proteins except Irgc seems to correlate with their role in countering infections by exogenous intracellular parasites [3]. The high constitutive expression of Irga6 in liver stands in contrast to that principle and presents something of a paradox. Certainly, constitutive expression of an early-acting resistance mechanism in barrier tissues may be considered preadaptive, and it was notable that a significant level of Irga6 expression was observed in the skin, although it could not be localised histologically to a specific cell layer and was in any case substantially lower than the liver-specific expression. Furthermore the apparent partial dependence of skin- specific Irga6 expression on IFN action implied by the reduced expression in IFN receptor-deficient mice may suggest that Irga6 expression in the skin is not primarily constitutive but rather due to continuous low-level stimulation of immunity in this barrier site. Low, IFN-dependent expression of Irga6 in lymphoid organs such as spleen and thymus presumably reflects low-level immune- related activity. Histologically, expression in red-pulp of spleen and in medulla of thymus suggests that macrophages may be the main sites of elevated Irga6 expression though this has not been investigated directly. However these considerations do not help to explain the genuinely constitutive expression of Irga6 at very high levels in liver parenchymal cells. Here, expression relative to the calnexin control was higher than in IFN-induced fibroblasts, both at the mRNA and protein levels, and was not detectably reduced by loss of either or both the type I and type II IFN receptors. The near exclusive use of the hepatocyte-specific 1B promoter for IFN- independent expression further confirms that this expression is indeed truly constitutive. To rationalize constitutive Irga6 expression in the liver, it may be argued that the liver is in some sense a barrier organ for pathogens such as L. monocytogenes and Plasmodium that above all favour liver parenchymal cells for their initial proliferative episode. Liver is an early site of infection in peroral T. gondii infection after dissemination from the wall of the intestine ([23] and Oliver Liesenfeld, personal communication) presumably via the hepatic portal system. However expression of Irga6 at high levels in hepatocytes in the absence of IFNc, as shown here, raises a further complex issue. We have shown elsewhere that both the resting localisation of Irga6 in cells and its ability to accumulate normally on the T. gondii parasitophorous vacuole is dependent on co- expression of multiple IFN-inducible members of the IRG protein family ([24]; Khaminets et al, in prep). At first sight, Irga6 expressed in resting hepatocytes appears to break this rule. The protein is accurately localised on the endoplasmic reticulum, and is able to accumulate on the T. gondii PVM. However the number of vacuoles showing detectable Irga6 accumulation is abnormally low, a number that is substantially restored towards expected levels by prior induction with IFNc. Furthermore, in the absence of IFNc induction, no disruption of the T. gondii vacuole was detected, suggesting that a high level of expression of Irga6 alone is not sufficient to complete the resistance mechanism. We considered the possibility that other IRG proteins may also be expressed at measurable levels in hepatocytes, even if not at the level of Irga6, thus enabling localisation of Irga6 to the ER and partially enabling access to the T. gondii PVM. Irgb6, Irgm1 and Irgd were all expressed at detectable though still relatively low levels. The striking exception is Irgm3, which was essentially undetectable in unstimulated primary hepatocytes but was strongly induced by IFNg. Interestingly, Irgm3 is expressed constitutively in whole liver, but is associated exclusively with scattered, much smaller cells probably associated with the sinusoids. These cells are lost during the hepatocyte purification. We reported inefficient Irga6 loading of the T. gondii PVM earlier Expression of Irga6 in Liver [24] in cells in which Irga6 alone of the effector IRG (GKS subfamily) proteins was co-expressed with the three regulator proteins, Irgm1, Irgm2 and Irgm3 [24]. We further showed that expression of all 3 Irgm proteins was required for loading. We have since shown that quantitatively normal PVM loading of Irga6 in such reconstruction experiments can be achieved by addition of normal levels of Irgb6 and/or Irgd (Khaminets et al, in prep). The anomalies of IRG loading in unstimulated hepatocytes may be therefore be attributable to the apparently complete absence of Irgm3 and the relatively low levels of other IRG proteins. Thus hepatocytes constitutively express an ‘‘unbalanced’’ collection of IRG proteins that supports only inefficient loading of the T. gondii PVM. Evidently, the complement of highly expressed IRG proteins would normalize in hepatocytes as soon as the IFNc levels begin to rise during infection, and we show that IFNg- induced primary hepatocytes are able to control T. gondii replication (Fig S3). Whether early expression of Irga6 alone accelerates the development of full cell-autonomous resistance is unclear. An adaptive explanation for the high and dedicated expression of Irga6 in hepatic parenchymal cells is therefore not yet available. An unexpected precedent exists for the complex regulation of expression of Irga6. The human and mouse RNA-specific adenosine deaminase, ADAR1, which is a classical Type I interferon-inducible gene associated with the dsRNA recognition and degradation pathway, is also transcribed from two distinct promoters, named exon 1A and exon 1B, each subtending its own untranslated 59 exon [25–27]. Like Irga6, one of the promoters is interferon-inducible while the other is expressed constitutively, in this case in embryos and in the adult brain. Despite the striking similarities there are also differences between the two situations. The two Irga6 promoters regulate the synthesis of identical protein products. The two ADAR promoters, on the other hand, control the expression of distinct products since the 1A exon behind the interferon-inducible promoter also contains an initiator methio- nine, while the constitutively expressed 1B exon does not and protein translation is initiated from a second methionine codon in exon 2, generating a shorter protein. Furthermore, there is evidence that the RNA editing activity of ADAR may be exercised on host cell mRNA, and that the constitutive form of ADAR1 is necessary for normal embryonic development. In contrast, mice carrying a genomic disruption in the Irga6 protein coding region show no developmental abnormalities and are perfectly viable and fertile, while showing a significant susceptibility to infection with an avirulent strain of T. gondii (unpublished results and Liesenfeld, O, personal communication). It is not excluded that the constitutive expression of Irga6 in the liver may contribute to a pathogen resistance function, but our data show that the high constitutive expression of Irga6 leads only to limited loading onto the T. gondii parasitophorous vacuole membrane and is unable to lead to disruption of the vacuolar membrane. For the time being, therefore, the functional meaning of constitutive expression of Irga6 in mouse liver remains to be identified. Materials and Methods Mice Specific pathogen-free C57BL/6 and CB20 mice were obtained from the mouse facility in the Institute for Genetics, University of Cologne. In Irga6-deficient (Irga62/2) mice the entire coding exon (exon 2) of the Irga6 gene was removed from the C57BL/6 genome by Cre-mediated recombination ([15] and unpublished results). Type I, Type II and double IFN receptor deficient mice (IFNAR [28], IFNGR ([29], IFNAGR [30]) on the 129SV PLoS ONE | www.plosone.org 10 August 2009 | Volume 4 | Issue 8 | e6787