Transcriptional Profiling Suggests T Cells Cluster around Neurons Injected with Toxoplasma gondii Proteins.

Toxoplasma gondii's tropism for and persistence in the central nervous system (CNS) underlies the symptomatic disease that T. gondii causes in humans. Our recent work has shown that neurons are the primary CNS cell with which Toxoplasma interacts and which it infects in vivo This predilection for neurons suggests that T. gondii's persistence in the CNS depends specifically upon parasite manipulation of the host neurons. Yet, most work on T. gondii-host cell interactions has been done in vitro and in nonneuronal cells. We address this gap by utilizing our T. gondii-Cre system that allows permanent marking and tracking of neurons injected with parasite effector proteins in vivo Using laser capture microdissection (LCM) and RNA sequencing using RNA-seq, we isolated and transcriptionally profiled T. gondii-injected neurons (TINs), Bystander neurons (nearby non-T. gondii-injected neurons), and neurons from uninfected mice (controls). These profiles show that TIN transcriptomes significantly differ from the transcriptomes of Bystander and control neurons and that much of this difference is driven by increased levels of transcripts from immune cells, especially CD8+ T cells and monocytes. These data suggest that when we used LCM to isolate neurons from infected mice, we also picked up fragments of CD8+ T cells and monocytes clustering in extreme proximity around TINs and, to a lesser extent, Bystander neurons. In addition, we found that T. gondii transcripts were primarily found in the TIN transcriptome, not in the Bystander transcriptome. Collectively, these data suggest that, contrary to common perception, neurons that directly interact with or harbor parasites can be recognized by CD8+ T cells.IMPORTANCE Like other persistent intracellular pathogens, Toxoplasma gondii, a protozoan parasite, has evolved to evade the immune system and establish a chronic infection in specific cells and organs, including neurons in the CNS. Understanding T. gondii's persistence in neurons holds the potential to identify novel, curative drug targets. The work presented here offers new insights into the neuron-T. gondii interaction in vivo By transcriptionally profiling neurons manipulated by T. gondii, we unexpectedly revealed that immune cells, and specifically CD8+ T cells, appear to cluster around these neurons, suggesting that CD8+ T cells specifically recognize parasite-manipulated neurons. Such a possibility supports evidence from other labs that questions the long-standing dogma that neurons are often persistently infected because they are not directly recognized by immune cells such as CD8+ T cells. Collectively, these data suggest we reconsider the broader role of neurons in the context of infection and neuroinflammation.

Aging with Toxoplasma gondii results in pathogen clearance, resolution of inflammation, and minimal consequences to learning and memory.

Persistent inflammation has been identified as a contributor to aging-related neurodegenerative disorders such as Alzheimer's disease. Normal aging, in the absence of dementia, also results in gradual cognitive decline and is thought to arise, in part, because of a chronic pro-inflammatory state in the brain. Toxoplasma gondii is an obligate intracellular parasite that establishes a persistent, asymptomatic infection of the central nervous system (CNS) accompanied by a pro-inflammatory immune response in many of its hosts, including humans and rodents. Several studies have suggested that the inflammation generated by certain strains of T. gondii infection can be neuroprotective in the context of a secondary insult like beta-amyloid accumulation or stroke. Given these neuroprotective studies, we hypothesized that a prolonged infection with T. gondii may protect against age-associated decline in cognition. To test this hypothesis, we infected young adult mice with either of two genetically distinct, persistent T. gondii strains (Prugniaud/type II/haplogroup 2 and CEP/type III/haplogroup 3) and monitored mouse weight, survival, and learning and memory over the ensuing 20 months. At the end of the study, we evaluated CNS inflammation and parasite burden in the surviving mice. We found that parasite infection had no impact on age-associated decline in learning and memory and that by 20 months post infection, in the surviving mice, we found no evidence of parasite DNA, cysts, or inflammation in the CNS. In addition, we found that mice infected with type III parasites, which are supposed to be less virulent than the type II parasites, had a lower rate of long-term survival. Collectively, these data indicate that T. gondii may not cause a life-long CNS infection. Rather, parasites are likely slowly cleared from the CNS and infection and parasite clearance neither positively nor negatively impacts learning and memory in aging.

Three-Dimensional Reconstruction of Toxoplasma-Neuron Interactions In Situ.

How tissue and cellular architecture affects host cell-microbe interactions in vivo remains poorly defined because imaging these interactions in complex tissue is difficult and standard in vitro cultures do not mimic whole organ architecture. Here we describe a method that combines new tissue clearing techniques, high-resolution imaging, and three-dimensional reconstruction to overcome these barriers and allow in situ imaging of host cell-microbe interactions in complex tissue. We use the interactions between neurons and Toxoplasma gondii, a ubiquitous, protozoan parasite that establish a lifelong central nervous system (CNS) infection in mice and humans, as a model for this technique. This method aims to provide an easy, reproducible way to visualize the complex relationship between host cells and intracellular pathogens within a whole organ.

Dissecting Amyloid Beta Deposition Using Distinct Strains of the Neurotropic Parasite Toxoplasma gondii as a Novel Tool.

Genetic and pathologic data suggest that amyloid beta (Aß), produced by processing of the amyloid precursor protein, is a major initiator of Alzheimer's disease (AD). To gain new insights into Aß modulation, we sought to harness the power of the coevolution between the neurotropic parasite Toxoplasma gondii and the mammalian brain. Two prior studies attributed Toxoplasma-associated protection against Aß to increases in anti-inflammatory cytokines (TGF-ß and IL-10) and infiltrating phagocytic monocytes. These studies only used one Toxoplasma strain making it difficult to determine if the noted changes were associated with Aß protection or simply infection. To address this limitation, we infected a third human amyloid precursor protein AD mouse model (J20) with each of the genetically distinct, canonical strains of Toxoplasma (Type I, Type II, or Type III). We then evaluated the central nervous system (CNS) for Aß deposition, immune cell responses, global cytokine environment, and parasite burden. We found that only Type II infection was protective against Aß deposition despite both Type II and Type III strains establishing a chronic CNS infection and inflammatory response. Compared with uninfected and Type I-infected mice, both Type II- and Type III-infected mice showed increased numbers of CNS T cells and microglia and elevated pro-inflammatory cytokines, but neither group showed a >2-fold elevation of TGF-ß or IL-10. These data suggest that we can now use our identification of protective (Type II) and nonprotective (Type III) Toxoplasma strains to determine what parasite and host factors are linked to decreased Aß burden rather than simply with infection.

Neurons are the Primary Target Cell for the Brain-Tropic Intracellular Parasite Toxoplasma gondii.

Toxoplasma gondii, a common brain-tropic parasite, is capable of infecting most nucleated cells, including astrocytes and neurons, in vitro. Yet, in vivo, Toxoplasma is primarily found in neurons. In vitro data showing that interferon-γ-stimulated astrocytes, but not neurons, clear intracellular parasites suggest that neurons alone are persistently infected in vivo because they lack the ability to clear intracellular parasites. Here we test this theory by using a novel Toxoplasma-mouse model capable of marking and tracking host cells that directly interact with parasites, even if the interaction is transient. Remarkably, we find that Toxoplasma shows a strong predilection for interacting with neurons throughout CNS infection. This predilection remains in the setting of IFN-γ depletion; infection with parasites resistant to the major mechanism by which murine astrocytes clear parasites; or when directly injecting parasites into the brain. These findings, in combination with prior work, strongly suggest that neurons are not incidentally infected, but rather they are Toxoplasma's primary in vivo target.

3-D imaging and analysis of neurons infected in vivo with Toxoplasma gondii.

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma establishes a lifelong persistent infection in the brain. While this brain infection is asymptomatic in most immunocompetent people, in the developing fetus or immunocompromised individuals such as acquired immune deficiency syndrome (AIDS) patients, this predilection for and persistence in the brain can lead to devastating neurologic disease. Thus, it is clear that the brain-Toxoplasma interaction is critical to the symptomatic disease produced by Toxoplasma, yet we have little understanding of the cellular or molecular interaction between cells of the central nervous system (CNS) and the parasite. In the mouse model of CNS toxoplasmosis it has been known for over 30 years that neurons are the cells in which the parasite persists, but little information is available about which part of the neuron is generally infected (soma, dendrite, axon) and if this cellular relationship changes between strains. In part, this lack is secondary to the difficulty of imaging and visualizing whole infected neurons from an animal. Such images would typically require serial sectioning and stitching of tissue imaged by electron microscopy or confocal microscopy after immunostaining. By combining several techniques, the method described here enables the use of thick sections (160 µm) to identify and image whole cells that contain cysts, allowing three-dimensional visualization and analysis of individual, chronically infected neurons without the need for immunostaining, electron microscopy, or serial sectioning and stitching. Using this technique, we can begin to understand the cellular relationship between the parasite and the infected neuron.