Human Intestinal Dendritic Cells Decrease Cytokine Release against Salmonella Infection in the Presence of Lactobacillus paracasei upon TLR Activation Miriam Bermudez-Brito1, Sergio Mun˜ oz-Quezada1, Carolina Gomez-Llorente1, Esther Matencio2, Mar´ıa J. Bernal2, Fernando Romero2, Angel Gil1* 1 Institute of Nutrition and Food Technology Jose´ Mataix, Biomedical Research Centre, Department of Biochemistry and Molecular Biology II, University of Granada, Granada, Spain, 2 Hero Institute for Infant Nutrition, Hero Spain, Alcantarilla, Murcia, Spain Abstract Probiotic bacteria have been shown to modulate immune responses and could have therapeutic effects in allergic and inflammatory disorders. However, little is known about the signalling pathways that are engaged by probiotics. Dendritic cells (DCs) are antigen-presenting cells that are involved in immunity and tolerance. Monocyte-derived dendritic cells (MoDCs) and murine DCs are different from human gut DCs; therefore, in this study, we used human DCs generated from CD34+ progenitor cells (hematopoietic stem cells) harvested from umbilical cord blood; those DCs exhibited surface antigens of dendritic Langerhans cells, similar to the lamina propria DCs in the gut. We report that both a novel probiotic strain isolated from faeces of exclusively breast-fed newborn infants, Lactobacillus paracasei CNCM I-4034, and its cell-free culture supernatant (CFS) decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with Salmonella. Interestingly, the supernatant was as effective as the bacteria in reducing pro-inflammatory cytokine expression. In contrast, the bacterium was a potent inducer of TGF-b2 secretion, whereas the supernatant increased the secretion of TGF-b1 in response to Salmonella. We also showed that both the bacteria and its supernatant enhanced innate immunity through the activation of Toll-like receptor (TLR) signalling. These treatments strongly induced the transcription of the TLR9 gene. In addition, upregulation of the CASP8 and TOLLIP genes was observed. This work demonstrates that L. paracasei CNCM I-4034 enhanced innate immune responses, as evidenced by the activation of TLR signalling and the downregulation of a broad array of pro-inflammatory cytokines. The use of supernatants like the one described in this paper could be an effective and safe alternative to using live bacteria in functional foods. Citation: Bermudez-Brito M, Mu n˜oz-Quezada S, Gomez-Llorente C, Matencio E, Bernal MJ, et al. (2012) Human Intestinal Dendritic Cells Decrease Cytokine Release against Salmonella Infection in the Presence of Lactobacillus paracasei upon TLR Activation. PLoS ONE 7(8): e43197. doi:10.1371/journal.pone.0043197 Editor: Benoit Foligne, Institut Pasteur de Lille, France Received May 15, 2012; Accepted July 20, 2012; Published August 14, 2012 Copyright: 2012 Bermudez-Brito et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by Hero Spain S.A. through the contract nu 3143 signed with the Fundaci o´n General Universidad de Granada Empresa and co- sponsored by the CDTI, a public entity of the Ministry of Economy and Competitiveness of the Spanish Government. Carolina Gomez-Llorente received a postdoctoral fellowship from Plan Propio of University of Granada. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Esther Matencio, Mar´ıa J. Bernal and Fernando Romero are members of the Hero Institute for Infant Nutrition, Hero Spain S. A. The sponsor had no role in the biological sample analysis, statistical analysis or data interpretation. This does not alter our adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: agil@ugr.es Introduction Probiotics are defined as ‘‘live microorganisms that, when administered in adequate amounts, confer a health benefit on the host’’ [1]. Bifidobacteria and lactic acid bacteria (LAB), primarily lactobacilli, are generally referred to as probiotics because of their health-promoting properties, such as the exclusion or inhibition of pathogens in the gut, the enhancement or maintenance of barrier function and the local and systemic modulation of the host immune system [2,3]. The clinical applications of lactobacilli and bifidobacteria include preventing and treating allergic diseases, particularly in relieving the symptoms of atopic eczema [4] and allergic rhinitis [5], reducing diarrhoea in children [6], preventing inflammatory bowel disease and viral infection and as adjuvants in vaccines [7]. Despite growing evidence of the immunomodulatory effects of probiotics, there is little information available regarding their mechanisms of action. Dendritic cells (DCs) are professional antigen-presenting cells and are essential mediators of immunity and tolerance [8,9]. The control of the immune response by DCs is particularly important in the gut, in which the immune system exists in intimate association with commensal bacteria, such as LAB. In their immature state, DCs reside in peripheral tissues, continuously sampling the microenvironment, sensing the presence of patho- gens and releasing chemokines and cytokines to amplify the immune response [10]. Furthermore, DCs interact directly with bacteria that have gained access via M cells [11]. Innate pattern- recognition receptors (PRRs), such as Toll-like receptors (TLRs), NOD-like receptors (NLRs) and C-type lectin receptors (CLRs), play crucial roles in the host recognition of probiotics and other microorganisms [12]. The binding of microbe-associated mole- cules to these receptors can activate antigen-presenting cells and modulate the activation of important transmission factors, such as nuclear factor kappa B (NFkB) and the production of different PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e43197