The study was conducted on female albino Wistar rats (Crl: WI; Charles River Laboratories, Wilmington, MA, USA) with an average weight of approximately 250 grams and an age range of 3 to 4 months. The animals were procured from the animal quarters of the Faculty of Biology and Environmental Protection at the University of Lodz. The experimental protocols received approval from the Local Ethics Committee for Experiments on Animals in Lodz (no 15/2021). Every effort was undertaken to minimize animal suffering. Prior to decapitation, all animals were anesthetized with isoflurane (BAXTER, Deerfield, IL, USA). Peritoneal cell suspensions were obtained using lavage with 50 mL of 1% Hank’s Balanced Salt Solution (HBSS; GIBCO, Gaithersburg, MD, USA) supplemented with 0.015% sodium bicarbonate (GIBCO). Following an abdominal massage of approximately 90 seconds, the cell suspension was extracted from the peritoneal cavity. The peritoneal cell suspension underwent two washes (150 x g, 5 minutes, 20°C) in complete Dulbecco’s Modified Eagle Medium (cDMEM), which consisted of DMEM (Biowest, Kansas City, MO, USA) supplemented with 10% fetal calf serum (FCS; GIBCO), 10 μg/mL gentamicin (GIBCO), and 2 mM glutamine (GIBCO). Isotonic 72.5% Percoll (Sigma-Aldrich, St. Louis, MO, USA) density gradient centrifugation (190 x g, 15 minutes, 20°C) was applied for the purification of MCs. Subsequently, the isolated MCs were centrifuged twice in cDMEM (150 x g, 5 minutes, 20°C). The isolation process for MCs lasted approximately 45 to 50 minutes. After washing, MCs were counted and resuspended in an appropriate volume of cDMEM (for quantitative RT-PCR, flow cytometry, and confocal microscopy analysis, as well as migration assays) or a medium specifically designed for rat MCs, which contained 137 mM NaCl (Sigma-Aldrich), 2.7 mM KCl (Sigma-Aldrich), 1 mM MgCl2 (Sigma-Aldrich), 1 mM CaCl2 (Sigma-Aldrich), 10 mM HEPES (Sigma-Aldrich), 5.6 mM glucose (Sigma-Aldrich), and 1 mg/mL bovine serum albumin (BSA; Sigma-Aldrich) (for histamine release assays, ELISA assays, and ROS generation), to achieve a MC concentration of 1.5 × 10⁶ cells/mL. A suitable number of animals was employed to attain the appropriate MC density and the required number of samples in a specific type of experiment. The MCs were prepared with a purity exceeding 98%, as measured by metachromatic staining using toluidine blue (Sigma-Aldrich). The viability of MCs was greater than 98%, determined through the trypan blue (Sigma-Aldrich) exclusion assay. The results obtained from the treated samples were compared to the control samples within the confines of the respective experiment.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was employed to assess constitutive and HMGB1-induced mRNA levels of receptors and cytokines/chemokines in MCs. Purified MCs, suspended in cDMEM, were stimulated with HMGB1 (Novus Biologicals, Centennial, CO, USA) at a final concentration of 1 μg/mL for 2 hours at 37°C in a humidified atmosphere containing 5% CO₂. For the control group, MCs were maintained under identical conditions without HMGB1. Total RNA was extracted from the cells utilizing the RNeasy^® Mini Kit (Qiagen, Valencia, CA, USA). Subsequently, complementary DNA (cDNA) was synthesized according to the manufacturer’s protocol for the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA). qRT-PCR was conducted utilizing the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories) in conjunction with the iTaq™ Universal SYBR^® Green Supermix (Bio-Rad Laboratories). The volume of the PCR reaction comprised 5 µL of iTaq™ Universal SYBR^® Green Supermix, 1 μL of cDNA, 2 μL of primers (500 nM), and 2 μL of PCR-grade water provided within the kit. The cycling conditions were established as follows: an initial denaturation at 95°C for 3 minutes, followed by 40 cycles consisting of denaturation at 95°C for 10 seconds, annealing at 60°C for 10 seconds, and extension at 72°C for 20 seconds. The fold changes in the tested samples were calculated using the Bio-Rad CFX Maestro Software, employing the ΔΔCt method. The expression levels of the receptor and cytokine/chemokine mRNAs were normalized based on the transcript level of the housekeeping gene, rat Actb. Unstimulated specimens were utilized as calibrator samples. The primer sequences are delineated in Table 1 .

The expression levels of Dectin-1, Dectin-2, TLR2, NOD1, and RIG-I induced by HMGB1, as well as their constitutive expression, were evaluated through flow cytometry and confocal microscopy. The constitutive expression of Dectin-1, Dectin-2, TLR2, NOD1, and RIG-I was analyzed in unstimulated MCs. Induced receptor expression was assessed in MCs that were incubated with HMGB1 at a final concentration of 10 ng/mL for durations of 1 or 3 hours at 37°C in a humidified atmosphere containing 5% CO₂. To ascertain the intracellular localization of the receptors, the MCs were fixed using CellFIX™ (BD Bioscience, San Jose, USA) solution for 15 minutes at 4°C and subsequently washed twice with 1 × PBS (Cayman Chemical, Ann Arbor, USA). Following this, the MCs were permeabilized with 0.1% saponin (Sigma-Aldrich) for 30 minutes at room temperature. The MCs were then resuspended in 1 × PBS and stained for 1 hour with goat anti-Dectin-1, goat anti-Dectin-2 (Invivogen, San Diego, CA, USA), rabbit anti-TLR2, goat anti-NOD1, or goat anti-RIG-I antibodies (Santa Cruz Biotechnology, Inc., Dallas, USA) at a dilution of 1:100. For the control, MCs were stained with goat or rabbit IgG isotype control (R&D Systems, Minneapolis, USA) exhibiting irrelevant specificity. The primary antibody was omitted from the sample to confirm the non-specific binding of the secondary antibody. Subsequently, the cells were washed with 1 × PBS and incubated with Alexa Fluor 488^® rabbit anti-goat IgG or Alexa Fluor 488^® goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, USA) at a dilution of 1:100 in 1 × PBS for 1 hour in the dark. To establish the surface localization of the receptors, the MCs were fixed with CellFIX™ (BD Bioscience, San Jose, USA) solution for 15 minutes at 4°C and washed twice with 1 × PBS (Cayman Chemical, Ann Arbor, USA). Following this procedure, the cells were washed twice and finally resuspended in 1 × PBS prior to receptor assessment. After each incubation period, the viability of the MCs was evaluated using the trypan blue exclusion test.

A total of ten thousand events from each sample were analyzed utilizing a BD FACSCalibur™ flow cytometer equipped with BD CellQuest™ software (BD Biosciences). The expression of HMGB1-dependent MC Dectin-1, Dectin-2, TLR2, NOD1, and RIG-I was presented as a percentage of the mean fluorescence intensity (MFI) of Dectin-1/Dectin-2/TLR2/NOD1/RIG-I, measured in unstimulated MCs, which is referred to as 100%.

The samples were affixed to microscope slides, and images were acquired using a Leica TCS SP8 microscope (Wetzlar, Germany) equipped with the HC PL APO CS2 63x/1.4 oil objective at the Laboratory of Microscopic Imaging and Specialized Biological Techniques at the University of Lodz. A 488 nm laser was employed to excite the fluorescence, and the emission was collected by a hybrid detector within the range of 505–550 nm. To facilitate visualization of the cells, the PMT transmission channel was utilized. LAS X 2.0.2.15022 software (Leica Microsystems, Wetzlar, Germany) was used for data analysis. All settings were maintained consistently throughout the experiments. All signals obtained from confocal microscopy were verified through profile view image analysis, along with diagrams presenting intensity values positioned beneath each microphotograph. The mean fluorescence intensity (expressed in arbitrary units, AU) was calculated for each sample. The calculations were conducted for a minimum of 40 different points, randomly selected within compartments exhibiting receptor expression.

In order to measure the generation of cytokines and chemokines, purified MCs suspended in a medium designed for rat MCs were incubated with HMGB1 at final concentrations of 0.1, 1, and 10 μg/mL, or with buffer alone to assess spontaneous cytokine and chemokine generation. This incubation occurred in a humidified environment containing 5% CO₂ for a duration of 3 hours at a temperature of 37°C. Subsequently, the supernatants were collected through centrifugation. The concentrations of TNF, TGF-β, IL-1β, CCL3, CCL4, and cysLTs in the supernatants were determined using ELISA kits – specifically, TNF and IL-1β from Wuhan Eiaab Science Inc., Wuhan, China; TGF-β, CCL3, and CCL4 from Biorbyt Ltd., Cambridge, UK; and cysLTs from Cayman Chemical, Michigan, USA—following the manufacturer’s instructions. The sensitivity of these assays was determined to be < 7 pg/mL, < 6 pg/mL, < 14 pg/mL, < 1 pg/mL, < 15 pg/mL, and < 20 pg/mL, respectively.

Purified MCs suspended in the medium specifically designed for rat MCs were incubated with HMGB1 at final concentrations of 0.1, 1, and 10 μg/mL, and compound 48/80 (Sigma-Aldrich), a well-established potent MC degranulation factor (30), at a final concentration of 5 µg/mL (positive control) or buffer alone (spontaneous histamine release) in a water bath for 30 minutes at 37°C with continuous stirring. Following the incubation period, the reaction was halted by adding 1.9 mL of cold medium. Subsequently, the cell suspension was subjected to centrifugation, and the supernatants were transferred into separate tubes. A total of 2 mL of distilled water was incorporated into each tube containing the cell pellets. The histamine content was quantified in both the cell pellets (residual histamine) and supernatants (released histamine) utilizing the spectrofluorometric method, as previously detailed (31). The histamine release was expressed as a percentage of the total cellular content of the amine.

The MC migratory response to HMGB1 was investigated utilizing a Boyden microchamber assay (Neuro Probe, Gaithersburg, USA) within a 48-well chemotaxis chamber (Neuro Probe). Thirty microliters of HMGB1 at final concentrations of 0.1, 1, and 10 μg/mL, or buffer alone (control for spontaneous migration), were placed into the lower compartments of the microchamber. These lower compartments were then covered with a polycarbonate membrane featuring an 8-μm pore size, and 50 μL of the cell suspensions were introduced into the upper compartments. Subsequently, the chemotaxis chamber was incubated for 3 hours in a humidified atmosphere containing 5% CO₂ at 37°C. After the incubation period, MCs that adhered to the upper surface of the membrane were removed by gently scraping with a rubber blade. Migrating cells that adhered to the lower surface of the membrane were fixed in 99.8% ethanol (Avantor Performance Materials, Poland), stained for 10 minutes with hematoxylin (Sigma-Aldrich), cleared in distilled water, and then mounted on a microscope slide. MC migration was quantified by counting the number of cells that traversed the membrane and adhered to the bottom surface of the filter. Ten high-power fields (HPF) were assessed in each assay (x 250). The spontaneous migration served as a control and was designated as 100%. The results were presented as a percentage of the control migration.

To determine the generation of reactive oxygen species (ROS), MCs suspended in a medium formulated for rat MCs were incubated with HMGB1 at final concentrations of 0.1, 1, and 10 μg/mL, or with the medium alone, for a duration of 1 hour within a humidified atmosphere containing 5% CO2 at 37°C. Subsequently, CellROX™ Green Reagent (Invitrogen, Carlsbad, CA, USA) was introduced at a final concentration of 5 µM and incubated with the cells at 37°C for an additional 30 minutes. The cells were then subjected to three washing cycles with 1 × PBS and subsequently analyzed using the FLUOstar Omega Microplate Reader (BMG Labtech, Ortenberg, Germany). The fluorescence intensity was assessed at excitation and emission wavelengths of 485/520 nm. ROS generation was quantified and expressed as mean signal intensity (MSI).

To ascertain the role of the HMGB1 receptor, the RAGE antagonist FPS-ZM1 (R&D Systems) was employed. Purified MCs underwent pretreatment with the antagonist for 15 minutes at 37°C in a water bath with continuous stirring prior to the execution of the principal procedures, which included ELISA for protein measurements, the histamine-release assay, the kit for intracellular ROS production measurement, and the migration assay. FPS-ZM1 was utilized at a concentration of 5 μM. The concentration of the antagonist was selected based on preliminary experiments conducted in accordance with the manufacturer’s guidelines.

For the analysis of cellular signaling pathways, purified MCs were pre-treated with various signaling molecule inhibitors or medium alone for 1 hour at 37°C prior to the commencement of the primary procedure. ERK1/2 inhibitor PD98059 (Sigma-Aldrich) was administered at a concentration of 50 μM, p38 inhibitor SB203580 (Sigma-Aldrich) at 10 μM, PI3K inhibitor LY294002 (Sigma-Aldrich) at 50 μM, and JAK2 inhibitor AG490 (Merck Millipore, Billerica, MA, USA) was used at 10 μM. MCs were also pre-incubated with 3 μM of NF-κB inhibitor MG-132 (InvivoGen) for 15 minutes at 37°C. The concentrations of all applied inhibitors were determined during preliminary experiments, in accordance with the manufacturer’s instructions. None of the inhibitors compromised MC viability, as confirmed by the trypan blue exclusion assay. Following incubation, the MCs were washed twice in 1 × PBS, re-suspended in appropriate medium, and utilized in the histamine-release assay, migration assay, and measurement of intracellular ROS production, as previously described.

The statistical analysis of the experimental data was conducted utilizing Statistica 13 software (Statsoft Inc., USA). The data are represented as mean ± standard deviation (SD). The normality of distribution was assessed using the Shapiro-Wilk test. The Student’s t-test and one-way ANOVA followed by post hoc Tukey’s test were employed to assess significant differences, with a P-value of < 0.05 regarded as statistically significant. The adjusted P-values are presented in Supplementary Data Tables S1 – S3 .

Initially, the capacity of HMGB1 to modulate the expression of PRRs on MCs was investigated. The qRT-PCR technique was employed to ascertain the mRNA expression levels of Dectin-1, Dectin-2, and TLR2 in unstimulated MCs compared to those stimulated with HMGB1. As illustrated in Figure 1A , HMGB1 led to a 1.3-fold upregulation of Dectin-1 mRNA expression (P < 0.05), while it did not affect the mRNA levels of Dectin-2 and TLR2. Subsequently, we explored whether HMGB1 influences the baseline expression levels of the selected surface PRRs utilizing flow cytometry and confocal microscopy. Receptor expression was assessed in unstimulated MCs and those exposed to HMGB1 (10 ng/mL) for durations of 1 hour or 3 hours. We observed that the baseline level of Dectin-1 expression was significantly (P < 0.05) upregulated following incubation with HMGB1 for both 1 hour and 3 hours, reaching values of 138.84 ± 13.42% and 140.56 ± 10.81% relative to control Dectin-1 expression, respectively ( Figures 1B, C ). Confocal and fluorescence intensity imaging indicated that cell surface signals for Dectin-1 peaked at 131.41 ± 5.81 arbitrary units (A.U.) (P < 0.05) after 1 hour and 133.15 ± 11.23 A.U. (P < 0.05) after 3 hours of incubation ( Figure 1D ). We established statistically significant differences determined by one-way ANOVA (P < 0.0001), too. Additionally, flow cytometry and confocal microscopy validated the presence of Dectin-2 and TLR2 on the surface of non-stimulated (NS) MCs, revealing no significant alterations in expression levels following stimulation with HMGB1.

The expression of intracellular PRRs by MCs – NOD1 and RIG-I is shown in Figure 2 . HMGB1 strongly affected both NOD1 (P < 0.001) and RIG-I (P < 0.001) mRNA expression ( Figure 2A ). HMGB1 induced enhancement of the intracellular NOD1 protein level, and the intensity of the signals reaching after 1 h 134.78 ± 1.30% (P < 0.05) and after 3 h 138.98 ± 2.42% (P < 0.01) of control receptor expression ( Figures 2B, C ). Using immunocytochemical staining, we visualized the presence of NOD1, mainly in the nuclear region ( Figure 2D ). Image analysis revealed that the receptor expression was upregulated upon incubation with this molecule (NS: 33.86 ± 6.56; HMGB1: 1h - 70.72 ± 15.17 A.U., 3h - 94.99 ± 15.57 A.U.) which was confirmed by a one-way ANOVA (P = 0.0026). The signals were strongly associated with the cytoplasm located near the cell nucleus.

The one-hour incubation of MCs with HMGB1 resulted in a statistically significant increase in the intracellular RIG-I level, with a P-value of less than 0.05, when compared to the control group of unstimulated MCs ( Figures 2B, C ). Extended incubation further enhanced the expression of the receptor, achieving 156.95 ± 26.17% of the baseline level, with a p-value of less than 0.001. Stimulation of MCs with HMGB1 led to an increase in RIG-I expression, as evidenced by intensity diagrams accompanying each microphotograph (NS: 23.17 ± 4.88; HMGB1: 1 h - 117.67 ± 11.30 A.U., 3 h - 200.34 ± 17.40 A.U.) (refer to Figure 2D ). We also found statistically significant differences, as determined by one-way (P < 0.0001). Significant enrichment in the signals was noted in the intracellular regions and beneath the cell surface.

Subsequently, to determine whether HMGB1 could elicit an inflammatory and immunoregulatory response from MCs, we assessed this factor’s capacity to influence the expression of chemokine/cytokine mRNA. qRT- PCR was conducted, and the fold change in cytokine/chemokine mRNA expression in HMGB1-stimulated (10 ng/mL) MCs compared to non-stimulated cells was evaluated. As illustrated in Figure 3A , among the chemokines/cytokines analyzed in HMGB1-stimulated MCs, the highest levels of mRNA expression were noted for TNF (9.8-fold increase), IL-1β (6.5-fold increase) (P < 0. 001), TGF-β (6.2-fold change), CCL3 (4.4-fold change), IL-18 (3.8- fold change) (P < 0. 01), CCL4 (3.5-fold change), and CCL5 (2.9-fold change) (P < 0. 05). In the subsequent phase, we investigated one chemokine and two cytokines exhibiting the greatest mRNA level increases for protein synthesis. For this purpose, MCs were stimulated with HMGB1 at concentrations ranging from 0.1 to 10 ng/mL for 3 hours, utilizing medium alone as a negative control and anti-IgE as the positive control. Additionally, our objective was to clarify the role of RAGE in the activation of MCs by HMGB1. The results of these experiments are presented in Figures 3B–D . The most pronounced secretion of the CCL3 chemokine was observed at 10 ng/mL of HMGB1, which increased to 102.56 ± 11.23 pg/1.5 × 10⁶ cells (P < 0.001; ANOVA P = 0.0057). Furthermore, we observed that HMGB1 also stimulated MCs to produce CCL3 at lower concentrations of 0.1 and 1 ng/mL (P < 0. 01) ( Figure 3B ). The statistical analysis indicated that HMGB1 at 10 ng/mL was the most potent inducer of IL-1β production (P < 0.05; ANOVA P = 0.0185) ( Figure 3C ). Conversely, in the case of MCs stimulated with HMGB1 across all concentrations, we recorded a significantly higher release of TNF (P < 0.001; ANOVA P = 0.0006) compared to non-stimulated cells ( Figure 3D ). To ascertain whether RAGE is implicated in the HMGB1-mediated response in MCs, the receptor antagonist FPS-ZM1 was employed. As demonstrated in Figures 3B–D , the pretreatment of MCs with FPS-ZM1 inhibited HMGB1-induced CCL3/IL-1β/TNF generation. We also established that HMGB1 at 1 (P < 0.01) and 10 (P < 0.001) ng/mL activated MCs to degranulation, assessed by histamine secretion ( Figure 4A ). There were statistically significant differences between means as determined by one-way ANOVA in this case (P < 0.0001). As shown in Figure 4B , HMGB1 did not stimulate cysLT production and release by MCs at 0.1 and 1 ng/mL but induced this mediator’s generation at 10 ng/mL (P < 0.05). Additionally, spectrofluorimetry was used to examine ROS generation by MCs in response to stimulation with HMGB1. As demonstrated in Figure 4C , MCs produced significant amounts of ROS after stimulation with HMGB1. Of the various molecule concentrations, the most significant ROS generation was observed at 10 ng/mL HMGB1, rising to 782.3 ± 18.45 MSI. We observed statistically significant differences, as determined by one-way ANOVA (P < 0.0001). Additionally, preincubation of MCs with FPS-ZM1 resulted in inhibition of HMGB1-induced histamine and ROS generation.

We also analyzed the potential of HMGB1 to initiate MC migration. Cells were incubated with HMGB1 for 3 h in a Boyden microchamber (at concentrations of 0.1–10 ng/mL) to determine its capability to induce MC migration. Results indicate that HMGB1 at a concentration of 10 ng/mL strongly influenced MC migration compared to spontaneous migration (578.89 ± 18.45% of control migration, P < 0.001) ( Figure 5 ). One-way ANOVA also revealed statistically significant differences (P < 0.0001). Alarmin at the highest concentration acted as a much more potent cell chemoattractant than a well-known chemotactic factor, i.e., TNF. Pretreatment with RAGE antagonist completely inhibited the migratory MC response to HMGB1.

We conducted inhibitory experiments utilizing ERK1/2 inhibitor PD98059, p38 inhibitor SB203580, PI3K inhibitor LY294002, NF-κB inhibitor MG-132, and JAK2 inhibitor AG490 ( Figure 6 ). Our documentation revealed that MC pretreatment with ERK1/2, p38, PI3K, and NF-κB inhibitors resulted in a markedly and statistically significant reduction in HMGB1-mediated histamine release ( Figure 6A ), ROS generation ( Figure 6B ), and migration ( Figure 6C ). Furthermore, pretreatment with JAK2 inhibitor AG490 elicited a statistically significant (P < 0.01) diminution in HMGB1-mediated histamine release ( Figure 6A ).