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Systems microscopy-based drug target discovery in pathogen-mediated inflammatory response signalling

Timeline

October 2017 – October 2021

Group and collaboration

Prof. Dr. Bob van de Water – Leiden University and Horizon2020 EU-ToxRisk
PhD student: Linda van den Berk

Project description

Infection caused by pathogens lead to an inflammatory response in both diseased tissue as at a systemic level. As a primary event, factors released from pathogens, so called pathogen-associated molecular patterns (PAMPs), bind to Toll-Like receptors (TLRs) and thereby activate intracellular signaling that impacts immune regulation and increases the capacity of the innate and adaptive immune system to fight the infection. This is mediated through a secondary event, where released pro-inflammatory cytokines, e.g. TNF-α and IL1β, bind to their respective receptors and initiate downstream signaling eventually leading to promote immune surveillance responses and cytoprotective measures against cell death both locally and systemically. Interestingly, the downstream signalling of TLRs and cytokine receptors is largely overlapping. For the most part it involves the activation of the NF-kB transcription factor that then mediates the transcription of pro-inflammatory and cytoprotective genes. Modulation of TLR and cytokine receptor signalling is likely to impact on the overall success of the immune system to fight pathogen infection. In the past, various simple high-throughput screens in tumor cell lines using luciferase-based reporters where performed, but this does not reflect endogenous signalling in normal differentiated cells. The aim of this project is to follow NF-kB activation and downstream signalling upon PAMP/cytokine exposure in differentiated cells and apply this for drug target and drug lead discovery. Our approach is to establish human induced pluripotent stem cell (hiPSC) fluorescent reporter cell lines via CRISPR/Cas9 for the NF-kB signalling pathway. Subsequently, the goal is to differentiate these cells in to different lineages, including hepatocytes, cardiomyocytes, sensory neurons and proximal tubular epithelial cells. In these different lineages we will quantitatively assess the dynamics of downstream signalling activation by different PAMPs/cytokines as well as tissue-specific differences in NF-kB signalling. In parallel, transcriptomic responses in the different lineages will be assessed using high-throughput RNAseq-based transcriptomics. In selected differentiated cell models we will unravel the signalling components that may impact on improving host defence against pathogens. This will be based on CRISPR/Cas9 approaches making advantage of previous identified candidate drug targets.


Disease Intervention Strategies is the overarching theme for more than 10 PhD tracks in NCOH projects to create new interdisciplinary, inter-thematic, and inter-institutional research collaborations.