Toxoplasma type II effector GRA15 has limited influence in vivo.

Toxoplasma gondii is an intracellular parasite that establishes a long-term infection in the brain of many warm-blooded hosts, including humans and rodents. Like all obligate intracellular microbes, Toxoplasma uses many effector proteins to manipulate the host cell to ensure parasite survival. While some of these effector proteins are universal to all Toxoplasma strains, some are polymorphic between Toxoplasma strains. One such polymorphic effector is GRA15. The gra15 allele carried by type II strains activates host NF-κB signaling, leading to the release of cytokines such as IL-12, TNF, and IL-1ß from immune cells infected with type II parasites. Prior work also suggested that GRA15 promotes early host control of parasites in vivo, but the effect of GRA15 on parasite persistence in the brain and the peripheral immune response has not been well defined. For this reason, we sought to address this gap by generating a new IIΔgra15 strain and comparing outcomes at 3 weeks post infection between WT and IIΔgra15 infected mice. We found that the brain parasite burden and the number of macrophages/microglia and T cells in the brain did not differ between WT and IIΔgra15 infected mice. In addition, while IIΔgra15 infected mice had a lower number and frequency of splenic M1-like macrophages and frequency of PD-1+ CTLA-4+ CD4+ T cells and NK cells compared to WT infected mice, the IFN-γ+ CD4 and CD8 T cell populations were equivalent. In summary, our results suggest that in vivo GRA15 may have a subtle effect on the peripheral immune response, but this effect is not strong enough to alter brain parasite burden or parenchymal immune cell number at 3 weeks post infection.

IFN-γ stimulated murine and human neurons mount anti-parasitic defenses against the intracellular parasite Toxoplasma gondii.

Dogma holds that Toxoplasma gondii persists in neurons because neurons cannot clear intracellular parasites, even with IFN-γ stimulation. As several recent studies questioned this idea, here we use primary murine neuronal cultures from wild type and transgenic mice in combination with IFN-γ stimulation and parental and transgenic parasites to reassess IFN-γ dependent neuronal clearance of intracellular parasites. We find that neurons respond to IFN-γ and that a subset of neurons clear intracellular parasites via immunity regulated GTPases. Whole neuron reconstructions from mice infected with parasites that trigger neuron GFP expression only after full invasion reveal that ~50% of these T. gondii-invaded neurons no longer harbor parasites. Finally, IFN-γ stimulated human pluripotent stem cell derived neurons show an ~50% decrease in parasite infection rate when compared to unstimulated cultures. This work highlights the capability of human and murine neurons to mount cytokine-dependent anti-T. gondii defense mechanisms in vitro and in vivo.

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.

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.