CONICET | Buscador de Institutos y Recursos Humanos

In the innate immune response the phagocytic cell (macrophages) has the pattern recognition receptors (PRRs) which recognize microbe-associated molecular patterns (MAMPs) to induce activation. Toll-like receptors (TLRs) are a family of PRRs that recognize a diversity of conserved molecules and can promote the production of proinflammatory cytokines and chemokines(1). Lactic acid bacteria (LAB) able to modulate the immune system and are known to play a beneficial role in the prevention and therapy of a variety of intestinal inflammatory disorders, including atopic and inflammatory bowel diseases. However, studies have shown that different strains of lactobacilli can evoke different responses in the host and therefore, the results from one strain cannot be generalized to others(2). The aim of the present study was to evaluate the importance of TLRs signaling inducing by probiotic stimulation in peritoneal (MQP), Peyer?s patches (MQPP) and spleen (MQS) macrophages of BALB/c mice. BALB/c mice received as probiotic bacteria (PB) Lactobacillus casei CRL431 and Lactobacillus paracasei CNCMI-1518 or probiotic fermented milk (PFM). Them, MQP, MQPP and MQS were taken and incubated with either TLR2 or TLR4 (TLRs) agonists and antagonists. The phagocytic activity (PA) was determined. The production of IL-6 and IL-10 was determined by ELISA in the supernatant of MQP, MQPP and MQS confronted with TLRs agonist or antagonist and them, stimulated with the PB or with PFM. The microbicidal activity (MA) from MQP and MQS was determined. Probiotics and PFM administration increased the PA of MQP, MQPP and MQS, this activity was enhanced with TLRs agonists (p≤0,01) and diminished with TLRs antagonists (pThe ability to affect the host immune response is a feature of importance in the application of the probiotic bacteria strains. The identifying and characterizing of bacterial components that stimulate the immune system is crucial for the elucidation of novel structure as oral adjuvant. Aim: To evaluate the impact of sub-cellular components: exopolysaccharide (EPS), intracellular components (IC) and wall cellular (WC) of probiotic bacteria by in vitro and ex vivo assays, on the activity of peritoneal macrophages (MQP) to be the sentinels of immune innate response. We compared with a commensal bacterium. Different fractions were obtained from two probiotic bacteria: Lactobacillus casei CRL 431 (Lc), Lactobacillus paracasei CNCMI-1518 (Lp) and a commensal bacteria: Lactobacillus acidophilus CRL 730 (La). In vitro, the production of IL-6 was determined by ELISA in the supernatant of MQP stimulated with the sub-cellular fractions. Ex vivo, BALB/c mice received by oral route: EPS, IC and WC of Lc and La for 7 days, and for Lp 5 days. Phagocytic and antimicrobial activities of MQP were determined. Results: In vitro, IL-6 levels increases significantly (P≤0.05) when the cells were stimulated with IC and WC for probiotics Lc and Lp, and with EPS for La. In vivo, phagoytic and antimicrobial activities of MQP were enhanced for the WC (P≤0.05) of both probiotic strains. Phagocytic activity increases also with IC from La. Conclusion: We showed that the main immunomodulatory effect of probiotic bacteria (Lc, Lp) are in the wall cellular and exopolysaccharide for the commensal bacteria (La). The characterization of probiotic components able to act as oral adjuvant is an approach to elucidate the molecules involved in such effect.The ability to affect the host immune response is a feature of importance in the application of the probiotic bacteria strains. The identifying and characterizing of bacterial components that stimulate the immune system is crucial for the elucidation of novel structure as oral adjuvant. Aim: To evaluate the impact of sub-cellular components: exopolysaccharide (EPS), intracellular components (IC) and wall cellular (WC) of probiotic bacteria by in vitro and ex vivo assays, on the activity of peritoneal macrophages (MQP) to be the sentinels of immune innate response. We compared with a commensal bacterium. Different fractions were obtained from two probiotic bacteria: Lactobacillus casei CRL 431 (Lc), Lactobacillus paracasei CNCMI-1518 (Lp) and a commensal bacteria: Lactobacillus acidophilus CRL 730 (La). In vitro, the production of IL-6 was determined by ELISA in the supernatant of MQP stimulated with the sub-cellular fractions. Ex vivo, BALB/c mice received by oral route: EPS, IC and WC of Lc and La for 7 days, and for Lp 5 days. Phagocytic and antimicrobial activities of MQP were determined. Results: In vitro, IL-6 levels increases significantly (P≤0.05) when the cells were stimulated with IC and WC for probiotics Lc and Lp, and with EPS for La. In vivo, phagoytic and antimicrobial activities of MQP were enhanced for the WC (P≤0.05) of both probiotic strains. Phagocytic activity increases also with IC from La. Conclusion: We showed that the main immunomodulatory effect of probiotic bacteria (Lc, Lp) are in the wall cellular and exopolysaccharide for the commensal bacteria (La). The characterization of probiotic components able to act as oral adjuvant is an approach to elucidate the molecules involved in such effect (p The ability to affect the host immune response is a feature of importance in the application of the probiotic bacteria strains. The identifying and characterizing of bacterial components that stimulate the immune system is crucial for the elucidation of novel structure as oral adjuvant. Aim: To evaluate the impact of sub-cellular components: exopolysaccharide (EPS), intracellular components (IC) and wall cellular (WC) of probiotic bacteria by in vitro and ex vivo assays, on the activity of peritoneal macrophages (MQP) to be the sentinels of immune innate response. We compared with a commensal bacterium. Different fractions were obtained from two probiotic bacteria: Lactobacillus casei CRL 431 (Lc), Lactobacillus paracasei CNCMI-1518 (Lp) and a commensal bacteria: Lactobacillus acidophilus CRL 730 (La). In vitro, the production of IL-6 was determined by ELISA in the supernatant of MQP stimulated with the sub-cellular fractions. Ex vivo, BALB/c mice received by oral route: EPS, IC and WC of Lc and La for 7 days, and for Lp 5 days. Phagocytic and antimicrobial activities of MQP were determined. Results: In vitro, IL-6 levels increases significantly (P≤0.05) when the cells were stimulated with IC and WC for probiotics Lc and Lp, and with EPS for La. In vivo, phagoytic and antimicrobial activities of MQP were enhanced for the WC (P≤0.05) of both probiotic strains. Phagocytic activity increases also with IC from La. Conclusion: We showed that the main immunomodulatory effect of probiotic bacteria (Lc, Lp) are in the wall cellular and exopolysaccharide for the commensal bacteria (La). The characterization of probiotic components able to act as oral adjuvant is an approach to elucidate the molecules involved in such effect (p ≤0,01). The IL-6 levels showed increased in the cells stimulated with PB and PFM, however with PFM the stimulation was more regulated (p≤0,02). The IL-10 in the MQP were increased when the cells were stimulated with PB or PFM; nevertheless in the MQPP and MQS were more regulated (p≤0,01). The MA of MQP and MQS were enhanced in mice given Lactobacillus casei CRL 431. The probiotics are able to activate the innate immune cells distant from the gut such as peritoneal and spleen macrophages. On the other hand, the localization of the cell is very important because it makes it more reactive to an antigen, and this can be explained by the different density of receptors on their surfaces, which are necessary for activation(3). In terms of cytokine production, there is an increased production of proinflammatory cytokines when stimulation is performed only with probiotic bacteria, while a fermented product containing a probiotic bacteria is more controlled response. However, they are also self-regulating since the production of IL-10 was also important and depends on the location of the cell. The microbicidal activity showed that the probiotic bacteria Lactobacillus casei CRL 431 is able to increase activity, this was reflected when macrophages were blocked with antagonists of TLRs, mainly TLR4 that engage LPS from Gram (-) bacteria. The Toll pathway is involved in this activity however this signal would not be the only as show the results with TLRs antagonists, and different strains of lactobacilli can elicit different responses and consequently, the results from one strain cannot be generalized to others

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