Modulatory Effect of Linoleic Acid During Brucella abortus 544 Infection in Murine Macrophage RAW264.7 Cells and Murine Model BALB/c Mice
1Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju, 52828, Republic of Korea
2College of Veterinary Medicine, Chonbuk National University, Iksan, 54596, Republic of Korea
J. Microbiol. Biotechnol. 2020; 30(5): 642-648
Published May 28, 2020
Copyright © The Korean Society for Microbiology and Biotechnology.
In our unpublished data, we performed metabolome profiling of plasma samples from
Materials and Methods
The animal procedures performed in this study were approved by the Animal Ethical Committee of Chonbuk National University (Authorization Number CBNU-2018-119).
Linoleic Acid (LA) Preparation
LA (molecular weight 280.45 g/mol; Sigma-Aldrich, USA) was dissolved in absolute ethanol (1 M) and further diluted in sterile phosphate-buffered saline solution (PBS, pH 7.4) containing 0.1% bovine serum albumin (BSA, GenDEPOT, USA).
A smooth, virulent
RAW 264.7 cells (ATCC TIB7-1, USA) were maintained at 37°C in 5% CO2 in RPMI 1640 containing 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (all provided by Gibco, USA) and were seeded (1 × 105 cells/ml in 96-well plates; 1 × 106 cells/ml in 6-well plates) in tissue culture plates overnight. Cells were incubated in fresh medium without antibiotics prior to all bacterial infection assays.
RAW264.7 cells were prepared in a 96-well plate overnight and then incubated at different concentrations of LA (0, 10, 20, 50, 100, 200, 500 µM) for 48 h. Cytotoxicity analysis was performed using MTT assay as previously reported . The control used contains 0.1% ethanol and 0.1% BSA in fresh medium without antibiotics in all in vitro assays.
Bacteria were grown to stationary phase and diluted using PBS (2 × 104 colony forming units, CFU). A 10 µl bacterial solution was added to different concentrations of LA (0, 10, 50, 100, 500 µM) for 0, 2, 24, and 48 h. Bactericidal analysis was performed using CFU counts as previously reported .
RAW264.76 cells were prepared in a 96-well plate overnight and then incubated with or without LA (10 µM) for at least 4 h. The cells were infected with
For internalization assay, RAW264.7 cells were prepared in a 96-well plate overnight and incubated with or without LA for at least 4 h as previously described . The cells were washed and then infected with
Eight-week-old, pathogen-free female BALB/c mice (Samtako Bio Co. Ltd., Korea) acclimatized for one week were randomly divided into four groups of five mice. The groups were further subdivided into non-infected and
Serum alanine aminotransferase 1 (ALT) concentration was quantified using an ALT (Mouse) ELISA Kit (BioVision Inc., USA) to monitor hepatocellular injury during LA treatment according to manufacturer’s instruction.
Culture supernatants and serum samples were processed to measure the different levels of cytokines involved in the course of brucellosis including IL-12p70, TNF, IFN-γ, MCP-1, IL-10, and IL-6 using a Cytometric Bead Array (CBA) mouse inflammation kit (BD Biosciences, USA) according to manufacturer’s instruction.
The data are expressed as the mean ± standard deviation (SD) of triplicate samples from at least three independent experiments. Student’s t-test was used to make statistical comparisons between groups using GraphPad InStat software version 3 (GraphPad Software, Inc., USA). Differences of
Effect of LA on Viability of RAW264.7 Cells and Survival of
Decreased OD values were observed in RAW264.7 cells treated with LA at concentrations of 20, 50, 100, 200, and 500 µM. OD values did not change in cells incubated at 10 µM (data not shown) compared to untreated controls, hence treatment with LA was applied at a concentration of 10 µM in the subsequent experiments. On the other hand, bacterial cells incubated with various concentrations of LA (10, 50, 100, and 500 µM) significantly inhibited the growth of
The bactericidal effect of the different concentrations of LA (0, 10, 50, 100, and 500 μM) againstData represent the mean ± SD of at least three replicates. Notes: * B. abortusincubated for 0, 2, 24, and 48 h. p< 0.05, *** p< 0.001, compared with control group.
Effect of LA on Internalization and Intracellular Survival of
RAW264.7 cells were pretreated with LA for at least 4 h prior to
The inhibitory effect of LA on (A) internalization and (B) intracellular growth efficiency ofData represent the mean ± SD of at least three replicates. Note: * B. abortusin RAW264.7 cells incubated at indicated times. p< 0.05, compared with control group.
Effect of LA on Nitrite Production in RAW264.7 Cells
RAW264.7 cells were pretreated with LA for at least 4 h and then infected with
The effect of LA on nitrite accumulation in RAW264.7 cells during (A) without infection and (B) B. abortusinfection at indicated times. Data represent the mean ± SD of at least three replicates. Note: * p< 0.05, compared with control group.
Effect of LA on Cytokine Production in RAW264.7 Cells
RAW264.7 cells were infected with
The effect of LA on cytokine production in RAW264.7 cells duringData represent the mean ± SD of at least three replicates. Note: * B. abortusinfection. p< 0.05, compared with control group.
Effect of LA on
B. abortus Infection in Mice
The mice were observed for any clinical symptoms during the entire treatment period. Body weight and serum ALT concentrations were checked at the end of the experiment and showed no differences between treated and untreated control groups without
The effect of LA on (A) spleen weight and (B) bacterial splenic proliferation duringData represent the mean ± SD of five mice. Note: ** B. abortusinfection in BALB/c mice. p< 0.01, compared with control group.
Effect of LA on Cytokine Production During
B. abortus Infection in Mice
In the uninfected groups, no significant differences in the serum level of cytokines were observed between LA-treated and untreated groups at 3 and 14 d post-infection. On the other hand, in the
The effect of LA on the production of serum cytokines in BALB/c mice at 3 and 14 d post-infection. Data are presented as the means ± SD for each group.Notes: * p< 0.05, ** p< 0.01, compared with B. abortus-infected group.
PUFAs have been suggested to function as endogenous anti-bacterial, anti-fungal, anti-viral, anti-parasitic and immunomodulating agents . It was also proposed that PUFAs hold inhibitory action against bacterial growth via cell membrane disruption . Dilika
Cell-mediated immunity and macrophage activation, both controlled by cytokine production during infection, are associated with host resistance to intracellular parasites . We therefore further investigated the immunomodulatory activities of LA on
This research was supported by a fund (Project Code No.Z-1543061-2019-20-01) by Research of Animal and Plant Quarantine Agency, South Korea.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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