Identification of Baicalin as an Immunoregulatory Compound by Controlling TH17 Cell Differentiation

TH17 cells have been implicated in a growing list of inflammatory disorders. Antagonism of TH17 cells can be used for the treatment of inflammatory injury. Currently, very little is known about the natural compound controlling the differentiation of TH17 cells. Here, we showed that Baicalin, a compound isolated from a Chinese herb, inhibited TH17 cell differentiation both in vitro and in vivo. Baicalin might inhibit newly generated TH17 cells via reducing RORγt expression, and together with up-regulating Foxp3 expression to suppress RORγt-mediated IL-17 expression in established TH17 cells. In vivo treatment with Baicalin could inhibit TH17 cell differentiation, restrain TH17 cells infiltration into kidney, and protect MRL/lpr mice against nephritis. Our findings not only demonstrate that Baicalin could control TH17 cell differentiation but also suggest that Baicalin might be a promising therapeutic agent for the treatment of TH17 cells-mediated inflammatory diseases.


Introduction
The T helper 17 (T H 17) lineage, a lineage of effector CD4 + T cells characterized by production of interleukin (IL)-17, is described based on developmental and functional features distinct from classical T H 1 and T H 2 lineages [1,2]. T H 17 cells are associated with the development and pathogenesis of a growing list of chronic inflammatory diseases, including rheumatic arthritis, psoriasis, atopic dermatitis, and asthma [3,4,5]. Our studies, as well as others, have shown that T H 17 cells also play a key role in the pathogenesis of systemic lupus erythematosus (SLE) [6,7,8,9,10,11]. Several studies have advocated that T H 17 cells might be a promising therapeutic target for chronic inflammatory injury [12,13].
The differentiation of T H 17 cells is initiated by transforming growth factor-b (TGF-b) and interleukin-6 (IL-6) in mice, and interleukin-23 (IL-23) is also required [14]. Signal transduction and activator of transcription 3 (STAT3), aryl hydrocarbon receptor (AHR) and the retinoic acid-receptor-related orphan receptor-ct (RORct) mediate T H 17 lineage commitment [15,16,17]. Several studies have indicated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), halofuginone, and retinoic acid could suppress the expression of these transcription factors, and subsequently inhibit the differentiation of T H 17 cells [18,19,20]. However, few natural compounds restraining T H 17 cells are known. Moreover, it is important to explore not only effective but also safe therapeutic agents for the treatment of T H 17 cellsmediated inflammatory injuries.
Baicalin, which is a main active ingredient originally isolated from the root of Huangqin (Scutellaria baicalensis Georgi), has safety records in clinic and has been used as an anti-inflammatory drug in traditional Chinese medicine [21,22]. Previous studies have showed that Baicalin could inhibit the proliferation of mononuclear cells, inhibit macrophage activation, inhibit the production of T H 1 related cytokines in different disease murine models [23,24]. Baicalin was shown to reduce the severity of experimental autoimmune encephalomyelitis (EAE) [23]. Since T H 17 cells are important inducer of EAE, we hypothesized that Baicalin might inhibit inflammatory injuries by suppressing effector T H 17 cells. Furthermore, previously published data confirmed that Baicalin inhibited the activation of AHR [25], which might has relevance to the proposed effect on T H 17 cell development.
In this study, we observed that Baicalin inhibited T H 17 cell differentiation in vitro. Detailed studies showed that Baicalin might inhibit newly generated T H 17 cells via suppressing RORct expression, and together with up-regulating Foxp3 expression to suppress RORct-mediated IL-17 expression in established T H 17 cells. Baicalin could inhibit the generation of T H 17 cells in vivo, reduce T H 17 cells infiltration into kidney via inhibition of the CCL20-CCR6 signaling pathway, and could protect lupus-prone MRL/lpr mice against nephritis. Taken together, these findings suggest that Baicalin might be a promising therapeutic agent for the treatment of T H 17 cells-mediated inflammatory diseases.

Baicalin inhibits T H 17 cell differentiation in vitro
Baicalin (7-glucuronic acid, 5, 6-dihydroxyflavone, molecular weight = 446.36. Figure S1A) is a flavonoid compound originally isolated from the Chinese Herb Huangqin (Scutellaria baicalensis Georgi). First, using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and flow cytometry, we observed that treatment with 20 mM Baicalin did not result in generalized inhibition of T cell proliferation and cell cycle ( Figure S1B and C), thus 20 mM Baicalin was used in most in vitro experiments. To determine whether Baicalin controls the differentiation of T H 17 cells, CD4 + CD25 2 T cells from B6 mice were isolated. Under T H 17 culture conditions (TGF-b plus IL-6 stimulation), IL-17 mRNA expression was increased 2.9-fold compared to control cells on day 2 and 10.7-fold compared to control cells on day 3. Following addition of 20 mM Baicalin to the culture, IL-17 mRNA expression was inhibited. In fact, IL-17 expression was decreased to 1.2-fold on day 2 and 2.5-fold on day 3 compared to controls ( Figure 1A). In addition, 20 mM Baicalin measurably inhibited IL-17 protein secretion ( Figure 1B). We further proved that the suppression of T H 17 cell differentiation was dependent on the dose of Baicalin ( Figure 1C). These results provide evidence that Baicalin can suppress the development of T H 17 cells.
Baicalin inhibits IL-6 receptor and RORct mRNA expression IL-6, an acute-phase protein induced during inflammation, may ''dictate'' T H 17 cell differentiation [26]. Thus, we next determine whether Baicalin-mediated inhibition of T H 17 cell differentiation is IL-6-dependent. CD4 + CD25 2 T cells from B6 mice were stimulated with anti-CD3, anti-CD28, and the indicated cytokines in the presence or absence of Baicalin. IL-6 receptor (IL-6R) mRNA expression was analyzed by real-time RT-PCR at the indicated times. As expected, IL-6R mRNA was suppressed by Baicalin ( Figure 2A). Further study confirmed that Baicalin could reduce IL-6R protein expression during Th17 cell differentiation ( Figure 2B). RORct, which is a key transcription factor involved in T H 17 cell differentiation, is elicited by IL-6 and TGF-b [17]. During T H 17 cell differentiation in vitro, addition of Baicalin reduced RORct expression ( Figure 2C). IL-23 expands the pool of T H 17 cells [27], but Baicalin failed to affect the expression of IL-23 receptor during T H 17 differentiation ( Figure S2A). TGF-b and IL-21 can induce STAT3-mediated IL-17 expression during T H 17 differentiation [16], while Baicalin did not restrain IL-21-induced STAT3 and IL-17 mRNA expression during T H 17 cell differentiation ( Figure S2B and 2C). These data suggest that Baicalin could reduce IL-6R and RORct expression during T H 17 cell differentiation, which imply that Baiclain might suppress de novo T H 17 cell differentiation via inhibition of IL-6-mediated RORct expression.  cells [19,28]. CD4 + CD25 2 T cells from B6 mice were stimulated with anti-CD3, anti-CD28, and the indicated cytokines, after 2 days stimulation, 20 mM Baicalin was added for additional 2 days. Foxp3 and IL-17 intracellular expression in CD4 + T cells were determined by flow cytometry. Surprisingly, T cells cultured with Baicalin under conditions that otherwise promoted IL-6-dependent T H 17 cell differentiation converted to Foxp3 + T cells with a concomitant decrease in T H 17 cell differentiation ( Figure 3A). In addition, Baicalin could inhibit RORct-mediated IL-17 mRNA expression in established T H 17 cells ( Figure S3A). Thus, we hypothesized that Baicalin could inhibit RORct transcriptional activity partly via up-regulation of endogenous Foxp3 expression, because previous report showed that Foxp3 could inhibit RORctmediated IL-17 expression and T H 17 cell differentiation [29]. To support this hypothesis, we further showed that Baicalin in synergy with TGF-b could up-regulate endogenous Foxp3 expression in CD4 + CD25 2 T cells ( Figure S3B), Baicalin could promote endogenous Foxp3 expression ( Figure 3B, middle panel) and reduce RORct expression during T H 17 cell differentiation ( Figure 3B, upper panel), and Baicalin together with forced expression of Foxp3 could inhibit RORct and IL-17 mRNA expression during T H 17 cell differentiation ( Figure 3C). Collectively, these data imply that Baicalin could inhibit RORct expression in established T H 17 cells, together with up-regulating Foxp3 inhibit RORct-mediated IL-17 expression.

Baicalin inhibits T H 17 cell differentiation in vivo
To determine whether Baicalin controls the development of T H 17 cells in vivo, lupus-prone MRL/lpr mice were treated with Baicalin or vehicle for nine weeks. Notably, mice without Baicalin treatment developed severe nephritis with increased urine protein, while mice receiving Baicalin were protected against nephritis with decreased urine protein ( Figure 4A-C). Baicalin also protected the survival and liver function of MRL/lpr mice (Table 1 and Figure S4). Furthermore, Baicalin reduced the spleen index and inhibited differentiation of T H 17 cells in spleens ( Figure 4D-F). Interestingly, Baicalin only slightly affected the frequency of T reg cells in vivo ( Figure 4F).
Further study showed that Baicalin reduced the infiltration of T H 17 cells into the kidneys ( Figure 5A). Inflamed tissue produces CCL20 to facilitate the migration of CCR6-expressing T H 17 cells to the inflamed tissues [30,31]. Baicalin treatment inhibited CCL20 mRNA expression in kidneys and CCR6 expression in T H 17 cells ( Figure 5B and C), which indicated that Baicalin might interfere T H 17 cell infiltration into kidneys via inhibition of the CCL20-CCR6 expression.

Baicalin inhibits IL-17 mediated gene expression of inflammatory molecules
IL-17 acts as a potent inflammatory cytokine, and mediates leukocyte infiltration and tissue destruction [2,32]. Baicalin inhibited expression of genes encoding inflammatory molecules (ICAM-1, VCAM-1, and IL-17) in HUVEC that were induced by exogenous IL-17 ( Figure 6A). In support of these results, Baicalin reduced IL-17-induced adhesion of T cells to HUVEC ( Figure 6B). In addition, Baicalin also suppressed gene expression of inflammatory mediators in MRL/lpr mouse kidney ( Figure S5). Together, these data indicate that Baicalin could partially inhibit IL-17-induced inflammation.

Discussion
Baicalin, which is a main active ingredient originally isolated from the root of Huangqin (Scutellaria baicalensis Georgi), has safety records in clinic and has been used as an anti-inflammatory drug in traditional Chinese medicine [21]. Baicalin has been found to possess anti-inflammatory, antioxidant and anti-allergic properties, and appears to contribute to the treatment of chronic inflammatory diseases, including hepatitis, allergic diseases, and EAE [22,23,33].
The binding of IL-6 with IL-6R plays a key role in the transcription of RORct during the development of T H 17 cells, and IL-6 blockade by treatment with an anti-IL-6R monoclonal antibody might inhibit the development of T H 17 cells [34,35]. IL-6-deficient mice do not express RORct and IL-17 [17]. Together, these data suggest that IL-6 is a key cytokine to induce the expression of RORct and the development of T H 17 cells. Our data showed that Baicalin treatment inhibited the expression of IL-6R and RORct under culture conditions promoting T H 17 cell differentiation. However, the down-regulation of IL-6R was not accompanied by decreased expression of STAT3. IL-21 is also a key cytokine for STAT3-mediated T H 17 cell differentiation [16], Baicalin did not suppress IL-21R and STAT3 mRNA expression induced by TGF-b and IL-6 ( Figure S2A), which might explain that reduced expression of IL-6R was not accompanied by decreased mRNA expression of STAT3. Baicalin also hardly restrain IL-21-induced STAT3 and IL-17 mRNA expression during T H 17 cell differentiation ( Figure S2B and C). But Baicalin could affect the STAT3 phosphorylation induced by TGF-b and IL-6 ( Figure S2D). Thus, these data implied that transcript levels of STAT3 did not mirror protein levels, and Baicalin might regulate T H 17 cell differentiation by affecting STAT3 phosphorylation but not the expression of STAT3. Furthermore, cytokine receptors like IL-4R and IL-12Rb2 have negative impacts on T H 17 cell differentiation, our supplemental data showed that 20 mM Baicalin did not affect the mRNA expression of IL-4R and IL-12Rb2 during T H 17 cell differentiation ( Figure S2A). These data implied that Baicalin might restrain de novo T H 17 cell differentiation by abrogating IL-6 mediated RORct transcription, and Baicalin-mediated inhibition of STAT3 activation might contribute to reduced STAT3-mediated gene expression, such as RORct and IL-17A [36]. Because previous study has proved that Foxp3 could interact with RORct and inhibit RORc-directed IL-17 expression during T H 17 cell differentiation [29]. To support this hypothesis, we showed that Baicalin together TGF-b could up-regulate endogenous Foxp3 mRNA and down-regulate RORct mRNA expression ( Figure 3A, B, and Figure S3B). In addition, exogenous over expressed Foxp3 could inhibit RORct-mediated IL-17 mRNA expression in T H 17 cells, Baicalin together with Foxp3 might augment inhibition of IL-17 mRNA expression ( Figure 3C). Interestingly, we also noticed that RORct mRNA expression was also inhibited by forced expression of Foxp3, and Baicalin together with exogenous Foxp3 have a additive inhibition of RORct expression ( Figure 3C). Whereas further study should be performed to make clear the mechanism of Foxp3-mediated inhibition of RORct expression. All together, these data implied that Baicalin could up-regulate Foxp3 expression and suppress RORct-mediated IL-17 expression in established T H 17 cells.
In our study, we unexpectedly found that Baicalin not only inhibited the differentiation of T H 17 cells but also promoted TGFb-mediated differentiation of T reg cells in vitro. Thus, Baicalin  appeared to play a dual role in T-cell differentiation by mediating a reciprocal balance of Foxp3 and RORct. IL-6 is a key cytokine to inhibit Foxp3 expression during T reg cell differentiation [19], inhibition of IL-6R expression could increase T reg cell differentiation [38] Thus, Baicalin might induce Foxp3 expression by restoring IL-6-mediated inhibition of Foxp3 expression in vitro. In contrast to the observation that Baicalin enhanced Foxp3 expression in vitro, Baicalin treatment in MRL/lpr mice only slightly affected CD4 + Foxp3 + T cells. This minor expansion of Foxp3 + T cells might stem from the strong inhibition of excessive inflammatory cytokines and lack of TGF-b in vivo [38,39,40]. Although we observed that 20 mM Baicalin did not affect cytokines expression during the differentiation of T H 1 and T H 2 cells in vitro ( Figure S6), further study should be done to explore different concentrations of Baicalin on the differentiation of T H 1 and T H 2 cells. The number of T H 17 cells was found to be increased in murine model of SLE, including BXD 2 [41], SNF 1 [42], NZB6NZW F 1 [43,44], and Ro52 knockout mice [45]. Our previous studies, as well as others, showed that there were expansion of T H 17 cells in MRL/lpr mice [9,46,47]. Our unpublished data also showed that treatment with anti-IL-17 antibody could protect MRL/lpr mice against disease onset. Together, these data suggested that T H 17 cells might play a key role in the pathogenesis of MRL/lpr mice. Here we observed that Baicalin could reduce IL-6R and RORct mRNA expression in spleens of MRL/lpr mice ( Figure 4E), and Baicalin could accordingly inhibit T H 17 cell differentiation in vivo ( Figure 4F). These results were consistent with in vitro study of Baicalin on the differentiation of T H 17 cells. Interestingly, we noticed that a high percentage of IL-17 producers was CD4 2 cells in MRL/lpr mice ( Figure 4F). Actually, T H 17 cells (CD4 + IL-17 + T cells) are main source of IL-17 during chronic inflammatory responses. However, in mice other subsets can also express IL-17, including CD8 + T cells, invariant natural killer T (NKT) cells, and cd T cells [48,49,50,51]. Thus, we hypothesized that CD4 2 L-17 + cells were also expanded in MRL/lpr mice duo to severe inflammatory responses, but further study should be performed to dissect the specific source and function of these groups of CD4 2 L-17 + cells.
Our data showed that Baicalin not only inhibited the differentiation of T H 17 cells in spleen but also reduced the infiltration of T H 17 cells into kidney, which might result from inhibition of the CCL20-CCR6 signaling pathway, since expression of CCL20 and CCR6 mRNA was found to be down-regulated by Baicalin. IL-17 is a key T H 17 cell-derived cytokine, which is implicated in leukocyte recruitment [32]. Treatment with Baicalin could suppress production of IL-17-mediated adhesion of T cells to HUVEC. Our in vivo studies further confirmed that Baicalin could inhibit IL-17-related inflammatory mediators, such as IL-22, IL-1, TNF-a, VCAM-1, and ICAM-1 ( Figure S5A). Through the potent inhibition of these adhesion molecules and inflammatory mediators, Baicalin might further impede the recruitment of T H 1, T H 2 or other effector cells in vivo. Thus, we are not making conclusions on the observed inhibition of T H 17 cells as the only function of Baicalin in vivo. Further study should be done to dissect the other specific subsets of effector cells affected by Baicalin in MRL/lpr mice. In addition, our unpublished data also showed that Baicalin could inhibit T H 17 cell differentiation in complete Freund's adjuvant induced inflammatory arthropathy mice, and ovalbumin immunized mice. Together, these data suggest that Baicalin could inhibit T H 17 cell differentiation in vivo, and exert therapeutic effects via inhibition of IL-17-mediated inflammation.
Our findings define a role of Baicalin in T H 17 lineage commitment, thereby linking this natural compound to adaptive immunity in a way that has important implications for immune homeostasis and inflammatory diseases. Taken together, these findings suggest that Baicalin might be a promising therapeutic agent for the treatment of T H 17 cells-mediated inflammatory diseases.

Mice and histopathology
C57BL/6 (B6) and lupus-prone MRL/lpr mice were purchased from the Shanghai Laboratory Animal Center (Chinese Academy of Sciences). The animal study was approved by the institutional animal care and use committee of Zhongshan Hospital, Fudan University (ZS0862701). All mice were maintained under pathogen-free conditions. The onset of autoimmune diseases in MRL/lpr mice was monitored by the assessment of proteinuria. After clinical onset of disease, Baicalin (100 mg/kg; Purity.98%, National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China; Baicalin was dissolved in phosphate-buffered saline prior to experimentation.) or PBS vehicle was given intraperitoneally every day for 9 weeks. For detection of urine protein, the total urine of 24 h were first collected, and performed according to the manufacturer's directions (Roche). Relative urine protein increases = urine protein (mg/L) at indicated time point -urine protein (mg/L) of week 0. At the time of sacrifice (9 weeks after treatment), the kidneys were fixed with formaldehyde, embedded in paraffin, stained with hematoxylin and eosin (H&E), and IL-17 (Santa Cruz Biotechnology, CA). The slides were read and interpreted in a blinded fashion, grading the kidneys for glomerular inflammation, proliferation, crescent formation, and necrosis. Interstitial changes and vasculitis were also noted. Scores from 0 to 3 were assigned for each of these features and then added together to yield a final renal scores. For example, glomerular inflammation was graded: 0, normal; 1, few inflammatory cells; 2, moderate inflammation; and 3, severe inflammation. Detailed pathological assessment was performed as described previously [52]. The spleens of MRL/lpr mice were collected to calculate the spleen index. Spleen index = spleen weight (g) divided by body weight (g).
CD4 + T cell isolation, culture conditions, and western blot

Intracellular cytokine staining and flow cytometry analysis
For detection of T H 17 cells, cells obtained from in vitro cultures or spleen cells from mice were incubated for 5 hours with 50 ng/ml phorbol myristate acetate (PMA) and 750 ng/ml ionomycin in the presence of 20 mg/ml brefeldin A (Sigma-Aldrich) in a tissue culture incubator at 37uC. Surface staining with FITCconjugated anti-CD4 (eBioscience) was first performed for 15 min, then cells were re-suspended in Fixation/Permeabilization solution according to the manufacturer's instructions (Invitrogen), intracellular staining of PE-conjugated anti-IL-17 or isotype control was performed according to the manufacturer's protocol (eBioscience). After staining, we first gated on CD4 + T cells, then CD4 + IL-17 + cells were analyzed in a CD4 + gate in a FACS-Calibur (BD-Bioscience, Biosciences, San Jose, CA), and followed by analysis with FlowJo software (Tree Star, San Carlos, CA).
For detection of T reg cells, cells were treated according to the Foxp3-staining kit protocol (eBioscience). Gating was on CD4 + T cells first, and then Foxp3 + cells were analyzed in CD4 + gate.

Cytokine production
Sorted CD4 + CD25 2 T cells from B6 mice were cultured under neutral conditions or in the presence of 5 ng/ml TGF-b plus 20 ng/ml IL-6 with or without 20 mM Baicalin for 3 days. IL-17 concentrations were determined by ELISA (R&D Systems, Minneapolis, MN).
HUVEC and T cell co-culture HUVEC was seeded into 12-well plates (10,000 cells/well) and allowed to adhere for 24 hours. Then HUVEC was stimulated with 50 ng/ml IL-17 (eBioscience) for 24 hours with or without 20 mM Baicalin. After stimulation, Jurkat cells were added to the HUVEC cultures at a 1:5 ratio, and the co-culture was extended for an additional 24 hours. The HUVEC was then washed twice to eliminate the non-adherent Jurkat cells. The adhesion of T cells was counted under 6200 magnification.

RNA isolation and real-time RT-PCR
Total RNA was prepared with the use of the Trizol reagent (Invitrogen). The cDNA was synthesized with a first-strand cDNA synthesis kit and oligo (dT) primers (Fermentas, Hanover, MD), and gene expression was examined with a Bio-Rad iCycler Optical System (Bio-Rad, Richmond, CA) using a SYBR green real-time PCR Master Mix (Toyobo, Osaka, Japan). The 2 2DDCt method was used to normalize transcription to human 18S or mus b-actin and calculate fold induction relative to controls. The primer pairs could be seen in Table 2.

Statistical analysis
The quantitative data were expressed as the means 6 standard deviation (SD). The statistical significance was determined by ANOVA followed by Bonferroni post-hoc test for multiple comparisons or the Student's t-test. A paired t-test was also used in some cases. All p values #0.05 were considered significant.