Immunobiotic Lactobacillus rhamnosus strains differentially modulate antiviral immune response in porcine intestinal epithelial and antigen presenting cells.

BACKGROUND: Previous findings suggested that Lactobacillus rhamnosus CRL1505 is able to increase resistance of children to intestinal viral infections. However, the intestinal cells, cytokines and receptors involved in the immunoregulatory effect of this probiotic strain have not been fully characterized. RESULTS: We aimed to gain insight into the mechanisms involved in the immunomodulatory effect of the CRL1505 strain and therefore evaluated in vitro the crosstalk between L. rhamnosus CRL1505, porcine intestinal epithelial cells (IECs) and antigen presenting cells (APCs) from swine Peyer's patches in order to deepen our knowledge about the mechanisms, through which this strain may help preventing viral diarrhoea episodes. L. rhamnosus CRL1505 was able to induce IFN-α and -ß in IECs and improve the production of type I IFNs in response to poly(I:C) challenge independently of Toll-like receptor (TLR)-2 or TLR9 signalling. In addition, the CRL1505 strain induced mRNA expression of IL-6 and TNF-α via TLR2 in IECs. Furthermore, the strain significantly increased surface molecules expression and cytokine production in intestinal APCs. The improved Th1 response induced by L. rhamnosus CRL1505 was triggered by TLR2 signalling and included augmented expression of MHC-II and co-stimulatory molecules and expression of IL-1ß, IL-6, and IFN-γ in APCs. IL-10 was also significantly up-regulated by CRL1505 in APCs. CONCLUSIONS: It was recently reviewed the emergence of TLR agonists as new ways to transform antiviral treatments by introducing panviral therapeutics with less adverse effects than IFN therapies. The use of L. rhamnosus CRL1505 as modulator of innate immunity and inductor of antiviral type I IFNs, IFN-γ, and regulatory IL-10 clearly offers the potential to overcome this challenge.

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

BACKGROUND: Some 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. OBJECTIVE: The 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. RESULTS: Nasally 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-ß 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. CONCLUSIONS: The 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.

Advanced application of bovine intestinal epithelial cell line for evaluating regulatory effect of lactobacilli against heat-killed enterotoxigenic Escherichia coli-mediated inflammation.

BACKGROUND: Previously, a bovine intestinal epithelial cell line (BIE cells) was successfully established. This work hypothesized that BIE cells are useful in vitro model system for the study of interactions of microbial- or pathogen-associated molecular patterns (MAMPs or PAMPs) with bovine intestinal epithelial cells and for the selection of immunoregulatory lactic acid bacteria (LAB). RESULTS: All toll-like receptor (TLR) genes were expressed in BIE cells, being TLR4 one of the most strongly expressed. We demonstrated that heat-stable PAMPs of enterotoxigenic Escherichia coli (ETEC) significantly enhanced the production of IL-6, IL-8, IL-1α and MCP-1 in BIE cells by activating both NF-κB and MAPK pathways. We evaluated the capacity of several lactobacilli strains to modulate heat-stable ETEC PAMPs-mediated inflammatory response in BIE cells. Among these strains evaluated, Lactobacillus casei OLL2768 attenuated heat-stable ETEC PAMPs-induced pro-inflammatory response by inhibiting NF-κB and p38 signaling pathways in BIE cells. Moreover, L. casei OLL2768 negatively regulated TLR4 signaling in BIE cells by up-regulating Toll interacting protein (Tollip) and B-cell lymphoma 3-encoded protein (Bcl-3). CONCLUSIONS: BIE cells are suitable for the selection of immunoregulatory LAB and for studying the mechanisms involved in the protective activity of immunobiotics against pathogen-induced inflammatory damage. In addition, we showed that L. casei OLL2768 functionally modulate the bovine intestinal epithelium by attenuating heat-stable ETEC PAMPs-induced inflammation. Therefore L. casei OLL2768 is a good candidate for in vivo studying the protective effect of LAB against intestinal inflammatory damage induced by ETEC infection or heat-stable ETEC PAMPs challenge in the bovine host.

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

BACKGROUND: Some 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. RESULTS: In 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 CD11b(high) 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. CONCLUSIONS: Results 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.

Additional copies of CBX2 in the genomes of males of mammals lacking SRY, the Amami spiny rat (Tokudaia osimensis) and the Tokunoshima spiny rat (Tokudaia tokunoshimensis).

Tokudaia osimensis (the Amami spiny rat) and Tokudaia tokunoshimensis (the Tokunoshima spiny rat) have a sex chromosome composition of XO/XO, no Y chromosome. The mammalian sex-determining gene, SRY, is also absent in these species, which indicates that these spiny rats exhibit a novel sex-determining mechanism that is independent of SRY. To identify a candidate gene that controls this mechanism, the copy numbers and chromosomal locations of 10 genes with important functions in gonadal differentiation were determined: ATRX, CBX2 (M33), DMRT1, FGF9, NR0B1 (DAX1), NR5A1 (Ad4BP/SF1), RSPO1, SOX9, WNT4, and WT1. Multiple bands were detected for NR0B1 in Southern blot analysis, which suggested the presence of multiple copies of the gene in the genomes of these two species. CBX2 was localized to two loci in both sexes of the two species by fluorescence in situ hybridization mapping: 3q24 and 6p11.2 in T. osimensis and 10q25-q26 and 14q12-q13.1 in T. tokunoshimensis. Quantification of copy numbers in the two species by quantitative real-time PCR indicated that there were two or three more copies of CBX2 per haploid genome in males (T. osimensis, n = 3; T. tokunoshimensis, n = 2) than in females (T. osimensis, n = 4; T. tokunoshimensis, n = 2), whereas NR0B1 was present as a single copy in both. The results suggest that additional copies of CBX2 in males might be involved in a novel sex-determining mechanism in species that lack SRY.

Immunobiotic lactic acid bacteria beneficially regulate immune response triggered by poly(I:C) in porcine intestinal epithelial cells.

This study analyzed the functional expression of TLR3 in various gastrointestinal tissues from adult swine and shows that TLR3 is expressed preferentially in intestinal epithelial cells (IEC), CD172a(+)CD11R1(high) and CD4(+) cells from ileal Peyer's patches. We characterized the inflammatory immune response triggered by TLR3 activation in a clonal porcine intestinal epitheliocyte cell line (PIE cells) and in PIE-immune cell co-cultures, and demonstrated that these systems are valuable tools to study in vitro the immune response triggered by TLR3 on IEC and the interaction between IEC and immune cells. In addition, we selected an immunobiotic lactic acid bacteria strain, Lactobacillus casei MEP221106, able to beneficially regulate the anti-viral immune response triggered by poly(I:C) stimulation in PIE cells. Moreover, we deepened our understanding of the possible mechanisms of immunobiotic action by demonstrating that L. casei MEP221106 modulates the interaction between IEC and immune cells during the generation of a TLR3-mediated immune response.

Immunobiotic lactobacilli reduce viral-associated pulmonary damage through the modulation of inflammation-coagulation interactions.

The exacerbated disease due to immune- and coagulative-mediated pulmonary injury during acute respiratory viruses infection results in severe morbidity and mortality. Identifying novel approaches to modulate virus-induced inflammation-coagulation interactions could be important alternatives for treating acute respiratory viruses infections. In this study we investigated the effect of the probiotic strain Lactobacillus rhamnosus CRL1505 on lung TLR3-mediated inflammation, and its ability to modulate inflammation-coagulation interaction during respiratory viral infection. Our findings reveal for the first time that a probiotic bacterium is able to influence lung immune-coagulative reaction triggered by TLR3 activation, by modulating the production of proinflammatory and anti-inflammatory cytokines as well as expression of tissue factor and thrombomodulin in the lung. We also demonstrated that the preventive treatment with the probiotic bacteria beneficially modulates the fine tune balance between clearing respiratory viruses (respiratory syncytial virus and influenza virus) and controlling immune-coagulative responses in the lung, allowing normal lung function to be maintained in the face of a viral attack. Our data also pinpoint a crucial role for IL-10 in the immune protection induced by L. rhamnosus CRL1505 during respiratory viral infections. These observations might be helpful to propose new preventive or therapeutic approaches to better control virus-inflammatory lung damage using probiotic functional foods.

Bifidobacterium breve MCC-117 Induces Tolerance in Porcine Intestinal Epithelial Cells: Study of the Mechanisms Involved in the Immunoregulatory Effect.

Bifidobacterium breve MCC-117 is able to significantly reduce the expression of inflammatory cytokines in porcine intestinal epithelial (PIE) cells and to improve IL-10 levels in CD4(+)CD25(high) Foxp3(+) lymphocytes in response to heat-stable enterotoxigenic Escherichia coli (ETEC) pathogen-associated molecular patterns (PAMPs), while the immunoregulatory effect of B. adolescentis ATCC15705 was significantly lower than that observed for the MCC-117 strain. Considering the different capacities of the two bifidobacterium strains to activate toll-like receptor (TLR)-2 and their differential immunoregulatory activities in PIE and immune cells, we hypothesized that comparative studies with both strains could provide important information regarding the molecular mechanism(s) involved in the anti-inflammatory activity of bifidobacteria. In this work, we demonstrated that the anti-inflammatory effect of B. breve MCC-117 was achieved by a complex interaction of multiple negative regulators of TLRs as well as inhibition of multiple signaling pathways. We showed that B. breve MCC-117 reduced heat-stable ETEC PAMP-induced NF-κB, p38 MAPK and PI3 K activation and expression of pro-inflammatory cytokines in PIE cells. In addition, we demonstrated that B. breve MCC-117 may activate TLR2 synergistically and cooperatively with one or more other pattern recognition receptors (PRRs), and that interactions may result in a coordinated sum of signals that induce the upregulation of A20, Bcl-3, Tollip and SIGIRR. Upregulation of these negative regulators could have an important physiological impact on maintaining or reestablishing homeostatic TLR signals in PIE cells. Therefore, in the present study, we gained insight into the molecular mechanisms involved in the immunoregulatory effect of B. breve MCC-117.

Advanced application of porcine intestinal epithelial cells for the selection of immunobiotics modulating toll-like receptor 3-mediated inflammation.

PURPOSE: In this study, we aimed to characterize toll-like receptor (TLR)-3-mediated inflammatory immune response in porcine intestinal epithelial (PIE) cells and in PIE-immune cell co-cultures and, to evaluate if these in vitro systems are useful for selecting immunomodulatory lactic acid bacteria. RESULTS: We demonstrated that these systems are valuable tools for the in vitro study of the inflammatory response triggered by TLR3 in intestinal epithelial cells (IECs) and of the interaction between IECs and immune cells. In addition, we showed that PIE cells could be used for the selection of immunobiotic lactobacilli strains with anti-inflammatory activities. We found that Lactobacillus casei MEP221114 is an immunobiotic candidate for modulation of TLR3-mediated inflammatory responses. CONCLUSION: The present study deepened our understanding of the mechanisms of immunobiotic action by demonstrating that the interaction between some lactobacilli strains and IECs can up-regulate the mRNA expression of TLR negative regulators and that this effect could help to regulate the production of inflammatory mediators during the generation of a TLR3-mediated immune response.

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.

Immunobiotic Lactobacillus rhamnosus improves resistance of infant mice against respiratory syncytial virus infection.

Previously we showed that orally administered Lactobacillus rhamnosus CRL1505 beneficially regulated the balance between pro- and anti-inflammatory mediators in the lungs of poly(I:C)-challenged mice, allowing an effective inflammatory response against the TLR3/RIG-I agonist but at the same time reducing tissue damage. The aim of the present study was to investigate whether oral administration of the CRL1505 strain was able to improve resistance against respiratory syncytial virus (RSV) infection in infant mice and to evaluate the immunological mechanisms involved in the immunobiotic effect. We demonstrated that treatment of 3-week old BALB/c mice with L. rhamnosus CRL1505 significantly reduce lung viral loads and tissue injuries after the challenge with RSV. Moreover, we showed that the protective effect achieved by the CRL1505 strain is related to its capacity to differentially modulate respiratory antiviral immune response. Our results shows that IFN-γ and IL-10 secreted in response to L. rhamnosus CRL1505 oral stimulation would modulate the pulmonary innate immune microenvironment conducting to the activation of CD103(+) and CD11b(high) dendritic cells and the generation of CD3(+)CD4(+)IFN-γ(+) Th1 cells with the consequent attenuation of the strong and damaging Th2 reactions associated with RSV challenge. Our results indicate that modulation of the common mucosal immune system by immunobiotics could favor protective immunity against respiratory viral pathogens with a high attack rate in early infancy, such as RSV.

A newly established bovine intestinal epithelial cell line is effective for in vitro screening of potential antiviral immunobiotic microorganisms for cattle.

We evaluated whether a bovine intestinal epithelial (BIE) cell line could serve as a useful in vitro model system for studying antiviral immune responses in bovine intestinal epithelial cells (IECs) and for the primary screening of immunobiotic microorganisms with antiviral protective capabilities. Immunofluorescent analyses revealed that toll-like receptor 3 (TLR3) was expressed in BIE cells, and the results of real-time quantitative PCR showed that these cells respond to stimulation with poly(I:C) by up-regulating pro-inflammatory cytokines and type I interferons. In addition, we demonstrated that BIE cells are useful for the primary screening of immunobiotic lactic acid bacteria strains which are able to beneficially modulate antiviral immune responses triggered by TLR3 activation in bovine IECs. The characterization of BIE cells performed in the present study represents an important step towards the establishment of a valuable bovine in vitro system that could be used for the development of immunomodulatory feed for bovine hosts.

Immunobiotic Lactobacillus jensenii modulates the Toll-like receptor 4-induced inflammatory response via negative regulation in porcine antigen-presenting cells.

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 Peyer's 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 ß 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.