Isolation and immunocharacterization of lactobacillus salivarius from the intestine of wakame-fed pigs to develop novel "Immunosynbiotics"

Emerging threats of antimicrobial resistance necessitate the exploration of effective alternatives for healthy livestock growth strategies. ?Immunosynbiotics?, a combination of immunoregulatory probiotics and prebiotics with synergistic effects when used together in feed, would be one of the most promising candidates. Lactobacilli are normal residents of the gastrointestinal tract of pigs, and many of them are able to exert beneficial immunoregulatory properties. On the other hand, wakame (Undaria pinnafida), an edible seaweed, has the potential to be used as an immunoregulatory prebiotic when added to livestock feed. Therefore, in order to develop a novel immunosynbiotic, we isolated and characterized immunoregulatory lactobacilli with the ability to utilize wakame. Following a month-long in vivo wakame feeding trial in 8-week-old Landrace pigs (n = 6), sections of intestinal mucous membrane were processed for bacteriological culture and followed by identification of pure colonies by 16S rRNA sequence. Each isolate was characterized in vitro in terms of their ability to assimilate to the wakame and to differentially modulate the expression of interleukin-6 (IL-6) and interferon beta (IFN-β) in the porcine intestinal epithelial (PIE) cells triggered by Toll-like receptor (TLR)-4 and TLR-3 activation, respectively. We demonstrated that feeding wakame to pigs significantly increased the lactobacilli population in the small intestine. We established a wakame-component adjusted culture media that allowed the isolation and characterization of a total of 128 Lactobacilli salivarius colonies from the gut of wakame-fed pigs. Interestingly, several L. salivarius isolates showed both high wakame assimilation ability and immunomodulatory capacities. Among the wakame assimilating isolates, L. salivarius FFIG71 showed a significantly higher capacity to upregulate the IL-6 expression, and L. salivarius FFIG131 showed significantly higher capacity to upregulate the IFN-β expression; these could be used as immunobiotic strains in combination with wakame for the development of novel immunologically active feeds for pigs.Fil: Masumizu, Yuki. Tohoku University; JapónFil: Zhou, Binghui. Tohoku University; JapónFil: Humayun Kober, AKM. Tohoku University; Japón. Chittagong Veterinary and Animal Sciences University; BangladeshFil: Islam, M. Aminul. Agricultural University; Bangladesh. Tohoku University; JapónFil: Iida, Hikaru. Tohoku University; JapónFil: Ikeda-Ohtsubo, Wakako. Tohoku University; JapónFil: Suda, Yoshihito. Department Of Food Agriculture, Miyagi University; JapónFil: Albarracín, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japón. Universidad Nacional de Tucumán; ArgentinaFil: Nochi, Tomonori. Tohoku University; JapónFil: Aso, Hisashi. Tohoku University; JapónFil: Suzuki, Keiichi. Tohoku University; JapónFil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; JapónFil: Kitazawa, Haruki. Tohoku University; Japó

Transcriptome Modifications in Porcine Adipocytes via Toll-Like Receptors Activation

Adipocytes are the most important cell type in adipose tissue playing key roles in immunometabolism. We previously reported that nine members of the Toll-like receptor (TLR) family are expressed in an originally established porcine intramuscular pre-adipocyte (PPI) cell line. However, the ability of TLR ligands to modulate immunometabolic transcriptome modifications in porcine adipocytes has not been elucidated. Herein, we characterized the global transcriptome modifications in porcine intramuscular mature adipocytes (pMA), differentiated from PPI, following stimulation with Pam3csk4, Poly(I:C) or LPS which are ligands for TLR2, TLR3, and TLR4, respectively. Analysis of microarray data identified 530 (218 up, 312 down), 520 (245 up, 275 down), and 525 (239 up, 286 down) differentially expressed genes (DEGs) in pMA following the stimulation with Pam3csk4, Poly(I:C), and LPS, respectively. Gene ontology classification revealed that DEGs are involved in several biological processes including those belonging to immune response and lipid metabolism pathways. Functionally annotated genes were organized into two groups for downstream analysis: immune response related genes (cytokines, chemokines, complement factors, adhesion molecules, and signal transduction), and genes involved with metabolic and endocrine functions (hormones and receptors, growth factors, and lipid biosynthesis). Differential expression analysis revealed that EGR1, NOTCH1, NOS2, TNFAIP3, TRAF3IP1, INSR, CXCR4, PPARA, MAPK10, and C3 are the top 10 commonly altered genes of TLRs induced transcriptional modification of pMA. However, the protein-protein interaction network of DEGs identified EPOR, C3, STAR, CCL2, and SAA2 as the major hub genes, which were also exhibited higher centrality estimates in the Gene-Transcription factor interaction network. Our results provide new insights of transcriptome modifications associated with TLRs activation in porcine adipocytes and identified key regulatory genes that could be used as biomarkers for the evaluation of treatments having immunomodularoty and/or metabolic functional beneficial effects in porcine adipocytes.Fil: Igata, Manami. Tohoku University; JapónFil: Islam, M. Aminul. Tohoku University; Japón. Bangladesh Agricultural University; BangladeshFil: Tada, Asuka. Tohoku University; JapónFil: Takagi, Michihiro. Tohoku University; JapónFil: Humayun Kober, AKM. Tohoku University; Japón. Chittagong Veterinary and Animal Sciences University; BangladeshFil: Albarracín, Leonardo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japón. Universidad Nacional de Tucumán. Facultad de Ciencias Exactas y Tecnología. Departamento de Ciencias de la Computación; ArgentinaFil: Aso, Hisashi. Tohoku University; JapónFil: Ikeda-Ohtsubo, Wakako. Tohoku University; JapónFil: Miyazawa, Kenji. Takanashi Milk Products Co.; JapónFil: Yoda, Kazutoyo. Takanashi Milk Products Co.; JapónFil: He, Fang. Takanashi Milk Products Co.; JapónFil: Takahashi, Hideki. Tohoku University; JapónFil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; JapónFil: Kitazawa, Haruki. Tohoku University; Japó

Evaluation of fat accumulation and adipokine production during the long-term adipogenic differentiation of porcine intramuscular preadipocytes and study of the influence of immunobiotics

The degree of fat accumulation and adipokine production are two major indicators of obesity that are correlated with increased adipose tissue mass and chronic inflammatory responses. Adipocytes have been considered effector cells for the inflammatory responses due to their capacity to express Toll-like receptors (TLRs). In this study, we evaluated the degree of fat accumulation and adipokine production in porcine intramuscular preadipocyte (PIP) cells maintained for in vitro differentiation over a long period without or with stimulation of either TNF-α or TLR2-, TLR3-, or TLR4-ligands. The cytosolic fat accumulation was measured by liquid chromatography and the expression of adipokines (CCL2, IL-6, IL-8 and IL-10) were quantified by RT-qPCR and ELISA at several time points (0 to 20 days) of PIP cells differentiation. Long-term adipogenic differentiation (LTAD) induced a progressive fat accumulation in the adipocytes over time. Activation of TLR3 and TLR4 resulted in an increased rate of fat accumulation into the adipocytes over the LTAD. The production of CCL2, IL-8 and IL-6 were significantly increased in unstimulated adipocytes during the LTAD, while IL-10 expression remained stable over the studied period. An increasing trend of adiponectin and leptin production was also observed during the LTAD. On the other hand, the stimulation of adipocytes with TLRs agonists or TNF-α resulted in an increasing trend of CCL2, IL-6 and IL-8 production while IL-10 remained stable in all four treatments during the LTAD. We also examined the influences of several immunoregulatory probiotic strains (immunobiotics) on the modulation of the fat accumulation and adipokine production using supernatants of immunobiotic-treated intestinal immune cells and the LTAD of PIP cells. Immunobiotics have shown a strain-specific ability to modulate the fat accumulation and adipokine production, and differentiation of adipocytes. Here, we expanded the utility and potential application of our in vitro PIP cells model by evaluating an LTAD period (20 days) in order to elucidate further insights of chronic inflammatory pathobiology of adipocytes associated with obesity as well as to explore the prospects of immunomodulatory intervention for obesity such as immunobiotics.Fil: Tada, Asuka. Tohoku University; JapónFil: Kober, Akm Humayun. Chittagong Veterinary and Animal Sciences University; Bangladesh. Tohoku University; JapónFil: Islam, Md Aminul. Bangladesh Agricultural University; Bangladesh. Tohoku University; JapónFil: Igata, Manami. Tohoku University; JapónFil: Takagi, Michihiro. Tohoku University; JapónFil: Suzuki, Masahiko. Tohoku University; JapónFil: Aso, Hisashi. Tohoku University; JapónFil: Ikeda Ohtsubo, Wakako. Tohoku University; JapónFil: Yoda, Kazutoyo. Takanashi Milk Products Co., Ltd.; JapónFil: Miyazawa, Kenji. Takanashi Milk Products Co., Ltd.; JapónFil: He, Fang. Takanashi Milk Products Co., Ltd.; JapónFil: Takahashi, Hideki. Tohoku University; JapónFil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; JapónFil: Kitazawa, Haruki. Tohoku University; Japó

Transcriptome Modifications in the Porcine Intramuscular Adipocytes during Differentiation and Exogenous Stimulation with TNF-α and Serotonin

Adipocytes are dynamic cells that have critical functions to maintain body energy homeostasis. Adipocyte physiology is affected by the adipogenic differentiation, cell program, as well as by the exogenous stimulation of biochemical factors, such as serotonin and TNF-α. In this work, we investigated the global transcriptome modifications when porcine intramuscular preadipocyte (PIP) was differentiated into porcine mature adipocyte (pMA). Moreover, we studied transcriptome changes in pMA after stimulation with serotonin or TNF-α by using a microarray approach. Transcriptome analysis revealed that the expression of 270, 261, and 249 genes were modified after differentiation, or after serotonin and TNF-α stimulation, respectively. Expression changes in APP, HNF4A, ESR1, EGR1, SRC, HNF1A, FN1, ALB, STAT3, CBL, CEBPB, AR, FOS, CFTR, PAN2, PTPN6, VDR, PPARG, STAT5A and NCOA3 genes which are enriched in the ‘PPAR signaling’ and ‘insulin resistance’ pathways were found in adipocytes during the differentiation process. Dose-dependent serotonin stimulation resulted in a decreased fat accumulation in pMAs. Serotonin-induced differentially expressed genes in pMAs were found to be involved in the significant enrichment of ′GPCR ligand-binding′, ‘cell chemotaxis’, ‘blood coagulation and complement’, ‘metabolism of lipid and lipoproteins’, ‘regulation of lipid metabolism by PPARA’, and ‘lipid digestion, mobilization and transport’ pathways. TNF-α stimulation also resulted in transcriptome modifications linked with proinflammatory responses in the pMA of intramuscular origin. Our results provide a landscape of transcriptome modifications and their linked-biological pathways in response to adipogenesis, and exogenous stimulation of serotonin- and TNF-α to the pMA of intramuscular origin.Fil: Tada, Asuka. Tohoku University; JapónFil: Islam, Md Aminul. Tohoku University; Japón. Bangladesh Agricultural University; BangladeshFil: Kober, Akm Humayun. Tohoku University; JapónFil: Fukuyama, Kohtaro. Tohoku University; JapónFil: Takagi, Michihiro. Tohoku University; JapónFil: Igata, Manami. Tohoku University; JapónFil: Albarracín, Leonardo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; JapónFil: Ikeda Ohtsubo, Wakako. Tohoku University; JapónFil: Miyazawa, Kenji. Takanashi Milk Products Co.; JapónFil: Yoda, Kazutoyo. Takanashi Milk Products Co.; JapónFil: He, Fang. Takanashi Milk Products Co.; JapónFil: Takahashi, Hideki. Tohoku University; JapónFil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; JapónFil: Aso, Hisashi. Tohoku University; JapónFil: Kitazawa, Haruki. Tohoku University; Japó

Paraimmunobiotic bifidobacteria modulate the expression patterns of peptidoglycan recognition proteins in porcine intestinal epitheliocytes and antigen presenting ells

Peptidoglycan recognition proteins (PGLYRPs) are a family of pattern recognition receptors (PRRs) that are able to induce innate immune responses through their binding to peptidoglycan (PGN), lipopolysaccharide, or lipoteichoic acid, or by interacting with other PRR-ligands. Recently, progress has been made in understanding the immunobiology of PGLYRPs in human and mice, however, their functions in livestock animals have been less explored. In this study, we characterized the expression patterns of PGLYRPs in porcine intestinal epithelial (PIE) cells and antigen-presenting cells (APCs) and their modulation by the interactions of host cells with PRR-ligands and non-viable immunomodulatory probiotics referred to as paraimmunobiotics. We demonstrated that PGLYRP-1, -2, -3, and -4 are expressed in PIE cells and APCs from Peyer?s patches, being PGLYPR-3 and -4 levels higher than PGLYRP-1 and -2. We also showed that PGLYRPs expression in APCs and PIE cells can be modulated by different PRR agonists. By using knockdown PIE cells for TLR2, TLR4, NOD1, and NOD2, or the four PGLYRPs, we demonstrated that PGLYRPs expressions would be required for activation and functioning of TLR2, TLR4, NOD1, and NOD2 in porcine epitheliocytes, but PGLYRPs activation would be independent of those PRR expressions. Importantly, we reported for the first time that PGLYRPs expression can be differentially modulated by paraimmunobiotic bifidobacteria in a strain-dependent manner. These results provide evidence for the use of paraimmunobiotic bifidobacteria as an alternative for the improvement of resistance to intestinal infections or as therapeutic tools for the reduction of the severity of inflammatory damage in diseases in which a role of PGLYRPs-microbe interaction has been demonstrated.Fil: Iida, Hikaru. Tohoku University; JapónFil: Tohno, Masanori. National Agriculture and Food Research Organization. Central Region Agricultural Research Centre; JapónFil: Islam, Md. Aminul. Tohoku University; Japón. Agricultural University. Faculty of Veterinary Science. Department of Medicine; BangladeshFil: Sato, Nana. Tohoku University; JapónFil: Kobayashi, Hisakazu. Tohoku University; JapónFil: Albarracín, Leonardo Miguel. Tohoku University; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Humayun Kober, AKM. Tohoku University; JapónFil: Ikeda-Ohtsubo, Wakako. Tohoku University; JapónFil: Suda, Yoshihito. Miyagi University. Department of Food, Agriculture and Environment; JapónFil: Aso, Hisashi. Tohoku University; JapónFil: Nochi, Tomonori. Tohoku University; JapónFil: Miyazaki, Ayako. National Institute of Animal Health. Viral Diseases and Epidemiology Research Division; JapónFil: Uenishi, Hirohide. National Agriculture And Food Research Organization; JapónFil: Iwabuchi, Noriyuki. Morinaga Milk Industry Co., Ltd.; JapónFil: Xiao, Jin-zhong. Morinaga Milk Industry Co., Ltd.; JapónFil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; JapónFil: Kitazawa, Haruki. Tohoku University; Japó

Immunomodulation Potential of Probiotics: A Novel Strategy for Improving Livestock Health, Immunity, and Productivity

Over the past decade, the use of probiotics as feed supplements in animal production has increased considerably due to the ban on antibiotic growth promoters in livestock. This review provides an overview of the current situation, limitation, and prospects for probiotic formulations applied to livestock. Recently, the use of probiotics in livestock has been suggested to significantly improve their health, immunity, growth performance, nutritional digestibility, and intestinal microbial balance. Furthermore, it was reported that the use of probiotics in animals was helpful in equilibrating their beneficial microbial population and microbial turnover via stimulating the host immune response through specific secretions and competitive exclusion of potentially pathogenic bacteria in the digestive tract. Recently, there has been great interest in the understanding of probiotics targeted diet and its ability to compete with harmful microbes and acquire their niches. Therefore, the present review explores the most commonly used probiotic formulations in livestock feed and their effect on animal health. In summary, this article provides an in-depth knowledge about the formulation of probiotics as a step toward a better alternative to antibiotic healthy growth strategies.Fil: Humayun Kober, AKM.. Tohoku University; JapónFil: Riaz Rajoka, Muhammad Shahid. Tohoku University; JapónFil: Mehwish, Hafiza Mahreen. Tohoku University; JapónFil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Kitazawa, Haruki. Tohoku University; Japó