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Infections of piglets during the weaning period can be fatal and thus leading to increased mortality. During that period, the protective effect of milk is missing and piglets are exposed to new antigens, as for instance feed antigens.
Furthermore, due to the essential change in intestinal mucosal function and morphology during that period, the intestine is extremely stressed. Regulation of gene expression is essential for growth and development of intestinal mucosa. A major class of mammalian gene modulators is constituted by miRNAs. It is known that miRNAs play a major role in cell differentiation processes, organ development and pathogen-host-interactions.
The aim of this study was to characterise the regulative influence of miRNAs during the porcine postnatal intestinal development focusing on the weaning phase.
For this purpose intestinal tissue samples (Ileum and ascending colon) from healthy piglets were collected within 7–56 d post natum. Gene expression analyses of mRNA and miRNA were conducted by microarray analysis, followed by integrative analysis of both datasets. For the first time it was shown, in this work, the expression of miRNAs in the porcine ileum and ascending colon. Particularly at weaning differentially regulated miRNAs were involved in hostpathogen-interactions and cell proliferation pathways.
Furthermore this study represents the first characterisation of major gene expression regulators during the porcine postnatal development, which are mainly involved in intestinal inflammation for example inflammatory bowel disease or cancer. Based on these findings approaches to treating diseases that occur during this critical phase of development of the intestine, could be found.
At birth and during the postnatal development with Salmonella infected pigs often remain infected in later development phases. Pigs are usually latently infected with Salmonella; thereby they are a major cause of food-borne infections in humans. Efforts to fight the infection in pigs are therefore desirable already at early stages of development. For this reason a further aim of this study was to characterise the regulative influence of miRNAs during S. Typhimurium infection.
This was done in animals with and without additional treatment with the probiotic strain E. faecium. Samples from both groups were taken 3 h–28 d post infection (28-56 d p.n.). For the first time, the miRNA expression profile has been described in porcine ileum and ascending colon during experimental Salmonella infection as well as after administration of the probiotic strain E. faecium. The results showed, for the first time, that the influence of Salmonella-specific pathways is time dependant and differentially regulated in both loci. Therefore both parts of intestine were considered separately.
In ascending colon the identified miRNAs mainly regulate the adaptive immunity.
RT-qPCR studies showed an increased miR-34a expression directly after Salmonella infection. In contrast the potential target Selectin-P-Ligand (SELPLG) showed a decreased expression. Reporter gene assays in human and porcine cell lines confirmed SELPLG as a miR-34a target and thus represents the first characterization of this interaction.
Integrated expression analysis with target prediction in ileum identified miRNAs that mainly regulate focal adhesion as well as actin cytoskeleton pathways.
MiR-29a was involved in most calculated interactions including Caveolin 2 (CAV2).
RT-qPCR experiments verified upregulation of miR-29a after infection while its predicted target CAV2 was significantly downregulated as shown by RT-qPCR and immuno detection. Reporter gene assays as well as RNAi experiments confirmed CAV2 as miR-29a target in human and porcine intestinal cells. This represents the first characterization of this interaction. Knock-down of CAV2 in human intestinal epithelial cells resulted in retarded proliferation and in increased bacterial uptake by regulating the activation state of the small Rho GTPase CDC42. A simultaneous application of the probiotic strain E. faecium resulted in no change in expression of miR-29a and CAV2 in the ileum.
Maybe the competition between E. faecium and Salmonella prevented physical contact of Salmonella with intestinal epithelial cells, accompanied by reduced expression of focal adhesion structures and associated reduced intake.
These results show, for the first time, that during Salmonella infection the influence of miRNA differs depending on the intestinal subdivision. The study provides the identification of new therapy strategies with respect to the inflammatory bowel disease during postnatal development by searching for molecules interrupting signaling pathways or the specific delivery of therapeutic siRNA. In addition this study gives new ideas for the control of Salmonella infection by the description of how miRNAs could regulate cellular uptake of Salmonella and by manipulation of host immunity.