Differential effect of Bifidobacterium species on cytokine production in a Caco-2/PBMC co-culture system

Abstract

Bifidobacterium species present in the gut microbiota have shown capacity to modulate cytokine production by intestinal epithelial cells, monocyte derived dendritic cells and peripheral blood mononuclear cells (PBMC) in in vitro experiments (1,2) . These findings may confer them the potential to be used as probiotics in the prevention and treatment of pathologies with underlying immune alterations, such as inflammatory bowel diseases, allergy and celiac disease.The aim of this study was to evaluate the effect of different bifidobacterial species (Bifidobacterium adolescentis ATCC15703; Bifidobacterium angulatum ATCC27535; Bifidobacterium breve ATCC15700; Bifidobacterium catenulatum LMG; Bifidobacterium longum biovar infantis LMG 11046T; B. longum biovar longum ATCC15707) on cytokine production in a Caco-2/PBMC co-culture system. Caco-2 monolayers were grown on 12 mm inserts (1 mm pore size) in 24-well cell culture plate assemblies (Millipore) and challenged by apical addition of 4 · 10 6 cfu/insert of a Bifidobacterium strain or a combination of strains corresponding to the species composition in faecal samples from breast fed (BF) and formula fed (FF) infants (3) , respectively. PBMCs isolated from healthy adult donors were added in the basal compartment of the culture well (1 · 10 6 cells/ml) for a 12-hour incubation. Thereafter, a further 36-h incubation was allowed after disassembly of the system in order to measure the cytokine production by the sensitised Caco-2 and PBMCs separately. IL-8 was measured in Caco-2 cells' basolateral medium by ELISA and IFN-g was measured in the PBMCs supernatant by high sensitivity Immunoassay Xmap Technology (Linco). Samples from 5 volunteers and duplicates of each experimental condition were used. Mann-Whitney test was employed for statistical analysis. B. breve, B. longum and the BF mixture induced a higher IL-8 production by Caco-2 cells than B. infantis and FF mixture. The rest of the Bifidobacterium species assayed showed intermediate levels and no differences with other strains were observed. A correlation between IL-8 production stimulated by B. breve and by BF was found, which can be explained by the singularly high relative presence of B. breve in the BF combination (22 %) compared to the FF mixture (7 %). The production of IFN-g by PBMCs was low in this system (range: 1-93 pg/ml and under the detection limit for one donor) and the effect of each strain seemed donor dependent. However, 3 donors showed stimulation of IFN-g with BF mixture and 2 of them also with B. breve. Significant differences between IFN-g production induced by BF mixture (349°161% control; Mean°SE) and B. adolescentis (-16°14 %control) were observed. The results suggest that B. breve is the most stimulatory strain and more so when combined with other Bifidobacterium strains, probably indicating synergistic effects. The presence or absence of particular bifidobacterial species as well as the overall composition of the bifidobacterial population in the infant gut could be key factors defining the i...

In Vitro Screening and Evaluation of Synbiotics

“…To better understand immunological responses, the co-culture system of IECs and peripheral blood mononuclear cells may be applied for studying cytokine modulation by different bacteria (Parlesak et al, 2004;Pozo et al, 2010;Pozo-Rubio et al, 2011) and could give valuable insight into immune-modulatory effects of synbiotics before testing in humans, which is especially relevant for at-risk groups such as premature infants, elderly, immunocompromised individuals suffering from, e.g., inflammatory bowel diseases.…”