High‐dose wogonin exacerbates DSS‐induced colitis by up‐regulating effector T cell function and inhibiting Treg cell

Abstract Wogonin exerts anti‐tumour activities via multiple mechanisms. We have identified that high‐dose wogonin (50 or 100 mg/kg) could inhibit the growth of transplanted tumours by directly inducing tumour apoptosis and promoting DC, T and NK cell recruitment into tumour tissues to enhance immune surveillance. However, wogonin (20–50 μM) ex vivo prevents inflammation by inhibiting NF‐κB and Erk signalling of macrophages and epithelial cells. It is elusive whether high‐dose wogonin promotes or prevents inflammation. To investigate the effects of high‐dose wogonin on murine colitis induced by dextran sodium sulphate (DSS), mice were co‐treated with DSS and various doses of wogonin. Intraperitoneal administration of wogonin (100 mg/kg) exacerbated DSS‐induced murine colitis. More CD4+ CD44+ and CD8+ CD44+ cells were located in the inflamed colons in the wogonin (100 mg/kg) treatment group than in the other groups. Frequencies of CD4+ CD25+ CD127− and CD4+ CD25+ Foxp3+ cells in the colons and spleen respectively, were reduced by wogonin treatment. Ex vivo stimulations with high‐dose wogonin (50–100 μg/ml equivalent to 176–352 μM) could synergize with IL‐2 to promote the functions of CD4+ and CD8+ cells. However, regulatory T cell induction was inhibited. Wogonin stimulated the activation of NF‐κB and Erk but down‐regulated STAT3 phosphorylation in the CD4+ T cells. Wogonin down‐regulated Erk and STAT3‐Y705 phosphorylation in the regulatory T cells but promoted NF‐κB and STAT3‐S727 activation. Our study demonstrated that high‐dose wogonin treatments would enhance immune activity by stimulating the effector T cells and by down‐regulating regulatory T cells.

The effects of wogonin on T cell function under different micro-environments remain ambiguous. Mid-dose (20 mg/kg) wogonin treatment significantly inhibited chronic colitis induced by dextran sodium sulphate (DSS) within 2 weeks through the downregulation of Th2-associated cytokine, particularly IL-4 and IL-10 secretion [23]. Wogonin also down-regulates OVA-induced Th2  immune responses, particularly IgE and IL-5 prediction [24]. However, IFN-c and IL-2 production of T cells co-stimulated by concanavalin A and wogonin has been shown to be significantly enhanced [23]. Wogonin also inhibits tumour-mediated induction of Treg cells by inhibiting TGF-b1 activity [25]. We found that wogonin administered at 50 and 100 mg/kg inhibited tumour growth and promoted the recruitment of DC, T, and NK cells in the tumour tissues in the xenograft tumour model of mice [26]. In the current study, the effect of high-dose wogonin on the onset of DSS-induced acute colitis was determined. Moreover, the effects of high-dose wogonin on the function of the effector T and regulatory T cell were examined.

Materials and methods
Animals and cell lines C57BL/6 mice, aged 6-8 weeks, were purchased from the Comparative Medicine Centre of Yangzhou University (Yangzhou, China). The mouse gastric cancer cell line (MFC) was from Shanghai cell bank of Chinese Academy of Sciences. MFC cells were adherent and subcultured every 3 days. The murine colon cancer cell line (MC-38) was kindly gifted by Dr. Hursting (University of Texas-Austin). Both cells were cultured in RPMI 1640 (Gibco, Grand Island, NY, USA) supplemented with 10% foetal bovine serum (FBS; Gibco), 100 U/ml penicillin, and 100 lg/ml streptomycin sulphate (Beyotime, Jiangsu, China). For storage, cell lines were suspended in complete growth medium supplemented with 5% (v/v) DMSO and located in liquid nitrogen vapour phase.

Drugs and reagents
Wogonin (purity >98%) purchased from Nanjing Zelang Medical Technology (Nanjing, Jiangsu, China) was dissolved in 1 M NaOH as a stock solution, stored at À20°C, and freshly diluted with RPMI 1640 medium to the final concentration. The working solution of NaOH was less than 0.1 lM. DSS (molecular weight: 36,000-50,000) was obtained from MP Biomedical (Solon, Ohio, USA). Lymphoprep was obtained from Axisshield (Oslo, Norway). Collagenase IV, Dnase I and Percoll were purchased from BIOSHARP (Hefei, Anhui, China). Dispase II was obtained from Roche (Basel, Switzerland).

DSS-induced colitis
Acute colitis was induced by administering DSS (2.5%) in drinking water from days 1 to 7. The mice were randomly divided into solvent control, DSS and solvent-treated, DSS and wogonin (20 mg/kg)-treated, DSS and wogonin (50 mg/kg)-treated, and DSS and wogonin (100 mg/kg)-treated groups. Wogonin or solvent was intraperitoneally injected daily from days 1 to 7. All mice were weighed every day. The mice were killed on day 8, and the spleens and intestinal tissues were removed for ex vivo analysis. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Yangzhou University.

Isolation and detection of colonic mononuclear cells of mice
The intestinal tissues of mice were washed repeatedly with phosphatebuffered saline (PBS) using 5-ml syringe until the intestines became translucent. The tissue was opened longitudinally, cut into 5-mm pieces, and incubated in 5 mM EDTA and 1 mM DTT in calcium-free and magnesium-free Hank's balanced salt solution for 20 min. at 37°C. The tissue was then centrifuged, and the supernatant was collected. The residual tissue was incubated again in a digestion solution containing 0.5 mg/ml of type IV collagenase or Dnase I, as well as 3 mg/ml of Dispase II for 30 min. at 37°C. The suspension combined with the previous supernatant was centrifuged, and the mononuclear cells were isolated by centrifugation in 35% Percoll solution [27]. The cell suspensions were incubated with 7-AAD to exclude dead cells stained with mAbs against CD4, CD25 and CD127. The cells were analysed by flow cytometry.

Real-time PCR
Total RNA was isolated from murine CD8 + T or CD4 + T cells after wogonin treatments by the TRIzol reagent (Life Technologies, Carlsbad, CA, USA). Reverse transcription was conducted for 15 min. at 42°C with 1 lg of total RNA using a QuantiTect Reverse Transcription Kit (QIA-GEN GmbH, Hilden, Germany). Quantitative RT-PCR reaction was monitored using the ABI 7500 (PE applied Biosystems, Carlsbad, CA, USA) and the results were analysed with the accompanying software. The SYBR Green PCR Kit (QIAGEN GmbH) was used for detection of mouse IFN-c and GAPDH. The primer pairs are shown below: 5 0 -CTGTTTCTG GCTGTTACTGC-3 0 and 5 0 -TGCTGATGGCCTGATTGT-3 0 (IFN-c);5 0 -CAAAA TGGTGAAGGTCGGTGTG-3 0 and 5 0 -TGATGTTAGTGGGGTCT CGCTC-3 0 (GAPDH).The relative fluorescence of IFN-c versus GAPDH was analysed by densitometry. Relative RNA expression was calculated by the 2 ÀMMCt method after normalizing expression levels of IFN-c mRNA to GAPDH mRNA.

Magnetic cell sorting
Splenic CD4 + CD25 À T cells of all groups of mice were obtained from a double-negative magnetic cell sorting (MACS). The Miltenyi CD4 cell isolation kit was used to label splenocytes with 100 ll of the biotinylated cocktail of antibodies (against CD8a, CD11b, CD11c, CD19, B220, CD49b, CD105, MHC-class II and Ter-119) specific for CD4 À cells diluted in 400 ll of RPMI 10% FBS for 15 min. at 4°C. Then, 200 ll of the anti-biotin antibody conjugated with microbeads was added, and the cells were incubated for 10 min. at 4°C. After washing, the cells were applied on an LD separation column (Miltenyi, Teterow, Germany). CD4 À cells remained in the column because of the magnetic beads attached to their surface. By contrast, CD4 + cells flowed through the column and could be collected in a tube. The untouched CD4 + cells were further indirectly labelled with an anti-CD25-PE and anti-PE conjugated with microbeads. Cells were flowed on an LS column, and CD4 + CD25 À cells were collected. The purity of CD4 + CD25 À T cells was more than 90% as identified by flow cytometry.  In vitro induction of CD4 + CD25 + regulatory T cells A 24-well plate was coated with 10 lg/ml anti-CD3 antibody in PBS at 37°C for 2 hrs and then washed once with PBS before cell plating. CD4 + CD25 À T cells obtained from MACS were resuspended in serumfree medium in anti-CD3-precoated wells without antibiotics. Sorted cells were stimulated with anti-CD28 antibody (2 lg/ml), IL-2 (20 U/ml) and TGF-b (5 ng/ml) for 5 days [28,29]. On day 6, cells were treated with various doses of wogonin for 24 hrs, and solvent was used as a negative control. Finally, the cells were collected and analysed by flow cytometry to detect the amount of CD4 + CD25 + Foxp3 + T cells.

Statistical analysis
Differences between groups were analysed using an unpaired, two-tailed Student's t-test. Data were evaluated by one-way ANOVA followed by Dunnett's test between control and multiple dose groups. Significance of differences was indicated when *P < 0.05, **P < 0.01, and ***P < 0.001.

Results
Wogonin exacerbates DSS-induced acute colitis of mice in association with T cell activation Treatment with 50 or 100 mg/kg wogonin accelerated the loss of bodyweight (Fig. 1A) and had the shortened colon length (Fig. 1B, C) of mice induced by DSS, particularly at 100 mg/kg. Treatment with DSS and 20 mg/kg wogonin did not show significant changes of weight and colon length compared with DSS alone. Intestinal bleeding and stenosis of mice in wogonin treatment groups (50 or 100 mg/kg) were more severe than those treated by solvent or DSS/solvent (Fig. 1B). Immunohistological analysis showed that more CD4 + and CD8 + T lymphocytes infiltrated the intestines of mice treated by 100 mg/kg wogonin (Fig. 1D), which indicated severe inflammation in the colons. Normal mice were also treated by wogonin (50 or 100 mg/kg) alone. There were not significant changes of bodyweight, colon length and histology of all groups of mice ( Figure S1).  Intestinal mononuclear cells were isolated and analysed on CD4 + and CD8 + T cells by flow cytometry. Percentages of viable cells were more than 90% confirmed by 7-AAD staining. The frequencies of colonic CD4 + cells and CD4 + CD44 + T cells were higher in mice treated with 100 mg/kg wogonin than in the solvent group. Moreover, the absolute CD4 + CD44 + T cell numbers in the colons of mice treated at the dosage were the highest among three groups (Fig. 1E). Meanwhile, the frequencies of CD8 + cells and CD8 + CD44 + T cells in the colons of mice treated with 100 mg/kg wogonin were significantly enhanced. Absolute numbers of colonic CD8 + CD44 + T cells in mice treated with 50 mg/kg wogonin were higher than in the solvent and 50 mg/kg wogonin-treated groups (Fig. 1F). The CD44 expression of T cells indicated the activation and migration capacity of T lymphocytes [30]. Thus, wogonin exacerbated DSS-induced colitis by recruiting activated CD4 + and CD8 + cells into the colons.

Wogonin treatment inhibited regulatory T cells in mice with DSS-induced colitis
Regulatory T cell is an important subset for homeostasis of intestine [31]. We observed the distributions of regulatory T cells in the colons and spleens of mice co-treated by DSS and wogonin. We avoided non-specific staining of Foxp3 by measuring the colonic regulatory T cells by flow cytometry gated on CD4 + CD25 + CD127 À cells [32]. Flow cytometric analysis of CD4 + CD25 + CD127 À cells of colons was shown in Figure 2A. The absolute number and frequency of CD4 + CD25 + CD127 À cell in total CD4 + cell was significantly decreased in mice cotreated by DSS and wogonin (100 mg/kg), contrary to those of treatments of DSS/solvent or DSS/wogonin (50 mg/kg) ( Fig. 2A). The frequencies of CD4 + CD25 + Foxp3 + (Fig. 2B) and CD4 + CD25 + CD127 À cells (Fig. 2C) in the spleens from mice with DSS-induced colitis were significantly down-regulated by wogonin (100 mg/kg) treatment, compared with those in the solvent and DSS/solvent groups.

Wogonin synergizes with IL-2 to stimulate CD4 + T and CD8 + T cells ex vivo
We observed the effects of wogonin on the biologic activities of CD4 + and CD8 + T cells in the presence of IL-2. Biologic function of CD8 + T cell is evaluated by its cytotoxicity against target cells and IFN-c production. Splenic CD8 + T cells in C57BL/6 mice were co-cultured with MFC gastric cancer cells and MC-38 colon cancer cells respectively. The cytotoxicity of CD8 + T cells was detected by the lactate dehydrogenase release assay. Compared with the solvent, wogonin treatments at 50 or 100 lg/ml concentration significantly enhanced the cytotoxicity of CD8 + T cells (Fig. 3A). Wogonin treatments at both dosages also promoted IFN-c secretion of CD8 + T cells (Fig. 3B) and IFN-ɣ transcription (Fig. 3C) detected by real-time PCR. Treatment with 100 lg/ml wogonin up-regulated IFN-c secretion, although 50 lg/ml wogonin showed no significant stimulatory effect on IFN-c production by CD4 + T cells (Fig. 3D). However, wogonin treatments at both dosages enhanced IFN-c transcription (Fig. 3E). Therefore, wogonin synergized with IL-2 to stimulate functions of CD4 + and CD8 + T cells ex vivo.

Wogonin inhibits the induction of regulatory T cells in vitro
Splenic CD4 + CD25 À T cells of normal mice were sorted using a double-negative MACS to observe whether wogonin could directly inhibit differentiation of Treg cells ex vivo. Sorted CD4 + CD25 À cells with 90% purity were cultured in the presence of IL-2, CD28 and TGF-b to induce CD4 + CD25 + Foxp3 + cells. The induction of CD4 + CD25 + Foxp3 + T was significantly suppressed by treatments of wogonin at 10, 40, and 160 lg/ml (Fig. 4). Thus, wogonin inhibited the induction of CD4 + CD25 + Foxp3 + T cell dose-dependently.
Wogonin down-regulates STAT3-Y705 and Erk, but activates STAT3-S727 and NF-jB of Treg cells We examined the effects of wogonin on the activation of NF-jB, Erk and STAT3 of Treg cells which were induced by IL-2, CD28 and TGF-b, to elucidate the molecular mechanisms of wogonin on the inhibition of Treg cells. The phosphorylated NF-jB p65 was up-regulated by wogonin treatment at both dosages (Fig. 6A). Conversely, wogonin (100 lg/ml) inhibited p44/42 (Erk1/2) phosphorylation, and wogonin at 50 and 100 lg/ml had no significant effects on the total p44/42 (Erk1/2) proteins (Fig. 6B). Wogonin also significantly down-regulated total STAT3 and the phosphorylation of STAT3 on Tyr705 dose-dependently, but promoted the phosphorylation of STAT3 on Ser724 of Treg cells. IL-6 had no effects of total STAT3, but inhibited STAT3-Y705 activation and increased STAT3-S727 activation. Wogonin co-cultured with IL-6 also significantly down-regulated STAT3-Y705 activation, but had no effects on the activation of STAT3-S727 (Fig. 6C, D).

Discussion
The anti-tumour and anti-inflammatory activities of wogonin have been widely demonstrated. We showed in this study that treatment with relatively high-dose wogonin (100 mg/kg) could stimulate the function of effector CD4 + T and CD8 + T cells to exacerbate symptoms of DSS-induced acute colitis in mice. Moreover, conventional CD4 + CD25 + Foxp3 + regulatory T cells were down-regulated in mice with colitis. High-dose wogonin treatment enhanced the biologic activities of CD4 + and CD8 + T cells by activating NF-jB and Erk and down-regulating STAT3-Y705 activation. Meanwhile, the flavone directly downregulated the induction of regulatory T cells ex vivo by down-regulating STAT3-Y705 and Erk and activating NF-jB and STAT3-S727. The study provided evidence that wogonin exhibits distinct effects on different subsets of T lymphocyte, and high-dose wogonin promotes inflammation.
Wogonin can promote IgA secretion, but down-regulate IgE levels of mesenteric lymph nodes from mice with DSS-induced colitis, which were orally administered with 20 mg/kg wogonin for 2 weeks [23]. In our study DSS-mice administrated by wogonin 20 mg/kg via peritoneal injection did not have protective effects. Oral drug is easier to induce tolerance [34]. The intraperitoneally injected wogonin is absorbed by gastrointestinal tissue and peritoneum, then blood concentration is increased rapidly. Thus, intraperitoneal injection of wogonin is more susceptible to induce inflammation. As a result of intestinal inflammation pre-induced by DSS, some inflammatory cytokines (eg, IL-2) will be released. Thus, T cells could be synergistically activated by IL-2 and wogonin to promote local inflammation.
Wogonin modulates the balance between Th1 and Th2 lymphocytes. This compound enhanced IFN-c and IL-2 production of T cells by co-treatment with concanavalin A [23]. Activation-induced IL-4, IL-5 and IL-10 secretions were lower in wogonin-fed mice compared with control mice with DSS-induced colitis [23]. In addition, relative low dose of wogonin (50 lM) suppressed IL-4 production of murine splenocytes in vitro and down-regulated OVA-induced Th2 immune responses, particularly IgE and IL-5 secretion in vivo [24]. Although Th2 cell-associated cytokines were not observed, our results confirmed that relatively high-dose wogonin treatment (50-100 lg/ml equivalent to 176-352 lM) promoted IFN-ɣ production of CD4 + and CD8 + cells in the presence of IL-2 ex vivo.
Wogonoside, as the glucuronide metabolite of wogonin, at dosages of 12.5, 25 or 50 mg/kg intragastrically, protects against DSS-induced experimental colitis in mice by inhibiting NF-jB and NLRP3 inflammasome activation [35]. Firstly, whether the effect of wogonoside is different from that of wogonin need further study. Next high dose of wogonin (50 or 100 mg/kg) were used intraperitoneally and mice were killed on day 8 in our study, which is different in colitis induction and wogonin treatment. In addition, infiltration of activated CD4 + and CD8 + T cells of colons was only enhanced in mice by 100 mg/kg wogonin treatment. Thus, wogonin may only play an antiinflammatory activity in mice with DSS-induced colitis if administered orally below 50 mg/kg, but exacerbates DSS-induced colitis by activating T lymphocytes when administered intraperitoneally with the dosage more than 50 mg/kg. Wogonin (<50 lM) mediates activities against inflammation by down-regulating the NF-jB pathway of macrophages and epithelial cells. We demonstrated that wogonin (176-352 lM) could stimulate effector T cell function by activating NF-jB p65 and Erk p44/42. The cell type, enhanced dose of wogonin and IL-2 co-stimulation would contribute to NF-jB p65 and Erk activation of T cells. The cytotoxicity and IFN-c production of effector T cells were stimulated by wogonin in the presence of IL-2 (20 U/ml). Wogonin alone may have slight stimulatory effects on T cell function but synergize with IL-2 or other non-specific stimulators, such as concanavalin A [23], to promote activities of T cells. Wogonin (60 lM) also inhibits Erk p44/42 activation induced by TGF-b1 of Treg cells [25], which is consistent with decreased Erk activation of Treg cells by wogonin treatment (Fig. 5B).
Wogonin down-regulated the activation of STAT3-Y705 in gastric cancer cells (SGC-7901) at dosages of 10, 40, 160, and 200 lg/ml but showed no significant effects on STAT3-S727 activation [26]. The phosphorylated tyrosine of STAT3 on position 705 was associated with TGF-b production and expression of B7H1 and MICA of tumour cells [36,37]. Wogonin (50 and 100 lg/mL) also inhibited the activation of STAT3-Y705 and STAT3-S727 of CD4 + T cells, and inhibited the activation of STAT3-Y705 of Treg cells like IL-6, which indicated that STAT3-Y705 activation is important for Treg cells. Effects and