A previous work demonstrated that L. casei CRL 431 administration induced activation of the immune cells associated to the small intestine of mice that received the probiotic strain . We also observed that this probiotic strain decreased the severity of S. Typhimurium infection in a mouse model, showing the continuous administration, the best effect. Continous probiotic administration decreased the mortality percentage (ten times) and the CFU/g of Salmonella in liver, spleen and large intestine for 7 and 10 days post- infection . In the present work, some immune mechanisms by which L. casei CRL 431 administration exerts its protective effect against Salmonella infection were analyzed, as the intestinal cytokine profile in the inductor (Peyer's patches) and effector sites (lamina propria) of the gut immune response. The modulation of TLRs expressions was also determined in the small intestine tissues.
Previous to the infection, analyzing the mononuclear cells isolated from Peyer's patches, it was observed that mice fed 7 days with L. casei CRL 431 significantly increased cytokines expression and also the release of IFNγ and IL-10 by these cells. The production of cytokines in Peyer's patches was maintained without significant differences in healthy animals that received the probiotic strain (Lc) during all the experiment. These cytokines were also studied 7 days post infection and it was observed that mice from infection control group (S) and the group fed continuously with the probiotic strain maintained increased expression of both TNFα and IFNγ in the cells isolated from Peyer's patches. Nevertheless, the release of IFNγ from these cell cultures was significantly higher in the infection control (S) than in the mice given probiotic (Lc-S-Lc group). The increases of these cytokines in Peyer's patches are important because they constitute the main inductor site for mucosal immune response. In S. Typhimurium infection, this site is one of the pathways that Salmonella uses to invade the host, although Salmonella infection can also occur through the intestinal epithelial cells along the small intestine . Therefore post infection, we also focused on the cytokine expression in cells from the lamina propria of the small intestine and the cytokines secretion into the intestinal lumen, due to this is the effector site of the gut immune response (Figure 1 and 2). TNFα is a pro-inflammatory cytokine that induces activation and recruitment of neutrophils involved in local inflammatory processes, and produces intestinal epithelial barrier dysfunction, contributing to the entry and colonization of pathogenic bacteria usually excluded from the subepithelial mucosa [15–17]. Seven days post infection, the probiotic administration (Lc-S and Lc-S-Lc grups) was able to maintain TNFα production in the lamina propria of the small intestine and its secretion to the intestinal fluid similar to the observed in the non infected groups (C and Lc groups). These values showed a tendency to decrease 10 days post challenge. In contrast, the infection control group significantly increased TNFα expression 7 days post challenge as well as its secretion 10 days post infection (Figure 2). The TNFα modulation by probiotic administration could be related with the lesser polymorphonuclear infiltration and inflammation degree in the lamina propria observed previously . Otherwise, the positive cells for this cytokine and its release from these cells were increased in Peyer's patches when the mice received continuously the probiotic strain compared to the untreated control (C). These increments could be related with the high number of activated macrophages present in these sites, suggesting that TNFα is required in the inductor site to maintain the immune response against Salmonella (Tables 1 and 2). IFNγ is implicated in the immune activation by probiotic bacteria and fermented milks. It contributes in the activation of macrophages to promote the effective killing of pathogens that can survive within them. In our model, the number of IFNγ (+) cells in small intestinal tissues was significantly lower in the group of mice from the infection control group (S) than in the group of mice given continuously L. casei CRL 431, which maintained the number of these positive cells similar to the Lc group (Figure 1B). As regard to the release of IFNγ to the intestinal fluid, the administration of the probiotic bacteria maintained the levels of this cytokine similar to the basal data, at difference of the S group, which showed a significant decrease of IFNγ concentration after infection (Figure 2B). IFNγ (+) cells also increased in healthy mice given probiotic bacteria in both inductor and effector sites of the immune response compared to the untreated control group (Figure 1B and Table 1). This is consistent with previous reports where the administration of probiotic suspensions or fermented milks was associated with increased number of IFNγ (+) cells in the small intestine of mice [4, 18]. Recent findings revealed an inhibitory effect of IFNγ on neutrophils trafficking and pro-inflammatory Th17 cells differentiation [19–21]. According to this observation, the increased levels of this cytokine in Lc-S-Lc group could be correlated with the reduced spread of Salmonella and the lower inflammation of small intestinal tissues observed previously . IL-6 was analyzed because promotes both B cell maturation  and pro-inflammatory activity . It was observed that 7 days after Salmonella challenge, the production of this cytokine in the small intestine tissues was significantly increased in the three infected groups compared with the untreated control (C), and 10 days post-challenge, only the group Lc-S-Lc maintained a number of IL-6 (+) cells higher than both control groups (C and S, Figure 1C). However, in the mice fed continuously with the probiotic (Lc-S-Lc group), the IL-6 release into the intestinal lumen remained stable 7 and 10 days post-infection. In contrast, the infection control group (S) significantly increased IL-6 secretion during all the experiment, compared with basal data (Figure 2C). These results showed that probiotic administration can down regulate the release of IL-6 but maintain increased production of this cytokine in the intestine which could be used by the host if it is required.
According with the results obtained for the mentioned cytokines, IL-10 was studied as an anti-inflammatory cytokine and similar to IL-6 is required to maintain the IgA (+) B cell population [24, 25]. In our work, 7 days post challenge the number of IL-10 (+) cells was significantly higher in infected mice that received probiotic administration than in mice from S group, (Figure 1D). As regard to this cytokine release, the concentration of IL-10 in the intestinal fluid was significantly decreased in the infected control group (S) throughout the study, while in mice from Lc-S group the significant decrease was observed 10 days post infection. At day 7 post-challenge, IL-10 release of Lc-S-Lc group was lower than absolute control (group C) and Lc group, but restored at day 10 post-challenge. These results highlight the importance of continuous probiotic administration in the modulation of the immune response (Figure 2D).
Previous results obtained in our group suggested that probiotic administration modulates the cytokine profile, mainly in the cells from the innate immune response through TLRs stimulation [4, 26]. According to this, and considering the differences observed for the cytokines, we analyzed the expression of TLRs in immune and epithelial cells of the small intestine in our infection model.
TLR2 was studied due this receptor recognize the peptidoglycan which is the principal component of the Gram+ bacteria such as Lactobacillus genus. Our results showed a significant increase of TLR2 (+) cells in the small intestine of healthy mice that received L. casei CRL 431 compared to the untreated control (Figure 3A) and significant increases were also observed, only for 7 days post infection, in the mice given continuously the probiotic bacteria (Lc-S-Lc group) compared to the infection control (S group). This result agrees with other findings describing a similar effect induced by two Lactobacillus strains, L. rhamnosus GG and L. plantarum BFE 1685, which enhanced TLR2 in vitro using human intestinal cells . We consider that the probiotic strain stimulates the TLR2 not only to increase the signals to produce cytokines, but also to increase the epithelial barrier because it was demonstrated TLR2 activation have an important role in enhancing trans-epithelial resistance to invading bacteria . Another receptor analyzed was TLR4, which recognizes the LPS present in the cell wall of the Gram(-) bacteria . It is known that TLR4 plays a significant role in the host defences against Salmonella infection in vivo [29–31]. In our model, L. casei CRL 431 administration to healthy mice increased the number of TLR4 (+) cells compared to the untreated control, which could be used as a surveillance mechanism against pathogen bacteria such as Salmonella. Recent findings suggest that the activation of this receptor initiates an innate immune response leading to the induction of pro-inflammatory mediators, to increase TLR2 expression, and to reduce its own expression, which leads to the recruitment of inflammatory cells and the initiation of the appropriate responses in the spleen leading control of the bacterial multiplication [29, 32]. This is consistent with the results obtained in our study where the enhancement of TLR4 was accompanied of increased number of TLR2 (+) cells previous and post infection (Figure 3). The early increase in the expression of TLR4 could be related with the decrease of the severity of the infection observed in the treated groups where the bacterial growth in the spleen and the liver decreased faster than in the infection control .
TLR5 was evaluated because flagellated bacteria, including E. coli and Salmonella, can interact with TLR5 to induce activation of pro-inflammatory gene programs for host protection [33–35]. In the present work, we observed that probiotic administration increased TLR5 (+) cells after Salmonella infection in both groups that received the probiotic strain for 7 days post challenge compared to untreated mice (C, Figure 3C). This finding agrees with other study where two lactobacilli were able to increase the cell surface expression of TLR5 in HT29 cells to respond to S. Typhimurium . In our model, this receptor could be also implicated in the protective effect of L. casei CRL 431 against S. Typhimurium infection.
Finally, in our study, it was observed that L. casei CRL 431 oral administration increased TLR9 expression in healthy mice (Figure 3D). Seven days post infection, the increase of TLR9 (+) cells was observed in both groups of mice given probiotic bacteria (Lc-S and Lc-S-Lc), but not in the infection control (S group), comparing with the untreated control group (C). This finding agrees with several works which affirm that CpG-TLR9 interaction can improve the resistance of normal adult mice to a variety of bacterial, viral and parasitic pathogens [36–38], including increased resistance to oral challenge with S. Typhimurium. TLR9 signalling is also required to mediate an anti-inflammatory effect induced by probiotics, in a mouse colitis model .