Wegner, Julia: Interplay of IKKs and IKK-related kinases in antiviral nucleic acid receptor signaling in human monocytes. - Bonn, 2022. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-67051
@phdthesis{handle:20.500.11811/9888,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-67051,
author = {{Julia Wegner}},
title = {Interplay of IKKs and IKK-related kinases in antiviral nucleic acid receptor signaling in human monocytes},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2022,
month = jun,

note = {Monocytes and monocyte-derived cells are equipped with pattern recognition receptors (PRRs) that detect conserved components of pathogens and in response initiate signaling cascades that result in the production of warning messenger substances like type I interferons (IFNs), chemokines, and proinflammatory cytokines. These mediators contribute decisively to pathogen clearance but may also harm the host if their release is not tightly restricted. The IκB kinases (IKKs) IKKα and IKKβ as well as the IKK-related kinases TBK1 and IKKε control antimicrobial gene transcription by directing the activation of the involved transcription factors of the IRF and NF-κB families and are therefore considered master regulators of PRR signaling. In this thesis, the individual functions of these kinases in different PRR pathways as well as their interplay in human monocytes were defined.
In this process, it was discovered that the monocyte model BLaER1 is not unconditionally suitable for studying PRR signaling cascades, as these cells harbor an epigenetic glycosylphosphatidylinositol anchor defect that abrogates surface expression of the toll-like receptor (TLR) coreceptor CD14. In consequence, THP1 and primary human monocytes were utilized to examine the roles of IKKs and IKK-related kinases.
Depletion of TBK1, IKKα, IKKβ, and IKKε in THP1 monocytes by CRISPR-Cas9 genome editing abolished RIG-I, cGAS, TLR8, and TLR2:TLR1 signaling, confirming the essential role of these four kinases in controlling PRR pathways. Comprehensive analysis of single, double, and triple kinase knockout cell lines unveiled redundant but also individual functions of TBK1 and IKKε as well as of IKKα and IKKβ in PRR signaling.
In this thesis, TBK1 and IKKε were found to inhibit TLR8 signaling by directly phosphorylating IKKβ and suppressing its activity. Depletion of catalytically active TBK1 and IKKε by CRISPRCas9-mediated knockout, small molecule inhibition, or expression of the NSs protein of SFTS virus, which sequesters TBK1 and IKKε in cytoplasmic granules, resulted in an amplified proinflammatory cytokine production via overactivation of NF-κB and IRF5. These findings demonstrate that TBK1 and IKKε are particularly important in preventing potentially harmful proinflammatory reactions and that viral inhibition of these kinases releases this restraint, which likely contributes to disease severity.
Although TBK1/IKKε are the main kinases responsible for IRF activation downstream of cytosolic nucleic acid receptors, RIG-I was found to induce an additional TBK1/IKKε-independent type I IFN response in human monocytes, which was identified to depend on IKKβ, IRF7, and partially IRF5. In consequence, viral inhibition of TBK1/IKKε abrogated the cGAS-induced type I IFN response, while RIG-I activation still resulted in substantial type I IFN production that triggered the upregulation of IFN-stimulated genes in bystander cells. These insights demonstrate that RIG-I induces type I IFN production via different pathways, enabling antiviral defense measures even if TBK1 and IKKε are inhibited by viral antagonists.
Furthermore, this thesis discovered an important mechanism of IKK-related kinase regulation. Independent of its enzymatic activities but dependent on its interaction with scaffold proteins, TBK1 was found to diminish IKKε expression by decreasing its protein stability. This regulatory mechanism was found pivotal to ensure unmitigated RIG-I- and cGAS-induced type I IFN production in conditions of TBK1 deficiency, which are compensated by IKKε upregulation. The findings indicate that monocytes are equipped with a backup mechanism that can prevent diminished antiviral responses upon viral degradation of TBK1.
Overall, this thesis has comprehensively characterized the individual activating and inhibitory functions of IKKs and IKK-related kinases in PRR signaling in human monocytes. The obtained insights contribute substantially to the understanding of kinase functions in dysregulated immune responses and in the clearance of viral infections, and the discovered mechanisms add new connections to the complex network of signaling pathways downstream of PRRs.},

url = {https://hdl.handle.net/20.500.11811/9888}
}

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