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Mar 5, 2014 - According to the Centers for Disease Control and Prevention,(1) salmonellosis is an infection by Salmonella bacteria that induces diarrh...
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A Polysaccharide Isolated from the Liquid Culture of Lentinus edodes (Shiitake) Mushroom Mycelia Containing Black Rice Bran Protects Mice against Salmonellosis through Upregulation of the Th1 Immune Reaction Sung Phil Kim,† Sun Ok Park,‡ Sang Jong Lee,‡ Seok Hyun Nam,*,† and Mendel Friedman*,§ †

Department of Biological Science, Ajou University, Suwon 443-749, Republic of Korea STR Biotech Company, Ltd., Chuncheon 200-160, Republic of Korea § Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, California 94710, United States ‡

ABSTRACT: The present study investigated the antibacterial effect of a bioprocessed polysaccharide (BPP) isolated from Lentinus edodes liquid mycelial culture supplemented with black rice bran against murine salmonellosis. BPP was not bactericidal in vitro, it did, however, stimulate uptake of the bacteria into RAW 264.7 murine macrophage cells, as indicated by increased colony-forming unit (CFU) counts of the contents of the lysed macrophages incubated with Salmonella Typhimurium for 30 and 60 min. Two hours postinfection, the bacterial counts drastically increased in the macrophages, but 4 and 8 h postinfection BPP extract-treated cells showed lower bacterial counts than the vehicle (saline phosphate pH 7.4 buffer, PBS)-treated control. BPP elicited altered morphology and markedly elevated inducible nitric oxide (NO) synthase (iNOS) mRNA and protein expression in the infected macrophage cells. BPP also activated leukocytes in S. Typhimurium-infected mice, as determined by spleen lymphocyte proliferation and IFN-γ levels in mice sera. ELISA analysis on cytokine production by Th1 and Th2 immune cells from splenocytes of infected mice showed significant increases in the levels of the following Th1 cytokines: IL-1β, IL-2, IL-6, and IL-12. Histology assays of the livers of mice infected with a sublethal dose (1 × 104 CFU) of S. Typhimurium showed that BPP, administered daily through an intraperitoneal (ip) or oral route, protected against necrosis of the liver, a biomarker of in vivo salmonellosis. The lifespan of mice similarly infected with a lethal dose of S. Typhimurium (1 × 105 CFU) was significantly extended by ip injection or oral administration of the BPP without side effects. These results suggest that the activity of BPP against bacterial infection in mice occurs mainly through the activation of macrophage-mediated immune response resulting from augmented Th1 immunity. The significance of the results for microbial food safety and human health and further research needs are discussed. KEYWORDS: antibacterial effect, polysaccharide, Lentinus edodes, black rice bran, salmonellosis, mushroom mycelia, prevention, immunostimulation

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find out whether BPP would also neutralize adverse effects of sublethal and lethal doses of Salmonella Typhimurium bacteria in infected mice via stimulation of the immune system. To demonstrate this possibility, the present study was designed to parallel our previous related studies on the protection against Salmonella-induced liver necrosis and mortality in mice by extracts of Hericium erinaceus mushrooms,5 the herbal medicine Herba Pogostemonis,6 and liquid rice hull smoke.7

INTRODUCTION According to the Centers for Disease Control and Prevention,1 salmonellosis is an infection by Salmonella bacteria that induces diarrhea, fever, and abdominal cramps. The illness is often severe in the elderly, infants, and individuals with impaired immune systems. In the United States, about one million individuals come down with the disease annually caused largely by the foodborne Salmonella Typhimurium and Enteritidis serotypes. In a recent publication in this journal,2 we describe the isolation of a new bioprocessed polysaccharide (BPP) from a mushroom mycelia culture of Lentinus edodes (Shiitake) with added black rice bran that protected mice against Salmonella lipopolysaccharide induced endotoxemia. The protective effect was associated with stimulation of the immune system and amelioration of endotoxemia-induced pathological effects in the liver, lung, and kidney tissues. Because the cause of endotoxemia (sepsis, septic shock) is largely associated with infections by Gram-negative bacteria such as Escherichia coli and Salmonella,3,4 it was of interest to © 2014 American Chemical Society

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MATERIALS AND METHODS

Materials. RPMI 1640 medium, Hanks balanced salt solution (HBSS), fetal bovine serum (FBS), and other miscellaneous cell culture reagents were purchased from Hyclone Laboratories (Logan, UT, USA). Potato dextrose agar medium (PDA) and nutrient agar medium (NA) were the products of Difco (Detroit, MI, USA) and Becton Dickinson (San Jose, CA, USA), respectively. Murine Received: Revised: Accepted: Published: 2384

November 19, 2013 February 18, 2014 February 24, 2014 March 5, 2014 dx.doi.org/10.1021/jf405223q | J. Agric. Food Chem. 2014, 62, 2384−2391

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cycle at 20−22 °C and relative humidity of 50 ± 10%. The mice were fed freely a pelletized commercial chow diet obtained from Orient Bio Inc. (catalog no. 5L79) and sterile tap water during the entire period. After acclimation for 1 week, the mice were then divided into three groups (n = 10), avoiding any intergroup difference in body weight. BPP was dissolved in water, and given to mice via the dietary (oral) or the intraperitoneal (ip) route. Two groups were given BPP, whereas one was given only water. The dose of the BPP was adjusted to 10 mg/kg body weight for both routes. After administration of the BPP for 14 consecutive days, the mice were infected ip with sublethal or lethal doses of S. Typhimurium (1 × 104 CFU or 1 × 105 CFU). Collection of blood samples and excision of organs including liver and spleen were carried out for further experiments. In Vivo Bactericidal Assay. After administration of BPP (10 mg/ kg) for 14 consecutive days, mice were infected ip with a sublethal dose of S. Typhimurium (1 × 104 CFU). Two days later, the mice were sacrificed by CO2 asphyxia, and the abdominal cavities were opened and repeatedly washed with a total PBS (10 mL) as described by Kodama et al.9 Bacterial counts in the pooled PBS wash were determined by plating technique to measure CFU levels described above. Serum Nitrite and Nitrate Levels. Serum nitrite and nitrate levels were measured primarily following the method of Misko et al.10 with some modification. Blood samples collected by cardiac puncture were left at 37 °C for 20 min to obtain the serum, followed by filtration of the serum through an Ultrafree-MC microcentrifuge filter unit (Millipore, Bedford, MA, USA) for 1 h at 14,000 rpm to remove the hemoglobin released by cell lysis. The serum (50 μL) was incubated in a reaction mixture [(40 mM Tris Cl, 40 μM reduced βnicotinamide adenine dinucleotide phosphate, 40 μM flavine adenine dinucleotide, and nitrate reductase (0.05 U/mL)] at pH 7.9 at 37 °C for 15 min. Reduced samples were incubated with an equal volume of Greiss reagent [1% sulfanilamide and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in 5% phosphoric acid] at room temperature for 15 min. The absorbance was read at 570 nm using a microplate reader (model 550, Bio-Rad, Hercules, CA, USA). Total nitrate/nitrite levels were determined by comparison with a reduced NaNO3 standard curve. Mitogen-Induced Lymphocyte Proliferation. Spleen cell suspensions were prepared by gentle pressing the spleen through a stainless steel mesh as described by Teixeira et al.11 Thereafter, the cells were suspended in RPMI 1649 medium supplemented with 5% FBS and seeded into 24-well culture plates in the presence of concanavalin A (Con A, 2.5 μg/mL) to stimulate T cells. After stimulation for 72 h at 37 °C in 5% CO2, cell proliferation levels were determined at 490 nm on a microplate. Cell numbers were determined using MTS [(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)] assay in the form of the CellTiter 96 aqueous nonradioactive cell proliferation assay kit (Promega, Madison, WI, USA) following the manufacturer’s instructions. The increase of cell growth was determined by setting as 1 the growth of spleen cells from pathogen-free mice in FBSsupplemented RPMI 1640 medium without Con A. ELISA of Cytokines. Th1 and Th2 cytokines (IL-1, IL-2, IL-6, and IL-12 vs IL-4, IL-5, and IL-10) in the culture supernatant of spleen cells from infected or mock-infected mice without Con A were determined by ELISA kits (Life Technologies, Carlsbad, CA, USA). The ELISA kit for IFN-γ was the product of Biosource Int. (Camarillo, CA, USA). After reactions, the absorbance of the final solution at 420 nm was read in a microplate reader (model 550, Bio-Rad, Hercules, CA, USA). Mice Salmonellosis Study. The salmonellosis assay was carried out by the method of Kim et al.,12 with some modification. Three groups of 10 mice each (water-treated control and BPP-treated experimental groups via ip and oral routes) were used for bacterial infection. Mice were infected ip with a lethal a dose of S. Typhimurium (1 × 105 CFU). After infection, the mice were treated with BPP (10 mg/kg) via the ip or oral routes every 24 h during the entire experimental period. To determine the survival rate, mice were observed for an additional 28 days after bacterial infection.

interferon-γ (IFN-γ) was purchased from PharMingen (San Diego, CA, USA). The AMV reverse transcriptase and dNTP mixture were obtained from Takara Bio (Kyoto, Japan). PCR primers were customsynthesized and purified by Bioneer (Daejon, Republic of Korea). Analytical reagents were purchased from Sigma Aldrich (St. Louis, MO, USA) and used without further purification. Preparation of Bioprocessed Polysaccharide (BPP). The isolation and characterization of BPP is described in our recent publication.2 The amount of β-glucan in each extract was determined using a Mixed-Linkage-β-glucan Kit (Megazyme Intern Treland Ltd., Wicklow, Ireland). Bacterial Strain and Culture Condition. Salmonella enterica subsp. enterica ser. Typhimurium (S. Typhimurium) ATCC 140 was obtained from the American Type Culture Collection (Manassas, VA, USA) and kept as frozen glycerol stock. Cells in frozen stock were streaked onto NA medium to produce cell colonies, from which a single colony was transferred to nutrient broth (NB) medium. For preparation of inocula, cells were grown for 20 h at 37 °C in NB. For infection, cultured bacterial cells were recovered by centrifugation at 13,000 rpm for 30 s and then washed with and resuspended in PBS. The turbidity of the cell suspensions was measured. The cell suspensions were diluted with PBS to the desired concentration of bacteria using a standard curve of optical density versus bacterial number determined as colony-forming units (CFU). In Vitro Bactericidal Assay. S. Typhimurium cells were diluted with PBS to a density of 2 × 104 CFU/mL, treated with three concentrations of BPP (1, 10, and 100 μg/mL), and incubated at 37 °C for 0, 2, 4, and 8 h. After incubation, the diluted culture (100 μL) was plated onto NA medium to assess bacterial CFU. Mammalian Cell Culture. Murine macrophage cell line RAW 264.7 from the American Type Culture Collection (Manassas, VA, USA) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS containing 100 U/mL penicillin and 100 μg/mL streptomycin. The cells were cultured at 37 °C in humidified air containing 5% CO2. Morphologic Changes in Macrophages. RAW 264.7 cells were grown in the presence of BPP (100 μg/mL) in 24-well cell culture plates with coverslips for 8 h. The coverslips were examined under a microscope (100×) to observe morphologic changes in macrophages. Cells were selected in six blindly chosen random fields, and the morphologically changed cells were then counted and recorded. This assay was performed for at least three individual experiments. Determination of Bacteria in Macrophages. To measure internalization and intracellular survival of bacteria in macrophages, RAW 264.7 cells were infected with S. Typhimurium following the method of Lu et al.8 For analysis of macrophage bacterial uptake efficiency, inoculum (10 μL) containing 1 × 104 CFU was added to RAW 264.7 macrophage cells (1 × 104 cells) pretreated with three concentrations of the BPP (1, 10, and 100 μg/mL) and incubated for 30 or 60 min in a 5% CO2 atmosphere. Cells were washed once with RPMI 1640 medium after incubation at 37 °C and then treated with the same medium containing 10% FBS and gentamicin (30 μg/mL) for 30 min to kill extracellular bacteria. For viable cell counting, the infected macrophage cells were washed three times and then lysed with distilled water. Aliquots of lysates were plated onto NA medium to measure bacterial CFU. To measure intracellular survival, S. Typhimurium cells (1 × 104 CFU) were added to serially diluted BPP-treated macrophage sample for 4 h. The samples were incubated at 37 °C for 1 h, followed by washing once with medium and subsequent treatment with RPMI 1640 medium containing 10% FBS with gentamicin (30 μg/mL) for 2, 4, and 8 h. Cell washing, lysis, and plating procedures onto NA medium were carried out following the same protocol as for the analysis of bacterial uptake efficiency. Mice and Treatments. The protocol for the mouse studies was approved by the Ethics Committee for Animal Care and Use, Ajou University, Republic of Korea. All experiments were performed in compliance with the relevant laws and institutional guidelines. Six- to eight-week-old female BALB/c mice were purchased from Orient Bio Inc. (Seongnam, Korea), and were hosted under a 12 h light/dark 2385

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Histology of Liver Tissue. For histological analysis, the liver tissue of the mice was fixed with 4% paraformaldehyde in 0.5 M phosphate buffer (pH 7.4). The tissues were rinsed with water, dehydrated with ethanol, and embedded in paraffin. The liver tissue was sectioned into 4 μm samples and mounted onto glass slides. The sections were then dewaxed using xylene and ethanol and stained with hematoxylin and eosin Y (H&E) to reveal the hemorrhagic necrosis in the liver. Histological changes were observed under a light microscope at 100× magnification. Reverse Transcription Polymerase Chain Reaction (RT-PCR) of Cellular RNA. Total cellular RNA was prepared following acid phenol guanidium thiocyanate−chloroform extraction.13 For reverse transcription, total RNA (1 μg) was incubated with AMV reverse transcriptase (5 U) and oligo (dT18) as primer (100 ng). DNA amplification was then primed in a reaction mixture containing dNTP mix (400 μM), Taq polymerase (2.5 U), and primer sets (20 μM each) representing target genes as follows: inducible nitric oxide synthase (iNOS) sense primer, 5′-ATGTCCGAAGCAAACATCAC-3′; iNOS antisense primer, 5′-TAATGTCCAGGAAGTAGGTG-3′; β-actin sense primer, GTGGGGCGCCCCAGGCACCA-3′; β-actin antisense primer, 5′-GTCCTTAATGTCACGCACGATTTC-3′. PCR was conducted using a thermocycler (model PTC-200, MJ Research Inc., Reno, NV) with one cycle for 5 min at 94 °C, followed by 30 cycles for 30 s at 94 °C, 45 s at 58 °C, and 45 s at 72 °C, and finally one cycle for 5 min at 72 °C. All amplified PCR products were subjected to 1.5% agarose gel electrophoresis and visualized with a UV illuminator. The intensity of the separated bands of DNA was quantified using a gel documentation system (model LAS-1000CH, Fuji Photo Film Co., Tokyo, Japan). Western Blot Analysis of Cell Proteins. The BPP-treated RAW 264.7 cells were lysed and extracted using RIPA buffer (50 mM Tris Cl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1 mM EDTA, pH 7.4). Protein concentrations were determined by the Bradford method using a Bio-Rad Protein kit. Bovine serum albumin (BSA) was used as standard. The cell extract containing proteins (30 μg) was separated on 10% SDS−polyacrylamide gels and electrophoretically transferred onto nitrocellulose membrane (Millipore, Billerica, MA, USA). After blocking with 5% skim milk, the membrane was incubated with rabbit anti-mouse iNOS polyclonal antibody (Cell signaling Tech., Danvers, MA, USA) or anti-mouse βactin monoclonal antibody (Millipore), followed by HRP-conjugated anti-IgG antibodies. Blots were developed using the ECL detection kit (Pierce, Rockford, IL, USA). The intensity of separated protein bands was quantified using a gel documentation system (model LAS1000CH, Fuji Photo Film Co., Tokyo, Japan). At least three separate replicates were determined for each experiment. Statistical Analysis. Results are expressed as the mean ± SD of three independent experiments. Significant differences between means were determined by ANOVA test using the Statistical Analysis Software package SAS (Cary, NC, USA). p < 0.05 is regarded as significant.

HPLC peak depends on the amount of the sample loaded on the chromatographic column. We observed sharper peaks with smaller amounts (results not shown). We cannot rule out the possibility that components other than glucan, possibly a bound protein as observed by Sanzen et al.14 in a related study, may contribute to the observed immunostimulation described below. Effect of BPP on in Vitro Growth of Salmonella. To find out whether BPP has antibacterial activity against S. Typhimurium, bacteria were incubated with serially diluted extracts in PBS (1, 10, and 100 μg/mL) at 37 °C for 0, 2, 4, and 8 h. Figure 1 shows that bacteria treated with the extracts at

Figure 1. Effects of BPP on growth of S. Typhimurium. Serially diluted BPP (1, 10, and 100 μg/mL) was incubated with S. Typhimurium (2 × 104 CFU) for 2, 4, and 8 h. Plotted values are mean ± SD of triplicate experiments.

each concentration for different incubation times showed no significant bactericidal activities until 8 h incubation compared to that of vehicle (PBS). The data show that BPP did not kill bacteria directly. We observed similar results with Hericium erinaceus mushroom extracts.5 Induction of Morphologic Changes in Macrophages. To determine whether BPP can induce changes in the morphology in the macrophage cells, RAW 264.7 cells were cultivated in the presence of the extract (100 μg/mL) for 2, 4, and 8 h. The microscopic observation of cell morphology revealed that the cells treated with BPP changed to dendritelike cells (Figure 2), reaching up to 3.6-, 10.3-, and 12.4-fold increases corresponding to the incubation times. Dendritic morphologic changes were not observed with PBS-treated controls regardless of incubation times. Phagocytotic Stimulatory Effects of BPP. To examine whether BPP enhances phagocytotic activity, RAW 264.7 cells were cultivated in the presence of three concentrations (1, 10, and 100 μg/mL) for 4 h before infection. Thereafter, the S. Typhimurium cells were incubated for 30 and 60 min before they were lysed and contents enumerated for bacteria. Figure 3 shows that, after 60 min of incubation, the internalization of bacteria into the macrophage cells increased in a dosedependent manner. The bacterial uptake rates of macrophages treated with 1, 10, and 100 μg/mL BPP were about 1.4-, 2.4-, and 3.5-fold greater than that of macrophages treated with the PBS control. The phagocytotic stimulatory effect of BPP was also evaluated in vivo in a mouse model. As described in Table 2, administration of the BPP via both oral and ip routes, respectively, elicited higher bactericidal effects than that of

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RESULTS AND DISCUSSION Structure of BPP. The method we used to prepare the polysaccharide is widely used for the isolation and purification of glucans. Results from our previous study,2 and in the present study (Table 1), suggest that BPP has a beta-glucan structure. Additional observations showed that the sharpness of the Table 1. β-Glucan Content of BPPsa sample

β-glucan (% of dry wt)

BPP from culture with black rice bran BPP from culture without black rice bran black rice bran only

0.75 ± 0.02 a 0.40 ± 0.01 b 0.03 ± 0.00 c

Values expressed as means ± SD (n = 3) in the column with the same letters are not significantly different at p < 0.05.

a

2386

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Figure 3. Changes in phagocytotic stimulatory effects of BPP on S. Typhimurium-infected macrophages. Murine macrophage cell line RAW 264.7 cells (1 × 104) were incubated with three concentrations of BPP (1, 10, and 100 μg/mL) for 4 h, and then infected with S. Typhimurium (1 × 104 CFU). Incubation continued at 37 °C for the indicated periods. After incubation, bacteria-infected macrophages were then cultured in the presence of gentamicin (30 μg/mL) for 30 min. Bacterial internalization efficiency by macrophages was determined by evaluating the protection of internalized bacteria from bactericidal action of the antibiotic gentamicin. Data are expressed as the mean ± SD of triplicate experiments. Bars sharing a common letter are not significantly different at p < 0.05.

Table 2. Effect of BPP on the Growth of S. Typhimurium Inoculated into Mouse Peritoneal Cavitya

Figure 2. Effects of BPP extract on morphologic changes in macrophages. Murine macrophage cell line RAW 264.7 cells were cultured in the presence of BPP (100 μg/mL) for 2, 4, and 8 h. (A) After 8 h incubation, the morphologic changes were photographed at 100× magnification. (B) One hundred cells in five blindly chosen random fields were examined per coverslip, and the rates of morphologically changed cells were scored. Data are expressed as the mean ± SD of triplicate experiments. Asterisks indicate statistical difference at p < 0.05, compared to each vehicle (PBS)-treated control.

treatment

colony numbers (×102 CFU/mL)

vehicle BPP ip (10 mg/kg) BPP oral (10 mg/kg)

5.02 ± 0.15 a 2.59 ± 0.08 c 2.83 ± 0.11 b

Values are expressed as the mean ± SD of triplicate experiments. Values with the same letters in the column are not significantly different at p < 0.05. a

vehicle (about 48 and 44% decreases in bacterial CFU by ip and oral administration, respectively). Effects of the BPP on Intracellular Bacterial Survival. To examine whether the BPP affects S. Typhimurium survival within macrophage cells, RAW 264.7 cells were treated with three concentrations of the extract (1, 10, and 100 μg/mL) for 4 h and then infected with the bacterial for 1 h. After removal of bacteria from the culture medium, infected macrophages were incubated for another 2, 4, or 8 h in the presence of gentamicin. Figure 4 shows that, 2 h after infection, the intracellular growth of S. Typhimurium within the cells treated with 1, 10, and 100 μg/mL BPP was about 6.9, 45.0, and 87.8% greater than that of the PBS-treated control, respectively. However, 8 h after infection, marked decreases in intracellular bacteria were observed in the cells treated with each concentration of BPP (about 12.2, 52.7, and 65.0% decreases by 1, 10, and 100 μg/ mL BPP treatments, respectively) compared with the PBStreated control. The apparent discrepancy might be due to the fact that at 2 h after bacterial infection, the amount of bacteria in BPP-treated cells was greater than in vehicle-treated cells due to enhanced engulfment of bacteria by the BPP treatment. In contrast, at 4 h and 8 h postinfection, bacteria cell levels in BPP-treated cells

Figure 4. Survival and/or proliferation of S. Typhimurium within the BPP-treated macrophages. Murine macrophage cell line RAW 264.7 cells (1 × 104) were incubated with three concentrations of the BPP (1, 10, and 100 μg/mL) for 4 h and then infected with S. Typhimurium (1 × 104 CFU) at 37 °C for 1 h. The cells were washed and then further incubated in the presence of gentamicin (30 μg/mL) for the indicated periods. At each time point, the cells were lysed and the number of viable intracellular bacteria counted. Data are expressed as the mean ± SD of triplicate experiments. Bars sharing a common letter are not significantly different at p < 0.05.

were markedly reduced compared with vehicle-treated cells, probably because of time-dependent enhancement of intracellular bacteriolysis by the BPP treatment through activation of Th1 immune reaction. 2387

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Salmonella−Immune System Relationships. To facilitate understanding the results and interpretations of the present study, we will briefly mention reported studies designed to define the role of the immune system following infection by Salmonella that complement our related studies with mushroom, herbal, and liquid rice hull extracts. • The innate host defense against intracellular pathogens seems to involve signaling pathways by which macrophages inhibit replication of Salmonella Typhimurium by triggering the MEK kinase cascade and NADPH oxidase activity.15 • Dendritic cells (DCs) link innate and adaptive immunity by recognizing pathogen-associated molecular patterns (PAMPs) to specialized receptors on their surface. Salmonella have developed molecular mechanisms based on virulence proteins to alter DC function and suppress the immune response to cause infection in the host.16 • A mechanism also exists that prevents virulent Salmonella from evading cell uptake, thus enhancing DC immunogenicity, bacterial degradation, and induction of innate and adaptive immune pathways.17,18 • Feeding mice a cornstarch-based diet with added fructooligosaccharide (FOS) or xylo-oligosaccharide (XOS) increased the translocation of S. Typhimurium SL1344 to internal organs, whereas feeding 10% apple pectin increased the intestinal and fecal content of this pathogen, suggesting that the mentioned prebiotics increased the severity of the Salmonella infection.19 The results of the present study indicate that the macrophage cells associated with the immune system seem to have a strong affinity for Salmonella bacteria, and that the BPP treatment induced internalization of the bacteria in the cells. Our results are consistent with the above-mentioned observations in the literature on the interactions of Salmonella with the immune system. Effects of BPP on Leukocyte Activation in Infected Mice. To evaluate whether BPP activates leukocytes in S. Typhimurium infected mice, we also determined mitogeninduced splenic lymphocyte proliferation and IFN-γ levels in sera. As shown in Table 3, administration of BPP markedly

Because NO generation by iNOS is an index of macrophage activation, RT-PCR was used to examine whether the BPP treatment elicited inducible NO synthase (iNOS) gene expression in RAW264.7 murine macrophage cells. Figure 5A

Figure 5. Effects of BPP on iNOS gene expression in S. Typhimuriuminfected macrophages. (A) iNOS mRNA expression profiles assessed by RT-PCR. Murine macrophage cell line RAW 264.7 cells (1 × 106) were cultured with or without BPP (100 μg/mL) for 8 h. Then, S. Typhimurium (1 × 104 CFU) was added to RAW 264.7 cells for 8 h. After incubation, total RNA was purified and iNOS mRNA expression was determined using RT-PCR analysis. Lanes: 1, PBS-treated without bacterial infection; 2, bacterial infection alone; 3, BPP-treated without bacterial infection; 4, BPP-treated with bacterial infection. (B) iNOS polypeptide expression profiles assessed by Western blot. RAW 264.7 cells (1 × 106) treated with BPP (100 μg/mL) for 24 h and subsequent infection of S. Typhimurium were lysed and iNOS protein in the cell lysate was identified by Western blot using anti-mouse iNOS polyclonal antibody. Lanes: 1, PBS-treated without bacterial infection; 2, bacterial infection alone; 3, BPP-treated without bacterial infection; 4, BPP-treated with bacterial infection. The relative proportions of iNOS mRNA and polypeptide are expressed as the R.E. (relative expression) values calculated from iNOS gene/β-actin gene expression. Figures represent results from at least three individual experiments.

shows that, for 8 h, iNOS expression by BPP-treated cells was ∼3.4-fold greater than in the cells infected with S. Typhimurium without BPP. The BPP treatment without S. Typhimurium infection induced iNOS expression to the same level in the bacteria-infected cells with BPP treatment (∼3.5fold increase). Western blot analysis shows that the iNOS protein expression profile was similar to that of the mRNA expression (Figure 5B). Effect of BPP on Th1 and Th2 Cytokine Production. To examine whether BPP has the capacity to regulate the differentiation pathway of CD4 T cells, changes in Th1 and Th2 cytokine production profiles were determined by ELISA assay. Table 4 shows that BPP administration (10 mg/kg) through both peritoneal and oral routes significantly increases Th1 cytokine production including IL-1β, IL-2, IL-6, and IL-12 from splenocyte in S. Typhimurium-infected mice. In contrast, BPP administration did not lead to the enhanced Th2 cytokine production including IL-4, IL-5, and IL-10 in the bacterial infected mice (Table 5). The results imply that BPP can differentiate native CD4 T cells into Th1 cells, thereby potentiating cell-mediated immunity,20 presumably by the above-described mechanisms.

Table 3. Effect of BPP on Spleen Lymphocyte Proliferation and Serum IFN-α Levels in S Typhimurium Infected Micea treatment

lymphocyte proliferation (foldincrease)

IFN-γ (pg/ mL)

vehicle BPP ip (10 mg/kg) BPP oral (10 mg/kg)

1.49 ± 0.11 b 2.18 ± 0.15 a 2.01 ± 0.12 a

309 ± 18 b 408 ± 21 a 375 ± 15 a

Values are expressed as the mean ± SD of triplicate experiments. Values with the same letters in the column are not significantly different at p < 0.05.

a

induced splenic lymphocyte proliferation by Con A stimulation (about 46.3 and 34.9% increases by the treatment by ip and oral routes, respectively). Serum IFN-γ level in S. Typhimuriuminfected mice was also increased by the treatment with BPP (about 32.0 and 21.3% increases by ip and oral treatment, respectively). As anticipated, the treatment through the ip route was more effective than oral administration to induce IFN-γ production, most probably by more effective peritoneal macrophage stimulation. 2388

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Table 4. Effect of BPP on the Th1 Cytokine Production from Splenocytes in Micea cytokines (pg/mL) treatment

IL-1β

IL-2

IL-6

IL-12

vehicle BPP ip (10 mg/kg) BPP oral (10 mg/kg)

188 ± 11 c 205 ± 12 c

Salmonella (−) 44.6 ± 2.3 d 60.6 ± 2.9 b 42.7 ± 3.3 d 61.0 ± 3.4 b

203 ± 15 c

45.3 ± 1.6 d

vehicle BPP ip (10 mg/kg) BPP oral (10 mg/kg)

235 ± 14 b 274 ± 16 a

Salmonella (+) 58.4 ± 2.0 c 71.2 ± 3.6 a 75.8 ± 3.4 a 78.0 ± 3.6 a

290 ± 18 b 358 ± 16 a

261 ± 11 a

66.1 ± 2.9 b

337 ± 16 a

58.5 ± 2.2 b

75.1 ± 2.7 a

252 ± 13 c 259 ± 10 c 263 ± 11 bc

Figure 6. Effect of the BPP on the formation of pathological lesions in liver of S. Typhimurium-infected mice. Liver specimens from BPPtreated mice (10 mg/kg) infected with S. Typhimurium (1 × 104 CFU) were fixed with 4% paraformaldehyde. The sections were then stained with hematoxylin and eosin (H&E). Magnification 100×. Arrows indicate representative hemorrhagic necrosis. Figures represent results from at least three individual experiments.

Values are expressed as the mean ± SD of triplicate experiments. Values with the same letters in the column are not significantly different at p < 0.05.

a

Table 5. Effect of BPP on the Th2 Cytokine Production from Splenocytes in Micea cytokines (pg/mL) treatment vehicle BPP ip (10 mg/kg) BPP oral (10 mg/kg) vehicle BPP ip (10 mg/kg) BPP oral (10 mg/kg)

IL-4 Salmonella (−) 56.0 ± 2.9 ab 55.7 ± 4.9 ab 52.6 ± 2.5 b Salmonella (+) 62.7 ± 3.1 a 63.6 ± 3.9 a 65.0 ± 4.4 a

IL-5

IL-10

283 ± 18 a 286 ± 16 a 283 ± 12 a

49.6 ± 1.1 a 50.0 ± 4.2 a 48.3 ± 1.2 a

302 ± 16 a 304 ± 22 a 305 ± 14 a

53.5 ± 3.1 a 54.0 ± 4.0 a 54.1 ± 4.4 a

Values are expressed as the mean ± SD of triplicate experiments. Values with the same letters in the column are not significantly different at p < 0.05.

a

Figure 7. Histogram showing effect of BPP on S. Typhimurium infection-induced lethality. Balb/c mice (10 mice per group) were infected with lethal doses of S. Typhimurium (1 × 105 CFU) through the intraperitoneal route. Then, the extract (10 mg/kg) was intraperitoneally or orally administered every 24 h during the entire experimental period. PBS was used as the vehicle in the control group. Plotted values are mean values of triplicate determinations.

Effects of BPP on Histopathology of Mouse Livers. To examine whether BPP can ameliorate liver injury induced by salmonellosis, mice were treated ip or orally with BPP (10 mg/ kg) every 24 h for 2 d after intraperitoneal infection of a sublethal dose (1 × 104 CFU) of S. Typhimurium. Figure 6 shows that extensive liver necrosis and hemorrhage were present in the tissues from Salmonella-infected control mice. However, liver tissues from bacteria-infected mice treated with BPP showed minimal damage, regardless of the administration route. These results show that BPP protected the livers against Salmonella-induced hepatic necrosis. Effects of BPP on Mortality of Infected Mice. To determine possible therapeutic effects of BPP on murine salmonellosis, mice were infected with lethal doses (1 × 105 CFU) of S. Typhimurium and assessed for mortality. Each group of 10 mice was treated with BPP (10 mg/kg) ip or orally every 24 h during the entire experimental period. Figure 7 shows that the mortality rate in the PBS-treated control group was 100% on day 7. However, the groups treated with BPP survived until at least day 23 or longer. Ip administration of BPP was more effective in prolonging the lifespan of the mice than administration by the oral route. These observations demonstrate the potential of BPP to protect mice against the

lethal effects of the bacteria, presumably via Th1 cell-mediated macrophage stimulation of the immune system. Beta-glucan might be the active component that induces Th1 cell-mediated macrophage stimulation. This suggestion is supported by the finding that β-glucan content was highest in BPP prepared from L. edodes mycelial culture with black rice bran compared with that from mycelia or black rice bran only (Table 1). Related Studies on Immunoprevention of Salmonella Infections. Here, we briefly mention several additional reported studies on the in vivo inhibition of Salmonella by natural compounds and plant extracts. • Beta-glucan acted as an immunomodulator of the innate immune response in immature chickens that decreased organ invasion of S. enterica.21 • A safety evaluation of a mushroom β-glucan showed that the compound was not mutagenic and exhibited no 2389

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observed adverse effect level (NOAEL) in rats at the highest dose of 2000 mg/kg/bw/day tested,22 suggesting that BPP, which also seems to have a β-glucan structure, might also be safe. • A study of the effect of an aqueous extract of the roots of the Indian herbal plant Withania somnifera on T helper (Th) immunity in mice using flow cytometry indicated selective Th1 upregulation, suggesting its use for selective Th1/Th2 modulation.23 • A water extract of the herb Houttuynia cordata extended the lifespan of mice infected with a lethal dose of S. Typhimurium in a dose-dependent manner, suggesting that the extract has the potential to be used in the treatment of bacterial infection.12 • A water extract of the roots of the Indian medicinal plant Asparagus racemosus containing steroidal saponins exhibited immunoadjuvant potential, as indicated by significant upregulation of Th1 (IL-2, IFN-γ, and IL-4) cytokines, suggesting its mixed Th1/Th2 adjuvant activity.20 It would be of interest to find out whether combinations of BPP with the above-mentioned natural compounds and plant extracts would exhibit additive or synergistic health-promoting effects. Summary and Research Needs. We evaluated the natural biopolymer, previously shown to protect mice against Salmonella lipopolysaccharide-induced endotoxemia, to protect mice against S. Typhimurium-induced sublethal and lethal infections. Administration of BPP by ip injection or by the oral route protected the mice against both liver necrosis and lethality. BPP administered by ip was more effective than that delivered by oral feeding. The protective effect was accompanied by stimulation of innate immune cells, as demonstrated by a series of in vitro and in vivo assays. Because the previous and present studies show that BPP can protect against both endotoxemia and infection without any adverse side effects in mice, it seems that the novel biopolymer has the potential to serve as a multifunctional food. Our observations suggest the need for additional studies that might further enhance the health-promoting characteristics of BPP. These include the following possibilities. • Because outbreaks and exposures associated with Salmonella enterica occur worldwide,24−26 it would be of interest to find out whether human consumption of BPP as part of a normal diet would protect indigenous populations and travelers against infection by Salmonella. • Because Salmonella is a major foodborne pathogen that develops resistance to medical antibiotics,27−29 it would be of interest to find out whether BPP would also protect against antibiotic-resistant Salmonella and other pathogens such as Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, and Vibrio cholerae. • It would be of interest to determine whether herbal or liquid rice hull extracts would also stimulate the production of BPP in mushroom mycelia. Because exposure of contaminated food to natural antimicrobials might be only partly effective in inactivating virulent foodborne pathogens30−32 and toxins33−38 produced by the pathogens and by plants, it would also be worthwhile to determine whether adding BPP to food would protect animals and humans against residual levels of pathogens and toxins after consumption via stimulation of the immune system.

Article

AUTHOR INFORMATION

Corresponding Authors

*S.H.N.: phone, 82-31-219-2619; fax, 82-31-219-1615; e-mail, [email protected]. *M.F.: e-mail, [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Carol E. Levin for facilitating preparation of the manuscript. ABBREVIATIONS USED BPP, bioprocessed polymer; CD-4, helper T cells that promote adhesion to antigen-presenting cells and to B cells in response to infection; CFU, colony-forming units; Con A, concanavalin A; DCs, dendritic cells of the immune system whose main function is to present antigen material to the surface of T cells; ELISA, enzyme-linked immunosorbent assay; dNTP, deoxyribonucleotide triphosphate; FBS, fetal bovine serum; FOS, fructo-oligosaccharide; HBSS, Hanks balanced salt solution; IgG, immunoglobulin G; IL-1β, interleukin 1β; IFN-γ, interferon-γ; iNOS, inducible nitric oxide (NO) synthase; ip, intraperitoneal; LPS, lipopolysaccharide; MEK, mitogenactivated protein kinase/extracellular signal-regulated kinase; MTS, (3-[(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)]; NA medium, nutrient agar medium; NADPH, reduced form of nicotine adenine dinucleotide phosphate; NB medium, nutrient broth medium; NOAEL, no observed adverse effect level; p-NPP, p-nitrophenyl phosphate; PAMPs, pathogen-associated molecular patterns; PBS, phosphate-saline buffer; PDA, potato dextrose agar medium; RPMI 1640 medium, Roswell Park Memorial Institute medium; RT-PCR, reverse transcription polymerase chain reaction; Th1/Th2 cells, functionally subclasses of helper T cells that recognize the presence of microorganisms and stimulate macrophages to destroy invading pathogens; XOS, xylo-oligosaccharide

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REFERENCES

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