Introduction: Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by relapsed eczema and serious pruritus. High-mobility group box 1 protein (HMGB1) is a nuclear-binding protein and serves as an alarmin to promote inflammatory responses. Methods: In this study, we established an AD mouse model by topical use of MC903 on ears and then used a specific HMGB1-binding peptide cIY8 and a HMGB1 inhibitor of glycyrrhizin to investigate HMGB1 on fibroblast activation in the pathogenesis of AD-like symptoms. Results: Topical use of cIY8 and oral use of glycyrrhizin significantly improved the MC903-induced AD-like symptoms and pathological changes of the ears and scratching behavior in an AD mouse model; cIY8 treatment inhibited the higher mRNAs of IL-1α, IL-4, IL-5, IL-13, and IL-31 in the ears. In human fibroblasts, HMGB1 caused nuclear translocation of NF-kB, and the nuclear translocation could be inhibited by pre-treatment of HMGB1 with cIY8, suggesting that NF-κB signaling pathway participates in the HMGB1-induced inflammation of AD in fibroblasts and that cIY8 effectively impedes the function of HMGB1. Glycyrrhizin inhibited the Ca2+ signaling induced by ionomycin in mouse primary fibroblasts. The fibroblast-related proteins of α-SMA, Hsp47, and vimentin and the pruritus-related proteins of IL-33 and periostin were increased in the ears of the AD mouse model, the ratio of EdU incorporation became higher in mouse fibroblasts treated with MC903, and the higher proliferation and inflammatory responses of the fibroblasts could be reversed by glycyrrhizin treatment. Conclusions: Fibroblast activation by HMGB1 is one of the critical processes in the development of inflammation and pruritus in the AD mouse model. The specific HMGB1-binding peptide cIY8 and the HMGB1 inhibitor glycyrrhizin inactivate skin fibroblasts to alleviate the inflammation and pruritus in the AD mouse model. Peptide cIY8 may be topically used to treat AD patients in the future.

Atopic dermatitis (AD) is a chronic skin disease causing inflamed skin and intractable itch commonly associated with allergic disorders [1]. Previous AD studies have primarily focused on skin barrier dysfunction, immune abnormalities, genetic correlations, and microbiome analysis [2]. T helper cells (Th2) mainly participate in the release of cytokines interleukin-(IL)-4, IL-13, and IL-31, directly inducing the associated itch sensation [2, 3]. Recent skin single-cell sequencing study has found that activation of fibroblasts in AD patients promotes the production of IL-4 and IL-13, which may amplify type 2 immunity and fibrotic remodeling in the inflammatory process [4].

High-mobility group protein B1 (HMGB1) is a highly conserved nucleoprotein widely distributed in mammalian cells [5]. Previous studies found that the expression levels of HMGB1 in serum and AD lesions were significantly increased compared to those in the healthy controls and were positively correlated with the severity of AD [6]. Currently known HMGB1 antagonists include glycyrrhizin, cisplatin, quercetin, ethyl pyruvate, triptolide, and others [7]. However, most of the antagonists mentioned above are proprietary Chinese medicines and have some side effects.

Dermal fibroblasts produce and organize the extracellular matrix of the dermis and closely communicate with keratinocytes and a variety of immune cells [8]. A recent study investigated the role of AD patient-derived fibroblasts on tissue homeostasis and inflammation in human skin equivalents and demonstrated that these fibroblasts triggered the hyperproliferation of keratinocytes and the chemotaxis of CD4+ T-cell migration [9]. Accordingly, understanding the role of HMGB1 in fibroblast is important for improving the present therapies of AD. We hypothesis that blocking HMGB1 in the immune microenvironment could directly inhibit the proliferation and activation of fibroblasts. To gain further insights into the processes, we developed a mouse AD model with calcipotriol (MC903) and used two HMGB1 blockers of glycyrrhizin and a novel HMBG1-binding peptide of cIY8 to explore dermal fibroblasts in signaling pathway and function of inflammatory factors in AD pruritus.

Mice

C57BL/6J mice (male, aged 8–10 weeks) were used in this research. All mice were housed in controlled temperature (23°C) and humidity (50 ± 10%) conditions with 12:12 h of light/dark cycles. Food and water were available ad libitum.

AD Model and Treatments

The AD mouse model was induced as previously described [10]. MC903 (2 nm) dissolved in ethanol was topically applied on mice ears daily to induce AD model [11]. Mice were gavaged with 0.9% saline or glycyrrhizin (2.5 mg/kg) once per day. Each group contained 6 to 8 mice. To investigate the impact of HMGB1 inhibitor on mice AD model, MC903 (2 nm) was topically applied on mice left ear as well as MC903 (2 nm) + HMGB1 inhibitor peptide (100 μm) were topically applied on mice right ear once a day for 7 days as a self-control study. Scratching behavior was quantified by recording the number of scratching bouts over a period of 60 min on day 3 and day 7. We photographed and documented mice ear lesions on day 7. The clinical score was evaluated in 4 possible signs: redness, bleeding, eruption and scaling (0 = no signs, 1 = mild, 2 = moderate, 3 = severe; maximum score is 12) [12].

HaCaT Cell Culture and Treatment

Human keratinocyte cell line HaCaT cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum in a humidified incubator at 37°C/5% CO2. HMGB1 (500 ng/mL; MCE, China) was first incubated with cIY8 or the control peptide IY8 (1 μm) for 30 min at room temperature and then added to the cell culture medium and incubated for 3 h.

Immunohistochemistry

The ear tissues were collected from sacrificed mice on day 7 and then fixed and embedded in paraffin. 6 μm tissue sections were deparaffinized and hydrated. After antigen retrieval, the tissues’ endogenous peroxidase and nonspecific sites were blocked with 3% H2O2 solution and normal goat serum. The tissues were then incubated with primary antibodies (anti-α-SMA, anti-Hsp47, anti-Vimentin, anti-Periostin) overnight at 4°C (antibody dilution information is shown in online suppl. Table S1; for all online suppl. material, see https://doi.org/10.1159/000534568). The sections were incubated with goat anti-mouse/rabbit IgG polymer for 30 min on the following day, after which the slices were stained with diaminobenzidine (DAB) for 2–5 min.

Primary Fibroblast Isolation and Culture

Neonatal mice (1–3 days of age) were euthanized with carbon dioxide and sterilized in 75% alcohol for 10 min. The back skins of mice were collected, and dermal-epidermal separation was performed with 0.25% dispase II solution. The pieces of dermis were incubated with a collagenase D solution in a shaker at 37°C for 1 h to obtain single fibroblast cell suspension. The resuspended cells were seeded into a cell culture flask with Dulbecco’s Modified Eagle Medium culture medium in an incubator at 37°C/5% CO2 [13].

EdU Staining

Primary fibroblasts were incubated with MC903 (100 ng/mL) in the absence and presence of glycyrrhizin (2 mm) for 48 h. To analyze cellular proliferation, EdU staining was conducted using the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 594 (Cat#: C0078S; Beyotime, Shanghai, China). EdU (10 μm) was added into the DMEM cell culture medium. The cells were incubated for 2 h at 37°C/5% CO2. After fixation and permeabilization, primary fibroblasts were incubated with the Click Reaction mixture for 30 min and stained with Hoechst 33,342. The cells were observed using a fluorescence microscope, and the proportion of EdU-positive cells was calculated as the number of Edu-positive cells out of the total cells.

Calcium Imaging

Calcium imaging was monitored using a confocal laser scanning microscope (Zeiss, LSM880). Time-lapse images capturing changes in fluorescence intensity were taken at 1 s time intervals for 3 min. Fluo-8-AM (5 μm) was used to load fibroblasts for 30 min [14]. The drugs were diluted to the required concentrations in an artificial cerebrospinal fluid buffer (140 mm NaCl, 2.4 mm CaCl2, 1.3 mm MgCl2, 4 mm KCl, 10 mm HEPES, and 5 mm glucose). The results are presented as ratios of ΔF/F0.

Real-Time Quantitative PCR

Total RNA was extracted from mouse skin tissues using the TRIzol Reagent (Invitrogen). cDNA was synthesized using the high-capacity cDNA reverse transcription kit (Applied Biosystems). cDNA was quantified with the SYBR Green Master Mix (Roche) using the Step OnePlus real-time PCR system (Applied Biosystems). The generated cycle threshold (Ct) value was normalized to the Ct value of GAPDH. The primers used are listed in online supplementary Table S2.

HMGB1-Binding Peptide cIY8 and Control Peptide IY8

Three-dimensional structures of HMGB1 and related proteins were downloaded from RCSB Protein Data Bank (RCSB PDB) and AlphaFold Protein Structure Database [15]. In our analysis, the similarity between human and mouse HMGB1 protein sequences is extremely high, at 99.07% (online suppl. Fig. S1). By peptide-protein binding prediction and force field-based molecular simulations, a novel peptide inhibitor targeting HMGB1 was identified. This inhibitory peptide, with the sequence CIPRFYLFYC (disulfide bond) and named cIY8, bears a cyclic structure by a disulfide bond formation with both ends. The control peptide IY8 has the identical sequence of IPRFYLFY to cIY8 but no cysteine at both ends, unable to form a cyclic structure. These peptides were synthesized by GenScript Biotech Co.

NF-κB Immunofluorescence Staining

NF-κB activation was performed with the NF-κB activation-nuclear translocation assay kit (Beyotime). Briefly, HFF-1 cells were seeded onto glass bottom dishes. HMGB1 (500 ng/mL, MCE) was incubated with cIY8 for 30 min at room temperature. The mixture was then added into cell culture medium and incubated for 1 h at 37°C/5% CO2. After fixation and blocking, cells were incubated with NF-κB p65 primary antibody at 4°C overnight. HFF-1 was incubated with Cy3-conjugated secondary antibody for 1 h at room temperature. Cells were stained with DAPI for 5 min at room temperature, washed, and then visualized by a confocal microscopy (Leica).

Statistical Analysis

Statistical analyses were performed using GraphPad Prism (version 9.0) for Student’s t test, unparied t test, or one-way analysis of variance (ANOVA) as indicated (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Numerical results were represented by mean ± standard error of the mean (SEM).

Novel HMGB1 Inhibitor Peptide cIY8 Improves AD Lesions

HMGB1 is a nuclear protein and triggers inflammation [16]. Extracellular HMGB1 is considered to bind TLR4 to initiate inflammation-related signaling pathway [17]. To investigate the functions of HMGB1, we identified a novel peptide inhibitor targeting HMGB1 by peptide-protein binding prediction and force field-based molecular simulations and validated its inhibition on cellular production of inflammatory factors and phenotype of skin inflammation in mice. This inhibitory peptide, named cIY8, bears a cyclic structure by disulfide bond formation (Fig. 1a). It is predicted to inhibit the fragment of HMGB1 A-box (Fig. 1b) and therefore has the potential to block HMGB1-mediated activation process of TLR4 signaling [17]. A self-controlled AD mouse model was established to study the efficacy of cIY8 (Fig. 1c). After cIY8 topical treatment for 7 days, the ear skin of the AD mouse model showed less redness, swelling, and eruption compared to the ear of the opposite side treated with MC903 only (Fig. 1d, f). Histopathological results also showed that cIY8 obviously inhibited hyperkeratosis and acanthosis in the epidermis and lymphocyte infiltration in the dermis (Fig. 1e). Compared with MC903-treated side, the number of scratch bouts was significantly reduced at the cIY8-treated side (Fig. 1g). However, control peptide IY8 could not change the inflammatory appearance, histopathology, clinical score, and scratching behavior of the ears in the AD mouse model (online suppl. Fig. S2). In addition, the mRNAs of type 2 cytokines, such as IL-1α, IL-4, and IL-13, decreased notably after cIY8 treatment by quantitative PCR (Fig. 1h). These data demonstrate that HMGB1 plays an important role in AD and cIY8 binding to HMGB1 attenuates the inflammation and pruritus sensation in the AD mouse model.

Fig. 1.

cIY8 inhibited AD-like symptoms in the AD mouse model. a Two-dimensional diagram and three-dimensional structure by molecular dynamics simulation of the peptide cIY8. b Predictive binding mode of peptide cIY8 (shown in yellow) and HMGB1 region (shown in cyan) with potential interactive regions presented (shown in marine). c Mice were topically applied with MC903 (2 nm) on left ears and MC903 (2 nm)+cIY8 (100 μm) topically applied on right ears once a day for 7 days. d Comparison of the ear appearance between MC903 side and MC903+cIY8 side. e cIY8 reduced the severity of AD lesions in mice by histopathology staining (scale bar = 100 μm). f Clinical scores (0–12) of MC903 side and MC903+cIY8 side. g Scratching records of mice in 60 min on day 7. h Relative levels of IL-1α, IL-4, IL-5, IL-13, and IL-31 mRNAs in the lesions of the two sides on day 7 (unpaired t tests, n = 3 mice per group, *p < 0.05, **p < 0.01).

Fig. 1.

cIY8 inhibited AD-like symptoms in the AD mouse model. a Two-dimensional diagram and three-dimensional structure by molecular dynamics simulation of the peptide cIY8. b Predictive binding mode of peptide cIY8 (shown in yellow) and HMGB1 region (shown in cyan) with potential interactive regions presented (shown in marine). c Mice were topically applied with MC903 (2 nm) on left ears and MC903 (2 nm)+cIY8 (100 μm) topically applied on right ears once a day for 7 days. d Comparison of the ear appearance between MC903 side and MC903+cIY8 side. e cIY8 reduced the severity of AD lesions in mice by histopathology staining (scale bar = 100 μm). f Clinical scores (0–12) of MC903 side and MC903+cIY8 side. g Scratching records of mice in 60 min on day 7. h Relative levels of IL-1α, IL-4, IL-5, IL-13, and IL-31 mRNAs in the lesions of the two sides on day 7 (unpaired t tests, n = 3 mice per group, *p < 0.05, **p < 0.01).

Close modal

Keratinocytes also have key roles in the pathogenesis of AD. To investigate the effects of HMGB1 and cIY8 on keratinocytes, we measured the mRNA levels of AD-related cytokines synthesized in keratinocytes by real-time qPCR, and the results showed that the mRNA expression levels of IL-1α, IL-17A, IL-33, and IFN-γ increased significantly after HMGB1 stimulation and could be inhibited notably by HMGB1 inhibitor peptide cIY8 but not by the control peptide IY8 (online suppl. Fig. S3).

cIY8 Inhibits the HMGB1-Mediated NF-κB Activation in Human Fibroblasts

We further explored the molecular pathway, by which cIY8 inhibited inflammation in AD. Human fibroblast cell line HFF-1 cells were stimulated with 500 ng/mL HMGB1, and a significant nuclear translocation of NF-kB was observed, suggesting the activation of NF-κB. Notably, cIY8 of 10 nm to 10 μm significantly inhibited the nuclear translocation of NF-κB (Fig. 2a), and this inhibition was in a cIY8 concentration-dependent manner (Fig. 2b). Therefore, HMGB1 activates the inflammation of AD through NF-κB pathway, and cIY8 binds HMGB1 and inhibits the HMGB1-mediated inflammation in skin fibroblasts through the blockade of NF-κB activation.

Fig. 2.

cIY8 peptide inhibited the NF-κB nuclear translocation by HMGB1 in human fibroblasts. a Representative images of NF-κB p65 (red) translocated in the nuclei of human fibroblasts HFF-1 treated with HMGB1 (500 ng/mL) in the absence and presence of cIY8 (1 μm) (scale bar = 100 μm). b cIY8 effectively inhibited the NF-κB nuclear translocation by HMGB1 in human fibroblasts (Student’s t test, n = 6 random areas per group, ***p < 0.001, ****p < 0.0001).

Fig. 2.

cIY8 peptide inhibited the NF-κB nuclear translocation by HMGB1 in human fibroblasts. a Representative images of NF-κB p65 (red) translocated in the nuclei of human fibroblasts HFF-1 treated with HMGB1 (500 ng/mL) in the absence and presence of cIY8 (1 μm) (scale bar = 100 μm). b cIY8 effectively inhibited the NF-κB nuclear translocation by HMGB1 in human fibroblasts (Student’s t test, n = 6 random areas per group, ***p < 0.001, ****p < 0.0001).

Close modal

HMGB1 Antagonist Glycyrrhizin Relieves the AD-Like Symptoms in the AD Mouse Model

Glycyrrhizin directly binds to the HMG boxes of HMGB1 and inhibits its chemotactic and mitogenic functions [18]. Glycyrrhizin treatment improved the thickening of collagen fibers in the superficial dermis in moderate-to-severe AD patients (online suppl. Fig. S4). To further confirm the therapeutic effects of glycyrrhizin on AD, we established a mouse model with AD-like symptoms [19] by topical application of MC903 for 7 days (Fig. 3a). Mice treated with oral glycyrrhizin displayed milder symptoms of erythema, dryness, edema, and excoriation and milder pathological changes of hyperkeratosis and acanthosis in the epidermis and lymphocyte infiltration in the dermis, as compared with those in the saline group (Fig. 3b, c). The number of scratch bouts were also less in the glycyrrhizin group than in the saline group (Fig. 3d). Immunohistochemistry of the ears found that the cell proliferation marker Ki67 was reduced in keratinocytes of the ears in the glycyrrhizin group (online suppl. Fig. S5). These results confirm that oral application of glycyrrhizin attenuates the inflammation in the AD mouse model.

Fig. 3.

Therapeutic effects of glycyrrhizin on AD mouse models. a Mice were orally administered with glycyrrhizin or 0.9% saline for 7 days. MC903 (2 nmoL/20 μL) was topically applied on mice ears once a day to induce AD model. Scratching bouts were recorded on day 3 and day 7. b Ear appearance of the saline group, glycyrrhizin group, and negative control group. c Pathological changes of the ears showing AD lesions reduced significantly in glycyrrhizin group (HE stain, scale = 100 μm). d Scratching bouts in 60 min on day 3 and day 7 in the glycyrrhizin group and saline group (unpaired t tests, n = 5–7, *p < 0.05).

Fig. 3.

Therapeutic effects of glycyrrhizin on AD mouse models. a Mice were orally administered with glycyrrhizin or 0.9% saline for 7 days. MC903 (2 nmoL/20 μL) was topically applied on mice ears once a day to induce AD model. Scratching bouts were recorded on day 3 and day 7. b Ear appearance of the saline group, glycyrrhizin group, and negative control group. c Pathological changes of the ears showing AD lesions reduced significantly in glycyrrhizin group (HE stain, scale = 100 μm). d Scratching bouts in 60 min on day 3 and day 7 in the glycyrrhizin group and saline group (unpaired t tests, n = 5–7, *p < 0.05).

Close modal

HMGB1 Antagonist Glycyrrhizin Inhibits Ca2+ Mobilization in Mouse Fibroblasts

To explore the inhibition of HMGB1 on Ca2+ mobilization in fibroblasts, we used mouse primary fibroblasts to quantify intracellular Ca2+ transients by calcium imaging. Ionomycin is a selective and effective calcium ionophore, which elicits Ca2+ current from extracellular fluid to cytoplasm and a strong intracellular Ca2+ response. We observed that the cytosolic Ca2+ response by ionomycin was almost abolished in the fibroblasts when glycyrrhizin was added to the medium (Fig. 4a). Ca2+ is a secondary messenger and participates in the modulation of various cellular biological processes, including survival, proliferation, apoptosis, and immune responses [20]. We demonstrated that glycyrrhizin restrained Ca2+ mobilization in fibroblasts (Fig. 4b, c), but the mechanism underlying the inhibition is yet unknown.

Fig. 4.

Glycyrrhizin inhibited Ca2+ mobilization in mouse primary fibroblasts. a Representative fluorescence images of Fluo-8 (5 μm) loaded fibroblasts pretreated with ionomycin (5 μm) or ionomycin + glycyrrhizin (17 μm). Ionomycin induced Ca2+ response in primary fibroblasts (top right), and the response was inhibited by adding glycyrrhizin (bottom right). b Representative traces showing intracellular Ca2+ responses elicited by ionomycin and ionomycin + glycyrrhizin. c The ratio of intracellular Ca2+ responsive fibroblasts in ionomycin group and ionomycin + glycyrrhizin group (Student’s t test, n = 3 mice per group, *p < 0.05).

Fig. 4.

Glycyrrhizin inhibited Ca2+ mobilization in mouse primary fibroblasts. a Representative fluorescence images of Fluo-8 (5 μm) loaded fibroblasts pretreated with ionomycin (5 μm) or ionomycin + glycyrrhizin (17 μm). Ionomycin induced Ca2+ response in primary fibroblasts (top right), and the response was inhibited by adding glycyrrhizin (bottom right). b Representative traces showing intracellular Ca2+ responses elicited by ionomycin and ionomycin + glycyrrhizin. c The ratio of intracellular Ca2+ responsive fibroblasts in ionomycin group and ionomycin + glycyrrhizin group (Student’s t test, n = 3 mice per group, *p < 0.05).

Close modal

HMGB1 Antagonist Glycyrrhizin Inhibits Proliferation and Secretion of Mouse Fibroblasts

We hypothesized that HMGB1 antagonists inhibit the proliferation and function of fibroblasts, through which inflammation and pruritus are improved in the AD mouse model. We used three biomarkers of α smooth muscle actin (α-SMA) [21], Hsp47, a collagen-specific chaperon with a crucial role in collagen folding [22], and vimentin, an integrator during wound healing and fibroblast proliferation [23], to assay the three proteins in the AD mouse model. Immunohistochemistry of the mouse ears showed that the ratios of α-SMA, Hsp47, and vimentin-positive cells were significantly reduced in glycyrrhizin group as compared to those in the saline group (Fig. 5a–c), suggesting the inhibition of fibroblast proliferation by glycyrrhizin treatment. The ex vivo experiments of EdU incorporation in mouse primary fibroblasts also demonstrated that MC903 stimulated the proliferation of the cells, and this proliferation effect could be inhibited in the presence of glycyrrhizin (Fig. 5d, e).

Fig. 5.

Glycyrrhizin inhibited proliferation of mouse primary fibroblasts in vivo and ex vivo (a, b, c) The immunoreactivity to anti-α-SMA, anti-Hsp47 and anti-vimentin antibodies increased notably in fibroblasts in the ears of saline group and decreased in the ears of glycyrrhizin group and control group (scale bar = 100 μm). d Representative images of EdU incorporation (red) into mouse primary fibroblasts treated with MC903 in the absence and presence of glycyrrhizin (2 mm) (scale bar = 100 μm). e MC903-induced proliferation of fibroblasts relative to the control group, and EdU+ fibroblasts decreased by glycyrrhizin treatment (one-way ANOVA followed by Tukey’s post hoc, *p < 0.05, **p < 0.01).

Fig. 5.

Glycyrrhizin inhibited proliferation of mouse primary fibroblasts in vivo and ex vivo (a, b, c) The immunoreactivity to anti-α-SMA, anti-Hsp47 and anti-vimentin antibodies increased notably in fibroblasts in the ears of saline group and decreased in the ears of glycyrrhizin group and control group (scale bar = 100 μm). d Representative images of EdU incorporation (red) into mouse primary fibroblasts treated with MC903 in the absence and presence of glycyrrhizin (2 mm) (scale bar = 100 μm). e MC903-induced proliferation of fibroblasts relative to the control group, and EdU+ fibroblasts decreased by glycyrrhizin treatment (one-way ANOVA followed by Tukey’s post hoc, *p < 0.05, **p < 0.01).

Close modal

IL-33 and the dermal matrix of periostin can be released from skin fibroblasts and take part in the pruritus sensation in AD. IL-33 and periostin as well as their mRNAs reduced significantly in the ears of the glycyrrhizin group (Fig. 6), consistent with the reduced scratch bouts in the glycyrrhizin group of the AD mouse model.

Fig. 6.

Periostin and IL-33 and their mRNAs expressed in the ears of the AD mouse model. a, b Periostin and IL-33 in the superficial dermis reduced significantly in glycyrrhizin and control groups and increased in saline group (scale bar = 100 μm). c Periostin and IL-33 mRNAs also reduced significantly in glycyrrhizin and control groups and increased in saline group (unpaired t tests, n = 6–8 mice per group, *p < 0.05, **p < 0.01).

Fig. 6.

Periostin and IL-33 and their mRNAs expressed in the ears of the AD mouse model. a, b Periostin and IL-33 in the superficial dermis reduced significantly in glycyrrhizin and control groups and increased in saline group (scale bar = 100 μm). c Periostin and IL-33 mRNAs also reduced significantly in glycyrrhizin and control groups and increased in saline group (unpaired t tests, n = 6–8 mice per group, *p < 0.05, **p < 0.01).

Close modal

We used a multidisciplinary approach including behavior, biochemistry, cell biology, and pathology methods to explore the role of skin fibroblasts in the pathogenesis of pruritus and inflammation in AD. We found that the peptide cIY8 and the HMGB1 antagonist glycyrrhizin targeted and inactivated the HMGB1 in dermal fibroblasts and effectively improved skin lesions and itch in the AD mouse model.

HMGB1 consists of two structurally similar domains, the pro-inflammatory box (B-box) and the anti-inflammatory box (A-box) [24]. HMGB1 A-box and B-box individually activate TLR4 signaling to release cytokines/chemokines. HMGB1 recognition cascade could be disrupted by blocking A-box with anti-HMGB1 mAb 2G7 [25]. We topically used the novel HMGB1-binding peptide cIY8 and demonstrated that blockade of HMGB1 significantly improved the symptoms and pruritus in the AD mouse model and well controlled the type 2 inflammation. The development of AD is related to Th1/Th2/Th17 immune imbalance. We tested mRNA levels of AD-related cytokines and found that HMGB1 widely stimulated the pro-inflammatory signals of IL-1α, Th1 (IFN-γ), Th2 (IL-33), and Th17 (IL-17A) in keratinocytes, and these inflammatory signals could be inhibited by the HMGB1-binding peptide cIY8. Previous studies have shown that extracellular HMGB1 interacts with TLR4 to activate NF-κB pathway [26]. TLR4 is prominently expressed in macrophages, dendritic cells, antigen-presenting cells, and fibroblasts [27]. Our ex vivo experiments confirmed that the extracellular HMGB1 promoted nuclear translocation of NF-κB in human fibroblast cell line HFF-1 cells, and cIY8 inhibited the NF-κB activation (Fig. 7). The important transcription factor NF-κB regulates the expression of many cytokines such as IL-4 and IFN-γ [28]. Human and murine HMGB1 are highly homologous. The remarkable therapeutic effects of peptide cIY8 in the AD mouse model and human skin fibroblasts imply the possibilities to develop new topical drugs for AD.

Fig. 7.

Diagram of the HMGB1 blockers of glycyrrhizin and peptide cIY8 to alleviate AD changes through the inhibition of NF-κB signaling pathway. The specific HMGB1-binding peptide cIY8 and the HMGB1 inhibitor glycyrrhizin activate skin fibroblasts to alleviate the inflammation and pruritus of AD. cIY8 and glycyrrhizin inhibit the release of Ca2+ from the endoplasmic reticulum and the activation of downstream NF-κB signaling pathway, which in turn inhibit the proliferation of fibroblasts and the release of inflammatory and itch-related factors from fibroblasts.

Fig. 7.

Diagram of the HMGB1 blockers of glycyrrhizin and peptide cIY8 to alleviate AD changes through the inhibition of NF-κB signaling pathway. The specific HMGB1-binding peptide cIY8 and the HMGB1 inhibitor glycyrrhizin activate skin fibroblasts to alleviate the inflammation and pruritus of AD. cIY8 and glycyrrhizin inhibit the release of Ca2+ from the endoplasmic reticulum and the activation of downstream NF-κB signaling pathway, which in turn inhibit the proliferation of fibroblasts and the release of inflammatory and itch-related factors from fibroblasts.

Close modal

Glycyrrhizin inhibits HMGB1 activity probably in two ways. First, glycyrrhizin binds to the nuclear HMGB1-DNA complex when HMGB1 is located in nucleus [29], thus reducing the combination of HMGB1 and DNA to inhibit the synthesis of inflammatory cytokines. Second, glycyrrhizin binds to the two HMGB1-DNA-binding domains when the protein is released into the cytoplasm or intercellular space, thus inhibiting the development of pro-inflammatory HMGB1 signals [30]. Lipopolysaccharide (LPS) is an important component of the outer membrane in gram-negative bacteria and a specific trigger for human umbilical vein endothelial cells to release HMGB1 [31]. LPS stimulates the release of HMGB1 in a TLR4- and caspase-11-dependent manner [32]. We used LPS to activate TLR4, the effective receptor that interacts with HMGB1 during pro-inflammatory responses, but detected no calcium responses in primary fibroblasts (data not shown), suggesting that the activation of TLR4 does not promote calcium mobilization. Ionomycin upregulates the concentration of cytosolic Ca2+ by mobilizing the intracellular storage in endoplasmic reticulum calcium pumps. Increased Ca2+ mobilization is a key component of the second messenger to promote proliferation, activation, and contractility [33]. In this study, we found that calcium signal was remarkably inhibited by glycyrrhizin in primary fibroblasts, in which the endogenous cell pathway is probably involved. Glycyrrhizin may attenuate Ca2+ mobilization and the release of periostin and IL-33 in mouse fibroblasts, but the specific pathways involved in this process remain unknown.

Periostin is a protein in the extracellular matrix secreted by fibroblasts mainly involved in tissue repair [34]. During the development of AD, fibroblasts have an increased ability to secrete extracellular matrix proteins including periostin, which activate the integrin αvβ3 receptor on sensory neurons and induce itch-like symptoms [35]. Periostin also participates in the communication between fibroblasts and other cells, enhancing the release of pruritogens and aggravating itch-like symptoms in AD patients. Keratinocytes can be activated by periostin and release TSLP through NK-κB signaling, which plays a significant role in pruritus and the progression of AD [36]. Single-cell RNA sequencing showed that a new subset of COL6A5+ and COL18A1+ fibroblasts was expressed in the lesional skin area of AD patients. This subset can secrete CCL19 and type 2 chemokine CCL2. In particular, a dendritic cell subset that expressed the CCL19 receptor CCR7 was unique to AD lesions, indicating a potential role that fibroblasts transmit signals to immune cells [4]. M2 macrophages are innate immune cells that release cytokines, such as IL-31 – an important pruritogen. We found that CD163 was downregulated in the AD mouse model after glycyrrhizin (data not shown) and HMGB1 inhibitor peptide cIY8 (online suppl. Fig. S6) treatment. Immune imbalance dominated by Th2-type immune responses plays an important role in the pathogenesis of AD. Our data showed no significant change in CD4+ T cells in the absence or presence of cIY8 in the AD mouse model (online suppl. Fig. S7). However, whether and how HMGB1 affects immune cells including M2 macrophages and T cells in the AD immune microenvironment remains to be elucidated. IL-33 disrupts the skin barrier by reducing the expression of filaggrin and claudin-1 in keratinocytes [37] and facilitates the production of type 2 cytokines, such as IL-4, IL-5. and IL-13, by group 2 innate lymphoid cells (ILC2s) [38]. Glycyrrhizin breaks chronic itch-scratch cycle by improving the Th2 immune microenvironment and repair of the skin barrier.

In conclusion, fibroblasts act as an amplifier of inflammation and pruritus in AD. As we know, there are no clinical trials employing HMGB1 antagonist to treat AD. Here we use glycyrrhizin and a novel HMGB1-binding peptide cIY8 to successfully treat the AD-like changes in an AD mouse model, providing a basis to explore new approaches for the treatment of AD patients.

All experimental procedures followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Studies Committee at the Peking University First Hospital, approval number J202172.

The authors state no conflict of interest.

This work was supported by CIBR Open Cooperation Fund for Science in China (Grant No. 2020-NKX-XM-08 to ZJH) and National Natural Science Foundation of China (Grant No. 81903213 to ZJH). This study also benefitted from the funding of a Joint Sino-German Research Project by the National Natural Science Foundation of China and the DFG (Grant No. GZ901 to ZZT) and the Beijing Municipal Science and Technology Project (Grant No. Z211100007921017 to ZZT).

Dr. Jiahui Zhao and Dr. Zuotao Zhao contributed to study design and supervised the experiments. Dr. Lingxuan Zhou, Dr. Xiaohui Yuan, Dr. Yongyan Hu, Dr. Siyu Zhu, Dr. Junxiang Li, and Dr. Chenyu Wang acquired the data. Dr. Lingxuan Zhou and Dr. Xiaohui Yuan performed the analyses and interpretation of the data. Dr. Jiahui Zhao, Dr. Lingxuan Zhou, and Dr. Xiaohui Yuan drafted the manuscript. Dr. Jiahui Zhao, Dr. Zuotao Zhao, Dr. Miao Jing, Dr. Zhe Xu, and Dr. Lingling Liu revised the manuscript.

Additional Information

LingXuan Zhou and Xiaohui Yuan contributed equally to this work.Edited by: H.-U. Simon, Bern.

All data generated or analyzed during this study are included in this article and its online supplementary material files. Further inquiries can be directed to the corresponding author.

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