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Immunomodulatory Effect of Fermented Vinegar on Cyclophosphamide-induced Immunosuppression Model

Hong Sun Hwa, Minjoo Kwon, Hak Yong Lee, Young Mi Park, Dong-Yeop Shin, Jin Seb Choi, Min-Jung Kim, Hye Jeong Yang*, Na-Rae Shin*

Korea Food Research Institute, 245, Nongsaengmyeong-ro, Iseo-myeon, Wanju-gun, Jeollabuk-do 55365, Republic of KoreaINVIVO Co. Ltd. Daehak-ro 121, Nonsan 32992, Republic of Korea

Abstract: Immunity is the defense against external invasion and breakdown of the immune system that causes various diseases. In this study, the immunomodulatory function of brown rice fermented vinegar (FV) was investigated using a cyclophosphamide (CP)-induced immunosuppression model. In the in vitro study, splenocytes were treated with CP to induce immunosuppression and to measure splenocyte proliferation and natural killer (NK) cell activity. Here, 5 mg/kg of CP was orally administered to induce immunosuppression in the animal model, and was then orally treated with 0.5 ml/kg, 1 ml/kg, and 2 ml/kg of FV each. The results from the in vitro studies showed that CP treatment decreased the activities of splenocytes, cytokines, and NK cells, which was later increased with FV treatment. Additionally, in the CP-induced immunosuppression animal model the number of immune cells, cytokines, and weights of the immune organs decreased, and the spleen tissues were destroyed compared to the normal control group. However, in the FV-treated groups, the immune cells and cytokines increased, in addition to the weights of the liver, thymus, and spleen, and the damaged spleen tissues recovered. Therefore, these results demonstrate that FV had an immunomodulatory function.

Keywords: fermented vinegar, immunomodulation, immunity, immunosuppression, cyclophosphamide

Received July 23, 2021; Revised August 30, 2021; Accepted September 10, 2021

Citation: Hong Sun Hwa, Minjoo Kwon, Hak Yong Lee, Young Mi Park, Dong-Yeop Shin, Jin Seb Choi, Min-Jung Kim, Hye Jeong Yang, Na-Rae Shin, Immunomodulatory Effect of Fermented Vinegar on Cyclophosphamide-induced Immunosuppression Model. Journal of Food and Nutrition Research, 9 (9). 469-476. January 2021. doi:10.12691/jfnr-9-9-3

1. Introduction

In modern society, chronic diseases are gradually increasing due to rapid development and economic growth, leading to stress along with changes in diet and exercise [1]. In addition, the number of immune-related diseases is increasing due to increased use of chemical substances and air pollution [2]. Thus, these diseases result in reduced immune responses, and research is being conducted to develop substances that can help enhance immunity [3,4].

Immunity is the defense mechanism against external invasion and is divided into innate and adaptive immunity according to the specificity of antigens [5]. Innate immunity plays a primary role in defense, and each type of immune cell is responsible for its own immune function. Cells responsible for innate immunity include natural killer (NK) cells, macrophages, neutrophils, monocytes, and dendritic cells [6]. Macrophages and dendritic cells very rapidly respond to foreign antigens and cause phagocytosis, leading to the production of various cytokines and physiologically active substances that are secreted and delivered along with T cells to maximize the immune response [7,8]. NK cells directly destroy cancer cells by releasing cytotoxic granules, and activating macrophages and T cells by secreting cytokines such as interferon (IFN)-γ [9]. Adaptive immunity is mediated by B lymphocytes and T lymphocytes, although it is primarily dependent on the activity of T lymphocytes. The activation of T lymphocytes is achieved helper (Th) cell 1 and Th2 by CD4+ receptors immune response. [10]. Th1 cell-induced cytokines, including interleukin (IL)-2, IL-12, tumor necrosis factor (TNF)-α, and INF-γ, activate cell-mediated immune functions [10]. Th2 cell-induced cytokines activate humoral immunity via IL-4, 5, 6, and 10 [11]. The balance between Th1 and Th2 regulates the immune system [12].

Vinegar is a traditional fermented condiment, which is used in both the East and West, and contains various nutrients, such as organic acids and polyphenols [13]. It is known to have various effects, such as anti-obesity, anti-diabetes, antioxidant, and anti-tumor effects [14,15,16]. Fermented vinegar (FV) has a variety of physiologically active substances due to chemical changes, compared to the substances present in the raw materials from which it has been produced [17]. The FV used in this experiment was obtained from fermented brown rice. Brown rice has a high content of various vitamins and essential fatty acids, and is known to have a beneficial effect on chronic diseases, and it reduces cholesterol levels [18,19]. Moreover, its contents was shown to be involved in immune regulation [20]. However, studies on the immunomodulation by brown rice FV have not yet been conducted.

In this study, a CP-induced immunosuppression model was used to investigate the immunomodulatory functions of brown rice FV. In the in vitro studies, cell proliferation, NK cell activity, and cytokine levels were evaluated. In the in vivo study, we performed histological analysis of the spleen tissues, and assessed the body weight, blood count, and level of cytokines.

2. Materials and Methods

2.1. Cell Culture

AR42J, a pancreatic epithelial cell line, was purchased from the Korea Cell Bank (KCLB, Seoul, Korea). The cells were cultured in a 10 % RPMI1640 (Gibco, USA) medium after adding 10 % FBS (Welgene, Gyeongsan, Korea) and 1 % antibiotic-antimycotic (Gibco, USA) in a cell incubator at 37°C and 5 % CO₂.

Spleens from Wister rats (Samtaco, Onsan, Korea, 4-5 weeks old) were excised to prepare a single suspension with RPMI1640 medium. The prepared suspension was centrifuged at 1000 rpm for 5 min at 4°C, washed three times, and treated with RBC lysis buffer (Roche) for 3 min to remove the red blood cells.

2.2. Cell Culture and Proliferation

Cell viability was assessed using the WST-1 reagent (EZ-Cytox, DoGen, Seoul, Korea). The splenocytes and AR42J cells were coated separately at 1 × 10⁶ cells/well and 2 × 10⁴ cells/well, respectively, onto a 96-well plate, and treated with FV of appropriate concentrations (1, 3, 5, 10, 30, 50, 100, 300, 500, 1000 μ/ml). After 24 h, 10 μl of the WST-1 reagent was added to each well, followed by incubation for 1 h, and the absorbance was measured at 450 nm using a Multi Detection Reader (Infinite 200, TECAN Group Ltd. Switzerland).

The analysis of cell proliferation using CP (Sigma Aldrich, USA) was performed by coating splenocytes onto a 96-well plate at 1×106 cells/well, followed by treatment with FV of appropriate concentrations after 4 h. After incubation for 1 h, the cells were treated with 1.2 mg/ml of CP. After 24 h, the cultured cells were treated with WST-1 cell proliferation reagent and the cell count was measured at 450 nm.

2.3. Assay for NK Cell Activity

AR42J cells were coated onto a 96-well plate at 5 × 10⁴ cells/well and cultured for 24 h. Splenocytes were coated onto 1 × 10⁶ cells/well and cultured for 4 h, followed by treatment with appropriate concentrations of FV. After 24 h, analyzed at 490 nm using the LDH Cytotoxicity Detection Kit (Takara Bio, Japan).

2.4. Experimental Animal Model

Male Wister rats (5 weeks old) were purchased from Orient Bio Inc. (Seong-Nam, Korea). The animals were provided with a standard diet and drinking water, and the experiment was conducted after a week of acclimatization. During the breeding period, a constant temperature and humidity (23 ± 1°C, 50 ± 5%) were maintained. All animal experiments were approved by the Institutional Animal Care and Use Committee of the INVIVO Co., Ltd. (IV-RB-17-2005-42).

The establishment of a model for inducing immunosuppression was performed by the oral administration of 5 mg/kg of CP. The experimental animals were separated into the following different groups (n = 10), after weighing each one of them: normal control group (NC), immunosuppression group (CP), immunosuppression + FV, 0.5 ml/kg (FV0.5), immunosuppression + FV, 1 ml/kg (FV1), immunosuppression + FV, 2 ml/kg (FV2). The samples were orally administered with distilled water in the NC group and the CP group, and 0.5 ml/kg, 1 ml/kg, and 2 ml/kg in the FV-treated groups. FV was taken over from Vinergar park Co. Ltd. (Boseong, Korea) and used. FV was that has been fermented with alcohol and acetic acid and aged using brown rice, rice koji, green tea and groundwater. Body weight was measured once per week for clinical evaluation.

2.5. Sample Collection

The animals were anesthetized using inhalation anesthesia (isoflurane), and the blood was collected through the abdominal vein. After blood collection, the animal was exsanguinated by severing the abdominal vena cava and artery, and the organs (liver, thymus, and spleen) were harvested. The harvested organs were washed with saline, weighed, and the spleen tissues alone were fixed in 10 % formalin solution.

2.6. Blood Analysis

The collected blood was divided into an EDTA tube and a 1.5 ml tube, and the blood was analyzed and the serum was separated. For hematological analysis, the blood contained in the EDTA tube was rotated in a roll mixer for 30 min, and the number of leukocytes, lymphocytes, and granulocytes was measured using a blood analyzer. The serum was coagulated at room temperature for approximately 30 min and separated at 3000 rpm for 10 min at 4°C in a centrifuge, and the supernatant was recovered and stored at - 80°C. The stored serum was utilized for analysis and the levels of inflammatory cytokines were measured using the respective ELISA kits (TNF-α, IFN-γ, IL-2; R&D system, USA, IL-12; CUSABIO, USA).

2.7. Histological Analysis

The spleen tissues were fixed in 10 % formalin, embedded in paraffin, sectioned to a thickness of 4 μm, and placed on the slide. The tissues were hydrated after the removal of paraffin, and stained with hematoxylin for 4 min and eosin for 2 min. The mounted slides were observed and photographed with an optical microscope (Olympus BX50 F4, Olympus, Japan).

2.8. Statistical Analysis

Statistical analysis is represented as mean ± SEM, and calculations were done using the Statistical Package For The Social Sciences program (SPSS ver.12.0, SPSS Inc., Chicago, IL, USA). The values were compared using ANOVA and Duncan Sau test. Statistical significance was considered at p < 0.05.

3. Results

3.1. Effects of FV on Immune Cells in the Splenocytes

To investigate the effect of FV on immune cells, the rates of cell viability and cell proliferation were measured using the splenocytes In the splenocytes, FV did not show any effect on the cell viability up to a concentration of 100 μg/ml, but the cell viability decreased with concentrations from 300 μg/ml (Figure 1A). Based on these results, the increase in cell proliferation caused by FV compared to CP treated cells was confirmed (Figure 1B). A concentration-dependent increase was observed with FV treatment compared to the group treated with CP alone, and the significant difference was observed from 5 μg/ml in particular.

Since the cytotoxicity of FV was confirmed in the AR42J cells (target cell), we investigated the effect of FV on the activity of NK cells, and it showed cytotoxicity from 300 μg/ml (Figure 1C). Based on this result, the experiment was conducted at a concentration without cytotoxicity, and the NK cell activity increased in the FV-treated group compared to the CP-treated group; however, the increase was not concentration-dependent (Figure 1D). The significant increase was observed at 30 μg/ml and 100 μg/ml.

3.2. Effect of FV on Cytokines among other CP-induced Immune Cells in the Splenocytes

Various cytokines are produced due to cellular activity and play an important role in immune responses [21]. Therefore, this study confirmed the effect of FV on CP-induced cytokines. In Table 1, it was shown that the level of cytokines in the CP-treated cells were reduced compared to the non-treated cells. However, the number of FV-treated cells generally increased, but the difference was not significant.

3.3. Effect of FV on Body Weight and Weight of the Immune Organs in the CP-induced Immunosuppression Animal Model

In Table 2, the mean weight of the rats in the CP group (226.23 g) decreased compared to the NC group (236.12 g). However, among the FV groups, the mean weight in the FV0.5 group increased to 230.30 g; FV1, 235.98 g; and FV2, 236.83 g.

Figure 1. FV increase immune cells and NK cell activity in splenocytes. (A) Cell viability, (B) Cell proliferation, (C) Cytotoxicity of FV in AR42J cells, (D) NK cell activity of FV. ‘a~g’ indicate significant difference at p<0.05. Data expressed as mean±SEM

Table 1. Effect of FV on CP-induced cytokines

Table 2. Effect of FV on body weight CP-induced immunosuppression model

Table 3. Effect of FV on organs weight CP-induced immunosuppression model

The mean weights of the immune organs (Table 3) show that the weight of the livers decreased to 6.18 g and 5.86 g in the NC and CP groups, respectively, although it increased in the FV group (FV0.5: 6.09 g, FV1: 6.19 g, FV2: 6.36 g). The mean weight of the thymus decreased significantly in the CP group (0.25 g) compared to the NC group (0.51 g), but FV treatment did not affect the weight of the thymus (FV0.5: 0.22 g, FV1: 0.20 g, FV2: 0.25 g). The mean weight of the spleen was significantly lower in the CP group (0.38 g) than in the NC group (0.60 g) and significantly increased in the FV group (FV0.5: 0.39 g, FV1: 0.43, FV2: 0.44 g).

3.4. Effect of FV on the Cytokines and other Immune Cells in the CP-induced Immunosuppression Model

CBC analysis was performed to determine the effect of FV on the cell count of immune cells in the CP-induced immunosuppression model (Figure 2). The number of leukocytes, lymphocytes, granulocytes, and MID cells significantly decreased in the CP group compared to the NC group. In contrast, the number of leukocytes and lymphocytes in the FV group increased in a concentration-dependent manner compared to those in the CP group. However, there were no difference in the number of granulocytes and MID cells between both the groups.

IL-2 and IFN-γ decreased in the CP group compared to the NC group, but there was no difference in the FV group (Figure 3A, D). However, reduced levels of IL-12 and TNF-α were recovered due to FV treatment; in particular, there was a significant difference in the level of TNF-α (Figure 3B, C).

Figure 2. FV increase immune cells on CP-induced animal model. WBC, white blood cell; GRA, neutrophilic granulocyte; LYM, lymphocyte; MID, basophil, monocyte and eosinophil. NC, normal control; CP, Cyclophosphamide (5mg/kg); FV0.5, Fermented vinegar (0.5ml/kg) + Cyclophosphamide (5mg/kg); FV1, Fermenter vinegar (1ml/kg) + Cyclophosphamide (5mg/kg); FV2, Fermenter vinegar (2ml/kg) + Cyclophosphamide (5mg/kg)

Figure 3. FV induction inflammatory cytokines on CP-induced animal model. (A) IL-2 in serum, (B) IL-12 in serum, (c) TNF-α in serum, (D) IFN-γ in serum. NC, normal control; CP, Cyclophosphamide (5mg/kg); FV0.5, Fermented vinegar (0.5ml/kg) + Cyclophosphamide (5mg/kg); FV1, Fermenter vinegar (1ml/kg) + Cyclophosphamide (5mg/kg); FV2, Fermenter vinegar (2ml/kg) + Cyclophosphamide (5mg/kg). ‘a’, ‘b’ and ‘c’ indicate significant difference at p<0.05. Data expressed as mean±SEM

Figure 4. FV recovery spleen tissue function on CP-induced animal model. Spleen tissue was stained hematoxylin and eosin. WP, white pule; RP, red pule; MZ, marginal zone; CV, central vein. NC, normal control; CP, Cyclophosphamide (5mg/kg); FV0.5, Fermented vinegar (0.5ml/kg) + Cyclophosphamide (5mg/kg); FV1, Fermenter vinegar (1ml/kg) + Cyclophosphamide (5mg/kg); FV2, Fermenter vinegar (2ml/kg) + Cyclophosphamide (5mg/kg). Scale bars present 100 µm

3.5. Effect of FV on Spleen Function in CP-induced Immunosuppression Model

In order to investigate the effect of FV on spleen function in the CP-induced immunosuppression model, we performed H & E staining of the spleen tissues (Figure 4). In the case of the NC group, the areas of RP and WP were clearly separated because of the clear boundary of the MZ, although the boundary of the MZ hardly appeared in the CP group, which confirmed the collapse of WP; the arrangement of RP was also irregular. However, compared to the CP group, in the FV group, the boundary of the MZ was observed and the collapse of WP was decreased, in addition to the recovery of the arrangement of RP.

4. Discussion

CP is one of the most commonly used alkylating anticancer drugs, and it acts on both periodic and intermittent cells and suppresses the activity of immune cells [22]. However, it causes side effects such as immunotoxicity, hematotoxicity, and mutagenesis [23]. In particular, due to the side effects of immunosuppression, drugs or food supplements with immunomodulatory functions are administered together [24]. The immunosuppression model using CP is widely used in studies of immunomodulation [25]. Therefore, in this study, the immunomodulatory function of FV was investigated in a CP-induced immunosuppression model.

Immune responses play a pivotal role in the prevention and treatment of diseases; hence, there is a growing interest in the research on immune regulation and immune stimulation [26]. In recent years, the development of healthy functional foods with immunomodulatory functions has increased owing to the increasing interest in enhancing immunity [27]. Since natural products have a low risk of side effects unlike chemical substances, healthy functional foods using natural products are primarily being developed [28].

The representative organs that are involved in immunity include the thymus and spleen [29]. The thymus is the central immune organ where the T lymphocytes develop and mature; hence, the weight of the thymus represents the thymic index [30]. In the spleen, NK cells, and B and T lymphocytes are deposited to participate in the immune response, and the cell differentiation and proliferation occur during immune activation [31]. NK cells are granular lymphocytes derived from the bone marrow that identify and eliminate external infections, injured cells and malignant cells [32]. NK cell activity is an important indicator of immunity because it is related to viral infections and tumors, and is also involved in allergic reactions [33,34]. In this study, the proliferation rate of splenocytes, NK cell activity, and the weights of the thymus and spleen were measured to determine the effect of FV on the functioning of immune organs. From the results, we confirmed that immune cell and NK cell activity decreased in the splenocytes treated with CP. In the CP-induced immunosuppression model, we confirmed that the weight of the thymus and spleen decreased. However, in the splenocytes treated with FV, splenocyte proliferation increased and NK cell activity increased. Additionally, the decreased weight of the spleen increased in a concentration-dependent manner. In the histological study, WP collapsed due to the loss of MZ in the CP group, but MZ and WP were recovered in the FV group. However, there was no change in the thymus from the FV group. These results show that FV can affect B cell immunity.

Leukocytes play an important role in the body’s defense system, and the number of leukocytes is used as an index to evaluate immune function [35]. Overexpression or reduction of leukocytes causes disorders in immune factors and significantly affects immune function [36]. Leukocytes include various immune cells involved in immunity, and these immune cells act as mediators of the immune response [37]. Therefore, the increase or decrease in the number of these immune cells depends on the immune response. In order to investigate the effect of FV on leukocyte count, we measured the leukocyte count in the CP-induced immunosuppression animal model. The level of leukocytes decreased compared to that in the NC group. However, the FV group showed a concentration-dependent increase compared to the CP group. The number of lymphocytes decreased in the CP group compared to the NC group, but the FV group showed similar results to that of leukocytes. However, the levels of granulocytes and MID cells increased, but not in a concentration-dependent manner. Thus, these results show that FV affects the number of leukocytes and regulates inactivated lymphocytes.

Cytokines are soluble proteins secreted by the activity of immune cells and play an important role in the immune response [21]. Therefore, a decrease in cytokines causes a disorder in the immune factor; thereby, decreasing immune function [38]. In this study, we evaluated the effects of FV on cytokines using CP-induced splenocytes in a CP-induced immunosuppression animal model. The CP-treated splenocytes showed decreased cytokine levels compared to untreated cells. In FV-treated cells, TNF-α and IL-12 levels increased, but the IL-2 and IFN-γ levels were not different. Similar to the results from the in vitro analysis, the levels of TNF-α and IL-12 in the CP group decreased compared to the NC group, and the FV group showed a concentration-dependent increase. These results show that FV regulates TNF-α and IL-12 during immune responses.

In summary, we evaluated the immunomodulatory function of FV in a CP-induced immunosuppression model. FV showed proliferation of immune cells, increased cytokine levels, and long-term functional recovery of splenocytes. Additionally, it showed recovery of immune tissue function in the CP-induced immunosuppression model. However, since the effect on the thymus and specific cytokines was unclear, FV is thought to be involved in the regulation of specific immune cells. Therefore, further research is needed to understand the regulation of specific immune cells by FV.

Conflict of Interests

There is no conflict of interest

Acknowledgements

This work was supported by “Food Functionality Evaluation program” under the Ministry of Agriculture, Food and Rural Affairs and partly Korea Food Research Institute.

Availability of Data and Materials

The data that support the findings of this study are included in this published article or are available from the corresponding author upon reasonable request.

Contributions of Authors

Conceived and designed the experiments: HJY, JSC, HYL. Performed the experiment: YMP, DYS. Analyzed the data: HSH, KMJ, YMP, DYS, NRS. Contributed reagents/ materials/ analysis tool: MJK, HJY. Wrote the paper: NRS.

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