Universität zu Köln

Kelepouras, Konstantinos ORCID: 0000-0003-4257-9404 (2025). Advancing the Understanding of Pathogenic Necroptotic Cell Death: Biomarker validation and Pathway Regulation Discovery in Immunity and Inflammatory Diseases. PhD thesis, Universität zu Köln.

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Abstract

Necroptosis is a regulated, lytic form of cell death that plays a pivotal role in inflammation and tissue damage, particularly in the context of impaired apoptotic signalling. While the molecular execution of necroptosis, mediated through RIPK3-dependent phosphorylation of MLKL, has been well defined in vitro, its in vivo relevance, regulation, and pathological outcomes have remained incompletely understood. This thesis addresses a major technical gap by establishing and validating immunohistochemistry and immunofluorescence-based method to detect phosphorylated MLKL at serine 345 (pMLKL-S345) in murine tissues, enabling high-resolution spatial mapping of necroptotic activity in vivo. Applying this method across various inflammatory models, including SARS-CoV infection, Sharpin-deficient dermatitis and TNFinduced systemic inflammatory response syndrome (SIRS), this work demonstrates distinct patterns of tissue-specific necroptosis. This approach not only differentiates necroptosis from apoptosis at a cellular level but also provides a foundation for dissecting its mechanistic drivers in pathophysiological settings, without the necessity of complex genetic knockout models traditionally required to assess the involvement of necroptosis in vivo. Building on this platform, this thesis investigates the upstream signalling mechanisms that trigger necroptosis in caspase-8 deficient epidermal keratinocytes (Casp8E-KO mice). Loss of caspase-8 leads to the accumulation of cytosolic DNA, which activates the cGAS-STING pathway and induces a type I interferon transcriptional response. This STING-driven cascade upregulates key necroptotic effectors, notably ZBP1 and MLKL, while the combined effects of caspase-8 deficiency and aberrant STING activation promote the accumulation of Znucleic acids. Together, these events converge to induce ZBP1-MLKL-mediated necroptosis via formation of a ZBP1-RIPK1-RIPK3 signalling complex. Notably, this pathway functions independently of the canonical FADD-RIPK1-RIPK3 complex, enabling TNF/TNFR1- independent execution of necroptosis. Collectively, these findings position STING as a central inducer and amplifier of necroptosis, coordinating both ZBP1- and TNFR1-mediated checkpoints and acting as a critical upstream regulator of inflammatory necroptotic cell death. Strikingly, this mechanism mirrors the pathogenesis observed in STING-associated vasculopathy with onset in infancy (SAVI), a severe human type I interferonopathy caused by gain-of-function mutations in the STING1 gene. Comparative transcriptomic analysis between Casp8E-KO mice and SAVI patients reveals a nearly identical necroptotic signature, implicating STING-induced necroptosis as a central pathological driver. This is further validated in a SAVI mouse model, where co-deletion of RIPK3 markedly rescues immunedriven tissue pathology. These findings establish STING-mediated necroptosis as a novel aetiological mechanism in both caspase-8-deficient inflammatory skin disease and SAVI, and identify this pathway as a potential therapeutic target in interferonopathies characterized by excessive necroptosis. In summary, this thesis bridges fundamental mechanistic insights with translational relevance by delineating how aberrant STING activation orchestrates a necroptotic program that underlies severe inflammatory pathology. It also provides a validated framework for detecting and studying necroptosis in vivo, enabling future investigations into its role across diverse disease contexts.

Item Type:	Thesis (PhD thesis)
Translated title:	Title Language Death: Biomarker validation and Pathway Regulation Discovery in Immunity and Inflammatory Diseases UNSPECIFIED
Creators:	Creators Email ORCID ORCID Put Code Kelepouras, Konstantinos kkelepo1@smail.uni-koeln.de https://orcid.org/0000-0003-4257-9404 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-794188
Date:	2025
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Medicine > Biochemie > Institut I für Biochemie
Subjects:	Natural sciences and mathematics
Uncontrolled Keywords:	Keywords Language cell death English inflammation English autoinflammatory diseases English Immunohistochemistry English pMLKL-S345 English cGAS/STING English
Date of oral exam:	28 November 2025
Referee:	Name Academic Title Walczak, Henning Prof. Dr. Liccardi, Gianmaria Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/79418