Transcriptomic profiling of TLR-7-mediated immune-challenge in zebrafish embryos in the presence and absence of glucocorticoid-induced immunosuppression.

Although numerous studies imply a correlation between chemical contamination and an impaired immunocompetence of wildlife populations, the assessment of immunomodulatory modes of action is currently not covered in the regulatory requirements for the approval of new substances. This is not least due to the complexity of the immune system and a lack of standardised methods and validated biomarkers. To tackle this issue, in this study, the transcriptomic profiles of zebrafish embryos were analysed in response to the immunosuppressive compound clobetasol propionate, a synthetic glucocorticoid, and/or the immunostimulatory compound imiquimod (IMQ), a TLR-7 agonist. Using IMQ, known for its potential to induce psoriasis-like effects in mice and human, this study additionally aimed at evaluating the usability of the zebrafish embryo model as an alternative and 3R conform system for the IMQ-induced psoriasis mouse model. Our study substantiates the suitability of previously proposed genes as possible biomarkers for immunotoxicity, such as socs3, nfkbia, anxa1c, fkbp5 and irg1l. Likewise, however, our findings indicate that these genes may be less suitable to distinguish a suppressive from stimulating fashion of action. In contrast, based on a differential regulation in opposite direction in response to both compounds, krt17, rtn4a, and1, smhyc1 and gmpr were identified as potential novel biomarkers with said power to differentiate. Observed IMQ-induced alterations in the expression of genes previously associated with the pathogenesis of psoriasis such as krt17, nfkbia, parp1, pparg, nfil3-6, per2, stat4, klf2, rtn4a, anxa1c and nr1d2 indicate the inducibility of psoriatic effects in the zebrafish embryo. Our work contributes to the establishment of an approach for a 3R-compliant investigation of immunotoxic mechanisms of action in aquatic vertebrates. The validated and newly identified biomarker candidates of specific immunotoxic effects can be used in future studies in the context of environmental hazard assessment of substances or also for human-relevant immunotoxicological questions.

Transcriptomic profiling of clobetasol propionate-induced immunosuppression in challenged zebrafish embryos.

In the ecotoxicological hazard assessment of chemicals, the detection of immunotoxicity is currently neglected. This is mainly due to the complexity of the immune system and the consequent lack of standardized procedures and markers for the comprehensive assessment of immunotoxic modes of action. In this study, we present a new approach applying transcriptome profiling to an immune challenge with a mixture of pathogen-associated molecular patterns (PAMPs) in zebrafish embryos, analyzing differential gene expression during acute infection with and without prior exposure to the immunosuppressive drug clobetasol propionate (CP). While PAMP injection itself triggered biological processes associated with immune activation, some of these genes were more differentially expressed upon prior exposure to CP than by immune induction alone, whereas others showed weaker or no differential regulation in response to the PAMP stimulus. All of these genes responding differently to PAMP after prior CP exposure showed additivity of PAMP- and CP-induced effects, indicating independent regulatory mechanisms. The transcriptomic profiles suggest that CP impaired innate immune induction by attenuating the response of genes involved in antigen processing, TLR signaling, NF-КB signaling, and complement activation. We propose this approach as a powerful method for detecting gene biomarkers for immunosuppressive modes of action, as it was able to identify alternatively regulated processes and pathways in a sublethal, acute infection zebrafish embryo model. This allowed to define biomarker candidates for immune-mediated effects and to comprehensively characterize immunosuppression. Ultimately, this work contributes to the development of molecular biomarker-based environmental hazard assessment of chemicals in the future.