RAIDD Mediates TLR3 and IRF7 Driven Type I Interferon Production.

BACKGROUND/AIMS: Viral infections represent a global health problem with the need for new viral therapies and better understanding of the immune response during infection. The most immediate and potent anti-viral defense mechanism is the production of type I interferon (IFN-I) which are activated rapidly following recognition of viral infection by host pathogen recognition receptors (PRR). The mechanisms of innate cellular signaling downstream of PRR activation remain to be fully understood. In the present study, we demonstrate that CASP2 and RIPK1 domain-containing adaptor with death domain (CRADD/RAIDD) is a critical component in type I IFN production. METHODS: The role of RAIDD during IFN-I production was investigated using western blot, shRNA mediated lentiviral knockdown, immunoprecipitation and IFN-I driven dual luciferase assay. RESULTS: Immunoprecipitation analysis revealed the molecular interaction of RAIDD with interferon regulatory factor 7 (IRF7) and its phosphorylating kinase IKKε. Using an IFN-4α driven dual luciferase analysis in RAIDD deficient cells, type I IFN activation by IKKε and IRF7 was dramatically reduced. Furthermore, deletion of either the caspase recruitment domain (CARD) or death domain (DD) of RAIDD inhibited IKKε and IRF7 mediated interferon-4α activation. CONCLUSION: We have identified that the adaptor molecule RAIDD coordinates IKKε and IRF7 interaction to ensure efficient expression of type I interferon.

Phototherapy Restores Deficient Type I IFN Production and Enhances Antitumor Responses in Mycosis Fungoides.

Transcriptional profiling demonstrated markedly reduced type I IFN gene expression in untreated mycosis fungoides (MF) skin lesions compared with that in healthy skin. Type I IFN expression in MF correlated with antigen-presenting cell-associated IRF5 before psoralen plus UVA therapy and epithelial ULBP2 after therapy, suggesting an enhancement of epithelial type I IFN. Immunostains confirmed reduced baseline type I IFN production in MF and increased levels after psoralen plus UVA treatment in responding patients. Effective tumor clearance was associated with increased type I IFN expression, enhanced recruitment of CD8+ T cells into skin lesions, and expression of genes associated with antigen-specific T-cell activation. IFNk, a keratinocyte-derived inducer of type I IFNs, was increased by psoralen plus UVA therapy and expression correlated with upregulation of other type I IFNs. In vitro, deletion of keratinocyte IFNk decreased baseline and UVA-induced expression of type I IFN and IFN response genes. In summary, we find a baseline deficit in type I IFN production in MF that is restored by psoralen plus UVA therapy and correlates with enhanced antitumor responses. This may explain why MF generally develops in sun-protected skin and suggests that drugs that increase epithelial type I IFNs, including topical MEK and EGFR inhibitors, may be effective therapies for MF.

Differential role for c-Rel and C/EBPbeta/delta in TLR-mediated induction of proinflammatory cytokines.

TLR stimulation triggers a signaling pathway via MyD88 and IL-1R-associated kinase 4 that is essential for proinflammatory cytokine induction. Although NF-kappaB has been shown to be one of the key transcriptional regulators of these cytokines, evidence suggests that other factors may also be important. In this study, we showed that MyD88-deficient macrophages have defective c-Rel activation, which has been linked to IL-12p40 induction, but not IL-6 or TNF-alpha. We also investigated other transcription factors and showed that C/EBPbeta and C/EBPdelta expression was limited in MyD88- or IL-1R-associated kinase 4-deficient macrophages treated with LPS. Importantly, the absence of both C/EBPbeta and C/EBPdelta resulted in the impaired induction of proinflammatory cytokines stimulated by several TLR ligands. Our results identify c-Rel and C/EBPbeta/delta as important transcription factors in a MyD88-dependent pathway that regulate the induction of proinflammatory cytokines.

DNA damage- and stress-induced apoptosis occurs independently of PIDD.

The p53-induced protein with a death domain, PIDD, was identified as a p53 target gene whose main role is to execute apoptosis in a p53-dependent manner. To investigate the physiological role of PIDD in apoptosis, we generated PIDD-deficient mice. Here, we report that, although PIDD expression is inducible upon DNA damage, PIDD-deficient mice undergo apoptosis normally not only in response to DNA damage, but also in response to various p53-independent stress signals and to death receptor (DR) engagement. This indicates that PIDD is not required for DNA damage-, stress-, and DR-induced apoptosis. Also, in the absence of PIDD, both caspase-2 processing and activation occur in response to DNA damage. Our findings demonstrate that PIDD does not play an essential role for all p53-mediated or p53-independent apoptotic pathways.

An image-based genetic assay identifies genes in T1D susceptibility loci controlling cellular antiviral immunity in mouse.

The pathogenesis of complex diseases, such as type 1 diabetes (T1D), derives from interactions between host genetics and environmental factors. Previous studies have suggested that viral infection plays a significant role in initiation of T1D in genetically predisposed individuals. T1D susceptibility loci may therefore be enriched in previously uncharacterized genes functioning in antiviral defense pathways. To identify genes involved in antiviral immunity, we performed an image-based high-throughput genetic screen using short hairpin RNAs (shRNAs) against 161 genes within T1D susceptibility loci. RAW 264.7 cells transduced with shRNAs were infected with GFP-expressing herpes simplex virus type 1 (HSV-1) and fluorescent microscopy was performed to assess the viral infectivity by fluorescence reporter activity. Of the 14 candidates identified with high confidence, two candidates were selected for further investigation, Il27 and Tagap. Administration of recombinant IL-27 during viral infection was found to act synergistically with interferon gamma (IFN-γ) to activate expression of type I IFNs and proinflammatory cytokines, and to enhance the activities of interferon regulatory factor 3 (IRF3). Consistent with a role in antiviral immunity, Tagap-deficient macrophages demonstrated increased viral replication, reduced expression of proinflammatory chemokines and cytokines, and decreased production of IFN-ß. Taken together, our unbiased loss-of-function genetic screen identifies genes that play a role in host antiviral immunity and delineates roles for IL-27 and Tagap in the production of antiviral cytokines.

Metalloproteinase axes increase beta-catenin signaling in primary mouse mammary epithelial cells lacking TIMP3.

Multiple cancers exhibit mutations in beta-catenin that lead to increased stability, altered localization or amplified activity. beta-catenin is situated at the junction between the cadherin-mediated cell adhesion and Wnt signaling pathways, and TIMP3 functions to alter beta-catenin signaling. Here we demonstrate that primary mouse embryonic fibroblasts (MEFs) and mammary epithelial cells (MECs) deficient in Timp3 have increased beta-catenin signaling. Functionally, the loss of TIMP3 exerted cell-type-specific effects, with Timp3(-/-) MEFs being more sensitive and Timp3(-/-) MECs more resistant to EGTA-induced cell detachment than the wild type. Timp3(-/-) MECs had higher dephosphorylated beta-catenin levels and increased beta-catenin transcriptional activity as measured by TCF/LEF-responsive reporter assays. Real-time PCR analysis of beta-catenin target genes in MEFs and MECs showed no alteration in Myc, decreased Ccnd1 (cyclin D1) and increased Mmp7 mRNA levels upon loss of TIMP3, with the latter occurring only in epithelial cells. Recombinant TIMP3 and synthetic metalloproteinase inhibitors reverted the increase in dephosphorylated beta-catenin, decrease in Ccnd1 gene expression and increase in Mmp7 gene expression. Physiologically, Timp3(-/-) mammary glands displayed accelerated mammary ductal elongation during pubertal morphogenesis. Gain-of-function studies using slow-release TIMP-containing pellets revealed distinct effects of individual TIMPs on ductal morphogenesis. Recombinant TIMP1, TIMP3 and TIMP4 inhibited ductal elongation whereas TIMP2 promoted this process.