Microglia are the resident macrophage of the central nervous system (CNS) and perform various functions, both inflammatory and regenerative, to control viral infection. The demyelination-inducing, neurotropic JHM strain of mouse hepatitis virus (JHMV), a beta-coronavirus, has proven instrumental in elucidating microglial functions within the immune landscape of CNS viral infections. This dissertation sets out to further explicate the roles of microglia in viral neurodegeneration of the CNS. What we firstly focused on in this regard was the role of microglia in controlling CNS infection of another beta-coronavirus, SARS-CoV-2. To achieve this, we employed the K18-hACE2 mouse model, which has been bred to express the human viral receptor, angiotensin-converting enzyme 2 (ACE2), and mice were fed either control chow or PLX5622 chow, a colony stimulating factor 1 receptor (CSF1R) inhibitor, to selectively ablate microglia prior to intranasal inoculation with SARS-CoV-2. Both control and PLX5622-treated mice displayed similar mortality and intense neuronal infection by the virus that was largely unaffected by microglial ablation. Summarily, this experiment highlighted an interesting limitation for the microglial capacity to control CNS viral disease, specifically of SARS-CoV-2, when previous studies have showcased the indispensability of these cells in other viral CNS infections.
In another PLX5622 study, this time using JHMV, we investigated the role of microglia following established demyelination by feeding mice either control or PLX5622 chow at 14 days post-infection (DPI). This experiment similarly spotlighted a limited ability for microglia to affect viral load and animal mortality, as neither significantly changed upon microglia ablation at this time point. However, a marked increase in demyelination was observed at 28 DPI in the PLX5622-treated mice, which was associated with a decrease in tissue-level remyelination-associated transcripts and an increase in the number of vacuoles. These data point to the important role of microglia in remyelination after 14 DPI through their phagocytosis of myelin debris.
Then, we focused on an important factor secreted by microglia, cystatin F (Cst7), which our previous single-cell RNA sequencing (scRNA-seq) data of JHMV-infected mice implicated as an important remyelination-associated microglial factor at 21 DPI. In fact, our germline knockout of this factor revealed significantly enhanced demyelination at 21 DPI, preceded by an enhanced CD8+ T cell response at 14 DPI. This study points to an important role for cystatin F, and for microglia as key producers of the factor within the CNS, in attenuating virus-induced demyelination through attenuation of the T cell response.
Finally, this dissertation touches on the role of another microglial factor, insulin-like growth factor 1 (IGF-1), which, like cystatin F, was also found to have increased microglial expression at 21 DPI in our previous scRNA-seq dataset. Knockout of this factor, which has important roles in myelinogenesis and aiding the maturation and differentiation of oligodendrocyte progenitor cells, was achieved via an inducible CreERT-Flox model, with Cre recombinase expression driven by the fractalkine receptor (Cx3cr1) promoter. Upon induction of this microglia-specific knockout of Igf1, mice experienced significantly worsened demyelination and consequent clinical disease, especially at 21 DPI. Like in the Cst7 knockout experiment, this heightened disease state was associated with an augmented CD8+ T cell response that suggests a role for microglial IGF-1 in controlling demyelination through dampening T cell activity. Further experimentation will attempt to show that this T cell-modulating effect of microglial IGF-1 comes as a result of the lack of IGF-1 signaling on microglia, skewing their phenotype toward a more inflammatory profile that may more readily recruits and reactivates CD8+ T cells.
