Hans Vergauwen

Intrauterine growth restriction in neonatal piglets affects small intestinal mucosal permeability and mRNA expression of redox-sensitive genes

Neonates with intrauterine growth restriction (IUGR) show lower efficiency of nutrient utilization compared to normal birth weight (NBW) newborns. This study was conducted using neonatal piglets as a model to test the hypothesis that IUGR affects the intestinal barrier function, intestinal structure, and antioxidant system development during the suckling period. The small intestinal mucosae were obtained from IUGR and NBW littermates in the suckling period (d 0, 3, 8, and 19 postnatal). The epithelial barrier function was assessed by FITC‐dextran 4 (FD4) and horseradish peroxidase (HRP) fluxes across the epithelium, histomorphologic measurements, and expression of tight‐junction proteins. Redox status represented by the glutathione disulfide/glutathione ratio and malondialdehyde concentrations was determined, whereas mRNA expressions of some redox‐sensitive proteins were quantified. Results showed that IUGR piglets exhibited a 2‐fold higher intestinal permeability in the proximal small intestine on d 0 (P < 0.05), and this difference between IUGR and NBW piglets was widened to 3 and 4 times for FD4 and HRP, respectively (P< 0.05), on d 3. In accordance, expression of occludin was downregulated at the transcriptional level in IUGR piglets at d 0 and 19 (P < 0.01). Furthermore, the transcription of heme oxygenase 1, catalase, and thioredoxin reductase genes was downregulated in IUGR piglets, mainly on postnatal d 0 and 19 (P < 0.01). It appears that IUGR subjects have a lower capacity to mount an antioxidant response in the early postnatal period. Collectively, these results add to our understanding of the mechanisms responsible for intestinal dysfunction in IUGR neonates.—Wang, W., Degroote, J., Van Ginneken, C., Van Poucke, M., Vergauwen, H., Dam, T. M. T., Vanrompay, D., Peelman, L. J., De Smet, S., Michiels, J. Intrauterine growth restriction in neonatal piglets affects small intestinal mucosal permeability and mRNA expression of redox‐sensitive genes. FASEB J. 30, 863–873 (2016). www.fasebj.org

Trolox and Ascorbic Acid Reduce Direct and Indirect Oxidative Stress in the IPEC-J2 Cells, an In Vitro Model for the Porcine Gastrointestinal Tract

Oxidative stress in the small intestinal epithelium is a major cause of barrier malfunction and failure to regenerate. This study presents a functional in vitro model using the porcine small intestinal epithelial cell line IPEC-J2 to examine the effects of oxidative stress and to estimate the antioxidant and regenerative potential of Trolox, ascorbic acid and glutathione monoethyl ester. Hydrogen peroxide and diethyl maleate affected the tight junction (zona occludens-1) distribution, significantly increased intracellular oxidative stress (CM-H2DCFDA) and decreased the monolayer integrity (transepithelial electrical resistance and FD-4 permeability), viability (neutral red) and wound healing capacity (scratch assay). Trolox (2 mM) and 1 mM ascorbic acid pre-treatment significantly reduced intracellular oxidative stress, increased wound healing capacity and reduced FD-4 permeability in oxidatively stressed IPEC-J2 cell monolayers. All antioxidant pre-treatments increased transepithelial electrical resistance and viability only in diethyl maleate-treated cells. Glutathione monoethyl ester (10 mM) pre-treatment significantly decreased intracellular oxidative stress and monolayer permeability only in diethyl maleate-treated cells. These data demonstrate that the IPEC-J2 oxidative stress model is a valuable tool to screen antioxidants before validation in piglets.

The IPEC-J2 Cell Line

IPEC-J2 cells are intestinal porcine enterocytes isolated from the jejunum of a neonatal unsuckled piglet. The IPEC-J2 cell line is unique as it is derived from the small intestine and is neither transformed nor tumorigenic in nature. IPEC-J2 cells mimic the human physiology more closely than any other cell line of non-human origin. Therefore, it is an ideal tool to study epithelial transport, interactions with enteric bacteria, effects of probiotic microorganisms and the effect of nutrients and other feedstuffs on a variety of widely used parameters (e.g. transepithelial electrical resistance (TEER), permeability, metabolic activity) reflecting epithelial functionality.

Osteopontin alters the functional profile of porcine microglia in vitro

OPN (osteopontin) is a secreted glycoprotein predominantly expressed in bone matrix and kidney tissue. More recently, a neuroprotective role has been attributed to this cytokine since it can be up‐regulated by microglia in neurodegeneration and inflammation. We demonstrate the expression of OPN within primary cultured microglia. Microglia incubated in vitro with different concentrations (0.1 fM–1 nM) of recombinant OPN showed increased proliferation at 10 fM. Moreover, conditioned medium of LLC‐PK1 cells, a pig renal epithelial cell line and a known source of secreted OPN, more than doubled the rate of proliferation of microglia. Addition of an anti‐OPN polyclonal antibody completely reversed this effect. Treatment with OPN dose‐dependently also inhibited microglial superoxide production. In contrast, phagocytosis of fluorescent‐labelled beads was enhanced by OPN. In conclusion, OPN shifts microglia, at least in vitro, to an alternative functional profile more fit to the immune‐balanced microenvironment of the CNS (central nervous system).

Effect of artificial rearing of piglets on the volume densities of M cells in the tonsils of the soft palate and ileal Peyer’s patches

The high prolificacy of modern hybrid sows has increased the mean litter size during the last decades. However, rearing large litters is challenging and has increased the use of alternative management strategies such as euthanasia of weak piglets, cross-fostering, supplementing piglets with milk, split-nursing and split-weaning. The latter includes artificial rearing on brooders where piglets have ad libitum access to milk replacer. The effect of this artificial rearing on the immune system of the piglet is the subject of various studies. The present study focused on the M cells in the tonsil of the soft palate and in the ileal Peyers patch (iPP). These epithelial cells are specialized in antigen sampling and play a pivotal role in the induction of adaptive immune responses. The volume densities of the M cells were assessed by stereological analysis of tissue samples from piglets of 0, 3, 8 and 19days of age. During the first three days, piglets suckled the sow, permitting them to ingest colostrum. At the third day, the piglets were either allowed to continue to suckle the sow or were transferred to brooders. The six experimental groups, each containing six piglets, thus consisted of newborn piglets, 3-day-old sow-suckled piglets, and conventionally and artificially reared piglets of 8 and 19days of age. To identify M cells, tissue samples were immersed in 4% phosphate-buffered paraformaldehyde and paraffin sections were immunohistochemically stained against cytokeratin 18. The volume densities of M cells in both the crypt epithelium of the tonsils of the soft palate and the follicle-associated epithelium of the iPPs did not show any difference between the various age groups of conventionally reared piglets. However, values were twice as high in the iPPs compared to the tonsils of the soft palate. In contrast, a decrease in volume densities of M cells was observed in the iPPs of piglets after they had been transferred to commercial brooders (P=0.05), resulting in significantly lower values (P=0.04) in comparison with the age-matched sow-suckled groups. However, this observation did not translate to values of the tonsils where M cell volume densities remained the same in all age and rearing groups. Based on these results, it appears that antigen sampling is possible from birth onwards and is more advanced in the small intestine than in the oropharynx, but possibly lags behind in artificially reared piglets.

In Vitro Investigation of Six Antioxidants for Pig Diets

Oxidative stress in the small intestinal epithelium can lead to barrier malfunction. In this study, the effect of rosmarinic acid (RA), quercetin (Que), gallic acid (GA), lipoic acid (LA), ethoxyquin (ETQ) and Se-methionine (SeMet) pre-treatments using 2 mM Trolox as a control on the viability and the generation of intracellular reactive oxygen species (iROS) of oxidatively (H2O2) stressed intestinal porcine epithelial cells (IPEC-J2) was investigated. A neutral red assay showed that RA (50–400 µM), Que (12.5–200 µM), GA (50–400 µM), ETQ (6.25–100 µM), and SeMet (125–1000 µM) pre-treatments but not LA significantly increased the viability of H2O2-stressed IPEC-J2 cells (p < 0.05). A 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) fluorescent probe showed that RA (100–600 µM), Que (25–800 µM), ETQ (3.125–100 µM) and SeMet (500–2000 µM) pre-treatments significantly reduced iROS in IPEC-J2 monolayers (p < 0.05). Moreover, RA and Que were most effective in reducing iROS. Therefore, the effects of RA and Que on barrier functioning in vitro were examined. RA and Que pre-treatments significantly decreased fluorescein isothiocyanate (FITC)-conjugated dextran-4 (4 kDa) permeability and transepithelial electrical resistance (TEER) of an IPEC-J2 cell monolayer (p < 0.05). These in vitro results of RA and Que hold promise for their use as antioxidants in pig feed.