Abstract:

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 pathogenassociated 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-1! and MCP-1 in BIE cells by activating both NF-"B 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-"B 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.

Author affiliation: Takanashi, Naoya. Food and Feed Immunology Group. Laboratory of Animal Products Chemistry. Graduate School of Agricultural Science. Tohoku University; Japan;

Author affiliation: Tomosada, Yohsuke. Food and Feed Immunology Group. Laboratory of Animal Products Chemistry. Graduate School of Agricultural Science. Tohoku University; Japan;

Author affiliation: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Tucuman. Centro de Referencia Para Lactobacilos (i); Food and Feed Immunology Group. Laboratory of Animal Products Chemistry. Graduate School of Agricultural Science. Tohoku University; Japan;

Author affiliation: Murata, Kozue. Food and Feed Immunology Group. Laboratory of Animal Products Chemistry. Graduate School of Agricultural Science. Tohoku University; Japan;

Author affiliation: Takahashi, Takuya. Food and Feed Immunology Group. Laboratory of Animal Products Chemistry. Graduate School of Agricultural Science. Tohoku University; Japan;

Author affiliation: Chiba, Eriko. Food and Feed Immunology Group. Laboratory of Animal Products Chemistry. Graduate School of Agricultural Science. Tohoku University; Japan;

Author affiliation: Tohno, Masanori. Food and Feed Immunology Group. Laboratory of Animal Products Chemistry. Graduate School of Agricultural Science. Tohoku University; Japan; National Agriculture and Food Research Organization. National Institute of Livestock and Grassland Science; Japan.;

Author affiliation: Tomoyuki Shimazu. Food and Feed Immunology Group. Laboratory of Animal Products Chemistry. Graduate School of Agricultural Science. Tohoku University; Japan; Laboratory of Animal Breading and Genetics. Graduate School of Agricultural Science; Japan.;

Author affiliation: Aso, Hisashi. Cell Biology Laboratory, Graduate School of Agricultural Science. Tohoku University; Japan.;

Author affiliation: Suda, Yoshihito. Department of Food, Agriculture and Environment. Miyagi University; Japan.;

Author affiliation: Ikegami, Shuji. Division of Research and Development. Food Science Institut. Meiji Dairies CoOdawara; Japan;

Author affiliation: Itoh, Hiroyuki. Division of Research and Development. Food Science Institut. Meiji Dairies CoOdawara; Japan;

Author affiliation: Kawai, Yasushi. Food and Feed Immunology Group. Laboratory of Animal Products Chemistry. Graduate School of Agricultural Science. Tohoku University; Japan;

Author affiliation: Tadao Saito. Food and Feed Immunology Group. Laboratory of Animal Products Chemistry. Graduate School of Agricultural Science. Tohoku University; Japan;

Author affiliation: Alvarez, Gladis Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); Argentina;

Author affiliation: Kitazawa, Haruki. Food and Feed Immunology Group. Laboratory of Animal Products Chemistry. Graduate School of Agricultural Science. Tohoku University; Japan;

Abstract:

We previously established a bovine intestinal epithelial cell line (BIE cells) and showed that BIE cells are useful in vitro model system for the study of interactions between pathogenic and beneficial microorganisms and bovine intestinal epithelial cells (IECs). In the present study we aimed to select potential immunomodulatory bifidobacteria that may be used to beneficially modulate the inflammatory response in bovine IECs. We also aimed to gain insight in the molecular mechanisms involved in the anti-inflammatory effect of bifidobacteria by evaluating the role of Toll-like receptor (TLR)-2 and TLR negative regulators in the regulation of proinflamatory cytokines production and MAPK, NF-κB and PI3K pathways activation in BIE cells. Five bifidobacteria strains were evaluated in this study and according to their capacity to modulate inflammatory response of BIE cells. Despite the unique effect of each strain, four common points were found when comparing the effect of the high and moderate anti-inflammatory strains: 1) Upregulation of TLR negative regulators and the intensity of that upregulation was related to the different immunomodulatory capacity of each bifidobacteria strain. 2) The balance between MAPK activation and MKP-1 upregulation affected the an- ti-inflammatory effect of bifidobacteria in BIE cells. 3) The inhibition of PI3K pathway was related to the an- ti-inflammatory effect of bifidobacteria. 4) The immunoregulatory effect of bifidobacteria in BIE cells is partially de- pendent on TLR2. This study shows that BIE cells can be used for the selection of immunoregulatory bifidobacteria and for studying the mechanisms involved in the protective activity of immunobiotics against TLR4-induced inflammatory damage. In addition, we have demonstrated that the anti-inflammatory effect of bifidobacteria was achieved by a com- plex interaction of multiple TLRs negative regulators as well as the inhibition/activation of multiple signaling pathways.

Author affiliation: Murata, Kozue. Tohoku University. Graduate School of Agricultural Science. Laboratory of Animal Products Chemistry. Food and Feed Immunology Group; Japón;

Author affiliation: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); Argentina; Tohoku University. Graduate School of Agricultural Science. Laboratory of Animal Products Chemistry. Food and Feed Immunology Group; Japón;

Author affiliation: Tomosada, Yohsuke. Tohoku University. Graduate School of Agricultural Science. Laboratory of Animal Products Chemistry. Food and Feed Immunology Group; Japón;

Author affiliation: Risa, Hara. Tohoku University. Graduate School of Agricultural Science. Laboratory of Animal Products Chemistry. Food and Feed Immunology Group; Japón;

Author affiliation: Chiba, Eriko. Tohoku University. Graduate School of Agricultural Science. Laboratory of Animal Products Chemistry. Food and Feed Immunology Group; Japón;

Author affiliation: Shimazu, Tomoyuki. Tohoku University. Graduate School of Agricultural Science. Laboratory of Animal Breeding and Genetics; Japón;

Author affiliation: Aso, Hisashi. Tohoku University. Graduate School of Agricultural Science. Cell Biology Laboratory; Japón;

Author affiliation: Suda, Yoshihito. Miyagi University. Department of Food, Agriculture and Environmental Science; Japón;

Author affiliation: Iwabuchi, Noriyuki. Morinaga Milk Industry Co. Ltd. Food Science and Technology Institute; Japón;

Author affiliation: Xiao, Jin-zhong. Morinaga Milk Industry Co. Ltd. Food Science and Technology Institute; Japón;

Author affiliation: Saito, Tadao. Tohoku University. Graduate School of Agricultural Science. Laboratory of Animal Products Chemistry. Food and Feed Immunology Group; Japón;

Author affiliation: Kitazawa, Haruki. Tohoku University. Graduate School of Agricultural Science. Laboratory of Animal Products Chemistry. Food and Feed Immunology Group; Japón;

Abstract:

Plant lectins, which are proteins/glycoproteins present in a wide range of vegetables, fruits, cereals and beans, are resistant to digestive enzymes and food cooking temperatures. They bind reversibly to specific glycosidic residues expressed on the membrane of intestinal epithelial cells (IEC) and cause anti-nutritional effects in humans and animals. Soybean lectin (SBA) has been detected in poultry diets, and its ability to bind to the intestinal epithelium has been reported. The development of new methods for removing SBA from feeds or to prevent interaction with the intestinal mucosa is of interest. In this study, the in vitro cytotoxicity of SBA on IEC of chicks was demonstrated for the first time. The LD50, assessed after 2 h exposure of IEC to SBA, was 6.13 μg mL-1. The ability of Bifidobacterium infantis CRL1395 to bind SBA on the bacterial envelope was confirmed, and prevention of IEC cytotoxicity by lectin removal was demonstrated. Safety of B. infantis CRL1395, resistance to gastrointestinal stress and adhesion were also determined. It was concluded that the early administration of B. infantis CRL1395 to chicks would effectively reduce the toxicity of SBA. Besides, it would favour the colonization of the gut with a beneficial microbiota.

Author affiliation: Babot, Jaime Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina

Author affiliation: Argañaraz Martínez, Fernando Eloy. Universidad Nacional de Tucumán; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Lorenzo Pisarello, Maria Jose. Universidad Nacional de Tucumán; Argentina. Universidad Nacional de Tucumán; Argentina

Author affiliation: Apella, Maria Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina. Universidad Nacional de Tucumán; Argentina

Author affiliation: Perez Chaia, Adriana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina. Universidad Nacional de Tucumán; Argentina

Abstract:

Previously, we reported that the non-viable immunomodulatory Bifidobacterium infantis MCC12 and Bifidobacterium breve MCC1274 strains (paraimmunobiotic bifidobacteria) were able to increase the protection against rotavirus infection in bovine intestinal epithelial (BIE) cells. In order to gain insight into the influence of paraimmunobiotic bifidobacteria on the innate antiviral immune response of BIE cells, their effect on the transcriptomic response triggered by Toll-like receptor 3 (TLR3) activation was investigated. By using microarray technology and qPCR analysis, we obtained a global overview of the immune genes involved in the innate antiviral immune response in BIE cells. Activation of TLR3 by poly(I:C) in BIE cells significantly increased the expression of interferon (IFN)-α and IFN-β, several interferon-stimulated genes, cytokines, and chemokines. It was also observed that both paraimmunobiotic bifidobacteria differently modulated immune genes expression in poly(I:C)-challenged BIE cells. Most notable changes were found in genes involved in antiviral defence (IFN-β, MX1, OAS1X, MDA5, TLR3, STAT2, STAT3), cytokines (interleukin (IL)-6), and chemokines (CCL2, CXCL2, CXCL6) that were significantly increased in bifidobacteria-treated BIE cells. B. infantis MCC12 and B. breve MCC1274 showed quantitative and qualitative differences in their capacities to modulate the innate antiviral immune response in BIE cells. B. breve MCC1274 was more efficient than the MCC12 strain to improve the production of type I IFNs and antiviral factors, an effect that could be related to its higher ability to protect against rotavirus replication in BIE cells. Interestingly, B. infantis MCC12 showed a remarkable anti-inflammatory effect. The MCC12 strain was more efficient to reduce the expression of inflammatory cytokines and chemokines (IL-16, IL-20, CX3CL1) when compared with B. breve MCC1274. These results provided valuable information for the deeper understanding of the antiviral immune response of intestinal epithelial cells as well as the host-paraimmunobiotic interaction in the bovine host.

Author affiliation: Albarracín, Leonardo Miguel. Tohoku University. Graduate School of Agricultural Science; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán; Argentina

Author affiliation: Komatsu, Ryoya. Tohoku University. Graduate School of Agricultural Science; Japón

Author affiliation: Garcia-Castillo, Valeria. Tohoku University. Graduate School of Agricultural Science; Japón. Universidad de Concepción; Chile

Author affiliation: Aso, Hisashi. Tohoku University. Graduate School of Agricultural Science; Japón

Author affiliation: Iwabuchi, Norisuke. Morinaga Milk Industry Co. Ltd; Japón

Author affiliation: Xiao, Jin-Zhong. Morinaga Milk Industry Co. Ltd; Japón

Author affiliation: Abe, Fumiaki. Morinaga Milk Industry Co. Ltd; Japón

Author affiliation: Takahashi, Hideki. Tohoku University. Graduate School of Agricultural Science; Japón

Author affiliation: Villena, Julio Cesar. Tohoku University. Graduate School of Agricultural Science; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina

Author affiliation: Kitazawa, Haruki. Tohoku University. Graduate School of Agricultural Science; Japón

Abstract:

Previous studies demonstrated that the extracellular polysaccharides (EPSs) produced by Lactobacillus delbrueckii OLL1073R-1 (LDR-1) improve antiviral immunity, especially in the systemic and respiratory compartments. However, it was not studied before whether those EPSs are able to beneficially modulate intestinal antiviral immunity. In addition, LDR-1-host interaction has been evaluated mainly with immune cells while its interaction with intestinal epithelial cells (IECs) was not addressed before. In this work, we investigated the capacity of EPSs from LDR-1 to modulate the response of porcine IECs (PIE cells) to the stimulation with the Toll-like receptor (TLR)-3 agonist poly(I:C) and the role of TLR2, TLR4, and TLR negative regulators in the immunoregulatory effect. We showed that innate immune response triggered by TLR3 activation in porcine IECs was differentially modulated by EPS from LDR-1. EPSs treatment induced an increment in the expression of interferon (IFN)-α and IFN-β in PIE cells after the stimulation with poly(I:C) as well as the expression of the antiviral factors MxA and RNase L. Those effects were related to the reduced expression of A20 in EPS-treated PIE cells. EPS from LDR-1 was also able to reduce the expression of IL-6 and proinflammatory chemokines. Although further in vivo studies are needed, our results suggest that these EPSs or a yogurt fermented with LDR-1 have potential to improve intestinal innate antiviral response and protect against intestinal viruses.

Author affiliation: Kanmani, Paulraj. Tohoku University; Japón

Author affiliation: Albarracín, Leonardo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japón

Author affiliation: Kobayashi, Hisakazu. Tohoku University; Japón

Author affiliation: Iida, Hikaru. Tohoku University; Japón

Author affiliation: Komatsu, Ryoya. Tohoku University; Japón

Author affiliation: Kober, Humayun A. K. M.. Tohoku University; Japón. Chittagong Veterinary and Animal Sciences University. Department of Dairy & Poultry Science; Bangladesh

Author affiliation: Ikeda-Ohtsubo, Wakako. Tohoku University; Japón

Author affiliation: Suda, Yoshihito. Miyagi University. Department of Food, Agriculture and Environment; Japón

Author affiliation: Aso, Hisashi. Tohoku University; Japón

Author affiliation: Makino, Seiya. Meiji Co., Ltd. Food Science Research Laboratory; Japón

Author affiliation: Kano, Hiroshi. Meiji Co., Ltd. Food Science Research Laboratory; Japón

Author affiliation: Saito, Tadao. Tohoku University; Japón

Author affiliation: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japón

Author affiliation: Kitazawa, Haruki. Tohoku University; Japón

Abstract:

In lactic acid bacteria, the synthesis of exopolysaccharides (EPS) has been associated with some favorable technological properties as well as health-promoting benefits. Research works have shown the potential of EPS produced by lactobacilli to differentially modulate immune responses. However, most studies were performed in immune cells and few works have concentrated in the immunomodulatory activities of EPS in non-immune cells such as intestinal epithelial cells. In addition, the cellular and molecular mechanisms involved in the immunoregulatory effects of EPS have not been studied in detail. In this work, we have performed a genomic characterization of Lactobacillus delbrueckii subsp. delbrueckii TUA4408L and evaluated the immunomodulatory and antiviral properties of its acidic (APS) and neutral (NPS) EPS in porcine intestinal epithelial (PIE) cells. Whole genome sequencing allowed the analysis of the general features of L. delbrueckii TUA4408L genome as well as the characterization of its EPS genes. A typical EPS gene cluster was found in the TUA4408L genome consisting in five highly conserved genes epsA-E, and a variable region, which includes the genes for the polymerase wzy, the flippase wzx, and seven glycosyltransferases. In addition, we demonstrated here for the first time that L. delbrueckii TUA4408L and its EPS are able to improve the resistance of PIE cells against rotavirus infection by reducing viral replication and regulating inflammatory response. Moreover, studies in PIE cells demonstrated that the TUA4408L strain and its EPS differentially modulate the antiviral innate immune response triggered by the activation of Toll-like receptor 3 (TLR3). L. delbrueckii TUA4408L and its EPS are capable of increasing the activation of interferon regulatory factor (IRF)-3 and nuclear factor κB (NF-κB) signaling pathways leading to an improved expression of the antiviral factors interferon (IFN)-β, Myxovirus resistance gene A (MxA) and RNaseL.

Author affiliation: Kanmani, Paulraj. Tohoku University. Graduate School of Agricultural Science. Food and Feed Immunology Group, Laboratory of Animal Products Chemistry; Japón

Author affiliation: Albarracín, Leonardo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University. Graduate School of Agricultural Science. Food and Feed Immunology Group, Laboratory of Animal Products Chemistry; Japón

Author affiliation: Kobayashi, Hisakazu. Tohoku University. Graduate School of Agricultural Science. Food and Feed Immunology Group, Laboratory of Animal Products Chemistry; Japón

Author affiliation: Hebert, Elvira Maria. Tohoku University. Graduate School of Agricultural Science. Food and Feed Immunology Group, Laboratory of Animal Products Chemistry; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina

Author affiliation: Saavedra, Maria Lucila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina

Author affiliation: Komatsu, Ryoya. Tohoku University. Graduate School of Agricultural Science. Food and Feed Immunology Group, Laboratory of Animal Products Chemistry; Japón

Author affiliation: Gatica, Brian. University of Concepcion. Departmento de Farmacología; Chile

Author affiliation: Miyazaki, Ayako. National Institute of Animal Health. Viral Diseases and Epidemiology Research Division; Japón

Author affiliation: Ikeda-Ohtsubo, Wakako. Tohoku University. Graduate School of Agricultural Science. Food and Feed Immunology Group, Laboratory of Animal Products Chemistry; Japón

Author affiliation: Suda, Yoshihito. Miyagi University. Department of Food, Agriculture, and Environment; Japón

Author affiliation: Aso, Hisashi. Tohoku University. Graduate School of Agricultural Science. Cell Biology Laboratory; Japón

Author affiliation: Egusa, Shintaro. Marusan-Ai Co. Research & Development Division; Japón

Author affiliation: Mishima, Takashi. Mie University. Graduate School of Regional Innovation Studies; Japón

Author affiliation: Salas-Burgos, Alexis. University of Concepcion. Departmento de Farmacología; Chile

Author affiliation: Takahashi, Hideki. Tohoku University. Graduate School of Agricultural Science. Laboratory of Plant Pathology; Japón

Author affiliation: Villena, Julio Cesar. Tohoku University. Graduate School of Agricultural Science. Food and Feed Immunology Group, Laboratory of Animal Products Chemistry; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina

Author affiliation: Kitazawa, Haruki. Tohoku University. Graduate School of Agricultural Science. Food and Feed Immunology Group, Laboratory of Animal Products Chemistry; Japón

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 (TLR)-4-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 pro-inflammatory cytokines and chemokines caused by ETEC or LPS challenge by downregulating TLR4-dependent nuclear factor-κB (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 MPK-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.

Author affiliation: Shimazu, Tomoyuki. Tohoku University. Graduate School of Agricultural Science. Food Immunology Group; Japón

Author affiliation: Villena, Julio Cesar. Tohoku University. Graduate School of Agricultural Science. Food Immunology Group; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina

Author affiliation: Tohno, Masanori. National Institute Of Livestock And Grassland Science; Japón

Author affiliation: Fujie, Hitomi. Tohoku University. Graduate School of Agricultural Science. Food Immunology Group; Japón. Josai University. Graduate School of Pharmaceutical Science. Department of Nutritional Physiology; Japón

Author affiliation: Hosoya, Shoichi. Tohoku University. Graduate School of Agricultural Science. Food Immunology Group; Japón

Author affiliation: Shimosato, Takeshi. Shinshu University. Fiber-Nanotech Young Researcher Empowerment Center; Japón

Author affiliation: Aso, Hisashi. Tohoku University. Graduate School of Agricultural Science. Cell Biology Laboratory; Japón

Author affiliation: Suda, Yoshihito. Miyagi University. Department of Food, Agriculture and Environmental Science; Japón

Author affiliation: Kawai, Yasushi. Tohoku University. Graduate School of Agricultural Science. Food Immunology Group; Japón

Author affiliation: Saito, Tadao. Tohoku University. Graduate School of Agricultural Science. Food Immunology Group; Japón

Author affiliation: Makino, Seiya. Meiji Dairies Corporation. Division of Research and Development; Japón

Author affiliation: Ikegami, Shuji. Meiji Dairies Corporation. Division of Research and Development; Japón

Author affiliation: Itoh, Hiroyuki. Meiji Dairies Corporation. Division of Research and Development; Japón

Author affiliation: Kitazawa, Haruki. Tohoku University. Graduate School of Agricultural Science. Food Immunology Group; Japón

Abstract:

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.

Author affiliation: Hosoya, Soichi. Tohoku University. Food Immunology Group; Japón

Author affiliation: Villena, Julio Cesar. Tohoku University. Food Immunology Group; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina

Author affiliation: Shimazu, Tomoyuki. Tohoku University. Food Immunology Group; Japón

Author affiliation: Tohno, Masanori. Tohoku University. Food Immunology Group; Japón

Author affiliation: Fujie, Hitomi. Tohoku University. Food Immunology Group; Japón

Author affiliation: Chiba, Eriko. Tohoku University. Food Immunology Group; Japón

Author affiliation: Shimosato, Takeshi. Tohoku University. Food Immunology Group; Japón

Author affiliation: Aso, Hisashi. Tohoku University. Food Immunology Group; Japón

Author affiliation: Suda, Yoshihito. Miyagi University. Department of Food Agriculture; Japón

Author affiliation: Kawai, Yasushi. Tohoku University. Food Immunology Group; Japón

Author affiliation: Saito, Tadao. Tohoku University. Food Immunology Group; Japón

Author affiliation: Alvarez, Gladis Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Bioquímica Aplicada; Argentina

Author affiliation: Ikegami, Shuji. Meiji Dairies Corporation Japan; Japón

Author affiliation: Itoh, Hiroyuki. Meiji Dairies Corporation Japan; Japón

Author affiliation: Kitazawa, Haruki. Tohoku University. Food Immunology Group; Japón

Abstract:

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-kB (NF-kB) 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-kB 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-kB 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.

Author affiliation: Tomosada, Yohsuke. Tohoku University; Japón;

Author affiliation: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Tucumán. Centro de Referencia Para Lactobacilos (i); Argentina;

Author affiliation: Morata, Kozue. Tohoku University; Japón;

Author affiliation: Chiba, Eriko. Tohoku University; Japón;

Author affiliation: Shimazu, Yomoyuki. Tohoku University; Japón;

Author affiliation: Aso, Hisashi. Tohoku University; Japón;

Author affiliation: Iwabuchi, Norisuke. Morinaga Milk Industry Co. Ltd.; Japón;

Author affiliation: Xiao, Jin-Zhong. Morinaga Milk Industry Co. Ltd.; Japón;

Author affiliation: Saito, Tadao. Tohoku University; Japón;

Author affiliation: Kitazawa, Haruki. Tohoku University; Japón;

Abstract:

The mucosal tissues of the gastrointestinal tract are the main portal entry of pathogens such as rotavirus (RV), which is a leading cause of death due to diarrhea among young children across the globe and a major cause of severe acute intestinal infection in livestock animals. The interactions between intestinal epithelial cells (IECs) and immune cells with RVs have been studied for several years, and now, it is known that the innate immune responses triggered by this virus can have both beneficial and detrimental effects for the host. It was demonstrated that natural RV infection in infants and experimental challenges in mice result in the intestinal activation of pattern recognition receptors (PRRs) such as toll-like receptor 3 (TLR3) and striking secretion of proinflammatory mediators that can lead to increased local tissue damage and immunopathology. Therefore, modulating desregulated intestinal immune responses triggered by PRRs activation are a significant promise for reducing the burden of RV diseases. The ability of immunoregulatory probiotic microorganisms (immunobiotics) to protect against intestinal infections, such as those caused by RVs, is among the oldest effects studied for these important group of beneficial microbes. In this review, we provide an update of the current status on the modulation of intestinal antiviral innate immunity by immunobiotics and their beneficial impact on RV infection. In addition, we describe the research of our group that demonstrated the capacity of immunobiotic strains to beneficially modulated TLR3-triggered immune response in IECs, reduce the disruption of intestinal homeostasis caused by intraepithelial lymphocytes, and improve the resistance to RV infections.

Author affiliation: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japón. Immunobiotics Research Group; Argentina

Author affiliation: Vizoso Pinto, María Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Tohoku University; Japón. Immunobiotics Research Group; Argentina

Author affiliation: Kitazawa, Haruki. Tohoku University; Japón