Lisa P. Daley-Bauer

RIP3 mediates the embryonic lethality of caspase-8-deficient mice

Apoptosis and necroptosis are complementary pathways controlled by common signalling adaptors, kinases and proteases; among these, caspase-8 (Casp8) is critical for death receptor-induced apoptosis. This caspase has also been implicated in non-apoptotic pathways that regulate Fas-associated via death domain (FADD)-dependent signalling and other less defined biological processes as diverse as innate immune signalling and myeloid or lymphoid differentiation patterns. Casp8 suppresses RIP3–RIP1 (also known as RIPK3–RIPK1) kinase complex-dependent necroptosis that follows death receptor activation as well as a RIP3-dependent, RIP1-independent necrotic pathway that has emerged as a host defence mechanism against murine cytomegalovirus. Disruption of Casp8 expression leads to embryonic lethality in mice between embryonic days 10.5 and 11.5 (ref. 7). Thus, Casp8 may naturally hold alternative RIP3-dependent death pathways in check in addition to promoting apoptosis. We find that RIP3 is responsible for the mid-gestational death of Casp8-deficient embryos. Remarkably, Casp8−/−Rip3−/− double mutant mice are viable and mature into fertile adults with a full immune complement of myeloid and lymphoid cell types. These mice seem immunocompetent but develop lymphadenopathy by four months of age marked by accumulation of abnormal T cells in the periphery, a phenotype reminiscent of mice with Fas-deficiency (lpr/lpr; also known as Fas). Thus, Casp8 contributes to homeostatic control in the adult immune system; however, RIP3 and Casp8 are together completely dispensable for mammalian development.

RIP3 Induces Apoptosis Independent of Pronecrotic Kinase Activity

Receptor-interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small-molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pronecrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound, whereas D161G, D143N, and K51A mutants, like wild-type, only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3(K51A/K51A)) are viable and fertile, in stark contrast to the perinatal lethality of Rip3(D161N/D161N) mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3.

RIP1 suppresses innate immune necrotic as well as apoptotic cell death during mammalian parturition

Significance The protein kinase receptor interacting protein 1 controls signaling via death receptors, Toll-like receptors, and retinoic acid-inducible gene 1-like receptors, dictating inflammatory outcomes as broad as cytokine activation and cell death. RIP1 makes a vital contribution during development, evident from the fact that RIP1-deficient mice die soon after birth. Here, we show that a kinase-independent function of RIP1 dampens the consequences of innate immune cell death. During parturition, RIP1 prevents the lethal consequences of RIP3-dependent necroptosis as well as caspase 8 (Casp8)-dependent apoptosis. In contrast to the RIP1-deficient phenotype, mice lacking a combination of RIP1, RIP3, and Casp8 are born and mature into viable, fertile, and immunocompetent adults. These results demonstrate the important protective role of RIP1 against physiologic and microbial death cues encountered at birth. 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.

True Grit: Programmed Necrosis in Antiviral Host Defense, Inflammation, and Immunogenicity

Programmed necrosis mediated by receptor interacting protein kinase (RIP)3 (also called RIPK3) has emerged as an alternate death pathway triggered by TNF family death receptors, pathogen sensors, IFNRs, Ag-specific TCR activation, and genotoxic stress. Necrosis leads to cell leakage and acts as a “trap door,” eliminating cells that cannot die by apoptosis because of the elaboration of pathogen-encoded caspase inhibitors. Necrotic signaling requires RIP3 binding to one of three partners—RIP1, DAI, or TRIF—via a common RIP homotypic interaction motif. Once activated, RIP3 kinase targets the pseudokinase mixed lineage kinase domain-like to drive cell lysis. Although necrotic and apoptotic death can enhance T cell cross-priming during infection, mice that lack these extrinsic programmed cell death pathways are able to produce Ag-specific T cells and control viral infection. The entwined relationship of apoptosis and necrosis evolved in response to pathogen-encoded suppressors to support host defense and contribute to inflammation.

Antiviral T Cell Response Triggers Cytomegalovirus Hepatitis in Mice

ABSTRACT One common sign of human cytomegalovirus infection is altered liver function. Murine cytomegalovirus strain v70 induces a rapid and severe hepatitis in immunocompetent mice that requires the presence of T cells in order to develop. v70 exhibits approximately 10-fold-greater virulence than the commonly used strain K181, resulting in a more severe, sustained, and lethal hepatitis but not dramatically higher viral replication levels. Hepatitis and death are markedly delayed in immunodeficient SCID compared to immunocompetent BALB/c mice. Transfer of BALB/c splenocytes to SCID mice conferred rapid disease following infection, and depletion of either CD4 or CD8 T cells in BALB/c mice reduced virus-induced hepatitis. The frequency of CD8 T cells producing gamma interferon and tumor necrosis factor in response to viral antigen was higher in settings where more severe disease occurred. Thus, virus-specific effector CD8 T cells appear to contribute to lethal virus-induced hepatitis, contrasting their protective role during sublethal infection. This study reveals how protection and disease during cytomegalovirus infection depend on viral strain and dose, as well as the quality of the T cell response.

Mouse cytomegalovirus M36 and M45 death suppressors cooperate to prevent inflammation resulting from antiviral programmed cell death pathways

Significance Caspase-8–mediated apoptotic and receptor-interacting protein (RIP)-dependent necroptotic signaling pathways are recognized host defense mechanisms that act by eliminating virus-infected cells. Cytomegalovirus-encoded inhibitors of apoptosis and necroptosis sustain infection and pathogenesis by preventing specific programmed cell death pathways. In the absence of viral inhibitors, combined apoptotic–necroptotic cell death signaling halts infection, preventing the virus from gaining a foothold in the host. We describe natural cooperation between apoptosis and necroptosis pathways in macrophages and within the host, resulting in robust proinflammatory cytokine responses not observed when infected cells die by either apoptosis or necroptosis alone. Thus, apoptosis combined with necroptosis serves a dual role in limiting herpesvirus persistence in the host. The complex interplay between caspase-8 and receptor-interacting protein (RIP) kinase RIP 3 (RIPK3) driving extrinsic apoptosis and necroptosis is not fully understood. Murine cytomegalovirus triggers both apoptosis and necroptosis in infected cells; however, encoded inhibitors of caspase-8 activity (M36) and RIP3 signaling (M45) suppress these antiviral responses. Here, we report that this virus activates caspase-8 in macrophages to trigger apoptosis that gives rise to secondary necroptosis. Infection with double-mutant ΔM36/M45mutRHIM virus reveals a signaling pattern in which caspase-8 activates caspase-3 to drive apoptosis with subsequent RIP3-dependent activation of mixed lineage kinase domain-like (MLKL) leading to necroptosis. This combined cell death signaling is highly inflammatory, greater than either apoptosis induced by ΔM36 or necroptosis induced by M45mutRHIM virus. IL-6 production by macrophages is dramatically increased during double-mutant virus infection and correlates with faster antiviral responses in the host. Collaboratively, M36 and M45 target caspase-8 and RIP3 pathways together to suppress this proinflammatory cell death. This study reveals the effect of antiviral programmed cell death pathways on inflammation, shows that caspase-8 activation may go hand-in-hand with necroptosis in macrophages, and revises current understanding of independent and collaborative functions of M36 and M45 in blocking apoptotic and necroptotic cell death responses.

Viral Replication Assay in Bone Marrow-Derived Macrophages

The selection of macrophages as a cell type for investigating virus-host interactions is based on cellular tropism of the virus during infection as well as contribution of these cells to pathogenesis in the host. In response to mouse cytomegalovirus (MCMV) infection, bone marrow-resident monocytes that mobilize to infected tissues to differentiate into macrophages and dendritic cells are hijacked in order to facilitate viral persistence. These cells contribute significantly to MCMV biology and, thus, are actively recruited by the virus-encoded chemokine. In this chapter, we provide detailed methodologies employed in our laboratory to assess MCMV replication in bone marrow-derived macrophages.

Growing Murine Bone Marrow-Derived Macrophages

Bone marrow-derived macrophages (BMDM) are primary macrophages obtained by in vitro differentiation of bone marrow cells in the presence of macrophage colony-stimulating factor (M-CSF or CSF1). They are easy to obtain in high yields, can be stored by freezing, and can be obtained from genetically modified mice strains. They are therefore widely used as prototypical macrophages for in vitro studies. In this chapter, we present the method for obtaining BMDMs and freezing them.

Remarkably Robust Antiviral Immune Response despite Combined Deficiency in Caspase-8 and RIPK3

Caspase-8 (Casp8)–mediated signaling triggers extrinsic apoptosis while suppressing receptor-interacting protein kinase (RIPK) 3–dependent necroptosis. Although Casp8 is dispensable for the development of innate and adaptive immune compartments in mice, the importance of this proapoptotic protease in the orchestration of immune response to pathogens remains to be fully explored. In this study, Casp8−/−Ripk3−/− C57BL/6 mice show robust innate and adaptive immune responses to the natural mouse pathogen, murine CMV. When young, these mice lack lpr-like lymphoid hyperplasia and accumulation of either B220+CD3+ or B220−CD3+CD4+ and CD8+ T cells with increased numbers of immature myeloid cells that are evident in older mice. Dendritic cell activation and cytokine production drive both NK and T cell responses to control viral infection in these mice, suggesting that Casp8 is dispensable to the generation of antiviral host defense. Curiously, NK and T cell expansion is amplified, with greater numbers observed by 7 d postinfection compared with either Casp8+/−Ripk3−/− or wild type (Casp8+/+Ripk3+/+) littermate controls. Casp8 and RIPK3 are natural targets of virus-encoded cell death suppressors that prevent infected cell apoptosis and necroptosis, respectively. It is clear from the current studies that the initiation of innate immunity and the execution of cytotoxic lymphocyte functions are all preserved despite the absence of Casp8 in responding cells. Thus, Casp8 and RIPK3 signaling is completely dispensable to the generation of immunity against this natural herpesvirus infection, although the pathways driven by these initiators serve as a crucial first line for host defense within virus-infected cells.