Temporal and spatial resolution of type I and III interferon responses in vivo.

Although the action of interferons (IFNs) has been extensively studied in vitro, limited information is available on the spatial and temporal activation pattern of IFN-induced genes in vivo. We created BAC transgenic mice expressing firefly luciferase under transcriptional control of the Mx2 gene promoter. Expression of the reporter with regard to onset and kinetics of induction parallels that of Mx2 and is thus a hallmark for the host response. Substantial constitutive expression of the reporter gene was observed in the liver and most other tissues of transgenic mice, whereas this expression was strongly reduced in animals lacking functional type I IFN receptors. As expected, the reporter gene was induced not only in response to type I (alpha and beta) and type III (lambda) IFNs but also in response to a variety of IFN inducers such as double-stranded RNA, lipopolysaccharide (LPS), and viruses. In vivo IFN subtypes show clear differences with respect to their kinetics of action and to their spatial activation pattern: while the type I IFN response was strong in liver, spleen, and kidney, type III IFN reactivity was most prominent in organs with mucosal surfaces. Infection of reporter mice with virus strains that differ in their pathogenicity shows that the IFN response is significantly altered in the strength of IFN action at sites which are not primarily infected as well as by the onset and duration of gene induction.

Tumor suppression by IFN regulatory factor-1 is mediated by transcriptional down-regulation of cyclin D1.

IFNs have been ascribed to mediate antitumor effects. IFN regulatory factor-1 (IRF-1) is a major target gene of IFNs. It inhibits cell proliferation and oncogenic transformation. Here, we show that 60% of all mRNAs deregulated by oncogenic transformation mediated by c-myc and H-ras are reverted to the expression levels of nontransformed cells by IRF-1. These include cell cycle-regulating genes. An indirect target is cyclin D1. Activation of IRF-1 decreased cyclin D1 expression and cyclin-dependent kinase 4 kinase activity concomitant with change in the levels of hyperphosphorylated retinoblastoma protein. These effects are mediated by inhibition of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway and a transcriptional repression of cyclin D1. As shown by in vitro assays and tumor growth in nude mice, IRF-1-mediated effects on cell cycle progression were found to be overridden by ectopic expression of cyclin D1. Conversely, decrease of cyclin D1 by RNA interference experiments prevents transformation and tumor growth. The data show that cyclin D1 is a key target for IRF-1-mediated tumor-suppressive effects.

Novel nonviral bioassays for mouse type I and type III interferon.

We used embryo fibroblasts from Mx2-Luc transgenic mice that express Firefly luciferase under control of the interferon (IFN)-regulated mouse Mx2 promoter to develop simple nonviral bioassays for type I and type III IFN. Since type III IFN is acid-labile, Mx2-Luc fibroblasts detected the presence of type I IFN in acid-treated biological samples with high sensitivity and selectivity. For selective detection of type III IFN, we employed embryo fibroblasts from Mx2-Luc mutant mice that lack functional receptors for type I IFN. The sensitivity of this latter assay remained comparatively low, presumably because type III IFN receptors are not abundantly present on fibroblasts. The main advantages of our novel IFN assays are that they are easy to perform, yield fast results, and can be used in laboratories that are not licensed for work with infectious agents. Further, the type I IFN assay has superior sensitivity than commercially available enzyme-linked immunosorbent assay systems.