INFLUENCE OF ACENOCOUMAROL ON THE INTERACTION BETWEEN Bifidobacterium AND THP-1 CELLS

ASAAD, S; FRAGOMENO, M; MINNAARD, J; PÉREZ, P.F

S.M Tucuman

Congreso; XII Congreso de Microbiología General; 2017

SAMIGE

Bifidobacteria are microorganisms widely used as probiotics. In previous studies we demonstrated the strain dependant immunomodulatory effects on THP-1 cells, a model of phagocytic cells. Moreover, studies have demonstrated that probiotics modify the bioavailability of certain drugs but there is no evidence of the action of the drugs on probiotic activity Our goal is to study the effect of acenocoumarol, one of the most used oral anticoagulant, on the interaction between Bifidobacteria and THP-1 cells.THP1 cells were differentiated with phorbol miristate acetate 200 nM in DMEM (10% Fetal bovine serum) for 48h at 37ºC/5% CO2. Bifidobacterium bifidum strain CIDCA 5310 and Bifidobacterium adolescentis strain CIDCA 5317 were cultured (48h;37ºC) in MRS broth in anaerobic conditions. To evaluate the effect of the drug on the phagocytosis activity, FITC-labeled bacteria were incubated with cells at MOI 10 (multiplicity of infection) and acenocoumarol at different concentrations for 1h at 37ºC/5% CO2. Phagocytosis was evaluated by flow cytometry. For quenching of non-internalized bacteria, trypan blue was used. UI (Uptake Index) = FL1(+) cells x mean fluorescence intensity, was calculated. A decrease of phagocytosis (11015.93 ± 707.46, P< 0.05) of CIDCA 5310 was observed in presence of the drug (471,64 uM) compared to the strain alone (14895.53 UI ± 331.61), while the association showed a reducing trend (16400.80 UI ± 2539.16 vs 21248.4 ± 920.59, P= 0.12). For strain CIDCA 5317, acenocoumarol did not affect neither association nor phagocytosis.Futhermore, intracellular localization of strain CIDCA 5310 was evaluated in presence of the drug in acid vesicles (Lysotracker DND-99) and in recycled endosomes (Transferrin Alexa-594) by confocal laser microscopy. In acid vesicules, the drug did not modified the localization percentage values (54 % and 50%). It may be inferred that the recycling endosomes are not used by this strain even in the presence of the drug. The expression of HLA-DR and TLR2 was measured by flow cytometry after 16hs of incubation with the strain CIDCA 5310 and the drug. Medium Fluorescence Intensity (IFM) was calculated. Positive controls HLA-DR (INF gamma) (1288,02 ± 127,28) and TLR2 (S. aureus) (1401,22 ± 4,33) were determinated. Strain CIDCA 5310 alone or with the drug diminished the expression of HLA-DR (263.89 ±10.65 and 481.32 ± 89.46, respectively). The lowest value of IFM was detected when THP-1 cells were incubated with INF-gamma in presence of the anticoagulant (41.40 ± 5.64). On the other hand, acenocoumarol decreased the expression of TLR2 when cells were incubated with strain CIDCA 5310 (573.04 ± 99.36) as compared with the strain alone (1832.05 ± 433.33) p