Tomaralimab (OPN-305; cat. no. NM-103), a humanized anti-TLR2 Ab, was provided by Neuramedy Co., Ltd. DNCB (cat. no. 237329), toluidine blue (TB; cat. no. 89640) and hematoxylin (cat. no. H3136) and eosin B (cat. no. 212954) were purchased from MilliporeSigma. Anti-thymic stromal lymphopoietin (TSLP) Ab (cat. no. NBP1-76754) was obtained from Novus Biologicals, LLC and anti-HMGB1 Ab (cat. no. ab18256) was obtained from Abcam. Anti-IL1β Ab (cat. no. 2022) and anti-F4/80 Ab (cat. no. 70076) were obtained from Cell Signaling Technology, Inc., and anti-IL33 Ab (cat. no. 12372-1-AP) was purchased from Proteintech Group, Inc. Anti-MPO Ab (code no. A0398) was obtained from Dako; Agilent Technologies, Inc. Anti-GAPDH (cat. no. sc-32233) and anti-IL17 (cat. no. sc-374218) Abs were purchased from Santa Cruz Biotechnology, Inc. Anti-IL31 Ab (cat. no. 701082) and secdondary Abs conjugated with horseradish peroxidase (anti-rabbit IgG, cat. no. 31460; anti-mouse IgG, cat. no. 31430) and rhodamine Red-X (cat. no. R6394) were obtained from Thermo Fisher Scientific, Inc.

A total of 18 male BALB/c mice (body weight range, 18-23 g), aged ~7 weeks, were purchased from Orient Bio, Inc. The mice were housed in a controlled environment at 22±2˚C with constant humidity (40-60%), maintained under a 12-h light-dark cycle. They were provided with unrestricted access to sterile water and food in a specific pathogen-free grade laboratory. In the induction of AD with DNCB in BALB/c mice, 4% SDS and 1% DNCB are typically used initially to create skin barrier disruption and sensitization, respectively (12,13). The commonly used dose of neutralizing Abs for intravenous (IV) administration in mice usually ranges from 1 to 25 mg/kg (14,15). In preliminary experiments, two doses of Tomaralimab were tested, 25 and 50 mg/kg. It was observed that administrating 25 mg/kg effectively reduced DNCB-induced skin inflammation, comparable to a higher dose of 50 mg/kg. Consequently, the 25 mg/kg dosage of Tomaralimab was selected for investigation. The mice were divided into three groups in a random manner: Group I, Control; Group II, DNCB + PBS; and Group III, DNCB + Tomaralimab (n=6 in each group). The method of inducing skin inflammation similar to AD through the topical application of DNCB was carried out as previously described (16). Group I mice were treated with a vehicle (acetone:olive oil, 3:1 v/v), and Group II and III mice were sensitized with 4% SDS on the dorsal skin to disrupt the skin barrier. After 4 h, SDS-sensitized areas were subjected to daily topical application of 1% DNCB in vehicle for 3 days. Following a 4-day break, 0.5% DNCB was applied topically to the same area seven times at 2-day intervals, and the mice were sacrificed the next day (on day 20). Group III mice received an IV dose of Tomaralimab (25 mg/kg) following each DNCB application. On day 20, all mice were euthanized in a chamber with CO₂ at a fill rate of 50% of the chamber volume per min. After visual confirmation of death, the mice were further exposed to CO₂ for an additional minute to ensure the absence of a heartbeat for confirmation of complete euthanasia and final death. The animal studies followed the guidelines approved (IACUC; approval number KU26036) by the Konkuk University Institutional Animal Care and Use Committee (Seoul, Korea).

Skin lesions from the dorsal surface were surgically excised and fixed overnight at 25˚C with 100% acetone, then embedded in paraffin. Paraffin blocks were sliced at a thickness of 5 µm with a microtome (Leica Microsystems GmbH). As previously described, paraffin-embedded tissues were deparaffinized with xylene and rehydrated using a graded series of ethanol (17). Rehydrated sections were immersed in hematoxylin solution for 1 min at 25˚C, rinsed under running tap water, and subsequently immersed in eosin solution for 30 sec at 25˚C. Dehydration was performed by passing the sections through a graded ethanol series (80, 90, 95 and 100%) for 30 sec each at 25˚C. The slides were then rinsed in xylene three times for 5 min each at 25˚C and mounted with coverslips.

The mast cells infiltrated were stained with 0.1% TB for 3 min at 25˚C. Stained images were observed using a light microscope (EVOS FL Auto; Thermo Fisher Scientific, Inc.). Epidermal thickness was measured using ImageJ 1.52a software (National Institutes of Health).

After blocking the intracellular peroxidase activity for 1 h at 25˚C with 3% hydrogen peroxide, tissue sections were incubated overnight at 4˚C with primary antibodies against F4/80 (1:200) and MPO (1:500), followed by avidin/biotin complex-mediated DAB staining using VECTASTAIN Elite ABC-HRP Kit (cat. no. PK-6101; Vector Laboratories, Inc.), and counterstained with hematoxylin, as previously described (11).

Fluorescent immunohistochemical staining was carried out as previously described (11). A secondary Ab labeled with rhodamine Red-X (1:500) was incubated for 1 h at 25˚C for the fluorescence staining. Hoechst 33258 (10 µg/ml) was employed to counterstain the nuclear DNA. Subsequently, the slides were treated with a fluorescence mounting medium (ProLong Gold Antifade Reagent; Invitrogen; Thermo Fisher Scientific, Inc.) after washing with PBS. Fluorescence images were taken with an EVOS FL Auto system, and the fluorescence levels were quantified using ImageJ software (National Institutes of Health).

Human keratinocyte HaCaT cells were obtained from the CLS Cell Line Service GmbH. The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (HyClone; Cytiva) and penicillin-streptomycin (Sigma-Aldrich; Merck KGaA) in a humidified atmosphere with 5% CO₂ at 37˚C. DNCB were treated with varying concentrations (0-50 nM) for 18 h (for detecting mRNA levels) and 24 h (for detecting protein levels), or 10 nM DNCB for various periods (0-36 h). Tomaralimab at concentrations of 0.5, 1, 5 and 10 µg/ml was pretreated 30 min before DNCB stimulation.

HaCaT cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.4, 400 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM NaF, 1 mM NaVO₄) as previously described (18). The proteins (20 µg/lane) were electrophoresed on 10% SDS-PAGE and transferred to nitrocellulose membranes. After incubation with the specific primary Abs overnight at 4˚C, HRP-conjugated secondary Abs (goat anti-rabbit IgG, 1:8,000; goat anti-mouse IgG, 1:8,000) were incubated for 1 h at 25˚C. The blots were visualized utilizing an enhanced chemiluminescence (ECL) detection system (cat. no. 34580; Thermo Fisher Scientific, Inc.). Densitometric analysis was performed using ImageJ 1.52a software (National Institutes of Health).

Total RNA isolation, cDNA synthesis, and PCR reaction were carried out as previously described (19). The PCR primers used in the present study were as follows: TSLP forward, 5'-TAGCAATCGGCCACATTGCCT-3' and reverse, 5'-GAAGCGACGCCACAATCCTTG-3'; IL1β forward, 5'-AAACAGATGAAGTGCTCCTTCCAGG-3' and reverse, 5'-TGGAGAACACCACTTGTTGCTCCA-3'; IL17 forward, 5'-CCATAGTGAAGGCAGGAA TC-3' and reverse, 5'-GAGGTGGATCGGTTGTAGTA-3'; IL31 forward, 5'-TCGAGGAATTACAGTCCCTCT-3' and reverse, 5'-TGTCGAGGTGCTCTATGATCTC-3'; IL33 forward, 5'-CAAAGAAGTTTGCCCCATGT-3' and reverse, 5'-AAGGCAAAGCACTCCACAGT-3'; HMGB1 forward, 5'-ATATGGCAAAAGCGGACAAG-3' and reverse, 5'-AGGCCAGGATGTTCTCCTTT-3'; and GAPDH forward, 5'-ACCCACTCCTCCACCTTTG-3' and reverse, 5'-CTCTTGTGCTCTTGCTGGG-3'. The amplified PCR products were visualized under UV transillumination.

Cell viability was measured using a EZ-Cytox assay kit (cat. no. EZ-3000; DoGenBio). HaCaT cells cultured in 96-well plates (5,000 cells/100 µl) were treated with varying concentrations of DNCB (0, 5, 10, 25 and 50 nM) for 24 h. The assay solution (10 µl per 100 µl culture medium) was added to each plate and incubate for 1 h in a 37˚C CO₂ incubator, after which the absorbance was measured at 450 nm using an Emax Endpoint ELISA Microplate Reader (Molecular Devices, LLC).

The data are presented as the average value ± standard deviation (SD). To compare multiple groups, a one-way ANOVA followed by Dunnett's multiple comparisons test or Tukey's multiple comparisons test was performed using GraphPad Prism V10.1.2 software (GraphPad Software Inc.; Dotmatics). P<0.05 was considered to indicate a statistically significant difference.

To assess the therapeutic effectiveness of systemic administration of Tomaralimab for allergenic hapten-induced AD, a mouse model was used, in which the allergenic hapten DNCB was applied topically to the back skin of BALB/c mice (11). Topical application of DNCB leads to typical AD-like skin inflammation with superficial redness and edema (11,20). Under these experimental conditions, Tomaralimab was injected IV through the lateral tail vein every other day until day 19 (Fig. S1). H&E staining revealed that DNCB significantly increased epidermal hyperplasia, which was reduced by systemic administration of Tomaralimab (Fig. 1A). Measurement of epidermal thickness using ImageJ demonstrated that Tomaralimab administration resulted in a significant reduction (P=0.043 by Dunnett's multiple comparisons test, n=6) in DNCB-induced epidermal thickness (Fig. 1B).

FLG is an epidermal protein responsible for maintaining skin barrier function (21), and its deficiency contributes critically to AD (22). TSLP is an epithelial-derived pro-inflammatory cytokine that triggers dendritic cells, T-lymphocytes and mast cells to stimulate the release of various inflammatory cytokines and is considered a crucial marker in the early pathogenesis of AD (23,24). Immunofluorescence staining revealed that administration of Tomaralimab led to the recovery of DNCB-induced suppression of FLG levels (Fig. 2A) and decreased DNCB-induced TSLP expression (Fig. 2B). These data suggest that systemic administration of Tomaralimab has a potential impact on the recovery of damaged skin barrier function and inhibiting the onset of allergic skin inflammation.

The increased infiltration of immune cells, including T lymphocytes and mast cells, at inflammatory sites is closely linked to the pathogenesis of AD (25). It was also observed that the administration of Tomaralimab significantly (P<0.001 by Dunnett's multiple comparisons test, n=6) reduced the population of infiltrated mast cells stained by TB (Fig. 3A and B). Furthermore, Tomaralimab led to a notable decrease in the presence of F4/80-positive macrophages (Fig. 3C) and myeloperoxidase (MPO)-positive immune cells, including basophils and neutrophils (Fig. 3D).

Keratinocytes play crucial roles in the pathogenesis of AD (26). To investigate how Tomaralimab impacts DNCB-induced inflammation on a cellular level,inflammatory responses in HaCaT keratinocytes were triggered using DNCB. Previous studies have demonstrated that keratinocytes release multiple pro-inflammatory cytokines involved in the pathogenesis of AD, including TSLP, IL-1β, IL-17 and IL-33 (16,27). Immunoblot analysis demonstrated that DNCB increased levels of TSLP, IL-1β, IL-17, IL-31 and IL-33 in a dose- (Fig. 4A) and time-dependent manner (Fig. 4B). When HaCaT cells were exposed to varying concentrations (0-50 nM) of DNCB, no cytotoxicity was observed; instead, DNCB exposure exhibited slight increases in survival rate (Fig. S2). Certain cytokines, such as IL-1β and IL-31, reached peak levels when the DNCB concentration exceeded 25 nM, and all tested cytokines approached sub-peak levels after 24 h of treatment. Cells were treated at a concentration of 10 nM for 24 h to maximize the inhibitory action of Tomaralimab. Under these experimental conditions, Tomaralimab treatment resulted in a dose-dependent decrease in mRNA levels of DNCB-induced inflammatory cytokines (Fig. 5A). In line with the RT-qPCR findings, the protein levels of inflammatory cytokines were reduced in a dose-dependent manner (Fig. 5B).

HMGB1 is a chromatin-binding nuclear protein involved in DNA bending and assembly of proteins at specific DNA sites (28). HMGB1 can be produced from necrotic cells and implicated as a mediator of inflammation by induction of various pro-inflammatory cytokines (29). AS HMGB1 is an endogenous TLR2 ligand involved in TLR2-induced inflammation (30), it was examined whether DNCB induces HMGB1 expression. It was observed that DNCB increased mRNA (Fig. 6A) and protein (Fig. 6B) levels in a time-dependent manner. Under this experimental condition, treatment with Tomaralimab dose-dependently reduced DNCB-induced HMGB1 expression at mRNA (Fig. 6C) and protein (Fig. 6D) levels. To evaluate the impact of Tomaralimab on HMGB1 expression in vivo, immunofluorescence analysis was conducted on skin tissues from BALB/c mice challenged with DNCB. Topical application of DNCB markedly increased the staining intensity of HMGB1 in the epidermis (Fig. 6E, left panel). By contrast, the administration of Tomaralimab led to a significant decrease in DNCB-induced HMGB1 intensity (P<0.05, n=6) (Fig. 6E, right graph). These data suggest that the decrease in HMGB1 levels may be associated with the inhibition of DNCB-induced skin lesions by the humanized anti-TLR2 Ab, Tomaralimab.