientific, Inc.) The HaCaT cells were washed with PBS and lysed with lysis buffer (0.025M pH=7.4 Tris, 0.15M NaCl, 0.001M EDTA, 1% NP40 and 5% glycerol) supplemented with a protease inhibitor cocktail (100: 1 (v/v)). After being centrifuged at 13,680 × g for 15 min at 4°C, the supernatant was collected, and 20 μl of the collected supernatant was used as input. The remaining cell lysate supernatant (0.5 mg per reaction) was incubated with 2 μg of IL-37 antibody (cat. no. ab278499; Abcam, IgG antibody was used as control) at 4°C overnight. The immunocomplex was captured using 10 μl of protein A+G beads at 37°C for 1 h. After incubation, the complex was separated on a magnetic rack for 10 sec, and then washed thrice using wash buffer (0.025 M pH=7.4 Tris, 0.15 M NaCl, 0.001 M EDTA, 1% NP40 and 5% glycerol) supplemented with a protease inhibitor cocktail. Western blotting was used for further analysis.

Statistical analysis

The data were analyzed using SPSS 18.0 (SPSS, Inc.) and GraphPad Prism 6 (Dotmatics) and expressed as mean ± SD. Differences between two groups were estimated by independent sample t test. Differences among multiple groups were estimated using one-way ANOVA with Bonferroni's post-hoc test. The correlations between serum IL-37 and IL-10, IL-6, and IL-17, respectively, were assessed using Pearson's correlation analysis. All experiments were conducted with three independent biological replicates. A post hoc power analysis was conducted using G*Power 3.1.9.2 (Heinrich Heine University Düsseldorf, Düsseldorf, Germany) to evaluate the sufficiency of the clinical sample size. Given α=0.05, n=15 per group, and IL-37 concentrations of 4.20±0.98 pg/ml (healthy control) vs. 10.52±2.87 pg/ml (PV), the achieved power was 1.00, which exceeds the conventional threshold of 0.9. P<0.05 was considered to indicate a statistically significant difference.

Results

IL-37 is augmented in PV and associated with inflammatory factors

To elucidate the pathophysiological role of IL-37 in PV, the present study analyzed the concentration of IL-37 in the serum and skin tissues from healthy controls and. Serum IL-37 levels were markedly higher in patients with PV compared with healthy controls (Fig. 1A). Similarly, IL-37 expression was markedly elevated in the skin tissues of patients with PV than in healthy controls, as indicated by immunohistochemistry assays (Fig. 1B and C). Furthermore, serum IL-37 levels showed a positive correlation with the anti-inflammatory factor IL-10 (Fig. 1D), and a negative correlation with the pro-inflammatory cytokines IL-6 and IL-17 (Fig. 1E and F).

Figure 1 IL-37 was augmented in pemphigus vulgaris and associated with inflammatory factor. (A) IL-37 concentration in the serum of healthy controls (n=15) and patients with pemphigus vulgaris (n=15) was detected by ELISA assay. Data are presented as mean ± SD. (B and C) IL-37 expression in skin tissue of healthy control and pemphigus vulgaris was analyzed by immunohistochemistry. (D) The correlation between serum IL-37 and IL-10 levels was assessed using Pearson's correlation analysis. (E) The correlation between IL-37 and IL-6 levels was assessed using Pearson's correlation analysis. (F) The correlation between IL-37 and IL-17 levels was assessed using Pearson's correlation analysis. *P<0.05. IL, interleukin; ELISA, enzyme-linked immunosorbent assay.

IL-37 protects HaCaT cells from Dsg3-induced keratinocyte dissociation and apoptosis

To investigate the functional role of IL-37 in PV, an in vitro PV model was established by treating HaCaT keratinocytes with the anti-Dsg3 antibody AK23. Immunofluorescence staining revealed that under control or with a low anti-Dsg3 antibody (1:2,000 dilution) treated conditions, Dsg3 exhibited a linear, continuous staining along the cell borders. By contrast, a higher anti-Dsg3 antibody concentration (1:1,000 dilution) resulted in markedly reduced Dsg3 staining, indicating impaired cell adhesion and successful modeling of PV-like acantholysis (Fig. 2A). HaCaT cells were treated with different concentration of IL-37 recombinant protein (0, 25, 50, 100 and 200 ng/ml). As shown in Fig. 2B, IL-37 at concentrations of 100 ng/ml and below had no effect on cell viability, whereas 200 ng/ml IL-37 markedly reduced cell viability. Therefore, 100 ng/ml IL-37 was selected for subsequent experiments. The anti-Dsg3 antibody treatment markedly increased the IL-37 levels (Fig. 2B and C). Co-treatment with 100 ng/ml of IL-37 and anti-Dsg3 antibody further enhanced IL-37 expression compared with anti-Dsg3 antibody alone (Fig. 2C and D). Additionally, the concentration and mRNA expression of IL-10 were markedly increased (Fig. 2E and F), while the concentration and mRNA expression of IL-6 (Fig. 2G and H) and IL-17 (Fig. 2I and J) were markedly decreased in IL-37 in combination with the anti-Dsg3 treated HaCaT group.

Figure 2 IL-37 protects HaCaT cells from Dsg3-induced keratinocyte dissociation and apoptosis. (A) The effect of different dilutions of Dsg-3 antibodies on cell dissociation was analyzed by immunofluorescence. (B) HaCaT cells were treated with different concentration of IL-37 recombinant protein (0, 25, 50, 100, and 200 ng/ml). Cell viability was detected by CCK-8 assay. (C) HaCaT cells were treated with the Dsg-3 antibodies, followed by administration of the IL-37 recombinant protein. The concentration of IL-37 was analyzed using an ELISA assay. (D) The relative mRNA expression of IL-37 was detected using RT-qPCR. The (E) concentration and (F) relative mRNA expression of IL-10 were measured using an IL-10 ELISA kit and RT-qPCR, respectively. The (G) oncentration and (H) relative mRNA expression of IL-6 were measured by the IL-6 ELISA kit and RT-qPCR, respectively. The (I) concentration and (J) relative mRNA expression of IL-17 were measured by the IL-17 ELISA kit and RT-qPCR, respectively. &P<0.05; *P<0.05 vs. Control group; #P<0.05 vs. anti-Dsg3 group. Data are presented as mean ± SD, n=3 biological independent replicates. One-way ANOVA with Bonferroni's post-hoc test was used for multiple group comparisons. IL, interleukin; Dsg3, desmoglein-3; ELISA, enzyme-linked immunosorbent assay; RT-qPCR, reverse transcription-quantitative PCR.

The dispase-based dissociation assays demonstrated a significant increase in the number of fragments in the anti-Dsg3 group compared with the control group (Fig. 3A). By contrast, IL-37 treatment reduced the number of fragments compared with the anti-Dsg3 group (Fig. 3A). Given that PV pathogenesis may be initiated by pro-apoptotic proteins and that the apoptotic pathway are activated prior to morphological signs of acantholysis (21,22), the present study further analyzed the apoptosis of the HaCaT cells. JC-1 assay revealed enhanced green fluorescence and reduced red fluorescence in the anti-Dsg3 group compared with the control group, indicating a transition from red to green fluorescence, characteristic of early apoptosis in the anti-Dsg3 group (Fig. 3B). However, IL-37 decreased the intensity of green fluorescence and increased the red fluorescence intensity, suggesting that IL-37 treatment increased mitochondrial membrane potential and inhibited cell apoptosis (Fig. 3B). Consistent with these findings, the flow cytometry analysis demonstrated that the apoptosis of HaCaT cells was increased in the anti-Dsg3 group compared with the control group, whereas the apoptosis of HaCaT cells in the anti-Dsg3+IL-37 group was decreased compared with that in the anti-Dsg3 group (Fig. 3C). Further analysis of apoptosis-related markers showed that anti-Dsg3 antibody exposure markedly decreased Bcl-2 expression and increased expression of Bax and cleaved-caspase 3 compared with the control group (Fig. 3D-G). IL-37 treatment reversed these changes, upregulating Bcl-2 and downregulating Bax and cleaved-caspase 3 expression (Fig. 3D-G).

Figure 3 IL-37 protects HaCaT cells from keratinocyte dissociation and apoptosis. (A) The effect of IL-37 on cell dissociation was evaluated using a cell dissociation assay. (B) Mitochondrial membrane potential was assessed by JC-1 staining. (C) Cell apoptosis was analyzed by flow cytometry. (D) Protein expression levels of Bcl-2, Bax, Cleaved-caspase 3 and Caspase 3 were determined by western blotting. Quantitative analysis of (E) Bcl-2, (F) Bax and (G) Cleaved-caspase 3 protein levels. *P<0.05 vs. Control group; #P<0.05 vs. anti-Dsg3 group. Data are presented as mean ± SD, n=3 biological independent replicates. One-way ANOVA with Bonferroni's post-hoc test was used for multiple group comparisons. IL, interleukin.

IL-37 protects HaCaT cells from keratinocyte dissociation and apoptosis through IL-1R8

As a ligand protein, IL-37 functions by binding to its receptor IL-1R8 (23). The co-immunoprecipitation assay confirmed the interaction of IL-37 and IL-1R8 in the HaCaT cells (Fig. 4A). To further investigate the functional role of this interaction, IL-1R8 and IL-18Rα were knocked down in the HaCaT cells through transfection with IL-1R8-specific siRNA and IL-18Rα-specific siRNA, respectively. IL-1R8 and IL-18Rα expression were markedly decreased in IL-1R8 siRNA (Fig. 4B) or IL-18Rα siRNA (Fig. 4C) transfected HaCaT cells. Knockdown of either IL-1R8 or IL-18Rα resulted in an increased number of cellular fragments compared with the negative control siRNA transfected HaCaT cells (Fig. 4D). Additionally, the apoptosis rate was markedly higher in the anti-Dsg3 + IL-37 + si-IL-1R8 group and anti-Dsg3 + IL-37 + si-IL-18Rα group than in the anti-Dsg3 + IL-37 + si-NC group (Fig. 4E and F). Consistent with these findings, the Bax expression was upregulated, while the Bcl-2 expression was downregulated in the anti-Dsg3 + IL-37 + si-IL-1R8 group and anti-Dsg3 + IL-37 + si-IL-18Rα group relative to the anti-Dsg3 + IL-37 + si-NC group (Fig. 4G).

Figure 4 IL-37/IL-1R8 protects HaCaT cells from keratinocyte dissociation and apoptosis through the ADAM17/EGFR pathway. (A) The interaction between IL-37 and IL-1R8 was analyzed using co-immunoprecipitation. (B) HaCaT cells were transfected with IL-1R8 siRNA and the transfection efficiency were detected by western blotting. (C) HaCaT cells were transfected with IL-18Rα siRNA and the transfection efficiency were detected by western blotting. (D) IL-1R8 siRNA or IL-18Rα transfected HaCaT were treated with anti-Dsg-3 and IL-37 recombinant protein. The number of fragments was analyzed by cell dissociation assay. (E and F) Cell apoptosis was analyzed by flow cytometry. (G) Protein expression levels of Bcl-2 and Bax were determined by western blotting. &P<0.05 vs. si-NC; *P<0.05 vs. anti-Dsg3 + IL-37 + si-NC group. Data are presented as mean ± SD, n=3 biological independent replicates. One-way ANOVA with Bonferroni's post-hoc test was used for multiple group comparisons. IL, interleukin; ADAM17, TNF-alpha-converting enzyme; EGFR, epidermal growth factor receptor; si, short interfering.

IL-37/IL-1R8 protects HaCaT cells from keratinocyte dissociation and apoptosis through the ADAM17/EGFR pathway

Following PV-IgG treatment, EGFR auto-phosphorylation, internalization and acantholysis were induced (24). It has been reported that IL-1R deficiency upregulates the expression of ADAM-17, a metalloproteinase responsible for the shedding and release of EGFR ligands (25). To further explore the potential mechanisms by which IL-37 administration induces keratinocyte dissociation and apoptosis, the present study examined the expression of ADAM17 and EGFR phosphorylation. Compared with the anti-Dsg3 group, ADAM17 expression, p-EGFR as well as EGFR downstream protein p-AKT, p-ERK1/2, and p-STAT3 levels were markedly decreased in the anti-Dsg-3 + IL-37 group, indicating a suppression of canonical EGFR signaling cascades (Fig. 5A). Furthermore, knockdown of IL-1R8 markedly increased the expression of ADAM17, p-EGFR, p-ERK1/2, p-AKT, and p-STAT3 levels in the anti-Dsg3 + IL-37 + si-IL-1R8 group when compared with that in the anti-Dsg3 + IL-37 + si-NC group (Fig. 5A).

Figure 5 IL-37/IL-1R8 protects HaCaT cells from keratinocyte dissociation and apoptosis through the ADAM17/EGFR pathway. (A) Protein expression levels of ADAM17, p-EGFR, total EGFR as well as EGFR downstream protein p-AKT, p-ERK1/2 and p-STAT3 levels were determined by western blotting. HaCaT cells were treated with EGFR activator NSC228155. (B) Cell dissociation was analyzed by cell dissociation assay. (C) Cell apoptosis was analyzed by flow cytometry. (D) Protein expression levels of Bcl-2 and Bax were analyzed by western blotting. (E) HaCaT was transfected with ADAM17 siRNA and the transfection efficiency was detected by western blotting. HaCaT cells were transfected with ADAM17 siRNA or treated with 1 μM of EGFR inhibitor AG1478 for 30 min followed by treated with AK23. (F) Cell dissociation was analyzed by cell dissociation assay. (G) Cell apoptosis was analyzed by flow cytometry. #P<0.05 vs. Control group; &P<0.05 vs. anti-Dsg3 group; @P<0.05 vs. anti- Dsg3 + IL-37 + si-NC group; *P<0.05; Data are presented as mean ± SD, n=3 biological indepen- dent replicates. One-way ANOVA with Bonferroni's post-hoc test was used for multiple group comparisons. IL, interleukin; ADAM17, TNF-alpha-converting enzyme; EGFR, epidermal growth factor receptor; p- phosphorylated; STAT, signal transducer and activator of transcription; si, short interfering.

Pharmacologically activation of EGFR using the EGFR activator NSC228155 enhanced epidermal fragment formation and apoptosis rates (Fig. 5B and C). Additionally, Bcl-2 expression was markedly decreased, whereas Bax protein expression was markedly increased in the anti-Dsg3 + IL-37 + NSC228155 group compared with the anti-Dsg3 + IL-37 group (Fig. 5D).

Subsequently, HaCaT was transfected with ADAM17 siRNA and western blotting showed a downregulation of ADAM17 after ADAM17 siRNA transfection, with ADAM17 siRNA #1 demonstrating the most pronounced reduction in protein level (Fig. 5E). This ADAM17 knockdown markedly decreased both epidermal fragment formation (Fig. 5F) and apoptosis rates (Fig. 5G) compared with anti-Dsg3+si-NC group. Notably, compared with the anti-Dsg3 group, EGFR inhibitor AG1478 also decreased epidermal fragment formation and apoptosis rates (Fig. 5F and G).

Discussion

The present study demonstrated that IL-37 is highly expressed in the serum and skin tissue of patients with PV. IL-37 regulates HaCaT cell dissociation and apoptosis in an IL-1R8-dependent manner. Furthermore, the IL-37/IL-1R8 axis inhibits HaCaT cell dissociation and apoptosis by negatively regulating the ADAM17/EGFR pathway.

It is well established that desmoglein-1 (Dsg1) and Dsg3, calcium-dependent cell adhesion molecules, serve as the primary autoantibodies targets in the pathogenesis of PV (26). Several non-Dsg antigens, including desmocollin 1 and 3, mitochondrial proteins, human leukocyte antigen molecules and various subtypes of nicotinic and muscarinic acetylcholine receptor, have also been implicated in PV (27), indicating a multifactorial etiology. In a previous study, we found that IL-37 inhibited keratinocyte dissociation and Dsg3 endocytosis by upregulating caveolin-1 and inhibiting the STAT3 pathway (8). The present study discovered that IL-37 recombination protein led to a loss of cell dissociation and a decrease in cell apoptosis. As a ligand protein, IL-37 functions by binding to its receptors, IL-1R8 and IL-18R. Previous reports indicate that IL-37b exerts anti-inflammatory effects through IL-1R8 by inhibiting the p38, ERK, JNK, and NF-κB pathways, and that IL-1R8 knockdown impairs the anti-inflammatory activity of IL-37 (28,29). Using a co-immunoprecipitation assay, the present study confirmed the interaction between IL-37 and IL-1R8. Knockdown of IL-1R8 reversed the protective effect of IL-37 on cell dissociation and apoptosis, suggesting that IL-37 acts through an IL-1R8-dependent mechanism.

Following PV IgG treatment, EGFR autophosphorylation, internalization and acantholysis are induced (24). In keratinocytes, inhibition of EGFR blocked PV IgG-triggered Dsg3 endocytosis, keratinocyte dissociation and blister formation, indicating cross-talk between EGFR and Dsg3 (24). Various factors, including G-protein-coupled receptor agonists and cytokines (such as IL-1β), can transactivate EGFR. This transactivation requires ADAM-17, a metalloproteinase that mediates the shedding and release of EGFR ligands (30). Previous studies have demonstrated that ADAM family members are associated with cell adhesion (31) and, specifically, ADAM10 and ADAM17 contribute to Dsg2 turnover (32). Additionally, ADAM17 can be activated by interleukin-15 (33). Notably, Wang et al (25) reported that IL-1R deficiency increased ADAM17 expression levels in a rat model of Alzheimer's disease. The present study found that IL-37 markedly decreased both ADAM17 expression and EGFR phosphorylation. Furthermore, activation of EGFR reversed the effects of IL-37 on HaCaT cell dissociation and apoptosis. It is well established that patients with PV exhibit a distinct autoantibody profile, leading to variations in intracellular signaling patterns (27,34). For instance, patients with PV and with anti-Dsg3 and anti-Dsg1 antibodies, but not anti-desmocollin3 antibodies, show increased levels of EGFR ligands such as EGF. These patients can benefit from treatment with EGF or EGFR inhibitors (34). Thus, patients with PV and with DSG1/3 antibodies might respond favorably to IL-37 therapy. Importantly, the function of IL-37 is concentration-dependent; at high concentrations, it forms dimers that inactivate it, thus inhibiting its anti-inflammatory function (35). This crucial mechanism underscores that its function is not linear. In the present study, the observed IL-37 levels appeared to fall within a therapeutic window where its net effect remained protective. Future studies are warranted to delineate the exact concentration threshold for dimerization in PV and to explore IL-37 as a potential therapeutic agent, where strategies to stabilize its active, monomeric form could be highly beneficial. Therefore, the concentration of IL-37 must be carefully considered in clinical applications aiming to inhibit EGFR in PV.

The present study used an anti-Dsg3 antibody (AK23) induced HaCaT cell model. However, this model does not fully recapitulate the complexity of PV, which involves multiple autoantibodies and patient heterogeneity. Validating key findings in a murine model of PV or patient-derived samples is needed in further studies.

In conclusion, the present study demonstrated that IL-37 inhibited ADAM17-mediated EGFR activation via its receptor IL-1R8, thereby inhibiting keratinocyte acantholysis and apoptosis. These findings provide new perspective on the treatment of pemphigus, suggesting that therapeutic strategies may extend beyond small-molecule EGFR inhibitors to include targeting the IL-37/IL-1R8 pathway.

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Author' contributions

FH was responsible for formal analysis, methodology, supervision, original draft preparation, writing, reviewing and editing. WC was responsible for formal analysis, supervision, original draft preparation, writing, reviewing and editing. QW was responsible for formal analysis and methodology. XZ was responsible for formal analysis and methodology. FX was responsible for formal analysis and methodology. JZ was responsible for conceptualization, supervision, writing, reviewing and editing. JL was responsible for conceptualization, supervision, writing, reviewing and editing.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of the People's Hospital of Xinjiang Uygur Autonomous Region (approval no. 2019030616). The procedures followed were in accordance with the Helsinki Declaration of 1975, as revised in 1983. Serum and skin tissues were collected from 15 patients with PV who were undergoing treatment in the Department of Dermatology in People's Hospital of Xinjiang Uygur Autonomous Region. Serum (15 samples) and skin tissue (5 samples) from healthy control were collected from participants who were undergoing skin plastic surgery in plastic surgery in the same hospital.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Acknowledgements

Not applicable.

Funding

The present study was supported by National Natural Science Foundation of China (grant no. 82260621), Natural Sciences of Xinjiang Uygur Autonomous Region (grant no. 2022D01C623) and Basic and Clinical Research Project of Stem Cell Biotherapy-People's Hospital of Xinjiang Uygur Autonomous Region (grant no. GXBZX-2023004).

References