Intestinal Upregulation of Melanin-Concentrating Hormone in TNBS-Induced Enterocolitis in Adult Zebrafish

Abstract

BackgroundMelanin-concentrating hormone (MCH), an evolutionarily conserved appetite-regulating neuropeptide, has been recently implicated in the pathogenesis of inflammatory bowel disease (IBD). Expression of MCH is upregulated in inflamed intestinal mucosa in humans with colitis and MCH-deficient mice treated with trinitrobenzene-sulfonic acid (TNBS) develop an attenuated form of colitis compared to wild type animals. Zebrafish have emerged as a new animal model of IBD, although the majority of the reported studies concern zebrafish larvae. Regulation MCH expression in the adult zebrafish intestine remains unknown.MethodsIn the present study we induced enterocolitis in adult zebrafish by intrarectal administration of TNBS. Follow-up included survival analysis, histological assessment of changes in intestinal architecture, and assessment of intestinal infiltration by myeloperoxidase positive cells and cytokine transcript levels. ResultsTreatment with TNBS dose-dependently reduced fish survival. This response required the presence of an intact microbiome, since fish pre-treated with vancomycin developed less severe enterocolitis. At 6 hours post-challenge, we detected a significant influx of myeloperoxidase positive cells in the intestine and upregulation of both proinflammatory and anti-inflammatory cytokines. Most importantly, and in analogy to human IBD and TNBS-induced mouse experimental colitis, we found increased intestinal expression of MCH and its receptor in TNBS-treated zebrafish. ConclusionsTaken together these findings not only establish a model of chemically-induced experimental enterocolitis in adult zebrafish, but point to effects of MCH in intestinal inflammation that are conserved across species.

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

“…However, the mortality observed in the TNBS-treated zebrafish in the present study was slightly lower than previously reported by Geiger et al [10], studying a comparable zebrafish model. Brugman et al [6] did not note reduced survival rates in adult zebrafish exposed to a low concentration (0.2%) oxazolone which is lower that the concentration (0.6%) used in this study.…”

“…In this study, transcript levels of IL-1β and TNF-α were significantly elevated in both colitis-groups. Previous zebrafish-based studies have reported an increase in gene expression of IL-1β and TNF-α in adult fish few hours after they had been exposed to oxazolone [6] or TNBS [10]. Similar increases have been reported using larval TNBS-models and qPCR/immunofluorescence techniques [7] [9] [11].…”

“…Two other anti-inflammatory cytokines, IL-10 and TGF-β, were also significantly up-regulated in the TNBS group, 72 hours after exposure; TGF-β also in the oxazolone group at the same time point. The anti-inflammatory effects have been documented in rodent models (reviewed in [36]) and IL-10 has also been studied in experimental zebrafish-colitis [6] [10]. In these acute inflammation models, intestinal IL-10 gene expression was elevated 5 hours after a single rectal exposure to oxazolone and TNBS, respectively, which is earlier observed than in the present study.…”

“…For oxazolone, this dosage was chosen based on pilot experiments which showed that 0.6% and 1.0% but not 0.2% oxazolone resulted in distinct goblet cell depletion when compared to control fish (Supplementary Material Figure S1). The concentration of TNBS used in the experiment was considered optimal based on the literature [10]. Control groups received 50% ethanol (oxazolone solvent), 30% ethanol (TNBS solvent) or tap water.…”

Inflammatory diseases modelling in zebrafish

“…A second model of intestinal inflammation in adult zebrafish used 2,4,6-trinitrobenzenesulfonic acid (TNBS) in a 30% ethanol solution injected intrarectally [48] . In mice, this model is widely used to study IBD because of clinical and histopathological findings resembling those seen in CD [49,50] .…”