Differential induction of TLR3-dependent innate immune signaling by closely related parasite species

Abstract

The closely related protozoan parasites Toxoplasma gondii and Neospora caninum display similar life cycles, subcellular ultrastructure, invasion mechanisms, metabolic pathways, and genome organization, but differ in their host range and disease pathogenesis. Type II (γ) interferon has long been known to be the major mediator of innate and adaptive immunity to Toxoplasma infection, but genome-wide expression profiling of infected host cells indicates that Neospora is a potent activator of the type I (α/β) interferon pathways typically associated with antiviral responses. Infection of macrophages from mice with targeted deletions in various innate sensing genes demonstrates that host responses to Neospora are dependent on the toll-like receptor Tlr3 and the adapter protein Trif. Consistent with this observation, RNA from Neospora elicits TLR3-dependent type I interferon responses when targeted to the host endo-lysosomal system. Although live Toxoplasma fail to induce type I interferon, heat-killed parasites do trigger this response, albeit much weaker than Neospora, and co-infection studies reveal that T. gondii actively suppresses the production of type I interferon. These findings reveal that eukaryotic pathogens can be potent inducers of type I interferon and that related parasite species interact with this pathway in distinct ways.

Figure 1. Differential induction of innate immune signaling by genetically related parasite species.

a) Microarray-based expression profiling of human foreskin fibroblasts (HFF) infected with Neospora caninum (NcLiv isolate) or Toxoplasma (GT1, Prugniaud or VEG strains). Heat map shows hierarchical clustering analysis of 822 genes differentially regulated relative to uninfected cells by ≥2-fold (FDR≤5%), in any of these experiments. Each row in heatmap represents average of duplicate (NcLiv and GT1) or triplicate arrays (VEG, PRU, uninf). Color pattern on heatmaps represents column Z-score. b) A cluster of 66 genes (Fig. 1a, asterisk) induced by Neospora but not any strain of Toxoplasma, including several well-known type I interferon response genes (arrows). c) Gene Ontology (GO) enrichment analysis of 66 Neospora-induced genes. Bar graph shows fold enrichment for top five GO Biological Process terms enriched at P≤0.05 and represented by ≥5 genes. Number of genes and GO term name is shown at the right of each bar. d) Fluorescence micrographs of uninfected HFF cells, or cells infected with Neospora or Toxoplasma, then challenged with GFP-tagged vesicular stomatitis virus. Representative images are shown. Experiment was repeated three times with similar results.

Figure 2. Active invasion is not required for innate recognition of parasites.

QPCR analysis of the expression of the antiviral gene Mx1 following (a) infection of HFF cells or (b) treatment of cells with heat-killed strains of Toxoplasma (open bars) or Neospora (shaded bars). (c) confocal fluorescence microscopy of HFF cells treated with live (top row) or heat killed (bottom row) Toxoplasma-mCherry. Lysosomes are stained with LysoTracker dye. Representative images are shown. Error bars indicate standard deviations for three biological replicates; * = P≤0.01. Experiments were repeated three times with similar results.

Figure 3. Toxoplasma actively suppresses anti-viral host responses via a soluble factor.

(a) co-infection assay in HFF cells showing Mx1 gene expression in Toxoplasma infected cultures (Tg), cultures inoculated with heat-killed Neospora (HK Nc; striped bar), or infected with Toxoplasma for one hr before challenge with heat-killed Neospora (black bar). (b) QPCR analysis of IRF7 (open bars) and HERC5 (striped bars) expression in HFF cells pre-treated with fresh media or conditioned media from T. gondii infected cells (1° treatment) prior to infection with N. caninum (2° treatment). c) HFF cells were pretreated with supernatants from experiment in panel a, then infected with VSV-GFP. Representative fluorescence micrographs are shown. Error bars indicate standard deviations for three biological replicates; * = P≤0.01. Experiment was repeated two times with similar results.

Figure 4. Macrophage transcriptional responses to Neospora infection are Ifnar1-dependent.

Microarray-based expression profiling of Neospora (Nc) or Toxoplasma (Tg) infected wild-type or Ifnar1-/- bone marrow-derived mouse macrophages. a) heat map shows hierarchical clustering analysis of 833 genes differentially regulated by either Nc or Tg on either cell type relative to uninfected cells by ≥2-fold (FDR≤5%). Two clusters of co-regulated genes are indicated. Each column in heatmap represents an average of duplicate arrays, and color pattern represents row Z-score. b) Venn diagrams showing number of differentially regulated genes for each condition. c and e) GO enrichment results for cluster 1 and 2, respectively. d and f) heatmaps showing top ten most strongly induced genes in cluster 1 and 2, respectively. Gene symbol and fold change of infected WT relative to uninfected WT control are shown for each gene.

Figure 5. The Type I interferon response to Neospora infection is dependent on Tlr3 and Trif.

QPCR analysis of Mx1 expression in Neospora infected bone marrow macrophages derived from wild-type (WT), Myd88^−/−, Myd88^−/−/Trif^−/−, Tlr2^−/−/Tlr4^−/− and Tlr3^−/− mice. Error bars indicate standard deviations for three biological replicates; * = P≤0.01. Experiment was repeated three times with similar results.

Figure 6. Neospora RNA elicits a TLR3-dependent type I interferon response.

a) QPCR analysis of Irf7 gene expression in wild-type mouse bone marrow-derived macrophages pretreated for 1 hr with either DMSO (control) or 100 nM bafilomycin A1 prior to 24 hr infection with live (Nc) or heat-killed Neospora (HK Nc). b) Irf7 expression in Myd88^−/− macrophages incubated with 1 µg/ml of either poly(I:C) or total RNA from HFF cells (host), or Neospora parasites, either alone (white) or in complex with DOTAP transfection reagent (striped), to enhance targeting of RNA to endosomes. c) Irf7 expression in Myd88^−/− macrophages incubated with 1 µg/ml of either Toxoplasma or Neospora RNA with DOTAP in cells pretreated with either DMSO or a small molecule inhibitor of TLR3/dsRNA complex formation. Error bars indicate standard deviations for three biological replicates; * = P≤0.01. Experiments were repeated two or three times with similar results.