Evaluation of The Number of CD4+ CD25+ FoxP3+TregCells in Normal Mice Exposed to AFB1 and Treated withAged Garlic Extract
Abstract
Objective:
Aflatoxin B1 (AFB1) suppresses the immune system. To decrease such suppressiveeffects on the immune system, a wide range of herbal medicines like garlic areutilized. Biological activities of garlic in vitro and in vivo have also been verified. Our previousstudies demonstrated that aged garlic (dry garlic bulbs preserved in the freezer forsix months at -20˚C) have increased immunostimulator fractions and reduced immunosuppressorfractions. This study focuses on the immunosuppressor activity of AFB1 andimmunostimulator activity of aged garlic extract (AGE) through the evaluation of CD4+CD25+ FoxP+ regulator cell (Treg) counts and the pattern of cytokine production in Balb/cnormal mice.
Materials and Methods:
In this experimental research, AFB1 was separated from Aspergillusflavus (PTCC 5004) by HPLC and AGE prepared using the Mantis method. TheDelayed-Type Hypersensitivity (DTH) test was carried out to determinate the effectivenessof different doses of AGE and AFB1, which can both have an effect on the immune system.Subsequent experiments were carried out on 20 Balb/c mice to estimate the effectsof AGE and AFB1 on the number of Treg cell in 4 groups: 10 µl/kg/day of AFB1 and AGEdiluents were administered for 4 consecutive days to group 1. AFB1, 2. control, 3. AGE+ AFB1 and 4. AGE via intraperitoneal (IP) route, respectively. Mice were sacrificed andsplenocytes harvested and the percentage of splenic Treg cells was measured by flowcytometry analysis. The ELISA method was utilized to measure Cytokine production.
Results:
The findings reveal that AGE increased the level of IFN-λ and IL-4 cytokines producedby splenocytes stimulated by specific tumor antigen and decreased the number ofTreg cells in the spleen (p<0.05). AFB1 increased the number Treg cells in the spleen anddecreased cytokine production (p<0.05). In groups 2 (control) and 4 (AGE) the number ofTreg cells decreased (p value<0.05) whereas in groups 1 and 3 the number of Treg cellsincreased (p<0.05).
Conclusion:
This study indicated that AGE is able to alter the cytokine production in normalmice into a Th1 protective pattern which is beneficial to the immune system in generaland anti-tumor immunity in particular. AFB1 is able to alter the cytokine production into aTh2 protective pattern. Therefore, AGE might be used as herbal medicine with few sideeffects as compared to chemotherapy in treating cancers caused by substances like AFB1.
Introduction
AFB1, a secondary metabolite of the fungusAspergillus flavus, is a hepatocarcinogen invarious animal species, including fish, birds,rodents, and nonhuman primates (1-4). It isalso a suspected human carcinogen and hasbeen shown to play a role in human hepatocarcinoma(5-7). When low dosages of AFB1 arereceived on a daily basis over a long time, thenumber of Treg cells (CD4+ CD25+ FoxP3+) ischanged in the human body (3,4). The authorspresent part of their immunotoxicity study,which aims to complement a larger, collaborativeeffort designed to assess potential biomarkersthat may have a role in the initiationand promotional stages of carcinogenesis (9,10), and which may be of relevance in the "riskassessment" process. In particular, the authorswere interested in the effects of AFB1 on cellsand the mechanisms of cell-mediated immunity(CMI) as this has been implicated as theimmune target (11, 12) with respect to carcinogenesis.AFB1 has unique chemical structures,which cause harm by reacting with the chemicalsin living organisms. The structure of AFB1is shown in figure 1.
The structure of AFB1
As a digestive stimulant, diuretic, and antispasmodic,garlic (Allium sativum, Liliaceae) isused by many people all over the world. Garlichas recently been reported to have antibioticproperties and benefits including antifungal(13) and antibacterial activities (14). It is alsoreported to have hypolipidemic, anti-atherosclerosis(15) and anti-carcinogenesis activities(16). Various research studies have indicatedthat garlic modulates immune responses (16,17).
The authors’ own previous studies have demonstratedthat garlic enhances natural killer(NK) cell activity (17) and T-lymphocyte proliferation(18-20). Garlic extract and a garlicprotein fraction were shown to augment theoxidative burst in peritoneal macrophages ofBalb/c mice (21). Ghazanfari et al. showed thatgarlic extract induces a shift in cytokine patternin Balb/c mice with a Leishmania majorinfection and an upshot in the immune responsewith regard to Th1 (IFN-λ, IL-2) (22, 23). At thesame time, a unique garlic preparation calledAGE has been reported to have a series of pharmacologiceffects including immunomodulation(20). In rodents, AGE and its constituentshave been reported to inhibit the developmentof chemically-induced tumors in the bladder,mammary glands (24, 25), colon, esophagus,lung, skin and stomach (26). Recent studieshave focused on the immunological behavior ofAGE and AFB1 (3). In the present study, the authorsinvestigated the stimulation and suppressionof the immune system of Balb/c mice invitro by AFB1 and AEG.
Materials and Methods
Materials
AFB1 preparation
Toxigenic Aspergillus flavus (PTCC 5004)was purchased from the Iranian Research Organizationfor Science and Technology (IROST)and tested for the generation of AFB1 byslide chromatography. The Aspergillus flavuswas then cultured in Aflatoxin production mediumto generate mycotoxin. AFB1 separatedof culture extract by HPLC method (1, 6).
Preparation of AGE
Fresh garlic bulbs were obtained from Hamadan, a city in western Iran and famous for its freshgarlic. Dry garlic bulbs were peeled and preservedin the freezer (-20˚C) for six months. Aqueousaged garlic extract was prepared using the Mantismethod (20). Garlic bulbs were homogenized withtwo parts of distilled water in a varying blender.The homogenized blend was filtered under vacuumthrough Whatman paper (No. 1) and the filtratewas centrifuged at 3400 g for 30 minutes. The clearsupernatant was collected. Twenty-seven grams ofNH4SO4 were added to one liter of the supernatantand centrifuged at 3400 g for 30 minutes. Theresidue was re-suspended in saline and dialyzedagainst buffer saline. AGE samples were then runon G 50 gel chromatography to measure proteinusing the Bradford assay and evaluated by sodiumdodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) (24).
SDS-PAGE electrophoresis
A 12% (weight/volume) polyacrylamide gelwas utilized to judge the purity of molecules andto estimate the molecular mass compacted withproteins. After electrophoresis, the gel was fixatedwith methanol and acetic acid formaldehyde for 60minutes and stained with coomassie blue.
The sample
The groups of inbred female Balb/c mice age4-6 weeks were purchased from the Pasteur Instituteof Iran. Four groups (five mice in each)were housed in a standard poly-propylene cage.The animals were kept under standard conditions(a cycle of 12/12 hour light/dark and atemperature of 20-22◦C) with free access towater and autoclaved standard mouse chow.Animal care and treatment were conducted inconformity with the guidelines of Animal Careand Research Committee of Tarbiat ModaresUniversity and in compliance with the Guidefor the Care and Use of Laboratory Animals[DHEW Publication No. (NIH) 85-23, Revised1985, Office of Science and Health Reports,DRR/NIH, Bethesda, MD 20205].
Methods
Delayed-type hypersensitivity (DTH) test
To evaluate DTH response, 20 female micewere randomly assigned into groups of five. Onday 0, 0.1 ml of a solution containing 1×108sheep red blood cells (sRBCs, Razi Institute,Tehran, Iran) suspended in PBS were subcutaneouslyinjected in the back of all mice. Threegroups received three doses (30, 15, 10 µl/kg/day) of AFB1 (0.1 ml) via the intraperitoneal(IP) route over a 5-day period The remaininggroup (the control group) received diluent [asolution of ethanol /PBS (40:60)] via the sameroute, dosage, and time interval. On the 5th day,the sensitized animals were subcutaneouslychallenged with 1×108 sRBCs in the left hindfoot pads. The increase in the foot pad thicknesswas measured with a vernier calliper (Mitutoyo,Japan) one,two and three days after thebooster injection of sRBCs. The results werecalculated according to the following formula(18, 19):
Increased DTH=(Left footpad challenged with sRBC-right footpad)×100Right foot padPreparation of the animal model
The first group in this experimental study wastreated with 0.1 ml of AFB1 at a daily dose of10 µl/kg via the IP route. This dose had alreadybeen defined by the DTH test (above) as theoptimal immunostimulatory dose. The secondgroup (negative control) also received the samevolume of diluents PBS via the IP route. Thethird group was treated with 0.1 ml of AFB1 (10µl/kg/day) and 0.1 ml of AGE (20 mg/kg/day)via the IP route (25) and the fourth group with0.1 ml of AGE (20 mg/kg/day) via the sameroute. The treatments were applied once in everydayin 7 day period (26).
Splenocyte cytokine production measurementthrough ELISA method
The isolated spleen mononuclear cells werecultured in 24-well plates (Nunc, Denmark) ina final concentration of 2×106 cells/ml. Threesamples were taken from each mouse in thegroup and each sample was analyzed in triplicate.Twenty microliters of purified tumorantigen and phythohemagglutinin (PHA) wereadded to each separately to stimulate the cells.
After three day incubation at 37˚C and 5% CO2, the supernatants were collected and frozenat -70˚C until analyzed by enzyme-linked immunosorbentmeasurement (ELISA). IFN-λ and IL-4concentrations were measured using the R&DAmerican DuoSet ELISA Development kit. Thetreatment mice were unconscious and medullaryand seperated spleen.
Separation of splenic mononuclear cells (MNC)
The control and treated animals were sacrificedby cervical dislocation on the 13th day;spleens were resected under sterile conditionsand were suspended in PBS. The splenic cellsuspension was RBC- lysed with 0.75% NH4Cland Tris buffer (0.02%) (pH=7.4). The cellswere washed and the single-cell suspension wasprepared in RPMI-1640 (Gibco, 51800-035,Stey cell Technology Company) containing stableglutamine (Cytogen, USA) and 10% heatinactivated fetal calf serum (Gibco, England).To define the viability and density of cells inthe suspension the Trypan blue dye exclusionmethod was used. The cells were counted usinghomocytometer light microscopy. The viabilityof the splenocytes was generally above 95%.After an additional washing, the suspensionwas adjusted to 4×106 cells per milliliters inRPMI-1640 supplemented with 10% FCS, 100µg/mL streptomycin, and 100 IU/mL penicillin(complete RPMI), and kept at 4˚C.
Three-color immunostaining and flow cytometryanalysis
After treating the mice during the 7-day periodas mentioned in 2.2, the MNCs purified from themice spleens were immunostained with the FITCanti-mouse CD4, PE-Cy5 anti-mouse CD25 (BD,eBioscience, USA), and subsequently with PECy5anti-mouse FoxP3+, according to the eBiosciencemouse regulatory Tcell staining kit’s instruction.Three samples were taken from each mouseand each was analyzed in triplicate. The sampleswere analyzed using a FACSCalibur flow cytometerat Tehran University and the results were analyzedwith WinMDI/25 software.
Statistical analyses
In this study each experiment was performed induplicate or triplicate and one-way analysis of variance(ANOVA) or Mann-Whitney non-parametrictests were used to determine the statistical significance(p<0.05) between values in the experimentaland control groups. The data were analyzed usingSPSS software version 16 and the results are expressedas measures of central tendency and dispersion(mean, SE, etc.).
Results
Bradford assay and SDS-PAGE electrophoresis
The results of the Bradford evaluation showedthat the quantity of the effective protein in AGEis 0.27 µg/ml. Gel electrophoresis was performed.The results are shown in figure 2.
Effect of AFB1 on DTH test
In order to estimate the effect of AFB1 on cellmediated immunity (CMI), twenty mice (fourgroups of five mice) were treated with AFB1and PBS as shown in figure 3. For five consecutivedays, the mice were sensitized usingsRBCs, treated with three doses of AFB1 (30,10, 5 µl/Kg/Day in three groups, and PBS: ethanol60:40 in the control group). A challengeusing sRBCs was then performed in the leftfoot pad.
The percentage of foot pad swelling was measuredusing a digital vernier capillier at intervalsof one, two and three days. There was a significant difference in mice treated with a doseof 10 µg/kg/day compared to control mice twoand three days after the foot pad challenge.The steady increase in the pad swelling twoand three days after injection (p value=0.01)showed that a dose of 10 µl/kg/day of AFB1significantly contributed to a greater DTH responseevery one day after the foot pad challengecompared to controls (p=0.01, 0.046 and0.021 respectively). This increase was not seenin other groups. As a consequence, the optimumdose of 10 µl/kg/day of AFB1 was used for therest of the investigations.
The results of delayed type hypersensitivity (DTH)assay. As shown in the graph a significant difference inthe degree of swelling was detected in the group treatedwith 10 µl of AFB1 (p=0.015 and 0.021 at two and threerespectively).
Effect of AFB1 and AGE on splenic CD4 + CD25 +FoxP3 + T cells
Concentration of IFN-λ and IL-4, typical cytokinesfor Th1 and Th2 (Previously you describedTh1 as follows: Th1 ((IFN-λ, IL-2)) andTh2 pattern has not been explained at all.) pattern,in treated and untreated mice was evaluatedby ELISA technique. The results demonstratedthat mice treated with AFB1 showed adecreased level of IFN-λ and an increased levelof IL-4, but in the case of mice treated withAGE, the results showed an increased level ofIFN-λ and a decreased level of IL-4. These differenceswere statistically significant (p<0.05).The results for the IFN-λ and IL-4 concentrationsare shown in figure 4 and figure 5.
Results of ELISA assessment showing the level ofIFN-λ and IL-4 cytokines produced from splenocytes stimulatedby specific tumor antigen. Results show a statisticallysignificant difference between the AGE-treated group andAFB1-treated group (p<0.05).
Results of ELISA assessment showing the level ofIFN-λ and IL-4 cytokines produced from splenocytes stimulatedby specific PHA. Results show a statistically significantdifference between the AGE-treated group and AFB1-treatedgroup (p<0.05).
Effect of AFB1 and AGE on splenic CD4 + CD25 +FoxP3 + T cells
The flow cytometry technique was used to definethe percentage of splenic Treg in Balb/c mice. Asshown in figure 6 (A and B) the results indicate astatistically significant difference between the percentageof splenic Treg cells in the AGE-treatedgroup and the AFB1-treated group and the controlgroup and the AFB1+ AGE-treated group. The percentageof splenic Treg cells in the AGE-treatedgroup was lower than the AFB1-treated group(p=0.049).
Results of flow cytometry measurement: A. Dot plots representing mean percentage of CD25+ and FoxP3+ expressing cellson the CD4+ lymphocyte gate of splenocytes. B. Mean ± SE spleen %Treg cells in treated and control groups. (p value=0.049).
Discussion
AFB1, a secondary metabolite of Aspergillus flavus,has unique chemical structures. These structuresreact and interact with the chemicals in livingorganisms causing harm. Aspergillus flavus is ahepatocarcinogen in humans and can invade tumorcells. If a human receives a small dose of AFB1 ondaily basis over a long period of time, it will resultin carcinoma (1, 5-7). Unfortunately, it is possiblefor humans to receive small doses of AFB1 whileconsuming contaminated food, specifically milk.AFB1 effects on the immune system can be eitherstimulatory or suppressive depending on the criticalexposure windows of dose and time (9, 10).The immune cells in the spleen, such as T-lymphocytesand macrophages, both significant mediatorsof inflammatory responses to tissue damage, havebeen shown to be differently affected by continuousand intermittent exposures to AFB1 (11, 12) .
During the past decade, medical researchers haveincreasingly focused on herbal medicine, especiallyGarlic. Garlic has been consumed for food andmedicinal purposes worldwide for thousands ofyears. Garlic’s beneficial effects on human healthare known to everyone (22, 24). Currently, thegarlic plant itself, as well as its numerous extracts,are commercially available as dietary supplements(20, 22, 24). Epidemiological studies suggest garlicconsumption has preventive effects in some typesof cancer (18). Various researchers have indicatedthat garlic modulates immune responses (17, 18).Previous studies showed that garlic enhances naturalkiller cell (NK) cell activity and T-lymphocyteproliferation (19). Also garlic extract and a garlic protein fraction have been shown to augmentthe oxidative burst in peritoneal macrophages ofBalb/c mice (26). Lau et al. showed that AGE isan efficient candidate as an immune modifier comparedto fresh garlic extract, which maintains thehomeostasis of immune functions (22). In the presentinvestigation, the authors explored the cytotoxityand immunomodulatory activities of AFB1 and AGE in vivo in a mouse model. First, in orderto select the optimal immunostimulatory dose ofAFB1, the researchers performed DTH measurements.The results demonstrated that a dose of 10µl/Kg/Day had a significant effect on the DTH testtwo and three after a foot pad challenge. Based onprevious research a dose of 20 mg/kg/Day of AGEwere used to perform these tests (17, 18, 20). Ourown findings and those of other studies (11, 24-25)found a dose of 10 µl/Kg/Day to be the optimalimmunostimulatory dose and this dose was usedin all further experimentation. Nevertheless,ourresults showed that AGE have effects of supportingof immune system. Figure 5 and figure 6 indicatethat in AFB1-treated groups the level of IFN-λcytokines decreased in the control group whereasin the AGE-treated groups the level of IFN-λ cytokinesincreased. Increased production of IFN-λand decreased IL-4 in turn have other anti-cancereffects including anti-angiogenesis, increased NKactivity and increased immune activity. Thereforeit seems that a continuous cascade of cytokineproduction and cellular activation, by definition,explain the AGE anti-cancer mechanisms and increasedactivation of the immune system. Thisfinding was confirmed in a previous study by Hassanet al. (22) and Noori et al. (23) which indicatedthat AGE is able to enhance the capacity of splenicleukocytes to produce IFN-λ and IL-4 followingmitogenic stimulation. The authors also showedthat AGE could decrease and that AFB1 could increasethe total number of splenic Treg cells in theexperimental group. Although the mechanism forTreg reduction by AGE is not yet fully understood,given that AGE has been shown to decrease tumorgrowth (24), decrease in the number of Tregcells may result in tumor size reduction. Moreover,some studies have reported that AGE can reducethe number of Treg cells via the inhibition of NOproduction "an inducer metabolite for Treg expansion"from macrophage cells (21). This finding isconfirmed in a previous study by Noori et al. (23)which showed that AGE is able to reduce Treg andinhibit tumor growth in vivo but AFB1 is able to increaseTreg and stimulation tumor growth in vivo.
Conclusion
Overall, based on the findings of this research andother studies, the authors measured and showed thespecific immunomodulatory properties of AFB1that are needed to suppress the immune system andthe specific immunomodulatory properties of AGEthat are needed to support the immune system. Immunecells in spleen such as T-lymphocytes andmacrophages, important mediators of inflammatoryresponses to tissue damage and cancer, wereaffected differently by continuous and intermittentexposure to AFB1. This study showed that AGEdecreases the production of Treg cells from splenocytes.Taken together, the findings suggest thatAGE may play a role in attenuating tumor growthby increasing cytokine production and decreasingTreg cells and anti-tumoral cell activation duringcell carcinogenesis. It is possible to speculate thatAGE could be used as a plant drug with few sideeffects during anticancer chemotherapy.
Acknowledgments
The present study received a grant from theParsroos Company. Immunological tests for thisresearch were performed at the Department ofImmunology at Tarbiat Modarres University ofMedical Sciences. The authors wish to expresstheir sincere appreciation for excellent technicalassistance from Mr. Mahdavi and Miss Langroodiin the immunological analysis, Mr. Tebyanian inflow cytometry and Dr. Mostaffaii in helping theauthors with statistical analyses of the obtaineddata. There is no conflict of interest in this article.
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