Development of a Zebrafish Sepsis Model for High-Throughput Drug Discovery

Abstract

Sepsis is a leading cause of death worldwide. Current treatment modalities remain largely supportive. Intervention strategies focused on inhibiting specific mediators of the inflammatory host response have been largely unsuccessful, a consequence of an inadequate understanding of the complexity and heterogeneity of the innate immune response. Moreover, the conventional drug-development pipeline is time-consuming and expensive, and the low success rates associated with cell-based screens underline the need for whole-organism screening strategies, especially for complex pathological processes. Here, we established a lipopolysaccharide (LPS)-induced zebrafish endotoxemia model, which exhibits the major hallmarks of human sepsis, including edema and tissue/organ damage, increased vascular permeability and vascular leakage accompanied by altered expression of cellular junction proteins, increased cytokine expression, immune cell activation and reactive oxygen species (ROS) production, reduced circulation and increased platelet aggregation. We tested the suitability of the model for phenotype-based drug screening using three primary readouts: mortality, vascular leakage and ROS production. Preliminary screening identified fasudil, a drug known to protect against vascular leakage in murine models, as a lead hit, thereby validating the utility of our model for sepsis drug screens. This zebrafish sepsis model has the potential to rapidly analyze sepsis-associated pathologies and cellular processes in the whole organism, as well as to screen and validate many compounds that can modify sepsis pathology in vivo.

Early life glucocorticoid exposure modulates immune function in zebrafish (Danio rerio) larvae

“…In line with results from other studies (Dios et al, 2014;Hsu et al, 2018;Novoa et al, 2009;Philip et al, 2017) LPS exposure produced phenotypical changes (tail fin oedema, swollen or damaged tail fins, and curved animals), mortality and increased expression levels of immune-related genes. To assess the molecular mechanisms underlying the effects of LPS exposure, we measured the expression of cldn5a, cldn2 and oclnb; genes of which the expression has been shown to be changed following LPS exposure (Hsu et al, 2018;Philip et al, 2017) and which are involved in endothelial van den Bos et al: immune function in larvae following early-life exposure to glucocorticoids barrier function (Kása et al, 2015;Odenwald and Turner, 2013;Shen et al, 2011;Yoseph et al, 2016). The strong increase in the expression of cldn2 at 3 hours in control-treated subjects is in line with data from other studies (Hsu et al, 2018;Philip et al, 2017).…”

“…First, we conducted a pilot study to assess the optimal LPS dose, exposure duration and parameters to be measured (protocols adapted from: Dios et al, 2014;Hsu et al, 2018;Novoa et al, 2009;Philip et al, 2017). Incubation for 30 minutes in 150 µg/ml LPS (B11.04; Sigma Aldrich, Zwijndrecht, the Netherlands) was effective in eliciting a robust increase in il1β expression (assessed using qPCR analysis), changes in tail fin morphology (swollen or damaged tails) and increased levels of reactive oxygen species (ROS; measured by a fluorescent labelling method according to Philip et al, 2017).…”

“…2.5 hrs after ending exposure; Novoa et al, 2009) was determined by qPCR analysis as described below. Genes of interest (see papers by: Hsu et al, 2018;Kanwal et al, 2013;Novoa et al, 2009;Philip et al, 2017) were genes encoding proteins involved in barrier function of the vascular endothelium (cldn5a, cldn2, oclnb), Tolllike receptors (tlr2, tlr4ba, tlr4bb, tlr5a, tlr5b), and regulators of the immune response (il1β, il10, myd88, cxcr4a, cxcr4b, ptpn6). Primer sequences are listed in Table 1.…”

“…27.5 hrs after ending exposure). Phenotypical changes included (see Philip et al, 2017): changes in tail fin morphology (normal, swollen (oedema) or damaged), presence of heart oedema and changes in shape (straight or curved). In the second experiment (that also served as replicate for the first experiment) survival and phenotypical changes were measured at 0, 0.5, 1, 3, 6 and 24 hrs (i.e.…”

“…Second, we used a sepsis model, which involved a challenge with lipopolysaccharide (LPS), the membrane component of Gram-negative bacteria, in 4 dpf larvae. We measured survival, phenotypical changes, and the expression of a series of LPS-responsive genes (Dios et al, 2014;Hsu et al, 2018;Novoa et al, 2009;Philip et al, 2017).…”