A Quantitative RNAi Screen for JNK Modifiers Identifies Pvr as a Novel Regulator of Drosophila Immune Signaling

Drosophila melanogaster responds to gram-negative bacterial challenges through the IMD pathway, a signal transduction cassette that is driven by the coordinated activities of JNK, NF-κB and caspase modules. While many modifiers of NF-κB activity were identified in cell culture and in vivo assays, the regulatory apparatus that determines JNK inputs into the IMD pathway is relatively unexplored. In this manuscript, we present the first quantitative screen of the entire genome of Drosophila for novel regulators of JNK activity in the IMD pathway. We identified a large number of gene products that negatively or positively impact on JNK activation in the IMD pathway. In particular, we identified the Pvr receptor tyrosine kinase as a potent inhibitor of JNK activation. In a series of in vivo and cell culture assays, we demonstrated that activation of the IMD pathway drives JNK-dependent expression of the Pvr ligands, Pvf2 and Pvf3, which in turn act through the Pvr/ERK MAP kinase pathway to attenuate the JNK and NF-κB arms of the IMD pathway. Our data illuminate a poorly understood arm of a critical and evolutionarily conserved innate immune response. Furthermore, given the pleiotropic involvement of JNK in eukaryotic cell biology, we believe that many of the novel regulators identified in this screen are of interest beyond immune signaling

Co-Regulation of NF-κB and Inflammasome-Mediated Inflammatory Responses by Myxoma Virus Pyrin Domain-Containing Protein M013

NF-κB and inflammasomes both play central roles in orchestrating anti-pathogen responses by rapidly inducing a variety of early-response cytokines and chemokines following infection. Myxoma virus (MYXV), a pathogenic poxvirus of rabbits, encodes a member of the cellular pyrin domain (PYD) superfamily, called M013. The viral M013 protein was previously shown to bind host ASC-1 protein and inhibit the cellular inflammasome complex that regulates the activation and secretion of caspase 1-regulated cytokines such as IL-1β and IL-18. Here, we report that human THP-1 monocytic cells infected with a MYXV construct deleted for the M013L gene (vMyxM013-KO), in stark contrast to the parental MYXV, rapidly induce high levels of secreted pro-inflammatory cytokines like TNF, IL-6, and MCP-1, all of which are regulated by NF-κB. The induction of these NF-κB regulated cytokines following infection with vMyxM013-KO was also confirmed in vivo using THP-1 derived xenografts in NOD-SCID mice. vMyxM013-KO virus infection specifically induced the rapid phosphorylation of IKK and degradation of IκBα, which was followed by nuclear translocation of NF-κB/p65. Even in the absence of virus infection, transiently expressed M013 protein alone inhibited cellular NF-κB-mediated reporter gene expression and nuclear translocation of NF-κB/p65. Using protein/protein interaction analysis, we show that M013 protein also binds directly with cellular NF-κB1, suggesting a direct physical and functional linkage between NF-κB1 and ASC-1. We further demonstrate that inhibition of the inflammasome with a caspase-1 inhibitor did not prevent the induction of NF-κB regulated cytokines following infection with vMyxM013-KO virus, but did block the activation of IL-1β. Thus, the poxviral M013 inhibitor exerts a dual immuno-subversive role in the simultaneous co-regulation of both the cellular inflammasome complex and NF-κB-mediated pro-inflammatory responses

XIAP Regulates Cytosol-Specific Innate Immunity to Listeria Infection

The inhibitor of apoptosis protein (IAP) family has been implicated in immune regulation, but the mechanisms by which IAP proteins contribute to immunity are incompletely understood. We show here that X-linked IAP (XIAP) is required for innate immune control of Listeria monocytogenes infection. Mice deficient in XIAP had a higher bacterial burden 48 h after infection than wild-type littermates, and exhibited substantially decreased survival. XIAP enhanced NF-κB activation upon L. monocytogenes infection of activated macrophages, and prolonged phosphorylation of Jun N-terminal kinase (JNK) specifically in response to cytosolic bacteria. Additionally, XIAP promoted maximal production of pro-inflammatory cytokines upon bacterial infection in vitro or in vivo, or in response to combined treatment with NOD2 and TLR2 ligands. Together, our data suggest that XIAP regulates innate immune responses to L. monocytogenes infection by potentiating synergy between Toll-like receptors (TLRs) and Nod-like receptors (NLRs) through activation of JNK- and NF-κB–dependent signaling

Steroid Hormone Control of Cell Death and Cell Survival: Molecular Insights Using RNAi

The insect steroid hormone ecdysone triggers programmed cell death of obsolete larval tissues during metamorphosis and provides a model system for understanding steroid hormone control of cell death and cell survival. Previous genome-wide expression studies of Drosophila larval salivary glands resulted in the identification of many genes associated with ecdysone-induced cell death and cell survival, but functional verification was lacking. In this study, we test functionally 460 of these genes using RNA interference in ecdysone-treated Drosophila l(2)mbn cells. Cell viability, cell morphology, cell proliferation, and apoptosis assays confirmed the effects of known genes and additionally resulted in the identification of six new pro-death related genes, including sorting nexin-like gene SH3PX1 and Sox box protein Sox14, and 18 new pro-survival genes. Identified genes were further characterized to determine their ecdysone dependency and potential function in cell death regulation. We found that the pro-survival function of five genes (Ras85D, Cp1, CG13784, CG32016, and CG33087), was dependent on ecdysone signaling. The TUNEL assay revealed an additional two genes (Kap-α3 and Smr) with an ecdysone-dependent cell survival function that was associated with reduced cell death. In vitro, Sox14 RNAi reduced the percentage of TUNEL-positive l(2)mbn cells (p<0.05) following ecdysone treatment, and Sox14 overexpression was sufficient to induce apoptosis. In vivo analyses of Sox14-RNAi animals revealed multiple phenotypes characteristic of aberrant or reduced ecdysone signaling, including defects in larval midgut and salivary gland destruction. These studies identify Sox14 as a positive regulator of ecdysone-mediated cell death and provide new insights into the molecular mechanisms underlying the ecdysone signaling network governing cell death and cell survival