Rainbow Trout (Oncorhynchus Mykiss) Intestinal Epithelial Cells as a Model for Studying Gut Immune Function and Effects of Functional Feed Ingredients

Abstract

The objective of this study was to evaluate the suitability of the rainbow trout intestinal epithelial cell line (RTgutGC) as an in vitro model for studies of gut immune function and effects of functional feed ingredients. Effects of lipopolysaccharide (LPS) and three functional feed ingredients [nucleotides, mannanoligosaccharides (MOS), and beta-glucans] were evaluated in RTgutGC cells grown on conventional culture plates and transwell membranes. Permeation of fluorescently-labeled albumin, transepithelial electrical resistance (TEER), and tight junction protein expression confirmed the barrier function of the cells. Brush border membrane enzyme activities [leucine aminopeptidase (LAP) and maltase] were detected in the RTgutGC cells but activity levels were not modulated by any of the exposures. Immune related genes were expressed at comparable relative basal levels as these in rainbow trout distal intestine. LPS produced markedly elevated gene expression levels of the pro-inflammatory cytokines il1b, il6, il8, and tnfa but had no effect on ROS production. Immunostaining demonstrated increased F-actin contents after LPS exposure. Among the functional feed ingredients, MOS seemed to be the most potent modulator of RTgutGC immune and barrier function. MOS significantly increased albumin permeation and il1b, il6, il8, tnfa, and tgfb expression, but suppressed ROS production, cell proliferation and myd88 expression. Induced levels of il1b and il8 were also observed after treatment with nucleotides and beta-glucans. For barrier function related genes, all treatments up-regulated the expression of cldn3 and suppressed cdh1 levels. Beta-glucans increased TEER levels and F-actin content. Collectively, the present study has provided new information on how functional ingredients commonly applied in aquafeeds can affect intestinal epithelial function in fish. Our findings suggest that RTgutGC cells possess characteristic features of functional intestinal epithelial cells indicating a potential for use as an efficient in vitro model to evaluate effects of bioactive feed ingredients on gut immune and barrier functions and their underlying cellular mechanisms.

Effects of increasing dietary level of organic acids and nature-identical compounds on growth, intestinal cytokine gene expression and gut microbiota of rainbow trout (Oncorhynchus mykiss) reared at normal and high temperature

“…The cytokine gene expression pattern obtained in this study may have been affected by the time course and the persistence of the stressor as previously suggested [80]. Furthermore, available literature suggests that feed additives may regulate inflammatory effects in an inconsistent pattern, possibly depending on the differences of composition, dosage, quality, route, and exposure time [81].…”

Effects of Yersinia ruckeri invasion on the proteome of the Chinook salmon cell line CHSE-214

“…In addition, the E-cadherin attachment protein was found to be differentially expressed, however, E-cadherin was not found to be present at significantly different level in our experiment (p = 0.433). On the other hand, the attachment proteins that were found to be differentially expressed in our experiment were not commented upon by Wang et al Similarly, these authors found the F-actin protein to be significantly over expressed following exposure to LPS, while in our experiment, the F-actin was not observed to be present at significantly different levels (p = 0.855) and HSP-70 that was found to be differentially expressed by Wang et al was not detected in our experiment 56 . The reason for these differences is difficult to ascertain.…”

“…Similarly, one of the findings recently reported by Wang et al 56 was that epithelial cells harvested from the intestine of rainbow trout and exposed to lipopolysaccharide (LPS) did express multiple inflammatory cytokines, including interleukin and tumour necrosis factors. In addition, the E-cadherin attachment protein was found to be differentially expressed, however, E-cadherin was not found to be present at significantly different level in our experiment (p = 0.433).…”

Microbiota in intestinal digesta of Atlantic salmon (Salmo salar), observed from late freshwater stage until one year in seawater, and effects of functional ingredients: a case study from a commercial sized research site in the Arctic region

“…Functional ingredients, such as nucleotides, and the so-called immunostimulants and prebiotics, are commonly used to improve fish health and disease resistance, in particular during stressful farming conditions [ 32 , 33 ]. They seem to have positive effects on gut health, at least under certain conditions [ 32 , 34 – 36 ], and their effects are suggested to be mediated primarily via modulation of the gut microbiota, such as increasing numbers of beneficial bacteria (e.g. lactic acid bacteria, LAB) [ 6 ].…”