Innate immune responses in NF-kappaB-repressing factor-deficient mice

Abstract

NF-kappaB-repressing factor (NRF) is a transcriptional silencer protein that specifically counteracts the basal activity of several NF-kappaB-dependent promoters by direct binding to specific neighboring DNA sequences. In cell culture experiments, the reduction of NRF mRNA leads to a derepression of beta interferon, interleukin-8, and inducible nitric oxide synthase transcription. The X chromosome-located single-copy NRF gene is ubiquitously expressed and encodes a protein of 690 amino acids. The N-terminal part contains a nuclear localization signal, the DNA-binding domain, and the NF-kappaB-repressing domain, while the C-terminal part is responsible for double-stranded RNA binding and nucleolar localization. To study the function of NRF in a systemic context, transgenic mice lacking the NRF gene were created. Against predictions from in vitro experiments, mice with a deletion of the NRF gene are viable and have a phenotype that is indistinguishable from wild-type mice, even after challenge with different pathogens. The data hint towards an unexpected functional redundancy of NRF.

FIG. 1.

Chromosomal localization of murine NRF by FISH analysis. The ES cell line 14-1 was hybridized with BAC DNA harboring the NRF gene. The NRF gene is labeled in green and marked by arrows. A. X chromosomal DNA is counterstained in red. B. Chromosomes of a different spread were counterstained with 4′,6′-diamidino-2-phenylindole.

FIG.2.

Targeted disruption of the mouse NRF gene. A. Schematic structures of the murine NRF gene, the targeting vector, and the genomic modifications in transgenic mice are shown. Homologous DNA is boxed. The orientation and locations of primers (arrows) and a probe fragment for genotyping (bars) are indicated. B. Southern blot analysis of DNA from transgenic mice. Genomic DNA isolated from tail biopsy samples was digested with SpeI and hybridized with the second exon (left panel) and 5′-end (right panel) specific probes as indicated. hom, homozygous; het, heterozygous mice. C. Schematic structure of WT, NRF-flox, and NRF-KO DNA forms and the expected mRNA. Note that RNA from NRF-KO is not detectable. D. Northern blot analysis. Poly(A)-RNA was isolated from WT, NRF-KO, and NRF-flox cells. The RNA blot was hybridized with a radioactively labeled NRF second exon-specific probe. For each sample, 2 μg RNA was loaded.

FIG. 2—

Continued.

FIG. 3.

Expression analysis of the NRF protein. A. Western blot analysis. Nuclear extracts were prepared from WT and NRF-KO primary cells as well as from the mouse C243 cell line either empty (-) or overexpressing the myc-tagged NRF protein. For primary cells 30 μg was loaded per lane, and for C243 cells 15 μg of protein was loaded per lane. The protein blot was incubated with anti-NRF antibody and treated as described in Materials and Methods. B. Immunofluorescence of the WT and NRF-KO cells using antibody directed against NRF. The secondary antibody was fluorescence labeled. Visualization was performed by confocal laser scanning microscopy (see Materials and Methods).

FIG. 4.

Histological analysis of tissue sections from WT and NRF-KO mice stained with hematoxylin and eosin. (A) Joint; (B) skin; (C) lymph nodes.

FIG. 5.

Composition of the peripheral blood cells. Lymphocytes obtained from WT, NRF-flox, and NRF-KO mice were analyzed by fluorescence-activated cell sorter. The percentage of the indicated population is indicated.

FIG. 6.

Expression of IFN-β and iNOS. A. IFN-β production was determined in nontreated and Newcastle disease virus (NDV)-injected WT, NRF-flox, and NRF-KO primary fibroblasts as indicated. B. RNA isolated from cultured adult skin fibroblasts from WT, NRF-flox, and NRF-KO mice or WT mouse embryonic fibroblasts (MEFs) was isolated. mRNA was reverse transcribed into cDNA and subjected to PCR analysis using IFN-β-specific primers. As a positive control, RNA isolated from MEFs was used. These cells are known for their ability to produce a low level of IFN-β in the nonstimulated state (R. Zawatzky, personal communication). C. iNOS activity in peritoneal macrophages. Freshly isolated macrophages from WT and NRF-KO animals were stimulated with 10 mg/ml LPS. NO₂ was measured in the supernatants 24 and 72 h after LPS stimulation using Griess reagent.

FIG. 7.

Survival rate of NRF-KO female mice and their heterozygous littermates after injection with 1.5 × 10⁴ CFU of L. monocytogenes. The condition of the mice was determined by daily measurement of animal body weight and observation of mouse behavior.