[논문]Immune-Enhancing Effect of Nanometric Lactobacillus plantarum nF1 (nLp-nF1) in a Mouse Model of Cyclophosphamide-Induced Immunosuppression

Choi, Dae-Woon; Jung, Sun Young; Kang, Jisu; Nam, Young-Do; Lim, Seong-Il; Kim, Ki Tae; Shin, Hee Soon

doi:10.4014/jmb.1709.09024

Immune-Enhancing Effect of Nanometric Lactobacillus plantarum nF1 (nLp-nF1) in a Mouse Model of Cyclophosphamide-Induced Immunosuppression 원문보기

Journal of microbiology and biotechnology, v.28 no.2, 2018년, pp.218 - 226

Choi, Dae-Woon (Food Biotechnology Program, Korea University of Science and Technology) , Jung, Sun Young (Food Biotechnology Program, Korea University of Science and Technology) , Kang, Jisu (Food Biotechnology Program, Korea University of Science and Technology) , Nam, Young-Do (Food Biotechnology Program, Korea University of Science and Technology) , Lim, Seong-Il (Division of Strategic Food Research, Korea Food Research Institute) , Kim, Ki Tae (Biogenics Korea Co. Ltd.) , Shin, Hee Soon (Food Biotechnology Program, Korea University of Science and Technology)

Abstract ▼ AI-Helper

Nanometric Lactobacillus plantarum nF1 (nLp-nF1) is a biogenics consisting of dead L. plantarum cells pretreated with heat and a nanodispersion process. In this study, we investigated the immune-enhancing effects of nLp-nF1 in vivo and in vitro. To evaluate the immunostimulatory effects of nLp-nF1, mice immunosuppressed by cyclophosphamide (CPP) treatment were administered with nLp-nF1. As expected, CPP restricted the immune response of mice, whereas oral administration of nLp-nF1 significantly increased the total IgG in the serum, and cytokine production (interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-${\alpha}$)) in bone marrow cells. Furthermore, nLp-nF1 enhanced the production of splenic cytokines such as IL-12, TNF-${\alpha}$, and interferon gamma (IFN-${\gamma}$). In vitro, nLp-nF1 stimulated the immune response by enhancing the production of cytokines such as IL-12, TNF-${\alpha}$, and IFN-${\gamma}$. Moreover, nLp-nF1 given a food additive enhanced the immune responses when combined with various food materials in vitro. These results suggest that nLp-nF1 could be used to strengthen the immune system and recover normal immunity in people with a weak immune system, such as children, the elderly, and patients.

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문제 정의

IgG can interact with macrophages, neutrophils, and natural killer cells, leading to activation of these cells [33]. In this study, we assessed serum IgG levels as an indicator of immune stimulation or immune enhancement. To investigate the immune-enhancing effect of nLp-nF1, the immunosuppressed mouse model (CPP-induced) was used.

제안 방법

Cell counting was performed by the trypan blue exclusion method, and the number of splenocytes was adjusted to a cell density of 5 × 106 cells/ml in RPMI 1640 medium (10% FBS).
ICR mice were divided into four groups consisting of naive, sham, low-dose nLp-nF1 (4 × 109 CFU/kg body weight), and highdose nLp-nF1 (4 × 1010 CFU/kg body weight) (n = 5).
CFU/kg body weight) (n = 5). Mice of all groups, except the naive group, were intraperitoneally injected with CCP (150 mg/kg/day) twice, once on day 0 and again on day 2, for induction of immunosuppression (Fig. 1). Mice in the nLp-nF1 group were orally administered with nLp-nF1 once a day on days 3 to 17 (15 days) .

대상 데이터

To measure IL-12, TNF-α, IL-1β, and IFN-γ, enzyme-linked immunosorbent assay (ELISA) kits were purchased from BD Biosciences (USA), and an IgG ELISA kit was obtained from Santa Cruz Biotechnology (USA). Anti-CD3 (FITC), anti-CD11b (APC), anti-CD11c (PE), and anti-CD335 (APC/Fire 750) antibodies were purchased from Biolegend (USA). CPP was purchased from Tokyo Chemical Industry Co.
The animals were housed in an air-conditioned room (23 ± 2℃) with a 12 h light/dark cycle in accordance with the guidelines for animal use and care of the Korea Food Research Institute (Permit No. KFRI-M-16044).

데이터처리

Statistical analysis of the differences between experimental groups were assessed by one-way analysis of variance followed by Fprotected Fisher’s least significant difference tests.

이론/모형

The cells were incubated on ice with Fc Block (anti-CD16/32) (BD Pharmagen, USA), and then they were stained with anti-CD3 (FITC), anti-CD11b (APC), anti-CD11c (PE), and anti-CD335 (APC/Fire 750) antibodies for 30 min. The presence of single cells was confirmed by flow cytometry, which was performed using CytoFLEX (Beckman Coulter, USA).

성능/효과

CPP is an immunosuppressor used in neoplastic and severe autoimmune disorders because it reduces a wide variety of immune responses such as those from macrophages, neutrophils, and effector T cells [34-36]. As expected, the CPP-injected group had reduced levels of IgG in sera compared with the control group, whereas the IgG levels in the group orally administered with nLp-nF1 were significantly higher than that in the CPP-treated group (Fig. 2). In general, an increase of IgG levels is able to connect with Th1-dominant immune responses, and the immune system maintains immune balance with Th1- and Th2-related immune responses [37].
To examine which of these cells was responsible for IL-12 and TNF-α production in our model, the population of immune cells in bone marrow, including CD3⁺ cells, CD11b⁺ cells, CD11c⁺ cells, and CD335⁺ cells, was investigated by flow cytometry. We found that high-dose administration of nLp-nF1 resulted in an increase in the CD11b⁺ cell population compared with the CPP-injected group (Fig. 4), but no changes in the CD3⁺ cell, CD11c⁺ cell, and CD335⁺ cell populations were detected (data not shown). In bone marrow, there are many kinds of CD11b-expressing cells such as monocytes, macrophages, dendritic cells, and granulocytes, and the proportion of CD11b⁺ cells accounts for 44% of bone marrow cells [42, 43].

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