Olive Mill Waste-Water Extract Enriched in Hydroxytyrosol and Tyrosol Modulates Host-Pathogen Interaction in IPEC-J2 Cells

Abstract

The dietary supplementation of olive oil by-products, including olive mill waste-water (OMWW) in animal diets, is a novel application that allows for their re-utilization and recycling and could potentially decrease the use of antibiotics, antimicrobial resistance risk in livestock species, and the occurrence of intestinal diseases. Salmonella serovar typhimurium is one of the most widespread intestinal pathogens in the world, causing enterocolitis in pigs. The aim of this study was to investigate the effect of an OMWW extract enriched in polyphenols (hydroxytyrosol and tyrosol) in the immune response of an intestinal porcine epithelial cell line (IPEC-J2) following S. typhimurium infection. Cells were pre-treated with OMWW-extract polyphenols (OMWW-EP, 0.35 and 1.4 µg) for 24 h and then infected with S. typhimurium for 1 h. We evaluated bacterial invasiveness and assayed IPEC-J2 gene expression with RT-qPCR and cytokine release with an ELISA test. The obtained results showed that OMWW-EP (1.4 µg) significantly reduced S. typhimurium invasiveness; 0.35 µg decreased the IPEC-J2 gene expression of IL1B, MYD88, DEFB1 and DEFB4A, while 1.4 µg down-regulated IL1B and DEFB4A and increased TGFB1. The cytokine content was unchanged in infected cells. This is the first study demonstrating the in vitro immunomodulatory and antimicrobial activity of OMWW extracts enriched in polyphenols, suggesting a protective role of OMWW polyphenols on the pig intestine and their potential application as feed supplements in farm animals such as pigs.

Keywords: IPEC-J2; Salmonella spp.; cytokine; defensin; immunomodulation; polyphenols.

Figure 1

IPEC-J2 viability after a 24 h exposition to the extract of liquid olive mill waste-water (OMWW) polyphenols (OMWW-EP: 0.35 μg; 0.7 μg; 1.4 μg; 7 µg; 14 µg; 70 µg; 140 µg). Cell viability was determined with XTT test. The number of living cells is expressed as optical density (OD) ± standard error (SE) and dots indicate samples included in each group. Statistical difference was calculated for all groups vs. Control (untreated cells): *** p < 0.001.

Figure 2

Effects of OMWW-extract polyphenols (OMWW-EP) on S. typhimurium penetration into IPEC-J2 cells. Data are expressed as log₁₀ CFU of penetrated, intracellular ST/3 × 10⁵ cells. The mean value of five replicates + standard error is presented, and dots indicate samples included in each group. The significant difference between S. typhimurium infected cells and pretreated with different concentrations of OMWW-EP (ST + 0.35 µg POL—pink; ST + 1.4 µg POL—fuchsia) and S. typhimurium infected cells (ST—blue) is indicated by * (* p < 0.05).

Figure 3

Effects of 24 h OMWW-extract polyphenols on IPEC-J2 gene expression. The RT-qPCR analysis was performed to evaluate CXCL8, IL18, MYD88, NFKB1, NFKB/p65, TGFB1 and NOS2 gene expression. Data are presented as bar plots displaying the mean value of normalized expression, standard error as error bars and dots indicate samples included in each group. For each gene and cytokine, differences between treated with polyphenols (0.35 μg POL—light green; 1.4 μg POL—gray) vs. untreated (Control—dark green) cells were evaluated through one-way ANOVA followed by a Dunnett’s test or a Kruskal–Wallis test followed by Dunn’s multiple comparison test; * p < 0.05, ** p < 0.01.

Figure 4

Effects of OMWW-extract polyphenols on IPEC-J2 cytokine release through multiplex ELISA measuring cytokine contents in culture supernatants (IL-1α, IL-1Ra, IL-6, IL-8, IL-10, IL-18). Data are presented as box and whisker plots displaying median and interquartile range (boxes) and minimum and maximum values (whiskers). For each gene and cytokine, differences between treated with polyphenols (0.35 μg POL—light green; 1.4 μg POL—gray) and untreated (Control—dark green) cells were evaluated through one-way ANOVA followed by a Dunnett’s test or a Kruskal–Wallis test followed by Dunn’s multiple comparison test; * p < 0.05.

Figure 5

Gene expression of IPEC-J2 cells in response to S. typhimurium infection with OMWW-extract polyphenol (OMWW-EP) pre-treatment. The tested conditions for IPEC-J2 cells were: uninfected and untreated cells (Control—dark green), infected with S. typhimurium (ST—blue), pre-treated with 0.35 µg OMWW-EP and infected (0.35 µg POL + ST—pink), and pre-treated with 1.4 µg OMWW-EP and infected (1.5 µg POL + ST—fuchsia). Data are reported as mean value and standard error, and dots indicate samples included in each group. Statistical tests were carried out comparing all conditions vs. ST. Significant differences: * p < 0.05, ** p < 0.01, *** p <0.001.

Figure 6

Cytokine release by IPEC-J2 cells in response to S. typhimurium infection after OMWW-extract polyphenol (OMWW-EP) pre-treatment. The tested conditions for IPEC-J2 cells were: uninfected and untreated cells (Control—dark green), infected with S. typhimurium (ST—blue), pre-treated with 0.35 µg OMWW-EP and infected (0.35 µg POL + ST—pink), and pre-treated with 1.4 µg OMWW-EP and infected (1.5 µg POL + ST—fuchsia). For each cytokine, differences between ST-infected cells and the other conditions were evaluated through one-way ANOVA followed by a Dunnett’s test or a Kruskal–Wallis test followed by Dunn’s multiple comparison test; * p < 0.05, *** p < 0.001; **** p < 0.0001.