Transcriptomic profiling of  TLR-7-mediated immune-challenge in zebrafish embryos in the presence and absence of glucocorticoid-induced immunosuppression
Within the regulatory framework on the approval of new substances, the assessment of immunotoxic modes of action (MoA) is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound-specific response of zebrafish embryos to two immunomodulative reference substances, clobetasol propionate (CP, CAS 25122-46-7) and imiquimod (IMQ, CAS 99011-02-6), on the global level of gene expression. The obtained results were used to (i) identify potential biomarker candidates for the assessment of immunotoxic MoAs, (ii) identify compound-specific molecular signatures, (iii) evaluate the reliability of data acquisition using OMICs-coupled approaches and (iv) to assess the suitability of the zebrafish embryo as an alternative model for the human-representative investigation of psoriatic effects. For this, the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP-induced immunosuppression in absence or presence of an IMQ-induced immune challenge, i.e. the simulation of a resting and an activated immune system.   In a modified version of the zebrafish embryo toxicity test (OECD 236), 15 fertilized eggs were exposed to either CP (250 nM) or to IMQ (4000 nM) or to a combination of both under semi static conditions. Exposure to CP started at 2 hours post fertilization (hpf) until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without CP nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf, all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit (Macherey-Nagel) following the manufacturer's protocol. RNA quality was assessed with a 2100 Bioanalyzer system (Agilent) before coding RNA was purified (PolyA selection with TruSeq RNA Library Prep Kit v2) and sequenced on an Illumina NovaSeq 6000 System (Illumina) in 150 bp paired-end mode, producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.