Immunosuppressive effect of hispidulin in allergic contact dermatitis

Male C57BL/6 mice, 7–10 weeks of age, were obtained from the National Laboratory Animal Center, Mahidol University, Salaya Campus, Nakhon Pathom, Thailand. All mice were housed in a 12-h/12-h light/dark specific pathogen-free condition, with free access to standard rodent feed and water. The care and treatment of study mice was in accordance with the guidelines of Mahidol University and the Office of the National Research Council of Thailand (NRCT). The total number of animals used in each experimental group was referred from the published article conducting the similar experiments [16]. All experimental protocols were approved by the Siriraj Animal Care and Use Committee (Si-ACUC), Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand (COA no. 015/2559).

Fluorescein isothiocyanate (FITC) conjugated antibody to mouse CD4, and purified antibodies to mouse-CD3, −IL-4, and -CD28 were purchased from eBioscience, Inc. (San Diego, CA, USA). Phycoerythrin (PE) conjugated antibody to mouse-IFN-γ, and recombinant mouse (rm) IL-12 were purchased from BioLegend, Inc. (San Diego, CA, USA). Recombinant mouse IL-2 was purchased from ImmunoTools GmbH (Friesoythe, Germany). 1-Fluoro-2,4-dinitrobenzene (DNFB), hispidulin (purity > 98%), dexamethasone, phorbol 12-myristate 13-acetate (PMA), and ionomycin were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Paraformaldehyde was purchased from Merck (Darmstadt, Germany). GolgiPlug™ Protein Transport Inhibitor and Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Propidium iodide (PI) solution was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Phosphate buffered saline (PBS) was purchased from AMERESCO, Inc. (Framingham, MA, USA. Roswell Park Memorial Institute (RPMI)-1640 Complete Medium (Gibco; Thermo Fisher Scientific, Waltham, MA, USA), fetal bovine serum (FBS) (Biochrom; Merck, Darmstadt, Germany), olive oil (ChemCruz™; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), acetone (Scharlau; Scharlab, S.L., Barcelona, Spain), and thiopental sodium (THIOPEN; Unique Pharmaceutical Laboratories Ltd., Ankleshwar, India) were also used.

Twenty-five mice were sensitized by painting 25 μL of 0.5% DNFB in a mixture solution of acetone and olive oil (4:1, v/v) on the shaved abdomen (day 0) with the average area of 2 × 2 cm. Five days after sensitization, mice were elicited by painting 20 μL of 0.3% DNFB in the same vehicle on the dorsal and ventral pinna of both ears. For the topical application of drug compounds, mouse pinna was applied with either hispidulin (10 or 30 μg/ear, treatment groups, n = 5 each concentration), dexamethasone (30 μg/ear, positive control, n = 5), or no treatment (the vehicle alone, a sensitized control, n = 5) starting on day 4 for 3 consecutive days. Mice sensitized and elicited with vehicle alone were used as a non-sensitized control (n = 5). Ear thickness of each ear of each individual mouse was measured at pre-challenge and at 6-, 24-, and 48-h post-challenge using a Vernier caliper (Mitutoyo, Kanagawa, Japan).

At 48-h post-challenge, all mice were sacrificed by intraperitoneal injection of thiopental sodium (50 mg/kg). The mouse pinna was resected, fixed with 4% paraformaldehyde, and embedded in paraffin. Sections (5 μm thick) were stained with hematoxylin and eosin (H & E) for histopathologic evaluation of CHS.

Total ribonucleic acid (RNA) was isolated from the excised pinna from one ear per mice at 24-h post-challenge using a Total RNA Extraction Kit (RBC Bioscience, New Taipei City, Taiwan). Complementary deoxyribonucleic acid (cDNA) was then synthesized from 1 μg of total RNA using an iScript™ Select cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Real-time polymerase chain reaction (real-time PCR) analysis was performed using a LightCycler® 480 Instrument II (Roche Applied Science, Penzberg, Germany). The final reaction mixture in each reaction tube consisted of 0.5 μL of cDNA; 0.4 μL of sense and antisense primer solutions, including IFN-γ (forward: 5′-AACGCTACACACTGCATCT-3′, reverse: 5′-TGCTCATTGTAATGCTTGG-3′) and β-actin (ACTB, forward: 5′-ATGGATGACGATATCGCT-3′, reverse: 5′- ATGAGGTAGTCTGTCAGGT-3′) (Integrated DNA Technologies, Coralville, IA, USA); 10 μL of KAPA SYBR® Fast qPCR Master Mix (2x) Kit (Kapa Biosystems, Wilmington, MA, USA); and, 8.7 μL of sterile Milli-Q® water (Merck). The conditions for reverse transcription and the PCR steps were according to the manufacturer’s instructions. The normalization and quantification of mRNA expression was performed using LightCycler® 480 software (Roche Applied Science, Rotkreuz, Switzerland).

Three naïve mice were sacrificed by intraperitoneal injection of thiopental sodium (50 mg/kg). Naïve CD4⁺ T cells were isolated from spleens by using CD4 MicroBeads and a MiniMACS™ Separator (Miltenyi Biotec) according to the manufacturer’s instructions. The isolated CD4⁺ T cells were then cultured at a cell density of 1 × 10⁵ cells/well in RPMI 1640 Complete Medium containing 10% FBS. Cells were also incubated in the absence or presence of hispidulin at different concentrations of 6.25 μM, 12.5 μM, 25 μM, and 50 μM for 7 days. After incubation, cells were stained with propidium iodide (PI) and analyzed using a CytoFLEX Flow Cytometer and CytExpert software (Beckman Coulter, Inc., Brea, California, US).

Isolated naïve CD4⁺ T cells were obtained from spleens of four naïve mice using CD4 MicroBeads and a MiniMACS™ Separator (Miltenyi Biotec), and then cultured in RPMI 1640 Complete Medium containing 10% FBS. For Th1 cell differentiation, isolated CD4⁺ T cells were stimulated with anti-CD3 (10 μg/mL) and anti-CD28 (5 μg/mL) for 2 consecutive days. Activated cells were then cultured in Th1-skewing condition consisting of recombinant mouse (rm) IL-2 (2500 U/mL), rm. IL-12 (10 ng/mL), and anti-IL-4 (10 μg/mL), together with/without hispidulin (6.25 μM, 12.5 μM, 25 μM, and 50 μM) for 7 days prior to intracellular cytokine staining for IFN-γ production.

For intracellular cytokine staining, cultured CD4⁺ T cells at 1 × 10⁶ cells/mL were stimulated with 50 ng/mL PMA and 1 μg/mL ionomycin in the presence of GolgiPlug™ Protein Transport Inhibitor containing brefeldin A (BFA). Stimulated cells were then incubated at 37 °C in a 5% CO₂ condition for 4 h before being surface stained with anti-mouse CD4 FITC at 4 °C for 30 min and then washed once. Stained cells were then fixed and permeabilized with 0.1 mL Cytofix/Cytoperm™ solution (BD Biosciences) at 4 °C for 20 min and washed with BD Perm/Wash™ Buffer (BD Biosciences) prior to centrifugation at 500 g for 5 min. Intracellular cytokine staining (ICS) was performed using anti-IFN-γ PE at 4 °C for 30 min before washing with BD Perm/Wash™ Buffer and resuspension in PBS. Stained samples were analyzed using a CytoFLEX Flow Cytometer and CytExpert software (Beckman Coulter).

To evaluate the immunosuppressive effect of hispidulin on ACD, a CHS mouse model was designed using DNFB for sensitization and elicitation (Fig. 1a). Data presented in Fig. 1b show that DNFB-mediated mice developed marked ear swelling when compared to sham immunized mice. Treatment with hispidulin at both the 10 and 30 μg concentrations was able to significantly reduce ear swelling within 6 h when compared to the sensitized control. The decrease in ear thickness after being treated with 30 μg/ear hispidulin was comparable to the decrease in ear thickness observed after treatment with dexamethasone at 6- and 24-h post-challenge. The observed suppression was then substantially decreased at 48-h post-challenge when compared to dexamethasone; however, the 30 μg hispidulin-treated ears still showed observably better suppression than the sensitized control.

Histopathological changes in the excised ears revealed dermal thickness and infiltration of immune cells were both markedly increased in DNFB-sensitized mouse controls [Fig. 2, Additional file 1] when compared to the non-sensitized mice [DNFB (−)]. After hispidulin treatment of 30 μg/ear, ear edema and spongiosis were reduced to almost the same levels as those observed after dexamethasone application [DNFB (+)/His30 vs. DNFB (+)/Dex]. Remnants of infiltrated inflammatory cells was observed in the dermis in both the hispidulin and dexamethasone groups.

As Th1 cells secreting IFN-γ play an important role, the mRNA expression of IFN-γ in excised ear tissue was determined using real-time PCR. Relative mRNA expression of IFN-γ to that of β-actin was considerably increased (p = 0.0002) after DNFB sensitization and challenge when compared to that of the non-sensitized group (Fig. 3). Hispidulin treatment of 30 μg/ear was able to significantly downregulate IFN-γ expression (p = 0.0236), with results comparable to those observed after treatment with dexamethasone.

For drug cytotoxicity, the tests revealed no change in the percentages of CD4⁺ T cell death compared to untreated controls (Fig. 4a). The total number of CD4⁺ T cells and the percentage of IFN-γ-producing CD4⁺ T cells cultured in the Th1-skewing condition were significantly higher than the total number and percentage of cells cultured in complete medium (Fig. 4b and c; p < 0.0001 and p = 0.0007, respectively), which suggests that the Th1-skewing condition successfully induced Th1 cell differentiation. Hispidulin at all concentrations effectuated marked suppression of CD4⁺ T cell proliferation when compared to the suppression observed in the untreated control (p < 0.01), and no difference was observed among the 4 different hispidulin concentrations (Fig. 4b). However, hispidulin significantly reduced frequencies of IFN-γ-producing CD4⁺ T cells in a dose-dependent manner when compared to those observed in the untreated control (Fig. 4c; p < 0.01 for 6.25 and 12.5 μM, p = 0.0010 for 25 μM and p = 0.0005 for 50 μM). These findings suggest that hispidulin effectively inhibited CD4⁺ T cell function, most notably IFN-γ production, without interrupting cell frequencies.