JW15 (KACC 91811P) isolated from Kimchi (Korean traditional fermented vegetables) was grown in De Man Rogosa and Sharpe (MRS) broth (BD, USA) at 37 °C for 18 hours and viable bacterial cells were counted on MRS plates. The cells were collected by centrifugation at 14 000 g for 10 minutes and the culture supernatant discarded. The pellet was washed twice with sterile phosphate-buffered saline (PBS, pH 7.2). The probiotic cells (1×108 CFU/mL) were heat-killed at 110 °C for 15 minutes and stored at −20 °C until use. The well-known Lactobacillus rhamnosus GG (LGG, ATCC 53103) was used as the reference strain.
The murine macrophage cell line RAW 264.7 (Korean Cell Line Bank, Korea) and RAW BLUE cells (InvivoGen, USA) were grown in Dulbecco's modified Eagle's medium (DMEM; HyClone, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco Laboratories, USA) and 100 units/mL of streptomycin and penicillin (Gibco Laboratories) at 37 °C in a humidified atmosphere with 5% CO2. After subculturing four to five times, RAW BLUE cells were cultured with 100 mg/mL of zeocin (InvivoGen).
RAW 264.7 macrophages (5×105 cells/well) and RAW BLUE (5×105 cells/well) cells were seeded in a 12-well plate. The viable and heat-killed JW15 (100 μL containing 5×108 or 1×108 CFU/mL) were added to each well. The probiotic concentration was adjusted such that each macrophage cell was exposed to either 20 or 100 probiotic cells at 37 °C and 5% CO2. Macrophages incubated with PBS were used as a negative control, while those treated with lipopolysaccharide (LPS) (100, 500, and 1 000 ng/mL; Sigma, USA) in PBS were used as a positive control. For experiments containing viable JW15, RAW 264.7 cells were cultured in gentamycin (50 μg/mL). After 48 hours, the culture supernatants were collected and the concentration of NO, NF-κB, and cytokines (IL-6 and TNF-α) in the supernatant were measured.
The cytotoxicity of the viable and heat-killed JW15 against RAW 264.7 cells was evaluated based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide MTT (Sigma-Aldrich, USA) method. RAW 264.7 cells were plated at a density of 1×105 cells in a 96-well plate, followed by their treatment with viable or heat-killed JW15 at 37 °C for 48 hours. Cells were washed twice with PBS and incubated with 0.5 mg/mL MTT for 4 hours. The purple-colored formazan was solubilized in dimethyl sulfoxide (DMSO) for at least 1 hour in the dark and the absorbance of the solution measured at 550 nm wavelength by an enzyme-linked immunosorbent assay (ELISA) reader (Tecan, Austria). Cell viability was calculated as follows: Cell viability=[D(550 nm) of sample]/[D(550 nm) of control]×100%.
The level of NO produced by macrophages was determined using Griess reagent (Promega, USA). A total of 50 μL cell culture supernatant or nitrite standard (0–100 μmol/L sodium nitrite) was treated with an equal volume of 1% sulfanilamide in 5% phosphoric acid and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride at room temperature (20 °C to 25 °C) in the dark for 10 minutes. The absorbance of the reaction solution was measured at 540 nm using a microplate reader. Samples were assayed in triplicates. NO concentrations were calculated using a nitrite standard curve.
To investigate whether JW15 activates NF-κB pathway, RAW BLUE cells stably transfected with the secreted alkaline phosphatase (SEAP) reporter gene placed under the transcriptional control of an NF-κB response element were used. SEAP was secreted in cell culture media upon NF-κB pathway activation. SEAP secretion was measured using alkaline phosphatase substrate QUANTI-Blue (InvivoGen) as per the manufacturer's instructions. The absorbance of the sample was measured at 620 nm with a microplate reader.
The production level of cytokines (IL-6 and TNF-α) in the culture supernatant was analyzed according to the ELISA assay protocol provided by the supplier (eBioscience, USA). Briefly, 96-well plates (SPL, Korea) were coated overnight with captured antibodies against IL-6 and TNF-α in a coating buffer at 4 °C. The plates were washed with PBS containing 0.05% (v/v) Tween 20 (PBST, BioShop, Canada) and incubated at room temperature for 1 hour to prevent any nonspecific protein binding. After washing with PBST, standard IL-6 and TNF-α and supernatant samples were incubated at room temperature for 2 hours. All standards and samples were tested in triplicates. The samples were treated with detection antibodies for 1 hour. After washing, the plate was treated with avidin-horseradish peroxidase (HRP) for 30 minutes and washed seven times with PBST. The wells were incubated with tetramethylbenzidine (TMB) in the dark for 15 minutes and the reaction was stopped with 2 N sulfuric acid (H2SO4). The absorbance was read at 450 nm with a microplate reader and the concentration of cytokines calculated from the standard curves of each cytokine standard (0–1 000 pg/mL for IL-6 and TNF-α).
RAW 264.7 cells cultured in a six-well plate for 24 hours were pretreated with viable and heat-killed JW15 for 24 hours at 37 °C and 5% CO2. After incubation, cells were washed in cold PBS. Total RNA was prepared with TRIzol reagent (Invitrogen, USA) and the concentration of total RNA measured by recording the absorbance at 260 nm using an Epoch Microplate Spectrophotometer (Bio Tek Instruments Inc., USA). One microgram of RNA was reverse transcribed into first-strand cDNAs using a Moloney murine leukemia virus (MMLV) reverse transcriptase (iNtRON Bio, Korea) and random primers (9-mers; TaKaRa Bio Inc., Japan). The relative expression of inducible nitric oxide synthase (iNOS), IL-1β, TNF-α, and IL-6 was detected by ABI 7300 Real-Time PCR system (Applied Biosystems, USA) according to the manufacturer's protocol and normalized to the level of β-actin as the reference gene. Primer sequences are listed in Table 1.
Gene Primer sequence (5′ → 3′) iNOS F: TCCCTTCCGAAGTTTCTGGC
IL-1β F: CCTTGGGCCTCAAAGGAAAGAATC
TNF-α F: GAACTGGCAGAAGAGGCACT
IL-6 F: TCCATCCAGTTGCCTTCTTG
β-actin F: ATCACTATTGGCAACGAGCG
Table 1. Primers for quantitative real-time PCR
RAW 264.7 macrophages were harvested by centrifugation at 6 000 g for 10 minutes and washed in cold PBS. Washed cell pellets were treated with Pro-prep solution (iNtRON Biotechnology, Korea) for 2 hours at 4 °C and the proteins collected by centrifugation at 14 000 g for 15 minutes. The supernatant was transferred to a new tube and the protein concentration was measured with the Bio-Rad protein assay reagent (Bio-Rad Laboratories Inc., USA). An equal amount of protein (50 μg) was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroblotted onto a polyvinylidene fluoride transfer membrane (Perkin Elmer Co., USA). The membrane was blocked with 5% skim milk at room temperature and incubated overnight with primary antibodies at 4 °C. After washing in Tris-buffered saline with Tween 20 (TBST), the membrane was incubated with secondary antibodies for 1 hour. The antibody-specific protein was detected with an enhanced chemiluminescence reagent (Amersham Biosciences, UK) with ChemiDoc equipment GeneGnome 5 (Syngene, UK). The density measurement of each band was carried out using NIH Image J software.
All data were analyzed using SPSS 12.0 for Windows Version (SPSS Inc., USA). Significant differences between groups were tested by ANOVA and compared using Duncan's test (P<0.05).
The immune-modulating effects of viable Weissella cibaria JW15 on RAW 264.7 macrophage cells
- Received Date: 2019-06-28
- Accepted Date: 2019-08-14
Abstract: The objective of this study is to investigate the immune-enhancing ability of viable and heat-killed Weissella cibaria JW15 (JW15) isolated from Kimchi in RAW 264.7 macrophages. The immune effects were evaluated by measuring the production of NO, cytokines, inflammatory enzyme, and activation of NF-κB. Viable JW15 executed higher activity on stimulating the release of TNF-α as well as activating NF-κB compared to that of heat-killed JW15. Additionally, viable and heat-killed JW15 significantly increased the production of NO, IL-6 and TNF-α more than that of Lactobacillus rhamnosus GG (LGG). Furthermore, viable JW15 induced higher production of iNOS compared with that of viable LGG. Collectively, our finding indicates that viable JW15 had similar, if not more, immune-enhancing activities as heat-killed JW15. In addition, viable JW15 had higher immune-enhancing activity than commercial strain LGG. Therefore, viable JW15 has the potential to be used as a functional food to improve the host immune response.