Insights into the Anti-Adipogenic and Anti-Inflammatory Potentialities of Probiotics against Obesity.

Functional foods with probiotics are safe and effective dietary supplements to improve overweight and obesity. Thus, altering the intestinal microflora may be an effective approach for controlling or preventing obesity. This review aims to summarize the experimental method used to study probiotics and obesity, and recent advances in probiotics against obesity. In particular, we focused on studies (in vitro and in vivo) that used probiotics to treat obesity and its associated comorbidities. Several in vitro and in vivo (animal and human clinical) studies conducted with different bacterial species/strains have reported that probiotics promote anti-obesity effects by suppressing the differentiation of pre-adipocytes through immune cell activation, maintaining the Th1/Th2 cytokine balance, altering the intestinal microbiota composition, reducing the lipid profile, and regulating energy metabolism. Most studies on probiotics and obesity have shown that probiotics are responsible for a notable reduction in weight gain and body mass index. It also increases the levels of anti-inflammatory adipokines and decreases those of pro-inflammatory adipokines in the blood, which are responsible for the regulation of glucose and fatty acid breakdown. Furthermore, probiotics effectively increase insulin sensitivity and decrease systemic inflammation. Taken together, the intestinal microbiota profile found in overweight individuals can be modified by probiotic supplementation which can create a promising environment for weight loss along enhancing levels of adiponectin and decreasing leptin, tumor necrosis factor (TNF)-α, interleukin (IL)-6, monocyte chemotactic protein (MCP)-1, and transforming growth factor (TGF)-ß on human health.

Immunobiotic Ligilactobacillus salivarius FFIG58 Confers Long-Term Protection against Streptococcus pneumoniae.

Previously, we isolated potentially probiotic Ligilactobacillus salivarius strains from the intestines of wakame-fed pigs. The strains were characterized based on their ability to modulate the innate immune responses triggered by the activation of Toll-like receptor (TLR)-3 or TLR4 signaling pathways in intestinal mucosa. In this work, we aimed to evaluate whether nasally administered L. salivarius strains are capable of modulating the innate immune response in the respiratory tract and conferring long-term protection against the respiratory pathogen Streptococcus pneumoniae. Infant mice (3-weeks-old) were nasally primed with L. salivarius strains and then stimulated with the TLR3 agonist poly(I:C). Five or thirty days after the last poly(I:C) administration mice were infected with pneumococci. Among the strains evaluated, L. salivarius FFIG58 had a remarkable ability to enhance the protection against the secondary pneumococcal infection by modulating the respiratory immune response. L. salivarius FFIG58 improved the ability of alveolar macrophages to produce interleukin (IL)-6, interferon (IFN)-γ, IFN-ß, tumor necrosis factor (TNF)-α, IL-27, chemokine C-C motif ligand 2 (CCL2), chemokine C-X-C motif ligand 2 (CXCL2), and CXCL10 in response to pneumococcal challenge. Furthermore, results showed that the nasal priming of infant mice with the FFIG58 strain protected the animals against secondary infection until 30 days after stimulation with poly(I:C), raising the possibility of using nasally administered immunobiotics to stimulate trained immunity in the respiratory tract.

Establishment of a porcine bronchial epithelial cell line and its application to study innate immunity in the respiratory epithelium.

In vitro culture models that precisely mirror the porcine respiratory epithelium are needed to gain insight into how pathogens and host interact. In this study, a new porcine bronchial epithelial cell line, designated as PBE cells, was established from the respiratory tract of a neonatal pig. PBE cells assumed a cobblestone-epithelial like morphology with close contacts between the cells when they reached confluence. The PBE cell line was characterized in terms of its expression of pattern recognition receptors (PRRs) and its ability to respond to the activation of the Toll-like receptor 3 (TLR3) and TLR4 signaling pathways, which are key PRRs involved in the defense of the respiratory epithelium against pathogens. PBE cells stimulated with poly(I:C) were able to up-regulate the expression of IFN-ß, IFN-λ1 (IL-29), IFN-λ3 (IL-28B), the antiviral factors Mx1, OAS1, and PKR, as well as the viral PRRs RIG-1 and MDA5. The expression kinetics studies of immune factors in PBE cells allow us to speculate that this cell line can be a useful in vitro tool to investigate treatments that help to potentiate antiviral immunity in the respiratory epithelium of the porcine host. In addition, poly(I:C) and LPS treatments increased the expression of the inflammatory cytokines TNF-α, IL-6, IL-8, and MCP-1/CCL2 and differentially modulated the expression of negative regulators of the TLR signaling pathways. Then, PBE cells may also allow the evaluation of treatments that can regulate TLR3- and TLR4-mediated inflammatory injury in the porcine airway, thereby protecting the host against harmful overresponses.

The Mucus Binding Factor Is Not Necessary for Lacticaseibacillus rhamnosus CRL1505 to Exert Its Immunomodulatory Activities in Local and Distal Mucosal Sites.

Both viable and non-viable orally administered Lacticaseibacillus rhamnosus CRL1505 modulate immunity in local (intestine) and distal (respiratory) mucosal sites. So, intestinal adhesion and colonization are not necessary for this probiotic strain to exert its immunomodulatory effects. In this work, a mucus-binding factor knockout CRL1505 strain (ΔmbfCRL1505) was obtained and the lack of binding ability to both intestinal epithelial cells and mucin was demonstrated in vitro. In addition, two sets of in vivo experiments in 6-week-old Balb/c mice were performed to evaluate ΔmbfCRL1505 immunomodulatory activities. (A) Orally administered ΔmbfCRL1505 prior to intraperitoneal injection of the Toll-like receptor 3 (TLR3) agonist poly(I:C) significantly reduced intraepithelial lymphocytes (CD3+NK1.1+CD8αα+) and pro-inflammatory mediators (TNF-α, IL-6 and IL-15) in the intestinal mucosa. (B) Orally administered ΔmbfCRL1505 prior to nasal stimulation with poly(I:C) significantly decreased the levels of the biochemical markers of lung tissue damage. In addition, reduced recruitment of neutrophils and levels of pro-inflammatory mediators (TNF-α, IL-6 and IL-8) as well as increased IFN-ß and IFN-γ in the respiratory mucosa were observed in ΔmbfCRL1505-treated mice when compared to untreated control mice. The immunological changes induced by the ΔmbfCRL1505 strain were not different from those observed for the wild-type CRL1505 strain. Although it is generally accepted that the expression of adhesion factors is necessary for immunobiotics to induce their beneficial effects, it was demonstrated here that the mbf protein is not required for L. rhamnosus CRL1505 to exert its immunomodulatory activities in local and distal mucosal sites. These results are a step forward towards understanding the mechanisms involved in the immunomodulatory capabilities of L. rhamnosus CRL1505.

Modulation of Alveolar Macrophages by Postimmunobiotics: Impact on TLR3-Mediated Antiviral Respiratory Immunity.

Beneficial microbes with immunomodulatory capacities (immunobiotics) and their non-viable forms (postimmunobiotics) could be effectively utilized in formulations towards the prevention of respiratory viral infections. In this study, novel immunobiotic strains with the ability to increase antiviral immunity in porcine alveolar macrophages were selected from a library of Lactobacillus gasseri. Postimmunobiotics derived from the most remarkable strains were also evaluated in their capacity to modulate the immune response triggered by Toll-like receptor 3 (TLR3) in alveolar macrophages and to differentially regulate TLR3-mediated antiviral respiratory immunity in infant mice. We provide evidence that porcine alveolar macrophages (3D4/31 cells) are a useful in vitro tool for the screening of new antiviral immunobiotics and postimmunobiotics by assessing their ability to modulate the expression IFN-ß, IFN-λ1, RNAseL, Mx2, and IL-6, which can be used as prospective biomarkers. We also demonstrate that the postimmunobiotics derived from the Lactobacillus gasseri TMT36, TMT39 and TMT40 (HK36, HK39 or HK40) strains modulate the innate antiviral immune response of alveolar macrophages and reduce lung inflammatory damage triggered by TLR3 activation in vivo. Although our findings should be deepened and expanded, the results of the present work provide a scientific rationale for the use of nasally administered HK36, HK39 or HK40 to beneficially modulate TLR3-triggerd respiratory innate immune response.

Ligilactobacillus salivarius Strains Isolated From the Porcine Gut Modulate Innate Immune Responses in Epithelial Cells and Improve Protection Against Intestinal Viral-Bacterial Superinfection.

Previously, we constructed a library of Ligilactobacillus salivarius strains from the intestine of wakame-fed pigs and reported a strain-dependent capacity to modulate IFN-ß expression in porcine intestinal epithelial (PIE) cells. In this work, we further characterized the immunomodulatory activities of L. salivarius strains from wakame-fed pigs by evaluating their ability to modulate TLR3- and TLR4-mediated innate immune responses in PIE cells. Two strains with a remarkable immunomodulatory potential were selected: L. salivarius FFIG35 and FFIG58. Both strains improved IFN-ß, IFN-λ and antiviral factors expression in PIE cells after TLR3 activation, which correlated with an enhanced resistance to rotavirus infection. Moreover, a model of enterotoxigenic E. coli (ETEC)/rotavirus superinfection in PIE cells was developed. Cells were more susceptible to rotavirus infection when the challenge occurred in conjunction with ETEC compared to the virus alone. However, L. salivarius FFIG35 and FFIG58 maintained their ability to enhance IFN-ß, IFN-λ and antiviral factors expression in PIE cells, and to reduce rotavirus replication in the context of superinfection. We also demonstrated that FFIG35 and FFIG58 strains regulated the immune response of PIE cells to rotavirus challenge or ETEC/rotavirus superinfection through the modulation of negative regulators of the TLR signaling pathway. In vivo studies performed in mice models confirmed the ability of L. salivarius FFIG58 to beneficially modulate the innate immune response and protect against ETEC infection. The results of this work contribute to the understanding of beneficial lactobacilli interactions with epithelial cells and allow us to hypothesize that the FFIG35 or FFIG58 strains could be used for the development of highly efficient functional feed to improve immune health status and reduce the severity of intestinal infections and superinfections in weaned piglets.

Evaluation of Porcine Intestinal Epitheliocytes as an In vitro Immunoassay System for the Selection of Probiotic Bifidobacteria to Alleviate Inflammatory Bowel Disease.

The use of in vitro systems that allow efficient selection of probiotic candidates with immunomodulatory properties could significantly minimize the use of experimental animals. In this work, we generated an in vitro immunoassay system based on porcine intestinal epithelial (PIE) cells and dextran sodium sulfate (DSS) administration that could be useful for the selection and characterization of potential probiotic strains to be used in inflammatory bowel disease (IBD) patients. Our strategy was based on two fundamental pillars: on the one hand, the capacity of PIE cells to create a monolayer by attaching to neighboring cells and efficiently mount inflammatory responses and, on the other hand, the use of two probiotic bifidobacteria strains that have been characterized in terms of their immunomodulatory capacities, particularly in mouse IBD models and patients. Our results demonstrated that DSS administration can alter the epithelial barrier created in vitro by PIE cells and induce a potent inflammatory response, characterized by increases in the expression levels of several inflammatory factors including TNF-α, IL-1α, CCL4, CCL8, CCL11, CXCL5, CXCL9, CXCL10, SELL, SELE, EPCAM, VCAM, NCF2, and SAA2. In addition, we demonstrated that Bifidobacterium breve M-16V and B. longum BB536 are able to regulate the C-jun N-terminal kinase (JNK) intracellular signalling pathway, reducing the DSS-induced alterations of the in vitro epithelial barrier and differentially regulating the inflammatory response in a strain-dependent fashion. The good correlation between our in vitro findings in PIE cells and previous studies in animal models and IBD patients shows the potential value of our system to select new probiotic candidates in an efficient way.

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.

Evaluation of the Immunomodulatory Ability of Lactic Acid Bacteria Isolated from Feedlot Cattle Against Mastitis Using a Bovine Mammary Epithelial Cells In Vitro Assay.

Bovine mastitis, the inflammation of the mammary gland, affects the quality and quantity of milk yield. Mastitis control relies on single or multiple combinations of antibiotic therapy. Due to increasing antibiotic resistance in pathogens, the intramammary infusion of lactic acid bacteria (LAB) has been considered as a potential alternative to antibiotics for treating and preventing bovine mastitis through the improvement of the host immunity. Probiotic effects are a strain-dependent characteristic; therefore, candidate LAB strains have to be evaluated efficiently to find out the ones with the best potential. Here, we investigated LAB strains originally isolated from feedlot cattle's environment regarding their ability in inducing the Toll-like receptor (TLR)-triggered inflammatory responses in bovine mammary epithelial (BME) cells in vitro. The BME cells were pre-stimulated with the LAB strains individually for 12, 24, and 48 h and then challenged with Escherichia coli-derived lipopolysaccharide (LPS) for 12 h. The mRNA expression of selected immune genes-interleukin 1 alpha (IL-1α), IL-1ß, monocyte chemotactic protein 1 (MCP-1), IL-8, chemokine (C-X-C motif) ligand 2 (CXCL2), and CXCL3 were quantified by real-time quantitative PCR (RT-qPCR). Results indicated that pretreatment with some Lactobacillus strains were able to differentially regulate the LPS inflammatory response in BME cells; however, strain-dependent differences were found. The most remarkable effects were found for Lactobacillus acidophilus CRL2074, which reduced the expression of IL-1α, IL-1ß, MCP-1, IL-8, and CXCL3, whereas Lactobacillus rhamnosus CRL2084 diminished IL-1ß, MCP-1, and IL-8 expression. The pre-stimulation of BME cells with the CRL2074 strain resulted in the upregulated expression of three negative regulators of the TLRs, including the ubiquitin-editing enzyme A20 (also called tumor necrosis factor alpha-induced protein 3, TNFAIP3), single immunoglobin IL-1 single receptor (SIGIRR), and Toll interacting protein (Tollip) after the LPS challenge. The CRL2084 pre-stimulation upregulated only Tollip expression. Our results demonstrated that the L. acidophilus CRL2074 strain possess remarkable immunomodulatory abilities against LPS-induced inflammation in BME cells. This Lactobacillus strain could be used as candidate for in vivo testing due to its beneficial effects in bovine mastitis through intramammary infusion. Our findings also suggest that the BME cells immunoassay system could be of value for the in vitro evaluation of the immunomodulatory abilities of LAB against the inflammation resulting from the intramammary infection with mastitis-related pathogens.

Efficient Selection of New Immunobiotic Strains With Antiviral Effects in Local and Distal Mucosal Sites by Using Porcine Intestinal Epitheliocytes.

Previously, we evaluated the effect of the immunobiotic strain Lactobacillus rhamnosus CRL1505 on the transcriptomic response of porcine intestinal epithelial (PIE) cells triggered by the challenge with the Toll-like receptor 3 (TLR-3) agonist poly(I:C) and successfully identified a group of genes that can be used as prospective biomarkers for the screening of new antiviral immunobiotics. In this work, several strains of lactobacilli were evaluated according to their ability to modulate the expression of IFNα, IFNß, RIG1, TLR3, OAS1, RNASEL, MX2, A20, CXCL5, CCL4, IL-15, SELL, SELE, EPCAM, PTGS2, PTEGES, and PTGER4 in PIE cells after the stimulation with poly(I:C). Comparative analysis of transcripts variations revealed that one of the studied bacteria, Lactobacillus plantarum MPL16, clustered together with the CRL1505 strain, indicating a similar immunomodulatory potential. Two sets of in vivo experiments in Balb/c mice were performed to evaluate L. plantarum MPL16 immunomodulatory activities. Orally administered MPL16 prior intraperitoneal injection of poly(I:C) significantly reduced the levels of the proinflammatory mediators tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and IL-15 in the intestinal mucosa. In addition, orally administered L. plantarum MPL16 prior nasal stimulation with poly(I:C) or respiratory syncytial virus infection significantly decreased the levels of the biochemical markers of lung tissue damage. In addition, reduced levels of the proinflammatory mediators TNF-α, IL-6, and IL-8 were found in MPL16-treated mice. Improved levels of IFN-ß and IFN-γ in the respiratory mucosa were observed in mice treated with L. plantarum MPL16 when compared to control mice. The immunological changes induced by L. plantarum MPL16 were not different from those previously reported for the CRL1505 strain in in vitro and in vivo studies. The results of this work confirm that new immunobiotic strains with the ability of stimulating both local and distal antiviral immune responses can be efficiently selected by evaluating the expression of biomarkers in PIE cells.

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.

Characterization of the immunomodulatory and anti-Helicobacter pylori properties of the human gastric isolate Lactobacillus rhamnosus UCO-25A.

The ability to form biofilms and the potential immunomodulatory properties of the human gastric isolate Lactobacillus rhamnosus UCO-25A were characterized in vitro. It was demonstrated that L. rhamnosus UCO-25A is able to form biofilms on abiotic and cell surfaces, and to modulate the inflammatory response triggered by Helicobacter pylori infection in gastric epithelial cells and THP-1 macrophages. L. rhamnosus UCO-25A exhibited a substantial anti-inflammatory effect in both cell lines and improved IL-10 levels produced by challenged macrophages. Additionally, UCO-25A protected AGS cells against H. pylori infection with a higher pathogen inhibition when a biofilm was formed. Given the importance of inflammation in H. pylori-mediated diseases, the differential modulation of the inflammatory response in the gastric mucosa by an autochthonous strain is an attractive alternative for improving H. pylori eradication and reducing the severity of the diseases that arise from the resulting chronic inflammation.

Screening and Characterization of Immunobiotic Lactic Acid Bacteria with Porcine Immunoassay Systems.

Microorganisms with the ability to modulate the immune system (immunobiotics) have shown to interact with different pattern recognition receptors (PRRs) expressed in nonimmune and immune cells and exert beneficial effects on host's health maintenance and promotion. Suitable assay systems are necessary for an efficient and rapid screening of potential immunobiotic strains. More than a decade of research have allowed us to develop efficient in vitro models based on porcine receptors and cells (porcine immunoassay systems) to study the immunomodulatory effects of lactic acid bacteria (LAB). In addition, detailed studies of model immunobiotic LAB strains with proved abilities to improve immune health in humans (Lactobacillus rhamnosus CRL1505) or pigs (Lactobacillus jensenii TL2937) allowed us to select the most suitable biomarkers that have to be evaluated in those porcine immunoassay systems. Our in vitro models based on transfectant cells expressing porcine PRRs as well as an originally established porcine intestinal epitheliocyte (PIE) cell line have shown to be useful in vitro tools for the selection of immunobiotics and for obtaining information to elucidate the molecular mechanisms behind their beneficial effects.

Genomic Characterization of Lactobacillus delbrueckii TUA4408L and Evaluation of the Antiviral Activities of its Extracellular Polysaccharides in Porcine Intestinal Epithelial Cells.

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)-ß, Myxovirus resistance gene A (MxA) and RNaseL.

Exopolysaccharides from Lactobacillus delbrueckii OLL1073R-1 modulate innate antiviral immune response in porcine intestinal epithelial cells.

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-ß 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.

Effects of mycoplasmal pneumonia of swine (MPS) lung lesion-selected Landrace pigs on MPS resistance and immune competence in three-way crossbred pigs.

To clarify the genetic influence of mycoplasmal pneumonia of swine (MPS) lesion-selected Landrace (La) on MPS resistance and immune characteristics in three-way crossbred pigs (LaWaDa), the LaWaDa pigs were compared with the non-selected crossbred (LbWbDb) and purebred (La) pigs. The MPS lesion score in the three lines was as follows: La line < LaWaDa line < LbWbDb line, with significant differences among the lines. The proportions of myeloid cells and T cells were lower and higher, respectively, in the LaWaDa pigs compared with those in the other two lines. Messenger RNA (mRNA) expression of interleukin (IL)-6, IL-10, transforming growth factor-ß, and interferon-γ in peripheral blood was significantly increased after vaccination in the La and LaWaDa lines. IL-4 mRNA expression in the LaWaDa line was intermediate to the La and LbWbDb lines. Furthermore, principal component analysis for immune traits and MPS lesions was executed to clarify the characteristics of each pig line. These findings suggest that the immune responses in the three pig lines are genetically distinct and that MPS resistance and some immunity characteristics from the La line were transmitted to the three-way crossbred pigs.

Immunobiotic Bifidobacteria Strains Modulate Rotavirus Immune Response in Porcine Intestinal Epitheliocytes via Pattern Recognition Receptor Signaling.

In this work, we aimed to characterize the antiviral response of an originally established porcine intestinal epithelial cell line (PIE cells) by evaluating the molecular innate immune response to rotavirus (RVs). In addition, we aimed to select immunomodulatory bacteria with antiviral capabilities. PIE cells were inoculated with RVs isolated from different host species and the infective titers and the molecular innate immune response were evaluated. In addition, the protection against RVs infection and the modulation of immune response by different lactic acid bacteria (LAB) strains was studied. The RVs strains OSU (porcine) and UK (bovine) effectively infected PIE cells. Our results also showed that RVs infection in PIE cells triggered TLR3-, RIG-I- and MDA-5-mediated immune responses with activation of IRF3 and NF-κB, induction of IFN-ß and up-regulation of the interferon stimulated genes MxA and RNase L. Among the LAB strains tested, Bifidobacterium infantis MCC12 and B. breve MCC1274 significantly reduced RVs titers in infected PIE cells. The beneficial effects of both bifidobacteria were associated with reduction of A20 expression, and improvements of IRF-3 activation, IFN-ß production, and MxA and RNase L expressions. These results indicate the value of PIE cells for studying RVs molecular innate immune response in pigs and for the selection of beneficial bacteria with antiviral capabilities.

Cyclophilin A is a new M cell marker of bovine intestinal epithelium.

Microfold (M) cells in the follicle-associated epithelium (FAE) of Peyer's patches contribute to the mucosal immune response by the transcytosis of microorganisms. The mechanism by which M cells take up microorganisms, and the functional proteins by which they do this, are not clear. In order to explore one such protein, we developed a 2H5-F3 monoclonal antibody (2H5-F3 mAb) through its binding to bovine M cells, and identified the antibody reactive molecule as cyclophilin A (Cyp-A). The localization patterns of Cyp-A were very similar to the localization pattern of cytokeratin (CK) 18-positive M cells. Cyp-A was identified at the luminal surface of CK18-positive M cells in bovine jejunal and ileal FAE. The membranous localization of Cyp-A in the bovine intestinal cell line (BIE cells) increased as cells differentiated toward M cells, as determined by flow cytometry analysis. Additionally, BIE cells released Cyp-A to the extracellular space and the differentiation of BIE cells to M cells increased the secretion of Cyp-A, as determined by western blotting. Accordingly, Cyp-A may be localized in M cells in the small intestinal epithelium of cattle. The rise of the membranous localization and secretion of Cyp-A by differentiation toward M cells indicates that Cyp-A has an important role in the function of M cells. While Cyp-A of the M cell membrane may contribute to the uptake of viruses with peptidyl-prolyl cis-trans isomerase activity, in the extracellular space Cyp-A may work as a chemokine and contribute to the distribution of immuno-competent cells.

Immunogenic properties and mycoplasmal pneumonia of swine (MPS) lung lesions in Large White pigs selected for higher peripheral blood immune capacity.

Immunogenic properties and mycoplasmal pneumonia of swine (MPS) lung lesions were compared between the immunity-selected Large White line and the non-selected Large White line. The selected Large White line showed a higher level of pulmonary MPS lesions compared with the non-selected Large White line. Subsequent to vaccination, the percentage of natural killer cells and T cells (CD3(+) CD4(+) CD8(-) and CD3(+) CD4(-) CD8(+) T cells) were significantly increased in the non-selected line but remained unchanged in the immunity-selected Large White line. Secretion of Mycoplasma hyopneumoniae vaccine-specific immunoblogulin G and phagocyte activity in peripheral blood were significantly higher in the immunity-selected Large White line than in the non-selected line. Expression of interleukin (IL)-4 and IL-6 messenger RNA in hilar lymph nodes was significantly lower in the immunity-selected Large White line than in the non-selected line. However, expression of IL-10 in all immune tissues was significantly higher in the immunity-selected Large White line. These results suggest that the selection for high immunity was not effective in increasing resistance to MPS lung lesions.

Oral delivery of Lactococcus lactis that secretes bioactive heme oxygenase-1 alleviates development of acute colitis in mice.

BACKGROUND: Mucosal delivery of therapeutic proteins using genetically modified strains of lactic acid bacteria (gmLAB) is being investigated as a new therapeutic strategy. METHODS: We developed a strain of gmLAB, Lactococcus lactis NZ9000 (NZ-HO), which secretes the anti-inflammatory molecule recombinant mouse heme oxygenase-1 (rmHO-1). The effects of short-term continuous oral dosing with NZ-HO were evaluated in mice with dextran sulfate sodium (DSS)-induced acute colitis as a model of inflammatory bowel diseases (IBD). RESULTS: We identified the secretion of rmHO-1 by NZ-HO. rmHO-1 was biologically active as determined with spectroscopy. Viable NZ-HO was directly delivered to the colon via oral administration, and rmHO-1 was secreted onto the colonic mucosa in mice. Acute colitis in mice was induced by free drinking of 3 % DSS in water and was accompanied by an increase in the disease activity index score and histopathological changes. Daily oral administration of NZ-HO significantly improved these colitis-associated symptoms. In addition, NZ-HO significantly increased production of the anti-inflammatory cytokine interleukin (IL)-10 and decreased the expression of pro-inflammatory cytokines such as IL-1α and IL-6 in the colon compared to a vector control strain. CONCLUSIONS: Oral administration of NZ-HO alleviates DSS-induced acute colitis in mice. Our results suggest that NZ-HO may be a useful mucosal therapeutic agent for treating IBD.