Probiotic Escherichia coli Nissle 1917-derived outer membrane vesicles enhance immunomodulation and antimicrobial activity in RAW264.7 macrophages

Abstract

Background: Probiotic Escherichia coli Nissle 1917 (EcN) has been widely studied for the treatment of intestinal inflammatory diseases and infectious diarrhea, but the mechanisms by which they communicate with the host are not well-known. Outer membrane vesicles (OMVs) are produced by Gram-negative bacteria and deliver microbial molecules to distant target cells in the host, which play a very important role in mediating bacteria-host communication. Here, we aimed to investigate whether EcN-derived OMVs (EcN_OMVs) could mediate immune regulation in macrophages.

Results: In this study, after the characterization of EcN_OMVs using electron microscopy, nanoparticle tracking and proteomic analyses, we demonstrated by confocal fluorescence microscopy that EcN_OMVs could be internalized by RAW 264.7 macrophages. Stimulation with EcN_OMVs at appropriate concentrations promoted proliferation, immune-related enzymatic activities and phagocytic functions of RAW264.7 cells. Moreover, EcN_OMVs induced more anti-inflammatory responses (IL-10) than pro-inflammatory responses (IL-6 and TNF-α) in vitro, and also modulated the production of Th1-polarizing cytokine (IL-12) and Th2-polarizing cytokine (IL-4). Treatments with EcN_OMVs effectively improved the antibacterial activity of RAW 264.7 macrophages.

Conclusions: These findings indicated that EcN_OMVs could modulate the functions of the host immune cells, which will enrich the existing body of knowledge of EVs as an important mechanism for the communication of probiotics with their hosts.

Keywords: Escherichia coli Nissle 1917; Extracellular vesicle; Macrophages; Microbiota-host communication; Outer membrane vesicles; Probiotics.

Fig. 1

Visualization and characterization of OMVs derived from probiotic Escherichia coli Nissle 1917 (EcN_OMVs). a The particle numbers of the resulting fractions (1–10) after EcN_OMVs purification by Optiprep density gradient ultracentrifugation. Representative scanning electron micrograph (b) and transmission electron micrograph (c) of purified EcN_OMVs (indicated by red arrows). d Size distribution and concentration of these vesicles determined by nanoparticle tracking analysis. e Subcellular localizations of EcN_OMVs proteins identified by proteomic analysis. f Identified proteins according to Clusters of Orthologous Groups of proteins (COG)

Fig. 2

Internalization of EcN_OMVs by RAW264.7 macrophages. DiI-labeled EcN_OMVs (red signal) were incubated with RAW264.7 cells for 16 h at 37 °C. The cell membrane was visualized by immunostaining with anti-F4/80 antibody (5 μg/mL), the macrophage marker, followed by Dylight Fluor-conjugated Goat Anti-Rat IgG (green signal), and the cell nuclei were stained with DAPI (blue signal). The samples were observed using the High-speed spinning-disk confocal microscope

Fig. 3

EcN_OMVs at moderate concentrations promote the proliferation of RAW264.7 cells. a Viability of RAW264.7 cells at 16 h after exposure to EcN_OMVs at various concentrations. b The LDH activity in the cell supernatant at 16 h after exposure to EcN_OMVs at various concentrations. Data are representative of three independent experiments. *P < 0.05; **P < 0.01; versus control

Fig. 4

EcN_OMVs modulate immune-related enzymatic and phagocytic activities in RAW264.7 cells. RAW264.7 cells were stimulated with EcN_OMVs and heat-killed EcN for 16 h. After these stimulations, cell supernatants and lysates were collected for examining the following indicators: a ACP activity in cell lysates; b NO production in cell supernatants; c iNOS activity in cell lysates. d Phagocytic activity of RAW264.7 cells to FITC-labeled dextran at 16 h after stimulations with EcN_OMVs and heat-killed EcN. Data are representative of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; NS., not significant; versus control

Fig. 5

Profile of cytokine secretion by RAW264.7 macrophages stimulated with EcN_OMVs. RAW264.7 cells were stimulated with EcN_OMVs and heat-killed EcN for 16 h. After these stimulations, cell supernatants were collected for determining the following cytokines: a IL-6; b TNF-α; c IL-10; d IL-12p40; e IL-4. Data are representative of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; versus control

Fig. 6

Antimicrobial activity of macrophages stimulated with EcN_OMVs. RAW264.7 cells stimulated with EcN_OMVs were incubated with three bacterial pathogens for 5 h, including two Gram-negative species, E. coli CVCC1554 and S. typhimurium CVCC3757, and one Gram-positive specie, S. aureus CVCC4265. After incubation, the number of viable bacteria in cell lysates was determined using the plate cultivation method. Data are representative of three independent experiments. ***P < 0.001