Anti-inflammatory effects of yeast-derived vacuoles on LPS-induced murine macrophage activation

Abstract

Saccharomyces cerevisiae is a single-celled fungal microorganism. S. cerevisiae-derived vacuoles are closely related to mammalian lysosomes, which play a role in the degradation of macromolecules by various hydrolytic enzymes. This study evaluated the anti-inflammatory efficacy of S. cerevisiae-vacuoles by inhibiting inflammatory mediators induced by lipopolysaccharide (LPS). The results showed that treatment with 5, 10, and 20 µg/mL of S. cerevisiae-derived vacuoles almost completely inhibited the LPS-induced expression of iNOS protein and mRNA. Moreover, vacuoles significantly reduced the mRNA expression of tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), and interleukin 6 (IL-6) in LPS-stimulated macrophages compared to the control cells. The immunofluorescence analysis confirmed that S. cerevisiae-derived vacuoles inhibited the translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in LPS-stimulated cells. Taken together, the treatment with S. cerevisiae-derived vacuoles alone activated macrophages, but LPS-activated macrophages modulated pro-inflammatory mediators by downregulating the NF-κB pathway. These results suggest that S. cerevisiae-derived vacuoles may have therapeutic potential in the treatment of inflammatory diseases. In conclusion, our study provides new insights into the immunomodulatory effects of S. cerevisiae-derived vacuoles and their potential as a novel anti-inflammatory agent. IMPORTANCE This study investigates the potential of using vacuoles derived from the yeast Saccharomyces cerevisiae as a new anti-inflammatory therapy. Inflammation is a natural response of the immune system to invading pathogens, but when it is dysregulated, it can lead to chronic diseases. The researchers found that treating macrophages with vacuoles significantly reduced the production of pro-inflammatory cytokines and iNOS, markers of inflammation when they were stimulated with lipopolysaccharide. The study also showed that vacuoles inhibited the NF-κB signaling pathway, which is involved in the induction of pro-inflammatory cytokines in macrophages. These findings suggest that S. cerevisiae-derived vacuoles may have potential as a new therapeutic agent for regulating the inflammatory response in various diseases. Further studies are needed to evaluate the efficacy and safety of vacuoles in vivo and to elucidate the underlying mechanisms of their anti-inflammatory effects.

Keywords: S. cerevisiae-derived vacuole; anti-inflammatory; lipopolysaccharide; macrophage.

Fig 1

Characterization of S. cerevisiae-derived vacuoles. (A) Bio-TEM images of the S. cerevisiae-derived vacuoles. (B) FE-SEM images of the vacuoles. (C) Size distribution curve of the vacuoles analyzed by NTA.

Fig 2

Effect of S. cerevisiae-derived vacuoles on the expression of iNOS in LPS-stimulated RAW 264.7 cells. (A) The cells were stimulated with 1 µg/mL LPS for 2 hours at the indicated doses, treated with dexamethasone or vacuoles for 22 hours, and then assessed for iNOS protein using Western blot analysis. (B) The relative ratio of iNOS to β-actin was determined using densitometry, with LPS being employed as a control.

Fig 3

S. cerevisiae-derived vacuoles inhibit LPS-induced pro-inflammatory cytokines such as (A) TNF-α and (B) IL-6 protein production. (C) S. cerevisiae-derived vacuole enzyme and pellet components change TNF-α cytokine production. Cells were stimulated with 1 µg/mL LPS at the indicated doses for 2 hours, treated with dexamethasone or vacuoles for 22 hours, and then analyzed using ELISA.

Fig 4

Inhibitory effect of S. cerevisiae-derived vacuoles on mRNA expression levels of (A) iNOS, (B) COX-2, (C) TNF-α, and (D) IL-6 in RAW 264.7 cells stimulated by LPS. The cells were stimulated with 1 µg/mL LPS for 2 hours, followed by treatment with dexamethasone or vacuoles for 22 hours. The mRNA levels were measured using reverse transcription quantitative PCR (RT-qPCR). The gene expression levels were normalized to the expression of the housekeeping gene GAPDH.

Fig 5

Effect of S. cerevise-derived vacuoles on the translocation of NF-κB p65 in LPS-stimulated RAW 264.7 cells. Cells were stimulated with 1 µg/mL LPS, and the nuclear and cytoplasmic localization of p65 was evaluated using an NF-κB p65 monoclonal antibody, a goat anti-rabbit IgG H&L antibody (green), and DAPI nuclear stain (blue). Scale bar = 10 µM.