“Set-up of an in vitro model to study intestinal health in pigs”
BACKGROUND AND AIM OF THE PROJECT:
Intestinal health is essential for the health of the body since the gastro-intestinal mucosa is the main site of interaction with the external environment, as well as the major area colonized by the microbiota. Intestinal health relies on proper barrier function, epithelial integrity and related mechanisms of protection (mucous layer, tight junctions, immune and inflammatory system).
Recently, the gastro-intestinal tract has been studied for the chemo-sensing properties sincespecialized epithelial cells (entero-endocrine cells) are able, through specific chemo-receptors on the apical side, to “sense” the lumen environment and nutrients concentration and then mediate the secretion of hormones or neurotransmitters. This “chemo-sensing” of lipids, sugars and proteins seems to be particularly important in regulating digestion and absorptive function but also involved in the regulation of the intestinal inflammation (Liou, 2013; Furness et al., 2013).
In pigs, during the weaning transition, intestinal inflammation and barrier integrity play a crucial role in regulating intestinal health and, consequently, pig’s health, growth and productivity. Several nutritional approaches and dietary interventions have been tested and proposed in order to control intestinal inflammation and improve growth performance of piglets in the peri-weaning period. However,very often it is difficult to elucidate the mechanisms of action at the intestinal level responsible for the auxinic effect observed. In this context, it would be of great interest to develop a reliable tool to investigate the nutritional modulation of the intestinal health.
Therefore, aim of this project is to set up an in vitro model to study the intestinal health of the pig
EXPERIMENTAL ACTIVITY:
The in vitro model will use IPEC-J2 cells, that are porcine non-transformed columnar epithelial cells isolated from neonatal piglet mid-jejunum. This cell line forms polarized monolayers expressing tight junctions proteins, cytoskeletal proteins, cytokines and toll-like receptors, as well as showing electro-physiological properties that closely mimic the porcine small intestine. For this reason, there has been a steadily increasing use of these cells for microbiological investigations aimed to assess the epithelial immune response to a wide variety of microorganisms (including pathogens or probiotic strains).
In this project, IPEC-J2 cells will be used as in vitro model of porcine intestinal health.
The project proposal can be divided into three steps:
1)In vitro culture and characterization of IPEC-J2:
IPEC-J2 will be cultured according to protocols in literature. The cells will be grown on porous filters and verified for electrophysiological parameters (such as trans-epithelial electrical resistance and para-cellular permeability) and for expression of tight junctions markers as index of intestinal barrier integrity along with inflammatory cytokines and immune molecules as index of the inflammatory status. In addition, IPEC-J2 cells will be investigated for the expression of chemo-sensing receptors involved in the detection of nutrients.
2)In vitro intestinal health perturbation induction:
The IPEC-J2 model will be used to study conditions that can alter the intestinal health. For this purpose cells will be tested under both microbial, chemical and inflammatory challenges. Particularly, object of the study will be those conditions that can simulate the alterations occurring at the intestinal level in the piglet around weaning.
3)Evaluation of bioactive molecules on the intestinal health perturbationin vitro:
The IPEC-J2 model of intestinal health perturbation will be used to study the effect of bioactive molecules at the intestinal level. Bioactive molecules supposed to positively modulate intestinal health will be tested with particular reference to barrier integrity, inflammatory status and chemo-sensing mechanisms. This step will help in understanding the molecular mechanisms of action of bioactive molecules at the cellular level in the intestinal epithelium.
The experimental activity of the project will use cell culture techniques for daily IPEC-J2 handling along to electrophysiological parameters assessment for barrier function test. Bio-molecular methods will be performed in order to assess the expression of markers of epithelial integrity, inflammation and chemo-sensing at both mRNA level (qPCR) and protein level (Western Blot, ELISA assay). Immunocytochemistry will be used to detect specific proteins and localize them in the epithelial layer.
LITERATURE CITED:
Liou (2013), J. Anim. Sci. 2013, 91:1946-1956.
Furness et al. (2013), Nat. Rev. Gastroenterol. Hepatol. 10:729-740.