Comparison of Two Chemically-Induced Colitis-Models in Adult Zebrafish, Using Optical Projection Tomography and Novel Transcriptional Markers

Abstract

Crohn's disease and ulcerative colitis-inflammatory bowel diseases (IBD)-are chronic conditions with an inadequately understood pathogenesis. Employing a set of novel molecular markers in a gene expression assay (qPCR), we have used adult zebrafish to investigate two acute inflammatory models, induced by the haptenizing agents oxazolone and TNBS. In addition, goblet cell dynamics in the scales and intestine and 5-HT (serotonin) in intestinal tissues were investigated through optical projection tomography. Gene expression studies revealed a distinct and significant upregulation of proinflammatory cytokines, acute-phase reactants and metalloprotease 9 in both chemical models, primarily after 72 hours. In comparison, transcription factors and cytokines associated with Th1 and Th17 (Crohn's) and Th2 (ulcerative colitis) were mainly not affected in this acute setting. However, elevated transcript levels were detected in Foxp3, IL-10 and T-bet, which are linked with tolerance and Tregs in mammals. Goblet cells in scales were depleted in both chemical models and in the intestine of oxazolone-treated fish. A marked 5-HT signal was noted in intestinal tissue of some chemically treated zebrafish. In conclusion, a distinct acute inflammatory reaction was induced in both chemical models. Further, oxazolone and TNBS did not result in clear-cut Th2 and Th1/Th17 pathway signaling at this early timepoint, but the applied molecular tools may provide further insight to the IBD pathogenesis and translational value of the IBD zebrafish model. How to cite this paper:

Cannabidiol effects on behaviour and immune gene expression in zebrafish (Danio rerio)

“…il17a/f2 expressed in zebrafish is homologous to IL-17A and IL-17F in humans [ 25 ] and acts as an inflammatory cytokine also in trout [ 26 ]. This observation is in line with upregulation of il1b which also is a pro-inflammatory cytokine in zebrafish [ 18 ]. In contrast other immune related genes were down regulated including ighm and cd4-1 .…”

“…Genes investigated encoded cytokines ( il1b , il13 , il4 , il6 , cxcl8a , il10 , il12a , il17a/f1 , il17a/f2 , il17a/f3 , il22 , ifng1 , tgfba , tnfa ), acute phase proteins ( saa ) cellular receptors ( cd4-1 , cd8a ), immunoglobulins ( ighm , ighz ), matrix metalloproteinase-9 ( mmp9 ), transcription factors ( gata3 , tbx21 ), calcium binding protein s100a10b and reference genes acbt2 , EF-1α and rpl13a . Primers and probes ( Table 1 ) were designed as described by Haarder et al [ 18 ]. The reaction volume 12.5 μL included: 2.5μL of cDNA, 6.25 μL Brilliant III Ultra-Fast QPCR Master Mix (Agilent Technologies, Denmark), 1μL of forward primer and 2.75 μL of RNase-free water.…”

Effect of ES products from Anisakis (Nematoda: Anisakidae) on experimentally induced colitis in adult zebrafish

“…Surprisingly, the gene encoding TNF‐α, a key pro‐inflammatory cytokine in IBD, was not significantly regulated in this study apart from an increase in the TNBS group compared to control fish. This has been reported previously from zebrafish‐colitis models . In this context, it is noteworthy that ES compounds from A. simplex comprise a long range of allergens which may counteract induced anti‐inflammatory processes .…”

“…Survival was around 40% after 24 hours and reached a plateau after 48 hours with slightly less than 20% of the initially exposed fish still alive. Also, similar values were derived from experiments using 160 mmol L −1 TNBS in adult zebrafish . Other studies using ES products from helminths including Anisakis have demonstrated a beneficial effect on experimentally induced colitis in rodent models, and the present investigation aimed at a more precise molecular characterization of the effect of Anisakis ES compounds in a zebrafish model.…”

“…We investigated the effects of excretory‐secretory (ES) products from the fish parasite Anisakis in an acute zebrafish‐colitis model. The disease was clearly induced by TNBS as previously demonstrated and it has been hypothesized that TNBS affects the integrity and barrier function of the intestinal wall associated with cell shedding and influx of inflammatory immune cells in both rodent and zebrafish models (eg). Mortality levels were comparable in zebrafish treated with TNBS alone or TNBS and ES products for the first 24 hours with around 60% survival in both groups.…”