Oral administration of a probiotic Lactobacillus modulates cytokine production and TLR expression improving the immune response against Salmonella enterica serovar Typhimurium infection in mice

Abstract

Background: Diarrheal infections caused by Salmonella, are one of the major causes of childhood morbidity and mortality in developing countries. Salmonella causes various diseases that range from mild gastroenteritis to enteric fever, depending on the serovar involved, infective dose, species, age and immune status of the host. Probiotics are proposed as an attractive alternative possibility in the prevention against this pathogen infection. Previously we demonstrated that continuous Lactobacillus casei CRL 431 administration to BALB/c mice before and after challenge with Salmonella enterica serovar Typhimurium (S. Typhimurium) decreased the severity of Salmonella infection. The aim of the present work was to deep into the knowledge about how this probiotic bacterium exerts its effect, by assessing its impact on the expression and secretion of pro-inflammatory (TNFα, IFNγ) and anti-inflammatory (IL-10) cytokines in the inductor and effector sites of the gut immune response, and analyzing toll-like receptor (TLR2, TLR4, TLR5 and TLR9) expressions in both healthy and infected mice.

Results: Probiotic administration to healthy mice increased the expression of TLR2, TLR4 and TLR9 and improved the production and secretion of TNFα, IFNγ and IL-10 in the inductor sites of the gut immune response (Peyer's patches). Post infection, the continuous probiotic administration, before and after Salmonella challenge, protected the host by modulating the inflammatory response, mainly in the immune effector site of the gut, decreasing TNFα and increasing IFNγ, IL-6 and IL-10 production in the lamina propria of the small intestine.

Conclusions: The oral administration of L. casei CRL 431 induces variations in the cytokine profile and in the TLRs expression previous and also after the challenge with S. Typhimurium. These changes show some of the immune mechanisms implicated in the protective effect of this probiotic strain against S. Typhimurium, providing an alternative way to reduce the severity of the infection.

Figure 1

Determination of cytokine (+) cells in the small intestine tissues. Positive cells were counted in histological sections from small intestine of mice fed 7 d with L. casei CRL 431 previous challenge with S. Typhimurium (Lc-S), and mice fed continuously (before and after infection) with the probiotic bacteria (Lc-S-Lc), compared to the infection control (S). Tissues from healthy mice fed or not with L. casei (Lc and C groups, respectively) were also analyzed. The samples were obtained the day of the infection (basal data) for Lc and C groups, and 7 and 10 days post challenge for all the groups. Representative microphotographs show the differences observed between C group (E and F), S group (G and H), and Lc-S-Lc group (I and J) in the number of IL-6 (+) cells (arrows), 7 days post challenge. The microphotographs E, G and I were obtained at 400× while F, H and J were taken at 1 000X. A difference of 1 cell at 1000× is related with 10 cells of difference in the final result. Means for each value without a common letter differ significantly (P < 0.01).

Figure 2

Determination of the concentration of TNFα, IFNγ IL-10 and IL-6 in intestinal fluid by ELISA. The samples were taken before the infection for the untreated (C) and L. casei CRL 431(Lc) groups, and 7 and 10 days post challenge for all the experimental groups. The results were expressed as the means ± SD of the concentration of each cytokine in pg/ml. Means for each value without a common letter differ significantly (P < 0.01).

Figure 3

Determination of TLRs (+) cells in histological sections of small intestine. The samples were obtained before the infection for the untreated control (C) and healthy mice given L. casei CRL431 (Lc group), and 7 and 10 days post challenge for all experimental groups. The number of fluorescent cells was counted in 30 fields of vision at 1 000X of magnification and the results were expressed as the number of positive cells counted per 10 fields. The microphotographs (400×) F and H show the increases of TLR2+ and TLR4+ cells, respectively (fluorescent cells) in mice from Lc group compared to the untreated control (C group: E for TLR2 and G for TLR4). Means for each value without a common letter differ significantly (P < 0.01).