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.

Caspase-8 restricts antiviral CD8 T cell hyperaccumulation.

The magnitude of CD8 T cell responses against viruses is checked by the balance of proliferation and death. Caspase-8 (CASP8) has the potential to influence response characteristics through initiation of apoptosis, suppression of necroptosis, and modulation of cell death-independent signal transduction. Mice deficient in CASP8 and RIPK3 (Casp8-/-Ripk3-/- ) mount enhanced peak CD8 T cell levels against the natural mouse pathogen murine cytomegalovirus (MCMV) or the human pathogen herpes simplex virus-1 compared with littermate control RIPK3-deficient or WT C57BL/6 mice, suggesting an impact of CASP8 on the magnitude of antiviral CD8 T cell expansion and not on contraction. The higher peak response to MCMV in Casp8-/-Ripk3-/- mice resulted from accumulation of greater numbers of terminally differentiated KLRG1hi effector CD8 T cell subsets. Antiviral Casp8-/-Ripk3-/- T cells exhibited enhanced proliferation when splenocytes were transferred into WT recipient mice. Thus, cell-autonomous CASP8 normally restricts CD8 T cell proliferation following T cell receptor activation in response to foreign antigen. Memory inflation is a hallmark quality of the T cell response to cytomegalovirus infection. Surprisingly, MCMV-specific memory inflation was not sustained long-term in Casp8-/-Ripk3-/- mice even though these mice retained immunity to secondary challenge. In addition, the accumulation of abnormal B220+CD3+ T cells in these viable CASP8-deficient mice was reduced by chronic MCMV infection. Combined, these data brings to light the cell death-independent role of CASP8 during CD8 T cell expansion in mice lacking the confounding impact of RIPK3-mediated necroptosis.

Cytomegalovirus impairs antiviral CD8+ T cell immunity by recruiting inflammatory monocytes.

Inflammatory monocytes are key early responders to infection that contribute to pathogen-host interactions in diverse ways. Here, we report that the murine cytomegalovirus-encoded CC chemokine, MCK2, enhanced CCR2-dependent recruitment of these cells to modulate antiviral immunity, impairing virus-specific CD8(+) T cell expansion and differentiation into effector cytotoxic T lymphocytes, thus reducing the capacity to eliminate viral antigen-bearing cells and slowing viral clearance. Adoptive transfer of inflammatory monocytes into Ccr2(-/-)Ccl2(-/-) mice impaired virus antigen-specific clearance. Cytomegalovirus therefore enhances a natural CCR2-dependent immune regulatory network to modulate adaptive immunity via nitric oxide production, reminiscent of the monocytic subtype of myeloid-derived suppressor cells primarily implicated in cancer immunomodulation.

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.

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

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.

Caspase-8-dependent control of NK- and T cell responses during cytomegalovirus infection.

Caspase-8 (CASP8) impacts antiviral immunity in expected as well as unexpected ways. Mice with combined deficiency in CASP8 and RIPK3 cannot support extrinsic apoptosis or RIPK3-dependent programmed necrosis, enabling studies of CASP8 function without complications of unleashed necroptosis. These extrinsic cell death pathways are naturally targeted by murine cytomegalovirus (MCMV)-encoded cell death suppressors, showing they are key to cell-autonomous host defense. Remarkably, Casp8-/-Ripk3-/-, Ripk1-/-Casp8-/-Ripk3-/- and Casp8-/-Ripk3K51A/K51A mice mount robust antiviral T cell responses to control MCMV infection. Studies in Casp8-/-Ripk3-/- mice show that CASP8 restrains expansion of MCMV-specific natural killer (NK) and CD8 T cells without compromising contraction or immune memory. Infected Casp8-/-Ripk3-/- or Casp8-/-Ripk3K51A/K51A mice have higher levels of virus-specific NK cells and CD8 T cells compared to matched RIPK3-deficient littermates or WT mice. CASP8, likely acting downstream of Fas death receptor, dampens proliferation of CD8 T cells during expansion. Importantly, contraction proceeds unimpaired in the absence of extrinsic death pathways owing to intact Bim-dependent (intrinsic) apoptosis. CD8 T cell memory develops in Casp8-/-Ripk3-/- mice, but memory inflation characteristic of MCMV infection is not sustained in the absence of CASP8 function. Despite this, Casp8-/-Ripk3-/- mice are immune to secondary challenge. Interferon (IFN)γ is recognized as a key cytokine for adaptive immune control of MCMV. Ifngr-/-Casp8-/-Ripk3-/- mice exhibit increased lifelong persistence in salivary glands as well as lungs compared to Ifngr-/- and Casp8-/-Ripk3-/- mice. Thus, mice deficient in CASP8 and RIPK3 are more dependent on IFNγ mechanisms for sustained T cell immune control of MCMV. Overall, appropriate NK- and T cell immunity to MCMV is dependent on host CASP8 function independent of RIPK3-regulated pathways.

Caspase-8 restricts natural killer cell accumulation during MCMV Infection.

Natural killer (NK) cells provide important host defense against herpesvirus infections and influence subsequent T cell control of replication and maintenance of latency. NK cells exhibit phases of expansion, contraction and memory formation in response to the natural mouse pathogen murine cytomegalovirus (MCMV). Innate and adaptive immune responses are tightly regulated in mammals to avoid excess tissue damage while preventing acute and chronic viral disease and assuring resistance to reinfection. Caspase (CASP)8 is an autoactivating aspartate-specific cysteine protease that initiates extrinsic apoptosis and prevents receptor interacting protein (RIP) kinase (RIPK)1-RIPK3-driven necroptosis. CASP8 also promotes death-independent signal transduction. All of these activities make contributions to inflammation. Here, we demonstrate that CASP8 restricts NK cell expansion during MCMV infection but does not influence NK memory. Casp8-/-Ripk3-/- mice mount higher NK response levels than Casp8+/-Ripk3-/- littermate controls or WT C57BL/6 J mice, indicating that RIPK3 deficiency alone does not contribute to NK response patterns. MCMV m157-responsive Ly49H+ NK cells support increased expansion of both Ly49H- NK cells and CD8 T cells in Casp8-/-Ripk3-/- mice. Surprisingly, hyperaccumulation of NK cells depends on the pronecrotic kinase RIPK1. Ripk1-/-Casp8-/-Ripk3-/- mice fail to show the enhanced expansion of lymphocytes observed in Casp8-/-Ripk3-/- mice even though development and homeostasis are preserved in uninfected Ripk1-/-Casp8-/-Ripk3-/- mice. Thus, CASP8 naturally regulates the magnitude of NK cell responses in response to infection where strong activation signals depend on another key regulator of death signaling, RIPK1. In addition, the strong NK cell response promotes survival of effector CD8 T cells during their expansion. Thus, hyperaccumulation of NK cells and crosstalk with T cells becomes amplified in the absence of extrinsic cell death machinery.

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.

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

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.

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.

Viral immunity: Basic mechanisms and therapeutic applications-a Keystone Symposia report.

Viruses infect millions of people each year. Both endemic viruses circulating throughout the population as well as novel epidemic and pandemic viruses pose ongoing threats to global public health. Developing more effective tools to address viruses requires not only in-depth knowledge of the virus itself but also of our immune system's response to infection. On June 29 to July 2, 2022, researchers met for the Keystone symposium "Viral Immunity: Basic Mechanisms and Therapeutic Applications." This report presents concise summaries from several of the symposium presenters.

Mesenchymal stromal cells in hematopoietic cell transplantation.

Mesenchymal stromal cells (MSCs) are widely recognized to possess potent immunomodulatory activity, as well as to stimulate repair and regeneration of diseased or damaged tissue. These fundamental properties suggest important applications in hematopoietic cell transplantation. Although the mechanisms of therapeutic activity in vivo are yet to be fully elucidated, MSCs seem to suppress lymphocytes by paracrine mechanisms, including secreted mediators and metabolic modulators. Most recently, host macrophage engulfment of apoptotic MSCs has emerged as an important contributor to the immune suppressive microenvironment. Although bone marrow-derived MSCs are the most commonly studied, the tissue source of MSCs may be a critical determinant of immunomodulatory function. The key application of MSC therapy in hematopoietic cell transplantation is to prevent or treat graft-versus-host disease (GVHD). The pathogenesis of GVHD reveals multiple potential targets. Moreover, the recently proposed concept of tissue tolerance suggests a new possible mechanism of MSC therapy for GVHD. Beyond GVHD, MSCs may facilitate hematopoietic stem cell engraftment, which could gain greater importance with increasing use of haploidentical transplantation. Despite many challenges and much doubt, commercial MSC products for pediatric steroid-refractory GVHD have been licensed in Japan, conditionally licensed in Canada and New Zealand, and have been recommended for approval by an FDA Advisory Committee in the United States. Here, we review key historical data in the context of the most salient recent findings to present the current state of MSCs as adjunct cell therapy in hematopoietic cell transplantation.

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.

Cytomegalovirus hijacks CX3CR1(hi) patrolling monocytes as immune-privileged vehicles for dissemination in mice.

Peripheral blood myelomonocytic cells are important for cytomegalovirus dissemination to distal organs such as salivary glands where persistent replication and shedding dictates transmission patterns. We find that this process is markedly enhanced by the murine cytomegalovirus (MCMV)-encoded CC chemokine, MCK2, which promotes recruitment of CX3CR1(hi) patrolling monocytes to initial infection sites in the mouse. There, these cells become infected and traffic via the bloodstream to distal sites. In contrast, inflammatory monocytes, the other major myelomonocytic subset, remain virus negative. CX3CR1 deficiency prevents patrolling monocyte migration on the vascular endothelium and interrupts MCMV dissemination to the salivary glands independent of antiviral NK and T cell immune control. In this manner, CX3CR1(hi) patrolling monocytes serve as immune-privileged vehicles to transport MCMV via the bloodstream to distal organs. MCMV commandeers patrolling monocytes to mediate systemic infection and seed a persistent reservoir essential for horizontal transmission.