Expression of Irga6 in Liver Figure 7. Constitutive and IFNc stimulated Irga6 proteins accumulate around T. gondii PVs in mouse primary hepatocytes. A. Immunofluorescence analysis of Irga6 protein accumulation at T. gondii vacuoles in murine primary hepatocytes. C57BL/6 murine primary hepatocytes were freshly isolated and either stimulated (+IFNc) for 12 h with 200 U/ml IFNc or left untreated and cultured for 3 hours (-IFN-c). The cells were inoculated with T. gondii (strain ME49) at a MOI of 3 for 2 h, fixed and immunostained. Double staining for Irga6 with mAb 10E7 (green) and GRA7 with anti-GRA7 mAb (red) was performed. Nuclei were counterstained with DAPI (blue). White arrowhead indicates a disrupting vacuole. B. Quantification of Irga6 positive T. gondii PVs in the experimental setup described in (A). Percentages of Irga6 positive PVs per total intracellular T. gondii vacuoles are given. Intracellular parasites were identified by the distinct GRA7 pattern. The means of two or three independent experiments doi:10.1371/journal.pone.0006787.g007 gondii vacuoles, at 10%, was much lower than previously observed in IFNc-induced fibroblasts and other cells, which is normally in the range from 50% to 70% [11]. In unstimulated primary hepatocytes Irga6 was the only IRG protein that was detected on the T. gondii PVM. Irgb6 and Irgd could not be detected at the PVM despite their significant basal expression levels (data not shown). In IFNc-stimulated primary hepatocytes, however, the normal pattern of PVM loading with these IRG proteins was observed, and the typical quantitative hierarchy of loading (Irgb6.Irga6.Irgd.Irgm3) described in detail elsewhere (Kha- minets et al, in preparation), was restored (not shown). In IFNc- treated cells, T. gondii PVs coated with IRG proteins became disrupted during the first few hours after infection [11,14]. No disruption of Irga6-coated T. gondii PVs was observed in primary hepatocytes without IFNc treatment, but disrupted vacuoles were readily seen in infected IFNc-induced primary hepatocytes (Fig. 7A). IFNg-induced primary hepatocytes were also able to control T. gondii replication (Fig. 7C). Discussion We have shown that Irga6, an IFN-inducible member of the immunity-related IRG protein family in the C57BL/6 mouse, is also expressed constitutively and strongly in normal mouse liver through use of a dedicated, liver-specific promoter. IFN-inducible and constitutive expression are initiated from separate promoters. Each promoter is upstream of a dedicated 59 exon that splices into the single long protein-coding exon 2 just upstream of the translation start. Thus the proteins derived from the two promoters are identical. Inducibility by interferons is a strong indication for a role in induced resistance to infection [1], and the strong IFN inducibility of Irga6 and all the other mouse IRG PLoS ONE | www.plosone.org 9 August 2009 | Volume 4 | Issue 8 | e6787