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.

Selection of Immunobiotic Ligilactobacillus salivarius Strains from the Intestinal Tract of Wakame-Fed Pigs: Functional and Genomic Studies.

In this article, Ligilactobacillus salivarius FFIG strains, isolated from the intestinal tract of wakame-fed pigs, are characterized according to their potential probiotic properties. Strains were evaluated by studying their interaction with porcine intestinal epithelial (PIE) cells in terms of their ability to regulate toll-like receptor (TLR)-3- or TLR4-mediated innate immune responses, as well as by assessing their adhesion capabilities to porcine epithelial cells and mucins. These functional studies were complemented with comparative genomic evaluations using the complete genome sequences of porcine L. salivarius strains selected from subgroups that demonstrated different "immune" and "adhesion" phenotypes. We found that their immunomodulatory and adhesion capabilities are a strain-dependent characteristic. Our analysis indicated that the differential immunomodulatory and adhesive activities of FFIG strains would be dependent on the combination of several surface structures acting simultaneously, which include peptidoglycan, exopolysaccharides, lipoteichoic acid, and adhesins. Of note, our results indicate that there is no correlation between the immunomodulatory capacity of the strains with their adhesion ability to mucins and epithelial cells. Therefore, in the selection of strains destined to colonize the intestinal mucosa and modulate the immunity of the host, both properties must be adequately evaluated. Interestingly, we showed that L. salivarius FFIG58 functionally modulated the innate immune responses triggered by TLR3 and TLR4 activation in PIE cells and efficiently adhered to these cells. Moreover, the FFIG58 strain was capable of reducing rotavirus replication in PIE cells. Therefore, L. salivarius FFIG58 is a good candidate for further in vivo studying the protective effect of lactobacilli against intestinal infections in the porcine host. We also reported and analyzed, for the first time, the complete genome of several L. salivarius strains that were isolated from the intestine of pigs after the selective pressure of feeding the animals with wakame. Further genomic analysis could be of value to reveal the metabolic characteristics and potential of the FFIG strains in general and of the FFIG58 strain, in particular, relating to wakame by-products assimilation.

Draft Genome Sequence of Ligilactobacillus salivarius FFIG58, Isolated from the Intestinal Tract of Wakame-Fed Pig.

Ligilactobacillus salivarius FFIG58 was isolated from the intestine of a wakame-fed pig and sequenced with an Illumina HiSeq system. FFIG58 genome sequencing revealed a genome size of 1,984,180 bp, with 1,994 protein-coding genes and a GC content of 32.9%. This draft genome sequence will contribute to a better understanding of the porcine gut microbiome.

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.

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.