Development of a chicken ileal explant culture model for measurement of gut inflammation induced by lipopolysaccharide.

Gut mucosa holds a single layer of epithelial cells and the largest mass of lymphoid tissue in the body. Although the epithelial cell culture model is widely used to assess intestinal barrier function, it has limitations for studying cellular interactions, in particular those of the immune system. In this study, a chicken ileal explant culture model was developed for investigating short-term gut inflammatory and secretory responses in an ex vivo environment. Initially, ileal explants from broilers at 21 d of age were cultured ex vivo up to 6 h. Explants cultured for a maximum of 2 h remained over 90% viable, based on lactate dehydrogenase (LDH) release assay. Morphologically, explants cultured for 2 h displayed normal morphology compared to those cultured longer, further confirming that short-term culture for up to 2 h duration is an acceptable model for studying ex vivo regulation of inflammation. Subsequently, lipopolysaccharide (LPS) dose-related responses were determined for explants cultured for 2 h. Results from LDH activity assay showed that the viability of explants was decreased (P ≤ 0.05) at an LPS dose higher than 50 µg/mL. A significant (P ≤ 0.05) nitric oxide release was observed at LPS concentrations of 10 and 20 µg/mL. In addition, the highest inflammatory and secretory responses were detected at 20 µg/mL LPS based on gene expression of TLR-4, IL-1ß, IL-8, MUC2, IgA, and pIgR (P ≤ 0.05). However, the gene expression of claudin-1 and claudin-4 were not increased at the determined LPS concentrations (P > 0.05). These results demonstrated the potential usefulness of this intestinal explant culture model for short-term study of biological factors in gut inflammatory and secretory responses, but not a sufficient duration for evaluation of tight junction responsiveness.

Effect of threonine on secretory immune system using a chicken intestinal ex vivo model with lipopolysaccharide challenge.

Secretory IgA (sIgA) and its transcytosis receptor, polymeric immunoglobulin receptor (pIgR), along with mucus, form the first lines of intestinal defense. Threonine (Thr) is a major component of intestinal mucins and IgA, which are highly secreted under lipopolysaccharide (LPS) induced inflammation. In the current study, the effect of Thr on the secretory immune system was determined in an ex vivo chicken ileal explant model. Results showed that a 2-hour Thr-deprivation of culture medium induced a compensatory increase in the mRNA expression of interleukin-8 (IL-8), mucin 2 (MUC2), and IgA during LPS challenge, and this increase was suppressed with Thr addition to the media (P ≤ 0.05), suggesting that Thr was required for mucin and IgA production after exposure to LPS. Similarly, a 2-hour culture of explants from birds fed a Thr adequate diet showed an increase in the mRNA abundance of IL-8, MUC2, and IgA with LPS treatment (P ≤ 0.003), which had a trend to be attenuated with Thr supplementation in the media (P ≤ 0.10). In contrast, explants from birds fed a Thr deficient diet had no response to LPS treatment. These results indicated that in vivo Thr deficiency induced impaired inflammatory and secretory immune responses in broiler chicks. Furthermore, our results revealed that induction of MUC2 and pIgR gene expression required nuclear factor-κB (NF-κB) activation. Additionally, IgA transcytosis may be dependent on extracellular-regulated protein kinase (ERK) activation, which may indirectly impact pIgR gene expression.