Extracellular vesicles derived from Lactobacillus gasseri GFC-1220 alleviate inflammation via the TLR4/NF-κB signaling pathway in LPS-stimulated RAW264.7 macrophages

Abstract

Studies on the impact of gram-positive extracellular vesicles have paved the way for novel medical advancements. These vesicles serve as efficient carriers of microbial molecules to target cells, thereby influencing human pathophysiological processes. Thus, this study aimed to investigate the anti-inflammatory properties of extracellular vesicles derived from Lactobacillus gasseri GFC-1220 (LEVs) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. LEVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and dynamic light scattering measurements to examine their morphology, size, and concentration. We further assessed the anti-inflammatory effects of LEVs and their underlying mechanism in LPS-stimulated RAW264.7 macrophages. Our findings revealed that LEVs did not cause cytotoxic effects and significantly decreased the level of mitochondria superoxide and reactive oxygen species production. In addition, these vesicles effectively inhibited the LPS-induced activation of TLR4/NF-κB signaling pathway, consequently suppressing the secretion and expression of various pro-inflammatory mediators and cytokines. These included a reduction in the production of NO and PGE₂, along with their corresponding producer enzymes iNOS and COX-2, as well as IL-6, TNF-α, and IL-1β cytokines. These findings strongly suggest that LEVs have significant potential for the development of new anti-inflammatory agents.

Keywords: Lactobacillus gasseri GFC-1220; Anti-inflammatory effects; Extracellular vesicles; Macrophages.

Fig. 1

Maximum likelihood tree based on 16S rRNA gene sequences of L. gasseri GFC-1220.

Fig. 2

Characterization of LEVs in terms of structure and quantity. Representative images of Cryo-EM analysis (A); the particle size and concentration of LEVs determined using NTA (B); zeta potential analysis (C).

Fig. 3

Effects of LEVs on cell viability. The effect of LEVs on the viability of LPS-stimulated RAW264.7 cells using MTT assay (A); and live/dead staining, with live cells (green color) and dead cells (red color) (B). Significant differences between groups are indicated by different superscripts, as determined by Duncan’s multiple range test (p < 0.05).

Fig. 4

Effects of LEVs on ROS production and Mito-SOX detection in LPS-stimulated RAW264.7 cells. Significant differences between groups are indicated by different superscripts, as determined by Duncan’s multiple range test (p < 0.05).

Fig. 5

Effects of LEVs on LPS-induced pro-inflammatory cytokines and mediators in RAW264.7 macrophages. The suppressed effect of LEVs on NO production (A); and the secretion of PGE₂ measured in LPS-induced RAW264.7 macrophages (B); The effect of LEVs on the LPS-induced gene expression of iNOS (C) and COX-2 (D). The inhibitory effect of LEVs on the IL-6, TNF-α, and IL-1β secretion (E) and gene expression (F) in the activated RAW264.7 cells. Significant differences between groups are indicated by different superscripts, as determined by Duncan’s multiple range test (p < 0.05).

Fig. 6

Effects of LEVs on the release and gene expression of pro-inflammatory cytokines in LPS-stimulated RAW264.7 cells treated with TAK-242 inhibitor. IL-6, TNF-α, and IL-1β secretion (A); IL-6, TNF-α, and IL-1β expression (B). Significant differences between groups are indicated by different superscripts, as determined by Duncan’s multiple range test (p < 0.05).

Fig. 7

Effects of LEVs on the TLR4/NF-κB signaling pathway in LPS-stimulated RAW264.7 cells. The inhibitory effects of LEVs on the gene expression analysis of TLR4/NF-κB pathway (A); and western blot analysis of NF-κB pathway (B). Samples for the same marker were obtained from the same blot. Original and full-length blots are presented in supplementary Fig. S2. Significant differences between groups are indicated by different superscripts, as determined by Duncan’s multiple range test (p < 0.05).