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

The cell-autonomous circadian clock regulates IgE- and IL-33-mediated mast cell activation, both of which are key events in the development of allergic diseases. Accordingly, clock modifiers could be used to treat allergic diseases, as well as many other circadian-related diseases, such as sleep and metabolic disorders. The nuclear receptors REV-ERB-α and -β (REV-ERBs) are crucial components of the circadian clockwork. Efforts to pharmacologically target REV-ERBs using putatively specific synthetic agonists, particularly SR9009, have yielded beneficial effects on sleep and metabolism. Here, we sought to determine whether REV-ERBs are functional in the circadian clockwork in mast cells and, if so, whether SR9009 affects IgE- and IL-33-mediated mast cell activation. Bone marrow-derived mast cells (BMMCs) obtained from wild-type mice expressed REV-ERBs, and SR9009 or other synthetic REV-ERBs agonists affected the mast cell clockwork. SR9009 inhibited IgE- and IL-33-mediated mast cell activation in wild-type BMMCs in association with inhibition of Gab2/PI3K and NF-κB activation. Unexpectedly, these suppressive effects of SR9009 were observed in BMMCs following mutation of the core circadian gene Clock. These findings suggest that SR9009 inhibits IgE- and IL-33-mediated mast cell activation independently of the functional circadian clock activity. Thus, SR9009 or other synthetic REV-ERB agonists may have potential for anti-allergic agents.


Introduction
The circadian clock controls a large proportion of genes in a cyclic manner, thereby regulating the timing of cellular activities [1,2]. The circadian clock consists of a cell-autonomous transcription-translation feedback loop involving several clock genes. Briefly, the transcription factors BMAL1 (Arntl) and CLOCK heterodimerize, bind to E-box motifs throughout the genome, and activate transcription of their own repressors Period (Per1-3) and Cryptochrome (Cry1, 2). The PER and CRY proteins form oligomers and enter the nucleus, where they inhibit BMAL1/CLOCK activity. This negative-feedback loop takes~24 h to be completed, with several post-transcriptional regulation. Accordingly, the circadian clock controls periodic expression of thousands of clock-controlled genes (CCGs) with E-box motifs in their promoter/enhancer regions other than Per and Cry.
Previously, we showed that in mouse mast cells, Clock binds to E-box motif in the promoter of β-subunit gene of the high-affinity IgE receptor FcεRIβ or IL-33 receptor ST2 in a circadian manner,
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.

SR9009 and Other Synthetic Agonists of REV-ERBs Inhibit IgE-and IL-33-Mediated Mast Cell Activation
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 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.
Unexpectedly, these suppressive effects of SR9009, SR9011, and GSK4112 were also observed in BMMCs derived from Clock-mutated mice (Figure 2a-c,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.
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.

SR9009 Inhibits IgE-and IL-33-Mediated Activation of the Gab2/PI3K and NF-κB Pathways 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.
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.
We found that SR9009 did not affect surface expression levels of FcεRI or IL-33 receptor ST2 in wild-type BMMCs ( Figure S8). 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.

SR9009 May Inhibit IgE-and IL-33-Mediated Mast Cell Activation Independently of REV-ERBs
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 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 IgEor 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 ( Figure S8). 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.

SR9009 May Inhibit IgE-and IL-33-Mediated Mast Cell Activation Independently of REV-ERBs
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. 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 4, c and 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.

Discussion
Molecular understanding of the circadian clock is opening new therapeutic frontiers for several diseases-including sleep and metabolic disorders, inflammatory diseases, and cancer-through pharmacological targeting of circadian clock components [23]. Given that IgE-and IL-33-mediated mast cell activation is under the control of the circadian clock [3,4] and synthetic REV-ERBs agonists, particularly SR9009, have exhibited many beneficial effects in animal models of circadian-related disorders [12][13][14][15], this study sought to determine whether mast cells express functional REV-ERBs and SR9009 affects IgE-and IL-33-mediated mast cell activation. The current results suggest that SR9009 or other synthetic REV-ERB agonists can synchronize the mast cell clockworks and inhibit IgE-and IL-33-mediated mast cell activation.
SR9009 significantly inhibited IgE-and IL-33-mediated mast cell activation ( Figure 2). The Gab2/PI3K pathway is critical in FcεRI signaling leading to degranulation in mast cells [22] and the NF-κB pathway mediates IgE-or IL-33-mediated transcriptonal activation of cytokine gene expression [5,6,20,21]. Thus, it is likely that SR9009 and other synthetic REV-ERB agonists inhibited IgE-mediated degranulation and IgE-and IL-33-mediated IL-6 and IL-13 expression through the suppression of the Gab2/PI3K and NF-kB pathways, respectively. Because the Gab2/PI3K pathway play a partial role in IgE-mediated cytokein expression [22], inhibition of this pathway by SR9009 may be also involved in the suppression of IgE-mediated IL-6 and IL-13 expression.

Discussion
Molecular understanding of the circadian clock is opening new therapeutic frontiers for several diseases-including sleep and metabolic disorders, inflammatory diseases, and cancer-through pharmacological targeting of circadian clock components [23]. Given that IgE-and IL-33-mediated mast cell activation is under the control of the circadian clock [3,4] and synthetic REV-ERBs agonists, particularly SR9009, have exhibited many beneficial effects in animal models of circadian-related disorders [12][13][14][15], this study sought to determine whether mast cells express functional REV-ERBs and SR9009 affects IgE-and IL-33-mediated mast cell activation. The current results suggest that SR9009 or other synthetic REV-ERB agonists can synchronize the mast cell clockworks and inhibit IgE-and IL-33-mediated mast cell activation.
SR9009 significantly inhibited IgE-and IL-33-mediated mast cell activation ( Figure 2). The Gab2/PI3K pathway is critical in FcεRI signaling leading to degranulation in mast cells [22] and the NF-κB pathway mediates IgE-or IL-33-mediated transcriptonal activation of cytokine gene expression [5,6,20,21]. Thus, it is likely that SR9009 and other synthetic REV-ERB agonists inhibited IgE-mediated degranulation and IgE-and IL-33-mediated IL-6 and IL-13 expression through the suppression of the Gab2/PI3K and NF-kB pathways, respectively. Because the Gab2/PI3K pathway play a partial role in IgE-mediated cytokein expression [22], inhibition of this pathway by SR9009 may be also involved in the suppression of IgE-mediated IL-6 and IL-13 expression.
It remains to be determined how SR9009 inhibits the Gab2/PI3K and NF-κB pathways. However, regarding the NF-κB pathway, there were several reports that address the inhibitory mechanisms of SR9009 on the NF-κB pathway, including transcriptional repression of NF-κB-related genes such as IL-6 [24], p65 [25], and induction of FABP4, an intracellular lipid-binding protein [26]. In contrast, it remains totally unclear how SR9009 inhibits IgE-mediated Gab2/PI3K activation in mast cells.
Pretreatment of Clock-mutated BMMCs with SR9009 and other synthetic REV-ERBs agonists for 1 h can suppress IgE-and IL-33-mediated activation, as in wild-type BMMCs ( Figure 2). Moreover, given that treatment of wild-type PER2 LUC BMMCs with SR9009, SR9011, or GSK4112 did not affect PER2 LUC expression 1 h after the addition of the agent (Figure 1b), the suppressive effects of SR9009 or other synthetic REV-ERB agonists may be independent of functional circadian clock activity and PER2 expression in mast cells. The observation that treatment of wild-type BMMCs with SR9009 for 1 h did not affect the expression of FcεRI or ST2 ( Figure S8) also support this idea, as expression of FcεRI and ST2 is under the control of mast cell clock activity [3,4].
Surprisingly, SR9009 inhibited IgE-or IL-33-mediated activation in REV-ERBs knocked-down BMMCs ( Figure 4). Thus, the inhibitory actions of SR9009 on IgE-and IL-33-mediated mast cell activation may be independent on its agonistic function through REV-ERBs. Most recently, Dierickx et al. reported that SR9009 has REV-ERB-independent effects on cell proliferation and metabolism [27]. They showed that SR9009 can decrease cell viability, rewire cellular metabolism, and alter gene transcription in hepatocytes and embryonic stem cells derived from REV-ERB-α and -β double knockout mice although the mechanisms remain unclear. Thus, SR9009 and possibly other SR9009-related synthetic REV-ERB agonists might inhibit IgE-and IL-33-mediated mast cell activation independently of REV-ERBs. However, it should be noted that REV-ERBs knocked-down BMMCs still express REV-ERB-α and REV-ERB-β mRNAs, albeit at very low levels and SR9009 could exert its function through the residual expression of REV-ERBs.
Clock mutation decreases IL-33-mediated IL-13 production in BMMCs (Figure 2b,d). Kawauchi et al. reported that IL-33-mediated IL-6 and IL-13 production exhibit a time-of-day-dependent variation in synchronized, but not Clock-mutated, BMMCs [4]. Thus, it is likely that Clock may be involved in the circadian regulation of IL-33-mediated IL-13 production, although the mechanisms involved remain to be determined.
In summary, our findings show that activation of REV-ERBs by SR9009 or other synthetic REV-ERBs agonists can inhibit IgE-and IL-33-mediated activation of mast cells in association with suppression of the Gab2/PI3K and NF-κB pathways. Thus, modulation of REV-ERB activity by synthetic REV-ERB agonists may have potential for broad ranges of allergic diseases.