We found that doses below 100 pg/l significantly decrease the production of TNF-�� by LPS-stimulated RAW 264.7 cells in vitro, while similarly prepared Lp did not (Figure 5A). Using the FACS analysis of cultured cells, we found that neither Lc nor Lp changes the viability of RAW 264.7 cells (data not shown). The treatment with either lysate of bacteria in the absence of LPS did not change the TNF-�� www.selleckchem.com/products/Y-27632.html production (data not shown), this data is in agreement with a study using L. casei 3260 [34]. As published by others [34], [35], this result suggests that Lc could interfere with the intracellular proinflammatory signaling cascade leading to activation of NF-��B transcription factor. To test this hypothesis, we isolated the nuclear extract from the untreated RAW 264.

7 cells or from cells treated with either LPS (1 mg/l), or LPS with Lc and measured the activity of the NF-��B signaling pathway. Lc significantly decreased the NF-��B/DNA binding activity of p65 subunit as compared to the LPS-only or Lp+LPS treated cells (Figure 5B). Figure 5 Lc exerts anti-inflammatory effect on LPS-activated macrophage cell line RAW 264.7. Since Lc treatment decreased production of TNF-�� by LPS-activated macrophages, we decided to characterize macrophages further by investigating their stage of polarization by FACS. We found that M2 phenotype marker, the mannose receptor CD206 was significantly upregulated and M1 phenotype marker IL-7R downregulated in LPS+Lc treated macrophages as compared to either LPS or LPS+Lp treated macrophages. Therefore, Lc seems to counteract the LPS mediated M1 polarization.

Neither Lc nor Lp without the addition of LPS changes the macrophage polarization. Discussion Oral treatment with probiotic bacteria has emerged recently as a potentially useful therapeutic strategy for human IBD [5], [36]. However, the clinical utility of such approach remains controversial, as the link between specific mechanisms of action and therapeutic effects of specific bacterium has been difficult to establish. We have shown previously that repeated oral administration of probiotic bacteria L. casei DN-114 001 protects BALB/c mice from severe forms of acute intestinal inflammation [21]. In this study we demonstrated that not only live probiotic bacteria, but also its lysate protects BALB/c, but not SCID, mice from severe forms of DSS-induced inflammation. The lack of protective effect in SCID mice suggests that mechanisms of adaptive immunity are essential for the beneficial effect of Lc. We did not find any changes neither in Lc-specific serum IgA, IgG and IgM, nor in gut SIgA during our experiments (data Cilengitide not shown), so we analyzed another mechanism executed by adaptive immune response, oral tolerance.