In-vitro studies about the efficacy of feed additives to fight intestinal inflammation or pathogens are usually performed on 2D primary enterocyte monolayers or immortalized cells (1). Enterocyte monolayers are useful but they lack the different epithelial components, present in the intestine, which are essential to better study the various properties of botanicals. For this purpose, intestinal organoids could be a valid alternative. Nash and colleagues (2021) published a study about the deep characterization of chicken apical-out enteroids (2) and recently, Joo and colleagues (2022) demonstrated that by sub-culturing basal-out pig enteroids on ultra-low attachment plates it is possible to obtain porcine apical-out enteroids (3), which are both ideal for in-vitro functional studies. The aim of this study was to investigate the ability of a thymol-based blend of botanicals (BOT) to reduce the disruptive effects of LPS-induced inflammation on apical-out chicken and porcine enteroids. Methods To obtain chicken enteroids, intestines of 18day-old chicken embryos were recovered and digested with collagenase type I. The digested tissue was filtered through cell-strainers to recover the >40 μm fraction. The obtained cell aggregates were then cultured in suspension for 4 days with a proper organoid floating medium to generate mature chicken enteroids. To obtain pig enteroids, intestinal crypts were obtained from 35 days-old pigs’ jejunum using EDTA. Recovered intestinal crypts were cultured embedded in extracellular matrix for 2 days to allow the formation of basal-out organoids. Then, on day 3 basal-out enteroids were recovered and cultured for 24h in suspension on ultra-low attachment plates to generate apical-out organoids. Mature enteroids of both species were then challenged with lipopolysaccharide (LPS) for 6h, in the absence or presence of BOT. FD4 paracellular permeability (PCP) and gene expression for selected markers were then evaluated. Results Chicken enteroids responded to the LPS challenge by showing a 60% increase in FD4 PCP, a near 60-fold increase in IL1β expression, a 4-fold increase in IL6, and IL8 levels (p<0.01), a 7-fold increase in tumor necrosis factor-α (TNFα) mRNA expression (p<0.01) and a 30-fold increase in interferon-γ (INFγ - p<0.01) expression. Moreover, a decrease in zonula occludens 1 (ZO1 - p<0.05), occludin (OCCL - p<0.05), and toll-like receptor 4 mRNA levels were observed (p<0.01). BOT showed anti-inflammatory properties in reducing all the effects connected to the LPS challenge. FD4 PCP was decreased by 40% (p<0.05), and the expression of all pro-inflammatory markers was significantly reduced compared to the challenged control. BOT restored the ZO1 and OCCL levels near the control levels (p<0.05). Moreover, BOT significantly increased the defensin beta 4a (DEFB4A) expression over the negative control (p<0.05). This could be an interesting effect to improve the defense ability of the host against pathogens. Furthermore, apical-out porcine enteroids also well responded to the inflammatory challenge by showing a 70% increase in FD4 PCP, a 2-fold increase in IL1β, IL8, and mucin 4 (MUC4) levels (p<0.01), and a 3-fold increase in IL6 mRNA expression (p<0.01). Moreover, a decrease in ZO1 and occludin OCCL levels was observed. BOT showed beneficial properties in reducing all the effects connected to the LPS challenge. FD4 PCP was decreased by 35% (p<0.05), and IL1β, IL6, IL8, and MUC4 expression were significantly reduced compared to the challenged control. Moreover, BOT increased the ZO1 and OCCL levels by 50% (p<0.05), acting as a barrier-reinforcing agent. Conclusion BOT showed interesting anti-inflammatory properties both on chicken and pig enteroids, being able to maintain epithelial integrity thus reducing LPS-induced damage in-vitro. This thymol-based blend has the potential to be further investigated as a feed additive to improve chickens and pigs ability to overcome stressful phases during their lifecycle.
A blend of botanicals can reduce LPS-induced inflammation and disruptive effects on apical-out chicken and porcine enteroids
Federico GHISELLI;Andrea PIVA;Ester GRILLI
2023
Abstract
In-vitro studies about the efficacy of feed additives to fight intestinal inflammation or pathogens are usually performed on 2D primary enterocyte monolayers or immortalized cells (1). Enterocyte monolayers are useful but they lack the different epithelial components, present in the intestine, which are essential to better study the various properties of botanicals. For this purpose, intestinal organoids could be a valid alternative. Nash and colleagues (2021) published a study about the deep characterization of chicken apical-out enteroids (2) and recently, Joo and colleagues (2022) demonstrated that by sub-culturing basal-out pig enteroids on ultra-low attachment plates it is possible to obtain porcine apical-out enteroids (3), which are both ideal for in-vitro functional studies. The aim of this study was to investigate the ability of a thymol-based blend of botanicals (BOT) to reduce the disruptive effects of LPS-induced inflammation on apical-out chicken and porcine enteroids. Methods To obtain chicken enteroids, intestines of 18day-old chicken embryos were recovered and digested with collagenase type I. The digested tissue was filtered through cell-strainers to recover the >40 μm fraction. The obtained cell aggregates were then cultured in suspension for 4 days with a proper organoid floating medium to generate mature chicken enteroids. To obtain pig enteroids, intestinal crypts were obtained from 35 days-old pigs’ jejunum using EDTA. Recovered intestinal crypts were cultured embedded in extracellular matrix for 2 days to allow the formation of basal-out organoids. Then, on day 3 basal-out enteroids were recovered and cultured for 24h in suspension on ultra-low attachment plates to generate apical-out organoids. Mature enteroids of both species were then challenged with lipopolysaccharide (LPS) for 6h, in the absence or presence of BOT. FD4 paracellular permeability (PCP) and gene expression for selected markers were then evaluated. Results Chicken enteroids responded to the LPS challenge by showing a 60% increase in FD4 PCP, a near 60-fold increase in IL1β expression, a 4-fold increase in IL6, and IL8 levels (p<0.01), a 7-fold increase in tumor necrosis factor-α (TNFα) mRNA expression (p<0.01) and a 30-fold increase in interferon-γ (INFγ - p<0.01) expression. Moreover, a decrease in zonula occludens 1 (ZO1 - p<0.05), occludin (OCCL - p<0.05), and toll-like receptor 4 mRNA levels were observed (p<0.01). BOT showed anti-inflammatory properties in reducing all the effects connected to the LPS challenge. FD4 PCP was decreased by 40% (p<0.05), and the expression of all pro-inflammatory markers was significantly reduced compared to the challenged control. BOT restored the ZO1 and OCCL levels near the control levels (p<0.05). Moreover, BOT significantly increased the defensin beta 4a (DEFB4A) expression over the negative control (p<0.05). This could be an interesting effect to improve the defense ability of the host against pathogens. Furthermore, apical-out porcine enteroids also well responded to the inflammatory challenge by showing a 70% increase in FD4 PCP, a 2-fold increase in IL1β, IL8, and mucin 4 (MUC4) levels (p<0.01), and a 3-fold increase in IL6 mRNA expression (p<0.01). Moreover, a decrease in ZO1 and occludin OCCL levels was observed. BOT showed beneficial properties in reducing all the effects connected to the LPS challenge. FD4 PCP was decreased by 35% (p<0.05), and IL1β, IL6, IL8, and MUC4 expression were significantly reduced compared to the challenged control. Moreover, BOT increased the ZO1 and OCCL levels by 50% (p<0.05), acting as a barrier-reinforcing agent. Conclusion BOT showed interesting anti-inflammatory properties both on chicken and pig enteroids, being able to maintain epithelial integrity thus reducing LPS-induced damage in-vitro. This thymol-based blend has the potential to be further investigated as a feed additive to improve chickens and pigs ability to overcome stressful phases during their lifecycle.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
