Partial Characterization and Immunomodulatory Effects of Exopolysaccharides from Streptococcus thermophilus SBC8781 during Soy Milk and Cow Milk Fermentation.

The immunomodulatory properties of exopolysaccharides (EPSs) produced by Streptococcus thermophilus have not been explored in depth. In addition, there are no comparative studies of the functional properties of EPSs produced by streptococci in different food matrices. In this work, EPSs from S. thermophilus SBC8781 were isolated after soy milk (EPS-s) or cow milk (EPS-m) fermentation, identified, and characterized in their abilities to modulate immunity in porcine intestinal epithelial cells. Fresh soy milk and cow milk were inoculated with S. thermophilus SBC8781 (7 log CFU/mL) and incubated at 37 °C for 24 h. The extraction of EPSs was performed by the ethanol precipitation method. Analytical techniques, including NMR, UV-vis spectroscopy, and chromatography, identified and characterized both biopolymer samples as polysaccharides with high purity levels and similar Mw. EPS-s and EPS-m had heteropolysaccharide structures formed by galactose, glucose, rhamnose, ribose, and mannose, although with different monomer proportions. On the other hand, EPS-s had higher quantities of acidic polymer than EPS-m. The biopolymer production of the SBC8781 strain from the vegetable culture broth was 200-240 mg/L, which was higher than that produced in milk, which reached concentrations of 50-70 mg/L. For immunomodulatory assays, intestinal epithelial cells were stimulated with 100 µg/mL of EPS-s or EPS-m for 48 h and then stimulated with the Toll-like receptor 3 agonist poly(I:C). EPS-s significantly reduced the expression of IL-6, IFN-ß, IL-8, and MCP-1 and increased the negative regulator A20 in intestinal epithelial cells. Similarly, EPS-m induced a significant reduction of IL-6 and IL-8 expressions, but its effect was less remarkable than that caused by EPS-s. Results indicate that the structure and the immunomodulatory activity of EPSs produced by the SBC8781 strain vary according to the fermentation substrate. Soy milk fermented with S. thermophilus SBC8781 could be a new immunomodulatory functional food, which should be further evaluated in preclinical trials.

Lactobacillus delbrueckii CRL 581 Differentially Modulates TLR3-Triggered Antiviral Innate Immune Response in Intestinal Epithelial Cells and Macrophages.

Lactobacillus delbrueckii subsp. lactis CRL 581 beneficially modulates the intestinal antiviral innate immune response triggered by the Toll-like receptor 3 (TLR3) agonist poly(I:C) in vivo. This study aimed to characterize further the immunomodulatory properties of the technologically relevant starter culture L. delbrueckii subsp. lactis CRL 581 by evaluating its interaction with intestinal epithelial cells and macrophages in the context of innate immune responses triggered by TLR3. Our results showed that the CRL 581 strain was able to adhere to porcine intestinal epithelial (PIE) cells and mucins. The CRL 581 strain also augmented the expression of antiviral factors (IFN-α, IFN-ß, Mx1, OAS1, and OAS2) and reduced inflammatory cytokines in PIE cells triggered by TLR3 stimulation. In addition, the influence of L. delbrueckii subsp. lactis CRL 581 on the response of murine RAW macrophages to the activation of TLR3 was evaluated. The CRL 581 strain was capable of enhancing the expression of IFN-α, IFN-ß, IFN-γ, Mx1, OAS1, TNF-α, and IL-1ß. Of note, the CRL 581 strain also augmented the expression of IL-10 in macrophages. The results of this study show that the high proteolytic strain L. delbrueckii spp. lactis CRL 581 was able to beneficially modulate the intestinal innate antiviral immune response by regulating the response of both epithelial cells and macrophages relative to TLR3 activation.

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.

Tailoring minimal synthetic receptors to reconstitute signaling properties through multiple tyrosine motifs.

As intracellular signal transduction is important for determining cell fate, artificial control of signaling properties through engineered receptors is attractive in the fields of synthetic biology and cell therapy. In this study, we tailored minimal synthetic receptors to reconstitute signaling properties by incorporating multiple tyrosine motifs. The size of molecular parts including the linker between the tyrosine motifs was minimized as much as possible to create the minimal synthetic receptors. By combining the membrane localization signal sequence, a mutant of FK506-binding protein, a JAK-binding domain, tyrosine motifs, and linkers, we successfully reconstituted simple receptor chains that were activated by dimerization via a synthetic small-molecule ligand capable of membrane permeation. Furthermore, up to four signaling molecules of interest were able to be recruited and activated by the minimal synthetic receptors. Thus, the tailored minimal synthetic receptors could be utilized to analyze the role of specific signaling molecules/pathways in controlling cell fate and to efficiently induce specific cell fate for therapeutic applications in the future.