Thymol and thyme essential oil can reduce LPS-induced inflammation and disruptive effects on apical-out chicken enteroids

In vitro studies on efficacy of feed additives in combating intestinal inflammation or pathogens are typically conducted on 2D enterocyte monolayers. While these monolayers are useful, they lack the different cell types present in the intestinal mucosa, which are essential for studying the diverse properties of botanicals. To address this issue, intestinal apical-out organoids could be a viable alternative. The objective of this study was to investigate the ability of thymol (THY) and thyme essential oil (TEO) to mitigate the disruptive effects of LPS-induced inflammation on apical-out chicken enteroids. Enteroids were obtained from the intestines of 18-day-old chicken embryos. The tissues were recovered, digested with collagenase and filtered through cell strainers to recover the >40 μm fraction. The obtained cell aggregates were then cultured in suspension for 4 days with an organoid floating medium to generate mature enteroids. They were then challenged with lipopolysaccharide (LPS – n=6) for 6 h, in the absence or presence of THY 10 ppm (n=6) and TEO 20 ppm (n=6). A negative control (CTR- - n=6) group, which was neither challenged nor treated, was also included. Fluorescein isothiocyanate-4 kDa dextran (FD4) paracellular permeability (PCP), reported as relative fluorescence units (RFU), and gene expression for selected markers were then evaluated. Data were analyzed using one-way ANOVA with Tukey’s multiple comparisons (p<0.05). The enteroids responded to the LPS challenge by exhibiting a 2.46-fold increase in FD4 PCP compared to the CTR- (CTR- 1680 ± 330 vs LPS 4121 ± 721 – p<0.0001) RFU, as well as an increase in mRNA levels of interleukin (IL)1β (60-fold), IL6, and IL8 (both 4-fold), tumor necrosis factor-α (7-fold), and interferon-γ (30-fold) (p<0.01). Moreover, a significant (p<0.05) decrease in zonula occludens 1 (ZO1) and occludin (OCCL) expression was observed. THY and TEO reduced the effects associated with the LPS challenge. FD4 PCP was significantly decreased by 30% with THY (LPS 4121 ± 721 vs THY 2896 ± 667 – p=0.0002) RFU and by 40% with TEO (LPS 4121 ± 721 vs TEO 2511 ± 371 – p<0.0001) RFU. Furthermore, the gene expression of all the pro-inflammatory markers was significantly reduced compared to LPS (p<0.05). THY and TEO restored ZO1 and OCCL mRNA levels to CTR- values (p<0.05). Finally, THY significantly increased the expression of defensin beta 4a, avian beta-defensin 3, and cathelicidin-2 mRNA expression closer to the CTR- (p<0.05). In conclusion, THY and TEO exhibited intriguing properties by reducing LPS-induced inflammation and damage in vitro. These two botanicals have the potential to be used as feed additives to help chickens overcome stressful phases during their lifecycle.