Modulators of Neuroimmune Interactions Induced by Toxoplasma gondii

Abstract

Toxoplasma gondii is a common, intracellular parasite that establishes a long-terminfection in neurons in the central nervous system (CNS). Infections are often asymptomatic; however, in individuals lacking a T cell response Toxoplasma infection can lead to uncontrolled neuroinflammation, and potentially death. A long-standing question in the field is how Toxoplasma survives long term within the host. One hypothesis suggests that long term persistence is enabled by parasites residing in neurons because neurons do not have the same cell-intrinsic immune capabilities to clear parasites as non-neuronal cells (i.e., neurons are thought to lack major histocompatibility complex as an immune receptor to interact with T cells and are considered unable to respond to cytokine). Another hypothesis is that Toxoplasma modifies neurons to allow persistence by manipulating neurons through injection of Toxoplasma effector proteins, known as ROPs and GRAs, which are secreted into host cells during infection and can alter host cell signaling. Given we know Toxoplasma modifies many signaling pathways through these effector proteins, we set out to identify novel pathways that may aid in parasite persistence by transcriptionally profiling neurons that have been injected with Toxoplasma proteins. We infected Cre reporter mice, which only express a green fluorescent protein (GFP) after Cre mediated recombination, with Toxoplasma strains that have Cre recombinase fused to an effector protein that is secreted into host cells prior to invasion. Therefore, in the brain we can detect Toxoplasma injected neurons (TINs). Laser capture microdissection allowed us to isolate these injected neurons from an infected brain and perform transcriptional analysis to compare the transcripts of these injected neurons to neighboring neurons that have not interacted with parasites (Bystander neurons). Pathway analysis indicated a high level of T cell transcripts within the TINs transcriptome compared to the Bystander neurons transcriptome. Analysis of immunofluorescently stained infected brain sections was consistent with T cells being in closer proximity to TINs compared to Bystander neurons. This extreme proximity suggested potential MHCI-T cell receptor interactions. To test this idea, we infected primary neuronal cultures with parasites expressing the model antigen, OVA, and cocultured antigen specific T cells with these infected cultures. We found T cells could be activated by the infected neuronal cultures, indicating neurons are capable of presenting peptide on MHCI to T cells. To explore the ability of neurons to clear parasites (a possible outcome of T cell-neuron interactions), we infected Cre reporter mice with a strain of Toxoplasma that only triggers Cre mediated recombination in host cells after a successful invasion (GCre). We generated 200μm thick brain sections from these mice, optically cleared the sections using PACT clearing, and generated 3D reconstructions of the GFP+ neurons to determine if they actively harbor parasites. Approximately 50% of rendered neurons did not harbor parasites, indicating that neurons are capable of clearing Toxoplasma in vivo. Lastly, within this dissertation, I explored the importance of a specific effector protein, GRA15, and how it contributes to parasite persistence. Analysis of the peripheral immune response and parasite burden in the brain indicates that GRA15—an effector that causes polarization to classically activated macrophages—is does not significantly influence parasite dissemination and persistence. Overall, this dissertation uses Toxoplasma as a tool to investigate the immune capacity of neurons. We find that neurons can interact with T cells and possess competence at clearing Toxoplasma. Future directions include describing how utilizing tools I generated will allow others to determine how T cell recognition of and activation by antigen generated at different life-stages of Toxoplasma contributes to parasite clearance vs persistence.