Investigating the influence of TREM2 on myeloid cell reactivity and the resolution of acute CNS inflammation

Abstract

Neuroinflammation can be considered a tightly coordinated network of events involving the engagement of CNS-resident microglia and the recruitment of leukocytes from the periphery following an injurious or noxious insult. This process involves an initial amplification of the inflammatory event transitioning to a pro-resolution response in order to restore a healthy brain parenchyma. Yet, an aberrant or sustained neuroinflammatory response underpins the pathological progression of a multitude of acute and chronic neurological conditions. Numerous studies have implicated the immunoglobulin receptor “Triggering receptor expressed on the surface of myeloid cells-2” (TREM2), most notably expressed on microglia and on monocyte-derived cell populations, as a master regulator of core myeloid cellular functions. Moreover, individuals with TREM2-variants exhibit an increased susceptibility to chronic neurodegenerative diseases including Alzheimer’s disease (AD) and frontotemporal dementia (FTD). Thus, given a substantial role of these cell subsets in many forms of neuroinflammation, this project sought to determine the influence of TREM2 on microglial and monocyte-derived cell reactivity and how it regulates inflammation resolution following an acute CNS inflammatory insult. To interrogate aspects of the neuroinflammatory cascade, both in vitro and in vivo models were employed. Firstly, an in vivo model of acute CNS inflammation, involving localised stereotaxic injections of lipopolysaccharide (LPS) into the striatum, showed an enrichment of the Trem2 transcript during the resolution phase highlighting a potential engagement of TREM2 function at this time. A bilateral version of a previously characterised a model of self-limiting acute CNS inflammation confirmed a defined neutrophil influx during the initiation of the inflammatory cascade followed by clearance during resolution. Moreover, the model demonstrated a symmetrical inflammatory response in both hemispheres enabling dual flow cytometry and immunohistochemistry analyses. Neutrophils were effectively cleared from the brain parenchyma in both WT and Trem2-/- mice after intrastriatal LPS injection. A reduction in the number of microglia and Ly6Clo monocyte-derived cell populations during resolution was observed in the absence of TREM2. Furthermore, this model identified an influx of potential CD11b-CD45hi lymphocyte populations which are maintained in the system during resolution in both genotypes. Additional analysis of the systemic immune blood and bone marrow compartments did not show an effect of TREM2 deficiency on myeloid cell populations in either location but did, however, identify an increase in T-cell proportions in the blood in Trem2-/- mice prompting speculation of an involvement of TREM2 in connecting innate and adaptive arms of the immune system at later stages of resolution. An in vitro primary adult murine microglial model was optimised and validated as a suitable platform to assess transcriptional responses to inflammatory stimuli, LPS and interleukin-4 (IL- 4), which mimic conditions associated with pro-inflammatory and pro-resolution aspects of the cascade, respectively. Application of these stimuli, to wildtype (WT) primary microglial cultures showed stimulus dependent induction of gene expression and, notably, the suppression of Trem2 in response to LPS. Moreover, WT and TREM2-deficent primary microglia and bone-marrow derived macrophages (BMDMs) were stimulated similarly. Markedly, transcriptional alterations in Trem2-/- BMDM cultures suggested the induction of compensatory transcriptional mechanisms associated with phagocytosis and lipid metabolism at baseline and the impaired induction of genes associated with regulating autophagy and apoptosis following LPS treatment. Taken together, the results within this thesis have furthered understanding of the multifaceted and complex nature in which TREM2 may regulate myeloid cell activity. Furthermore, these data suggest that TREM2 may influence an innate-to-adaptive transition in the immune response during acute CNS inflammation - a theory that requires more targeted and in-depth investigation in future studies.