Exopolysaccharides from Lactobacillus rhamnosus RW-9595M stimulate TNF, IL-6 and IL-12 in human and mouse cultured immunocompetent cells, and IFN-$\gamma$ in mouse splenocytes

Abstract

International audienceExopolysaccharides (EPS) from Lactobacillus rhamnosus RW- 9595M have been prepared from bacterial cultures, isolated, concentrated, fractionated and tested in vitro for their possible modulating properties on mouse splenocytes from the C57Bl/6 and BALB/c strains, on the murine RAW 264.7 macrophage-like cell line and on human Peripheral Blood Mononuclear Cells (PBMC) from a total of 14 healthy donors. A first step of EPS fractionation was attempted, using membranes with different molecular weight cut-off. Fractions were as follows: F1: $> $1000 kg$\cdot$mol$^{-1}$; F2: 1000-100 kg$\cdot$mol$^{-1}$; F3: 100-10 kg$\cdot$mol$^{-1}$; F4: $< $10 kg$\cdot$mol$^{-1}$. Total EPS, as well as F1, appeared slightly mitogenic in both mouse splenocytes and human PBMC in 2-3 d cultures, and F3 also exhibited such a property on human PBMC. Unfractionated concentrated ("total" ) EPS, as well as F1, elicited TNF, IL-6 and IL-12 p40 both in the mouse and human cells, in 6 h and 24 h cultures, with important variability depending on the cell source. In 24 h cultures, total EPS or F1 elicited bio-active IFN-$\gamma$ in both C57Bl/6 and BALB/c splenocytes, and this IFN-$\gamma$ secretion was sustained until at least 3 d of culture. In human PBMC, no IFN-$\gamma$ production was observed despite high IL-12p40 secretion. These results suggest the possibility of enhancing the immune system through EPS from lactic acid bacteria, in individuals responsive to such a stimulus.Productions de TNF, IL-6, IL-12 et IFN-g chez des cellules immunocompétentes traitées avec des exopolysaccharides du Lactobacillus rhamnosus RW-9595M. Différence entre les réponses de cellules de sang périphérique humain et de splénocytes de souris. Les exopolysaccharides (EPS) du Lactobacillus rhamnosus RW- 9595M ont été préparés à partir de cultures bactériennes, isolés, concentrés, fractionnés et testés in vitro quant à leur potentiel immunomodulateur sur des splénocytes de souris C57Bl/6 et BALB/c, sur la lignée macrophagique murine RAW.264.7 et sur des cellules mononuclées du sang humain provenant de 14 donneurs. Nous avons fractionné ces EPS en fonction des tailles moléculaires, avec des membranes filtrantes de différentes porosités. Les fractions correspondent aux tailles suivantes : F1 : $> $ 1000 kg$\cdot$mol$^{-1}$, F2 : 1000-100 kg$\cdot$mol$^{-1}$, F3 : 100-10 kg$\cdot$mol$^{-1}$ et F4 : $< $ 10 kg$\cdot$mol$^{-1}$. Les EPS concentrés, non fractionnés, ainsi que F1, se sont avérés légèrement mitogènes sur des splénocytes de la souris, ou des cellules sanguines humaines, cultivées 2-3 jours. Dans le dernier cas, F3 manifeste également cette propriété. Les EPS non fractionnés, ainsi que F1, sont capables de provoquer la production de TNF, IL-6, IL-12p40 après 6 ou 24 h de culture, l'origine des cellules étant importante pour l'intensité de la réponse. Après 24, 48 et 72 h de culture, les EPS non fractionnés, ainsi que F1, ont provoqué la production d'interféron gamma par des splénocytes de souris C57Bl/6 et BALB/c. Au contraire, dans le ca...

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