Induction of interferon-stimulated genes by IRF3 promotes replication of Toxoplasma gondii.

Innate immunity is the first line of defense against microbial insult. The transcription factor, IRF3, is needed by mammalian cells to mount innate immune responses against many microbes, especially viruses. IRF3 remains inactive in the cytoplasm of uninfected cells; upon virus infection, it gets phosphorylated and then translocates to the nucleus, where it binds to the promoters of antiviral genes and induces their expression. Such genes include type I interferons (IFNs) as well as Interferon Stimulated Genes (ISGs). IRF3-/- cells support enhanced replication of many viruses and therefore, the corresponding mice are highly susceptible to viral pathogenesis. Here, we provide evidence for an unexpected pro-microbial role of IRF3: the replication of the protozoan parasite, Toxoplasma gondii, was significantly impaired in IRF3-/- cells. In exploring whether the transcriptional activity of IRF3 was important for its pro-parasitic function, we found that ISGs induced by parasite-activated IRF3 were indeed essential, whereas type I interferons were not important. To delineate the signaling pathway that activates IRF3 in response to parasite infection, we used genetically modified human and mouse cells. The pro-parasitic signaling pathway, which we termed PISA (Parasite-IRF3 Signaling Activation), activated IRF3 without any involvement of the Toll-like receptor or RIG-I-like receptor pathways, thereby ruling out a role of parasite-derived RNA species in activating PISA. Instead, PISA needed the presence of cGAS, STING, TBK1 and IRF3, indicating the necessity of DNA-triggered signaling. To evaluate the physiological significance of our in vitro findings, IRF3-/- mice were challenged with parasite infection and their morbidity and mortality were measured. Unlike WT mice, the IRF3-/- mice did not support replication of the parasite and were resistant to pathogenesis caused by it. Our results revealed a new paradigm in which the antiviral host factor, IRF3, plays a cell-intrinsic pro-parasitic role

IRF3 facilitates intracellular <i>T</i>. <i>gondii</i> replication regardless of parasite virulence type.

<p><b>A,</b> Stunted parasite replication in IRF3 -/- cells. Isogenic wild type and IRF3 -/- MEF cells were infected by <i>T</i>. <i>gondii</i> RH at an m.o.i. of 0.2, and at the indicated times (4, 18, 24 h) stained for parasites (green), cytoplasmic actin (red) and nucleus (DAPI, blue) and visualized by confocal microscopy. For each sample, several individual PVs are shown; number of parasites in 12–14 PVs was counted and the mean ± SD values are shown. <b>B,</b> Flow cytometry measurement of parasite growth. DCs isolated from wild type and IRF3 -/- mice were infected with <i>T</i>. <i>gondii</i> RH at 0.3 m.o.i., and cells were permeabilized and dual-stained for CD11c and parasitic SAG1 at 18 h post-infection, with uninfected wild type cells as control. Gating was set to count infected cells with 2 or more parasites per PV, as marked by contour plot (left), and the percentage of such cells in the total population is shown. <b>C,</b> IRF3 is important for optimal growth of diverse <i>T</i>. <i>gondii</i> strains. The indicated <i>T</i>. <i>gondii</i> strains, represented the three major types: GT1 (type I), ME49 (type II), and VEG (type III). Infection was performed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004779#ppat.1004779.g001" target="_blank">Fig. 1A</a>, and total DNA was isolated at the indicated times. The amount of <i>T</i>. <i>gondii</i> DNA was quantified by qPCR of the genomic DNA as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004779#ppat.1004779.g001" target="_blank">Fig. 1</a>.</p

Our working model for PISA.

<p>In this model, a parasitic PAMP (possibly DNA) is sensed by the PRR, cGAS; the CDN product of cGAS then activates the STING-TBK1 kinase complex, which phosphorylates IRF3. The phospho-IRF3 translocates into the nucleus and leads to the induction of specific, parasite-friendly host gene(s), which facilitate <i>T</i>. <i>gondii</i> replication, thereby causing pathology and death.</p

Requirement of IRF3-induced host genes for <i>T</i>. <i>gondii</i> replication.

<p><b>A,</b> Infection and immunoblotting was performed with HDAC6 KO and wild type MEF cells. <b>B</b>, MEF cells (wild type or IFNAR KO) were infected with <i>T</i>. <i>gondii</i> and the indicated proteins were measured by immunoblotting. <b>C</b>, <i>T</i>. <i>gondii</i> growth measured in IRF3 KO MEF cells; where indicated, recombinant murine IFN-β (2,000 U/ml, R&D Systems) was added, and parasite was added 6 h later. Immunoblotting was performed as above. Where shown, parasite growth was also quantified by qPCR of genomic DNA, and ISG56 (Ifit1) mRNA was quantified by qRT-PCR as described before [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004779#ppat.1004779.ref066" target="_blank">66</a>].</p

Essential role of a TLR3/4-independent cGAS-STING signaling axis in PISA and optimal <i>T</i>. <i>gondii</i> replication.

<p>Parasite infection and growth analyses were performed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004779#ppat.1004779.g001" target="_blank">Fig. 1A</a>. <b>A,</b> MEFs (WT, RIG-I-/-, TLR3-/-) and primary macrophages (MyD88-/-, TLR4-/-) were used for comparing PISA (<i>Tg</i>SAG1 expression or IRF3 phosphorylation) upon <i>T</i>. <i>gondii</i> infection. <b>B/C,</b> STING-/- MEF cells (B) or human 293T cells (C) were transiently transfected with wild type or mutant (mt) FLAG-cGAS and/or HA-STING plasmids, and infected as shown. <b>D,</b> Parasite growth in various conditions used in the other panels, quantified by qPCR; error bars are from three data sets.</p