Microglia are the resident innate immune cells that are critical for innate and adaptive immune responses within the CNS. They recognize and are activated by pathogen-associated molecular patterns (PAMPs) present on the surface of pathogens. β-glucans, the major PAMP present within fungal cell walls, are recognized by Dectin-1, which mediates numerous intracellular events invoked by β-glucans in various immune cells. Previously, we showed that Dectin-1 mediates phagocytosis of β-glucan and subsequent superoxide production in microglia. Here, we report that the guanine nucleotide exchange factor Vav1 as well as phosphoinositide-3 kinase (PI3K) are downstream mediators of what is now recognized as the Dectin-1 signaling pathway. Both Vav1 and PI3K are activated upon stimulation of microglia with β-glucans, and the two proteins are required for phagocytosis of the glucan particles and for subsequent superoxide production. We also show that Vav1 functions upstream of PI3K and is required for activation of PI3K. Together, our results provide an important insight into the mechanistic aspects of microglial activation in response to β-glucans.

Microglia, the resident immune cells of the brain, are activated in response to any kind of CNS injury, and their activation is critical for maintaining homeostasis within the CNS. However, during inflammatory conditions, sustained microglial activation results in damage to surrounding neuronal cells. β-Glucans are widely recognized immunomodulators, but the molecular mechanisms underlying their immunomodulatory actions have not been fully explored. We previously reported that β-glucans activate microglia through Dectin-1 without inducing significant amount of cytokines and chemokines. Here, we show that particulate β-glucans attenuate cytokine production in response to TLR stimulation; this inhibitory activity of β-glucan is mediated by Dectin-1 and does not require particle internalization. At the molecular level, β-glucan suppressed TLR-mediated NF-κB activation, which may be responsible for the diminished capacity of microglia to produce cytokines in response to TLR stimulation. Overall, these results suggest that β-glucans may be used to prevent or treat excessive microglial activation during chronic inflammatory conditions.

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A major challenge in the treatment of major depressive disorder (MDD) is relapse, which is defined as the return of depressive symptoms during a period of remission. Relapse rates in MDD are high, with approximately 50% of individuals relapsing following treatment of their first depressive episode, therefore early intervention to prevent relapse is crucial. Evidence suggests that immune dysregulation may be linked to longitudinal changes in depressive severity. However, it is currently unknown whether inflammation can predict future relapse in MDD. The objective of this project was to identify potential immune predictors of relapse in participants that responded to a treatment or a combination of treatments for MDD. A secondary objective was to investigate immune composition in efforts to stratify MDD individuals into more homogenous groups and further explore these groups in relation to clinical symptoms. This project is part of the Wellness Monitoring for Major Depressive Disorder longitudinal study (NCT02934334) of responders to antidepressant treatment conducted at 6 clinical sites across Canada. Montgomery Asberg Depression Rating Scale (MADRS) scores were used to assess depression severity and to categorize participants into ultrastable, unstable, and relapse groups. Plasma immune profiles were generated using the LEGENDplex Human Th Cytokine Panel immunoassay. Principal Component Analysis and Kruskal-Wallis tests of individual immune cytokines did not show differences between ultrastable, unstable, or relapse groups. Principal Component Analysis did reveal two cytokine clusters. Hierarchical Clustering analysis identified three distinct immune biotypes characterized by differing levels of Th cytokines and validated the presence of the cytokine clusters. Neither of these outcomes was predictive of relapse in this cohort. Our findings have shown that immune composition may serve as an important factor in parsing heterogeneity that is observed in this disorder through identification of distinct immune biotypes and highly interconnected cytokine subnetworks in major depression. The potential for immune biotypes for optimizing treatment regimens and relapse prevention necessitates further investigation and replication. / Thesis / Master of Science (MSc)

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Microglia Purinergic Receptor-Mediated Neuroinflammation In Alzheimer’s Disease Neurodegeneration involves a complicated cascade of homeostatic dysfunction that converges on neuron loss and cognitive decline, involving complex immune, metabolic, and cell cell crosstalk pathways. The complicated interplay and heterogeneous nature of these factors in the brain make therapeutic development challenging. Recent advances have placed the immune system as an important driver of neurodegeneration both mechanistically and genetically. Microglia are the professional phagocytes that inhabit the brain and direct these inflammatory pathways, which can have reparative or destructive outcomes on the brain parenchyma. While various genetic risk factors for neurodegeneration reside in microglia, how these trigger and facilitate disease requires further investigation. In the present dissertation, I investigate inflammatory activation in microglia upon various damage or pathology-associated stimuli by utilizing a primary human monocyte-derived microglia-like cell (MDMi) model from a diverse donor cohort, which allows for the examination of genetically driven differences. I find that MDMi stimulated through ATP-mediated P2RX7 activation display reduced phagocytic function for amyloid beta uptake, and this pathway is also influenced by individual donors’ SPI1 genotype which has been associated with Alzheimer’s disease in previous computational studies. These experiments demonstrate functional outcomes related to AD genetics in immune cells. Previous computational studies have identified cognitive-decline associated gene modules expressed in human brain tissues from late-stage AD. I conducted in vitro follow up experiments to interrogate these genetic findings which is crucial for validating RNA sequencing data in a biological model. To interrogate differential MDMi inflammatory pathways, I treated cells with the toxic immunostimulatory molecule lipopolysaccharide (LPS), or its non-toxic derivative monophosphoryl lipid A (MPLA) which has positive immune properties currently utilized in vaccine adjuvants. My results indicated that individual gene expression in this module does not shift in a uniform manner upon LPS or MPLA challenge, suggesting more nuanced in vitro interrogation is required to identify conditions propagating this end stage disease phenotype. Microglia serve as the primary immune cells of the brain but also interact closely with astrocytes, large glial cells that facilitate neuronal homeostasis and are central players in AD due to their high apolipoprotein (APOE) production. Given the newly appreciated role of cellular crosstalk in neurological disease pathogenesis, I sought to optimize a protocol for isolation of primary mouse astrocytes for coculture with MDMi and investigation of non-direct cell contact interactions through astrocyte supernatants. Described in this dissertation is my optimized protocol for purified mouse astrocyte isolation from mice expressing humanized APOE2, APOE3, or APOE4. By developing this model, I was able to discern differential changes to MDMi gene expression in the presence of APOE2, 3, or 4 astrocyte supernatants. Verification of these tools allows further exploration of APOE genotype on glial crosstalk and downstream AD pathology. Overall, this work uncovers important mechanisms of human microglia activation through AD genetics and extracellular P2RX7 receptor behavior. By interrogating these scientific questions in a human microglia model derived from donors of various genetic and age backgrounds, we can assess how real biological variation modulates canonical inflammatory pathways. This adds powerful clinical relevance as AD and other neurodegenerative conditions can present a very heterogenous phenotype pathologically and therefore may require the nuance of more personalized medicine therapeutically.

Autoimmunity is a failure to tolerate circulating or cell surface expressed self antigens,leading to activation of the immune system and attack of self tissues. Muscle-specific kinase (MuSK) myasthenia gravis (MG) is a disease caused by antibodies to MuSK and hallmarked by fatigable muscle weakness. MuSK is a tyrosine kinase that interacts with low-density lipoprotein receptor-related protein 4 (LRP4), resulting in maintenance of the high density of acetylcholine receptors (AChRs) at the neuromuscular junction; this high density is essential for efficient transmission of signals from nerve to muscle, and MuSK antibodies impair this transmission. MuSK antibodies are predominantly IgG4, a subclass that does not induce immunological damage. Thus how these antibodies cause neuromuscular junction dysfunction is not clear. Potential mechanisms of the MuSK antibodies were explored in in vitro experiments. Plasmas from fourteen MuSK-MG patients were studied. IgG antibodies and IgG subclass profiles were measured with flow cytometry. Total IgG, Fabs, IgG4 and IgG1-3 subclass antibodies were prepared and purified; these were used to investigate the effects on MuSK surface expression, binding of LRP4 to MuSK, and agrin-LRP4-MuSK-induced AChR clustering in C2C12 mouse myotubes. No evidence for MuSK endocytosis by MuSK IgG, IgG1-3 or IgG4 antibodies was found. The predominant IgG4 subclass, and the monovalent IgG Fabs, blocked binding between LRP4 and MuSK but both IgG4 and IgG1-3 subclass antibodies were equally able to disperse pre-formed and newly-induced AChR clusters in C2C12 myotubes. The block of LRP4-MuSK interaction by IgG4 antibodies is likely to be a major pathogenic mechanism in MuSK-MG, which may lead to disrupted signal transduction, reduced AChR aggregation and neuromuscular transmission failure at the neuromuscular junction. In addition, MuSK IgG1-3, until now described as nonpathogenic, may also contribute to the reduced AChR density and neuromuscular dysfunction in MuSK-MG. These results provide new evidence concerning the pathogenic antibodies and their mechanisms in MuSK-MG.