Wageningen Academic  P u b l i s h e r s

Beneficial Microbes, 2015; 6(5): 697-705

http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0159 - Tuesday, September 26, 2017 7:27:10 PM - Göteborgs Universitet IP Address:130.241.16.16

Evaluation of the potential anti-allergic effects of heat-inactivated Lactobacillus paracasei V0151 in vitro, ex vivo, and in vivo Y.W. Liu1#, T.Y. Fu1,2#, W.S. Peng1,2, Y.H. Chen1,2, Y.M. Cao3, C.C. Chen3, W.L. Hung3 and Y.C. Tsai1,2* 1Institute of Biochemistry and Molecular Biology, National Yang-Ming University, No. 155, Li-Nong St., Sec. 2, BeiTou Dist.,

Taipei 11221, Taiwan, R.O.C.; 2Probiotics Research Center, National Yang-Ming University, No. 155, Li-Nong St., Section 2, BeiTou Dist., Taipei 11221, Taiwan, R.O.C.; 3Want Want China Holdings Ltd., No.1088, East Hond Song Rd., Shanghai 201103, China P.R.; [email protected]; #These two authors contributed equally to this study Received: 9 November 2014 / Accepted: 8 April 2015 © 2015 Wageningen Academic Publishers

RESEARCH ARTICLE Abstract The efficacy of Lactobacillus paracasei V0151 (V0151), isolated from the faeces of a child, to modulate immune responses was investigated. In RAW 264.7 cells expressing an inducible nitric oxide synthase (iNOS)-directed luciferase gene, heat-inactivated V0151 stimulated iNOS expression followed by nitric oxide production. V0151 significantly elevated interferon gamma, interleukin (IL)-10, tumour necrosis factor alpha, and IL-1β production in human peripheral blood mononuclear cells. In splenocytes isolated from ovalbumin (OVA)-sensitised BALB/c mice treated with OVA and V0151 at different bacterium-to-cell ratios (1:1, 10:1, and 20:1) for 96 h, IL-2, IL-4, IL-5, and IL-13 production was dose-dependently downregulated, whereas IL-12 was dose-dependently upregulated. Collectively, our findings indicate that V0151 might regulate pro-inflammatory factors in macrophages and splenocytes. Furthermore, the T helper 1/T helper 2 (Th1/Th2) balance was also skewed toward Th1 dominance through the elevation of Th1 cytokine production. Keywords: Lactobacillus paracasei, immune modulation, macrophages, splenocytes, ovalbumin

1. Introduction The immune system comprises innate and adaptive immunity. Macrophages play an important role in the innate immune response, as they can engulf pathogenic microorganisms and produce pro-inflammatory cytokines, such as tumour necrosis factor alpha (TNF-α), interleukin (IL)-6, and IL-1β, which further regulate the immune system (Wang et al., 2013b). The adaptive immune responses can be classified into two types, T helper (Th)1 and Th2, according to the different profiles of cytokines produced. IL-2, IL-12 and interferon gamma (IFN-γ) are secreted by Th1 cells, whereas IL-4, IL-5, and IL-13 are secreted by Th2 cells (Delcenserie et al., 2008). The balance of Th1 and Th2 responses is believed to be crucial for maintaining appropriate immune responses for host defence. The beneficial effects of lactic acid bacteria (LAB) have been widely reported. Some examples include preventing the

recurrence of cancer (Naito et al., 2008), reducing the risk of cancer (Larsson et al., 2008), decreasing the prevalence of allergies (De Vrese and Schrezenmeir, 2008), enhancing phagocytosis (Ji et al., 2013), ameliorating influenza virus infection (Iwabuchi et al., 2011), inducing dendritic cell maturation (Elawadli et al., 2014), and maintaining gastrointestinal homeostasis (Duerr and Hornef, 2012). The results of recent meta-analysis of probiotic studies confirmed the beneficial effects of probiotics. The daily consumption of greater than 1011 cfu of probiotics for more than 8 weeks induced moderate blood pressure elevation (Khalesi et al., 2014). A probiotic intervention in children and adults with acute respiratory infections decreased the numbers of days of illness and of absence from day care/ school/work (King et al., 2014). Bifidobacteria and lactobacilli are the most widely used probiotics, and their effects on the immune response have been extensively investigated. Bifidobacterium longum

ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2014.0159697

http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0159 - Tuesday, September 26, 2017 7:27:10 PM - Göteborgs Universitet IP Address:130.241.16.16

Y.W. Liu et al.

BB536 (BB536) supplementation increased the number of bifidobacteria cells in the intestinal microbiota and modulated immune function in the elderly fed by enteral tube feeding (Akatsu et al., 2013). The oral administration of Lactobacillus casei Shirota (LcS) to ovalbumin (OVA)sensitised mice upregulated the production of Th1 cytokines and downregulated the production of Th2 cytokines (Matsuzaki, 1998; Matsuzaki et al., 1998). Lactobacillus rhamnosus GG was reported to decrease the proinflammatory responses induced by Escherichia coli lipopolysaccharide (LPS) in the developing infant rat small intestine, plasma, lung and liver (Zhang et al., 2006). Both viable and heat-killed Lactobacillus paracasei 33 (LP33) improved allergic rhinitis symptoms (Costa et al., 2014; Peng and Hsu, 2005). These studies indicated the modulatory effects of probiotics on immune responses; furthermore, the effects seemed to be strain dependent. L. paracasei V0151 (V0151) was isolated from the faeces of a Taiwanese child. In the current study, we used different assay platforms to investigate the immunomodulatory activities of V0151. The effects of heat-inactivated V0151 on innate immunity were examined in two cell line models, RAW 264.7 cells expressing an inducible nitricoxide synthase (iNOS) directed luciferase gene and naïve RAW 264.7 cells. Splenocytes from BALB/c mice orally administered V0151 were cultured with LPS to investigate the inhibition of pro-inflammatory cytokine production. The anti-allergic potential of V0151 was evaluated by determining the production of Th1 and Th2 cytokines in OVA-sensitised mice splenocytes treated with OVA or V0151.

2. Materials and methods Materials and chemicals LPS and phosphate-buffered saline (PBS) were purchased from Sigma (St. Louis, MO, USA). Dulbecco’s modified Eagle medium (DMEM) and RPMI-1640 medium were purchased from Gibco BRL (Grand Island, NY, USA). Ficoll reagent was purchased from GE Healthcare (Uppsala, Sweden), and de Man-Rogosa-Sharpe (MRS) broth was purchased from BD (Sparks, MD, USA). All other chemicals used in this study were purchased from Merck (Darmstadt, Germany).

Bacterial strains and culture conditions V0151 was isolated from the faeces of a Taiwanese child and was deposited under the Budapest Treaty at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany) with the DSMZ Accession No. DSM 27446, provided by the International Depositary Authority. LcS was isolated from Yakult drink (Yakult, Tokyo, Japan). Bacteria were inoculated in MRS broth and cultured at 37 °C for 20 h. 698

Then, 1% volume of cultured broth was inoculated in fresh MRS broth for further activation. Bacteria were collected by centrifugation (3,000 rpm, 10 min), washed twice with sterile PBS, and resuspended to a density of 1010 cfu/ml. For the heat-inactivating treatment, bacteria were heattreated at 80 °C for 20 min and were then stored at -20 °C.

Treatment of RAW 264.7 cells stably expressing an iNOSdirected luciferase gene and naïve RAW 264.7 cells RAW 264.7 macrophages stably expressing an iNOS reporter, provided by Dr. Shu-Chen Hsieh (Wang et al., 2013a), were maintained in DMEM containing 10% (v/v) foetal bovine serum (FBS). Briefly, 5×105 cells/well was seeded into a 12-well culture plate. After 24 h of culture, the medium was removed and replaced with fresh medium. The cells were treated with heat-inactivated V0151 (1×107 cfu/well; bacterium-to-cell ratio, 20:1) in the presence or absence of LPS (1 μg/ml) and assayed for luciferase activity. After a 6 h-incubation, the cells were washed three times with PBS and lysed with lysis buffer. The luciferase activity was measured using the Luciferase Assay System (Promega, Madison, WI, USA) according to a published report (Wang et al., 2013b). For normalising, the value of recorded luminescence was divided by the content of protein for each individual sample. The data are presented as the fold change relative to the control, which was assigned a value of 1. Naïve RAW 264.7 cells were treated for 24 h, and the supernatants were collected for nitrite level analysis.

Peripheral blood mononuclear cells isolation and lactic acid bacteria treatment Human peripheral blood mononuclear cells (hPBMCs) were isolated from the heparinized blood by a Ficoll gradient centrifugation method, according to our previous report (Liu et al., 2011). The usage of samples from human was granted under IRB 15-006-B1 provided by Institutional Review Board of Antai Medical Care Corporation Aggregate, Taiwan R.O.C. Four independent assays were carried out using cells from donors with a median age of 28 years. First, 106 cells/well of hPBMCs were seeded into a 24-well cell culture plate and co-cultured with 107 cfu/ well of heat-inactivated V0151 for 48 h (bacterium-to-cell ratio, 10:1). The supernatant was collected and stored at -80 °C for further cytokine and NO analyses.

Animals Male BALB/c (6-8 weeks old) mice were supplied by BioLASCO Taiwan Co., Ltd. (Hsin Chu, Taiwan R.O.C.). The animals were maintained in an individually ventilated cage (IVC) system in 12 h light/12 h dark conditions. Food and water were provided ad libitum. The use of animals and the procedures for animal handling and treatments

Beneficial Microbes 6(5)



Immunomodulatory activities of V0151

In the V0151 oral administration model, mice (n=8) were given tube feedings containing 109 cfu/mouse/day of live or heat-inactivated V0151 for 28 days. The OVA sensitisation of BALB/c mice was performed according to a previous report, with slight modification (Liu et al., 2014). The OVA sensitisation was carried out on days 0 and 14 with an intraperitoneal injection of 20 μg OVA and 2 mg aluminium hydroxide (Al(OH)3) in 200 μl PBS. From day 16 to day 21, the mice (n=3) were used for splenocyte preparation.

Splenocyte isolation and ex vivo culture Mouse splenocytes were prepared according to our previous report (Liu et al., 2014). Briefly, after euthanising the mice, the spleens were removed for splenocyte isolation. The splenocyte concentration was adjusted to 2×106 cells/ml in RPMI-1640 medium (Gibco BRL, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% FBS. Cells were cultured with OVA (50 μg/ml), LPS (1 μg/ml), or heat-inactivated V0151 at a bacterium-to-cell ratio of 1:1, 10:1, or 20:1 for 96 h. Culture medium samples were then collected and stored at -80 °C for further cytokine measurements.

Cytokines and nitric oxide measurement The levels of cytokines and NO in the culture medium were measured by ELISA. The concentrations of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, TNF-α, transforming growth factor beta (TGF-β) and IFN-γ were measured according

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to the manufacturer’s instructions (ELISA MAX™ Deluxe Sets; BioLegend, San Diego, CA, USA). The level of NO in the culture medium was measured by the Griess reagent method (Liu et al., 2007). Briefly, equal volumes of cultured supernatant and Griess reagent were added to each well and incubated for 15 min at room temperature. The absorbance at 530 nm was measured, and sodium nitrite (0-200 μM) was used to plot the standard curve.

Statistical analysis The results are expressed as the mean ± standard deviation. The differences between the mean values were tested for statistical significance using one-way ANOVA followed by Tukey’s post-hoc test. A P-value of