Immunomodulatory effects of lactobacilli from green tea leaf in murine macrophages and intestinal epithelial cells: the role of bacterial RNA

MASAHIKO SUZUKI; TSUKIHO HIURA; HIDEO SATSU; YUJI TSUJIKAWA; IWAO SAKANE; JULIO VILLENA; HARUKI KITAZAWA

Conferencia; The 11th Asian Conference for Lactic Acid Bacteria; 2021

The immunomodulatory effects of lactic acid bacteria (LAB) were reported to depend on each specific strain. In addition, although great advances were made in understanding the specific molecules involved in the lactobacilli´s modulating effects, studies are still needed to deepen this knowledge. Thus, the purpose of this study was to evaluate the immunomodulatory activities of LAB isolated form green tea leaf by using murine in vitro models and investigate the potential role of bacterial DNA and RNA molecules in such effect. In the first experiments, several heat-killed LAB from green tea leaf (LOC strains) were evaluated in mouse macrophage-like J774.1 cells according their ability to enhance IL-12 production. Furthermore, heat-killed LAB treated with DNase or RNase were also used to evaluate the effect of those molecules in macrophages´ IL-12 secretion. The well-characterized immunomodulatory strains Lactiplantibacillus plantarum CRL1506 (1) and Lacticaseibacillus rhamnosus CRL1505 (2) were used for comparisons. Among the several LAB strains evaluated, Lactiplantibacillus plantarum LOC1 showed the highest capacity to improve the IL-12 secretion in macrophages. The abilities of LOC1, CRL1505 and CRL1506 strains to enhance IL-12 were unchanged by DNase treatment, while they were significantly decreased by RNase treatment. The capacity of L. plantarum LOC1 to modulate macrophages was further evaluated by studying the expression of TNF-a, IL-12, CSF2, CSF3, SOCS1, IL-10 and IL-27 after the challenge with LPS. The LOC1 strain improved the expression of several mRNAs in LPS-challenged macrophages. In the second experiments, the LOC1 strain was evaluated in a co-culture system of mouse macrophage-like RAW264.7 cells and murine intestinal epithelial (MIE) cells (3). MIE cells were pre-cultured on transwell inserts and stimulated with heat-killed LAB. The transwell insert plates with MIE cells were added into multiplate wells preloaded with RAW264.7 macrophages and then, heat-killed lactobacilli were applied to the apical sides of individual wells. After 24 hours, macrophages were directly stimulated with LPS and the expression of several mRNAs were evaluated in macrophages. The most immunological factors were not modified with the treatment of LOC1 when compared to LPS-challenged macrophages. The results presented here demonstrated that L. plantarum LOC1 is able to modify macrophages function when it interacts directly with these cells, while this capacity is reduced when the interaction is made indirectly through the intestinal epithelial cells. The data presented here also demonstrate that RNA would be a key bacterial molecule involved in the ability of the LOC1 strain to modulate macrophages function.