Evaluation of The Number of CD4+ CD25+ FoxP3+ Treg
Cells in Normal Mice Exposed to AFB1 and Treated with
Aged Garlic Extract
Aflatoxin B1 (AFB1) suppresses the immune system. To decrease such suppressive
effects on the immune system, a wide range of herbal medicines like garlic are
utilized. Biological activities of garlic
Materials and Methods:
In this experimental research, AFB1 was separated from Aspergillus
flavus (PTCC 5004) by HPLC and AGE prepared using the Mantis method. The
Delayed-Type Hypersensitivity (DTH) test was carried out to determinate the effectiveness
of 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 effects
of AGE and AFB1 on the number of
The findings reveal that AGE increased the level of IFN-λ and IL-4 cytokines produced
by splenocytes stimulated by specific tumor antigen and decreased the number of
This study indicated that AGE is able to alter the cytokine production in normal mice into a Th1 protective pattern which is beneficial to the immune system in general and anti-tumor immunity in particular. AFB1 is able to alter the cytokine production into a Th2 protective pattern. Therefore, AGE might be used as herbal medicine with few side effects as compared to chemotherapy in treating cancers caused by substances like AFB1.
AFB1, a secondary metabolite of the fungus
As a digestive stimulant, diuretic, and antispasmodic, garlic (Allium sativum, Liliaceae) is used by many people all over the world. Garlic has recently been reported to have antibiotic properties and benefits including antifungal (13) and antibacterial activities (14). It is also reported to have hypolipidemic, anti-atherosclerosis (15) and anti-carcinogenesis activities (16). Various research studies have indicated that garlic modulates immune responses (16, 17).
The authors’ own previous studies have demonstrated that garlic enhances natural killer (NK) cell activity (17) and T-lymphocyte proliferation (18-20). Garlic extract and a garlic protein fraction were shown to augment the oxidative burst in peritoneal macrophages of Balb/c mice (21). Ghazanfari et al. showed that garlic extract induces a shift in cytokine pattern in Balb/c mice with a Leishmania major infection and an upshot in the immune response with regard to Th1 (IFN-λ, IL-2) (22, 23). At the same time, a unique garlic preparation called AGE has been reported to have a series of pharmacologic effects including immunomodulation (20). In rodents, AGE and its constituents have been reported to inhibit the development of chemically-induced tumors in the bladder, mammary glands (24, 25), colon, esophagus, lung, skin and stomach (26). Recent studies have focused on the immunological behavior of AGE and AFB1 (3). In the present study, the authors investigated the stimulation and suppression of the immune system of Balb/c mice in vitro by AFB1 and AEG.
Materials and Methods
Preparation of AGE
Fresh garlic bulbs were obtained from Hamadan, a city in western Iran and famous for its fresh garlic. Dry garlic bulbs were peeled and preserved in the freezer (-20˚C) for six months. Aqueous aged garlic extract was prepared using the Mantis method (20). Garlic bulbs were homogenized with two parts of distilled water in a varying blender. The homogenized blend was filtered under vacuum through Whatman paper (No. 1) and the filtrate was centrifuged at 3400 g for 30 minutes. The clear supernatant was collected. Twenty-seven grams of NH4SO4 were added to one liter of the supernatant and centrifuged at 3400 g for 30 minutes. The residue was re-suspended in saline and dialyzed against buffer saline. AGE samples were then run on G 50 gel chromatography to measure protein using the Bradford assay and evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (24).
A 12% (weight/volume) polyacrylamide gel was utilized to judge the purity of molecules and to estimate the molecular mass compacted with proteins. After electrophoresis, the gel was fixated with methanol and acetic acid formaldehyde for 60 minutes and stained with coomassie blue.
The groups of inbred female Balb/c mice age 4-6 weeks were purchased from the Pasteur Institute of 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 a temperature of 20-22◦C) with free access to water and autoclaved standard mouse chow. Animal care and treatment were conducted in conformity with the guidelines of Animal Care and Research Committee of Tarbiat Modares University and in compliance with the Guide for the Care and Use of Laboratory Animals [DHEW Publication No. (NIH) 85-23, Revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20205].
Delayed-type hypersensitivity (DTH) test
To evaluate DTH response, 20 female mice were randomly assigned into groups of five. On day 0, 0.1 ml of a solution containing 1×108 sheep red blood cells (sRBCs, Razi Institute, Tehran, Iran) suspended in PBS were subcutaneously injected in the back of all mice. Three groups 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 remaining group (the control group) received diluent [a solution of ethanol /PBS (40:60)] via the same route, dosage, and time interval. On the 5th day, the sensitized animals were subcutaneously challenged with 1×108 sRBCs in the left hind foot pads. The increase in the foot pad thickness was measured with a vernier calliper (Mitutoyo, Japan) one,two and three days after the booster injection of sRBCs. The results were calculated according to the following formula (18, 19):
Preparation of the animal model
The first group in this experimental study was treated with 0.1 ml of AFB1 at a daily dose of 10 µl/kg via the IP route. This dose had already been defined by the DTH test (above) as the optimal immunostimulatory dose. The second group (negative control) also received the same volume of diluents PBS via the IP route. The third group was treated with 0.1 ml of AFB1 (10 µl/kg/day) and 0.1 ml of AGE (20 mg/kg/day) via the IP route (25) and the fourth group with 0.1 ml of AGE (20 mg/kg/day) via the same route. The treatments were applied once in everyday in 7 day period (26).
Splenocyte cytokine production measurement through ELISA method
The isolated spleen mononuclear cells were cultured in 24-well plates (Nunc, Denmark) in a final concentration of 2×106 cells/ml. Three samples were taken from each mouse in the group and each sample was analyzed in triplicate. Twenty microliters of puriﬁed tumor antigen and phythohemagglutinin (PHA) were added to each separately to stimulate the cells.
After three day incubation at 37˚C and 5% CO2 , the supernatants were collected and frozen at -70˚C until analyzed by enzyme-linked immunosorbent measurement (ELISA). IFN-λ and IL-4 concentrations were measured using the R&D American DuoSet ELISA Development kit. The treatment mice were unconscious and medullary and seperated spleen.
Separation of splenic mononuclear cells (MNC)
The control and treated animals were sacrificed by cervical dislocation on the 13th day; spleens were resected under sterile conditions and were suspended in PBS. The splenic cell suspension was RBC- lysed with 0.75% NH4Cl and Tris buffer (0.02%) (pH=7.4). The cells were washed and the single-cell suspension was prepared in RPMI-1640 (Gibco, 51800-035, Stey cell Technology Company) containing stable glutamine (Cytogen, USA) and 10% heat inactivated fetal calf serum (Gibco, England). To define the viability and density of cells in the suspension the Trypan blue dye exclusion method was used. The cells were counted using homocytometer light microscopy. The viability of the splenocytes was generally above 95%. After an additional washing, the suspension was adjusted to 4×106 cells per milliliters in RPMI-1640 supplemented with 10% FCS, 100 µg/mL streptomycin, and 100 IU/mL penicillin (complete RPMI), and kept at 4˚C.
Three-color immunostaining and flow cytometry analysis
After treating the mice during the 7-day period as mentioned in 2.2, the MNCs purified from the mice spleens were immunostained with the FITC anti-mouse CD4, PE-Cy5 anti-mouse CD25 (BD, eBioscience, USA), and subsequently with PECy5 anti-mouse FoxP3+, according to the eBioscience mouse regulatory Tcell staining kit’s instruction. Three samples were taken from each mouse and each was analyzed in triplicate. The samples were analyzed using a FACSCalibur flow cytometer at Tehran University and the results were analyzed with WinMDI/25 software.
In this study each experiment was performed in duplicate or triplicate and one-way analysis of variance (ANOVA) or Mann-Whitney non-parametric tests were used to determine the statistical significance (p<0.05) between values in the experimental and control groups. The data were analyzed using SPSS software version 16 and the results are expressed as measures of central tendency and dispersion (mean, SE, etc.).
Effect of AFB1 on DTH test
In order to estimate the effect of AFB1 on cell mediated immunity (CMI), twenty mice (four groups of five mice) were treated with AFB1 and PBS as shown in figure 3. For five consecutive days, the mice were sensitized using sRBCs, treated with three doses of AFB1 (30, 10, 5 µl/Kg/Day in three groups, and PBS: ethanol 60:40 in the control group). A challenge using sRBCs was then performed in the left foot pad.
The percentage of foot pad swelling was measured using a digital vernier capillier at intervals of one, two and three days. There was a significant difference in mice treated with a dose of 10 µg/kg/day compared to control mice two and three days after the foot pad challenge. The steady increase in the pad swelling two and three days after injection (p value=0.01) showed that a dose of 10 µl/kg/day of AFB1 significantly contributed to a greater DTH response every one day after the foot pad challenge compared to controls (p=0.01, 0.046 and 0.021 respectively). This increase was not seen in other groups. As a consequence, the optimum dose of 10 µl/kg/day of AFB1 was used for the rest of the investigations.
Effect of AFB1 and AGE on splenic CD4 + CD25 + FoxP3 + T cells
Concentration of IFN-λ and IL-4, typical cytokines for Th1 and Th2 (Previously you described Th1 as follows: Th1 ((IFN-λ, IL-2)) and Th2 pattern has not been explained at all.) pattern, in treated and untreated mice was evaluated by ELISA technique. The results demonstrated that mice treated with AFB1 showed a decreased level of IFN-λ and an increased level of IL-4, but in the case of mice treated with AGE, the results showed an increased level of IFN-λ and a decreased level of IL-4. These differences were statistically significant (p<0.05). The results for the IFN-λ and IL-4 concentrations are shown in figure 4 and figure 5.
Effect of AFB1 and AGE on splenic CD4 + CD25 + FoxP3 + T cells
The flow cytometry technique was used to define
the percentage of splenic
AFB1, a secondary metabolite of
During the past decade, medical researchers have
increasingly focused on herbal medicine, especially
Garlic. Garlic has been consumed for food and
medicinal purposes worldwide for thousands of
years. Garlic’s beneficial effects on human health
are known to everyone (22, 24). Currently, the
garlic plant itself, as well as its numerous extracts,
are commercially available as dietary supplements
(20, 22, 24). Epidemiological studies suggest garlic
consumption has preventive effects in some types
of cancer (18). Various researchers have indicated
that garlic modulates immune responses (17, 18).
Previous studies showed that garlic enhances natural
killer cell (NK) cell activity and T-lymphocyte
proliferation (19). Also garlic extract and a garlic protein fraction have been shown to augment
the oxidative burst in peritoneal macrophages of
Balb/c mice (26). Lau et al. showed that AGE is
an efficient candidate as an immune modifier compared
to fresh garlic extract, which maintains the
homeostasis of immune functions (22). In the present
investigation, the authors explored the cytotoxity
and immunomodulatory activities of AFB1 and AGE
Overall, based on the findings of this research and
other studies, the authors measured and showed the
specific immunomodulatory properties of AFB1
that are needed to suppress the immune system and
the specific immunomodulatory properties of AGE
that are needed to support the immune system. Immune
cells in spleen such as T-lymphocytes and
macrophages, important mediators of inflammatory
responses to tissue damage and cancer, were
affected differently by continuous and intermittent
exposure to AFB1. This study showed that AGE
decreases the production of
The present study received a grant from the Parsroos Company. Immunological tests for this research were performed at the Department of Immunology at Tarbiat Modarres University of Medical Sciences. The authors wish to express their sincere appreciation for excellent technical assistance from Mr. Mahdavi and Miss Langroodi in the immunological analysis, Mr. Tebyanian in flow cytometry and Dr. Mostaffaii in helping the authors with statistical analyses of the obtained data. There is no conflict of interest in this article.