Year: 2024
Type: dissertation
Abstract: Hemophagocytic lymphohistiocytosis (HLH) is a severe and often fatal medical condition linked to germline mutations that disrupt immune function, referred to as inborn errors of immunity (IEI). HLH clinically manifests as an extreme systemic inflammatory response, where pathogens can trigger an abnormal activation of T cells and macrophages, leading to a life-threatening cytokine storm. Recent studies in the Pachlopnik lab have demonstrated a correlation between pathogenic variants in the RNA sensor ZNFX1 and the occurrence of HLH. Zinc finger NFX1-type containing 1 (ZNFX1) is an interferon-stimulated cytosolic receptor for nucleic acids, which can autonomously activate interferon-stimulated genes (ISGs) and further amplify the innate immune response. \nIn patients with ZNFX1 deficiency, the mRNA of ISGs exhibited an extended half-life, leading to inflammatory bursts resembling HLH episodes. This suggests that ZNFX1 may have a pivotal role in early sensing of the nucleic acids in the cytosol and later re-establishing immune homeostasis following pathogen-induced inflammation (question of time). It is expected that in the absence of ZNFX1, late-onset negative regulation of the type 1 interferon pathway might be inadequately activated or engaged, contributing to prolonged inflammation. Furthermore, we propose that ZNFX1 is crucial in processing mRNAs in the cytoplasm, which are sequestered into membraneless organelles in response to pathogenic stimuli (question of space). In its absence, it is expected that mRNA species cannot be efficiently sequestered into membraneless organelles, where they can be degraded. Free in the cytoplasm, these RNAs continuously trigger an inflammatory response. \nTo explore these hypotheses, we employed advanced techniques, including 4i high- throughput imaging and computational image analysis, to capture spatiotemporal data from thousands of cells. Complementary methods such as flow cytometry, molecular biology, and cellular assays were also utilized. During this master’s, a stable ZNFX1 knock-out cell line was developed based on lung adenocarcinoma cells (A549). Further, a 4i staining protocol and an image analysis pipeline were established. Preliminary data revealed variations in mRNA expression and differentially expressed mediators of type I interferon signaling. Comprehensive spatiotemporal analysis of all the imaged markers is expected to elucidate the functional role of ZNFX1. Moreover, the plan is to use the established experimental setup with other physiologically relevant model systems, such as A549 cells with and without ZNFX1 expressing the ACE2 receptor to facilitate viral infections with SARS-CoV-2 coronavirus. The established experimental workflow and the image analysis pipeline can be used to explore the spatiotemporal role of ZNFX1 upon pathogenic encounters and the consequences of its absence, and it can be extrapolated to study other IEI.
DOI: 10.5167/uzh-264545
Author: Häfeli, Joël
Language: English
Subfield: Hematology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W6929690940
Cites:
Cited by:
Related to: