cytokines [13–15]. Therefore, this recognition provides a platform for modulation of the local innate and systemic adaptive immune response in the host [16–19]. Immune assays have shown that the in vitro immune response to probiotics is both species- and strain- specific [12]. Interestingly, some probiotics secrete antimicrobial factors that affect both virulence gene expression in pathogenic bacteria [2,20] and gene expression in the host epithelium [21]. In a previous study, a novel LAB strain was isolated from the faeces of exclusively breast-fed newborn infants and selected based on its probiotic properties, such as adhesion to intestinal mucus, sensitivity to antibiotics and resistance to gastrointestinal juices, biliary salts, NaCl and low pH. We identified this strain as Lactobacillus paracasei CNCM I-4034 [22]. The aim of the present study was to investigate the capacity of L. paracasei CNCM I-4034 and its cell-free culture supernatant (CFS) to activate human intestinal DCs, to determine how they respond to pathogenic bacteria, specifically Salmonella typhi, and to elucidate the molecular mechanisms involved in these interactions. The expression of genes involved in TLR signalling and cytokine secretion was analysed. Results DCs Co-cultured with the Probiotic and the Enteropathogen Show a Markedly Reduced Pro- inflammatory Response The immunomodulatory effects of L. paracasei CNCM I-4034 were studied in human DCs. The DCs were incubated with the probiotic (live bacteria or CFS) and the pathogen, either individually or together. Our data indicate that probiotic bacteria and their CFS can induce cytokine secretion; this induction was similar in response to probiotic or CFS stimulation. As shown in Figures 1 and 2, the addition of pathogenic bacteria (S. typhi CECT 725) or LPS to DCs markedly increased the secretion of pro- inflammatory cytokines. In response to stimulation with the probiotic or its supernatant and the enteropathogen, the secretion of all of the pro-inflammatory cytokines (Figures 1, 2) such as IL-6 and TNF-a and chemokines (Figure 3) including MCP-1/CCL2 and RANTES/CCL5 was drastically reduced. TGF-b levels clearly showed that L. paracasei is a potent inducer of TGF-b. As shown in figure 3, this bacteria increased TGF- b2 production, while the probiotic supernatant increased the secre- tion of TGF-b1 in response to Salmonella. We did not detect the production of TGF-b3 under any of the treatment tested conditions (data not shown). Interestingly, the supernatant of L. paracasei was as effective as the live probiotic bacteria in reducing the secretion of pro- inflammatory cytokines, suggesting that this probiotic secretes metabolites or factors with anti-inflammatory properties. The Probiotic Strain L. paracasei CNCM I-4034 Induces the Expression of TLR Signalling Pathway Genes in Human DCs We also evaluated the expression patterns of genes within the TLR signalling pathway. These receptors regulate a variety of different genes, including those that function as transcriptional activators, such as NFkB, whose induction results in the expression of a wide variety of cytokines [23]. As shown in Figures 4, 5, 6, 7, the exposure of DCs to S. typhi for 4 hours resulted in an upregulation of TLR9 (Figure 5), which was accompanied by an increase in the expression of TLR1, TLR2, TLR4, TLR5, IRAK4, TAK1, JNK (Figures 4, 5, 6) and IL-10 Modulation of Human DCs by a Lactobacillus Strain (Figure 7). In addition, it was found that Salmonella downregulated CASP8 (Figure 6) and TNF-a (Figure 7) gene expression. The probiotic bacterium L. paracasei CNCM I-4034 and its CFS exhibited similar abilities to regulate TLR pathways (Figures 4, 5, 6, 7). They both induced strong and sustained TLR9 transcription (Figure 5). Regarding TLR, the probiotic strain alone activated TLR1, TLR2 and TLR4, whereas its supernatant increased TLR5 expression (Figure 4). Interestingly, in response to stimulation with CFS and Salmonella, TLR1, TLR2, TLR3, TLR4, TLR5 and TLR9 expression was increased, while the exposure of DCs with the probiotic and Salmonella downregulated TLR1–TLR5 gene expres- sion (Figure 4, 5). In response to stimulation with the probiotic or its CFS and Salmonella, TOLLIP (Figure 5), CASP8 and TAK1 (Figure 6) and TNF-a expression was increased (Figure 7). Discussion There is growing evidence that probiotics, especially lactobacilli and bifidobacteria, have immunomodulatory properties [24]. DCs provide an interface between the innate and adaptive immune systems, acting as professional antigen-presenting cells [8]. Innate PRRs, such as TLRs, are found on epithelial and immune cell surfaces [25] and play a crucial role in the host recognition of bacteria, such as probiotics [10]. During inflammation, these receptors interact with microbe-associated molecules, such as LPS and bacterial DNA, resulting in DC activation. These pathways affect various DC functions, including cytokine production, and the cytokine profile determines the type of T-cell response that will develop [26]. Therefore, this study mainly focused on comparing the expression patterns of genes involved in the TLR signalling pathway and the cytokine profile mediated by exposure to a probiotic or its CFS in the presence or absence of S. typhi. We employed an in vitro culture system to study the immunological effects and anti-inflammatory properties of the novel probiotic strain L. paracasei CNCM I-4034, which was isolated from the faeces of breast-fed newborns. Specific probiotic strains have been shown to interact with DCs and induce strain-specific effects [27]. In this context, L. paracasei CNCM I-4034 and its supernatant dramatically reduced the production of all of the evaluated pro-inflammatory cytokines and chemokines in the presence of S. typhi. The co-incubation of human DCs with Salmonella and L. paracasei (live bacteria) significantly reduced the ability of Salmonella to induce IL-6, IL-8, IL-12p 70 and TNF-a. Interestingly, L. paracasei was not able to induce IL-6 secretion. This effect was observed in the presence or absence of pathogens. This observation contrasts with that of Weiss et al. who reported that lactobacilli and bifidobacteria are potent inducers of IL-6 [28]. However, this author reported these effects in murine DCs cells and as we mentioned above murine DCs are quite different from human gut DCs in several respects. In this paper, we used human DCs differentiated from umbilical cord blood CD34+ progenitor cells (hematopoietic stem cells). These human DCs are equivalent to those DCs found in Langerhans islets, which extend dendrites and sample antigens, akin to lamina propria DCs in the gut. In addition, as mentioned, L. paracasei decreased TNF-a production in response to Salmonella. During the inflammatory process, TNF- a functions at the apex of the inflammatory cascade [29] as a consequence of NFkB activation; NFkB regulates the transcrip- tional activation of a number of genes involved in immune and pro-inflammatory responses [30]. Therefore, this probiotic strongly inhibited the inflammatory response of human DCs to Salmonella. In addition, Mileti et al. demonstrated that Lactobacillus PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e43197