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 has 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, utilizing transfectant cells expressing PRRs along with an established porcine intestinal epitheliocyte (PIE) cell line, have proven to be valuable tools for immunobiotic selection and for gaining insights into the molecular mechanisms responsible for their beneficial effects.

Immunobiotic Lactobacillus jensenii TL2937 Alleviates Dextran Sodium Sulfate-Induced Colitis by Differentially Modulating the Transcriptomic Response of Intestinal Epithelial Cells.

Immunobiotics have emerged as a promising intervention to alleviate intestinal damage in inflammatory bowel disease (IBD). However, the beneficial properties of immunobiotics are strain dependent and, therefore, each strain has to be evaluated in order to demonstrate its potential application in IBD. Our previous in vitro and in vivo studies demonstrated that Lactobacillus jensenii TL2937 attenuates gut acute inflammatory response triggered by Toll-like receptor 4 activation. However, its effect on colitis has not been evaluated before. In this work, we studied whether the TL2937 strain was able to protect against the development of colitis in a dextran sodium sulfate (DSS)-induced mouse model and we delved into the mechanisms of action by evaluating the effect of the immunobiotic bacteria on the transcriptomic response of DSS-challenged intestinal epithelial cells. L. jensenii TL2937 was administered to adult BALB/c mice before the induction of colitis by the administration of DSS. Colitis and the associated inflammatory response were evaluated for 14 days. Mice fed with L. jensenii TL2937 had lower disease activity index and alterations of colon length when compared to control mice. Reduced myeloperoxidase activity, lower production of pro-inflammatory (TNF-α, IL-1, CXCL1, MCP-1, IL-15, and IL-17), and higher levels of immunoregulatory (IL-10 and IL-27) cytokines were found in the colon of TL2937-treated mice. In addition, the treatment of porcine intestinal epithelial (PIE) cells with L. jensenii TL2937 before the challenge with DSS differentially regulated the activation of the JNK pathway, leading to an increase in epithelial cell integrity and to a differential immunotranscriptomic response. TL2937-treated PIE cells had a significant reduction in the expression of inflammatory cytokines (TNF-α, IL-1α, IL-1ß, IL-6, IL-15), chemokines (CCL2, CCL4, CCL8, CXCL4, CXCL5, CXCL9, CXCL10), adhesion molecules (SELE, SELL, EPCAM), and other immune factors (NCF1, NCF2, NOS2, SAA2) when compared to control cells after the challenge with DSS. The findings of this work indicate that (a) L. jensenii TL2937 is able to alleviate DSS-induced colitis suggesting a potential novel application for this immunobiotic strain, (b) the modulation of the transcriptomic response of intestinal epithelial cells would play a key role in the beneficial effects of the TL2937 strain on colitis, and (c) the in vitro PIE cell immunoassay system could be of value for the screening and selection of new immunobiotic strains for their application in IBD.

Exopolysaccharides From Streptococcus thermophilus ST538 Modulate the Antiviral Innate Immune Response in Porcine Intestinal Epitheliocytes.

It was reported that exopolysaccharides (EPSs) from lactobacilli are able to differentially modulate mucosal antiviral immunity. Although research has described the ability of EPSs derived from Streptococcus thermophilus to modulate the mucosal immune system, their impact on antiviral immunity was less explored. In this work, we investigated the capacity of the EPS-producing S. thermophilus ST538 to modulate the innate antiviral immune response triggered by the activation of the Toll-like receptor 3 (TLR3) in porcine intestinal epitheliocytes (PIE cells). Moreover, in order to study the immunomodulatory potential of S. thermophilus ST538 EPS, we successfully developed two mutant strains through the knockout of the epsB or epsC genes. High-performance liquid chromatography and scanning electron microscopy studies demonstrated that the wild type (WT) strain produced as high as 595 µg/ml of EPS in the skim milk medium, while none of the mutant strains (S. thermophilus ΔepsB and ΔepsC) were able to produce EPS. Studies in PIE cells demonstrated that the EPS of S. thermophilus ST538 is able to significantly improve the expression of interferon ß (IFN-ß), interleukin 6 (IL-6), and C-X-C motif chemokine 10 (CXCL10) in response to TLR3 stimulation. The role of EPS in the modulation of antiviral immune response in PIE cells was confirmed by comparative studies of cell free culture supernatants and fermented skim milks obtained from S. thermophilus ΔepsB and ΔepsC. These results suggest that S. thermophilus ST538 could be used as an immunobiotic strain for the development of new immunologically functional foods, which might contribute to improve resistance against viral infections.

Evaluation of the Immunomodulatory Activities of the Probiotic Strain Lactobacillus fermentum UCO-979C.

Lactobacillus fermentum UCO-979C, a strain isolated from a human stomach, was previously characterized by its potential probiotic properties. The UCO-979C strain displayed the ability to beneficially regulate the innate immune response triggered by Helicobacter pylori infection in human gastric epithelial cells. In this work, we conducted further in vitro studies in intestinal epithelial cells (IECs) and in vivo experiments in mice in order to characterize the potential immunomodulatory effects of L. fermentum UCO-979C on the intestinal mucosa. Results demonstrated that the UCO-979C strain is capable to differentially modulate the immune response of IECs triggered by Toll-like receptor 4 (TLR4) activation through the modulation of TLR negative regulators' expression. In addition, we demonstrated for the first time that L. fermentum UCO-979C is able to exert its immunomodulatory effect in the intestinal mucosa in vivo. The feeding of mice with L. fermentum UCO-979C significantly increased the production of intestinal IFN-γ, stimulated intestinal and peritoneal macrophages and increased the number of Peyer's patches CD4+ T cells. In addition, L. fermentum UCO-979C augmented intestinal IL-6, reduced the number of immature B220+CD24high B cells from Peyer's patches, enhanced the number of mature B B220+CD24low cells, and significantly increased intestinal IgA content. The results of this work revealed that L. fermentum UCO-979C has several characteristics making it an excellent candidate for the development of immunobiotic functional foods aimed to differentially regulate immune responses against gastric and intestinal pathogens.

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.

Immunoregulatory Effects Triggered by Lactic Acid Bacteria Exopolysaccharides: New Insights into Molecular Interactions with Host Cells.

Researchers have demonstrated that lactic acid bacteria (LAB) with immunomodulatory capabilities (immunobiotics) exert their beneficial effects through several molecules, including cell wall, peptidoglycan, and exopolysaccharides (EPS), that are able to interact with specific host cell receptors. EPS from LAB show a wide heterogeneity in its composition, meaning that biological properties depend on the strain and. therefore, only a part of the mechanism of action has been elucidated for these molecules. In this review, we summarize the current knowledge of the health-promoting actions of EPS from LAB with special focus on their immunoregulatory actions. In addition, we describe our studies using porcine intestinal epithelial cells (PIE cells) as a model to evaluate the molecular interactions of EPS from two immunobiotic LAB strains and the host cells. Our studies showed that EPS from immunobiotic LAB have anti-inflammatory capacities in PIE cells since they are able to reduce the production of inflammatory cytokines in cells challenged with the Toll-like receptor (TLR)-4-agonist lipopolysaccharide. The effects of EPS were dependent on TLR2, TLR4, and negative regulators of TLR signaling. We also reported that the radioprotective 105 (RP105)/MD1 complex, a member of the TLR family, is partially involved in the immunoregulatory effects of the EPS from LAB. Our work described, for the first time, that LAB and their EPS reduce inflammation in intestinal epithelial cells in a RP105/MD1-dependent manner. A continuing challenge for the future is to reveal more effector-receptor relationships in immunobiotic-host interactions that contribute to the beneficial effects of these bacteria on mucosal immune homeostasis. A detailed molecular understanding should lead to a more rational use of immunobiotics in general, and their EPS in particular, as efficient prevention and therapies for specific immune-related disorders in humans and animals.

The toll-like receptor family protein RP105/MD1 complex is involved in the immunoregulatory effect of exopolysaccharides from Lactobacillus plantarum N14.

The radioprotective 105 (RP105)/MD1 complex is a member of the Toll-like receptor (TLR) family. It was reported that RP105/MD1 cooperates with the lipopolysaccharide (LPS) receptor TLR4/MD2 complex and plays a crucial role in the response of immune cells to LPS. This work evaluated whether RP105, TLR4 or TLR2 were involved in the immunoregulatory capacities of Lactobacillus plantarum N14 (LP14) or its exopolysaccharides (EPS). EPS from LP14 were fractionated into neutral (NPS) and acidic (APS) EPS by anion exchange chromatography. Experiments with transfectant HEK(RP105/MD1) and HEK(TLR2) cells demonstrated that LP14 strongly activated NF-κB via RP105 and TLR2. When we studied the capacity of APS to activate NF-κB pathway in HEK(RP105/MD1) and HEK(TLR4) cells; we observed that APS strongly stimulated both transfectant cells. Our results also showed that LP14 and APS were able to decrease the production of pro-inflammatory cytokines (IL-6, IL-8 and MCP-1) in porcine intestinal epithelial (PIE) cells in response to enterotoxigenic Escherichia coli (ETEC) challenge. In order to confirm the role of TLR2, TLR4 and RP105 in the immunoregulatory effect of APS from LP14, we used small interfering RNA (siRNA) to knockdown these receptors in PIE cells. The capacity of LP14 and APS to modulate pro-inflammatory cytokine expression was significantly reduced in PIE(RP105-/-) cells. It was also shown that LP14 and APS were capable of upregulating negative regulators of the TLR signaling in PIE cells. This work describes for the first time that a Lactobacillus strain and its EPS reduce inflammation in intestinal epithelial cells in a RP105/MD1-dependend manner.

Lactobacillus delbrueckii TUA4408L and its extracellular polysaccharides attenuate enterotoxigenic Escherichia coli-induced inflammatory response in porcine intestinal epitheliocytes via Toll-like receptor-2 and 4.

SCOPE: Immunobiotics are known to modulate intestinal immune responses by regulating Toll-like receptor (TLR) signaling pathways, which are responsible for the induction of cytokines and chemokines in response to microbial-associated molecular patterns. However, little is known about the immunomodulatory activity of compounds or molecules from immunobiotics. METHODS AND RESULTS: We evaluated whether Lactobacillus delbrueckii subsp. delbrueckii TUA4408L (Ld) or its extracellular polysaccharide (EPS): acidic EPS (APS) and neutral EPS (NPS), modulated the response of porcine intestinal epitheliocyte (PIE) cells against Enterotoxigenic Escherichia coli (ETEC) 987P. The roles of TLR2, TLR4, and TLR negative regulators in the immunoregulatory effects were also studied. ETEC-induced inflammatory cytokines were downregulated when PIE cells were prestimulated with both Ld or EPSs. Ld, APS, and NPS inhibited ETEC mediated mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) activation by upregulating TLR negative regulators. The capability of Ld to suppress inflammatory cytokines was diminished when PIE cells were blocked with anti-TLR2 antibody, while APS failed to suppress inflammatory cytokines when cells were treated with anti-TLR4 antibody. Induction of Ca²âº fluxes in TLR knockdown cells confirmed that TLR2 plays a principal role in the immunomodulatory action of Ld, while the activity of APS is mediated by TLR4. In addition, NPS activity depends on both TLR4 and TLR2. CONCLUSION: Ld and its EPS have the potential to be used for the development of anti-inflammatory functional foods to prevent intestinal diseases in both humans and animals.

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

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