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1.  Immunobiotic Lactobacillus jensenii as immune-health promoting factor to improve growth performance and productivity in post-weaning pigs 
BMC Immunology  2014;15:24.
Immunoregulatory probiotics (immunobiotics) have been proposed to improve piglets’ immune system to avoid intestinal infections and reduce unproductive inflammation after weaning. Previously, it was demonstrated that Lactobacillus jensenii TL2937 (LjTL2937) attenuated the inflammatory response triggered by activation of Toll-like receptor 4 (TLR-4) in porcine intestinal epithelial (PIE) cells and antigen presenting cells (APCs) from porcine Peyer’s patches (PP).
In view of the critical importance of PIE-APCs interactions in the regulation of intestinal immune responses, we aimed to examine the effect of LjTL2937 on activation patterns of APCs from swine PPs in co-cultures with PIE cells. In addition, we investigated whether LjTL2937 was able to beneficially modulate intestinal immunity of piglets after weaning to improve immune-health status.
Stimulation of PIE-APCs co-cultures with LjTL2937 increased the expression of MHC-II, CD80/86, IL-10, and Bcl-3 in CD172a+CD11R1- and CD172a+CD11R1high APCs. In addition, the TL2937 strain caused the upregulation of three negative regulators of TLR4 in PIE cells: MKP-1, Bcl-3 and A20. These changes significantly reduced the inflammatory response triggered by TLR4 activation in PIE-APCs co-cultures. The in vivo experiments using castrated male piglets (crossbreeding (LWD) with Landrace (L), Large Yorkshire (W) and Duroc (D))of 3 weeks of age demonstrated that feeding with LjTL2937 significantly reduced blood complement activity and C reactive protein concentrations while no changes were observed in blood leukocytes, ratio of granulocytes to lymphocyte numbers, macrophages’ activity and antibody levels. In addition, treatment with LjTL2937 significantly improved growth performance and productivity, and increased carcass quality.
We demonstrated that the use of immunobiotics strains like LjTL2937, as supplemental additives for piglets feedings, could be used as a strategy to maintain and improve intestinal homeostasis; that is important for the development of the pig and for health and performance throughout the productive life of the animal.
PMCID: PMC4068960  PMID: 24943108
Immunobiotics; Immune performance; Productivity; Piglets; Lactobacillus jensenii TL2937; TLR4; TLRs negative regulators
2.  Immunobiotic Lactobacillus rhamnosus strains differentially modulate antiviral immune response in porcine intestinal epithelial and antigen presenting cells 
BMC Microbiology  2014;14:126.
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.
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 –β 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-1β, IL-6, and IFN-γ in APCs. IL-10 was also significantly up-regulated by CRL1505 in APCs.
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.
PMCID: PMC4035899  PMID: 24886142
Lactobacillus rhamnosus; Poly(I:C); Antiviral immunity; PIE cells; Intestinal antigen presenting cells; TLR2
3.  Effect of Peripheral 5-HT on Glucose and Lipid Metabolism in Wether Sheep 
PLoS ONE  2014;9(2):e88058.
In mice, peripheral 5-HT induces an increase in the plasma concentrations of glucose, insulin and bile acids, and a decrease in plasma triglyceride, NEFA and cholesterol concentrations. However, given the unique characteristics of the metabolism of ruminants relative to monogastric animals, the physiological role of peripheral 5-HT on glucose and lipid metabolism in sheep remains to be established. Therefore, in this study, we investigated the effect of 5-HT on the circulating concentrations of metabolites and insulin using five 5-HT receptor (5HTR) antagonists in sheep. After fasting for 24 h, sheep were intravenously injected with 5-HT, following which-, plasma glucose, insulin, triglyceride and NEFA concentrations were significantly elevated. In contrast, 5-HT did not affect the plasma cholesterol concentration, and it induced a decrease in bile acid concentrations. Increases in plasma glucose and insulin concentrations induced by 5-HT were attenuated by pre-treatment with Methysergide, a 5HTR 1, 2 and 7 antagonist. Additionally, decreased plasma bile acid concentrations induced by 5-HT were blocked by pre-treatment with Ketanserin, a 5HTR 2A antagonist. However, none of the 5HTR antagonists inhibited the increase in plasma triglyceride and NEFA levels induced by 5-HT. On the other hand, mRNA expressions of 5HTR1D and 1E were observed in the liver, pancreas and skeletal muscle. These results suggest that there are a number of differences in the physiological functions of peripheral 5-HT with respect to lipid metabolism between mice and sheep, though its effect on glucose metabolism appears to be similar between these species.
PMCID: PMC3913723  PMID: 24505376
4.  Modulation of Respiratory TLR3-Anti-Viral Response by Probiotic Microorganisms: Lessons Learned from Lactobacillus rhamnosus CRL1505 
Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants and young children. Host immune response is implicated in both protective and immunopathological mechanisms during RSV infection. Activation of Toll-like receptor (TLR)-3 in innate immune cells by RSV can induce airway inflammation, protective immune response, and pulmonary immunopathology. A clear understanding of RSV–host interaction is important for the development of novel and effective therapeutic strategies. Several studies have centered on whether probiotic microorganisms with the capacity to stimulate the immune system (immunobiotics) might sufficiently stimulate the common mucosal immune system to improve defenses in the respiratory tract. In this regard, it was demonstrated that some orally administered immunobiotics do have the ability to stimulate respiratory immunity and increase resistance to viral infections. Moreover, during the last decade scientists have significantly advanced in the knowledge of the cellular and molecular mechanisms involved in the protective effect of immunobiotics in the respiratory tract. This review examines the most recent advances dealing with the use of immunobiotic bacteria to improve resistance against viral respiratory infections. More specifically, the article discuss the mechanisms involved in the capacity of the immunobiotic strain Lactobacillus rhamnosus CRL1505 to modulate the TLR3-mediated immune response in the respiratory tract and to increase the resistance to RSV infection. In addition, we review the role of interferon (IFN)-γ and interleukin (IL)-10 in the immunoregulatory effect of the CRL1505 strain that has been successfully used for reducing incidence and morbidity of viral airways infections in children.
PMCID: PMC4026741  PMID: 24860569
Lactobacillus rhamnosus CRL1505; TLR3; respiratory immunity; respiratory syncytial virus; immunobiotics
5.  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+CD25high 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.
PMCID: PMC4034327  PMID: 24936377
bifidobacteria; anti-inflammatory activity; porcine intestinal epithelial cells; Toll-like receptors negative regulators; Toll-like receptor 2
6.  Regulation of Toll-Like Receptors-Mediated Inflammation by Immunobiotics in Bovine Intestinal Epitheliocytes: Role of Signaling Pathways and Negative Regulators 
Intestinal epithelial cells (IECs) detect bacterial and viral associated molecular patterns via germline-encoded pattern-recognition receptors (PRRs) and are responsible for maintaining immune tolerance to the communities of resident commensal bacteria while being also capable to mount immune responses against pathogens. Toll-like receptors (TLRs) are a major class of PRRs expressed on IECs and immune cells, which are involved in the induction of both tolerance and inflammation. In the last decade, experimental and clinical evidence was generated to support the application of probiotics with immunoregulatory capacities (immunobiotics) for the prevention and treatment of several gastrointestinal inflammatory disorders in which TLRs exert a significant role. The majority of these studies were performed in mouse and human cell lines, and despite the growing interest in the bovine immune system due to the economic importance of cattle as livestock, only few studies have been conducted on cattle. In this regard, our group has established a bovine intestinal epithelial (BIE) cell line originally derived from fetal bovine intestinal epitheliocytes and used this cell line to evaluate the impact of immunobiotics in TLR-mediated inflammation. This review aims to summarize the current knowledge of the beneficial effects of immunobiotics in the regulation of intestinal inflammation/infection in cattle. Especially, we discuss the role of TLRs and their negative regulators in both the inflammatory response and the beneficial effects of immunobiotics in bovine IECs. This review article emphasizes the cellular and molecular interactions of immunobiotics with BIE cells through TLRs and gives the scientific basis for the development of immunomodulatory feed for bovine healthy development.
PMCID: PMC4151153  PMID: 25228903
immunobiotics; TLR4; intestinal immunity; inflammation; bovine intestinal epitheliocytes; TLR negative regulators; lactobacilli; bifidobacteria
7.  Nasally administered Lactobacillus rhamnosus strains differentially modulate respiratory antiviral immune responses and induce protection against respiratory syncytial virus infection 
BMC Immunology  2013;14:40.
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.
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.
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-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.
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.
PMCID: PMC3751766  PMID: 23947615
Lactobacillus rhamnosus; Nasal treatment; Poly(I:C); Sntiviral immunity; Respiratory tract; Respiratory syncytial virus
8.  Immunoregulatory Effect of Bifidobacteria Strains in Porcine Intestinal Epithelial Cells through Modulation of Ubiquitin-Editing Enzyme A20 Expression 
PLoS ONE  2013;8(3):e59259.
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.
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.
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.
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.
PMCID: PMC3608626  PMID: 23555642
9.  Advanced application of bovine intestinal epithelial cell line for evaluating regulatory effect of lactobacilli against heat-killed enterotoxigenic Escherichia coli-mediated inflammation 
BMC Microbiology  2013;13:54.
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).
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).
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.
PMCID: PMC3605377  PMID: 23497067
Bovine intestinal epithelial cells; Immunobiotic; ETEC PAMPs; TLRs negative regulators; Lactobacillus casei OLL2768
10.  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 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.
PMCID: PMC3393362  PMID: 22573738
11.  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.
PMCID: PMC3890654  PMID: 24459463
Lactobacillus jensenii TL2937, TLR4; intestinal immunity; inflammation; immunobiotics
12.  Class I/II hybrid inhibitory oligodeoxynucleotide exerts Th1 and Th2 double immunosuppression 
FEBS Open Bio  2012;3:41-45.
We designed class I/II hybrid inhibitory oligodeoxynucleotides (iODNs), called iSG, and found that the sequence 5′-TTAGGG-3′, which has a six-base loop head structure, and a 3′-oligo (dG)3–5 tail sequence are important for potent immunosuppressive activity. Interestingly, splenocytes isolated from ovalbumin (OVA)-immunized mice and treated with iSG3 showed suppression of not only interleukin (IL)-6, IL-12p35, IL-12p40, and interferon (IFN) γ mRNA expression, but also IL-4 and IL-13 mRNA expression. Thus, both Th2 and Th1 immune responses can be strongly suppressed by iODNs in splenocytes from allergen-immunized mice, suggesting usefulness in the treatment of diseases induced by over-active immune activation.
▸ Inhibitory oligodeoxynucleotides (iODNs) suppress some innate immune responses. ▸ We designed a class I/II hybrid iODN that includes the telomeric motif 5′-TTAGGG-3′. ▸ The telomere motif and a 3′-(G)3–5 polytail sequence are required for potent immunosuppression. ▸ Class I/II hybrid iODNs exert a novel effect of Th1 and Th2 double immunosuppression.
PMCID: PMC3668506  PMID: 23847756
iODN; Class I iODN; Class II iODN; Class I/II hybrid iODN; immunosuppression; ODN, oligodeoxynucleotide; IL, interleukin; TLR, Toll-like receptor; PS, phosphorothioate; PO, phosphodiester; OVA, ovalbumin; ELISA, enzyme-linked immunosorbent assay; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IFN, interferon; Th1 cell, type 1 helper T cell; Th2 cell, type 2 helper T cell; STAT, signal transducer and activator of transcription
13.  Orally administered Lactobacillus rhamnosus modulates the respiratory immune response triggered by the viral pathogen-associated molecular pattern poly(I:C) 
BMC Immunology  2012;13:53.
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.
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 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.
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.
PMCID: PMC3460727  PMID: 22989047
L. rhamnosus CRL1505; Poly(I:C); Antiviral immunity; Respiratory tract
14.  Immunobiotic Lactobacillus jensenii Elicits Anti-Inflammatory Activity in Porcine Intestinal Epithelial Cells by Modulating Negative Regulators of the Toll-Like Receptor Signaling Pathway 
Infection and Immunity  2012;80(1):276-288.
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.
PMCID: PMC3255675  PMID: 22083706
15.  Enzymatic digestion of the milk protein β-casein releases potent chemotactic peptide(s) for monocytes and macrophages 
International immunopharmacology  2007;7(9):1150-1159.
Proteins in the milk release biologically active peptides upon enzymatic digestion. In the present study, we report the identification of novel monocyte/macrophage chemotactic peptides derived from enzymatically digested bovine β-casein, a casein family member that is a major constituent of the milk. β-casein fragments generated by actinase E showed potent chemotactic activity for human and mouse monocytes/macrophages, but not neutrophils, T lymphocytes or dendritic cells. The fragment-induced migration of human monocytes was inhibited by pertussis toxin and was not desensitized by a variety of known chemoattractants, suggesting that the digests activate a unique G protein-coupled receptor(s). The digests were further fractionated and purified to yield 3 small peptides. One peptide Q1 designated as “β-casochemotide-1” with the amino acid sequence of YPVEP (f114-118 of β-casein) induced high levels of macrophage chemotaxis. It also promoted calcium mobilization in macrophages, another indication of cell activation. Our study suggests that biologically active peptides released by actinase-digested milk β-casein may promote innate host immune responses by inducing macrophage migration and activation.
PMCID: PMC3205927  PMID: 17630193
β-casein; monocyte; macrophage; chemotaxis; peptide; β-casochemotide
16.  Immunobiotic lactic acid bacteria beneficially regulate immune response triggered by poly(I:C) in porcine intestinal epithelial cells 
Veterinary Research  2011;42(1):111.
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+CD11R1high 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.
PMCID: PMC3220634  PMID: 22046952
17.  Transcytosis of Murine-Adapted Bovine Spongiform Encephalopathy Agents in an In Vitro Bovine M Cell Model▿ † #  
Journal of Virology  2010;84(23):12285-12291.
Transmissible spongiform encephalopathies (TSE), including bovine spongiform encephalopathy (BSE), are fatal neurodegenerative disorders in humans and animals. BSE appears to have spread to cattle through the consumption of feed contaminated with BSE/scrapie agents. In the case of an oral infection, the agents have to cross the gut-epithelial barrier. We recently established a bovine intestinal epithelial cell line (BIE cells) that can differentiate into the M cell type in vitro after lymphocytic stimulation (K. Miyazawa, T. Hondo, T. Kanaya, S. Tanaka, I. Takakura, W. Itani, M. T. Rose, H. Kitazawa, T. Yamaguchi, and H. Aso, Histochem. Cell Biol. 133:125-134, 2010). In this study, we evaluated the role of M cells in the intestinal invasion of the murine-adapted BSE (mBSE) agent using our in vitro bovine intestinal epithelial model. We demonstrate here that M cell-differentiated BIE cells are able to transport the mBSE agent without inactivation at least 30-fold more efficiently than undifferentiated BIE cells in our in vitro model. As M cells in the follicle-associated epithelium are known to have a high ability to transport a variety of macromolecules, viruses, and bacteria from gut lumen to mucosal immune cells, our results indicate the possibility that bovine M cells are able to deliver agents of TSE, not just the mBSE agent.
PMCID: PMC2976421  PMID: 20861256
18.  DNA Sequencing and Homologous Expression of a Small Peptide Conferring Immunity to Gassericin A, a Circular Bacteriocin Produced by Lactobacillus gasseri LA39▿  
Gassericin A, produced by Lactobacillus gasseri LA39, is a hydrophobic circular bacteriocin. The DNA region surrounding the gassericin A structural gene, gaaA, was sequenced, and seven open reading frames (ORFs) of 3.5 kbp (gaaBCADITE) were found with possible functions in gassericin A production, secretion, and immunity. The deduced products of the five consecutive ORFs gaaADITE have homology to those of genes involved in butyrivibriocin AR10 production, although the genetic arrangements are different in the two circular bacteriocin genes. GaaI is a small, positively charged hydrophobic peptide of 53 amino acids containing a putative transmembrane segment. Heterologous expression and homologous expression of GaaI in Lactococcus lactis subsp. cremoris MG1363 and L. gasseri JCM1131T, respectively, were studied. GaaI-expressing strains exhibited at least sevenfold-higher resistance to gassericin A than corresponding control strains, indicating that gaaI encodes an immunity peptide for gassericin A. Comparison of GaaI to peptides with similar characteristics found in the circular bacteriocin gene loci is discussed.
PMCID: PMC2648179  PMID: 19114506
19.  Structural and Functional Differences in Two Cyclic Bacteriocins with the Same Sequences Produced by Lactobacilli 
Lactobacillus gasseri LA39 and L. reuteri LA6 isolated from feces of the same human infant were found to produce similar cyclic bacteriocins (named gassericin A and reutericin 6, respectively) that cannot be distinguished by molecular weights or primary amino acid sequences. However, reutericin 6 has a narrower spectrum than gassericin A. In this study, gassericin A inhibited the growth of L. reuteri LA6, but reutericin 6 did not inhibit the growth of L. gasseri LA39. Both bacteriocins caused potassium ion efflux from indicator cells and liposomes, but the amounts of efflux and patterns of action were different. Although circular dichroism spectra of purified bacteriocins revealed that both antibacterial peptides are composed mainly of α-helices, the spectra of the bacteriocins did not coincide. The results of d- and l-amino acid composition analysis showed that two residues and one residue of d-Ala were detected among 18 Ala residues of gassericin A and reutericin 6, respectively. These findings suggest that the different d-alanine contents of the bacteriocins may cause the differences in modes of action, amounts of potassium ion efflux, and secondary structures. This is the first report that characteristics of native bacteriocins produced by wild lactobacillus strains having the same structural genes are influenced by a difference in d-amino acid contents in the molecules.
PMCID: PMC404377  PMID: 15128550

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