Detection of environmental glucocorticoids and investigation of their effects using the zebrafish embryo as a model

Synthetic glucocorticoids (GCs) are widely used anti-inflammatory drugs. They mimic cortisol, the natural stress hormone in humans and in fish. Cortisol binds and activates the glucocorticoid receptor (GR), which regulates genes governing development, immune response, osmoregulation, and behavior. Therefore, the presence of GCs in the aquatic environment may cause endocrine disruption through impairing these essential physiological processes in fish. The aim of this thesis was to assess the effects of environmentally relevant concentrations of GCs in teleost fish, focusing on the inflammatory response and molecular mechanisms of GC action on different levels, from mRNA to proteins to metabolites. To confirm the environmental presence of GCs, GC activity was determined along a Swiss freshwater stream impacted by a wastewater treatment plant, using effect-directed analysis. The effluent samples showed the highest potency to activate the GR, as determined by the GR-CALUX® assay. The chemical analysis found clobetasol propionate (CP), a highly potent GC, to be responsible for 63% of the total GR activity in the sample. CP was selected as a model chemical to study GC effects in fish. Embryos of zebrafish (Danio rerio) aged until 5 days post fertilization (dpf) were used as a model organism; working with embryonic stages is a refinement of animal experiments. We demonstrated that CP is readily taken up by the embryos. Bacterial lipopolysaccharides (LPS) induce one of the main inflammatory cascades. As observed in LPS challenge assays, the inflammatory response of the embryos was significantly reduced after exposure to ¿0.1 nM CP. We showed that mRNA expression of several LPS mechanism- and GC action-related genes was regulated by CP. Among them, Annexin a1b (anxa1b) was significantly down-regulated after exposure to ¿0.05 nM CP. Sensitive regulation of anxa1b by the non-steroidal anti-inflammatory drug diclofenac (DCF) suggested that this gene product is a potential biomarker of steroidal and non-steroidal immunosuppressive effects. An LC-MS/MS-based targeted proteomics technique was developed to measure proteins, as it is these molecules that actually carry out cellular functions. 12 out of 40 GC action-related protein targets were detectable in control embryo digests and were subsequently monitored after CP, DCF and grab water sample exposures. We found that the muscle protein Myhz2 was regulated only after DCF exposure, while I¿B¿, an anti-inflammatory protein, was regulated only by CP, in agreement with mRNA level data. Global and targeted metabolomics analyses were performed in zebrafish embryos exposed to CP from 4 to 5 dpf, in collaboration with M. Lamoree (VU Amsterdam). Several of the detected compounds showed significant response to CP: lysine, nicotinamide, choline, hypoxanthine, tyrosine and tryptophan. To conclude, this thesis demonstrates that GCs are present in the aquatic environment and may suppress the inflammatory response of fish at environmentally relevant concentrations.

Clobetasol propionate causes immunosuppression in zebrafish (Danio rerio) at environmentally relevant concentrations

Anita Orsolya Hidasi, Kristin Schirmer

Synthetic glucocorticoids (GCs) are potential endocrine disrupting compounds that have been detected in the aquatic environment around the world in the low ng/L (nanomolar) range. GCs are used as immunosuppressants in medicine. It is of high interest whether clobetasol propionate (CP), a highly potent GC, suppresses the inflammatory response in fish after exposure to environmentally relevant concentrations. Bacterial lipopolysaccharide (LPS) challenge was used to induce inflammation and thus mimic pathogen infection. Zebrafish embryos were exposed to ≤1000 nM CP from ~1 h post fertilization (hpf) to 96 hpf, and CP uptake, survival after LPS challenge, and expression of inflammation-related genes were examined. Our initial experiments were carried out using 0.001% DMSO as a solvent vehicle, but we observed that DMSO interfered with the LPS challenge assay, and thus masked the effects of CP. Therefore, DMSO was not used in the subsequent experiments. The internal CP concentration was quantifiable after exposure to ≥10 nM CP for 96 h. The bioconcentration factor (BCF) of CP was determined to be between 16 and 33 in zebrafish embryos. CP-exposed embryos showed a significantly higher survival rate in the LPS challenge assay after exposure to ≥0.1 nM in a dose dependent manner. This effect is an indication of immunosuppression. Furthermore, the regulation pattern of several genes related to LPS challenge in mammals supported our results, providing evidence that LPS-mediated inflammatory pathways are conserved from mammals to teleost fish. Anxa1b, a GC-action related anti-inflammatory gene, was significantly down-regulated after exposure to ≥0.05 nM CP. Our results show for the first time that synthetic GCs can suppress the innate immune system of fish at environmentally relevant concentrations. This may reduce the chances of fish to survive in the environment, as their defense against pathogens is weakened.

Elsevier2017

Gene expression analysis of metastases and IL-1R1 characterization in a NALP3 mouse model

Heidi Fournier

Tumor microenvironment contributes strongly to tumor evolution toward metastasis, a final stage in cancer progression. It provides essential clues to promote migration of cancer cells in metastatic sites. Moreover, inflammatory cells and immunomodulatory mediators present in the microenvironment are able to polarize the host immune response, thereby potentiating primary tumor development. The reciprocal and dynamic interactions between microenvironment and tumor cells orchestrate critical steps of evolution toward metastasis. To address the role of inflammation in tumor progression, a mouse model of downregulated inflammation was used: the NALP3 gene in these mice is knocked-out, causing a impaired processing of the two inflammatory cytokines Interleukin-1[beta] and IL-18 which are essential for the recruitment and activation of immune cells. In this case, a decrease in tumor growth and metastasis was observed in previous studies, leading us to investigate the molecular and cellular interactions of tumor and immune cells in inflammatory and non-inflammatory contexts. Gene expression analysis was performed on two steps of mouse tumor cells: the B16-F10 melanoma cells seem to be more dependent on the immune cells recruitment to metastasize in vivo, while the Lewis Lung Carcinomas cells showed a quite equivalent growth in both wild-type or NALP3 knock-out mice. Different techniques were used to do so, such as standard in vitro assays and mRNA expression analyses, and in vivo tests with extraction of tumoral mRNA by Laser Capture Microdissection. Gene expression analyses of tumor cells were performed to address the influence of the inflammatory background. Comparison of these two gene expression profiles may reveal genes related to the specific reactive inflammatory response surrounding the given metastases. Another point that was investigated was the importance of the IL-1R1, receptor of interleukin-1. Since it represents the counterpart of IL-1[beta] signaling, its expression by immune and tumor cells may correlate with degree of tumor growth and invasion. In vitro and in vivo assays allowed better understanding the importance of this receptor. While a downregulation of this receptor impairs proliferation of in vitro cell cultures, at the in vivo level, the number of metastases is decreased too. These results provide potential clues indicating the important role played by the inflammatory cells, and how they can support and promote tumor progression, in particular through the expression of NALP3. The adaptation mechanisms of tumor cells to their microenvironment were also assessed, opening different insights for targeting the tumor itself as well as the inflammatory microenvironment.

2011

Elucidating regulatory mechanisms shaping the transcriptome and proteome of the gut immune response in Drosophila melanogaster

Michael Vincent Frochaux

Work on Drosophila melanogaster paved the way to our current understanding of modern genetics. Since then, this model organism has contributed greatly to various fields such as neurobiology, development, and immunology. The discovery and analysis of the various pathways of the Drosophila immune response led the way to the in-depth characterization of the gut immune response. Furthermore, a genome-wide association study on a population of Drosophila identified resistance to infection by the entomopathogenic bacteria Pseudomonas entomophila (P.e.) as a complex phenotype, with highly resistant and susceptible lines, and highlighted genes and pathways mediating inter-individual differences. However, the molecular mechanisms mediating the resistance to enteric infection remain largely uncharacterized. This study uses the same Drosophila population to analyze what are the molecular mechanisms mediating the resistance to enteric infection. We analyzed the genetic determinants of gene expression variation among the most resistant and susceptible lines in response to infection using expression quantitative trait loci (eQTL) analysis. We also characterized the mode of action of these eQTLs into cis- and trans-acting. Furthermore, we identified the gene nutcracker (ntc) as the only differentially-expressed gene between resistance classes. We then demonstrate that loss of function ntc mutants are significantly more susceptible to infection and then identify a single nucleotide polymorphism (SNP) located in a transcription factor binding site (TFBS) which affects the binding affinity of the transcription factor Broad leading to allele-specific expression variation of the gene ntc. If the transcriptomic response to infection has been thoroughly characterized, it is not the case for the proteomic response. We thus sought to characterize for the first time the proteomic response of the Drosophila to enteric infection. We performed mass spectrometry and RNA sequencing on dissected guts from flies infected by two Gram-negative bacteria, Erwinia carotovora carotovora 15 (Ecc15) or Pseudomonas entomophila, 4h and 16h after infection. We found that a large portion of the measurable proteome (12%) varies after infection and that protein changes are strongly time- and infection-dependent. We showed the relatively poor correlation between gene expression and protein abundance in the Drosophila enteric immune response. Finally, we performed a screen of several proteins that were identified in our proteomics work but had previously not been found when assessing the gene expression response to infection using loss-of-function mutants and identifying 7 genes that modulate overall susceptibility to P.e. infection. In summary, this work analyze the genetic determinants of gene expression variation in the DGRP population upon infection and characterize them according to their mode of action. Furthermore, we describe how a non-coding variant lowers resistance to infection by modulating ntc gene expression through altered Broad repressor binding. Finally, it provides the first comprehensive characterization of the Drosophila gut proteome upon infection by Gram-negative bacteria which can be the basis of future proteomic analysis of the enteric immune response

EPFL2019