RIPK1 and RIPK3 Kinases Promote Cell-Death-Independent Inflammation by Toll-like Receptor 4.

Macrophages are a crucial component of the innate immune system in sensing pathogens and promoting local and systemic inflammation. RIPK1 and RIPK3 are homologous kinases, previously linked to activation of necroptotic death. In this study, we have described roles for these kinases as master regulators of pro-inflammatory gene expression induced by lipopolysaccharide, independent of their well-documented cell death functions. In primary macrophages, this regulation was elicited in the absence of caspase-8 activity, required the adaptor molecule TRIF, and proceeded in a cell autonomous manner. RIPK1 and RIPK3 kinases promoted sustained activation of Erk, cFos, and NF-κB, which were required for inflammatory changes. Utilizing genetic and pharmacologic tools, we showed that RIPK1 and RIPK3 account for acute inflammatory responses induced by lipopolysaccharide in vivo; notably, this regulation did not require exogenous manipulation of caspases. These findings identified a new pharmacologically accessible pathway that may be relevant to inflammatory pathologies.

Kinase Activities of RIPK1 and RIPK3 Can Direct IFN-ß Synthesis Induced by Lipopolysaccharide.

The innate immune response is a central element of the initial defense against bacterial and viral pathogens. Macrophages are key innate immune cells that upon encountering pathogen-associated molecular patterns respond by producing cytokines, including IFN-ß. In this study, we identify a novel role for RIPK1 and RIPK3, a pair of homologous serine/threonine kinases previously implicated in the regulation of necroptosis and pathologic tissue injury, in directing IFN-ß production in macrophages. Using genetic and pharmacologic tools, we show that catalytic activity of RIPK1 directs IFN-ß synthesis induced by LPS in mice. Additionally, we report that RIPK1 kinase-dependent IFN-ß production may be elicited in an analogous fashion using LPS in bone marrow-derived macrophages upon inhibition of caspases. Notably, this regulation requires kinase activities of both RIPK1 and RIPK3, but not the necroptosis effector protein, MLKL. Mechanistically, we provide evidence that necrosome-like RIPK1 and RIPK3 aggregates facilitate canonical TRIF-dependent IFN-ß production downstream of the LPS receptor TLR4. Intriguingly, we also show that RIPK1 and RIPK3 kinase-dependent synthesis of IFN-ß is markedly induced by avirulent strains of Gram-negative bacteria, Yersinia and Klebsiella, and less so by their wild-type counterparts. Overall, these observations identify unexpected roles for RIPK1 and RIPK3 kinases in the production of IFN-ß during the host inflammatory responses to bacterial infection and suggest that the axis in which these kinases operate may represent a target for bacterial virulence factors.

Phosphorylation of STAT2 on serine-734 negatively regulates the IFN-α-induced antiviral response.

Serine phosphorylation of STAT proteins is an important post-translational modification event that, in addition to tyrosine phosphorylation, is required for strong transcriptional activity. However, we recently showed that phosphorylation of STAT2 on S287 induced by type I interferons (IFN-α and IFN-ß), evoked the opposite effect. S287-STAT2 phosphorylation inhibited the biological effects of IFN-α. We now report the identification and characterization of S734 on the C-terminal transactivation domain of STAT2 as a new phosphorylation site that can be induced by type I IFNs. IFN-α-induced S734-STAT2 phosphorylation displayed different kinetics to that of tyrosine phosphorylation. S734-STAT2 phosphorylation was dependent on STAT2 tyrosine phosphorylation and JAK1 kinase activity. Mutation of S734-STAT2 to alanine (S734A) enhanced IFN-α-driven antiviral responses compared to those driven by wild-type STAT2. Furthermore, DNA microarray analysis demonstrated that a small subset of type I IFN stimulated genes (ISGs) was induced more by IFNα in cells expressing S734A-STAT2 when compared to wild-type STAT2. Taken together, these studies identify phosphorylation of S734-STAT2 as a new regulatory mechanism that negatively controls the type I IFN-antiviral response by limiting the expression of a select subset of antiviral ISGs.

Differential requirement for the IKKß/NF-κB signaling module in regulating TLR- versus RLR-induced type 1 IFN expression in dendritic cells.

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 IκB kinase ß (IKKß)/NF-κB pathway regulates early IFN-ß expression, but not the magnitude of type 1 IFN expression following RLR engagement. In this study, we use IKKß inhibition and mice deficient in IKKß or canonical NF-κB subunits (p50, RelA/p65, and cRel) to demonstrate that the IKKß/NF-κB 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 IKKß/NF-κB in TLR- but not RLR-induced expression of type 1 IFNs and inflammatory cytokines. Together, these findings reveal a previously unappreciated specificity of the IKKß/NF-κB 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.

Interferon-induced RIP1/RIP3-mediated necrosis requires PKR and is licensed by FADD and caspases.

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 (α/ß) and type II (γ) 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.

Identification of STAT2 serine 287 as a novel regulatory phosphorylation site in type I interferon-induced cellular responses.

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-α. S287A-STAT2 increased cell growth inhibition, prolonged protection against vesicular stomatitis virus infection and enhanced transcriptional responses following exposure of cells to IFN-α. 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.

The interferon-inducible antiviral protein Daxx is not essential for interferon-mediated protection against avian sarcoma virus.

BACKGROUND: 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 (α/ß) 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. FINDINGS: Using an ASV reporter system, we show that type I interferons are potent inhibitors of ASV replication. We demonstrate that, while Daxx is necessary to silence ASV gene expression in the absence of interferons, type I interferons are fully-capable of inducing an antiviral state in the absence of Daxx. CONCLUSIONS: These results provide evidence that Daxx is not essential for the anti-ASV interferon response in mammalian cells, and that interferons deploy multiple, redundant antiviral mechanisms to protect cells from ASV.

Distinct roles for the NF-kappa B RelA subunit during antiviral innate immune responses.

Production of type I interferons (IFNs; prominently, IFN-α/ß) following virus infection is a pivotal antiviral innate immune response in higher vertebrates. The synthesis of IFN-ß proceeds via the virus-induced assembly of the transcription factors IRF-3/7, ATF-2/c-Jun, and NF-κB on the ifnß promoter. Surprisingly, recent data indicate that the NF-κB subunit RelA is not essential for virus-stimulated ifnß expression. Here, we show that RelA instead sustains autocrine IFN-ß signaling prior to infection. In the absence of RelA, virus infection results in significantly delayed ifnß induction and consequently defective secondary antiviral gene expression. While RelA is not required for ifnß 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-ß signaling in uninfected cells, facilitates inflammatory and adaptive immune responses following infection, and promotes infected-cell survival during this process.

Characterization of age-related changes in natural killer cells during primary influenza infection in mice.

The current investigation examined the importance of natural killer (NK) cells during the innate immune response to primary influenza infection in young and aged mice. Young (6-8 weeks) and aged (22 months) C57BL/6 mice were infected intranasally with influenza A virus, and NK cell-mediated cytotoxicity was determined in lung and spleen during the first 4 days of infection. Aged mice demonstrated both a decrease in influenza-inducible NK activity and a reduction in the percentage and number of NK1.1+ cells in response to primary influenza infection, relative to young mice. In order to further establish a role for NK cells in controlling influenza infection, young mice were depleted of NK cells in vivo by injecting rabbit anit-NK1.1 antibody 2 days and 1 day prior to influenza infection. Young mice depleted of NK cells exhibited increased weight loss and lung virus titers during the course of infection, compared to young mice infected with influenza virus. These data indicate that NK cell function is impaired in response to primary influenza infection in aged mice. More importantly, these results underscore the essential role of NK cells in controlling virus titers in lung during the early course of influenza infection, regardless of age.

Energy restriction impairs natural killer cell function and increases the severity of influenza infection in young adult male C57BL/6 mice.

Energy restriction (ER) without malnutrition extends lifespan in mice and postpones age-related changes in immunity. However, we have previously shown that aged (22 mo old) ER mice exhibit increased mortality, impaired viral clearance, and reduced natural killer (NK) cell cytotoxicity following influenza infection compared with aged mice that consumed food ad libitum (AL). To determine whether the detrimental effects of ER in response to influenza infection occur independently of advanced age, young adult (6 mo) male C57BL/6 mice consuming an AL or ER diet were infected with influenza A virus (H1N1, PR8). Young adult ER mice exhibited increased mortality (P < 0.05) and weight loss (P < 0.01) in response to infection. ER mice exhibited decreased total (P < 0.001) and NK1.1+ lymphocytes (P < 0.05) in lung and reduced influenza-induced NK cell cytotoxicity in both lung (P < 0.01) and spleen (P < 0.05). Importantly, the mRNA expression of interferon (IFN)alpha/beta (P < 0.05) was also reduced in the lungs of ER mice in response to infection, and in vitro stimulation of NK cells from ER mice with type I IFN resulted in cytotoxicity comparable to that in NK cells from AL mice. In contrast, NK cell activation was enhanced in ER mice, determined as an increase in the percentage of NK cells expressing B220 (P < 0.001) and increased intracellular production of IFNgamma (P < 0.01). These data describe an age-independent and detrimental effect of ER on the innate immune response to influenza infection and suggest that a decrease in NK cell number and alterations in the NK cell-activating environment may contribute to decreased innate immunity in ER mice.

Analysis of Cytokine- and Influenza A Virus-Driven RIPK3 Necrosome Formation.

In multicellular organisms, regulated cell death plays a vital role in development, adult tissue homeostasis, and clearance of damaged or infected cells. Necroptosis is one such form of regulated cell death, characterized by its reliance on receptor-interacting protein kinase 3 (RIPK3). Once activated, RIPK3 nucleates a protein complex, termed the "necrosome," which includes the adaptors RIPK1 and FADD, and the effector protein MLKL. From the necrosome, RIPK3 phosphorylates MLKL to drive necroptosis, and can also induce RIPK1/FADD-mediated apoptosis, via caspase-8. Assembly of the necrosome thus serves as a useful readout of RIPK3 activation. In this chapter, we describe molecular methods for examining necrosome activation in response to the cytokines TNF-α, IFN-ß, and IFN-γ, and upon infection with influenza A virus (IAV).

Phenytoin inhibits necroptosis.

Receptor-interacting protein kinases 1 and 3 (RIPK1/3) have best been described for their role in mediating a regulated form of necrosis, referred to as necroptosis. During this process, RIPK3 phosphorylates mixed lineage kinase domain-like (MLKL) to cause plasma membrane rupture. RIPK3-deficient mice have recently been demonstrated to be protected in a series of disease models, but direct evidence for activation of necroptosis in vivo is still limited. Here, we sought to further examine the activation of necroptosis in kidney ischemia-reperfusion injury (IRI) and from TNFα-induced severe inflammatory response syndrome (SIRS), two models of RIPK3-dependent injury. In both models, MLKL-ko mice were significantly protected from injury to a degree that was slightly, but statistically significantly exceeding that of RIPK3-deficient mice. We also demonstrated, for the first time, accumulation of pMLKL in the necrotic tubules of human patients with acute kidney injury. However, our data also uncovered unexpected elevation of blood flow in MLKL-ko animals, which may be relevant to IRI and should be considered in the future. To further understand the mode of regulation of cell death by MLKL, we screened a panel of clinical plasma membrane channel blockers and we found phenytoin to inhibit necroptosis. However, we further found that phenytoin attenuated RIPK1 kinase activity in vitro, likely due to the hydantoin scaffold also present in necrostatin-1, and blocked upstream necrosome formation steps in the cells undergoing necroptosis. We further report that this clinically used anti-convulsant drug displayed protection from kidney IRI and TNFα-induces SIRS in vivo. Overall, our data reveal the relevance of RIPK3-pMLKL regulation for acute kidney injury and identifies an FDA-approved drug that may be useful for immediate clinical evaluation of inhibition of pro-death RIPK1/RIPK3 activities in human diseases.

Correction to: Phenytoin inhibits necroptosis.

The name of the one of the authors was misspelt. The author's surname is Rodriguez, not Rodriquez as originally published. This has been corrected in both the PDF and HTML versions of the Article.

RIPK3 Is Largely Dispensable for RIG-I-Like Receptor- and Type I Interferon-Driven Transcriptional Responses to Influenza A Virus in Murine Fibroblasts.

The kinase RIPK3 is a key regulator of cell death responses to a growing number of viral and microbial agents. We have found that influenza A virus (IAV)-mediated cell death is largely reliant on RIPK3 and that RIPK3-deficient mice are notably more susceptible to lethal infection by IAV than their wild-type counterparts. Recent studies demonstrate that RIPK3 also participates in regulating gene transcription programs during host pro-inflammatory and innate-immune responses, indicating that this kinase is not solely an inducer of cell death and that RIPK3-driven transcriptional responses may collaborate with cell death in promoting clearance of IAV. Here, we carried out DNA microarray analyses to determine the contribution of RIPK3 to the IAV-elicited host transcriptional response. We report that RIPK3 does not contribute significantly to the RLR-activated transcriptome or to the induction of type I IFN genes, although, interestingly, IFN-ß production at a post-transcriptional step was modestly attenuated in IAV-infected ripk3-/- fibroblasts. Overall, RIPK3 regulated the expression of <5% of the IAV-induced transcriptome, and no genes were found to be obligate RIPK3 targets. IFN-ß signaling was also found to be largely normal in the absence of RIPK3. Together, these results indicate that RIPK3 is not essential for the host antiviral transcriptional response to IAV in murine fibroblasts.

DAI Senses Influenza A Virus Genomic RNA and Activates RIPK3-Dependent Cell Death.

Influenza A virus (IAV) is an RNA virus that is cytotoxic to most cell types in which it replicates. IAV activates the host kinase RIPK3, which induces cell death via parallel pathways of necroptosis, driven by the pseudokinase MLKL, and apoptosis, dependent on the adaptor proteins RIPK1 and FADD. How IAV activates RIPK3 remains unknown. We report that DAI (ZBP1/DLM-1), previously implicated as a cytoplasmic DNA sensor, is essential for RIPK3 activation by IAV. Upon infection, DAI recognizes IAV genomic RNA, associates with RIPK3, and is required for recruitment of MLKL and RIPK1 to RIPK3. Cells lacking DAI or containing DAI mutants deficient in nucleic acid binding are resistant to IAV-triggered necroptosis and apoptosis. DAI-deficient mice fail to control IAV replication and succumb to lethal respiratory infection. These results identify DAI as a link between IAV replication and RIPK3 activation and implicate DAI as a sensor of RNA viruses.

RIPK3 Activates Parallel Pathways of MLKL-Driven Necroptosis and FADD-Mediated Apoptosis to Protect against Influenza A Virus.

Influenza A virus (IAV) is a lytic virus in primary cultures of many cell types and in vivo. We report that the kinase RIPK3 is essential for IAV-induced lysis of mammalian fibroblasts and lung epithelial cells. Replicating IAV drives assembly of a RIPK3-containing complex that includes the kinase RIPK1, the pseudokinase MLKL, and the adaptor protein FADD, and forms independently of signaling by RNA-sensing innate immune receptors (RLRs, TLRs, PKR), or the cytokines type I interferons and TNF-α. Downstream of RIPK3, IAV activates parallel pathways of MLKL-driven necroptosis and FADD-mediated apoptosis, with the former reliant on RIPK3 kinase activity and neither on RIPK1 activity. Mice deficient in RIPK3 or doubly deficient in MLKL and FADD, but not MLKL alone, are more susceptible to IAV than their wild-type counterparts, revealing an important role for RIPK3-mediated apoptosis in antiviral immunity. Collectively, these results outline RIPK3-activated cytolytic mechanisms essential for controlling respiratory IAV infection.

Structure guided design of potent and selective ponatinib-based hybrid inhibitors for RIPK1.

RIPK1 and RIPK3, two closely related RIPK family members, have emerged as important regulators of pathologic cell death and inflammation. In the current work, we report that the Bcr-Abl inhibitor and anti-leukemia agent ponatinib is also a first-in-class dual inhibitor of RIPK1 and RIPK3. Ponatinib potently inhibited multiple paradigms of RIPK1- and RIPK3-dependent cell death and inflammatory tumor necrosis factor alpha (TNF-α) gene transcription. We further describe design strategies that utilize the ponatinib scaffold to develop two classes of inhibitors (CS and PN series), each with greatly improved selectivity for RIPK1. In particular, we detail the development of PN10, a highly potent and selective "hybrid" RIPK1 inhibitor, capturing the best properties of two different allosteric RIPK1 inhibitors, ponatinib and necrostatin-1. Finally, we show that RIPK1 inhibitors from both classes are powerful blockers of TNF-induced injury in vivo. Altogether, these findings outline promising candidate molecules and design approaches for targeting RIPK1- and RIPK3-driven inflammatory pathologies.

Anti-CD70 immunocytokines for exploitation of interferon-γ-induced RIP1-dependent necrosis in renal cell carcinoma.

Metastatic renal cell carcinoma (RCC) is an incurable disease in clear need of new therapeutic interventions. In early-phase clinical trials, the cytokine IFN-γ 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-γ, and its direct anti-tumor effects, including its ability to kill tumor cells, remains mostly unexploited. We have previously shown that IFN-γ induces RIP1 kinase-dependent necrosis in cells lacking NF-κB survival signaling. RCC cells display basally-elevated NF-κB activity, and inhibiting NF-κB in these cells, for example by using the small-molecule proteasome blocker bortezomib, sensitizes them to RIP1-dependent necrotic death following exposure to IFN-γ. While these observations suggest that IFN-γ-mediated direct tumoricidal activity will have therapeutic benefit in RCC, they cannot be effectively exploited unless IFN-γ 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-γ is fused to an antibody targeting the putative metastatic RCC biomarker CD70. These immunocytokines display high levels of species-specific IFN-γ activity and selective binding to CD70 on human RCC cells. Importantly, the IFN-γ immunocytokines function as well as native IFN-γ 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-γ-driven tumoricidal activity in RCC.

NF-κB inhibition by bortezomib permits IFN-γ-activated RIP1 kinase-dependent necrosis in renal cell carcinoma.

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-γ 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-γ as a monotherapy for RCCs, were less promising. Notably, these trials were designed to exploit the indirect immunomodulatory effects of IFN-γ, 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-γ. Mechanistically, we show that bortezomib functions, at least in part, by inhibiting prosurvival NF-κB signaling. In the absence of this signal, IFN-γ triggers programmed necrosis (or "necroptosis") dependent on the kinase RIP1. When taken together with the observation that NF-κB signaling is elevated in RCCs, these results provide rationale for the combined use of IFN-γ and bortezomib in the treatment of metastatic RCCs.

Differential effects of stimulatory factors on natural killer cell activities of young and aged mice.

Age-associated influences on natural killer (NK) cell functions following cytokine stimulation were examined in splenocytes from C57BL/6 mice. NK cells of both young and aged mice exhibited significantly increased: interferon-γ production after interleukin (IL)-12 or IL-15 alone or any combination of IL-12, IL-18, and IL-2; cytotoxicity after IL-2 or IL-15; and granzyme B expression after IL-15. The only significant age-associated differences were observed in interferon-γ production after IL-15 or IL-12 + 18 + 2 and in granzyme B expression following IL-2 or IL-15. Perforin expression did not increase following stimulation; however, NK cells from aged mice expressed significantly higher levels than young mice. These results underscore the complexity of the cytokine-induced functional activities of NK cells and illustrate the differential response of NK cells from young and aged mice to cytokine stimulation.