Defining neuronal responses to the neurotropic parasite Toxoplasma gondii

Abstract

A select group of pathogens infects neurons in the brain. Prior dogma held that neurons were "defenseless" against infecting microbes, but many studies suggest that neurons can mount anti-microbial defenses. However, a knowledge gap in understanding how neurons respond in vitro and in vivo to different classes of microorganisms remains. To address this gap, we compared a transcriptional data set derived from primary neuron cultures (PNCs) infected with the neurotropic intracellular parasite Toxoplasma gondii with a data set derived from neurons injected with T. gondii protein in vivo. These curated responses were then compared to the transcriptional responses of PNCs infected with the single-stranded RNA viruses, West Nile virus or Zika virus. These analyses highlighted a conserved response to infection associated with chemokines (Cxcl10, Ccl2) and cytokines (interferon signaling). However, T. gondii had diminished IFN-α signaling in vitro compared to the viral data sets and was uniquely associated with a decrease in neuron-specific genes (Snap25, Slc17a7, Prkcg). These data underscore that neurons participate in infection-induced neuroinflammation and illustrate that neurons possess both pathogen-specific and pathogen-conserved responses.IMPORTANCEThough neurons are commonly the target of pathogens that infect the central nervous system (CNS), few data sets assess the neuronal response to infection. This paucity of data is likely because neurons are perceived to have diminished immune capabilities. However, to understand the role of neurons in neuroinflammation and their immune capabilities, their responses must be investigated. Here, we analyzed publicly accessible, neuron-specific data sets to compare neuron responses to a eukaryotic pathogen vs two Orthoflaviviruses. A better understanding of neuron responses to different infections will allow us to develop methods for inhibiting pathways that lead to neuron dysfunction, enhancing those that limit pathogen survival, and mitigating infection-induced damage to the CNS.

Keywords: RNA-seq; T. gondii; Toxoplasma gondii; central nervous system infections; host response; neurons; transcriptomics.

Fig 1

T. gondii-infected neurons from in vitro (PNC) and in vivo (LCM) systems were captured and analyzed for differentially expressed genes. (A) Experimental schematic of neurons captured by laser capture microdissection and infected primary murine neuronal cultures. (B) Volcano plots of differentially expressed genes in both data sets. Horizontal bars indicate adjusted P values ≤0.1, and vertical bars indicate log₂ fold change ≥1 for up- and downregulated genes.

Fig 2

Pathway analysis reveals the neuronal response to T. gondii infection involves an increase in proinflammatory cytokines and a decrease in neuron function. (A) The top 14 enriched pathways between LCM data set and PNCs were selected out of 975 upregulated pathways. (B) Quantification of the most represented genes in the 14 most enriched pathways with a heatmap of their log₂FC. (C) Representative signature pathways between PNCs and LCM with normalized enrichment scores (NES). (D) Venn diagram of 28 downregulated pathways in LCM and PNC data sets. (E) Enrichment scores of downregulated neuron pathways in T. gondii data sets. GSEA, gene set enrichment analysis.

Fig 3

WNV- and ZKV-infected PNCs and the LCM data set show the upregulation of IFN-α response genes, unlike T. gondii-infected PNCs. (A) Volcano plots of WNV and ZKV-infected PNCs. Horizontal bars indicate adjusted P values ≤ 0.1, and vertical bars indicate log2 fold change ≥ 1 for up- and downregulated genes. (B) Upset plot of upregulated gene set enrichment analysis pathways. (C) Relative expression of inflammatory pathways across data sets. (D) IFN-α response genes expressed in LCM, T. gondii, WNV, and ZKV infected in log₂FC. (E) Count per million of IFN-α response genes in LCM and T. gondii PNC data sets with raw values shown. ND, not detected. ND genes in both LCM and PNCs in panel D are not included in panel E.

Fig 4

Cxcl10 is upregulated across acute and subacute data sets. Other conserved genes include Irf7, Icam1, Gbp3, Usp18, Stat1, and Ifih1.

Fig 5

Despite Irf7 upregulation in all data sets, T. gondii-infected neurons fail to upregulate and secrete IFN-α. (A) WNV and T. gondii activate PRR receptors leading to an intracellular cascade that results in IFN-α production through IRF7 phosphorylation. IFN-α binds IFNAR and upregulates Stat1 and other subsequent IFN-α response genes in WNV-infected neurons. T. gondii inhibits Stat1 in this pathway in vitro with the T. gondii effector protein TgIST. The lack of Ifn-α upregulation in both T. gondii data sets indicates that the parasite may inhibit the action of phosphorylated IRF7 through an unknown mechanism, either before full invasion or after. (B) In vivo, Stat1 may be upregulated through alternative stimulation pathways, such as IFN-γ. ISRE = interferon-stimulated response element, GAS = gamma interferon activation site.