Host innate-immune responses are tailored by cell type to control and eradicate specific infectious agents. For example, an acute RNA virus infection can result in high-level expression of type 1 IFNs by both conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs), but whereas cDCs preferentially use RIG-I-like receptor (RLR) signaling to produce type 1 IFNs, pDCs predominantly use TLRs to induce these cytokines. We previously found that the I?B kinase ? (IKK?)/NF-?B pathway regulates early IFN-? expression, but not the magnitude of type 1 IFN expression following RLR engagement. In this study, we use IKK? inhibition and mice deficient in IKK? or canonical NF-?B subunits (p50, RelA/p65, and cRel) to demonstrate that the IKK?/NF-?B axis is critical for virus-induced type 1 IFN expression in pDCs, but not in cDCs. We also reveal a crucial and more general requirement for IKK?/NF-?B in TLR- but not RLR-induced expression of type 1 IFNs and inflammatory cytokines. Together, these findings reveal a previously unappreciated specificity of the IKK?/NF-?B signaling axis in regulation of antimicrobial responses by different classes of pattern recognition receptors, and therefore by individual cell types reliant on particular pattern recognition receptors for their innate-immune transcriptional responses.
The pronecrotic kinase, receptor interacting protein (RIP1, also called RIPK1) mediates programmed necrosis and, together with its partner, RIP3 (RIPK3), drives midgestational death of caspase 8 (Casp8)-deficient embryos. RIP1 controls a second vital step in mammalian development immediately after birth, the mechanism of which remains unresolved. Rip1(-/-) mice display perinatal lethality, accompanied by gross immune system abnormalities. Here we show that RIP1 K45A (kinase dead) knockin mice develop normally into adulthood, indicating that development does not require RIP1 kinase activity. In the face of complete RIP1 deficiency, cells develop sensitivity to RIP3-mixed lineage kinase domain-like-mediated necroptosis as well as to Casp8-mediated apoptosis activated by diverse innate immune stimuli (e.g., TNF, IFN, double-stranded RNA). When either RIP3 or Casp8 is disrupted in combination with RIP1, the resulting double knockout mice exhibit slightly prolonged survival over RIP1-deficient animals. Surprisingly, triple knockout mice with combined RIP1, RIP3, and Casp8 deficiency develop into viable and fertile adults, with the capacity to produce normal levels of myeloid and lymphoid lineage cells. Despite the combined deficiency, these mice sustain a functional immune system that responds robustly to viral challenge. A single allele of Rip3 is tolerated in Rip1(-/-)Casp8(-/-)Rip3(+/-) mice, contrasting the need to eliminate both alleles of either Rip1 or Rip3 to rescue midgestational death of Casp8-deficient mice. These observations reveal a vital kinase-independent role for RIP1 in preventing pronecrotic as well as proapoptotic signaling events associated with life-threatening innate immune activation at the time of mammalian parturition.
The antiviral protein Daxx acts as a restriction factor of avian sarcoma virus (ASV; Retroviridae) in mammalian cells by promoting epigenetic silencing of integrated proviral DNA. Although Daxx is encoded by a type I (?/?) interferon-stimulated gene, the requirement for Daxx in the interferon anti-retroviral response has not been elucidated. In this report, we describe the results of experiments designed to investigate the role of Daxx in the type I interferon-induced anti-ASV response.
Interferons (IFNs) are cytokines with powerful immunomodulatory and antiviral properties, but less is known about how they induce cell death. Here, we show that both type I (?/?) and type II (?) IFNs induce precipitous receptor-interacting protein (RIP)1/RIP3 kinase-mediated necrosis when the adaptor protein Fas-associated death domain (FADD) is lost or disabled by phosphorylation, or when caspases (e.g., caspase 8) are inactivated. IFN-induced necrosis proceeds via progressive assembly of a RIP1-RIP3 "necrosome" complex that requires Jak1/STAT1-dependent transcription, but does not need the kinase activity of RIP1. Instead, IFNs transcriptionally activate the RNA-responsive protein kinase PKR, which then interacts with RIP1 to initiate necrosome formation and trigger necrosis. Although IFNs are powerful activators of necrosis when FADD is absent, these cytokines are likely not the dominant inducers of RIP kinase-driven embryonic lethality in FADD-deficient mice. We also identify phosphorylation on serine 191 as a mechanism that disables FADD and collaborates with caspase inactivation to allow IFN-activated necrosis. Collectively, these findings outline a mechanism of IFN-induced RIP kinase-dependent necrotic cell death and identify FADD and caspases as negative regulators of this process.
Advanced renal cell carcinoma (RCC) is an invariably fatal cancer. Currently, small-molecule inhibitors that target cell growth, angiogenesis, or nutrient-sensing pathways represent the primary pharmacologic interventions for this disease, but these inhibitors only delay tumor progression and are not curative. The cytokine IFN-? showed the potential to provide lasting remission in several phase I/II trials for advanced RCCs, but subsequent trials, including a multicenter phase III study using IFN-? as a monotherapy for RCCs, were less promising. Notably, these trials were designed to exploit the indirect immunomodulatory effects of IFN-?, whereas its direct antitumor properties--including its ability to trigger programmed cell death in tumors-remain mostly untapped. Here, we show that the proteasome inhibitor bortezomib (PS-341, Velcade) sensitizes otherwise resistant RCC cells to direct necrotic death by IFN-?. Mechanistically, we show that bortezomib functions, at least in part, by inhibiting prosurvival NF-?B signaling. In the absence of this signal, IFN-? triggers programmed necrosis (or "necroptosis") dependent on the kinase RIP1. When taken together with the observation that NF-?B signaling is elevated in RCCs, these results provide rationale for the combined use of IFN-? and bortezomib in the treatment of metastatic RCCs.
Advanced renal cancer is an incurable malignancy in need of novel therapeutic avenues. We have generated interferon ? (IFN?)-based fusion antibodies (immunocytokines) that target CD70, a putative biomarker of renal cancer. These immunocytokines efficiently labeled renal cancer cells, and, when combined with the proteasome inhibitor bortezomib, killed them by activating a RIP1-dependent necrotic pathway.
Interferon (IFN)-?-like the well-known antitumor biotherapeutic IFN-?-is a powerful antiproliferative and immune modulatory cytokine, but mixed results from clinical trials, together with issues of systemic toxicity, have dampened enthusiasm for its use in the treatment of cancer. We suggest that at least 2 factors reduce the antitumor efficacy of IFN-?: (1) poorly understood survival mechanisms that protect most tumor cells from IFN-?-induced direct cytotoxicity, and (2) the short half-life of IFN-? in serum. In this review, we outline avenues to overcome both these limitations. First, we have identified the transcription factor nuclear factor-kappa B (NF-?B) as a protective mechanism against IFN-?-induced necrosis, and disabling NF-?B allows IFN-? to trigger RIP1 kinase-dependent programmed necrosis (or necroptosis) in otherwise resistant cells. Second, we propose that fusing IFN-? to tumor-specific antibodies will stabilize IFN-? in serum and target this cytokine to tumor cells. We expect that such IFN-?-antibody chimeras (called immunocytokines), when combined with agents that neutralize tumor-intrinsic survival signals such as NF-?B, will exert potent tumoricidal activity with minimized systemic side effects. Although this review will focus on exploiting IFN-?-induced necrosis for treatment of renal cell carcinoma, these approaches are also directly applicable to several human cancers in which IFNs have shown therapeutic potential.
The RIG-I like receptor pathway is stimulated during RNA virus infection by interaction between cytosolic RIG-I and viral RNA structures that contain short hairpin dsRNA and 5 triphosphate (5ppp) terminal structure. In the present study, an RNA agonist of RIG-I was synthesized in vitro and shown to stimulate RIG-I-dependent antiviral responses at concentrations in the picomolar range. In human lung epithelial A549 cells, 5pppRNA specifically stimulated multiple parameters of the innate antiviral response, including IRF3, IRF7 and STAT1 activation, and induction of inflammatory and interferon stimulated genes - hallmarks of a fully functional antiviral response. Evaluation of the magnitude and duration of gene expression by transcriptional profiling identified a robust, sustained and diversified antiviral and inflammatory response characterized by enhanced pathogen recognition and interferon (IFN) signaling. Bioinformatics analysis further identified a transcriptional signature uniquely induced by 5pppRNA, and not by IFN?-2b, that included a constellation of IRF7 and NF-kB target genes capable of mobilizing multiple arms of the innate and adaptive immune response. Treatment of primary PBMCs or lung epithelial A549 cells with 5pppRNA provided significant protection against a spectrum of RNA and DNA viruses. In C57Bl/6 mice, intravenous administration of 5pppRNA protected animals from a lethal challenge with H1N1 Influenza, reduced virus titers in mouse lungs and protected animals from virus-induced pneumonia. Strikingly, the RIG-I-specific transcriptional response afforded partial protection from influenza challenge, even in the absence of type I interferon signaling. This systems approach provides transcriptional, biochemical, and in vivo analysis of the antiviral efficacy of 5pppRNA and highlights the therapeutic potential associated with the use of RIG-I agonists as broad spectrum antiviral agents.
Nuclear factor kappa B (NF-?B) and type 1 interferon (T1-IFN) signaling are innate immune mechanisms activated upon viral infection. However, the role of NF-?B and its interplay with T1-IFN in antiviral immunity is poorly understood. We show that NF-?B is essential for resistance to ectromelia virus (ECTV), a mouse orthopoxvirus related to the virus causing human smallpox. Additionally, an ECTV mutant lacking an NF-?B inhibitor activates NF-?B more effectively in vivo, resulting in increased proinflammatory molecule transcription in uninfected cells and organs and decreased viral replication. Unexpectedly, NF-?B activation compensates for genetic defects in the T1-IFN pathway, such as a deficiency in the IRF7 transcription factor, resulting in virus control. Thus, overlap between the T1-IFN and NF-?B pathways allows the host to overcome genetic or pathogen-induced deficiencies in T1-IFN and survive an otherwise lethal poxvirus infection. These findings may also explain why some pathogens target both pathways to cause disease.
Epithelial ovarian cancer (EOC) accounts for the most gynecologic malignancy-associated deaths in the United States. Enhancer of zeste homolog 2 (EZH2), which silences gene expression through generating trimethylation on lysine 27 residue of histone H3 (H3K27Me3), is often overexpressed in EOCs and has been suggested as a therapeutic target. However, the mechanism underlying EZH2 overexpression in EOCs is unknown. Here, we show that EZH2 is upregulated at the transcription level, and two CCAAT boxes in the proximal regions of the human EZH2 gene promoter are critical for its transcription in EOC cells. Indeed, NF-YA, the regulatory subunit of the CCAAT-binding transcription factor NF-Y, is expressed at higher levels in human EOCs than in primary human ovarian surface epithelial (HOSE) cells. In addition, there is a positive correlation between expression of NF-YA and EZH2 in EOCs. Notably, high NF-YA expression predicts shorter overall survival in patients with EOCs. The association of NF-YA with the promoter of the human EZH2 gene is enhanced in human EOC cells compared with primary HOSE cells. Significantly, knockdown of NF-YA downregulates EZH2, decreases H3K27Me3 levels, and suppresses the growth of human EOC cells both in vitro and in a xenograft mouse model. Notably, NF-YA knockdown induces apoptosis of EOC cells and ectopic EZH2 expression partially rescues apoptosis induced by NF-YA knockdown. Together, these data reveal that NF-Y is a key regulator of EZH2 expression and is required for EOC cell proliferation, thus representing a novel target for developing EOC therapeutics.
To determine the expression patterns of NF-?B regulators and target genes in clear cell renal cell carcinoma (ccRCC), their correlation with von Hippel Lindau (VHL) mutational status, and their association with survival outcomes.
Metastatic renal cell carcinoma (RCC) is an incurable disease in clear need of new therapeutic interventions. In early-phase clinical trials, the cytokine IFN-? showed promise as a biotherapeutic for advanced RCC, but subsequent trials were less promising. These trials, however, focused on the indirect immunomodulatory properties of IFN-?, and its direct anti-tumor effects, including its ability to kill tumor cells, remains mostly unexploited. We have previously shown that IFN-? induces RIP1 kinase-dependent necrosis in cells lacking NF-?B survival signaling. RCC cells display basally-elevated NF-?B activity, and inhibiting NF-?B in these cells, for example by using the small-molecule proteasome blocker bortezomib, sensitizes them to RIP1-dependent necrotic death following exposure to IFN-?. While these observations suggest that IFN-?-mediated direct tumoricidal activity will have therapeutic benefit in RCC, they cannot be effectively exploited unless IFN-? is targeted to tumor cells in vivo. Here, we describe the generation and characterization of two novel immunocytokine chimeric proteins, in which either human or murine IFN-? is fused to an antibody targeting the putative metastatic RCC biomarker CD70. These immunocytokines display high levels of species-specific IFN-? activity and selective binding to CD70 on human RCC cells. Importantly, the IFN-? immunocytokines function as well as native IFN-? in inducing RIP1-dependent necrosis in RCC cells, when deployed in the presence of bortezomib. These results provide a foundation for the in vivo exploitation of IFN-?-driven tumoricidal activity in RCC.
Necroptosis represents a form of alternative programmed cell death that is dependent on the kinase RIP1. RIP1-dependent necroptotic death manifests as increased reactive oxygen species (ROS) production in mitochondria and is accompanied by loss of ATP biogenesis and eventual dissipation of mitochondrial membrane potential. Here, we show that tumor necrosis factor alpha (TNF-?)-induced necroptosis requires the adaptor proteins FADD and NEMO. FADD was found to mediate formation of the TNF-?-induced pronecrotic RIP1-RIP3 kinase complex, whereas the I?B Kinase (IKK) subunit NEMO appears to function downstream of RIP1-RIP3. Interestingly, loss of RelA potentiated TNF-?-dependent necroptosis, indicating that NEMO regulates necroptosis independently of NF-?B. Using both pharmacologic and genetic approaches, we demonstrate that the overexpression of antioxidants alleviates ROS elevation and necroptosis. Finally, elimination of BAX and BAK or overexpression of Bcl-x(L) protects cells from necroptosis at a later step. These findings provide evidence that mitochondria play an amplifying role in inflammation-induced necroptosis.
Interferons (IFNs) are cytokines with well-described immunomodulatory and antiviral properties, but less is known about the mechanisms by which they promote cell survival or cell death. Here, we show that IFN-? induces RIP1 kinase-dependent necroptosis in mammalian cells deficient in NF-?B signaling. Induction of necroptosis by IFN-? was found to depend on Jak1 and partially on STAT1. We also demonstrate that IFN-? activates I?B kinase ? (IKK?)-dependent NF-?B to regulate a transcriptional program that protects cells from necroptosis. IFN-? induced progressive accumulation of reactive oxygen species (ROS) and eventual loss of mitochondrial membrane potential in cells lacking the NF-?B subunit RelA. Whole-genome microarray analyses identified sod2, encoding the antioxidant enzyme manganese superoxide dismutase (MnSOD), as a RelA target and potential antinecroptotic gene. Overexpression of MnSOD inhibited IFN-?-mediated ROS accumulation and partially rescued RelA-deficient cells from necroptosis, while RNA interference (RNAi)-mediated silencing of sod2 expression increased susceptibility to IFN-?-induced cell death. Together, these studies demonstrate that NF-?B protects cells from IFN-?-mediated necroptosis by transcriptionally activating a survival response that quenches ROS to preserve mitochondrial integrity.
Hepatitis delta virus (HDV) infects hepatocytes, the major cell type of the liver. Infection of the liver may be either transient or chronic. The prognosis for patients with chronic HDV infection is poor, with a high risk of cirrhosis and hepatocellular carcinoma. The best antiviral therapy is weekly administration for at least one year of high doses of interferon alpha. This efficacy of interferon therapy has been puzzling in that HDV replication in transfected cell lines is reported as insensitive to administration of interferon alpha or gamma. Similarly, this study shows that even when an interferon response was induced by transfection of poly(IC) into a cell line, HDV RNA accumulation was only modestly inhibited. However, when the HDV replication was initiated by infection of primary human hepatocytes, simultaneous addition of interferons alpha or gamma at 600 units/ml, a concentration comparable to that achieved in treated patients, the subsequent HDV RNA accumulation was inhibited by at least 80%. These interferon treatments were shown to produce significant time-dependent increases of host response proteins such as for Stat-1, phosphoStat-1, Mx1/2/3 and PKR, and yet interferon pretreatment of hepatocytes did not confer an increased inhibition of HDV replication over interferon treatment at the time of (or after) infection. These and other data support the interpretation that interferon action against HDV replication can occur and is largely mediated at the level of entry into primary human hepatocytes. Thus in vivo, the success of long-term interferon therapy for chronic HDV, may likewise involve blocking HDV spread by interfering with the initiation of productive infection of naïve hepatocytes.
Production of type I interferons (IFNs; prominently, IFN-?/?) following virus infection is a pivotal antiviral innate immune response in higher vertebrates. The synthesis of IFN-? proceeds via the virus-induced assembly of the transcription factors IRF-3/7, ATF-2/c-Jun, and NF-?B on the ifn? promoter. Surprisingly, recent data indicate that the NF-?B subunit RelA is not essential for virus-stimulated ifn? expression. Here, we show that RelA instead sustains autocrine IFN-? signaling prior to infection. In the absence of RelA, virus infection results in significantly delayed ifn? induction and consequently defective secondary antiviral gene expression. While RelA is not required for ifn? expression after infection, it is nonetheless essential for fully one-fourth of double-stranded RNA (dsRNA)-activated genes, including several mediators of inflammation and immune cell recruitment. Further, RelA directly regulates a small subset of interferon-stimulated genes (ISGs). Finally, RelA also protects cells from dsRNA-triggered RIP1-dependent programmed necrosis. Taken together, our findings suggest distinct roles for RelA in antiviral innate immunity: RelA maintains autocrine IFN-? signaling in uninfected cells, facilitates inflammatory and adaptive immune responses following infection, and promotes infected-cell survival during this process.
RNA virus infection results in expression of type 1 IFNs, especially IFN-alpha/beta, which play a crucial role in host antivirus responses. Type 1 IFNs are induced in a cell type-specific manner through TLR and RIG-I-like receptor pathways, both of which activate IFN regulatory factors (IRFs) and NF-kappaB transcription factors. Although NF-kappaB activation and association with the IFN-beta promoter after RNA virus infection is well documented, our previous work showed that, surprisingly, NF-kappaB is not essential for IFN-beta gene expression. Thus, the actual function of NF-kappaB in IFN-beta expression and virus replication is not clear. In this study, we found Newcastle disease virus and vesicular stomatitis virus replication is enhanced in mouse embryonic fibroblasts (MEFs) lacking the NF-kappaB RelA subunit. Increased virus replication was traced to a specific requirement for RelA in early virus-induced IFN-beta expression. At these time points, when IFN-beta expression is ~100-fold less than peak levels, impaired IFN-beta production delayed IFN-induced gene expression, resulting in increased virus replication in RelA(-/-) MEFs. Importantly, our results show that RelA requirement is crucial only when IRF3 activation is low. Thus, high levels of activated IRF3 expression are sufficient for induction of IFN-beta in RelA(-/-) MEFs, transcriptional synergism with the coactivator CREB-binding protein, and rescue of susceptibility to virus. Together, these findings indicate that NF-kappaB RelA is not crucial for regulating overall IFN-beta production, as previously believed; instead, RelA is specifically required only during a key early phase after virus infection, which substantially impacts the host response to virus infection.
STAT2 is a positive modulator of the transcriptional response to type I interferons (IFNs). STAT2 acquires transcriptional function by becoming tyrosine phosphorylated and imported to the nucleus following type I IFN receptor activation. Although most STAT proteins become dually phosphorylated on specific tyrosine and serine residues to acquire full transcriptional activity, no serine phosphorylation site in STAT2 has been reported. To find novel phosphorylation sites, mass spectrometry of immunoprecipitated STAT2 was used to identify several phosphorylated residues. Of these, substitution of serine 287 with alanine (S287A) generated a gain-of-function mutant that enhanced the biological effects of IFN-?. S287A-STAT2 increased cell growth inhibition, prolonged protection against vesicular stomatitis virus infection and enhanced transcriptional responses following exposure of cells to IFN-?. In contrast, a phosphomimetic STAT2 mutant (S287D) produced a loss-of-function protein that weakly activated IFN-induced ISGs. Our mechanistic studies suggest that S287A-STAT2 likely mediates its gain-of-function effects by prolonging STAT2/STAT1 dimer activation and retaining it in transcriptionally active complexes with chromatin. Altogether, we have uncovered that in response to type I IFN, STAT2 is serine phosphorylated in the coiled-coil domain that when phosphorylated can negatively regulate the biological activities of type I IFNs.
Ribosomal protein (RP) mutations in diseases such as 5q- syndrome both disrupt hematopoiesis and increase the risk of developing hematologic malignancy. However, the mechanism by which RP mutations increase cancer risk has remained an important unanswered question. We show here that monoallelic, germline inactivation of the ribosomal protein L22 (Rpl22) predisposes T-lineage progenitors to transformation. Indeed, RPL22 was found to be inactivated in ? 10% of human T-acute lymphoblastic leukemias. Moreover, monoallelic loss of Rpl22 accelerates development of thymic lymphoma in both a mouse model of T-cell malignancy and in acute transformation assays in vitro. We show that Rpl22 inactivation enhances transformation potential through induction of the stemness factor, Lin28B. Our finding that Rpl22 inactivation promotes transformation by inducing expression of Lin28B provides the first insight into the mechanistic basis by which mutations in Rpl22, and perhaps some other RP genes, increases cancer risk.
Neurons are chiefly nonrenewable; thus, cytolytic immune strategies to clear or control neurotropic viral infections could have lasting neurologic consequences. IFN-? is a potent antiviral cytokine that is critical for noncytolytic clearance of multiple neurotropic viral infections, including measles virus (MV); however, the downstream pathways through which IFN-? functions in neurons have not been defined. Unlike most cell types studied to date in which IFN-? affects gene expression via rapid and robust activation of STAT1, basal STAT1 levels in primary hippocampal neurons are constitutively low, resulting in attenuated STAT1 activation and consequently slower kinetics of IFN-?-driven STAT1-dependent gene expression. Given this altered expression and activation of STAT1 in neurons, we sought to determine whether STAT1 was required for IFN-?-mediated protection from infection in neurons. To do so, we evaluated the consequences of MV challenge of STAT1-deficient mice and primary hippocampal neurons explanted from these mice. Surprisingly, the absence of STAT1 did not restrict the ability of IFN-? to control viral infection either in vivo or ex vivo. Moreover, the canonical IFN-?-triggered STAT1 gene expression profile was not induced in STAT1-deficient neurons, suggesting that IFN-? regulates neuronal STAT1-independent pathways to control viral replication.
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