Julio Villena

Immunomodulatory activity of Lactobacillus rhamnosus strains isolated from goat milk: impact on intestinal and respiratory infections.

The immune stimulation induced by Lactobacillus rhamnosus CRL1505 (Lr05) and L. rhamnosus CRL1506 (Lr06) on the resistance to infection with an intestinal pathogen (Salmonella typhimurium) and a respiratory pathogen (Streptococcus pneumoniae) was studied in swiss-albine mice experimental models. The cytokine profiles that induced the innate and specific immune response in both infectious processes were investigated. Both strains were able to improve resistance against the intestinal pathogen. Only Lr05 was able to induce a significant decrease in the number of S. pneumoniae in the lung, prevent its dissemination into the blood and induce a significant increase in Th1 (INF-gamma) and Th2 (IL-6, IL-4 and IL-10) cytokine levels in the bronchoalveolar lavages (BAL). The changes in the cytokines profiles in BAL were associated with an increase in the number and activity of phagocytic cells and with the increase in specific antibodies in serum and BAL, which would explain the increased resistance to the challenge. The administration of Lr06 did not induce significant effects at the respiratory mucosal level. The results described in the present paper showed that certain LAB strains can share certain functional properties, although some of them can perform a functional role better than others, so that it is important to perform careful studies on specific strains, according to their therapeutic use.

Immunobiotic Lactobacillus jensenii Elicits anti-inflammatory activity in porcine intestinal epithelial cells by modulating negative regulators of the toll-like receptor signaling pathway

ABSTRACT The effect of Lactobacillus jensenii TL2937 on the inflammatory immune response triggered by enterotoxigenic Escherichia coli (ETEC) and lipopolysaccharide (LPS) in a porcine intestinal epitheliocyte cell line (PIE cells) was evaluated. Challenges with ETEC or LPS elicited Toll-like receptor 4 (TLR4)-mediated inflammatory responses in cultured PIE cells, indicating that our cell line may be useful for studying inflammation in the guts of weaning piglets. In addition, we demonstrated that L. jensenii TL2937 attenuated the expression of proinflammatory cytokines and chemokines caused by ETEC or LPS challenge by downregulating TLR4-dependent nuclear factorκB (NF-κB) and mitogen-activated protein kinase (MAPK) activation. Furthermore, we demonstrated that L. jensenii TL2937 stimulation of PIE cells upregulated three negative regulators of TLRs: A20, Bcl-3, and MKP-1, deepening the understanding of an immunobiotic mechanism of action. L. jensenii TL2937-mediated induction of negative regulators of TLRs would have a substantial physiological impact on homeostasis in PIE cells, because excessive TLR inflammatory signaling would be downregulated. These results indicated that PIE cells can be used to study the mechanisms involved in the protective activity of immunobiotics against intestinal inflammatory damage and may provide useful information for the development of new immunologically functional feeds that help to prevent inflammatory intestinal disorders, including weaning-associated intestinal inflammation.

Modulation of Intestinal TLR4-Inflammatory Signaling Pathways by Probiotic Microorganisms: Lessons Learned from Lactobacillus jensenii TL2937

The intestinal mucosa plays a critical role in the host’s interactions with innocuous commensal microbiota and invading pathogenic microorganisms. Intestinal epithelial cells (IECs) and gut associated immune cells recognize the bacterial components via pattern-recognition receptors (PRRs) and are responsible for maintaining tolerance to the large communities of resident luminal bacteria while being also able to mount inflammatory responses against pathogens. Toll-like receptors (TLRs) are a major class of PRRs that are present on IECs and immune cells which are involved in the induction of both tolerance and inflammation. A growing body of experimental and clinical evidence supports the therapeutic and preventive application of probiotics for several gastrointestinal inflammatory disorders in which TLRs exert a significant role. This review aims to summarize the current knowledge of the beneficial effects of probiotic microorganisms with the capacity to modulate the immune system (immunobiotics) in the regulation of intestinal inflammation in pigs, which are very important as both livestock and human model. Especially we discuss the role of TLRs, their signaling pathways, and their negative regulators in both the inflammatory intestinal injury and the beneficial effects of immunobiotics in general, and Lactobacillus jensenii TL2937 in particular. This review article emphasizes the cellular and molecular interactions of immunobiotics with IECs and immune cells through TLRs and their application for improving animal and human health.

Nasally administered Lactobacillus rhamnosus strains differentially modulate respiratory antiviral immune responses and induce protection against respiratory syncytial virus infection

BackgroundSome studies have shown that nasally administered immunobiotics had the potential to improve the outcome of influenza virus infection. However, the capacity of immunobiotics to improve protection against respiratory syncytial virus (RSV) infection was not investigated before.ObjectiveThe aims of this study were: a) to evaluate whether the nasal administration of Lactobacillus rhamnosus CRL1505 (Lr05) and L. rhamnosus CRL1506 (Lr06) are able to improve respiratory antiviral defenses and beneficially modulate the immune response triggered by TLR3/RIG-I activation; b) to investigate whether viability of Lr05 or Lr06 is indispensable to modulate respiratory immunity and; c) to evaluate the capacity of Lr05 and Lr06 to improve the resistance of infant mice against RSV infection.ResultsNasally administered Lr05 and Lr06 differentially modulated the TLR3/RIG-I-triggered antiviral respiratory immune response. Lr06 administration significantly modulated the production of IFN-α, IFN-β and IL-6 in the response to poly(I:C) challenge, while nasal priming with Lr05 was more effective to improve levels of IFN-γ and IL-10. Both viable Lr05 and Lr06 strains increased the resistance of infant mice to RSV infection while only heat-killed Lr05 showed a protective effect similar to those observed with viable strains.ConclusionsThe present work demonstrated that nasal administration of immunobiotics is able to beneficially modulate the immune response triggered by TLR3/RIG-I activation in the respiratory tract and to increase the resistance of mice to the challenge with RSV. Comparative studies using two Lactobacillus rhamnosus strains of the same origin and with similar technological properties showed that each strain has an specific immunoregulatory effect in the respiratory tract and that they differentially modulate the immune response after poly(I:C) or RSV challenges, conferring different degree of protection and using distinct immune mechanisms. We also demonstrated in this work that it is possible to beneficially modulate the respiratory defenses against RSV by using heat-killed immunobiotics.

Orally administered Lactobacillus rhamnosus modulates the respiratory immune response triggered by the viral pathogen-associated molecular pattern poly(I:C)

BackgroundSome studies have shown that probiotics, including Lactobacillus rhamnosus CRL1505, had the potential to beneficially modulate the outcome of certain bacterial and viral respiratory infections. However, these studies did not determine the mechanism(s) by which probiotics contribute to host defense against respiratory viruses.ResultsIn this work we demonstrated that orally administered Lactobacillus rhamnosus CRL1505 (Lr1505) was able to increase the levels of IFN-γ, IL-10 and IL-6 in the respiratory tract and the number of lung CD3+CD4+IFN-γ+ T cells. To mimic the pro-inflammatory and physiopathological consecuences of RNA viral infections in the lung, we used an experimental model of lung inflammation based on the administration of the artificial viral pathogen-associated molecular pattern poly(I:C). Nasal administration of poly(I:C) to mice induced a marked impairment of lung function that was accompanied by the production of pro-inflammatory mediators and inflammatory cell recruitment into the airways. The preventive administration of Lr1505 reduced lung injuries and the production of TNF-α, IL-6, IL-8 and MCP-1 in the respiratory tract after the challenge with poly(I:C). Moreover, Lr1505 induced a significant increase in lung and serum IL-10. We also observed that Lr1505 was able to increase respiratory IFN-γ levels and the number of lung CD3+CD4+IFN-γ+ T cells after poly(I:C) challenge. Moreover, higher numbers of both CD103+ and CD11bhigh dendritic cells and increased expression of MHC-II, IL-12 and IFN-γ in these cell populations were found in lungs of Lr1505-treated mice. Therefore, Lr1505 treatment would beneficially regulate the balance between pro-inflammatory mediators and IL-10, allowing an effective inflammatory response against infection and avoiding tissue damage.ConclusionsResults showed that Lr1505 would induce a mobilization of cells from intestine and changes in cytokine profile that would be able to beneficially modulate the respiratory mucosal immunity. Although deeper studies are needed using challenges with respiratory viruses, the results in this study suggest that Lr1505, a potent inducer of antiviral cytokines, may be useful as a prophylactic agent to control respiratory virus infection.

Nasal Immunization with Lactococcus lactis Expressing the Pneumococcal Protective Protein A Induces Protective Immunity in Mice

ABSTRACT Nisin-controlled gene expression was used to develop a recombinant strain of Lactococcus lactis that is able to express the pneumococcal protective protein A (PppA) on its surface. Immunodetection assays confirmed that after the induction with nisin, the PppA antigen was predictably and efficiently displayed on the cell surface of the recombinant strain, which was termed L. lactis PppA. The production of mucosal and systemically specific antibodies in adult and young mice was evaluated after mice were nasally immunized with L. lactis PppA. Immunoglobulin M (IgM), IgG, and IgA anti-PppA antibodies were detected in the serum and bronchoalveolar lavage fluid of adult and young mice, which showed that PppA expressed in L. lactis was able to induce a strong mucosal and systemic immune response. Challenge survival experiments demonstrated that immunization with L. lactis PppA was able to increase resistance to systemic and respiratory infection with different pneumococcal serotypes, and passive immunization assays of naïve young mice demonstrated a direct correlation between anti-PppA antibodies and protection. The results presented in this study demonstrate three major characteristics of the effectiveness of nasal immunization with PppA expressed as a protein anchored to the cell wall of L. lactis: it elicited cross-protective immunity against different pneumococcal serotypes, it afforded protection against both systemic and respiratory challenges, and it induced protective immunity in mice of different ages.

Immunobiotic Lactobacillus jensenii Modulates the Toll-Like Receptor 4-Induced Inflammatory Response via Negative Regulation in Porcine Antigen-Presenting Cells

ABSTRACT Previously, we demonstrated that Lactobacillus jensenii TL2937 attenuates the inflammatory response triggered by activation of Toll-like receptor 4 (TLR-4) in porcine intestinal epithelial cells. In view of the critical importance of antigen-presenting cell (APC) polarization in immunoregulation, the objective of the present study was to examine the effect of strain TL2937 on the activation patterns of APCs from swine Peyers patches (PPs). We demonstrated that direct exposure of porcine APCs to L. jensenii in the absence of inflammatory signals increased expression of interleukin-10 (IL-10) and transforming growth factor β in CD172a+ APCs and caused them to display tolerogenic properties. In addition, pretreatment of CD172a+ APCs with L. jensenii resulted in differential modulation of the production of pro- and anti-inflammatory cytokines in response to TLR4 activation. The immunomodulatory effect of strain TL2937 was not related to a downregulation of TLR4 but was related to an upregulation of the expression of three negative regulators of TLRs: single immunoglobulin IL-1-related receptor (SIGIRR), A20, and interleukin-1 receptor-associated kinase M (IRAK-M). Our results also indicated that TLR2 has an important role in the anti-inflammatory activity of L. jensenii TL2937, since anti-TLR2 antibodies blocked the upregulation of SIGIRR and IRAK-M in CD172a+ APCs and the production of IL-10 in response to TLR4 activation. We performed, for the first time, a precise functional characterization of porcine APCs from PPs, and we demonstrated that CD172a+ cells were tolerogenic. Our findings demonstrate that adherent cells and isolated CD172a+ cells harvested from swine PPs were useful for in vitro study of the inflammatory responses in the porcine gut and the immunomodulatory effects of immunobiotic microorganisms.

Lactic acid bacteria in the prevention of pneumococcal respiratory infection: Future opportunities and challenges

Lactic acid bacteria (LAB) are technologically and commercially important and have various beneficial effects on human health. Several studies have demonstrated that certain LAB strains can exert their beneficial effect on the host through their immunomudulatory activity. Although most research concerning LAB-mediated enhanced immune protection is focused on gastrointestinal tract pathogens, recent studies have centered on whether these immunobiotics might sufficiently stimulate the common mucosal immune system to provide protection to other mucosal sites as well. In this sense, LAB have been used for the development of probiotic foods with the ability to stimulate respiratory immunity, which would increase resistance to infections, even in immunocompromised hosts. On the other hand, the advances in the molecular biology of LAB have enabled the development of recombinant strains expressing antigens from respiratory pathogens that have proved effective to induce protective immunity. In this review we examine the current scientific literature concerning the use of LAB strains to prevent respiratory infections. In particular, we have focused on the works that deal with the capacity of probiotic and recombinant LAB to improve the immune response against Streptococcus pneumoniae. Research from the last decade demonstrates that LAB represent a promising resource for the development of prevention strategies against respiratory infections that could be effective tools for medical application.

Immunoregulatory Effect of Bifidobacteria Strains in Porcine Intestinal Epithelial Cells through Modulation of Ubiquitin-Editing Enzyme A20 Expression

Background We previously showed that evaluation of anti-inflammatory activities of lactic acid bacteria in porcine intestinal epithelial (PIE) cells is useful for selecting potentially immunobiotic strains. Objective The aims of the present study were: i) to select potentially immunomodulatory bifidobacteria that beneficially modulate the Toll-like receptor (TLR)-4-triggered inflammatory response in PIE cells and; ii) to gain insight into the molecular mechanisms involved in the anti-inflammatory effect of immunobiotics by evaluating the role of TLR2 and TLR negative regulators in the modulation of proinflammatory cytokine production and activation of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways in PIE cells. Results Bifidobacteria longum BB536 and B. breve M-16V strains significantly downregulated levels of interleukin (IL)-8, monocyte chemotactic protein (MCP)-1 and IL-6 in PIE cells challenged with heat-killed enterotoxigenic Escherichia coli. Moreover, BB536 and M-16V strains attenuated the proinflammatory response by modulating the NF-κB and MAPK pathways. In addition, our findings provide evidence for a key role for the ubiquitin-editing enzyme A20 in the anti-inflammatory effect of immunobiotic bifidobacteria in PIE cells. Conclusions We show new data regarding the mechanism involved in the anti-inflammatory effect of immunobiotics. Several strains with immunoregulatory capabilities used a common mechanism to induce tolerance in PIE cells. Immunoregulatory strains interacted with TLR2, upregulated the expression of A20 in PIE cells, and beneficially modulated the subsequent TLR4 activation by reducing the activation of MAPK and NF-κB pathways and the production of proinflammatory cytokines. We also show that the combination of TLR2 activation and A20 induction can be used as biomarkers to screen and select potential immunoregulatory bifidobacteria strains.

Stimulation of macrophages by immunobiotic Lactobacillus strains : influence beyond the intestinal tract

Lactobacillus rhamnosus CRL1505 (Lr1505), L. rhamnosus CRL1506 (Lr1506) and L. casei CRL431 (Lc431) are able to stimulate intestinal immunity, but only Lr1505 and Lc431 are able to stimulate immunity in the respiratory tract. With the aim of advancing the understanding of the immunological mechanisms involved in stimulation of distant mucosal sites, this study evaluated the effects of orally administered probiotics on the functions of alveolar and peritoneal macrophages. Compared to a control group, these three lactobacilli were able to significantly increase phagocytic and microbicidal activities of peritoneal macrophages. After intraperitoneal challenge with pathogenic Candida albicans, mice treated with immunobiotics had significantly lower pathogen counts in infected organs. Moreover, lactobacilli‐treated mice had a stronger immune response against C. albicans. On the other hand, only Lc1505 and Lc431 were able to improve activity of and cytokine production by alveolar macrophages. Only in these two groups was there better resistance to respiratory challenge with C. albicans, which correlated with improved respiratory immune response. The results of this study suggest that consumption of some probiotic strains could be useful for improving resistance to infections in sites distant from the gut by increasing the activity of macrophages at those sites.