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.

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.

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.

Kinase Activities of RIPK1 and RIPK3 Can Direct IFN-β 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-β. 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-β production in macrophages.

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-β), evoked the opposite effect. S287-STAT2 phosphorylation inhibited the biological effects of IFN-α.

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.

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.

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.

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.

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.

Differential requirement for the IKKβ/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 β (IKKβ)/NF-κB pathway regulates early IFN-β expression, but not the magnitude of type 1 IFN expression following RLR engagement.

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/β) 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.

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/β) 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.

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.

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.

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.

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.

Interferon impedes an early step of hepatitis delta virus infection.

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.

NF-kappaB protects cells from gamma interferon-induced RIP1-dependent 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-γ induces RIP1 kinase-dependent necroptosis in mammalian cells deficient in NF-κB signaling. Induction of necroptosis by IFN-γ was found to depend on Jak1 and partially on STAT1.

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

Production of type I interferons (IFNs; prominently, IFN-α/β) following virus infection is a pivotal antiviral innate immune response in higher vertebrates. The synthesis of IFN-β proceeds via the virus-induced assembly of the transcription factors IRF-3/7, ATF-2/c-Jun, and NF-κB on the ifnβ promoter. Surprisingly, recent data indicate that the NF-κB subunit RelA is not essential for virus-stimulated ifnβ expression.

Low-dose supplementation with active hexose correlated compound improves the immune response to acute influenza infection in C57BL/6 mice.

Supplementation with mushroom-derived active hexose correlated compound (AHCC) modulates immunity and increases survival in response to a broad spectrum of acute infections, including influenza virus infection. However, dose-response data are nonexistent. Therefore, the aims of this study were to evaluate AHCC supplementation at various doses and determine the effects of low-dose supplementation on the immune response in a mouse model of influenza virus infection.

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).

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.

Supplementation with active hexose correlated compound increases the innate immune response of young mice to primary influenza infection.

The emergence of H5N1 avian influenza and the threat of new or adapted viruses in bioterrorism have created an urgent interest in identifying agents to enhance the immune response to primary virus infection. Active hexose correlated compound (AHCC) is a natural mushroom extract reported to increase natural killer (NK) cell activity, survival, and bacterial clearance in young mice. However, the effects of AHCC on the response to viral infections have not been studied.

Age-related changes in the immune response to influenza vaccination in a racially diverse, healthy elderly population.

Proliferation to phytohemagglutinin (PHA) and cell-mediated responses to influenza vaccine (FLU) were assessed in healthy elderly Caucasians, African Americans, Latinos and young Caucasians before and after immunization. Both PHA- and FLU-induced proliferation were reduced in elderly subgroups. FLU-induced proliferation increased post-vaccination in all elderly, except African Americans, but responses were lower than young.