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

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.

L-methionine supplementation modulates IgM+ B cell responses in rainbow trout.

The interest in dietary amino acids (AAs) as potential immunomodulators has been growing the recent years, since specific AAs are known to regulate key metabolic pathways of the immune response or increase the synthesis of some immune-related proteins. Methionine, tryptophan and lysine are among the ten essential AAs for fish, meaning that they cannot be produced endogenously and must be provided through the diet. To date, although dietary supplementation of fish with some of these AAs has been shown to have positive effects on some innate immune parameters and disease resistance, the effects that these AAs provoke on cells of the adaptive immune system remained unexplored. Hence, in the current study, we have investigated the effects of these three AAs on the functionality of rainbow trout (Oncorhynchus mykiss) IgM+ B cells. For this, splenic leukocytes were isolated from untreated adult rainbow trout and incubated in culture media additionally supplemented with different doses of methionine, tryptophan or lysine in the presence or absence of the model antigen TNP-LPS (2,4,6-trinitrophenyl hapten conjugated to lipopolysaccharide). The survival, IgM secreting capacity and proliferation of IgM+ B cells was then studied. In the case of methionine, the phagocytic capacity of IgM+ B cells was also determined. Our results demonstrate that methionine supplementation significantly increases the proliferative effects provoked by TNP-LPS and also up-regulates the number of cells secreting IgM, whereas tryptophan or lysine have either minor or even negative effects on rainbow trout IgM+ B cells. This increase in the number of IgM-secreting cells in response to methionine surplus was further verified in a feeding experiment, in which the beneficial effects of methionine on the specific response to anal immunization were also confirmed. The results presented demonstrate the beneficial effects of dietary supplementation with methionine on the adaptive immune responses of fish.