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

Abstract

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-1β, IL-12p35; in moDCs, decreased mRNA levels for IL-1β). 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-1β, 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.

Newly isolated Limosilactobacillus reuteri B1/1 modulates cell response in porcine ex vivo model mirroring the gut

“…Structural components of the LAB cell wall, as well as the EPS they are able to produce and release to the surrounding environment, are capable to interact with TLRs in the gut (Oerlemans et al, 2021). In our previous study with the IPEC-J2/moDCs co-culture model, we demonstrated the ability of L. reuteri Biocenol™ derived EPS applied to increase mRNA levels in dendritic cell monocultures (Kiššová et al, 2023). In the present work we used live lactobacilli, where by treating apically deposited IPEC-J2 cells with L. fermentum CCM 7158, we observed an increase in gene expression for the gene encoding the TLR4 receptor not only in directly treated enterocytes but also in basolaterally deposited MDM cells.…”

“…IPEC-J2 cells were used from 25th to 35th passages, regularly controlled for mycoplasma contamination using PCR analysis (Young et al, 2010). For co-culture experiments the IPEC-J2 cells were handled according to the protocol described previously by Kiššová et al, (2023). Brie y, the IPEC-J2 cells were seeded on the top of collagenased cell culture inserts (Transwell® system, 12 mm diameter, 1.12 cm 2 , 0.4 µm pore size; Costar, Corning BV, The Netherlands) at a density of 2.5 × 10 5 cells per cell culture insert and were cultivated under a humidi ed atmosphere (5% CO 2 at 37°C) for 15 days.…”