Decreased Levels of Spleen Tissue CD4+CD25+Foxp3+ Regulatory T Lymphocytes in Mice Exposed to Berberine + MODEL Available online at www sciencedirect com Journal of Acupuncture and Meridian Studies jo[.]
+ MODEL J Acupunct Meridian Stud 2016; (-): e Available online at www.sciencedirect.com Journal of Acupuncture and Meridian Studies journal homepage: www.jams-kpi.com RESEARCH ARTICLE Decreased Levels of Spleen Tissue CD4DCD25DFoxp3D Regulatory T Lymphocytes in Mice Exposed to Berberine Gholamreza Karimi 1, Mahmoud Mahmoudi 2, Mahdi Balali-Mood 3, Maryam Rahnama 4, Shahrzad Zamani Taghizadeh Rabe 4, Nafiseh Tabasi 4, Bamdad Riahi-Zanjani 3,* Pharmaceutical Research Center, Pharmacy School, Mashhad University of Medical Sciences, Mashhad, Iran Immunology Research Center, Department of Immunology and Allergy, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran Medical Toxicology Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran Immunology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran Available online - - - Received: Jun 18, 2016 Revised: Oct 4, 2016 Accepted: Oct 20, 2016 KEYWORDS berberine; BALB/c mice; spleen; Treg cells Abstract The effects of isoquinoline alkaloid berberine (BER) on spleen tissue CD4ỵCD25ỵFoxp3ỵ regulatory T (Treg) cells were evaluated in BALB/c mice Here, BER was administered daily by intraperitoneal injection at doses of mg/kg and 10 mg/kg for 14 days Following the exposure, mice spleen cellularities, IL-10 production by splenocytes, and spleen Treg/CD4ỵ 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 Treg cells as well as decrease the production of IL-10 by splenocytes in the treated mice (p < 0.05) BER at 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 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited * Corresponding author Tel.: ỵ98 5138002458; fax: ỵ98 5138002467 E-mail: riahib@mums.ac.ir (B Riahi-Zanjani) pISSN 2005-2901 eISSN 2093-8152 http://dx.doi.org/10.1016/j.jams.2016.10.003 Copyright ª 2016, Medical Association of Pharmacopuncture Institute Please cite this article in press as: Karimi G, et al., Decreased Levels of Spleen Tissue CD4ỵCD25ỵFoxp3ỵ Regulatory T Lymphocytes in Mice Exposed to Berberine, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.10.003 + MODEL G Karimi et al 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 Introduction CD4ỵCD25ỵFoxp3ỵ regulatory T (Treg) cells are vital for preserving self-tolerance [1,2] and play critical roles to control immune system homeostasis [2] Growing documents indicate that Treg 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 Treg 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 Treg 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) [9e11], type 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 bloodebrain/intestinal epithelial barriers, and downregulating inflammatory cytokines and antibodies However, little is known about the role of natural compounds in controlling the differentiation and functions of Treg cells Because Treg cells regulate the functions of effector T cells, we hypothesized that some therapeutic herbs may suppress inflammation by promoting Treg cell differentiation, thus inhibiting inflammation and preventing autoimmunity Therefore, in this study, we aimed to evaluate the subacute effects of BER on CD4ỵCD25ỵFoxp3ỵ Treg cells of the BALB/c mice spleen Materials and methods 2.1 Animals Male BALB/c mice (19e21 g; 6e8 weeks old) were purchased from the Pasteur Institute (Tehran, Iran) and housed in standard laboratory conditions (25 2 C and 40e70% 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 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 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 mL 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 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 mg/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 mL of RBC lysing buffer containing 0.83% NH4Cl in 100mM Tris buffer, pH 7.4 and kept at room temperature for minutes The cells were washed thrice with the media and suspended into 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; 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 e70 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 Please cite this article in press as: Karimi G, et al., Decreased Levels of Spleen Tissue CD4ỵCD25ỵFoxp3ỵ Regulatory T Lymphocytes in Mice Exposed to Berberine, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.10.003 + MODEL The Effects of Berberine on Mice spleen Tregs 2.5 Treg and CD4Dcell subtyping Levels (relative percentages) of splenic Treg cells were determined using a FACSCalibur flow cytometer (BD, San Jose, CA) and a mouse Treg cell staining kit (with isotype control; FITC-anti-CD4, PE-Cy5- anti-CD25, and PE-Cy5anti-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 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 CD4ỵCD25ỵFoxP3ỵ Treg 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 CD4ỵ cells in each treatment group’s cell population analyzed as a 100% baseline, the relative percentages of spleen CD4ỵCD25ỵFoxP3ỵ Treg 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 mg/kg did not cause any significant change in these percentages On the other hand, the absolute number of spleen CD4ỵ T cells and their relative percentages based on whole splenocyte levels in the sample set as a 100% value (from both mg and10 mg BER/kgtreated mice) did not show any significant changes compared with the values from vehicle control group The effects of the treatment regimens on PHAstimulated splenocyte IL-10 production are shown in Fig Significant decreases (in comparison with values from cells from vehicle control mice) in IL-10 production Table Figure Effects of subacute exposure to berberine on ex vivo IL-10 production by mouse spleen cells BER Z berberine Value is significantly different versus vehicle control at **p < 0.01 or ***p < 0.001 were noted in cultures of splenocytes from the mice treated with 10 mg/kg dose of BER or with cyclophosphamide 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 Treg cells Surprisingly, our results showed a decreased level of mice spleen Treg 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 Treg 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 Effects of subacute exposure to berberine on mouse spleen CD4ỵ and CD4ỵCD25ỵFoxp3ỵ cells Parameter Spleen cellularity (107) CD4ỵ cell (%) CD4ỵ content (107) CD4ỵCD25ỵFoxp3ỵ cell (Treg; % of CD4ỵ cells) CD4ỵCD25ỵFoxp3ỵ cell (Treg; content [107]) Vehicle control BER mg/kg BER 10 mg/kg Cyclophosphamide 8.52 1.36 27.55 1.97 2.35 0.42 14.23 1.31 8.16 1.33 31.82 2.42 2.59 0.43 16.22 2.72 6.12 0.59 26.70 1.71 1.64 0.12 10.00 1.25* 5.77 1.52* 29.25 5.19 1.68 0.39 9.08 1.04* 0.33 0.02 0.42 0.03 0.16 0.02* 0.15 0.04* Value is significantly different versus vehicle control at *p < 0.05 BER Z berberine Please cite this article in press as: Karimi G, et al., Decreased Levels of Spleen Tissue CD4ỵCD25ỵFoxp3ỵ Regulatory T Lymphocytes in Mice Exposed to Berberine, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.10.003 + MODEL 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 Treg 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 CD25ỵ and FoxP3ỵ expressing Treg cells [26,27] Therefore, a significant decrease in the spleen relative percentage/absolute number of Treg 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 CD4ỵ cells that must be converted to Treg cells Moreover, Niedbala et al reported that NO helped to induce the expansion of Treg cell populations in situ Thus, based on both the sets of findings, it is plausible to believe that the observed decreases in Treg 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 Treg 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 Treg 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 Treg 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 CD4ỵCD25ỵFoxp3ỵ Treg cells [29], whereas in our study, it is shown that BER reduces the number of Treg cells Therefore, the combination of BER with acupuncture (also known as herbal acupuncture) may G Karimi et al 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 Disclosure statement The authors declare that they have no conflicts of interest and no financial interests related to the material of this manuscript Acknowledgments The authors would like to acknowledge the research council and vice chancellor for research and Mashhad University of Medical Sciences, Mashhad, Iran (911174), for their financial support References [1] Aluvihare VR, Kallikourdis M, Betz AG Regulatory T cells mediate maternal tolerance to the fetus Nat Immunol 2004; 5:266e271 [2] Sakaguchi S Naturally arising Foxp3-expressing CD25ỵCD4ỵ regulatory T cells in immunological tolerance to self and nonself Nat Immunol 2005;6:345e352 [3] Stummvoll GH, DiPaolo RJ, Huter EN, Davidson TS, Glass D, Ward JM, et al Th1, Th2, and Th17 effector T cell-induced autoimmune gastritis differs in pathological pattern and in susceptibility to suppression by regulatory T cells J Immunol 2008;181:1908e1916 [4] Sakaguchi S Naturally arising CD4ỵ regulatory t cells for immunologic self-tolerance and negative control of immune responses Annu Rev Immunol 2004;22:531e562 [5] Yang J, Chu Y, Yang X, Gao D, Zhu L, Yang X, et al Th17 and natural Treg cell population dynamics in systemic lupus erythematosus Arthritis Rheumatol 2009;60:1472e1483 [6] Glisic S, Klinker M, Waukau J, Jailwala P, Jana S, Basken J, et al Genetic association of HLA DQB1 with CD4ỵCD25ỵ(high) T-cell apoptosis in type diabetes Genes Immun 2009;10: 334e340 [7] Hasani-Ranjbar S, Larijani B, Abdollahi M A systematic review of the potential herbal sources of future drugs effective in oxidant-related diseases Inflamm Allergy Drug Targets 2009; 8:2e10 [8] Kheir MM, Wang Y, Hua L, Hu J, Li L, Lei F, et al Acute toxicity of berberine and its correlation with the blood concentration in mice Food Chem Toxicol 2010;48:1105e1110 [9] Jiang Y, Wu A, Zhu C, Pi R, Chen S, Liu Y, et al The protective effect of berberine against neuronal damage by inhibiting matrix metalloproteinase-9 and laminin degradation in experimental autoimmune encephalomyelitis Neurol Res 2013;35:360e368 [10] Ma X, Jiang Y, Wu A, Chen X, Pi R, Liu M, et al Berberine attenuates experimental autoimmune encephalomyelitis in C57 BL/6 mice PLoS One 2010;5:e13489 [11] Qin X, Guo BT, Wan B, Fang L, Lu L, Wu L, et al Regulation of Th1 and Th17 cell differentiation and amelioration of experimental autoimmune encephalomyelitis by natural product compound berberine J Immunol 2010;185:1855e1863 [12] Cui G, Qin X, Zhang Y, Gong Z, Ge B, Zang YQ Berberine differentially modulates the activities of ERK, p38 MAPK, and JNK to suppress Th17 and Th1 T cell differentiation in type diabetic mice J Biol Chem 2009;284:28420e28429 Please cite this article in press as: Karimi G, et al., Decreased Levels of Spleen Tissue CD4ỵCD25ỵFoxp3ỵ Regulatory T Lymphocytes in Mice Exposed to Berberine, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.10.003 + MODEL The Effects of Berberine on Mice spleen Tregs [13] Hu Z, Jiao Q, Ding J, Liu F, Liu R, Shan L, et al Berberine induces dendritic cell apoptosis and has therapeutic potential for rheumatoid arthritis Arthritis Rheumatol 2011;63:949e959 [14] Wang XH, Jiang SM, Sun QW Effects of berberine on human rheumatoid arthritis fibroblast-like synoviocytes Exp Biol Med 2011;236:859e866 [15] Marinova EK, Nikolova DB, Popova DN, Gallacher GB, Ivanovska ND Suppression of experimental autoimmune tubulointerstitial nephritis in BALB/c mice by berberine Immunopharmacology 2000;48:9e16 [16] Yan F, Wang L, Shi Y, Cao H, Liu L, Washington MK, et al Berberine promotes recovery of colitis and inhibits inflammatory responses in colonic macrophages and epithelial cells in DSS-treated mice Am J Physiol Gastrointest Liver Physiol 2012;302:G504eG514 [17] Tsang CM, Cheung YC, Lui VW, Yip YL, Zhang G, Lin VW, et al Berberine suppresses tumorigenicity and growth of nasopharyngeal carcinoma cells by inhibiting STAT3 activation induced by tumor associated fibroblasts BMC cancer 2013;13:1e11 [18] Rahnama M, Mahmoudi M, Zamani Taghizadeh Rabe S, BalaliMood M, Karimi G, Tabasi N, et al Evaluation of anti-cancer and immunomodulatory effects of carnosol in a BALB/c WEHI-164 fibrosarcoma model J Immunotoxicol 2015;12: 231e238 [19] Farsam V, Hassan ZM, Zavaran-Hosseini A, Noori S, Mahdavi M, Ranjbar M Antitumor and immunomodulatory properties of artemether and its ability to reduce CD4ỵ CD25ỵ FoxP3ỵ T reg cells in vivo Int Immunopharmacol 2011;11:1802e1818 [20] Abuharfeil N, Sarsour E, Hassuneh M The effect of sodium nitrite on some parameters of the immune system Food Chemical Toxicol 2001;39:119e124 [21] Riahi B, Rafatpanah H, Mahmoudi M, Memar B, Brook A, Tabasi N, et al Immunotoxicity of paraquat after subacute exposure to mice Food Chem Toxicol 2010;48:1627e1631 [22] Li H, Li XL, Zhang M, Xu H, Wang CC, Wang S, et al Berberine ameliorates experimental autoimmune neuritis by suppressing both cellular and humoral immunity Scand J Immunol 2014; 79:12e19 [23] Mahmoudi M, Zamani Taghizadeh Rabe S, Balali-Mood M, Karimi G, Memar B, Rahnama M, et al Immunotoxicity induced in mice by subacute exposure to berberine J Immunotoxicol 2016;13:255e262 [24] Griffiths HR ROS as signalling molecules in T cellseevidence for abnormal redox signalling in the autoimmune disease, rheumatoid arthritis Redox Rep 2005;10:273e280 [25] Los M, Droge W, Stricker K, Baeuerle PA, Schulze-Osthoff K Hydrogen peroxide as a potent activator of T lymphocyte functions Eur J Immunol 1995;25:159e165 [26] Chatenoud L, Bach JF Adaptive human regulatory T cells: myth or reality? Journal Clin Invest 2006;116:2325e2327 [27] Yang XO, Nurieva R, Martinez GJ, Kang HS, Chung Y, Pappu BP, et al Molecular antagonism and plasticity of regulatory and inflammatory T cell programs Immunity 2008;29:44e56 [28] Niedbala W, Cai B, Liu H, Pitman N, Chang L, Liew FY Nitric oxide induces CD4ỵCD25ỵ Foxp3 regulatory T cells from CD4ỵCD25 T cells via p53, IL-2, and OX40 Proceedings of the National Academy of Sciences of the United States of America 2007;104:15478e15483 [29] Kwon Y, Sohn S-H, Lee G, Kim Y, Lee H, Shin M, et al Electroacupuncture Attenuates Ovalbumin-Induced Allergic Asthma via Modulating CD4ỵCD25ỵ Regulatory T Cells EvidBased Compl Alt Med 2012;2012:1e10 Please cite this article in press as: Karimi G, et al., Decreased Levels of Spleen Tissue CD4ỵCD25ỵFoxp3ỵ Regulatory T Lymphocytes in Mice Exposed to Berberine, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.10.003 ... redox state of the immune cells, to elucidate and determine how BER functions to impart the toxicity effects demonstrated here and in other studies Introduction CD4ỵCD25ỵFoxp3ỵ regulatory T (Treg)... further studies are needed to prove this hypothesis Disclosure statement The authors declare that they have no conflicts of interest and no financial interests related to the material of this... suggests that Treg 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