Exopolysaccharides from Limosilactobacillus reuteri: their influence on in vitro activation of porcine monocyte-derived dendritic cells - brief report.

The aim of this study was to evaluate the immunomodulatory potential of two α-D-glucans from Limosilactobacillus reuteri L26 Biocenol™ (EPS-L26) and L. reuteri DSM17938 (EPS-DSM17938), with respect to their influence on in vitro activation of porcine dendritic cells (DCs). We used immature DCs differentiated from porcine blood monocytes under in vitro conditions. Based on the surface expression of MHC II and costimulatory CD80/86 molecules, we showed that both used EPSs favour the maturation of monocyte-derived DCs (MoDCs) similarly to the commonly used stimulant tumour necrosis factor α (TNF-α). In contrast to TNF-α stimulation, MoDCs treated with both used EPSs significantly up-regulated the mRNA levels not only for interleukin (IL)-10 (P < 0.0001 for EPS-DSM17938; P = 0.0037 for EPS-L26), but also for IL-12 (P = 0.0176 for EPS-DSM17938; P = 0.0019 for EPS-L26). These cytokines are known to regulate T-cell kinetics and play a key role in maintaining immune homeostasis. Interestingly, only relatively linear α-D-glucan (EPS-DSM17938) significantly increased gene expression of the major pro-inflammatory cytokine IL-1ß (P = 0.0011) and the "SOS" cytokine IL-6 (P = 0.0127). However, it is important to highlight the need for further studies aimed at cytokine kinetics in DCs, as well as a co-culture study with allogenic T-lymphocytes.

Newly Isolated Limosilactobacillus reuteri B1/1 Modulates the Expression of Cytokines and Antimicrobial Proteins in a Porcine ex Vivo Model.

BACKGROUND: The epithelia of the intestine perform various functions, playing a crucial role in providing a physical barrier and an innate immune defense against infections. By generating a "three-dimensional" (3D) model of cell co-cultures using the IPEC-J2 cell line and porcine blood monocyte-derived macrophages (MDMs), we are getting closer to mimicking the porcine intestine ex vivo.Methods: The effect of Limosilactobacillus reuteri B1/1 and Limosilactobacillus fermentum CCM 7158 (indicator strain) on the relative gene expression of interleukins (IL-1ß, IL-6, IL-8, IL-18 and IL-10), genes encoding receptors for TLR4 and TLR2, tight junction proteins such as claudin-1 (CLDN1), occludin (OCLN) and important antimicrobial proteins such as lumican (LUM) and olfactomedin-4 (OLMF-4) was monitored in this model. RESULTS: The results obtained from this pilot study point to the immunomodulatory potential of newly isolated L. reuteri B1/1, as it was able to suppress the enhanced pro-inflammatory response to lipopolysaccharide (LPS) challenge in both cell types. L. reuteri B1/1 was even able to up-regulate the mRNA levels of genes encoding antimicrobial proteins LUM and OLFM-4 and to increase tight junction (TJ)-related genes CLDN1 and OCLN, which were significantly down-regulated in LPS-induced IPEC-J2 cells. Conversely, L. fermentum CCM 7158, chosen as an indicator lactic acid bacteria (LAB) strain, increased the mRNA levels of the investigated pro-inflammatory cytokines (IL-18, IL-6, and IL-1ß) in MDMs when LPS was simultaneously applied to basally deposited macrophages. Although L. fermentum CCM 7158 induced the production of pro-inflammatory cytokines, synchronous up-regulation of the anti-inflammatory cytokine IL-10 was detected in both LAB strains used in both cell cultures. CONCLUSIONS: The obtained results suggest that the recently isolated LAB strain L. reuteri B1/1 has the potential to alleviate epithelial disruption caused by LPS and to influence the production of antimicrobial molecules by enterocytes.

Immunomodulatory effect of probiotic exopolysaccharides in a porcine in vitro co-culture model mimicking the intestinal environment on ETEC infection.

The aim of this study was to evaluate the immunomodulatory effect of EPS-L26 isolated from the probiotic strain Lactobacillus (Limosilactobacillus) reuteri L26 Biocenol™, in a model of infection with an enterotoxigenic E. coli (ETEC) by establishing monocultures consisting of the IPEC-J2 cell line or monocyte-derived dendritic cells (moDCs) and creating a 3D model of cell co-cultures established with IPEC-J2 cells and moDCs. The immunomodulatory and immunoprotective potential of used EPS-L26 was confirmed in monocultures in an experimental group of pretreated cells, where our study showed that pretreatment of cells with EPS-L26 and subsequent exposure to infection resulted in significantly down-regulated mRNA levels of genes encoding inflammatory cytokines compared to ETEC challenge in single cell cultures (in IPEC-J2, decreased mRNA levels for TNF-α, IL-6, IL-1ß, IL-12p35; in moDCs, decreased mRNA levels for IL-1ß). Similar to monocultures, we also demonstrated the immunostimulatory potential of the ETEC strain in the co-culture model on directly treated IPEC-J2 cells cultivated on insert chambers (apical compartment) and also on indirectly treated moDCs cultivated in the lower chamber (basolateral compartment), however in the co-culture model the expression of inflammatory cytokines was attenuated at the mRNA level compared to monocultures. Pretreatment of the cells on the insert chambers pointed to the immunoprotective properties of EPS-L26, manifested by decreased mRNA levels in both cell lines compared to ETEC challenge (in IPEC-J2 decreased mRNA levels for IL-12p35; in moDCs decreased mRNA levels for IL-1ß, IL-6). Our results suggest intercellular communication via humoral signals derived from IPEC-J2 cells by influencing the gene expression of indirectly treated moDC cells located in the basolateral compartment.