EMSA Eritin Drives Expansion of Regulatory T Cells and Promotes T Cells Differentiation in Irradiated Mice

Abstract

Sublethal irradiation therapy in cancer treatment causes generalized immunosuppression, which results in a range of DNA damage. We examined the significance of a polyherbal medicine called “EMSA Eritin” on immunological responses in sublethally irradiated mice focusing on the involvement of Treg, naïve T cell, and also the development and differentiation of T cells in thymus. Normal BALB/c mice were sublethally irradiated with dose of 600 rad. The irradiated mice were then orally administered by EMSA Eritin once a day at different doses: 1.04, 3.12, 9.37 mg/g body weight. The treatment was performed for 14 days. On day 15, immunological responses were observed by analyzing the status of Treg and differentiation of T cells in thymus. The administration of EMSA Eritin to irradiated mice resulted in a significant increase of pre T cells, Treg cells, and naïve T cells, which in general could maintain and normalize healthy condition in mice.

Ionizing irradiation has often been used in bone marrow transplants and also for cancer treatment. ^1,2 One of the consequences of irradiation therapy is immune suppression. Immune suppression causes a serious and immediate concern for patients of sublethal irradiation exposure, which in turn becomes apparent in the composition of peripheral lymphoid populations and in the long term causes impairment of immune responses. ^3,4 Patients with high-dose irradiation therapy showed depletion in all of naïve T cell subsets, with normal CD4⁺, and increased CD8⁺ memory T cell populations. ⁴ Thus, γ-radiation exposure could potentially cause an inhibition or depletion of adaptive Tregs, which could address the immunologic lesions.

Herbal medicine is the principle health care resource of people all over the world. The importance of herbal medicine has led to the discovery and adoption of plant extracts that were commonly used in traditional medicine, as an alternative source of remedy. EMSA Eritin is a polyherbal that consists of red rice, soybeans, and coconut water. EMSA Eritin has a broad range of important contents such as genistein, cytokinin, nicotinic acid, pantothenic acid, biotin, riboflavin, folic acid, thiamine B₁, vitamin C, pyridoxine, daidzein, glycitein, phenolic acids, and anthocyanins. ^{5 –8}

Here, we reported a novel observation that oral gavage of polyherbal medicine called EMSA Eritin resulted in modulating Treg cells and also normalized T cells development and differentiation in mice after irradiation with a dose of 600 rad based on the level of regulatory T cells, naïve T cells in spleen, and also CD4⁺CD8⁺ T cells in thymus.

Materials and Methods

Completely random factorial experiments were conducted with 4 replications in 6 groups: the healthy control group, the irradiated group, the irradiated followed by Hemapo Epoetin alfa group, and the irradiated group followed by of EMSA Eritin administration with 3 different doses, D1, D2, and D3.

Mice

Balb/c mice arrived at 4 to 6 weeks of age and were quarantined on arrival. The mice were between 7 and 8 weeks old at the beginning of the study. In this experiment, we used normal Balb/c mice that were obtained from Gadjah Mada University, Yogyakarta, Indonesia, and maintained in a pathogen-free facility. The experimental protocol was approved by ethical clearance from the Research Ethics Committee (Animal Care and Use Committee), University of Brawijaya (No. 255-KEP-UB).

Total Body Irradiation

Total body irradiation was performed using Co-60 Teletherapy NPICEM with a dose of 6 Gy. Mice were placed in a box of size 10 × 10 cm², and each box contained 5 mice. The dose was measured at the midpoint within the field for 50 × 50 mm² at 80 cm SSD (source to surface distance) in the machine GWXJ80 of NPICEM China installed at Dr Saiful Anwar Hospital, Malang. The gantry and collimator angles of the ⁶⁰Co teletherapy units were kept at 0° for these measurements.

EMSA Eritin and Hemapo Epoetin Alfa Treatment

Determination of EMSA Eritin doses for in vivo experiments were based on the human consumption of 60 kg of body weight that consumes as much as 15 g of EMSA Eritin. Polyherbal EMSA Eritin was grouped into 3 doses: D1 (1.04 mg/g body weight), D2 (3.125 mg/g body weight), and D3 (9.37 mg/g body weight). We also used EPO or Hemapo Epoetin alfa 0.21 mg/g body weight and injected intraperitoneally twice a week. EMSA Eritin was dissolved in distilled water until 1 mL and administrated in mice by oral gavage (force-feeding) in 24 hours after radiation exposure once a day for 2 weeks.

Lymphocyte Isolation and Flow Cytometry Analysis

The mice were sectioned on day 15. Spleen were isolated and pressed clockwise with a syringe base. Aggregates were separated by gentle pipetting, and debris was discarded by passaging the suspension through a cell strainer (100-μm Nylon). The suspension in propylene was added with phosphate-buffered saline up to 10 mL and then centrifuged at 1500 rpm, 4°C for 5 minutes. The supernatant was removed and the pellet was resuspended with 1 mL of sterile phosphate-buffered saline, which was co-incubated with monoclonal antibodies: fluorescein isothiocyanate anti-mouse CD-4 (clone RM4-4), phycoerythrin (PE) anti-mouse CD-25 (clone PC61), PE/Cy5 anti-mouse CD62L (clone MEL-14), PE anti-mouse CD-8a (clone 53-6.7) for 15 minutes. Antibodies were purchased from BioLegend, Inc (San Diego, CA). The pellet was then resuspended in 500 μL phosphate-buffered saline and assessed via a BD FACS Calibur flow cytometer (BD Biosciences, San Jose, CA). The data were then processed using the BD CellQuest Pro software (BD Biosciences).

Statistical Analysis

One-way ANOVA was used to analyze the data. The differences between groups were considered significant at P < .05. All results are presented as the mean ± standard deviation values of 4 mice in each group. This was followed by a post hoc Tukey’s honestly significant difference test to determine major changes and differences among the CD4⁺CD25⁺CD62L⁺ T cells, CD4⁺CD62L⁺ and CD4⁺CD8⁺ T cells relative to the density mean values.

Results and Discussion

EMSA Eritin Treatment Augments T_reg Expression In Vivo

A previous study revealed that BALB/c mice are very sensitive to high-dose irradiation. When BALB/c mice receive irradiation at a dose higher than 880 rad, they will develop considerable radiation-induced sickness (inappetence, diarrhea, lethargy) and may lead to death. To avoid unnecessary irradiation-induced deaths in BALB/c mice, milder irradiation doses should be used. ^{9 –12} The irradiation dose of 600 rad was considered a sublethal dose in BALB/c mice. To determine whether the mechanism by which EMSA Eritin protects mice from the deleterious effect caused by irradiation might involve induction or enhancement of T_reg, we analyzed the relative number of CD4⁺CD25⁺CD62L⁺ T cells by flow cytometry. The flow cytometry analysis of the spleen showed significant depletion in the relative number of CD25⁺CD62L⁺ T cells (13.08%; P > .05) among CD4⁺ cells during the irradiation treatment compared with the controls (25.06%). Treatment with Hemapo Epoetin alfa showed increasing number (19.17%) compared with irradiated mice. Furthermore, the number of CD4⁺CD25⁺CD62L⁺ T cells in irradiated mice with EMSA Eritin treatment was markedly increased from D1 (lower dose) to D3 (higher dose), respectively (15.03%, 23.94%, 69.68%; Figure 1), compared with irradiated mice. The highest dose of EMSA Eritin (D3) was considered as a therapeutic dose that correlated to its ability to significantly increase the level of CD25⁺CD62L⁺ cells among CD4⁺ cells that protected mice from the deleterious effect caused by irradiation.

Figure 1.

Prevalence of CD4⁺CD25⁺CD62L⁺ regulatory T cells is elevated in irradiated mice following EMSA Eritin administration. Sublethally irradiated (6 Gy) mice were orally administered with EMSA Eritin for 15 days with different concentration as indicated or EPO injection (according to company protocol). Spleen cells (2 × 106) were obtained from the treated mice, and then subjected to cell surface staining with anti-CD4, anti-CD25, and anti-CD62L antibodies and analyzed by flow cytometry. Controls are normal-type mice without manipulation. Percentage of spleen cells expressing CD4⁺CD25⁺CD62L⁺ are presented in the panels. Data are mean ± standard deviation values of 4 mice in each group.

EMSA Eritin Modulates T-Cell Development and Differentiation After Irradiation

The development and differentiation of pre-T cell into CD4 and CD8 T cells was inhibited when BALB/c mice received irradiation at a dose of 600 rad. Examination of T-cell development and differentiation showed marked reduction in the number of CD4⁺CD8⁻ cells in splenic cells of mice after 600 rad irradiation compared to healthy control (3.35% vs 12.32%, P < .05). Furthermore, the number of CD4⁺CD8⁻ cells in the irradiated mice with EMSA Eritin was markedly increased from D1 to D3, respectively (6.56%, 17.43%, 28.54%), compared with irradiated mice and mice with Hemapo Epoetin alfa treatment (4.23%). On the other hand, EMSA Eritin also could increase the level of CD4⁻CD8⁺ from D1 to D3, respectively (1.76%, 2.45%, 3.35%), compared with irradiated mice and mice with Hemapo Epoetin alfa treatment (0.51%; Figure 2). These data suggested the importance of EMSA Eritin to trigger CD4 and CD8 T-cell development. In this study, we demonstrated an increase in T-cell maturation in line with the dose of EMSA Eritin.

Figure 2.

The development and differentiation of CD4 T cells and CD8 T cells in thymus is increased after EMSA Eritin treatment. Representative bars show the calculation of the number of CD4 T cells and CD8 T cells in thymus. CD4 T cells and CD8 T cells in thymus was increased in all dose of EMSA Eritin treatments compared with EPO treatment and irradiated mice. Thymic cells (2 × 106) were obtained from the treated mice and then subjected to cell surface staining with anti-CD4 and anti-CD8 antibodies and analyzed by flow cytometry. Controls are normal type mice without manipulation. Data are mean ± standard deviation values of 4 mice in each group.

EMSA Eritin Prevents T Cells Differentiation Into Effector Phase by Upregulating CD62L Expression

CD62L molecule is essential to mediate homing T cells in peripheral lymphoid organs. This study revealed that the CD62L molecule is depleted in irradiated mice compared with normal mice (13.33% vs 58%). The addition of Hemapo Epoetin alfa could not help increase the expression of CD62L compared with irradiated mice (17.80%), but EMSA Eritin is able to trigger the expression of CD62L in all doses (37.8%, 35.66%, 31.83%; Figure 3). The L-selectin adhesion molecule (CD62L) acts as a mediator in lymphocyte recirculation through peripheral lymphoid tissues and recruits leukocytes to inflammatory sites, thereby playing a fundamental role in immune homeostasis.

Figure 3.

EMSA Eritin prevents T cells differentiation into effector phase. Sublethally irradiated (600 rad) mice were orally administered with EMSA Eritin for 15 days with different concentration as indicated or EPO injection (according to company protocol). Spleen cells (2 × 106) were obtained from the treated mice and then subjected to cell surface staining with anti-CD4 and anti-CD62L antibodies and analyzed by flow cytometry. Controls are normal-type mice without manipulation. Percentage of spleen cells positively stained with CD4 and CD62L (CD4⁺CD62⁺L) are presented in the panels. Data are mean ± standard deviation values of 4 mice in each group.

Discussion

CD4⁺CD25⁺ regulatory T cells (Tregs) consist of 5% to 10% of the circulating CD4⁺ T cell population and powerfully suppress immune responses. Various studies reported that these cells play a significant role in a host of clinically relevant areas, such as self-tolerance, transplantation, allergy, and tumor/microbial immunity. ¹³ In the present work, we have identified that EMSA Eritin act as a protolerance regulator of the Treg compartment. The ability of EMSA Eritin to protect mice from deleterious effects of irradiation has been showed with its ability to augment CD25⁺CD62L⁺ expression in the CD4⁺ T cell compartment.

Sublethally total body irradiation significantly decreases the lymphocyte pool, and the various lymphocyte subsets have different sensitivities to irradiation including Th1/Th2 balance. ^{14 –16} Recently, it was reported that immunosuppressive CD4⁺CD25⁺ Treg cells displayed different radiation sensitivities compared with CD4⁺CD25⁻ T effector cells in vivo. ^{17 –20} In stabilizing immune regulation, the survival of Tregs relative to T effectors following sublethal irradiation could have significant effects on the composition and function of T cell populations for a prolonged time after exposure. ^{21 –24} Overall, immune cells are sensitive to radiation-induced damage and readily induce apoptosis in response to small doses of radiation. Gamma irradiation causes apoptosis in human monocyte derived DCs. Monocytes are activated through the presence of molecular signals provided by structures of immunopathogenic agents, or inflammatory mediators and chemotactic factors such as cytokine or chemokine released by adjacent cells. Adhesion molecules on leukocytes and endothelial cells play an important role in recruitment of leukocytes to the inflammation site. This process is promoted by vasodilatation and by expression of selectins. The selectin CD62L (L-selectin) is expressed on leukocytes and is crucial in the initial attachment between leukocytes and endothelial cells. ²⁴

The isoflavones found in soybean, genistein, daidzein, and glycitein (and their respective glycosides) have been reported to have anti-inflammatory properties. Chacko et al ²⁵ reported that genistein has been shown to have a potentially anti-inflammatory action through inhibiting monocyte adhesion to cytokine-activated endothelial cells. The potential beneficial effects of genistein in the human population are supported by similar observations in experimental animals. In this study, we have found that EMSA Eritin could potentially increase the level of Treg and naïve T cells in spleen. However, this study has an opposite result with Yellayi et al, ²⁶ who had revealed that genistein, an estrogenic soy isoflavone, causes thymic atrophy and inhibits both humoral and cell-mediated immunity in mice; these effects are only partially inhibited by anti-estrogens. We could speculate that this different result is caused by the difference in the use of compounds as EMSA Eritin comprises various compounds from soybean, coconut water, and red rice, which help stabilize the immune function in mice after sublethal γ-irradiation.

The mechanisms of EMSA Eritin in increasing T-cells maturation in thymus is unknown, but one of the hypothetical scenarios attributed this phenomenon to improved T-cell development and differentiation due to the capacity of antioxidant in EMSA Eritin in neutralizing free radicals after sublethal irradiation. ^27,28 In this study, we demonstrated an increase in T-cell maturation in line with the dose of EMSA Eritin. Another study found that soy isoflavonoids may inhibit the diffusion of free radicals and lower the reaction kinetics of free radicals that can stabilize the structure of cell membranes. ²⁹ EMSA Eritin also contains red rice and coconut water extract. The pigment in red rice has the potential to act as a free radical scavenger, including radicals of oxygen and nitrogen derivatives. ³⁰ Coconut water contains various cytokinins such as kinetin and trans-zeatin. Kinetin in coconut water can reduce the formation of reactive oxygen species. Kinetin was shown to act as a strong antioxidant both in vitro and in vitro. Moreover, kinetin riboside also significantly inhibits the growth of human hepatoma and mammary carcinoma. ³¹

Conclusion

In summary, the results of the present study demonstrated that EMSA Eritin administration after irradiation with a dose of 600 rad plays a role in increasing the survival and promoting the expansion of Treg cells and also normalized T cells development and differentiation. However, EMSA Eritin may be a protective agent to minimize or prevent damage from irradiation. Although our investigations might provide some information basis for the possible use of EMSA Eritin as a radioprotector of the immune system, further studies are necessary to determine the mechanism of its radioprotective action.