The Putatively Specific Synthetic REV-ERB Agonist SR9009 Inhibits IgE- and IL-33-Mediated Mast Cell Activation Independently of the Circadian Clock

First, we investigated whether REV-ERBs are expressed and functional in mast cells. For this purpose, we examined the kinetics of the mRNA levels of REV-ERB-α and REV-ERB-β as well as two other major clock genes, Per2 and Bmal1, in bone marrow-derived mast cells (BMMCs) from wild-type mice. REV-ERB-α and REV-ERB-β mRNAs were expressed at considerable levels comparable to Per2 and Bmal1 in wild-type BMMCs (Threshold Cycle (Ct value) of each gene in the real-time quantitative PCR experiments were as follows; REV-ERB-α: 32~34, REV-ERB-β: 30~32, Per2: 31~33, Bmal1: 30~32). REV-ERB-α, but not REV-ERB-β, mRNA exhibited oscillations (REB-ERB-α: p = 4.15 × 10⁻⁵, REV-ERB-β: p = 0.26, one-way ANOVA) with a peak at 18 h following a medium change to synchronize the mast cell clock (Figure 1a). Per2 and Bmal1 mRNA levels exhibited circadian oscillations (Per2: p = 9.44 × 10⁻⁹, Bmal1: p = 9.89 × 10⁻⁷, One-way ANOVA), as previously reported (Figure 1a) [16]. Because no good anti-REV-ERB-α or -β antibody is available, we were unable to confirm REV-ERB-α and -β expression in BMMCs at the protein level. Consistent with a model in which transcription of REV-ERBs is activated by the BMAL1/CLOCK heterodimer [1,2], BMMCs from Clock-mutated mice [17] expressed significantly much lower levels of REV-ERB-α and REV-ERB-β mRNA expression than BMMCs from wild-type mice (Figure S1).

We next examined the effects of SR9009 and other synthetic REV-ERBs agonists SR9011 [12] and GSK4112 [14] on the mast cell clockwork in vitro. We confirmed that treatment of wild-type BMMCs with SR9009, SR9011, or GSK4112 for 24 h at a concentration of 1 or 10 µM did not affect cell viability, as judged by a metabolic assay (NAD(P)H-based: WST-1 assay) (Figure S2a) and expression of Annexin V (Figure S2b); by contrast, a dose of 50 µM exerted cytotoxicity. Therefore, in this study, we used 10 μM, the most commonly used concentration among published studies [12,14,15].

We previously showed that the mast cell clockwork (i.e., the kinetics of PER2 expression) can be evaluated in vitro [3], based on monitoring of bioluminescent emission of BMMCs from Per2^Luc knock-in mice, which express PER2 as a luciferase fusion protein [18] (PER2^LUC BMMCs). A simple medium change triggers synchronization of the circadian clocks in peripheral cells in vitro [19]. Accordingly, the mast cell clockwork (as reflected by the oscillation of PER2^LUC) was observed from 0 to 72 h after a media change, as previously described (Figure 1b) [3]. The PER2^LUC oscillation may have been limited to 0–72 h due to a lack of oscillator coupling in dissociated cell cultures without internal zeitgebers (‘time givers’ in German), leading to damping of the ensemble rhythm at the population level [3]. We found that addition of 10 μM SR9009, SR9011, or GSK4112 72 h after the medium change recovered the mast cell clockwork (i.e., PER2^LUC oscillation) for another 48 h (Figure 1b), suggesting that activation of REV-ERBs by these reagents can synchronize the mast cell clockwork at the population level. Collectively, these results suggested that mast cells express functional REV-ERBs, and that SR9009 or other synthetic agonists can affect the mast cell clockwork.

We next examined the effects of SR9009 and other synthetic REV-ERBs agonists on IgE- and IL-33-mediated activation in mast cells. Pretreatment of wild-type BMMCs for 1 h with 10 µM SR9009, SR9011, or GSK4112 inhibited IgE-mediated degranulation, as judged by β-hexosaminidase release, histamine release, and CD63 expression (Figure 2a, Figure S3). IgE-mediated IL-6 and IL-13 protein expression in wild-type BMMCs was also suppressed by pretreatment with 10 µM SR9009, SR9011, and GSK4112 for 1 h (Figure 2b). Importantly, intraperitoneal administration of SR9009 inhibited passive cutaneous anaphylactic (PCA) reaction, a classical in vivo model of IgE/mast cell-mediated skin allergic response, in wild-type mice (Figure 2c). Similarly, pretreatment of wild-type BMMCs for 1 h with 10 µM SR9009, SR9011, or GSK4112 inhibited IL-33-mediated IL-6 and IL-13 protein expression in wild-type BMMCs (Figure 2d). We also found that pretreatment of wild-type BMMCs for 1 h with 10 µM SR9009, SR9011, or GSK4112 inhibited LPS-mediated IL-6 and IL-13 protein expression in wild-type BMMCs (Figure S4a).

Similar to wild-type BMMCs, 10 μM SR9009, SR9011, and GSK4112 inhibited IgE-mediated degranulation and IgE- and IL-33-mediated IL-6 and IL-13 expression in wild-type fetal skin-derived mast cells (FSMCs), mouse connective tissue-type skin-derived mast cells (). Figure S5

Unexpectedly, these suppressive effects of SR9009, SR9011, and GSK4112 were also observed in BMMCs derived from Clock-mutated mice (Figure 2a–d), suggesting that suppression of IgE- and IL-33-mediated mast cell activation by SR9009 or other synthetic REV-ERB agonists does not require functional circadian clock activity. In addition, we found that IL-33-mediated IL-13 production was significantly lower in Clock-mutated BMMCs than in wild-type BMMCs (Figure 2b,d), suggesting that Clock mutation can affect IL-13 production via IL-33 in mast cells.

To investigate the mechanisms by which SR9009 suppresses IgE- and IL-33-mediated mast cell activation, we examined the drug’s effects on IgE- and IL-33–induced intracellular signaling pathways leading to degranulation or cytokine expression such as the Gab2/PI3K pathway and NF-κB and p38 MAPK pathways [5,6,20,21].

The Gab2/PI3K pathway is critical in FcεRI signaling leading to degranulation in mast cells [22]. Briefly, stimulation of FcεRI phosphorylates Gab2 probably by Syk and Src family protein-tyrosine kinases Syk or Lyn. Then, phosphorylated Gab2 binds to the p85 subunit of PI3K and recruits PI3K to its substrate lipids, thereby leading to PLCγ activation and degranulation. Interestingly, SR9009 inhibited IgE-mediated phosphorylation of Gab2 and the p55 subunit of PI3K in wild-type BMMCs (Figure 3a). SR9009 did not affect Gab2 mRNA expression in wild-type BMMCs (Figure S6). In contrast to IgE stimulation, IL-33 did not induce phosphorylaton of Gab2 in wild-type BMMCs.

Both the NF-κB and p38 MAPK pathways mediate IgE- or IL-33-mediated transcriptonal activation of cytokine gene expression [5,6,20,21]. SR9009 inhibited IgE- and IL-33–induced phosphorylation of p65, a subunit of NF-κB, but did not affect IgE- or IL-33–induced phosphorylation of p38 MAPK in wild-type BMMCs (Figure 3a). In addition, a reporter assay showed that SR9009 suppressed IgE- or IL-33-mediated transcriptional activation of NF-κB in BMMCs (Figure 3b). SR9009 also inhibited LPS-mediated transcriptional activation of NF-κB in BMMCs (Figure S4b). Consistent with these findings, SR9009 inhibited IgE-, IL-33-, and LPS-mediated IL-6 and IL-13 mRNA expression in wild-type BMMCs (Figure S7).

We found that SR9009 did not affect surface expression levels of FcεRI or IL-33 receptor ST2 in wild-type BMMCs (). Together, these results suggest that inhibition of the Gab2/PI3K and NF-κB pathways, but not p38 MAPK, contributes to the suppressive effects of SR9009 on IgE- or IL-33-mediated degranulation and cytokine gene expression of mast cells. Figure S8

The inhibitions of mast cell activation by SR9009 appeared to be independent of the functional circadian clock activity (Figure 2). Thus, we asked whether the inhibitory actions of SR9009 depended on its agonistic function. For this purpose, the effects of SR9009 on IgE- or IL-33-mediated mast cell activation were examined when REV-ERBs expression were knocked-down by specific siRNAs in wild-type BMMCs.

Both REV-ERBα and β mRNA expressions were significantly downregulated using the specific siRNAs in wild-type BMMCs by ~80% compared to those in wild-type BMMCs using the control siRNAs (Figure 4a). Unexpectedly, pretreatment of the REV-ERBs knocked-down BMMCs for 1 h with 10 µM SR9009 inhibited IgE-mediated degranulation, as judged by β-hexosaminidase release and CD63 expression (Figure 4b). IgE- and IL-33-mediated IL-13 protein expression was also suppressed by pretreatment with 10 µM SR9009 for 1 h in REV-ERBs knocked-down BMMCs (Figure 4c,d). These results suggest that the inhibitory actions of SR9009 on IgE- and IL-33-mediated mast cell activation may not depend on its agonistic function through REV-ERBs.

Reagents used in this study were acquired from the indicated suppliers: SR9009 (Merck Millipore, Burlington, MA, USA); SR9011, GSK4112, LPS, and Evans blue (Sigma-Aldrich, St. Louis, MO, USA); recombinant mouse IL-3 (PeproTech, Rocky Hill, NJ, USA); recombinant mouse IL-33 (R & D Systems, Minneapolis, MN, USA); anti-TNP IgE, anti-DNP mouse IgE mAb, anti-mouse CD16/32, PE-conjugated anti-mouse c-kit Ab, and APC-conjugated anti-mouse ST2 Ab (BD Bioscience, San Jose, CA, USA); DNP-BSA (Cosmo Bio, Tokyo, Japan); APC-conjugated anti-mouse CD63 Ab, FITC-conjugated anti-mouse FcεRIα (BioLegend, San Diego, CA, USA); anti-phospho-NF-κB p65 Ab (Ser536; #3033), anti-phospho-p38 MAPK Ab (Thr180/Thy182; #4511), anti-phospho-Gab2 Ab (Tyr452; #3882), anti-phosph-PI3 Kinase p85 (Tyr458)/p55 (Tur199) Ab (#4228), and anti-β-actin Ab (#4970) (Cell Signaling Technology, Danvers, MA, USA).

Male 6–8-week-old C57BL/6 mice (Japan SLC, Tokyo, Japan), Per2^Luciferase (Per2^Luc) knock-in mice (C57BL/6 background) where PERIOD2 (PER2) is expressed as a luciferase fusion protein [18], and C57BL/6 Clock^Δ19/Δ19 mice [17] were kept under 12-hour light / 12-hour dark conditions. Clock^Δ19/Δ19 mice have an A-to-T point mutation in the 5’ splice site of intron 19 and consequently an in-frame deletion of the whole exon 19 (Clock^Δ19/Δ19), resulting in the loss of normal transcriptional activity [17]. All animal experiments were approved by the Institutional Review Board (IRB) of the University of Yamanashi and carried out according to IRB guidelines (ethics committee: Koji Moriishi, Toshiyuki Oda, Hiroaki Nagatomo, Hiroyuki Narita, Jiang Ling, Junichi Miyazaki, Kazuhiro Mori, and Teruhiko Wakayama, approval code: A28-36, 14 August 2019).

BMMCs were prepared from femoral bone marrow cell suspensions from male C57BL/6 mice, Per2^Luc mice, or Clock^Δ19/Δ19 mice as previously described [3]. Briefly, crude bone marrow cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 mM nonessential amino acids, penicillin–streptomycin, 10 mM sodium pyruvate, and 50 μM 2-ME (complete RPMI 1640) in the presence of 10 ng/mL recombinant mouse IL-3 (rmIL-3). Floating cells were refreshed twice per week, and further expanded for 2–4 weeks in fresh complete RPMI 1640 supplied with rmIL-3. Finally, the cells (>90% FcεR1⁺c-kit⁺) were used as BMMCs without further purification.

Fetal skin mast cells (FSMCs) were generated from fetal skin of C57BL/6 mice on day 14, as described previously [28]. Briefly, fetal skin was treated with trypsin diluted in medium to form a single cell suspension. Then the cells were cultured in complete medium containing 20 ng/mL rmIL-3 and 20 ng/mL recombinant mouse SCF. After 2 weeks, non-adherent and loosely-adherent cells were collected, and mast cells were collected by Percoll density-gradient centrifugation. Finally, the cells (>90% FcεR1⁺c-kit⁺) were used as FSMCs.

Total RNA was isolated from BMMCs using the RNeasy Mini Kit (QIAGEN, Valencia, CA, USA). RNA was quantified on a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized using the ReverTra Ace RT-PCR Kit (TOYOBO, Osaka, Japan). Quantitative real-time PCR (qPCR) was performed on a Step One Plus Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) using qPCR Master Mix (Applied Biosystems) with specific primers and probes against mouse REV-ERB-α, IL-13 (Applied Biosystems), REV-ERB-β, IL-6, and GAPDH (IDT; Coralville, IA, USA). qPCR data were normalized against the corresponding levels of GAPDH mRNA.

BMMCs generated from Per2^Luc knock-in mice were centrifuged at 1500 rpm for 5 min, placed in a 35 mm Petri dish and incubated at 37 °C. As previously described, bioluminescence was monitored for 120 h at 10-min intervals using a dish-type luminometer (Kronos DioAB-2550; ATTO, Tokyo, Japan) [3]. After 72 h of medium change for synchronization, 10 μM REV-ERB agonists were added to the culture.

BMMCs were stained with PE-conjugated anti-mouse c-kit, FITC-conjugated anti-mouse FcεRI, APC-conjugated anti-mouse ST2, or APC-conjugated anti-mouse CD63 in the presence of rat anti-mouse CD16/32. After washing with PBS, the stained cells were analyzed on a BD Accuri C6 flow cytometer (BD Biosciences). Flow cytometry data were analyzed using the CellQuest software (BD Biosciences).

BMMCs or FSMCs were incubated with 1 µg/mL anti-DNP mouse IgE mAb for 1 h at 4 °C and, then, incubated with or without REV-ERB agonists for 1 h at 37 °C. Then, these cells were stimulated with 1 µg/mL of anti-mouse IgE antibody for 1 h at 37 °C (for β-Hexosaminidase or histamine measurement) or for 6 h at 37 °C (for IL-6 or IL-13 measurement). BMMCs or FSMCs were incubated with or without REV-ERB agonists for 1 h at 37 °C and then stimulated with 1 ng/mL IL-33 or 1 µg/mL LPS for 6 h at 37 °C, followed by measurement of IL-6 and IL-13 in the culture supernatants.

Concentrations of IL-6, IL-13, and histamine in supernatants of cell cultures were determined by ELISA. Kits for mouse IL-6 (R & D Systems), mouse histamine (Oxford Biomedical Research, Rochester Hills, MI, USA), mouse IL-13 (eBioscience/Thermo Fisher Scientific) were obtained from the indicated suppliers.

BMMCs or FSMCs were incubated with 1 µg/mL anti-DNP mouse IgE mAb for 1 h at 4 °C, and then stimulated with 1 µg/mL of anti-mouse IgE antibody with or without REV-ERB agonists for 1 h at 37 °C. Total release was obtained by adding 1% Triton buffer for 40 min. Supernatants were collected from each well and mixed with p-nitrophenyl-N-acetyl-β-d-glucosaminide to determine the enzymatic activity of the released β-hexosaminidase. After 90 min at 37 °C, the reaction was stopped by addition of 0.2 M glycine solution, and OD (405 nm) was measured on a spectrophotometer.

Ten minutes after various stimulations, BMMCs were lysed in RIPA buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% Triton ×100, 1% sodium deoxycholate, 0.1% SDS) with protease inhibitor cocktail (Merck Millipore, Burlington, MA, USA) and vanadate (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Cell lysate was dissolved in sample buffer containing 50 mM dithiothreitol and bromophenol blue, and then boiled for 5 min. Protein concentrations were measured on a NanoDrop ND-1000. Proteins were subjected to SDS-PAGE and transferred to polyvinylidene fluoride membranes. Blots were immersed in 5% milk blocking solution for 1 h at room temperature (RT), followed by incubation with primary antibody solution overnight at 4 °C. Membranes were washed three times with TBS/T, and then incubated in a secondary antibody solution for 1 h at RT. Immunoreactive proteins were visualized using ECL Prime (GE Healthcare).

Using the Mouse Macrophage Nucleofector kit (VPA-1009; Lonza, Basel, Switzerland), BMMCs were transiently transfected with the pNFκB-Luc reporter plasmid, with pRL-CMV as an internal control. After 24 h, the transfected BMMCs were stimulated with IL-33 (1 ng/mL) or anti-mouse IgE antibody (1 μg/mL) or LPS (1 μg/mL) in the absence or presence of REV-ERBs agonists. Relative NF-κB luciferase activity was normalized against transfection efficiency.

After stimulations with synthetic REV-ERB agonists for 24 h, cell viability was monitored using the Cell Counting Kit-8 (Dojindo Laboratory, Kumamoto, Japan) and PE-Annexin V apoptosis detection kit (BD Bioscience, San Jose, CA, USA).

Mouse anti-TNP IgE (100 ng) was intradermally injected into dorsal skin. After 24 h, 15% Cremophor (vehicle) or SR9009 (100 mg/kg) was intraperitoneally injected. After 1 h, the mice were challenged by intravenous injection with 2.5mg/kg of DNP-BSA in PBS containing 0.2% Evans blue dye. Vascular permeability was visualized 40 min later as blue staining of the injection areas on the inside of the skin. Staining sites were digitized using a high-resolution color camera, and images were saved JPEG files. The images analyzed using ImageJ 1.43 (NIH, Bethesda, MD, USA) as previously described [3]. Briefly, the color-scale images (the upper panels) were converted to HSB (hue/saturation/brightness) stack images, which were then split into hue, saturation, and brightness images. The blue-stained areas were selected from the hue image using the threshold tool, afterwards these images were combined with the saturation image. The density values for the blue-stained areas were measured using the analyze tool.

All siRNAs were purchased from Invitrogen (now Thermo Fisher, Waltham, MA, USA). Specific siRNAs against NR1D1 (stealth^TM RNAi; Nr1d1MSS211361 [3_RNAI]) or NR1D2 (stealth^TM RNAi: Nr1d2MSS221355 [3_RNAI]) were used.

Transfection of wild-type BMMCs were performed with Mouse Macrophage Nucleofector Kit (VPA-1009; Lonza, Basel, Switzerland). BMMCs were suspended in Nucleofector Solution to a final concentration of 2 × 10⁶ cells/100μL. The cells were transfected with 500 nM negative control or 250 nM specific siRNA of NR1D1 and NR1D2, respectively, using the Nucleofector II (Amaxa Biosystems, now Lonza) program Y-001. Subsequently, the transferred cells were placed in a 20% FBS medium and cultured in a 24-well plate. After 24 h, the transfected BMMCs were stimulated with anti-mouse IgE antibody (1μg/mL), IL-33 (1 ng/mL), or LPS (1 μg/mL) in the absence or presence of 10 μM SR9009.

Statistical analyses were performed using the unpaired Student’s t-test for two-group comparisons. Multigroup comparisons were analyzed by one-way ANOVA with Tukey–Kramer post hoc test. A value of p < 0.05 was considered to be significant.