Milk fermentation products of L. helveticus R389 activate calcineurin as a signal to promote gut mucosal immunity

<p/> <p>Background</p> <p>Fermented milks containing probiotic bacteria are a way of delivering bioactive constituents to targets in the gastrointestinal tract. We reported previously that the fermentation of milk at constant pH 6 by <it>L. helveticus </it>R389 increased its content of peptide fractions, and the oral administration of the non-bacterial fraction (FMSpH6) to mice increased total secretory IgA in the intestinal lumen and enhanced the number of IgA and various cytokines producing cells as well as the secretion of IL-6 by small intestine epithelial cells. We also demonstrated that this FMSpH6 was effective for the prevention of <it>Salmonella typhimurium </it>infection in mice. In this work, we studied in mice the impact of the oral administration of the supernatant of milk fermented by <it>L. helveticus </it>R389 on the gut physiology by measuring parameters such as calcium channels and E-cadherin expression, the activation of the biological signal calcineurin and mast and goblet cells, as a way to determine some mechanisms involved in the immunomodulating effects of the milk fermentation products, observed in previous studies. We analyzed the impact of the supernatant of milk fermented by <it>L. helveticus </it>R389 at pH6-controlled on the expression of calcineurin and on the reinforcement of the ephitelial barrier, measuring parameters such as calcium channels and E-cadherin expression and in the reinforcement of the non-specific immunity determining mast cells and goblet cells associated to the gut.</p> <p>Results</p> <p>We observed an enhanced expression of TRPV6 channels in the duodenum, indicating an improved capacity for dietary Ca2+ uptake. We demonstrated an enhanced expression of calcineurin in the small intestine, able to upregulate immune parameters such as IL-2 and TNF production, with an increase in the number of these cytokines secreting cells. We determined an increase in the number of mucosal mast cells and goblet cells, which would mean an improved state of mucosal surveillance at sites of infection.</p> <p>Conclusion</p> <p>The oral administration of the supernatant of milk fermented by <it>L. helveticus </it>R389 enhanced the gut mucosal immunity by improving the mechanisms that reinforce the epithelial and non-specific barriers and the gut functioning at sites of infection, with an improvement in the expression of the enzyme calcineurin, an important signal in the network that activates the gut immune system. The results of this work contribute to revealing the mechanisms underlying the immunomodulation of the gut immune function by fermented milks with probiotic bacteria.</p

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

<p>Abstract</p> <p>Background</p> <p>Diarrheal infections caused by <it>Salmonella</it>, are one of the major causes of childhood morbidity and mortality in developing countries. <it>Salmonella </it>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 <it>Lactobacillus casei </it>CRL 431 administration to BALB/c mice before and after challenge with <it>Salmonella enterica </it>serovar Typhimurium (<it>S</it>. Typhimurium) decreased the severity of <it>Salmonella </it>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.</p> <p>Results</p> <p>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 <it>Salmonella </it>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.</p> <p>Conclusions</p> <p>The oral administration of <it>L. casei </it>CRL 431 induces variations in the cytokine profile and in the TLRs expression previous and also after the challenge with <it>S</it>. Typhimurium. These changes show some of the immune mechanisms implicated in the protective effect of this probiotic strain against <it>S</it>. Typhimurium, providing an alternative way to reduce the severity of the infection.</p

Study of the effect exerted by fructo-oligosaccharides from yacon (Smallanthus sonchifolius) root flour in an intestinal infection model with Salmonella Typhimurium

Beneficial effects of prebiotics like inulin and fructo-oligosaccharides (FOS) have been proven in health and nutrition. Yacon (Smallanthus sonchifolius), an Andean crop, contains FOS (50 – 70 % of its dry weight) and, therefore, is considered a prebiotic. Commercial FOS can up- regulate total secretory IgA (S-IgA) in infant mice, prevent infection with Salmonella in swine or enhance immune response for Salmonella vaccine in a mouse model. Previously, we found that administration of yacon root flour regulates gut microbiota balance and has immu- nomodulatory effects without inflammatory responses. The aim of the present paper is to analyse if yacon prevents enteric infection caused by a strain of Salmonella enteritidis serovar Typhimurium (S. Typhimurium) in a mouse model. BALB/c mice were supplemented with yacon flour (45d), challenged with S. Typhimurium and killed to study pathogen translocation, total and specific IgA production by ELISA, presence of IgA and other cytokines and Toll-like receptor 4 (TLR4) and clustor of differentiation 206 (CD206) receptors positive cells by immunofluorescence and histological changes. Yacon flour administration had a protective effect from 15 to 30 d of treatment. We found a peak of total S-IgA production without translocation of the pathogen for these periods. At 30 d, there was an increase in IL-6 and macrophage inflammatory proteins-1aþ cells and expression of the receptors CD206 and TLR4. Yacon flour did not have incidence in pathogen-specific S-IgA production. Longer periods (45 d) of administration had no protective effect. Therefore, yacon can prevent enteric infection caused by S. Typhimurium when given up to 30 d; this effect would be mediated by enhancing non-specific immunity, such as total S-IgA, that improves the immunological intestinal barrier.Fil: Velez, Eva Maria del Mar. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Castillo, Natalia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Mesón, Oscar Enrique. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; ArgentinaFil: Grau, Alfredo. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales e Instituto Miguel Lillo. Laboratorio de Investigaciones Ecológicas de las Yungas; ArgentinaFil: Bibas Bonet, María Eugenia. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Microbiología; ArgentinaFil: Perdigón, Gabriela del Valle. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentin

Impact of a probiotic fermented milk in the gut ecosystem and in the systemic immunity using a non-severe protein-energy-malnutrition model in mice

<p>Abstract</p> <p>Background</p> <p>Malnutrition affects the immune response, causing a decrease of defence mechanisms and making the host more susceptible to infections. Probiotics can reconstitute the intestinal mucosa and stimulate local and systemic immunity. The aim of this work was evaluate the effects of a probiotic fermented milk as a complement of a re-nutrition diet, on the recovery of the intestinal barrier, and mucosal and systemic immune functions in a murine model of non-severe protein-energy-malnutrition. Its potential protection against <it>Salmonella enterica </it>serovar Typhimurium (<it>S</it>. Typhimurium) infection was also analyzed.</p> <p>Methods</p> <p>Mice were undernourished and divided into 3 groups according to the dietary supplement received during re-nutrition (milk, probiotic fermented milk or its bacterial free supernatant) and compared to well-nourished and malnourished mice. They were sacrificed previous to the re-nutrition and 5 days post re-nutrition. The phagocytic activity of macrophages from spleen and peritoneum and the changes in the intestinal histology and microbiota were evaluated. Different immune cell populations and cytokine productions were analyzed in the small intestine tissues. The effect of the re-nutrition supplements on the systemic immunity using OVA antigen and against an infection with <it>S. </it>Typhimurium was also studied.</p> <p>Results</p> <p>Probiotic fermented milk was the most effective re-nutrition diet that improved the intestinal microbiota. Its administration also increased the number of IgA+ cells, macrophages and dendritic cells. The production of different cytokine (IFN-γ, TNF-α, IL-12) by these cells and the phagocytic activity in peritoneum and spleen was also increased. This re-nutrition diet also stimulated the systemic immune response against OVA antigen which was diminished after the malnutrition period and also improved the host response against <it>S. </it>Typhimurium, decreasing the spread of pathogenic bacteria to the liver and the spleen. The importance of the metabolites released during milk fermentation was also demonstrated through the analysis of the bacterial free supernatant obtained from the probiotic fermented milk, but the whole product showed the best effects in the parameters evaluated in this study.</p> <p>Conclusions</p> <p>The administration of probiotic fermented milk as a dietary supplement during the re-nutrition process in a murine immunodeficiency model by malnutrition could be a good adjuvant diet to improve the gut and systemic immune response for the protection against <it>Salmonella </it>infection.</p