Background
Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders that are elicited by the breakdown of immune tolerance. IBDs are generally divided into two primary forms: ulcerative colitis (UC), which is mediated by a T helper type (Th)2-dominant condition, and Crohn’s disease (CD), which is mediated by a Th1-dominant condition [
1]. Animal models for IBD have been established using dextran sulfate sodium (DSS), trinitrobenzene sulfonic acid (TNBS), and the adoptive transfer of naive T cells and these animal models quite faithfully reproduced the clinical symptoms of human IBD [
1]. Host immune systems profoundly contribute to maintaining intestinal homeostasis and also controlling the pathogenesis of IBD. For example, Th17 cells are critically involved in the pathogenesis of colitis, whereas regulatory T (Treg) cells play central roles in the protection against colitis [
2]. Furthermore, intestinal dendritic cells (DCs) can induce the differentiation of Th17 cells, but when stimulated by retinoic acid (RA) DCs can induce Treg cells [
3]. The differentiation of intestinal forkhead box P3 (Foxp3)
+ Treg cells is induced by RA-producing DCs, and consequently these Treg cells produce the anti-inflammatory cytokine interleukin 10 (IL10) for gut homeostasis [
4]. In colitis, M1 macrophages that are polarized by interferon γ (IFNγ) or lipopolysaccharide (LPS) are associated with inflammation and gut destruction, whereas M2 macrophages that are polarized by IL4 have anti-inflammatory functions that are associated with tissue repair, which indicates that the modulation of macrophage polarization could be a therapeutic strategy for IBD [
5].
Tabebuia avellanedae Lorentz ex Griseb., a typical tree of the Brazilian savannah, is indigenous to South America from Brazil to northern Argentina, and the products from this tree are named “ipe-roxo”, “lapacho” or “taheebo” [
6]. Among these names, “taheebo” is most widely used, so we use this term hereafter. These products were used as traditional folk remedies for treating a variety of diseases [
7]. Moreover, these products have been reported to possess various functions, including anti-inflammatory [
8,
9], anti-bacterial [
10], and anti-ulcer [
11]. However, the effects of taheebo water extract (TWE) on mucosal immune responses have not been explored. Thus, in this study, we investigated whether oral administration of TWE can modulate intestinal inflammatory immune responses by employing DSS-induced murine colitis model.
Methods
Mice and reagents
C57BL/6 (B6) wild type (WT) mice were purchased from Jung Ang Lab Animal, Inc. (Seoul, Korea). The IL4/GFP (4get) and IFNγ/YFP (yeti) cytokine reporter mice were kindly provided by Dr. R. Locksley (University of California at San Francisco, CA, USA). Foxp3/GFP reporter B6 mice and CD11c-diphtheria toxin receptor (DTR) transgenic (tg) mice were obtained from Dr. Rho H. Seong and Dr. E. Choi, respectively (Seoul National University, Seoul, Korea). All of the mice were on a B6 genetic background, were maintained at Sejong University, and were used for experiments at 6–12 weeks of age. They were maintained on a 12-h light/12-h dark cycle in a temperature-controlled barrier facility with free access to food and water. These mice were fed with a γ-irradiated sterile diet and autoclaved tap water. In this study, age- and sex-matched mice were used for all the experiments. The animal experiments were approved by the Institutional Animal Care and Use Committee at Sejong University (SJ-20130802). Our experiments were conducted in a blinded and randomized trial. For the induction of colitis, DSS with a molecular weight of 36–50 kDa was purchased from MP Biomedicals (Solon, OH, USA). LPS derived from Escherichia coli (serotype 0111:B4) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
Preparation of TWE
Taheebo, the dried inner bark of Tabebuia avellanedae Lorentz ex Griseb., was purchased from Frontier Natural Products Co-op. (Norway, IA, USA). To prepare the TWE, five hundred grams of taheebo was stirred in 5 L cold water at 4 °C for 24 h. After filtration, the solution was reduced to 10% of the original volume with a 40 °C rotary evaporator under vacuum. The extracts were freeze-dried, ground to a fine powder, and stored at 4 °C until use.
Standardization of TWE by HPLC
TWE was standardized with 6-O-(3,4-dimethoxybenzoyl)-ajugol and 6-O-(
p-dimethoxybenzoyl)-ajugol known as constituents in
Tabebuia avellanedae Lorentz ex Griseb. [
12], using high-performance liquid chromatography (HPLC; SCL-10A, Shimadzu, Japan) installed with a UV-VIS detector (SPD-10A; Shimadzu, Japan). Sample filtration was performed using membrane filter (Maidstone, Kent, UK) with pore size 0.45 μm prior to injection. Sunfire C18 column (4.6 mm × 250 mm; Waters, USA) was used in reversed-phase chromatography. The temperature within the chamber was maintained at 30 °C. A 10 μl aliquot of the sample was injected onto a C18 reversed-phase column and the absorbance was detected at 260 nm. Sample concentration was calculated from a standard calibration curve obtained with 6-O-(3,4-dimethoxybenzoyl)-ajugol and 6-O-(
p-dimethoxybenzoyl)-ajugol. 6-O-(3,4-dimethoxybenzoyl)-ajugol and 6-O-(
p-dimethoxybenzoyl)-ajugol were found in TWE at a mean level of 12.29 mg/g and 16.25 mg/g, respectively.
Flow Cytometry
The following monoclonal antibodies (mAbs) from BD Bioscience were used: fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-Cy7, or allophycocyanin (APC)-conjugated anti-CD3ε (clone 145-2C11); PE- or APC-conjugated anti-CD11c (clone HL3); PE-conjugated anti-cytotoxic T lymphocyte-associated protein 4 (CTLA4) (clone UC10-4F10–11); PE-Cy7-conjugated anti-CD11b (clone M1/70); APC-conjugated anti-F4/80 (clone BM8); PE-Cy7- or APC-conjugated anti-CD4 (RM4–5); APC-conjugated anti-CD25 (clone PC61); PE-conjugated anti-MHC II (clone M5/114.15.2); biotin-conjugated anti-CD86 (clone GL1); PE-conjugated anti-tumor necrosis factor α (TNFα) (clone MP6-XT22); PE-conjugated anti-IL6 (clone MP5-20F3); PE-conjugated anti-IL10 (clone JES5-16E3); PE-conjugated anti-IL12p40 (clone C15.6); and PE-conjugated anti-IFNγ (clone XMG1.2). The following mAbs were obtained from eBioscience (San Diego, USA): FITC- or PE-conjugated anti-IL4 (clone BVD6-24G2); FITC- or PE-conjugated anti-IL17A (clone eBio17B7); PE-conjugated anti-inducible nitric oxide synthase (iNOS) (clone CXNFT); and FITC- or PE-conjugated anti-Foxp3 (clone NRRF-30). The following mAb from R&D Systems was used: PE-conjugated anti-arginase-1. To perform surface staining, the cells were harvested and washed twice with 0.5% BSA-containing cold PBS (FACS buffer). To block the Fc receptors, the cells were incubated with anti-CD16/CD32 mAbs on ice for 10 min and were subsequently stained with fluorescence-labeled mAbs.
Intracellular cytokine staining
To perform intracellular staining, splenocytes were incubated with brefeldin A, an intracellular protein transport inhibitor (10 μg/ml), in RPMI medium for 2 h at 37 °C. The cells were stained for cell surface markers, fixed with 1% paraformaldehyde, washed once with cold FACS buffer, and permeabilized with 0.5% saponin. The permeabilized cells were then stained for an additional 30 min at room temperature with the indicated mAbs (FITC-conjugated anti-IL17 or anti-IL4; PE-conjugated anti-IFNγ, anti-IL4, anti-IL10, anti-IL6, anti-IL12, anti-TNFα, anti-iNOS, or anti-arginase-1; FITC- or PE-conjugated isotype control rat IgG mAbs). Fixation and permeabilization were performed using a Foxp3 staining kit (eBioscience) with the indicated mAbs (FITC-conjugated anti-Foxp3; PE-conjugated anti-Foxp3; or FITC- or PE-conjugated isotype control rat IgG mAbs). More than 5000 cells per sample were acquired using a FACSCalibur, and the data were analyzed using the FlowJo software package (Tree Star, Ashland, OR, USA).
Cell isolation by magnetic activated cell sorting (MACS) and culture
Splenic CD4+ T cells were isolated from mice using a MACS system (Miltenyi Biotec, Bergisch Gladbach, Germany), following the manufacturer’s instructions. The purity of CD4+ T cells was >97% after MACS. Mesenteric lymph nodes (MLNs) were aseptically removed, and single-cell suspensions of the MLN were obtained by homogenization and passing through a 70 μm nylon cell strainer. Primary macrophages and the RAW264.7 macrophage cell line were cultured in RPMI 1640 (Gibco BRL, USA) culture media supplemented with 10% FBS, 10 mM HEPES, 2 mM L-glutamine, 100 units/mL penicillin-streptomycin, and 5 mM 2-mercaptoethanol.
In vivo depletion of DCs
In order to deplete CD11c+ DC populations, CD11c-DTR tg recipient mice were intraperitoneally injected with diphtheria toxin (DT) (120 ng/mouse) twice 3 days apart and subsequently mice were sacrificed for experiments 2 days after the second injection.
Determination of cell viability
To examine the effect of TWE on cell viability of RAW264.7 cells, RAW264.7 cells were seeded in 24-well plates at a cell density of 1 × 106 cells/ml (2 × 105 cells/well) and stimulated with either TWE (100, 300, 900, and 2700 μg/ml) or LPS (1 μg/ml) for 12 h at 37 °C in a CO2 incubator. Total cells were washed in PBS, and 100 μl annexin V buffer (BD Pharmingen, San Diego, CA) containing 10 μl 7-amino actinomycin (7-AAD) was added for 15 min at room temperature in the dark. Cells were resuspended in 300 μl annexin V buffer and were analyzed within 1 h by flow cytometry. The relative cell viability (%) was expressed as a percentage (7-AAD negative) relative to the control cells.
Polarization of macrophages with M1 or M2 stimuli
To examine the effect of TWE on the polarization towards the M2 phenotype, RAW264.7 cells (1 × 106 cells) were seeded in 24-well plates and stimulated with either TWE (100, 300, and 900 μg/ml) or vehicle in the presence of IL4 (20 ng/ml) for 12 h at 37 °C in a CO2 incubator. In addition, to test the effect of TWE on M1 polarization, RAW264.7 cells were stimulated with either TWE (100, 300, and 900 μg/ml) or vehicle in the presence of IFNγ (100 ng/ml) for 12 h at 37 °C in a CO2 incubator. LPS (1 μg/ml) was used as positive control in the same culture conditions as above. After treatment, the polarization status of the RAW264.7 cells was determined by evaluating the expression of either iNOS for M1 or arginase-1 for M2 by flow cytometry.
Induction of colonic inflammation
Before DSS treatment, two groups of B6 mice were fed with either TWE-containing or control water for 5 days. Mice in the TWE-treating group were fed with approximately 2 mg of TWE per day. Subsequently, these mice were fed with 3% (w/v) DSS in either TWE-containing or control water ad libitum for 5 days. After 5 days of DSS administration, both groups of mice were given normal control water for 5 days until sacrifice for experiments. To evaluate the clinical symptoms of DSS-induced colitis, the mice were monitored for a change in the percentage of body weight (0, none; 1, 1–10%; 2, 11–20%; 3, >20%), stool consistency (0, normal; 1, loose stool; 2, diarrhea), and bleeding (0, normal; 1, hemoccult positive; 2, gross bleeding) on a daily basis during colitis induction for 10 days. The body weight was expressed as a percentage of weight change for each individual mouse and was calculated relative to the starting body weight on day 0. These data were used to calculate a disease activity index (DAI).
Histology
Distal colonic sections were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 6 μm sections using a microtome (RM 2235, Leica, Germany). The sections were then stained with H&E for the analysis of histological changes. The histological score of each individual mouse was measured as follows: epithelial damage (E), 0 = none; 1 = minimal loss of goblet cells; 2 = extensive loss of goblet cells; 3 = minimal loss of crypts and extensive loss of goblet cells; 4 = extensive loss of crypts; and infiltration (I), 0 = no infiltrate; 1 = infiltrate around the crypt basis; 2 = infiltrate reaching the muscularis mucosa; 3 = extensive infiltration reaching the muscularis mucosa and thickening of the mucosa with abundant edema; 4 = infiltration of the submucosa. The total histological score was calculated as E + I.
Statistical analysis
Statistical significance was determined using the Excel software (Microsoft, USA). To compare two groups, Student’s t-test was performed. *P < 0.05, **P < 0.01, and ***P < 0.001 were considered significant.
Discussion
As public interest in natural products as a source of potential candidates for medicine is increasing, complementary and alternative medicines that use scientifically verified natural products have rapidly grown over the last decades in both the Eastern and Western world [
14]. Bark products from the taheebo tree have been one of the natural products used as a traditional folk remedy for various diseases such as arthritis. Although many people drink taheebo as a tea, TWE has been noted as a source of natural products that can provide a health benefit beyond basic nutrition. Although the in vitro and in vivo anti-inflammatory effects of taheebo have previously reported [
8,
9], the effects of TWE on intestinal immune responses have been poorly understood. Herein, we demonstrate that the oral administration of TWE led to the induction of Th2 and Treg differentiation via alterations in APCs, such as M2 polarization, and resulted in the inhibition of Th1 cells and IFNγ production by CD8
+ T cells in the MLN. Furthermore, by employing the DSS-induced murine model of colitis, we clearly showed that TWE treatment was capable of attenuating the clinical symptoms of colitis in mice, which suggests that TWE could be an excellent reagent as natural products to improve the clinical symptoms of IBD.
Adaptive immune responses are controlled by innate immune cells such as DCs. Depending on the types of cytokines they secrete, DCs can influence the differentiation of CD4
+ T cells that play critical roles in adaptive immunity. Tolerogenic DCs produce immunosuppressive IL10 rather than IL12 and express high levels of costimulatory molecules, such as MHC II and B7, when induced by thymic stromal lymphopoietin (TSLP). Because it has been shown that tolerogenic DCs can prime Th2 and Treg cells and also contribute to preventing excessive inflammation in the mucosal environment [
15], we inferred that TWE could affect the differentiation of CD4
+ T cells towards the Th2 and Treg cells via a DC-mediated pathway. However, our results showed that Th2 and Treg cells could be still induced upon TWE treatment, even in the case of DC depletion. Although DCs are the most potent APCs at initiating adaptive immune responses, it could be possible that other professional APCs such as macrophages are responsible for the modulation of T helper cells by TWE treatment. For example, it has been reported that lamina propria macrophages inhibit Th1 and Th17 differentiation but induce Treg differentiation during the intestinal immune response [
3,
16]. Furthermore, gut-resident macrophages play a central role in oral tolerance and the expansion of Treg cells during the intestinal immune response to orally administered food antigen, which implies that macrophages act as local APCs in the intestine [
17]. Based on the above reports, macrophages rather than DCs might be involved in tolerance in the intestine. Conversely, one recent study has reported that both DCs and macrophages are essential for optimal Th1 polarization during IBD-like pathogenesis in mouse infection models [
18], which implies that DCs and macrophages participate synergically to induce Th2 and Treg polarization upon TWE treatment.
Foxp3
+ Treg cells are known to play a critical role in maintaining tolerance against self-antigens. Recently, some studies have reported the reciprocal relationship between the Treg cell number and the severity of mucosal inflammation. For example, a decreased number of Treg cells was correlated with active IBD in patients [
19], whereas an increased number of Treg cells inhibited the severity of an experimental colitis model [
20,
21]. Thus, our finding that TWE treatment can induce Treg cells can explain how the oral administration of TWE could prevent DSS-induced colitis in mice. In addition, a couple of studies have demonstrated that Th2 polarization using helminths can be an effective way to control mucosal inflammation [
22,
23]. Such alternative therapeutic approaches to induce the mucosal Th2 immune response have been shown to suppress the Th1 response, consequently protecting against colitis in mice. Therefore, the expansion of Th2 and Treg cells by TWE treatment might be one of the key mechanisms that contribute to inhibiting DSS-induced colitis.
Our results suggest that macrophages are among the key players for the TWE-mediated inhibition of colitis. Classically activated M1 (stimulated by IFNγ) and alternatively activated M2 (stimulated by IL4) are two major subsets of macrophage in the intestine [
5]. Recently, it has been reported that the up-regulation of M2 macrophages suppressed the severity of colitis via IL10 receptor signaling [
13] but that the down-regulation of M2 macrophages increased the susceptibility to colitis [
24,
25]. In addition, M2 macrophages have been shown to be involved in Th2 polarization, and the consequent reciprocal IL4 production by Th2 cells can affect the phenotypic alteration of macrophages from M1 to M2 [
26]. Because M2 macrophages were increased in response to TWE stimulation, our findings suggest that the mutual interaction between M2 and Th2 cells might account for the suppressive activities of TWE in colitis. Emerging evidence supports the idea that the interaction between gut microbiota and intestinal immunity plays a central role in maintaining intestinal homeostasis [
2]. Intriguingly, Jang et al. showed that
Lactobacillus spp. belonging to the Firmicutes phylum ameliorated colitis in mice by polarizing M1 macrophages to the M2 phenotype [
27,
28]. Because one previous study demonstrated that the major component of TWE helped to maintain lactic acid-producing beneficial bacteria, such as
Lactobacillus spp. and
Bifidobacterium spp., but preferentially inhibited the growth of harmful bacteria such as
Clostridia and
E. coli [
29], we cannot exclude the possibility that the inhibition of DSS-induced colitis by TWE treatment could be attributed to the distinct effects of TWE on gut microbiota. Further investigations need to be done to address this issue.
The TWE used in this study was prepared by extracting the water-soluble portions from taheebo barks immersed in 4 °C cold water for 24 h, as described in the Methods section. Thus, TWE is not a pure compound and instead consists of several active components, including naphthoquinone, β-lapachol, and polyphenols, which all have anti-inflammatory activities. For example, naphthoquinone induced the decreased production of NO (M1 phenotype) in macrophages stimulated with LPS [
30] and promoted Th2 immune responses (M2 inducer) in conditions of antigen-related lung inflammation [
31,
32]. β-lapachol displayed anti-inflammatory effects in EAE [
33] and polyphenols had potent anti-inflammatory activities [
34,
35]. Therefore, it will be of interest to examine which components of TWE are primarily responsible for its anti-inflammatory activity in colitis.
Considering our results, we might anticipate a couple of concerns about TWE-mediated Th2-deviation. First, there is one type of colitis called UC that is known to be mediated by Th2-dominant immune responses. Unlike Th1-mediated CD, Th2-mediated UC actually became worse after helminth infection [
36]. If TWE prevents colitis via a Th2-polarized manner, it could worsen UC upon TWE treatment. However, although it has been reported that UC pathogenesis is dependent on IL13 production by natural killer T (NKT) cells, we did not find that TWE treatment activated NKT cells to produce cytokines such as IL13 (data not shown). This finding implies that TWE may not have much influence in the pathogenesis of UC, though it should be investigated to confirm whether TWE has any side effects on UC in the future. Second, TWE treatment might increase the induction of Th2-mediated allergic responses, such as atopic dermatitis (AD) and asthma. Recently, very interesting studies have reported on the correlation between allergy and mucosal immune responses. Feeding food allergens with Th2-skewing adjuvants alone did not promote allergic responses in the skin [
37] and acute diarrhea in the intestine [
38] without repeated prior skin sensitization. In addition, intestinal allergic responses were suppressed by orally ingested food allergen before skin sensitization occurred [
37]. Thus, we speculate that orally administered TWE has little influence on causing skin allergic diseases such as AD. In support of our speculation, we found that the oral administration of TWE did not affect the severity of AD in NC/Nga mice (unpublished preliminary data).