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The multifaceted and paradoxical roles of Toll-like receptors and their signaling adaptors in modulating microglia innate immune response to neurotropic betacoronaviruses
Dissertation   Open access

The multifaceted and paradoxical roles of Toll-like receptors and their signaling adaptors in modulating microglia innate immune response to neurotropic betacoronaviruses

Elijah Davis
Doctor of Philosophy (Ph.D.), Drexel University
Apr 2024
DOI:
https://doi.org/10.17918/00010619
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Abstract

Antiviral Betacoronavirus MDA5 Microglia Proinflammatory Toll-like receptors Virology
Despite recent advances in medicine, viral infections of the central nervous system (CNS) constitute 20% of diagnosed neurological disorders. Left untreated, these infections can prove fatal and up to 50% of survivors develop long-term neurological complications. Inappropriate inflammation is considered the hallmark of CNS disease. Thus, understanding the mechanisms underlying virus-associated neuroinflammation, also known as viral encephalitis (VE), is crucial for the development of neuroprotective treatments. Microglia, CNS-resident innate immune cells, play a pivotal role in limiting viral replication within the CNS, protecting against lethal VE, and mediating neuronal repair. However, direct infection or aberrant activation can dysregulate microglia activity and generate a pro-inflammatory microenvironment that promotes neurotoxicity, worsening neuropathogenesis. In this study, we demonstrate in vitro that microglia facilitate the replication of Mouse Hepatitis virus (MHV) A59 and -JHM, neurotropic beta coronaviruses that model acute VE with or without chronic demyelination. Efficient MHV infection was partially dependent on Toll-like receptor (TLR) -2, -3, -4, and -7 signaling and deficiency these receptors reduced viral titers, protein production, and double-stranded RNA synthesis. This suppression was more pronounced in microglia expressing nonfunctional TLR signaling adaptors, which disrupts multiple TLR pathways. Mechanistically, we show that the refractory nature of adaptor-deficient microglia is partially mediated by amplified IFN[beta] responses. ELISA and microarray analyses of the innate immune secretome revealed that TLRs were the primary regulators of microglia secretory responses during MHV infection, though the role of each TLR greatly varied. Given that peripheral viral infections may be linked to lipopolysaccharide (LPS)-induced neuroinflammation, we investigated how LPS exposure influenced microglial immune responses to MHV. Stimulation with TLR4-specific LPS prior and/or after viral adsorption significantly impaired MHV replication through the induction of IFN [beta]. Surprisingly, pharmacological TLR4 inhibition similarly restricted MHV replication while elevating IFN [beta] expression. Remarkably, LPS stimulation increased expression of the cytosolic dsRNA sensor melanoma differentiation-associated protein 5 (MDA5) and activated the endosomal dsRNA sensor TLR3. TLR3 signaling was demonstrated to selectively contribute to LPS-induced inflammation and was required for the upregulation of MDA5 during MHV infection. Taken together, these novel discoveries unveil a complex and interconnected microglial PRR network that can either antagonize or promote MHV replication while also modulating neuroinflammation during acute and peripheral disease.

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