Decreased Levels of Spleen Tissue CD4⁺CD25⁺Foxp3⁺ Regulatory T Lymphocytes in Mice Exposed to Berberine

Abstract

The effects of isoquinoline alkaloid berberine (BER) on spleen tissue CD4⁺CD25⁺Foxp3⁺ regulatory T (T_reg) cells were evaluated in BALB/c mice. Here, BER was administered daily by intraperitoneal injection at doses of 5 mg/kg and 10 mg/kg for 14 days. Following the exposure, mice spleen cellularities, IL-10 production by splenocytes, and spleen T_reg/CD4⁺ cell profiles were studied in all the test groups of animals. The results showed that a high dose of BER (10 mg/kg) could decrease both the absolute and relative percentages of spleen T_reg cells as well as decrease the production of IL-10 by splenocytes in the treated mice (p < 0.05). BER at 5 mg/kg did not appear to affect any of these parameters. Based on the finding here, it would seem that BER has effective immunostimulatory properties, which contradicts the results from other studies indicating immunosuppressive effects of BER. Depending on the doses of BER used, it might have a broad spectrum from immunosuppressive to stimulatory effects. Further studies, including more doses, are required to better evaluate the effects of this natural product. Mechanistic studies are required, particularly in case of redox state of the immune cells, to elucidate and determine how BER functions to impart the toxicity effects demonstrated here and in other studies.

1. Introduction

CD4⁺CD25⁺Foxp3⁺ regulatory T (T_reg) cells are vital for preserving self-tolerance [1,2] and play critical roles to control immune system homeostasis [2]. Growing documents indicate that T_reg cells are able to inhibit the function of Th1, Th2, Th17, and other effector cells, inhibit inflammation, and prevent autoimmunity [3,4]. Therefore, the lack or dysfunction of T_reg cells often leads to autoimmune diseases, such as systemic lupus erythematosus [5], type I diabetes, and inflammatory bowel disease [2,6]. As a result, it seems that conditions in which T_reg cells are being promoted may consequently alleviate the severity of autoimmune diseases.

Since ancient times, people have looked for cures for illness in nature. The use of natural medicines has intensified in recent times because of the low level of side effects, cost, and efficacy against several human illnesses [7]. Berberine (2,3-methylenedioxy-9,10-dimethoxyproto-berberine chloride; BER) is an isoquinoline alkaloid with a broad spectrum of pharmacologic and biochemical effects and is the main active component in plants, such as Berberis spp. and goldenseal [8]. In traditional medicine, BER has been repeatedly applied as an anticancer, antihypertension, antidiarrhea, antiarrhythmia, and antibiotic agent. Moreover, there are several studies suggesting the possibility of its application for the treatment of autoimmune diseases. For instance, BER has been reported to play a role in the management of immune inflammatory diseases, such as experimental autoimmune encephalomyelitis (an animal model of multiple sclerosis) [9–11], type 1 diabetes [12], rheumatoid arthritis [13,14], experimental autoimmune tubulointerstitial nephritis [15], and colitis [16]. In these studies, BER has been reported to affect by activating dendritic cell apoptosis, inhibiting Th1 and Th17 differentiation, decreasing the permeability of blood—brain/intestinal epithelial barriers, and down-regulating inflammatory cytokines and antibodies. However, little is known about the role of natural compounds in controlling the differentiation and functions of T_reg cells. Because T_reg cells regulate the functions of effector T cells, we hypothesized that some therapeutic herbs may suppress inflammation by promoting T_reg cell differentiation, thus inhibiting inflammation and preventing autoimmunity. Therefore, in this study, we aimed to evaluate the subacute effects of BER on CD4⁺CD25⁺Foxp3⁺ T_reg cells of the BALB/c mice spleen.

2. Materials and methods

2.1. Animals

Male BALB/c mice (19–21 g; 6–8 weeks old) were purchased from the Pasteur Institute (Tehran, Iran) and housed in standard laboratory conditions (25±2°C and 40–70% relative humidity) with a 12-hour day/night lighting cycle throughout the experimental period. The mice were kept in spacious polypropylene cages and provided ad libitum access to standard rodent chow and filtered water. All mice were acclimatized for 1 week prior to usage. The Ethnic Council of Mashhad University of Medical Sciences (Mashhad, Iran) approved all protocols used in this study.

2.2. Doses and treatment schedules

Twenty four mice were randomly divided into four groups (six mice per group). Mice in the BER experimental groups were injected intraperitoneally (IP) daily with a BER solution (98%; Sigma, St. Louis, MO) prepared in dimethyl sulfoxide (DMSO)/phosphate-buffered saline (PBS, pH 7.4) solution (1:20, v/v) in order to receive 5 mg or 10 mg BER/kg/d. Mice in the positive control group received cyclophosphamide (Sigma) at 20 mg/kg/d. Mice in the vehicle control group received only DMSO/PBS injections. No injection volume ever exceeded 100 µL. Mice were treated daily for 14 days. The doses of BER used here were based on the study of Tsang et al, demonstrating the anticarcinogenic effects of doses of 5 mg or 10 mg BER/kg in mice [17]. The dose of cyclophosphamide was selected based on the studies of Farsam et al and Rahnama et al [18,19].

2.3. Preparation of single-cell suspension

The spleen was placed in a small petri dish including 10 mL RPMI-1640 media supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2mM glutamine. The spleen was teased, and the tissue dispersion was centrifuged at 1200 rpm at 4°C for 10 minutes. The supernatant was discarded, and the pellet was resuspended in 3 mL of RBC lysing buffer containing 0.83% NH₄Cl in 100mM Tris buffer, pH 7.4 and kept at room temperature for 3 minutes. The cells were washed thrice with the media and suspended into 1 mL of the media containing 10% fetal bovine serum. Viability of cells was performed using the trypan blue exclusion method [20].

2.4. Measurement of IL-10 production

In brief, aliquots of the isolated splenocytes (2 × 10⁶ cells/well) were placed into each well of 96-well plates, and phytohemagglutinin-A (PHA; 5 mg/mL; final concentration) was subsequently added to stimulate the cells. The cells were incubated for 72 h at 37°C before well supernatants were collected and frozen at −70°C until testing. Commercial ELISA assay kit (R&D Systems, Minneapolis, MN) was used to measure the levels of interleukin IL-10, according to manufacturer’s instructions.

2.5. T_reg and CD4⁺cell subtyping

Levels (relative percentages) of splenic T_reg cells were determined using a FACSCalibur™ flow cytometer (BD, San Jose, CA) and a mouse T_reg cell staining kit (with isotype control; FITC-anti-CD4, PE-Cy5- anti-CD25, and PE-Cy5-anti-Foxp3; eBioscience, San Diego), according to the manufacturer’s protocol. For each sample, a minimum of 10,000 events were captured. The absolute number of each cell type in each spleen was determined by multiplying the differential ratio of the subtypes by the total spleen cell contents [21].

2.6. Statistical analysis

Data were statistically analyzed using Student t test to determine significant differences in the data of the groups. A p value < 0.05 was considered significant. Values were expressed as mean ±S.E.

3. Results

Of all the treatments, only spleen cellularity decreased significantly (p < 0.05) in cyclophosphamide-treated mice compared with that in vehicle-treated mice. The absolute (total) number of spleen tissue CD4⁺CD25⁺FoxP3⁺ T_reg cells associated with 10 mg/kg BER group was significantly decreased compared with the corresponding values in tissues recovered from vehicle control mice (Table 1). Using the total numbers of CD4⁺ cells in each treatment group’s cell population analyzed as a 100% baseline, the relative percentages of spleen CD4⁺CD25⁺FoxP3⁺ T_reg cells in samples from the mice treated with 10 mg BER/kg and with 20 mg cyclophosphamide/kg were also significantly lower than those in tumors from the vehicle control mice. However, BER at the dose of 5 mg/kg did not cause any significant change in these percentages. On the other hand, the absolute number of spleen CD4⁺ T cells and their relative percentages based on whole splenocyte levels in the sample set as a 100% value (from both 5 mg and10 mg BER/kg-treated mice) did not show any significant changes compared with the values from vehicle control group.

Table 1 Effects of subacute exposure to berberine on mouse spleen CD4⁺ and CD4⁺CD25⁺Foxp3⁺ cells.

The effects of the treatment regimens on PHA-stimulated splenocyte IL-10 production are shown in Fig. 1. Significant decreases (in comparison with values from cells from vehicle control mice) in IL-10 production were noted in cultures of splenocytes from the mice treated with 10 mg/kg dose of BER or with cyclophosphamide.

Figure 1

Effects of subacute exposure to berberine on ex vivo IL-10 production by mouse spleen cells. BER = berberine. Value is significantly different versus vehicle control at **p < 0.01 or ***p < 0.001.

4. Discussion

Since recent studies have suggested the possibility of BER usage as a therapeutic agent for some autoimmune diseases, it is possibly thought that BER may be able to increase the level of T_reg cells. Surprisingly, our results showed a decreased level of mice spleen T_reg cells among the 10 mg/kg BER-treated mice group. In addition, mice treatment with BER at a dose of 10 mg/kg/d significantly (p < 0.05) decreased the ex vivo IL-10 production of their splenocytes in response to PHA (p < 0.01) relative to that by control mice cells. Initially, it seems that our results contradict the other researches regarding antiinflammatory/autoimmunity effects of BER because evidence suggests that T_reg cells are capable of inhibiting the function of Th1, Th2, Th17, and other effector cells, inhibiting inflammation, and preventing autoimmunity [3,4]. There is a research in which BER ameliorates experimental autoimmune neuritis by suppressing both cellular and humoral Immunity [22]. In a comprehensive screening of the subacute effects of BER on mice immune system, the results showed that a high dose of BER (10 mg/kg) could suppress both cellular and humoral immune functions in the treated mice. According to that study, it would seem that BER has effective immunosuppressive properties due to its antioxidative effects leading to changes in T and B cell redox states required to act correctly [23].

The equilibrium between oxidizing and reducing agents within T cells controls their redox state. Transient controlled changes in the redox state, such as elevated production of reactive oxygen species (ROS), are critical for signaling and induction of various biological processes. Low levels of ROS are reportedly vital for T cell function [24]. A study has reported that small amounts of ROS are pivotal for inducing transcription of nuclear factor kB and gene expression of cytokines and receptors required for T cell proliferation, highlighting an important role for cellular redox environment on T cell function [25]. On the other hand, inducible T_reg cells originate as CD4 single-positive cells from the thymus, following adequate antigenic stimulation in the presence of cognate antigen and specialized immunoregulatory cytokines, such as IL-10, differentiate into CD25⁺ and FoxP3+ expressing T_reg cells [26,27]. Therefore, a significant decrease in the spleen relative percentage/absolute number of T_reg cells as well as in IL-10 level in supernatant of splenocytes cultivation may be due to the antioxidative effects of BER in neutralizing ROS and consequently dysfunction of CD4⁺ cells that must be converted to T_reg cells. Moreover, Niedbala et al reported that NO helped to induce the expansion of T_reg cell populations in situ. Thus, based on both the sets of findings, it is plausible to believe that the observed decreases in T_reg cell levels in 10mg/kg BER mice here might be associated with an inhibition of NO production possibly as a result of the antioxidative effects of BER [28]. Of course, further studies are needed to investigate those aforementioned hypotheses.

Finally, the present study showed that BER at a high dose (10 mg/kg/d for 14 days) imparted an inhibitory effect on the mouse spleen T_reg cells, whereas BER at a lower dose (5 mg/kg/d for 14 days) did not seem to affect this parameter. Further studies, including far more doses, are required to better evaluate the effects of this natural product. In addition, mechanistic studies are needed to elucidate exactly how BER functions to impart T_reg toxicity demonstrated here. Further studies should be carried out to determine the levels of ROS generated by mice spleen T lymphocytes after BER administration and changes in the activation of pathways in which ROS operate as a signaling mediator for converting these cells to T_reg cells. Additionally, further researches should be performed concerning BER to develop its potential use as an effective immunomodulator or coadjuvant in the treatment of diseases originated from immune system dysfunction. As a recommendation, in future studies, BER could be considered as a good choice to be combined with acupuncture technique since acupuncture has been demonstrated to enhance the function and number of CD4⁺CD25⁺Foxp3⁺ T_reg cells [29], whereas in our study, it is shown that BER reduces the number of T_reg cells. Therefore, the combination of BER with acupuncture (also known as herbal acupuncture) may be considered as a good strategy to modulate and shift immune system to our desirable direction for the treatment of some diseases. However, further studies are needed to prove this hypothesis.