Publications by authors named "Siddharth Balachandran"

66 Publications

Vaccinia virus E3 prevents sensing of Z-RNA to block ZBP1-dependent necroptosis.

Cell Host Microbe 2021 Jun 24. Epub 2021 Jun 24.

Department of Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322, USA. Electronic address:

Necroptosis mediated by Z-nucleic-acid-binding protein (ZBP)1 (also called DAI or DLM1) contributes to innate host defense against viruses by triggering cell death to eliminate infected cells. During infection, vaccinia virus (VACV) protein E3 prevents death signaling by competing for Z-form RNA through an N-terminal Zα domain. In the absence of this E3 domain, Z-form RNA accumulates during the early phase of VACV infection, triggering ZBP1 to recruit receptor interacting protein kinase (RIPK)3 and execute necroptosis. The C-terminal E3 double-strand RNA-binding domain must be retained to observe accumulation of Z-form RNA and induction of necroptosis. Substitutions of Zα from either ZBP1 or the RNA-editing enzyme double-stranded RNA adenosine deaminase (ADAR)1 yields fully functional E3 capable of suppressing virus-induced necroptosis. Overall, our evidence reveals the importance of Z-form RNA generated during VACV infection as a pathogen-associated molecular pattern (PAMP) unleashing ZBP1/RIPK3/MLKL-dependent necroptosis unless suppressed by viral E3.
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http://dx.doi.org/10.1016/j.chom.2021.05.009DOI Listing
June 2021

Cell-specific activation of RIPK1 and MLKL after intracerebral hemorrhage in mice.

J Cereb Blood Flow Metab 2021 07 19;41(7):1623-1633. Epub 2020 Nov 19.

Department of Pediatrics, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA.

Receptor-interacting protein kinase-1 (RIPK1) is a master regulator of cell death and inflammation, and mediates programmed necrosis (necroptosis) via mixed-lineage kinase like (MLKL) protein. Prior studies in experimental intracerebral hemorrhage (ICH) implicated RIPK1 in the pathogenesis of neuronal death and cognitive outcome, but the relevant cell types involved and potential role of necroptosis remain unexplored. In mice subjected to autologous blood ICH, early RIPK1 activation was observed in neurons, endothelium and pericytes, but not in astrocytes. MLKL activation was detected in astrocytes and neurons but not endothelium or pericytes. Compared with WT controls, RIPK1 kinase-dead () mice had reduced brain edema (24 h) and blood-brain barrier (BBB) permeability (24 h, 30 d), and improved postinjury rotarod performance. Mice deficient in MLKL () had reduced neuronal death (24 h) and BBB permeability at 24 h but not 30d, and improved post-injury rotarod performance vs. WT. The data support a central role for RIPK1 in the pathogenesis of ICH, including cell death, edema, BBB permeability, and motor deficits. These effects may be mediated in part through the activation of MLKL-dependent necroptosis in neurons. The data support development of RIPK1 kinase inhibitors as therapeutic agents for human ICH.
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http://dx.doi.org/10.1177/0271678X20973609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8221773PMC
July 2021

Somatic Epigenetic Silencing of Inactivates Necroptosis and Contributes to Chemoresistance in Malignant Mesothelioma.

Clin Cancer Res 2021 Feb 17;27(4):1200-1213. Epub 2020 Nov 17.

Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania.

Purpose: Receptor-interacting protein kinase 3 (RIPK3) phosphorylates effector molecule MLKL to trigger necroptosis. Although RIPK3 loss is seen in several human cancers, its role in malignant mesothelioma is unknown. This study aimed to determine whether RIPK3 functions as a potential tumor suppressor to limit development of malignant mesothelioma.

Experimental Design: RIPK3 expression was examined in 66 malignant mesothelioma tumors and cell lines. Promoter methylation and siRNA studies were performed to assess the mode of silencing in RIPK3-deficient malignant mesothelioma cells. Restoration of RIPK3 expression in RIPK3-negative malignant mesothelioma cells, either by treatment with 5-aza-2'-deoxycytidine or lentiviral expression of cDNA, was performed to assess effects on cell viability, necrosis, and chemosensitization.

Results: Loss of RIPK3 expression was observed in 42/66 (63%) primary malignant mesotheliomas and malignant mesothelioma cell lines, and RT-PCR analysis demonstrated that downregulation occurs at the transcriptional level, consistent with epigenetic silencing. RIPK3-negative malignant mesothelioma cells treated with 5-aza-2'-deoxycytidine resulted in reexpression of RIPK3 and chemosensitization. Ectopic expression of RIPK3 also resulted in chemosensitization and led to necroptosis, the latter demonstrated by phosphorylation of downstream target MLKL and confirmed by rescue experiments. Mining of expression and survival outcomes among patients with malignant mesothelioma available from The Cancer Genome Atlas repository revealed that promoter methylation of is associated with reduced expression and poor prognosis.

Conclusions: These data suggest that RIPK3 acts as a tumor suppressor in malignant mesothelioma by triggering necroptosis and that epigenetic silencing of by DNA methylation impairs necroptosis and contributes to chemoresistance and poor survival in this incurable disease.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-3683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7887036PMC
February 2021

Netrin G1 Promotes Pancreatic Tumorigenesis through Cancer-Associated Fibroblast-Driven Nutritional Support and Immunosuppression.

Cancer Discov 2021 Feb 30;11(2):446-479. Epub 2020 Oct 30.

Cancer Biology Program, Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania.

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multiplex data from patient tissue, three-dimensional coculturing assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. We found that NetG1 cancer-associated fibroblasts (CAF) support PDAC survival, through a NetG1-mediated effect on glutamate/glutamine metabolism. Also, NetG1 CAFs are intrinsically immunosuppressive and inhibit natural killer cell-mediated killing of tumor cells. These protumor functions are controlled by a signaling circuit downstream of NetG1, which is comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally, blocking NetG1 with a neutralizing antibody stunts tumorigenesis, suggesting NetG1 as potential target in PDAC. SIGNIFICANCE: This study demonstrates the feasibility of targeting a fibroblastic protein, NetG1, which can limit PDAC tumorigenesis by reverting the protumorigenic properties of CAFs. Moreover, inhibition of metabolic proteins in CAFs altered their immunosuppressive capacity, linking metabolism with immunomodulatory function...
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http://dx.doi.org/10.1158/2159-8290.CD-20-0775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7858242PMC
February 2021

SARS-CoV2-mediated suppression of NRF2-signaling reveals potent antiviral and anti-inflammatory activity of 4-octyl-itaconate and dimethyl fumarate.

Nat Commun 2020 10 2;11(1):4938. Epub 2020 Oct 2.

Department of Biomedicine, Aarhus Research Center for Innate Immunology, Aarhus University, Aarhus, Denmark.

Antiviral strategies to inhibit Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and the pathogenic consequences of COVID-19 are urgently required. Here, we demonstrate that the NRF2 antioxidant gene expression pathway is suppressed in biopsies obtained from COVID-19 patients. Further, we uncover that NRF2 agonists 4-octyl-itaconate (4-OI) and the clinically approved dimethyl fumarate (DMF) induce a cellular antiviral program that potently inhibits replication of SARS-CoV2 across cell lines. The inhibitory effect of 4-OI and DMF extends to the replication of several other pathogenic viruses including Herpes Simplex Virus-1 and-2, Vaccinia virus, and Zika virus through a type I interferon (IFN)-independent mechanism. In addition, 4-OI and DMF limit host inflammatory responses to SARS-CoV2 infection associated with airway COVID-19 pathology. In conclusion, NRF2 agonists 4-OI and DMF induce a distinct IFN-independent antiviral program that is broadly effective in limiting virus replication and in suppressing the pro-inflammatory responses of human pathogenic viruses, including SARS-CoV2.
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http://dx.doi.org/10.1038/s41467-020-18764-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7532469PMC
October 2020

Necroptosis restricts influenza A virus as a stand-alone cell death mechanism.

J Exp Med 2020 11;217(11)

Blood Cell Development and Function Program, Fox Chase Cancer Center, Philadelphia, PA.

Influenza A virus (IAV) activates ZBP1-initiated RIPK3-dependent parallel pathways of necroptosis and apoptosis in infected cells. Although mice deficient in both pathways fail to control IAV and succumb to lethal respiratory infection, RIPK3-mediated apoptosis by itself can limit IAV, without need for necroptosis. However, whether necroptosis, conventionally considered a fail-safe cell death mechanism to apoptosis, can restrict IAV-or indeed any virus-in the absence of apoptosis is not known. Here, we use mice selectively deficient in IAV-activated apoptosis to show that necroptosis drives robust antiviral immune responses and promotes effective virus clearance from infected lungs when apoptosis is absent. We also demonstrate that apoptosis and necroptosis are mutually exclusive fates in IAV-infected cells. Thus, necroptosis is an independent, "stand-alone" cell death mechanism that fully compensates for the absence of apoptosis in antiviral host defense.
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http://dx.doi.org/10.1084/jem.20191259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596817PMC
November 2020

Influenza Virus Z-RNAs Induce ZBP1-Mediated Necroptosis.

Cell 2020 03;180(6):1115-1129.e13

Blood Cell Development and Function Program, Fox Chase Cancer Center, Philadelphia, PA, USA. Electronic address:

Influenza A virus (IAV) is a lytic RNA virus that triggers receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated pathways of apoptosis and mixed lineage kinase domain-like pseudokinase (MLKL)-dependent necroptosis in infected cells. ZBP1 initiates RIPK3-driven cell death by sensing IAV RNA and activating RIPK3. Here, we show that replicating IAV generates Z-RNAs, which activate ZBP1 in the nucleus of infected cells. ZBP1 then initiates RIPK3-mediated MLKL activation in the nucleus, resulting in nuclear envelope disruption, leakage of DNA into the cytosol, and eventual necroptosis. Cell death induced by nuclear MLKL was a potent activator of neutrophils, a cell type known to drive inflammatory pathology in virulent IAV disease. Consequently, MLKL-deficient mice manifest reduced nuclear disruption of lung epithelia, decreased neutrophil recruitment into infected lungs, and increased survival following a lethal dose of IAV. These results implicate Z-RNA as a new pathogen-associated molecular pattern and describe a ZBP1-initiated nucleus-to-plasma membrane "inside-out" death pathway with potentially pathogenic consequences in severe cases of influenza.
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http://dx.doi.org/10.1016/j.cell.2020.02.050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7153753PMC
March 2020

Benefits and Perils of Necroptosis in Influenza Virus Infection.

J Virol 2020 04 16;94(9). Epub 2020 Apr 16.

Blood Cell Development and Function Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA

Influenza A viruses (IAV) are lytic viruses that have recently been found to activate necroptosis in many of the cell types they infect. Necroptotic cell death is potently immunogenic and limits IAV spread by directly eliminating infected cells and by mobilizing both innate and adaptive immune responses. The benefits of necroptosis to the host, however, may sometimes be outweighed by the potentially deleterious hyperinflammatory consequences of activating this death modality in pulmonary and other tissues.
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http://dx.doi.org/10.1128/JVI.01101-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7163144PMC
April 2020

ZBP1/DAI-Dependent Cell Death Pathways in Influenza A Virus Immunity and Pathogenesis.

Curr Top Microbiol Immunol 2020 Jan 23. Epub 2020 Jan 23.

Blood Cell Development and Function Program, Fox Chase Cancer Center, Room 224 Reimann Building, 333 Cottman Ave., 19111, Philadelphia, PA, USA.

Influenza A viruses (IAV) are members of the Orthomyxoviridae family of negative-sense RNA viruses. The greatest diversity of IAV strains is found in aquatic birds, but a subset of strains infects other avian as well as mammalian species, including humans. In aquatic birds, infection is largely restricted to the gastrointestinal tract and spread is through feces, while in humans and other mammals, respiratory epithelial cells are the primary sites supporting productive replication and transmission. IAV triggers the death of most cell types in which it replicates, both in culture and in vivo. When well controlled, such cell death is considered an effective host defense mechanism that eliminates infected cells and limits virus spread. Unchecked or inopportune cell death also results in immunopathology. In this review, we discuss the impact of cell death in restricting virus spread, supporting the adaptive immune response and driving pathogenesis in the mammalian respiratory tract. Recent studies have begun to shed light on the signaling pathways underlying IAV-activated cell death. These pathways, initiated by the pathogen sensor protein ZBP1 (also called DAI and DLM1), cause infected cells to undergo apoptosis, necroptosis, and pyroptosis. We outline mechanisms of ZBP1-mediated cell death signaling following IAV infection.
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http://dx.doi.org/10.1007/82_2019_190DOI Listing
January 2020

Nrf2 Negatively Regulates Type I Interferon Responses and Increases Susceptibility to Herpes Genital Infection in Mice.

Front Immunol 2019 6;10:2101. Epub 2019 Sep 6.

Department of Biomedicine, Aarhus Research Center for Innate Immunology, Aarhus University, Aarhus, Denmark.

Herpes simplex virus-2 (HSV-2) is a leading cause of sexually transmitted infections for which no effective vaccines or prophylactic treatment currently exist. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor involved in the detoxification of reactive oxygen species (ROS) and has been more recently shown to regulate inflammatory and antiviral responses. Here, we evaluated the importance of Nrf2 in the control of HSV-2 genital infection, and its role in the regulation of HSV-induced innate antiviral immunity. Comparison of antiviral gene expression profile by RNA-sequencing analysis of wild type and -mutant ( ) murine macrophages showed an upregulation at the basal level of the type I interferon-associated gene network. The same basal increased antiviral profile was also observed in the spleen of mice. Interestingly, the lack of Nrf2 in murine cells was sufficient to increase the responsiveness to HSV-derived dsDNA and protect cells from HSV-2 infection . Surprisingly, there was no indication of an alteration in STING expression in murine cells as previously reported in cells of human origin. Additionally, genetic activation of Nrf2 in mouse embryonic fibroblasts increased HSV-2 infectivity and replication. Finally, using an vaginal herpes infection model, we showed that Nrf2 controlled early innate immune responses to HSV-2 without affecting STING expression levels. mice exhibited reduced viral replication that was associated with higher level of type I interferons in vaginal washes. mice also displayed reduced weight loss, lower disease scores, and higher survival rates than wild type animals. Collectively, these data identify Nrf2 as a negative regulator of the interferon-driven antiviral response to HSV-2 without impairing STING mRNA and protein expression levels in murine cells.
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http://dx.doi.org/10.3389/fimmu.2019.02101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6742979PMC
October 2020

ZBP1/DAI Drives RIPK3-Mediated Cell Death Induced by IFNs in the Absence of RIPK1.

J Immunol 2019 09 29;203(5):1348-1355. Epub 2019 Jul 29.

Blood Cell Development and Function Program, Fox Chase Cancer Center, Philadelphia, PA 19111;

Receptor-interacting protein kinase 1 (RIPK1) regulates cell fate and proinflammatory signaling downstream of multiple innate immune pathways, including those initiated by TNF-α, TLR ligands, and IFNs. Genetic ablation of results in perinatal lethality arising from both RIPK3-mediated necroptosis and FADD/caspase-8-driven apoptosis. IFNs are thought to contribute to the lethality of -deficient mice by activating inopportune cell death during parturition, but how IFNs activate cell death in the absence of RIPK1 is not understood. In this study, we show that Z-form nucleic acid binding protein 1 (ZBP1; also known as DAI) drives IFN-stimulated cell death in settings of RIPK1 deficiency. IFN-activated Jak/STAT signaling induces robust expression of ZBP1, which complexes with RIPK3 in the absence of RIPK1 to trigger RIPK3-driven pathways of caspase-8-mediated apoptosis and MLKL-driven necroptosis. In vivo, deletion of either or core IFN signaling components prolong viability of mice for up to 3 mo beyond parturition. Together, these studies implicate ZBP1 as the dominant activator of IFN-driven RIPK3 activation and perinatal lethality in the absence of RIPK1.
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http://dx.doi.org/10.4049/jimmunol.1900216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702065PMC
September 2019

Bayonets over bombs: RIPK3 and MLKL restrict without triggering necroptosis.

J Cell Biol 2019 Jun 16;218(6):1773-1775. Epub 2019 May 16.

Blood Cell Development and Function Program, Fox Chase Cancer Center, Philadelphia, PA

RIPK3 induces necroptosis by phosphorylating MLKL, which then induces plasma membrane rupture and necrotic cell death. In this issue, Sai et al. (2019. https://doi.org/10.1083/jcb.201810014) show that RIPK3-MLKL signaling in epithelial cells promotes clearance by directly suppressing cytosolic bacterial replication, without activating cell death.
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http://dx.doi.org/10.1083/jcb.201905047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548124PMC
June 2019

Molecular model linking Th2 polarized M2 tumour-associated macrophages with deaminase-mediated cancer progression mutation signatures.

Scand J Immunol 2019 May 18;89(5):e12760. Epub 2019 Mar 18.

GMDxCo Pty Ltd, Melbourne, Victoria, Australia.

A new and diverse range of somatic mutation signatures are observed in late-stage cancers, but the underlying reasons are not fully understood. We advance a "combinatorial association model" for deaminase binding domain (DBD) diversification to explain the generation of previously observed cancer-progression associated mutation signatures. We also propose that changes in the polarization of tumour-associated macrophages (TAMs) are accompanied by the expression of deaminases with a new and diverse range of DBDs, and thus accounting for the generation of new somatic mutation signatures. The mechanism proposed is molecularly reminiscent of combinatorial association of heavy (H) and light (L) protein chains following V(D)J recombination of immunoglobulin molecules (and similarly for protein chains in heterodimers α/β and γ/δ of V(D)Js of T Cell Receptors) required for pathogen antigen recognition by B cells and T cells, respectively. We also discuss whether extracellular vesicles (EVs) emanating from tumour enhancing M2-polarized macrophages represent a likely source of the de novo deaminase DBDs. We conclude that M2-polarized macrophages extruding EVs loaded with deaminase proteins or deaminase-specific transcription/translation regulatory factors and like information may directly trigger deaminase diversification within cancer cells, and thus account for the many new somatic mutation signatures that are indicative of cancer progression. This hypothesis now has a plausible evidentiary base, and it is worth direct testing in future investigations. A long-term objective would be to identify molecular biomarkers predicting cancer progression (or metastatic disease) and to support the development of new drug targets before metastatic pathways are activated.
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http://dx.doi.org/10.1111/sji.12760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6850162PMC
May 2019

Nrf2 negatively regulates STING indicating a link between antiviral sensing and metabolic reprogramming.

Nat Commun 2018 08 29;9(1):3506. Epub 2018 Aug 29.

Department of Biomedicine, Aarhus Research Center for Innate Immunology, Aarhus University, Bartholin Alle 6, 8000, Aarhus, Denmark.

The transcription factor Nrf2 is a critical regulator of inflammatory responses. If and how Nrf2 also affects cytosolic nucleic acid sensing is currently unknown. Here we identify Nrf2 as an important negative regulator of STING and suggest a link between metabolic reprogramming and antiviral cytosolic DNA sensing in human cells. Here, Nrf2 activation decreases STING expression and responsiveness to STING agonists while increasing susceptibility to infection with DNA viruses. Mechanistically, Nrf2 regulates STING expression by decreasing STING mRNA stability. Repression of STING by Nrf2 occurs in metabolically reprogrammed cells following TLR4/7 engagement, and is inducible by a cell-permeable derivative of the TCA-cycle-derived metabolite itaconate (4-octyl-itaconate, 4-OI). Additionally, engagement of this pathway by 4-OI or the Nrf2 inducer sulforaphane is sufficient to repress STING expression and type I IFN production in cells from patients with STING-dependent interferonopathies. We propose Nrf2 inducers as a future treatment option in STING-dependent inflammatory diseases.
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http://dx.doi.org/10.1038/s41467-018-05861-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6115435PMC
August 2018

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

Methods Mol Biol 2018 ;1857:93-99

Blood Cell Development and Function Program, Fox Chase Cancer Center, Philadelphia, PA, USA.

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-β, and IFN-γ, and upon infection with influenza A virus (IAV).
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http://dx.doi.org/10.1007/978-1-4939-8754-2_9DOI Listing
May 2019

Correction to: Phenytoin inhibits necroptosis.

Cell Death Dis 2018 May 24;9(6):622. Epub 2018 May 24.

Division of Nephrology, Medical Clinic 3, University Hospital Carl Gustav Carus, Technical University Dresden, Dresden, Germany.

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.
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http://dx.doi.org/10.1038/s41419-018-0638-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5967310PMC
May 2018

A Nonpyroptotic IFN-γ-Triggered Cell Death Mechanism in Nonphagocytic Cells Promotes Clearance In Vivo.

J Immunol 2018 05 13;200(10):3626-3634. Epub 2018 Apr 13.

Blood Cell Development and Function Program, Fox Chase Cancer Center, Philadelphia, PA 19111;

The cytokine IFN-γ has well-established antibacterial properties against the bacterium in phagocytes, but less is known about the effects of IFN-γ on -infected nonphagocytic cells, such as intestinal epithelial cells (IECs) and fibroblasts. In this article, we show that exposing human and murine IECs and fibroblasts to IFN-γ following infection with triggers a novel form of cell death that is neither pyroptosis nor any of the major known forms of programmed cell death. Cell death required IFN-γ-signaling via STAT1-IRF1-mediated induction of guanylate binding proteins and the presence of live in the cytosol. In vivo, ablating IFN-γ signaling selectively in murine IECs led to higher bacterial burden in colon contents and increased inflammation in the intestine of infected mice. Together, these results demonstrate that IFN-γ signaling triggers release of from the -containing vacuole into the cytosol of infected nonphagocytic cells, resulting in a form of nonpyroptotic cell death that prevents bacterial spread in the gut.
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http://dx.doi.org/10.4049/jimmunol.1701386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6034694PMC
May 2018

Phenytoin inhibits necroptosis.

Cell Death Dis 2018 03 2;9(3):359. Epub 2018 Mar 2.

Division of Nephrology, Medical Clinic 3, University Hospital Carl Gustav Carus, Technical University Dresden, Dresden, Germany.

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.
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http://dx.doi.org/10.1038/s41419-018-0394-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834524PMC
March 2018

Pandemic H1N1 influenza A viruses suppress immunogenic RIPK3-driven dendritic cell death.

Nat Commun 2017 12 5;8(1):1931. Epub 2017 Dec 5.

Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.

The risk of emerging pandemic influenza A viruses (IAVs) that approach the devastating 1918 strain motivates finding strain-specific host-pathogen mechanisms. During infection, dendritic cells (DC) mature into antigen-presenting cells that activate T cells, linking innate to adaptive immunity. DC infection with seasonal IAVs, but not with the 1918 and 2009 pandemic strains, induces global RNA degradation. Here, we show that DC infection with seasonal IAV causes immunogenic RIPK3-mediated cell death. Pandemic IAV suppresses this immunogenic DC cell death. Only DC infected with seasonal IAV, but not with pandemic IAV, enhance maturation of uninfected DC and T cell proliferation. In vivo, circulating T cell levels are reduced after pandemic, but not seasonal, IAV infection. Using recombinant viruses, we identify the HA genomic segment as the mediator of cell death inhibition. These results show how pandemic influenza viruses subvert the immune response.
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http://dx.doi.org/10.1038/s41467-017-02035-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5715119PMC
December 2017

Protein Kinase C Epsilon Is a Key Regulator of Mitochondrial Redox Homeostasis in Acute Myeloid Leukemia.

Clin Cancer Res 2018 02 10;24(3):608-618. Epub 2017 Nov 10.

Fox Chase Cancer Center, Philadelphia, Pennsylvania.

The intracellular redox environment of acute myeloid leukemia (AML) cells is often highly oxidized compared to healthy hematopoietic progenitors and this is purported to contribute to disease pathogenesis. However, the redox regulators that allow AML cell survival in this oxidized environment remain largely unknown. Utilizing several chemical and genetically-encoded redox sensing probes across multiple human and mouse models of AML, we evaluated the role of the serine/threonine kinase PKC-epsilon (PKCε) in intracellular redox biology, cell survival and disease progression. We show that RNA interference-mediated inhibition of PKCε significantly reduces patient-derived AML cell survival as well as disease onset in a genetically engineered mouse model (GEMM) of AML driven by MLL-AF9. We also show that PKCε inhibition induces multiple reactive oxygen species (ROS) and that neutralization of mitochondrial ROS with chemical antioxidants or co-expression of the mitochondrial ROS-buffering enzymes SOD2 and CAT, mitigates the anti-leukemia effects of PKCε inhibition. Moreover, direct inhibition of SOD2 increases mitochondrial ROS and significantly impedes AML progression Furthermore, we report that PKCε over-expression protects AML cells from otherwise-lethal doses of mitochondrial ROS-inducing agents. Proteomic analysis reveals that PKCε may control mitochondrial ROS by controlling the expression of regulatory proteins of redox homeostasis, electron transport chain flux, as well as outer mitochondrial membrane potential and transport. This study uncovers a previously unrecognized role for PKCε in supporting AML cell survival and disease progression by regulating mitochondrial ROS biology and positions mitochondrial redox regulators as potential therapeutic targets in AML. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-2684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5796864PMC
February 2018

RIPK3-driven cell death during virus infections.

Immunol Rev 2017 05;277(1):90-101

Blood Cell Development and Function Program, Fox Chase Cancer Center, Philadelphia, PA, USA.

The programmed self-destruction of infected cells is a powerful antimicrobial strategy in metazoans. For decades, apoptosis represented the dominant mechanism by which the virus-infected cell was thought to undergo programmed cell death. More recently, however, new mechanisms of cell death have been described that are also key to host defense. One such mechanism in vertebrates is programmed necrosis, or "necroptosis", driven by receptor-interacting protein kinase 3 (RIPK3). Once activated by innate immune stimuli, including virus infections, RIPK3 phosphorylates the mixed lineage kinase domain-like protein (MLKL), which then disrupts cellular membranes to effect necroptosis. Emerging evidence demonstrates that RIPK3 can also mediate apoptosis and regulate inflammasomes. Here, we review studies on the mechanisms by which viruses activate RIPK3 and the pathways engaged by RIPK3 that drive cell death.
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http://dx.doi.org/10.1111/imr.12539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7537726PMC
May 2017

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

J Immunol 2017 06 1;198(11):4435-4447. Epub 2017 May 1.

Medical Scientist Training Program, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, MA 02111;

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. Using genetic and pharmacologic tools, we show that catalytic activity of RIPK1 directs IFN-β synthesis induced by LPS in mice. Additionally, we report that RIPK1 kinase-dependent IFN-β 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-β production downstream of the LPS receptor TLR4. Intriguingly, we also show that RIPK1 and RIPK3 kinase-dependent synthesis of IFN-β is markedly induced by avirulent strains of Gram-negative bacteria, and , and less so by their wild-type counterparts. Overall, these observations identify unexpected roles for RIPK1 and RIPK3 kinases in the production of IFN-β 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.
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http://dx.doi.org/10.4049/jimmunol.1601717DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5471631PMC
June 2017

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

J Cell Sci 2016 11 19;129(22):4190-4199. Epub 2016 Oct 19.

Dept. of Medical Genetics and Molecular Biochemistry, Lewis Katz School of Medicine, Temple University, Philadelphia, PA 19140, USA

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. 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.
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http://dx.doi.org/10.1242/jcs.185421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6518330PMC
November 2016

TAX1BP1 Restrains Virus-Induced Apoptosis by Facilitating Itch-Mediated Degradation of the Mitochondrial Adaptor MAVS.

Mol Cell Biol 2017 01 19;37(1). Epub 2016 Dec 19.

Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, Maryland, USA

The host response to RNA virus infection consists of an intrinsic innate immune response and the induction of apoptosis as mechanisms to restrict viral replication. The mitochondrial adaptor molecule MAVS plays critical roles in coordinating both virus-induced type I interferon production and apoptosis; however, the regulation of MAVS-mediated apoptosis is poorly understood. Here, we show that the adaptor protein TAX1BP1 functions as a negative regulator of virus-induced apoptosis. TAX1BP1-deficient cells are highly sensitive to apoptosis in response to infection with the RNA viruses vesicular stomatitis virus and Sendai virus and to transfection with poly(I·C). TAX1BP1 undergoes degradation during RNA virus infection, and loss of TAX1BP1 is associated with apoptotic cell death. TAX1BP1 deficiency augments virus-induced activation of proapoptotic c-Jun N-terminal kinase (JNK) signaling. Virus infection promotes the mitochondrial localization of TAX1BP1 and concomitant interaction with the mitochondrial adaptor MAVS. TAX1BP1 recruits the E3 ligase Itch to MAVS to trigger its ubiquitination and degradation, and loss of TAX1BP1 or Itch results in increased MAVS protein expression. Together, these results indicate that TAX1BP1 functions as an adaptor molecule for Itch to target MAVS during RNA virus infection and thus restrict virus-induced apoptosis.
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http://dx.doi.org/10.1128/MCB.00422-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5192085PMC
January 2017

Interferon-γ in Salmonella pathogenesis: New tricks for an old dog.

Cytokine 2017 10 20;98:27-32. Epub 2016 Oct 20.

Blood Cell Development and Function Program, Fox Chase Cancer Center, Philadelphia, PA 19111, United States. Electronic address:

Salmonella enterica is a facultative intracellular bacterium that is the leading cause of food borne illnesses in humans. The cytokine IFN-γ has well-established antibacterial properties against Salmonella and other intracellular microbes, for example its capacity to activate macrophages, promote phagocytosis, and destroy phagocytosed microbes by free radical-driven toxification of phagosomes. But IFN-γ induces the expression of hundreds of uncharacterized genes, suggesting that this cytokine deploys additional antimicrobial strategies that await discovery. Recently, one such mechanism, mediated by a family of IFN-inducible small GTPases called Guanylate Binding Proteins (GBPs) has been uncovered. GBPs were shown to facilitate the pyroptotic clearance of Salmonella from infected macrophages by rupturing the protective intracellular vacuole this microbe forms around itself. Once this protective vacuole is lost, exposed Salmonella activates pyroptosis, which destroys the infected cell. In this review, we summarize such emerging roles for IFN-γ in restricting Salmonella pathogenesis.
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http://dx.doi.org/10.1016/j.cyto.2016.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5398957PMC
October 2017

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

Cell Host Microbe 2016 Nov 13;20(5):674-681. Epub 2016 Oct 13.

Blood Cell Development and Function Program, Fox Chase Cancer Center, Philadelphia, PA 19111, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.chom.2016.09.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5687825PMC
November 2016

Immune-Mediated Nephropathy and Systemic Autoimmunity in Mice Does Not Require Receptor Interacting Protein Kinase 3 (RIPK3).

PLoS One 2016;11(9):e0163611. Epub 2016 Sep 26.

Department of Medicine/Rheumatology Section, Lewis Katz School of Medicine, Temple University, 3500 N. Broad Street, Philadelphia, PA, 19140, United States of America.

Immune mediated nephropathy is one of the most serious manifestations of lupus and is characterized by severe inflammation and necrosis that, if untreated, eventually leads to renal failure. Although lupus has a higher incidence in women, both sexes can develop lupus glomerulonephritis; nephritis in men develops earlier and is more severe than in women. It is therefore important to understand the cellular and molecular mechanisms mediating nephritis in each sex. Previous work by our lab found that the absence or pharmacological inhibition of Poly [ADP-ribose] polymerase 1 (PARP-1), an enzyme involved in DNA repair and necrotic cell death, affects only male mice and results in milder nephritis, with less in situ inflammation, and diminished incidence of necrotic lesions, allowing for higher survival rates. A second pathway mediating necrosis involves Receptor-Interacting Serine-Threonine Kinase 3 (RIPK3); in this study we sought to investigate the impact of RIPK3 on the development of lupus and nephritis in both sexes. To this end, we used two inducible murine models of lupus: chronic graft versus host disease (cGvHD) and pristane-induced lupus; and nephrotoxic serum (NTS)-induced nephritis as a model of immune mediated nephropathy. We found that the absence of RIPK3 has neither positive nor negative impact on the disease development or progression of lupus and nephritis in all three models, and in both male and female mice. We conclude that RIPK3 is dispensable for the pathogenesis of lupus and immune mediated nephropathy as to accelerate, worsen or ameliorate the disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0163611PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5036882PMC
September 2016

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

Immunity 2016 07 5;45(1):46-59. Epub 2016 Jul 5.

Program in Pharmacology and Experimental Therapeutics, Sackler Graduate School, Tufts University, Boston, MA 02111, USA.

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.
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http://dx.doi.org/10.1016/j.immuni.2016.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4956514PMC
July 2016

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

PLoS One 2016 8;11(7):e0158774. Epub 2016 Jul 8.

Blood Cell Development and Function Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of America.

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-β 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-β 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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0158774PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4938532PMC
August 2017
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