With the exception of Irgc ([10] and Rohde in preparation), all mouse IRG proteins are probably involved in a cell-autonomous resistance mechanism against intracellular pathogens. Irga6 contributes to interferon-induced immunity against Toxoplasma gondii since expression of a dominant negative mutant of the protein inhibits interferon-induced resistance to the parasite [11]. Furthermore, two independent mouse strains with disruption or loss of the Irga6 gene show higher susceptibility to infection with the avirulent ME49 strain of T. gondii (unpublished results). We show here that endogenous Irga6, constitutively expressed in hepatocytes, is able to accumulate on the parasitophorous vacuole membrane of infecting ME49 strain T. gondii, but is ineffective in initiating the vacuolar rupture typical of complex IFNc-induced responses in which multiple IRG proteins are expressed at high levels [11–14]. Results Constitutive expression of Irga6 in liver When Irga6 was first described [4] we showed by Northern blot that the transcript was massively induced in the liver 24 h after infection with Listeria monocytogenes. The autoradiographic exposure of that Northern blot was optimized to obtain the best resolution between transcript levels from uninfected and infected liver, and was not normalized to any other tissue or cell type. The image conveyed the impression that Irga6 is transcribed at very low levels, if at all, in the uninfected liver compared with the infected liver. This is what we believed, and the conclusion was fully compatible with parallel data obtained from tissue culture cells stimulated with IFNc for 24 h or left unstimulated. Again, the autoradiographic image was optimized to obtain clear resolution between IFNc-induced and uninduced transcript levels and not normalized to the liver data. We have now revisited that experiment and present a new Northern blot of the same liver RNA pool that was used in the previous study, compared with RNA from IFNc-induced and uninduced mouse fibroblasts, in this case normalized to GAPDH signal intensity in the two assay systems. The Northern blot and its quantitation are shown in Fig. 1A. The new analysis shows that Irga6 is indeed induced in fibroblasts from a very low expression level, but also shows that the expression level in normal liver from uninfected mice is already even higher than the level reached in IFNc-induced fibroblasts. This already high level was further enhanced another 10-fold 24 h after L. monocytogenes infection. The elevated expression in liver following L. monocytogenes infection is due to stimulation by IFNc, since no such effect followed L. monocytogenes infection of IFNc-receptor-deficient mice. In addition, however, the high basal expression of Irga6 in liver was not significantly affected by loss of the IFNc receptor, providing a strong indication that basal Irga6 transcription in the liver is not dependent on intrinsic IFNc stimulation of whatever cause. The high level of constitutive transcription of Irga6 in the liver was also found to be associated with a high level of Irga6 protein expression in this organ, as shown by Western blot in Fig. 1B. Protein levels relative to calnexin were equal to or higher than those in IFNc-induced L929 fibroblasts. Constitutive Irga6 protein expression in the liver was also independent of immunological maturity: levels in 123 day old mice were no different from levels in mature mice. By in situ hybridization (Fig. 1C) and immunohistology (Fig. 1D) the expression of both RNA and protein of Irga6 was confirmed in liver sections, controlled in the case of in situ hybridization by the sense probe and in immunohistology on liver sections from an Irga6-deficient mouse derived by gene targeting [15]. The results of both assays Expression of Irga6 in Liver confirmed the expression of Irga6 in the liver and both assays also clearly demonstrated that the protein is universally expressed in hepatic parenchymal cells. Constitutive expression of Irga6 in other organs By Western blot (Fig. 2A) Irga6 is also expressed in untreated mice at widely different levels in other organs, notably heart, skin, spleen and thymus, although nowhere as strongly as in the liver. By immunohistology (Fig. 2B), Irga6 expression was found in some cardiac myocytes but not others. In spleen, Irga6 was expressed exclusively in the red pulp, and in the thymus in the medulla. Surprisingly, an earlier immunohistological analysis of the spleen using a monoclonal antibody (5D9) directed against Irga6 did not report the expression of Irga6 in the red pulp [6]. Occasional patches of Irga6 expression were found in kidney tubular epithelium. No defined compartment expressing Irga6 could be identified in skin despite significant expression in this organ (not shown and Fig. 2A). We considered the possibility that at least some of this apparently constitutive expression of Irga6 might be due to local expression of IFNs, although our observations (Fig. 1) showed this was not the case for the liver. Western blots on organ lysates from single IFNc-receptor-deficient (IFNGR), single IFN- type I receptor deficient (IFNAR) and double-deficient (IFNAGR) mice were compared with wild type, normalized to calnexin (Fig. 3). As expected, very high constitutive Irga6 expression in the liver was unaffected by single or double IFN receptor deficiency and high expression also persisted in the heart. In skin, spleen, intestine and thymus, however, loss of the IFN receptors markedly reduced the detectable Irga6 signal, with the greatest reduction being in the double receptor deficient mouse. We conclude that Irga6 expression in normal, not intentionally infected, mice arises from two causes, genuinely constitutive expression in liver parenchymal cells and probably also cardiac myocytes, and expression induced by local production of type I and Type II IFNs in skin, spleen and thymus in unidentified cell types. Genomic and transcriptional structure of Irga6: two alternative 59 untranslated exons The exceptionally high, apparently IFN-independent, expres- sion of Irga6 in liver parenchymal cells suggested that Irga6 transcription in this organ must be under completely different regulation from that typical of fibroblasts and other tissues where interferons appeared to dominate control of Irga6 expression. The genomic organization of the Irga6 gene was reported earlier [10]. In that study we noted that transcription of Irga6 was alternatively initiated upstream of two distinct and widely separated 59- untranslated exons, which we here name exon 1A and exon 1B respectively for the upstream and downstream exons. These observations suggested that a closer analysis of the 59 region of the Irga6 gene could be of interest in the present context. Alignments of ESTs from the public databases allowed us to identify Irga6 exons 1A and 1B and infer probable start-points for transcription (Figure S1). The sequences of exons 1A and 1B diverge considerably in the 59 region but converge towards near identity close to the splice donor site (Figure S1). Both exons normally splice to the same splice acceptor upstream of the long coding exon 2, 20 bp upstream of the initiator codon (Figure S1). Further short untranslated regions can be found very occasionally introduced between Exon 1A or Exon 1B and the ORF-encoding exon 2 (unpublished results). These unique events are too rare to be dignified by the term ‘‘alternative splicing’’ and too inconsistent to earn the regions concerned the titles of ‘‘exons’’. A peculiar case recently reported [16] was an Irga6 cDNA carrying both exon 1A PLoS ONE | www.plosone.org 2 August 2009 | Volume 4 | Issue 8 | e6787