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<strong>4. DISCUSSION</strong> <p>The mucosal immune system is continuously exposed to dietary and microbial antigens and needs to react with appropriate immune responses. Compelling evidence suggested that appropriate microbial colonization of the gut is important in providing signals to prevent the overexpression of Th2-dominated atopic responses [22]. In the last few years, the potential role of selected probiotic strains in the prevention of allergic diseases has become more evident. Nevertheless the mechanism underlying these properties remains unknown. Intestinal epithelial cells permanently interact with the luminal content, including the commensal flora, and the cellular network of immune cells. Because of their location at mucosal sites and their implication in the initiation of the immune reaction, DC play a key role in this process. In order to evaluate the involvement of DC intestinal environment on their function, we used a well-established model of epithelial cells andDC coculture [10, 21, 23].We analyzed the effect of a selected L. casei strain, which was previously demonstrated to reduce allergen-specific Th2 secretion in vitro [20]. In the present study, we demonstrated that monocyte-derived DC from healthy or allergic donors take up nonpathogenic L. casei through an intestinal epithelium. The analysis of the FITC-bacteria uptake in the Transwellmodel showed that the process is time-dependent, increasing between 24 and 48 h, and higher in healthy donors compared to allergic ones. The number of DC which have captured bacteria through the epithelium was low even after 48 h in comparison with DC directly interacting with L. casei (without the epithelium). Using fluorescent beads, we checked that beads incubated in the upper chamber did not pass through the epithelium. Moreover, because no change in TER was observed, we hypothesize that in our conditions DC open tight junctions and sample bacteria by extending their dendrites through the epithelium without disrupting epithelial integrity as it was previously demonstrated in the same model [10]. This active process may explain the time required to capture bacteria efficiently in the presence of the epithelium. By confocal analysis, we clearly confirmed the bacteria uptake by DC as L. casei colocalized with adherent DC.</p> <p>By producing cytokines and/or by expressing specific costimulatory molecules, DC can modulate the development of T cell response and are considered as pivotal cells in the development or the limitation of the allergic reaction. DC phenotype and functions are dependent upon their mucosal environment. In particular, intestinal epithelial cells condition DC to become “noninflammatory” in vitro and may be involved in their capacity to maintain gut homeostasis [21, 24]. Here we showed that DC from both allergic and healthy donors acquired an intermediate maturation status characterized by a moderate expression of cell-surface costimulatory molecules and maturation markers upon stimulation with L. casei in the presence of the intestinal epithelium. This intermediate maturation status has previously been described in different experimental settings. The ability of two Gram-positive bacteria, pathogenic Streptococcus pyogenes, and nonpathogenic Lactobacillus rhamnosus to induce monocyte-derived DC maturation has been compared. S. pyogenes was shown to be a more potent stimulator of DC maturation than L. rhamnosus. Indeed, this lactic acid bacteria induced a partial DC maturation, as evidenced by the moderate expression of costimulatory molecules CD80, CD83, and CD86 and a weak cytokine and chemokine response [25]. The same observations were reported with activation by direct contact of human DC by a Gram-negative pathogenic bacteria compared to L. rhamnosus and with activation of murine DC with various strains of Lactobacilli [18, 26]. Similarly, in amodel of intestinal epithelium, activation with L. plantarum induced a lower expression of CD80 and CD83 compared with activation with Gram-negative pathogenic bacteria [24]. As costimulatory molecules are involved in contact between DC and T cells during antigen presentation, a moderate expression of these molecules may result in orientation toward a T regulatory pathway [27]. The cytokine microenvironment plays a critical role during T cell polarization. For example, IL-12 is important to skew T cell differentiation towards Th1-type response, and IL-10 is associated with immunoregulatory processes [28, 29]. Here we showed that L. casei-induced IL-10, and IL-12 production by DC from allergic donors was lower compared to healthy donors, which may be related to a lower adherence to the epithelium and a lower number of DC which have captured L. casei. However, as this lower production of IL-10 and IL-12 by DC from allergic donors was also obtained after L. casei stimulation in the absence of the epithelium, this suggests a dysfunction of DC from allergic compared to healthy donors. Both cytokines were shown to be produced by murine DC stimulated by a mixture of bifidobacteria and lactobacilli (VSL#3) [30]. We also noticed that, in the presence of the intestinal epithelium, DC from allergic patients produced higher levels of IL-12 and IL-10 compared to cells directly in contact with bacteria. The interactions of L. casei with epithelial cells might have an effect on IL-12 and IL-10 secretion by DC from allergic patients, therefore balancing the potential defect in their production. Indeed, some mediators released by epithelial cells might induce “noninflammatory DC,” which contribute to maintain gut immune homeostasis, and limit excessive inflammatory reactions like allergy [31]. Consequently, in allergic patients, stimulation with nonpathogenic L. casei associated with the anti-inflammatory effect of the epithelium might favor the development of semimature DC, characterized by moderate expression of costimulatorymolecules and low cytokine production that could counterbalance the Th2-biased immune response [27, 32].</p> <p>T cell activation and polarization of the immune response may be influenced by DC maturation status and DC cytokine production induced by L. casei. In the present study, we showed that the epithelium increased the cytokine production by autologous T cells instructed by L casei-activated DC derived from healthy and allergic donors, and especially the IFN-γ. Increased IFN-γ induced by LAB has previously been shown in a system of direct contact with bacteria using DC from allergic and healthy donors [20, 33]. In vivo, another LAB strain (L. plantarum) exhibited Th1 promoting capacity in a murine model of birch pollen allergy [34]. IL-5 and IL-10 production were measured to analyze the effect of LAB activation on the Th2 or on the immunoregulatory responses, respectively. L. casei-pulsed adherent DC conditioned by the intestinal epithelium moderately increased the secretion of IL-5 and IL-10 in both allergic and healthy donors. The increase in IL-5 production is in agreement with a previous study using DC derived from healthy donors and activated by L. plantarum in the presence of an intestinal epithelial monolayer [24]. However, in the absence of the epithelium, L. plantarum has been shown to reduce the Th2 response induced by DC from allergic donors stimulated by the relevant allergen; whilst it did not modify the response in healthy donors [20]. This suggests that LAB may differentially regulate the immune response according to their environment. Selective lactobacilli strains (L. casei and L. reuteri) have been demonstrated to drive the generation of regulatory IL-10+ T cells through DC activation in vitro [35]. Moreover, L. rhamnosus has been shown to induce a peripheral hyporesponsiveness in stimulated CD4+ T cells [36]. IL-10 is a cytokine known to limit an inflammation due to IFN-γ or IL-5 production. Even though, in our experiment, the moderate increased secretion of IL-10 in the coculture of T cells with L. casei-pulsed adherent DC may be due to increased production by DC, the balance between IL-10 and IFN-γ or IL-5 might regulate potential immune regulatory pathways. Thus, strains of lactobacilli might differentially affect DC and lead to the development of different T cell responses but each mechanism seems to control the immune homeostasis. Moreover, although DC nonadherent to the filter, that is, not in direct contact with the epithelium, exhibited a similar maturation status compared to adherent DC, nonadherent DC from both allergic and healthy donors failed to increase T cell cytokine production upon L. casei stimulation (data not shown). These results indicate that the epithelium plays a role in the capacity of L. casei-activated DC to regulate T cells, even though the sole presence of the epithelium decreases the Th1 response as previously demonstrated [31]. DC in contact with L. plantarum and epithelial cells in the Transwell model have been shown to be more “inflammatory” because of their capacity to release IL-12, IL- 10 and to promote T cell proliferation, compared to DC activated only by epithelial mediators [24]. The crosstalk between epithelial cells, DC, and LAB might allow on the one hand the establishment of a protective effect against any antigen encountered, vital for combating pathogenic organisms, and on the other hand the limitation of potentially damaging inflammatory immune reactions against endogenousmicroflora. In conclusion, our results demonstrate efficient interactions between L. casei and DC through the intestinal epithelium. By modulating DC function and by preserving its capacity to react against harmful pathogenic organisms, L. casei may contribute to maintain the homeostasis of the gut immune system. Finally, by limiting inappropriate immune activation, L. casei might prevent or reduce inflammatory responses such as the development of an allergic inflammatory response.</p> <p><strong><em>ACKNOWLEDGMENTS</em></strong></p> <p><em>The authors are grateful to Jo¨elle Dewulf (BLIM, IPL) and Philippe Marquillies (Inserm U416) for technical assistance and to Bruno Pot for advice. The authors also thank the personnel of the Calmette Hospital for the selection of patients and the blood collection. C´eline Ratajczak is a recipient of a grant fromConseil R´egional Nord-Pas-de-Calais and Institut Pasteur de Lille. Catherine Duez is a recipient of a grant from the Danone Institute. Jo¨el Pestel is a recipient of a grant from Syndifrais.</em></p>
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