Supernatant from Bifidobacterium Differentially Modulates Transduction Signaling Pathways for Biological Functions of Human Dendritic Cells

International audienceBACKGROUND:Probiotic bacteria have been shown to modulate immune responses and could have therapeutic effects in allergic and inflammatory disorders. However, the signaling pathways engaged by probiotics are poorly understood. We have previously reported that a fermentation product from Bifidobacterium breve C50 (BbC50sn) could induce maturation, high IL-10 production and prolonged survival of DCs via a TLR2 pathway. We therefore studied the roles of mitogen-activated protein kinases (MAPK), glycogen synthase kinase-3 (GSK3) and phosphatidylinositol 3-kinase (PI3K) pathways on biological functions of human monocyte-derived DCs treated with BbC50sn.METHODOLOGY/PRINCIPAL FINDINGS:DCs were differentiated from human monocytes with IL-4 and GM-CSF for 5 days and cultured with BbC50sn, lipopolysaccharide (LPS) or Zymosan, with or without specific inhibitors of p38MAPK (SB203580), ERK (PD98059), PI3K (LY294002) and GSK3 (SB216763). We found that 1) the PI3K pathway was positively involved in the prolonged DC survival induced by BbC50sn, LPS and Zymosan in contrast to p38MAPK and GSK3 which negatively regulated DC survival; 2) p38MAPK and PI3K were positively involved in DC maturation, in contrast to ERK and GSK3 which negatively regulated DC maturation; 3) ERK and PI3K were positively involved in DC-IL-10 production, in contrast to GSK3 that was positively involved in DC-IL-12 production whereas p38MAPK was positively involved in both; 4) BbC50sn induced a PI3K/Akt phosphorylation similar to Zymosan and a p38MAPK phosphorylation similar to LPS.CONCLUSION/SIGNIFICANCE:We report for the first time that a fermentation product of a bifidobacteria can differentially activate MAPK, GSK3 and PI3K in order to modulate DC biological functions. These results give new insights on the fine-tuned balance between the maintenance of normal mucosal homeostasis to commensal and probiotic bacteria and the specific inflammatory immune responses to pathogen bacteria

The Dual Effect of Rac2 on Phospholipase D2 Regulation That Explains both the Onset and Termination of Chemotaxis ▿

We document a biphasic effect of Rac2 on the activation and inhibition of PLD2. Cells overexpressing Rac2 and PLD2 simultaneously show a robust initial (<10 min) response toward a chemoattractant that is later (>30 min) greatly diminished over PLD2-only controls. The first phase is due to the presence of a Rac2-PLD2 positive-feedback loop. To explain the mechanism for the Rac2-led PLD2 inhibition (the second phase), we used leukocytes from wild-type (WT) and Rac2−/− knockout mice. Rac2−/− cells displayed an enhanced PLD2 (but not PLD1) enzymatic activity, confirming the inhibitory role of Rac2. Late inhibitory responses on PLD2 due to Rac2 were reversed in the presence of phosphatidylinositol 4,5-bisphosphate (PIP2) both in vitro (purified GST-PH-PLD2, where GST is glutathione S-transferase and PH is pleckstrin homology) and in vivo. Coimmunoprecipitation and immunofluorescence microscopy indicated that PLD2 and Rac2 remain together. The presence of an “arc” of Rac2 at the leading edge of leukocyte pseudopodia and PLD2 physically posterior to this wave of Rac2 was observed in late chemotaxis. We propose Rac-led inhibition of PLD2 function is due to sterical interference of Rac with PLD2's PH binding site to the membrane and deprivation of the PIP2. This work supports the importance of functional interactions between PLD and Rac in the biological response of cell migration