Wogonin Suppresses IL-10 Production in B Cells via STAT3 and ERK Signaling Pathway

Wogonin (5,7-dihydroxy-8-methoxyflavone) is an ingredient of the extracts from Scutellaria baicalensis, which has documented a wide spectrum of anti-inflammatory and antitumor activities, including inhibiting regulatory T cells, regulating effector T cell functions, and mediating macrophage immunity. However, the potential effect of Wogonin on B cells has not been fully understood. Here, our results showed that Wogonin inhibited IL-10 secretion in B cells. When purified B cells were activated by lipopolysaccharide (LPS) in vitro, the amount of IL-10 production in supernatant was decreased by Wogonin significantly. The protective role of B cells on dextran sulfate sodium- (DSS-) induced colitis was alleviated after exposure to Wogonin. Furthermore, administration of Wogonin on LPS-treated B cells suppressed phosphorylation of STAT3 and ERK, but not AKT. Interestingly, among those IL-10 signaling-associated transcription factors, mRNA and protein levels of Hif-1α were specifically decreased by Wogonin. Overall, our study indicates that Wogonin suppresses potentially IL-10 production in B cells via inhibition of the STAT3 and ERK signaling pathway as well as inhibition of mRNA and protein levels of the transcription factor Hif-1α. These results provide novel and potential molecular targets of Wogonin in B cells and help us further understand its mechanism of action, which could potentially improve its clinical application in the future.

Conventionally, B cells were deemed as the humoral immune cells executing antigen presentation and antibody secretion. However, progressive proof displayed immunosuppressive cytokine production from B cells, such as IL-35, TGF-β, and IL-10. Further investigation defined several sub-populations of B cells retaining regulatory function, such as peripheral circulating CD19 + CD1d hi CD5 + , peritoneal CD5 +-B1a, CD19 + CD23 hi CD21 hi IgM + , CD19 + CD24 hi CD38 hi , CD19 + B220 low , and CD19 + CD138 + plasma cells. Although they have different phenotypes, development pathways, anatomical distribution, and pathogenic implications, they all possess an immunoregulatory activity, which put them onto center stage by basic and clinical scientists [10,11].
Wogonin repelled the production of immunoglobulins and cytokines (such as IL-4, IL-5, and IL-10) from mesenteric lymphocytes in DSS-induced colitis in vivo [12]. The anti-inflammatory effect of Wogonin associated with its suppression the production of nitric oxide, cytokines, and chemokines in dsRNA-induced macrophages via the calcium-STAT pathway [13]. Dandawate et al. [6] also described the inhibition of glioma-induced, TGF-β1-mediated regulatory T cell activity by Wogonin in vitro. In this work, Wogonin inhibited the IL-10 secretion of regulatory T cells. Instrumentally, Wogonin repressed Smad-3, GSK-3β, and ERK1/2 signaling in regulatory T cells. Meanwhile, P38 phosphorylation was markedly boosted, implying that Wogonin may regulate T cells' function via Smad and non-Smad signaling pathways. However, whether and how Wogonin perturb the production of IL-10 in B cells are still unknown.
In this study, we aimed to investigate the response and mechanism of Wogonin on IL-10 production of LPStreated B cells. Our data suggested that Wogonin impeded IL-10 production from LPS-treated B cells in vitro and weakened the immunoregulatory effect of B cells on DSS-induced murine colitis. In further investigation in LPS-treated B cells, Wogonin was shown to constrain phosphorylation of ERK and STAT3 and Hif-1α transcription. Our studies suggested that Wogonin might inhibit IL-10 production from B cells The body weight changes of mice after DSS induction of colitis. * * P < 0:01; * * * P < 0:001 for comparison with the DSS+B group. (c) Representative colonic length of mice was measured in four groups. (d) Quantification of colonic length of mice in four groups was shown. Data are presented as mean ± SD (n = 6 per group). * * * P < 0:001; * * * * P < 0:0001.

DSS-Induced Acute Colitis and Abdominal B Cell
Adoptive Transfer. Mice were divided randomly into four groups, six in each group. 5% DSS (MP Biomedicals, USA) in drinking water was administered to induce acute colitis. Peritoneal cavity cells from mouse were isolated and resuspended in PBS according to a published protocol [29]. After being cultured for 2 h in the incubator, macrophages attached to the plate surface, suspension B cells were harvested in the supernatant. After being treated with/without 50 μM Wogonin for 6 h, 2 × 10 6 B cells were suspended in 200 μL of PBS and transferred intraperitoneally into recipient mice on day 1 of colitis induction. Body weight was supervised every day. All mice were terminated on the ninth day. The colon length was measured to evaluate the colitis. Skeptical procedure was described in Figure 1(a).

Flow Cytometry for Phenotyping and Cytokine Secretion.
Flow cytometry analysis for cell phenotype and intracellular cytokine secretion has been described previously [30]. Briefly, cells were washed twice and maintained in 100 μL of PBS containing 0.1% BSA and 0.05% sodium azide. For phenotyping, cells were stained with corresponding Abs for 30 min at 4°C in the dark. For the analysis of intracellular cytokines, after surface staining as described above, cells were fixed with 4% paraformaldehyde and permeabilized in staining buffer containing 0.1% saponin (Sigma-Aldrich, St Louis, MO, USA) for at least 2 h or overnight at 4°C. After being washed twice, cells were incubated with the respective cyto-kine Abs for 30 min at 4°C. Samples were acquired on FACS Arial II, and data were analyzed by FlowJo software (Tree Star, San Carlos, CA, USA). The antibodies used are shown in Table 1.

Cell Viability Measurement.
To avoid dead cell-induced false positive, cell viability of LPS-activated B cells was investigated. 2 × 10 6 /mL B cells were cultured in 96-well plates with or without LPS and Wogonin for 6 h, 12 h, 24 h, and 48 h. The cell viability was measured by a CCK8 kit (Beyotime, Shanghai, China) according to the supplier's instructions.
2.6. ELISA. Purified B cells were suspended in complete RPMI 1640 medium at a density of 1 × 10 6 /mL and stimulated with/without LPS and Wogonin for 6 h, 12 h, 24 h, and 48 h in 24-well plates. Cell-free supernatants were collected for the

Western
Blot. CD19 + B cells were challenged by LPS for 6 h with/without Wogonin (12.5, 25, and 50 μM). Cells were lysed in RIPA cell buffer (Beyotime) supplemented with PMSF (Solarbio) and protease inhibitors (Roche) on ice for 30 min. 50 μg of protein sample in the lysate was separated in 10% SDS-PAGE gels and transferred to PVDF membranes (Millipore). Membranes were blocked in TBST (Triton-containing Tris-buffered saline) with 5% milk for 2 h at room temperature and then incubated with specific primary antibodies overnight at 4°C. The secondary antibodies were added to the membranes and incubated for 1 h. Blot bands were visualized by enhanced chemiluminescence. The quantitative analysis was evaluated using ImageJ software according to the grey value of western blot bands.

Effect of Wogonin on the Surface Molecules of B Cells.
After investigation on IL-10 secretion, the phenotype of B cells was also assessed under different conditions of Wogonin administration. Frequencies of typical B cell markers, such as CD5, CD24, CD21, CD38, CD23, MHCII, IgD, IgM, CD80, and CD86, were analyzed by flow cytometry. We found that the expression amount of most surface markers did not obviously change by Wogonin ( Figure S3); only frequencies of CD80 and CD86 were significantly decreased by Wogonin after LPS stimulation (Figures 3(a)-3(c)). These observations indicated that Wogonin might regulate antigen presentation capability of B cells, which could be interesting for immunotherapy of PD-1/PDL-1 Ab in different clinical settings.

Effect of Wogonin on B Cells in Mouse with Acute Colitis.
To validate our observations in vitro, the response of B cells to Wogonin challenge was evaluated in vivo. Isolated B cells from mouse peritoneal cavity were challenged with/without Wogonin, and then, their impingement on DSS-induced colitis was examined. As shown in Figure 1(a), the body weights of DSS-treated mice were significantly decreased from day 5, whereas intraperitoneal injection of B cells significantly attenuated the loss of body weight in comparison with the DSS group, which suggested the immunological regulation of adoptive transferred B cells, and this regulation function was lost in Wogonin-treated B cells (Figure 1(b)). Colon length was assessed among these 4 groups of mice, which echoed weight loss (Figures 1(c) and 1(d)). These results suggested that Wogonin treatment abrogated immunological regulation of B cells in vivo.
To further verify the role of Wogonin on adoptive transferred B cells in vivo, in situ histopathological analysis of colon tissues was investigated among all 4 groups of animals. Inflammation, mucosal and submucosa damage degree, epithelial intact, distortion of crypts, and percentage were compiled into histology score. Similar to weight loss and colon loss, significant colon damage caused by DSS administration was attenuated by transferred B cells, but this debilitation was suppressed by Wogonin treatment (Figures 4(a) and 4(b)).

Effect of Wogonin on the STAT3 and ERK Signaling
Pathway of LPS-Mediated B Cells. To explore the potential molecular mechanisms of Wogonin impinge on B cells, we assessed signaling pathways involved in IL-10 production. Strikingly, phosphorylation of STAT3 and ERK was decreased via Wogonin treatment, and this suppression showed doseeffect tendency. On the contrary, AKT phosphorylation was kept intact on Wogonin treatment (Figures 5(a)-5(d)). This investigation strongly suggested Wogonin suppression on IL-10 production from B cells involved in STAT3/ERK signaling pathway, instead of AKT/ERK.  Foxp3, c-maf, JunB, Gata3, Egr2, Ahr, and Batf, Hif-1α was the only transcription factor whose mRNA amount was significantly abrogated by Wogonin treatment, and this suppression showed a dose-effect curve (Figures 6(a)-6(d)).
In order to verify the change of Hif-1α caused by Wogonin, Hif-1α protein expression was also assessed by western blot. As expected, Wogonin significantly decreased the protein level of Hif-1α in B cells after LPS stimulation in a dose-dependent manner, which was consistent with the change of mRNA level (Figures 7(a) and 7(b)). Hence, our data suggested that Hif-1α was a potential target of Wogonin in B cells.

Discussion
Wogonin is a flavonoid-like compound, purified from Scutellaria baicalensis. Originally, Wogonin showed anxiolytic properties in mice [35]. Later on, extensive investigations on different tumor models proved the therapeutic effect of Wogonin [36], which is due to their regulation in different signaling pathways [37].
Recently, the anti-inflammatory and immune regulatory effect from Wogonin was also documented [7,38]. Wogonin shaped phenotype and function of macrophage, effector and regulatory T cells via different signaling pathway. The progress indicated comprehensive clinical implications of this compound and drew broad attention on this extract.
Paralleling to Treg, a small subset of B cells participating in immunomodulation of immune responses has been described in the models of inflammation, autoimmune dis-eases, transplantation, and antitumor immunity. IL-10 from Breg is one of the main regulatory molecules in these pathogenic processes. Hence, we investigated the effect of Wogonin in B cells, focusing on IL-10 production. To our knowledge, this is the first report on this topic. TLR agonists including LPS (TLR4), R848 (TLR7, 8), or CpG (TLR9) can potently activate B cells and induce IL-10 production [39]. Therefore, we observed the frequency of IL-10 production of B cells stimulated by LPS before and after Wogonin treatment. In an in vitro model, Wogonin significantly decreased the IL-10 secretion of B cells. Interestingly, Wogonin was also shown to inhibit IL-10 secretion from regulatory T cells specifically [6]. Furthermore, the protective role of B cells on DSS-induced colitis was alleviated after exposure to Wogonin in vivo. These results indicated that Wogonin executed regulatory effect on B cells via inhibition of IL-10 secretion. To validate the specificity of Wogonin on B cells, we investigated the consequence of Wogonin on B cells' phenotype. The intact amount of CD5, CD24, CD21, CD38, CD23, MHCII, IgD, and IgM on B cells verified the tolerance of B cells under current experimental conditions, which highlighted the specificity of Wogonin on IL-10 secretion from B cells. Moderately, Wogonin decreased the frequencies of CD80 and CD86. At the moment, it is difficult to determine that this change was due to direct implementation of Wogonin on B cells or their regulation via monocytes/macrophage. However, the influence of Wogonin on these checkpoint molecules indicated potential preclinical combinative regime.
Mechanically, TLR4 signaling switched on IL-10 via both ERK and AKT pathways. Previous studies also have shown that the activation of STAT3 and ERK is required for TLRinduced IL-10 production in human B cells [21]. However, our data showed that Wogonin treatment diminished STA-T3/ERK signaling only, it did not abate AKT phosphorylation under current conditions. On the contrary, Parajuli et al. [40] reported that Scutellaria flavonoids could inhibit the phosphorylation of Akt and GSK-3β in malignant gliomas. Further investigation may uncover a novel target of Wogonin in B cells.
In brief, on a different model, we showed that Wogonin treatment suppressed Hif-1α expression, STAT3/ERK signaling, and IL-10 secretion from B cells; this suggested novel molecular and cellular mechanism of Wogonin in B cells and their potential effect on immunomodulation.

Conclusions
Our study suggests that Wogonin may inhibit IL-10 production from B cells via modulating Hif-1α and the ERK, STAT3 signaling pathway, which provides novel and potential molecular targets of Wogonin in B cells and helps us further understand its mechanism and function in immune regulation, potentially improving its clinical application in the future.

Conflicts of Interest
The authors declare that they have no conflicts of interest.