Immunomodulatory properties of Bacillus subtilis extracellular vesicles on rainbow trout intestinal cells and splenic leukocytes

Abstract

Extracellular vesicles (EVs) are cell-derived membrane-surrounded vesicles that carry bioactive molecules. Among EVs, outer membrane vesicles (OMVs), specifically produced by Gram-negative bacteria, have been extensively characterized and their potential as vaccines, adjuvants or immunotherapeutic agents, broadly explored in mammals. Nonetheless, Gram-positive bacteria can also produce bilayered spherical structures from 20 to 400 nm involved in pathogenesis, antibiotic resistance, nutrient uptake and nucleic acid transfer. However, information regarding their immunomodulatory potential is very scarce, both in mammals and fish. In the current study, we have produced EVs from the Gram-positive probiotic Bacillus subtilis and evaluated their immunomodulatory capacities using a rainbow trout intestinal epithelial cell line (RTgutGC) and splenic leukocytes. B. subtilis EVs significantly up-regulated the transcription of several pro-inflammatory and antimicrobial genes in both RTgutGC cells and splenocytes, while also up-regulating many genes associated with B cell differentiation in the later. In concordance, B. subtilis EVs increased the number of IgM-secreting cells in splenocyte cultures, while at the same time increased the MHC II surface levels and antigen-processing capacities of splenic IgM⁺ B cells. Interestingly, some of these experiments were repeated comparing the effects of B. subtilis EVs to EVs obtained from another Bacillus species, Bacillus megaterium, identifying important differences. The data presented provides evidence of the immunomodulatory capacities of Gram-positive EVs, pointing to the potential of B. subtilis EVs as adjuvants or immunostimulants for aquaculture.

Keywords: B cells; Bacillus subtilis; RTgutGC; cell line; extracellular vesicles (EVs); probiotics.

Figure 1

Characterization of the isolated EVs. (A) Protein profile of isolated EVs (M, marker; C, control consisting in the same volume of LB medium without bacteria that was subjected to purification process; EVs=purified EVs by ultracentrifugation at 100,000 x g for 1 h, 18 μg of protein were loaded). (B) Dynamic light scattering (DLS) profile of isolated EVs obtained in a Zetasizer Ultra device. (C) Transmission electron microscopy image of EVs of B. subtilis (representative EVs indicated with arrowheads).

Figure 2

Transcriptional response of RTgutGC cells to different doses of B. subtilis EVs. RTgutGC cells were exposed to 3 x 10⁵, 1.5 x 10⁶ or 3 x 10⁶ per ml of B. subtilis EVs for 24, 48 and 72 h at 19°C. Thereafter, RNA was extracted and the levels of transcription of different genes analyzed by real-time PCR. Data are shown as the relative expression levels of the different genes normalized with the housekeeping gene b-actin (mean + SEM; n = 6). Asterisks denote transcription levels significantly different than those observed in cells exposed to the same volume of culture media subjected to the same purification process (control) (*p ≤ 0.05 and **p ≤ 0.01).

Figure 3

Transcriptional response of splenic leukocytes to different doses of B. subtilis EVs. Splenic leukocytes were exposed to 3 x 10⁵, 1.5 x 10⁶ or 3 x 10⁶ per ml of B. subtilis EVs and incubated for 24 h at 19°C. Thereafter, RNA was extracted and the levels of transcription of different genes analyzed by real-time PCR. Data are shown as relative expression levels of the different genes normalized with the housekeeping gene b-actin (mean + SEM; n = 6 independent fish). Asterisks denote significantly different transcription levels in treated groups compared to controls exposed to the same volume of culture media subjected to the same purification process (control) (*p ≤ 0.05 and **p ≤ 0.01).

Figure 4

Flow cytometry analysis of B cell subsets after exposure to B. subtilis EVs. Splenic leukocytes stimulated or not for 72 h with the different EVs concentrations were labelled with specific mAbs anti-trout IgM and IgD and analysed by flow cytometry. (A) Graphs showing mean percentages of IgM⁺IgD⁺, IgM⁺IgD^- and IgD⁺IgM^- B cells among total lymphoid cells (mean + SEM; n =12 independent fish). (B) Representative dot plot from one fish in which the different B cell subsets are shown after incubation with the different EV concentrations. Asterisks denote significantly different values between control cells (exposed to the same volume of culture media subjected to the same purification process) and cells treated with EVs (**p ≤ 0.01).

Figure 5

MHC II surface expression and antigen-processing capacity of splenic IgM⁺ cells after exposure to B. subtilis EVs. (A) Splenic leukocytes stimulated or not for 72 h with the different EV concentrations were labelled with mAbs anti-trout IgM, IgD and MHC II and analyzed by flow cytometry. A graph showing the mean fluorescence intensity (MFI) values of MHC II surface expression in IgM⁺IgD⁺ B cells (mean + SEM; n =12 independent fish) is shown along with a representative histogram. (B) In other experiments, these splenic leukocytes were incubated with DQ-casein (5 μg/mL) for 1 h at 19°C. Thereafter, cells were labelled with a mAb anti-trout IgM and analysed by flow cytometry. A representative histogram is shown along with a graph displaying the DQ-casein mean fluorescence intensity (MFI) values among IgM⁺ B cells (mean + SEM; n = 6 independent fish). Asterisks denote significantly different values between in cells treated with EVs when compared to control cells (exposed to the same volume of culture media subjected to the same purification process) (*p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001).

Figure 6

Quantification of IgM-secreting cells in splenocyte cultures after exposure to B. subtilis EVs. Splenic leukocytes (1x10⁵ cells) stimulated or not for 72 h with B. subtilis EVs were transferred to ELISpot plates pre-coated with a mAb anti-trout IgM for 24 h. After incubation, cells were washed and a biotinylated mAb anti-trout IgM was used to detect the number of spot-forming cells. Graph indicating mean number of spot-forming cells among 1 x 10⁵ cells (mean + SEM; n=12 independent fish) together with wells from a representative individual. Asterisks denote significantly different values between in cells treated with EVs when compared to control cells (exposed to the same volume of culture media subjected to the same purification process) (***p ≤ 0.001).

Figure 7

Proliferative effect of EVs on splenic IgM⁺ B cells. Splenic leukocytes were stimulated or not for 72 h with the different B. subtilis EV concentrations. After this time, the proliferation of IgM⁺ B cells and IgM^- cells was determined using Click-IT™ EdU cell proliferation kit Alexa Fluor™. The percentage of proliferating cells (EdU⁺) among IgM⁺ B cells and IgM^- cells is shown in graphs (mean + SEM; n = 12 independent fish) along with representative dot plots obtained in one individual. Asterisks denote significantly different values in treated groups compared to controls (exposed to the same volume of culture media subjected to the same purification process) (*p ≤ 0.05 and ***p ≤ 0.001).

Figure 8

Comparative effects of EVs obtained from two Bacillus species. (A) Comparative transcriptional effects of B. subtilis or B. megaterium EVs on RTgutGC cells. For this, cells were incubated with 1.5 x 10⁶ particles/ml or with the same volume of control media subjected to the same purification process for 48 h at 19°C. Thereafter, RNA was extracted and the levels of transcription of different genes were analyzed by real-time PCR. Data are shown as relative expression levels of the different genes normalized with the housekeeping gene b-actin (mean + SEM; n = 5 independent fish). Different lowercase letters indicate significant differences among groups (p ≤ 0.05). (B) Quantification of IgM-secreting cells by ELISpot in splenocyte cultures exposed to B. megaterium, B. subtilis or to control media as described for (A) Cells were transferred to ELISpot plates pre-coated with mAb anti-trout IgM for 24 h. After incubation and washing cells, a biotinylated mAb anti-trout IgM was used to detect the number of spot-forming cells. A graph showing the quantification of spot-forming cells (mean + SEM; n = 9 independent fish) is included along with representative wells from one individual. Different lowercase letters indicate significant differences among groups (p ≤ 0.05).