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. 2022 Nov 13;10(11):2247.
doi: 10.3390/microorganisms10112247.

Immunostimulatory Activity of Lactic Acid Bacteria Cell-Free Supernatants through the Activation of NF-κB and MAPK Signaling Pathways in RAW 264.7 Cells

Jaekoo Lee  1 , Seonyoung Kim  1 , Chang-Ho Kang  1
Affiliations

Immunostimulatory Activity of Lactic Acid Bacteria Cell-Free Supernatants through the Activation of NF-κB and MAPK Signaling Pathways in RAW 264.7 Cells

Jaekoo Lee et al. Microorganisms. .

Abstract

Lactic acid bacteria (LAB) can improve host health and has strong potential for use as a health functional food. Specific strains of LAB have been reported to exert immunostimulatory effects. The primary goal of this study was to evaluate the immunostimulatory activities of novel LAB strains isolated from humans and foods and to investigate the probiotic properties of these strains. Cell-free supernatants (CFS) obtained from selected LAB strains significantly increased phagocytosis and level of nitric oxide (NO) and pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 in RAW264.7 macrophage cells. The protein expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2, which are immunomodulators, was also upregulated by CFS treatment. CFS markedly induced the phosphorylation of nuclear factor-κB (NF-κB) and MAPKs (ERK, JNK, and p38). In addition, the safety of the LAB strains used in this study was demonstrated by hemolysis and antibiotic resistance tests. Their stability was confirmed under simulated gastrointestinal conditions. Taken together, these results indicate that the LAB strains selected in this study could be useful as probiotic candidates with immune-stimulating activity.

Keywords: RAW264.7 macrophage cells; immune-stimulating activity; lactic acid bacteria; probiotics.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effect of CFS from LAB strains on the phagocytosis of RAW264.7 macrophage cells. Cells were treated with LPS (10 ng/mL) or CFS (5 mg/mL) for 24 h, and phagocytosis was evaluated by adding neutral red dye for 2 h. (a) The morphology of the untreated (control) or treated cells was visualized by phase-contrast light microscopy at a 20× magnification. (b) Phagocytic activity was quantified by measuring the logarithmic intensity using a microplate reader. Data are presented as the mean ± SD of three independent experiments (n = 3). Different letters indicate significant differences between means at p < 0.05 based on Duncan’s multiple range test.
Figure 2
Figure 2
Effect of CFS from LAB strains on the protein expression levels of iNOS and COX-2 in RAW264.7 cells. Representative Western blot and expression of iNOS (a,c) and COX-2 (b,d). Cells were treated with LPS (10 ng/mL) or CFS (5 mg/mL) for 24 h. The protein expression levels were quantified by densitometry, and the data were normalized relative to the intensity of β-actin. Untreated cells (control) were treated with DMEM only. Data are presented as the mean ± SD of three independent experiments (n = 3). Different letters indicate significant differences between means at p < 0.05 based on Duncan’s multiple range test.
Figure 3
Figure 3
Effect of CFS from LAB strains on cytokine production in RAW264.7 cells. Cells were treated with LPS (10 ng/mL) or CFS (5 mg/mL) for 24 h. The amounts of TNF-α (a) and IL-6 (b) secreted by cells were measured using an ELISA kit. Data are presented as the mean ± SD of three independent experiments (n = 3). Different letters indicate significant differences between means at p < 0.05 based on Duncan’s multiple range test.
Figure 4
Figure 4
Effect of CFS from LAB strains on NF-κB activation in RAW264.7 cells. Representative Western blot (a) and activation levels of NF-κB (b). Cells were treated with LPS (10 ng/mL) or CFS (5 mg/mL) for 24 h. The NF-κB activation levels were quantified using densitometry. Data are presented as the mean ± SD of three independent experiments (n = 3). Different letters indicate significant differences between means at p < 0.05 based on Duncan’s multiple range test.
Figure 5
Figure 5
Effect of CFS from LAB strains on MAPK activation in RAW264.7 cells. Cells were treated with LPS (10 ng/mL) or CFS (5 mg/mL) for 24 h. Representative Western blot (a) and the activation levels of MAPK; ERK (b), JNK (c), and p38 (d) were quantified by densitometry. Data are presented as the mean ± SD of three independent experiments (n = 3). Different letters indicate significant differences between means at p < 0.05 based on Duncan’s multiple range test.
Figure 6
Figure 6
Hemolysis of LAB strains. (a) L. reuteri MG5462, (b) Lc. lactis MG4668, (c) Lc. lactis MG5278, (d) Lc. lactis MG5474, (e) L. fermentum MG4263, (f) L. fermentum MG4268, and (g) L. fermentum MG4282.
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