Recently, an absolute IRF-3 dependency of the in vitro IFN-ab response of bone marrow derived macrophages to Ad has been reported [63] . In our study IRF-3 deficiency had no significant effect on the levels of IFN-ab induced in Ad-infected mice. In addition, in Ad-infected macrophages, the relatively low level of IFN-a4 mRNA and its negative regulation by the autocrine Figure 11 . Adenovirus triggered IFN-ab production is the mediator of LPS hypersensitivity. Wild-type and IFN-abR 2/2 mice (4-6 mice/ group) were infected i. p. with 1610 10 Ad3 particles or left uninfected. 16 h later the animals received 1 mg LPS i. p. or diluent only. TNF-a was determined in plasma 2 h after challenge (A). One representative experiment of three is shown. n.d.: not detectable. IRF-7 2/2 and TLR9 2/2 mice were infected i. p. with 2610 9 Ad5 GFP particles and injected 16 h later with 1 mg LPS i. p. TNF-a was determined in plasma 2 h later (B). IFN-ab signaling is required for Ad infection augmented LPS lethality. Wt and IFNabR 2/2 mice were infected with 1610 10 Ad5 GFP particles i. p. or left uninfected. 16 h later mice were injected with 3.5 mg LPS/gr. bw. The number of dead/total animals are shown at the indicated time after LPS challenge (C). Ad infection increases mCD14 expression on splenic macrophages in an IFN-ab signaling dependent manner. Wt and IFN-abR 2/2 mice were infected with 1610 10 Ad5 GFP particles i. p. or left uninfected. Expression of mCD14 was measured on the surface of F4/80 + splenic macrophages by FACS 16 h after infection (D). Ad infection increases acetylation of histone H4 in wt macrophages at the TNF promoter. Wt and IFNabR 2/2 BMMs were mock-infected or infected with 5400 Ad5 GFP particles/cell and levels of acetylation of histone H4 were measured at the TNF-a promoter with ChIP assays (E). Representative experiments of two are shown. doi:10.1371/journal.ppat.1000208.g011 feedback was different from our in vivo findings (strong induction and positive regulation by feedback). This is in agreement with the concept that in vivo macrophages make no significant contribution to the IFN-ab response to Ad. A likely explanation is that the induction of IFN-ab in macrophages requires the high numbers of virions used in vitro, and that such multiplicities were never reached in vivo. Interestingly however, in contrast to IFN-ab induction, the induction of IL-6 proceeded in the spleen of infected mice in both DC and non-DC populations. In accordance, the depletion of IFN-ab-producing mDCs in mice prior to infection lowered, but did not entirely prevent the IL-6 response. It is possible that the pathways leading to the induction of IFN-ab and IL-6 by Ad are different, at least in different cell types. Alternatively, in vivo, part of the IL-6 formed is induced indirectly via secondary mechanisms. In this context it is interesting that blockade of the p38 MAPK in mDCs in vitro had no effect on Ad-induced IFN-ab production, but partially inhibited the production of IL-6. Our data showing that IFNabR 2/2 mice exhibit strongly impaired IL-6 responses to Ad is in agreement with a previous report [10] and shows that IFN-ab is a positive regulator of IL-6 production. Moreover, our data indicate that this effect could be explained at least in part by the positive regulatory effect of IFN-ab on Ad-induced IL-6 transcription and is achieved by the alteration of the chromatin structure at the IL-6 promoter.