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Inborn errors of TLR3-dependent type I IFN immunity in cortical neurons underlie forebrain herpes simplex virus-1 (HSV-1) encephalitis (HSE) due to uncontrolled viral growth and subsequent cell death. We report an otherwise healthy patient with HSE who was compound heterozygous for nonsense (R422*) and frameshift (P493fs9*) RIPK3 variants. Receptor-interacting protein kinase 3 (RIPK3) is a ubiquitous cytoplasmic kinase regulating cell death outcomes, including apoptosis and necroptosis. In vitro, the R422* and P493fs9* RIPK3 proteins impaired cellular apoptosis and necroptosis upon TLR3, TLR4, or TNFR1 stimulation and ZBP1/DAI-mediated necroptotic cell death after HSV-1 infection. The patient’s fibroblasts displayed no detectable RIPK3 expression. After TNFR1 or TLR3 stimulation, the patient’s cells did not undergo apoptosis or necroptosis. After HSV-1 infection, the cells supported excessive viral growth despite normal induction of antiviral IFN-β and IFN-stimulated genes (ISGs). This phenotype was, nevertheless, rescued by application of exogenous type I IFN. The patient’s human pluripotent stem cell (hPSC)–derived cortical neurons displayed impaired cell death and enhanced viral growth after HSV-1 infection, as did isogenic RIPK3-knockout hPSC-derived cortical neurons. Inherited RIPK3 deficiency therefore confers a predisposition to HSE by impairing the cell death–dependent control of HSV-1 in cortical neurons but not their production of or response to type I IFNs.

During aging, environmental stressors and mutations along with reduced DNA repair cause germ cell aneuploidy and genome instability, which limits fertility and embryo development. Benevolent commensal microbiota and dietary plants secrete indoles, which improve healthspan and reproductive success, suggesting regulation of germ cell quality. We show that indoles prevent aneuploidy and promote DNA repair and embryo viability, which depends on age and genotoxic stress levels and affects embryo quality across generations. In young animals or with low doses of radiation, indoles promote DNA repair and embryo viability; however, in older animals or with high doses of radiation, indoles promote death of the embryo. These studies reveal a previously unknown quality control mechanism by which indole integrates DNA repair and cell death responses to preclude germ cell aneuploidy and ensure transgenerational genome integrity. Such regulation affects healthy aging, reproductive senescence, cancer, and the evolution of genetic diversity in invertebrates and vertebrates.

Z-DNA Binding protein 1 (ZBP1) activates Receptor Interacting Protein Kinase 3 (RIPK3) - dependent cell death during lytic infection by members of the orthomyxovirus, herpesvirus and poxvirus families. ZBP1 possesses two Zα domains capable of selective binding to Z-DNA, as well as to Z-RNA. We have now unveiled Z-RNA as the ligand that activates ZBP1 in cells infected with orthomyxoviruses (influenza A and B viruses) and the poxvirus vaccinia virus (VACV). Orthomyxovirus Z-RNA is sensed by ZBP1 in the nucleus of infected cells, resulting in nuclear activation of RIPK3, consequent rupture of the nucleus, and hyper-inflammatory ‘nuclear necroptosis’. VACV-generated Z-RNA accumulates in the cytoplasm, where it is sequestered from ZBP1 by E3, the viral E3L gene product. In viruses where the E3 Zα domain has been mutated, ZBP1 senses Z-RNA and triggers RIPK3-dependent necroptosis in the cytoplasm. Z-RNA is thus a new viral pathogen-associated molecular pattern (PAMP).

RIPK3 kinase activity triggers cell death via necroptosis, while scaffold function supports protein binding and cytokine production. To determine if RIPK3 kinase or scaffold domains mediate pathology during Pseudomonas aeruginosa infection, control mice and those with deletion or mutation of RIPK3 and associated signaling partners were subjected to Pseudomonas pneumonia and followed for survival or sacrificed for biologic assays. Murine immune cells were studied in vitro for Pseudomonas-induced cytokine production and cell death, and RIPK3 binding interactions were blocked with the viral inhibitor M45. Human tissue effects were assayed by infecting airway epithelial cells with Pseudomonas and measuring cytokine production after siRNA inhibition of RIPK3. Deletion of RIPK3 reduced inflammation and decreased animal mortality following Pseudomonas pneumonia. RIPK3 kinase inactivation did neither. In cell culture, RIPK3 was dispensable for cell killing by Pseudomonas and instead drove cytokine production that required the RIPK3 scaffold domain but not kinase activity. Blocking the RIP homotypic interaction motif (RHIM) with M45 reduced the inflammatory response to infection in vitro. Similarly, siRNA knockdown of RIPK3 decreased infection-triggered inflammation in human airway epithelial cells. Thus, the RIPK3 scaffold drives deleterious pulmonary inflammation and mortality in a relevant clinical model of Pseudomonas pneumonia. This process is distinct from kinase-mediated necroptosis, requiring only the RIPK3 RHIM. Inhibition of RHIM signaling is a potential strategy to reduce lung inflammation during infection.

Invasion of the brain by herpes simplex virus 1 (HSV1) can lead to the development of herpes simplex encephalitis (HSE) that is often associated with significant morbidity and mortality regardless of therapeutic intervention. Both virus and host immune factors dictate HSE onset and progression. Because programmed cell death pathways including necroptosis are important antiviral defense mechanisms in HSV1-associated peripheral diseases, they might also play critical roles in HSV1 neuropathogenesis. HSV1-encoded ICP6 prevents receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis during infection of human cells, but it also acts as a species-dependent inducer of necroptosis in murine cells and thereby restricts virus replication. We therefore used an established mouse model of HSE to investigate RIPK3-mediated necroptosis impact on HSV1 neuropathogenesis. Following corneal HSV1 inoculation, RIPK3 knockout mice showed increased susceptibility to HSE when compared with wildtype mice indicating RIPK3 helps to limit HSE progression. RIPK3-mediated defense against HSE was found to be independent of the kinase domain necessary to drive necroptosis implicating that a death independent function of RIPK3 protects against HSE. Conversely the pro-necroptotic kinase function RIPK3 served to limit viral replication in corneal tissue implicating a tissue-specific RIPK3 function in limiting HSV1. Further evaluation of the kinase-independent mechanism to restrict HSE revealed that the RIPK3 signaling partner, caspase 8, contributes to limiting HSE neuropathogenesis. Increased HSE susceptibility from loss of caspase 8 and RIPK3 correlated with decreased levels of chemokines, cytokines, and antiviral lymphocytes recruitment to the brain. We conclude that RIPK3 contributes toward host control of HSV1 replication in a tissue-specific fashion. Whereas RIPK3-mediated necroptosis restricts virus replication within the cornea, kinase-independent induction of inflammation by RIPK3 in collaboration with caspase 8 restricts virus replication within the brain during HSE neuropathogenesis.

Infections caused by members of the mycobacterium tuberculosis complex [MTC] and nontuberculous mycobacteria [NTM] can induce widespread morbidity and mortality in people. Mycobacterial infections cause both a delayed immune response, which limits rate of bacterial clearance, and formation of granulomas, which contain bacterial spread, but also contribute to lung damage, fibrosis, and morbidity. Granulomas also limit access of antibiotics to bacteria, which may facilitate development of resistance. Bacteria resistant to some or all antibiotics cause significant morbidity and mortality, and newly developed antibiotics readily engender resistance, highlighting the need for new therapeutic approaches. Imatinib mesylate, a cancer drug used to treat chronic myelogenous leukemia [CML] that targets Abl and related tyrosine kinases, is a possible host-directed therapeutic [HDT] for mycobacterial infections, including those causing TB. Here, we use the murine Mycobacterium marinum [Mm] infection model, which induces granulomatous tail lesions. Based on histological measurements, imatinib reduces both lesion size and inflammation of surrounding tissue. Transcriptomic analysis of tail lesions indicates that imatinib induces gene signatures indicative of immune activation and regulation at early time points post infection that resemble those seen at later ones, suggesting that imatinib accelerates but does not substantially alter anti-mycobacterial immune responses. Imatinib likewise induces signatures associated with cell death and promotes survival of bone marrow-derived macrophages [BMDMs] in culture following infection with Mm. Notably, the capacity of imatinib to limit formation and growth of granulomas in vivo and to promote survival of BMDMs in vitro depends upon caspase 8, a key regulator of cell survival and death. These data provide evidence for the utility of imatinib as an HDT for mycobacterial infections in accelerating and regulating immune responses, and limiting pathology associated with granulomas, which may mitigate post-treatment morbidity.

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 KLRG1(hi) 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.

Programmed cell death pathways eliminate infected cells and regulate infection-associated inflammation during pathogen invasion. Cytomegaloviruses encode several distinct suppressors that block intrinsic apoptosis, extrinsic apoptosis, and necroptosis, pathways that impact pathogenesis of this ubiquitous herpesvirus. Here, we expanded the understanding of three cell autonomous suppression mechanisms on which murine cytomegalovirus relies: (i) M38.5-encoded viral mitochondrial inhibitor of apoptosis (vMIA), a BAX suppressor that functions in concert with M41.1-encoded viral inhibitor of BAK oligomerization (vIBO), (ii) M36-encoded viral inhibitor of caspase-8 activation (vICA), and (iii) M45-encoded viral inhibitor of RIP/RHIM activation (vIRA). Following infection of bone marrow-derived macrophages, the virus initially deflected receptor-interacting protein kinase (RIPK)3-dependent necroptosis, the most potent of the three cell death pathways. This process remained independent of caspase-8, although suppression of this apoptotic protease enhances necroptosis in most cell types. Second, the virus deflected TNF-mediated extrinsic apoptosis, a pathway dependent on autocrine TNF production by macrophages that proceeds independently of mitochondrial death machinery or RIPK3. Third, cytomegalovirus deflected BCL-2 family protein-dependent mitochondrial cell death through combined TNF-dependent and -independent signaling even in the absence of RIPK1, RIPK3, and caspase-8. Furthermore, each of these cell death pathways dictated a distinct pattern of cytokine and chemokine activation. Therefore, cytomegalovirus employs sequential, non-redundant suppression strategies to specifically modulate the timing and execution of necroptosis, extrinsic apoptosis, and intrinsic apoptosis within infected cells to orchestrate virus control and infection-dependent inflammation. Virus-encoded death suppressors together hold control over an intricate network that upends host defense and supports pathogenesis in the intact mammalian host.

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.

The squalene-based oil-in-water emulsion (SE) vaccine adjuvant MF59 has been administered to more than 100 million people in more than 30 countries, in both seasonal and pandemic influenza vaccines. Despite its wide use and efficacy, its mechanisms of action remain unclear. In this study we demonstrate that immunization of mice with MF59 or its mimetic AddaVax (AV) plus soluble antigen results in robust antigen-specific antibody and CD8 T cell responses in lymph nodes and non-lymphoid tissues. Immunization triggered rapid RIPK3-kinase dependent necroptosis in the lymph node which peaked at 6 hr, followed by a sequential wave of apoptosis. Immunization with alum plus antigen did not induce RIPK3-dependent signaling. RIPK3-dependent signaling induced by MF59 or AV was essential for cross-presentation of antigen to CD8 T cells by Batf3-dependent CD8+ DCs. Consistent with this, RIPK3 deficient or Batf3 deficient mice were impaired in their ability to mount adjuvant-enhanced CD8 T cell responses. However, CD8 T cell responses were unaffected in mice deficient in MLKL, a downstream mediator of necroptosis. Surprisingly, antibody responses were unaffected in RIPK3-kinase or Batf3 deficient mice. In contrast, antibody responses were impaired by in vivo administration of the pan-caspase inhibitor Z-VAD-FMK, but normal in caspase-1 deficient mice, suggesting a contribution from apoptotic caspases, in the induction of antibody responses. These results demonstrate that squalene emulsion-based vaccine adjuvants induce antigen-specific CD8 T cell and antibody responses, through RIPK3-dependent and-independent pathways, respectively.

Proteasome inhibitors have achieved clinical success because they trigger intrinsic and extrinsic cell death to eliminate susceptible human cancers. The ubiquitin-proteasome protein degradation system regulates signaling pathways by controlling levels of components such as cellular inhibitor of apoptosis (cIAP)1 and cIAP2 in TNF-mediated cell death. Here, we sought to evaluate the contribution of necroptosis to the cell death pattern induced by the specific proteasome inhibitor Carfilzomib (Cf). Proteasome inhibitor-sensitive multiple myeloma cell lines die in response to Cf by apoptosis in combination with serine protease-dependent death, without any contribution of RIPK3-dependent necroptosis. Proteasome inhibition leads to the induction of apoptotic markers such as activated caspase-3 rather than necroptotic markers such as phosphorylated-MLKL in all cell lines tested. In HT-29 cells, Cf attenuates the late RIPK1 interaction with TNFR1 during TNF-induced necroptosis without altering the sensitivity of cIAP antagonists. Cf treatment results in decreased translocation of death signaling components RIPK1, FADD, caspase-8, cFLIP, and RIPK3 to detergent insoluble fractions. Our results show that proteasome inhibition with Cf impairs necroptosis and favors apoptosis even in cells with intact necroptotic machinery. Following the induction of TNFR1-mediated necroptosis, proteasome activity stabilizes effective aggregation and activation of ripoptosome/necrosome complexes.

Vaccinia virus (VACV) encodes an innate immune evasion protein, E3, which contains an N-terminal Z-nucleic acid binding (Zα) domain that is critical for pathogenicity in mice. Here we demonstrate that the N terminus of E3 is necessary to inhibit an IFN-primed virus-induced necroptosis. VACV deleted of the Zα domain of E3 (VACV-E3LΔ83N) induced rapid RIPK3-dependent cell death in IFN-treated L929 cells. Cell death was inhibited by the RIPK3 inhibitor, GSK872, and infection with this mutant virus led to phosphorylation and aggregation of MLKL, the executioner of necroptosis. In 293T cells, induction of necroptosis depended on expression of RIPK3 as well as the host-encoded Zα domain-containing DNA sensor, DAI. VACV-E3LΔ83N is attenuated in vivo, and pathogenicity was restored in either RIPK3- or DAI-deficient mice. These data demonstrate that the N terminus of the VACV E3 protein prevents DAI-mediated induction of necroptosis.

Necroptosis mediated by Z-nucleic acid binding protein (ZBP)1 (also known as DAI and DLM1) provides innate host defense against viruses. Unless blocked by vaccinia virus (VACV) protein E3, ZBP1 triggers RIP-homotypic interaction motif (RHIM)-dependent activation of RIPK3 to execute cell death, cut short replication and limit pathogenesis. Here, Z-form RNA is shown to be sequestered in the cytoplasm dependent on the Z-nucleic acid binding domain of E3. Mutating the Zα motif within this domain permits accumulation of free Z-RNA during the early phase of VACV infection that triggers ZBP1-mediated VACV-induced necroptosis. Zα from either ZBP1 or the RNA editing enzyme double-stranded RNA adenosine deaminase (ADAR)1 are functionally interchangeable with E3 Zα and suffice to block virus-induced necroptosis as a component of chimeric viruses. Overall, the evidence shows that Z-form RNA generated during VACV infection unleashes ZBP1-RIPK3-dependent necroptosis and unveils the mechanism through which viral E3 suppresses sensing of this PAMP.

Viral immune evasion is currently understood to focus on deflect-ing CD8 T cell recognition of infected cells by disrupting antigen presentation pathways. We evaluated viral interference with the ultimate step in cytotoxic T cell function, the death of infected cells. The viral inhibitor of caspase-8 activation (vICA) conserved in human cytomegalovirus (HCMV) and murine CMV (MCMV) pre-vents the activation of caspase-8 and proapoptotic signaling. We demonstrate the key role of vICA from either virus, in deflecting antigen-specific CD8 T cell-killing of infected cells. vICA-deficient mutants, lacking either UL36 or M36, exhibit greater susceptibility to CD8 T cell control than mutants lacking the set of immunoeva-sins known to disrupt antigen presentation via MHC class I. This difference is evident during infection in the natural mouse host infected with MCMV, in settings where virus-specific CD8 T cells are adoptively transferred. Finally, we identify the molecular mech-anism through which vICA acts, demonstrating the central contribu-tion of caspase-8 signaling at a point of convergence of death receptor-induced apoptosis and perforin/granzyme-dependent cytotoxicity.

Z-nucleic acid binding protein (ZBP)1 (also known as DAI and DLM1) is a pathogen sensor activated by double-strand character RNA to recruit receptor-interacting protein (RIP) kinase via a RIP homotypic interaction motif. The activation of receptor-interacting protein kinase (RIPK)3 and initiation of virus-induced necroptosis were initially reported in a landmark publication Upton et al. (Cell Host Microbe 11: 290, 2012) employing the DNA virus murine cytomegalovirus (MCMV). M45-encoded viral inhibitor of RIP activation prevents virus-induced necroptosis. Additional virus-encoded suppressors of necroptosis were then identified, including herpes simplex virus ICP6 and vaccinia virus E3L. Caspase-8 suppressors encoded by these DNA viruses block apoptosis, unleashing necroptosis mediated through Z-nucleic acid binding protein 1 (ZBP1) recruitment of RIPK3. These studies all utilized ZBP1-deficient mice generated by the Akira Lab (Zbp1-/- AK ) to bring the significance of virus-induced necroptosis to light. C57BL/6 mice were chosen as controls based on the assumption that mutant mice were congenic; however, these mice were recently found to display an unexpected innate immune deficit, lacking C57BL/6-specific NK1.1 and Ly49H natural killer cell subpopulations important in the early control of MCMV infection. Short nucleotide polymorphism analysis of Zbp1-/- AK breeders revealed a mixed genetic background (~ 71% C57BL/6 DNA and ~ 29% 129). Even though this level of 129 strain background does not alter ZBP1 cell-autonomous function as a sensor and mediator of necroptosis, it confounds innate immune response characteristics. In the future, genetic background must be carefully controlled before implicating ZBP1 function in response characteristics that shape immunity, inflammation, metabolism, and pathogenesis.

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 in order 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/6J 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. Casp8(−/−)Ripk3(−/−) mice fail to show the enhanced expansion of lymphocytes observed in Casp8(−/−)Ripk3(−/−) mice even though development and homeostasis is 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.

Tumor necrosis factor receptor-1 (TNFR1) signaling, apart from its pleiotropic functions in inflammation, plays a role in embryogenesis as deficiency of varieties of its downstream molecules leads to embryonic lethality in mice. Caspase-8 noncleavable receptor interacting serine/threonine kinase 1 (RIPK1) mutations occur naturally in humans, and the corresponding D325A mutation in murine RIPK1 leads to death at early midgestation. It is known that both the demise of Ripk1D325A/D325A embryos and the death of Casp8−/− mice are initiated by TNFR1, but they are mediated by apoptosis and necroptosis, respectively. Here, we show that the defects in Ripk1D325A/D325A embryos occur at embryonic day 10.5 (E10.5), earlier than that caused by Casp8 knockout. By analyzing a series of genetically mutated mice, we elucidated a mechanism that leads to the lethality of Ripk1D325A/D325A embryos and compared it with that underlies Casp8 deletion-mediated lethality. We revealed that the apoptosis in Ripk1D325A/D325A embryos requires a scaffold function of RIPK3 and enzymatically active caspase-8. Unexpectedly, caspase-1 and caspase-11 are downstream of activated caspase-8, and concurrent depletion of Casp1 and Casp11 postpones the E10.5 lethality to embryonic day 13.5 (E13.5). Moreover, caspase-3 is an executioner of apoptosis at E10.5 in Ripk1D325A/D325A mice as its deletion extends life of Ripk1D325A/D325A mice to embryonic day 11.5 (E11.5). Hence, an unexpected death pathway of TNFR1 controls RIPK1 D325A mutation-induced lethality at E10.5., A study of mice expressing a caspase-8 non-cleavable RIPK1 mutant during embryonic development reveals an unexpected TNFR1-triggered death pathway involving RIPK3, caspase-8, and caspases -1, -11 and -3.

During infectious disease, pathogen load drives inflammation and immune response that together contribute to tissue injury often resulting in organ dysfunction. Pulmonary failure in SARS-CoV2-infected hospitalized COVID-19 patients is one such prominent example. Intervention strategies require characterization of the host-pathogen interaction by accurately assessing all of the above-mentioned disease parameters. To study infection in intact mammals, mice are often used as essential genetic models. Due to humane concerns, there is a constant unmet demand to develop studies that reduce the number of mice utilized while generating objective data. Here, we describe an integrated method of evaluating lung inflammation in mice infected with Pseudomonas aeruginosa or murine gammaherpesvirus (MHV)-68. This method conserves animal resources while permitting evaluation of disease mechanisms in both infection settings. Lungs from a single euthanized mouse were used for two purposes-biological assays to determine inflammation and infection load, as well as histology to evaluate tissue architecture. For this concurrent assessment of multiple parameters from a single euthanized mouse, we limit in-situ formalin fixation to the right lung of the cadaver. The unfixed left lung is collected immediately and divided into several segments for biological assays including determination of pathogen titer, assessment of infection-driven cytokine levels and appearance of cell death markers. In situ fixed right lung was then processed for histological determination of tissue injury and confirmation of infection-driven cell death patterns. This method reduces overall animal use and minimizes inter-animal variability that results from sacrificing different animals for different types of assays. The technique can be applied to any lung disease study in mice or other mammals.

We have revealed a critical role for innate immune signaling in nuclear reprogramming to pluripotency, and in the nuclear reprogramming required for somatic cell transdifferentiation. Activation of innate immune signaling causes global changes in the expression and activity of epigenetic modifiers to promote epigenetic plasticity. In our previous articles, we focused on the role of toll-like receptor 3 (TLR3) in this signaling pathway. Here, we define the role of another innate immunity pathway known to participate in response to viral RNA, the retinoic acid-inducible gene 1 receptor (RIG-1)-like receptor (RLR) pathway. This pathway is represented by the sensors of viral RNA, RIG-1, LGP2, and melanoma differentiation-associated protein 5 (MDA5). We first found that TLR3 deficiency only causes a partial inhibition of nuclear reprogramming to pluripotency in mouse tail-tip fibroblasts, which motivated us to determine the contribution of RLR. We found that knockdown of interferon beta promoter stimulator 1, the common adaptor protein for the RLR family, substantially reduced nuclear reprogramming induced by retroviral or by modified messenger RNA expression of Oct 4, Sox2, KLF4, and c-MYC (OSKM). Importantly, a double knockdown of both RLR and TLR3 pathway led to a further decrease in induced pluripotent stem cell (iPSC) colonies suggesting an additive effect of both these pathways on nuclear reprogramming. Furthermore, in murine embryonic fibroblasts expressing a doxycycline (dox)-inducible cassette of the genes encoding OSKM, an RLR agonist increased the yield of iPSCs. Similarly, the RLR agonist enhanced nuclear reprogramming by cell permeant peptides of the Yamanaka factors. Finally, in the dox-inducible system, RLR activation promotes activating histone marks in the promoter region of pluripotency genes. To conclude, innate immune signaling mediated by RLR plays a critical role in nuclear reprogramming. Manipulation of innate immune signaling may facilitate nuclear reprogramming to achieve pluripotency.

Background Human cytomegalovirus (HCMV) infection causes disease in newborns and transplant recipients. A HCMV vaccine (Towne) protects transplant recipients. Methods The genomes of Towne and the nonattenuated Toledo strain were recombined, yielding 4 Towne/Toledo chimera vaccines. Each of 36 HCMV-seronegative men received 1 subcutaneous dose of 10, 100, or 1000 plaque-forming units (PFU) in cohorts of 3. Safety and immunogenicity were evaluated over 12 weeks after immunization and for 52 weeks for those who seroconverted. Results There were no serious local or systemic reactions. No subject had HCMV in urine or saliva. For chimera 3, none of 9 subjects seroconverted. For chimera 1, 1 of 9 seroconverted (the seroconverter received 100 PFU). For chimera 2, 3 subjects seroconverted (1 received 100 PFU, and 2 received 1000 PFU). For chimera 4, 7 subjects seroconverted (1 received 10 PFU, 3 received 100 PFU, and 3 received 1000 PFU). All 11 seroconverters developed low but detectable levels of neutralizing activity. CD4+ T-cell responses were detectable in 1 subject (who received 100 PFU of chimera 4). Seven subjects receiving chimera 2 or 4 had detectable CD8+ T-cell responses to IE1; 3 responded to 1-2 additional antigens. Conclusions The Towne/Toledo chimera vaccine candidates were well tolerated and were not excreted. Additional human trials of chimeras 2 and 4 are appropriate. Clinical trials registration NCT01195571.

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

Receptor-interacting protein (RIP) kinase 3 (RIPK3)–dependent necroptosis directs inflammation and tissue injury, as well as anti-viral host defense. In human cells, herpes simplex virus 1 (HSV1) UL39-encoded ICP6 blocks RIP homotypic interacting motif (RHIM) signal transduction, preventing this leakage form of cell death and sustaining viral infection. TNF receptor 1 (TNFR1)-induced necroptosis is known to require the formation of a RIPK1–RIPK3–mixed lineage kinase domain–like pseudokinase (MLKL) signaling complex (necrosome) that we find compartmentalizes exclusively to caveolin-1–associated detergent-resistant membrane (DRM) vesicles in HT-29 cells. Translocation proceeds in the presence of RIPK3 kinase inhibitor GSK′840 or MLKL inhibitor necrosulfonomide but requires the kinase activity, as well as RHIM signaling of RIPK1. ICP6 impedes the translocation of RIPK1, RIPK3, and MLKL to caveolin-1–containing DRM vesicles without fully blocking the activation of RIPK3 or phosphorylation of MLKL. Consistent with the important contribution of RIPK1 RHIM-dependent recruitment of RIPK3, overexpression of RHIM-deficient RIPK3 results in phosphorylation of MLKL, but this does not lead to either translocation or necroptosis. Combined, these data reveal a critical role of RHIM signaling in the recruitment of the MLKL-containing necrosome to membrane vesicle–associated sites of aggregation. A similar mechanism is predicted for other RHIM-containing signaling adaptors, Z-nucleic acid–binding protein 1 (ZBP1) (also called DAI and DLM1), and TIR domain–containing adapter–inducing interferon-β (TRIF).

Summary The execution of shock following high dose E. coli lipopolysaccharide (LPS) or bacterial sepsis in mice required pro-apoptotic caspase-8 in addition to pro-pyroptotic caspase-11 and gasdermin D. Hematopoietic cells produced MyD88- and TRIF-dependent inflammatory cytokines sufficient to initiate shock without any contribution from caspase-8 or caspase-11. Both proteases had to be present to support tumor necrosis factor- and interferon-β-dependent tissue injury first observed in the small intestine and later in spleen and thymus. Caspase-11 enhanced the activation of caspase-8 and extrinsic cell death machinery within the lower small intestine. Neither caspase-8 nor caspase-11 was individually sufficient for shock. Both caspases collaborated to amplify inflammatory signals associated with tissue damage. Therefore, combined pyroptotic and apoptotic signaling mediated endotoxemia independently of RIPK1 kinase activity and RIPK3 function. These observations bring to light the relevance of tissue compartmentalization to disease processes in vivo where cytokines act in parallel to execute diverse cell death pathways.

Necroptosis complements apoptosis as a host defense pathway to stop virus infection. Herpes simplex virus shows a propensity to trigger necroptosis of mouse cells and mice even though cell death is blocked in human cells through UL39-encoded ICP6. This ribonucleotide reductase large subunit (R1) nucleates RHIM-dependent oligomerization of RIP3 kinase (RIPK3, also known as RIP3) in mouse cells but inhibits activation in cells from the natural human host. By interrogating the comparative behavior of ICP6-deficient viruses in mouse and human cells, here we unveil virus-induced necroptosis mediated by Z-DNA-binding protein 1 (ZBP1, also known as DAI). ZBP1 acts as a pathogen sensor to detect nascent RNA transcripts rather than input viral DNA or viral DNA generated through replication. Consistent with the implicated role of virus-induced necroptosis in restricting infection, viral pathogenesis is restored in Zbp1−/−, Ripk3−/− and Mlkl−/− mice. Thus, in addition to direct activation of RIPK3 via ICP6, HSV1 infection in mice and mouse cells triggers virus-induced necroptosis through ZBP1. Importantly, virus-induced necroptosis is also induced in human HT-29 cells by ICP6 mutant viruses; however, ZBP1 levels must be elevated for this pathway to be active. Thus, our studies reveal a common, species-independent role of this nucleic acid sensor to detect the presence of this virus. HSV1 ICP6 functions as a bona fide RHIM signaling inhibitor to block virus-induced necroptosis in its natural host. Altogether, ZBP1-dependent restriction of herpesvirus infection emerges as a potent antiviral armament of the innate immune system.

We recently described the induction of noncanonical IL-1β processing via caspase-8 recruited to ripoptosome signaling platforms in myeloid leukocytes. Here, we demonstrate that activated NLRP3·ASC inflammasomes recruit caspase-8 to drive IL-1β processing in murine bone marrow-derived dendritic cells (BMDC) independent of caspase-1 and -11. Sustained stimulation (2 h) of LPS-primed caspase-1-deficient (Casp1/11(-/-)) BMDC with the canonical NLRP3 inflammasome agonist nigericin results in release of bioactive IL-1β in conjunction with robust caspase-8 activation. This IL-1β processing and caspase-8 activation do not proceed in Nlrp3(-/-) or Asc(-/-) BMDC and are suppressed by pharmacological inhibition of caspase-8, indicating that caspase-8 can act as a direct IL-1β-converting enzyme during NLRP3 inflammasome activation. In contrast to the rapid caspase-1-mediated death of wild type (WT) BMDC via NLRP3-dependent pyroptosis, nigericin-stimulated Casp1/11(-/-) BMDC exhibit markedly delayed cell death via NLRP3-dependent apoptosis. Biochemical analyses of WT and Casp1/11(-/-) BMDC indicated that caspase-8 is proteolytically processed within detergent-insoluble ASC-enriched protein complexes prior to extracellular export during nigericin treatment. Although nigericin-stimulated caspase-1 activation and activity are only modestly attenuated in caspase-8-deficient (Casp8(-/-)Rip3(-/-)) BMDC, these cells do not exhibit the rapid loss of viability of WT cells. These results support a contribution of caspase-8 to both IL-1β production and regulated death signaling via NLRP3 inflammasomes. In the absence of caspase-1, NLRP3 inflammasomes directly utilize caspase-8 as both a pro-apoptotic initiator and major IL-1β-converting protease. In the presence of caspase-1, caspase-8 acts as a positive modulator of the NLRP3-dependent caspase-1 signaling cascades that drive both IL-1β production and pyroptotic death.

Like apoptosis, necroptosis is an innate immune mechanism that eliminates pathogen-infected cells. Receptor interacting protein kinase (RIP)3 (also called RIPK3) mediates necrotic death by phosphorylating an executioner protein, MLKL, leading to plasma membrane leakage. The pathway is triggered against viruses that block caspase 8. In murine CMV, the viral inhibitor of caspase 8 activation (vICA) prevents extrinsic apoptosis but also has the potential to unleash necroptosis. This virus encodes the viral inhibitor of RIP activation (vIRA) to prevent RIP homotypic interaction motif (RHIM)-dependent signal transduction and necroptosis. Recent investigations reveal a similar mechanism at play in the human alphaherpesviruses, herpes simplex virus (HSV)1 and HSV2, where RHIM competitor function and caspase 8 suppression are carried out by the virus-encoded large subunit of ribonucleotide reductase (R1). In human cells, R1 inhibition of caspase 8 prevents TNF-induced apoptosis, but sensitizes to TNF-induced necroptosis. The RHIM and caspase 8 interaction domains of R1 collaborate to prevent RIP3-dependent steps and enable both herpesviruses to deflect host cell death machinery that would cut short infection. In mouse cells, HSV1 infection by itself triggers necroptosis by driving RIP3 protein kinase activity. HSV1 R1 contributes to activation of RIP3 adaptor function in mice, a popular host animal for experimental infection. Based on these studies, infection of RIP3-kinase inactive mice should be explored in models of pathogenesis and latency. The necrotic death pathway that is suppressed during infection in the natural host becomes a cross-species barrier to infection in a non-natural host.

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.

Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, primarily manifesting as neurological disorders. HCMV infection alters expression of cellular microRNAs (miRs) and induces cell cycle arrest, which in turn modifies the cellular environment to favor virus replication. Previous observations found that HCMV infection reduces miR-21 expression in neural progenitor/stem cells (NPCs). Here, we show that infection of NPCs and U-251MG cells represses miR-21 while increasing the levels of Cdc25a, a cell cycle regulator and known target of miR-21. These opposing responses to infection prompted an investigation of the relationship between miR-21, Cdc25a, and viral replication. Overexpression of miR-21 in NPCs and U-251MG cells inhibited viral gene expression, genome replication, and production of infectious progeny, while shRNA-knockdown of miR-21 in U-251MG cells increased viral gene expression. In contrast, overexpression of Cdc25a in U-251MG cells increased viral gene expression and production of infectious progeny and overcame the inhibitory effects of miR-21 overexpression. Three viral gene products—IE1, pp71, and UL26—were shown to inhibit miR-21 expression at the transcriptional level. These results suggest that Cdc25a promotes HCMV replication and elevation of Cdc25a levels after HCMV infection are due in part to HCMV-mediated repression of miR-21. Thus, miR-21 is an intrinsic antiviral factor that is modulated by HCMV infection. This suggests a role for miR-21 downregulation in the neuropathogenesis of HCMV infection of the developing CNS.IMPORTANCEHuman cytomegalovirus (HCMV) is a ubiquitous pathogen and has very high prevalence among population, especially in China, and congenital HCMV infection is a major cause for birth defects. Elucidating virus-host interactions that govern HCMV replication in neuronal cells is critical to understanding the neuropathogenesis of birth defects resulting from congenital infection. In this study, we confirm that HCMV infection downregulates miR-21 but upregulates Cdc25a. Further determined the negative effects of cellular miRNA miR-21 on HCMV replication in neural progenitor/stem cells and U-251MG glioblastoma/astrocytoma cells. More importantly, our results provide the first evidence that miR-21 negatively regulates HCMV replication by targeting Cdc25a, a vital cell cycle regulator. We further found that viral gene products of IE1, pp71, and UL26 play roles in inhibiting miR-21 expression, which in turn causes increases in Cdc25a and benefits HCMV replication. Thus, miR-21 appears to be an intrinsic antiviral factor that represents a potential target for therapeutic intervention.

Necroptosis is an important form of lytic cell death triggered by injury and infection, but whether mixed lineage kinase domain-like (MLKL) is sufficient to execute this pathway is unknown. In a genetic selection for human cell mutants defective for MLKL-dependent necroptosis, we identified mutations in IPMK and ITPK1, which encode inositol phosphate (IP) kinases that regulate the IP code of soluble molecules. We show that IP kinases are essential for necroptosis triggered by death receptor activation, herpesvirus infection, or a pro-necrotic MLKL mutant. In IP kinase mutant cells, MLKL failed to oligomerize and localize to membranes despite proper receptor-interacting protein kinase-3 (RIPK3)-dependent phosphorylation. We demonstrate that necroptosis requires IP-specific kinase activity and that a highly phosphorylated product, but not a lowly phosphorylated precursor, potently displaces the MLKL auto-inhibitory brace region. These observations reveal control of MLKL-mediated necroptosis by a metabolite and identify a key molecular mechanism underlying regulated cell death.

A universal cytomegalovirus (CMV) vaccination promises to reduce the burden of the developmental damage that afflicts up to 0.5% of live births worldwide. An effective vaccination that prevents transplacental transmission would reduce CMV congenital disease and CMV-associated still births and leave populations less susceptible to opportunistic CMV disease. Thus, a vaccination against this virus has long been recognized for the potential of enormous health-care savings because congenital damage is life-long and existing anti-viral options are limited. Vaccine researchers, industry leaders, and regulatory representatives have discussed the challenges posed by clinical efficacy trials that would lead to a universal CMV vaccine, reviewing the links between infection and disease, and identifying settings where disrupting viral transmission might provide a surrogate endpoint for disease prevention. Reducing the complexity of such trials would facilitate vaccine development. Children and adolescents are the targets for universal vaccination, with the expectation of protecting the offspring of immunized women. Given that a majority of females worldwide experience CMV infection during childhood, a universal vaccine must boost natural immunity and reduce transmission due to reactivation and re-infection as well as primary infection during pregnancy. Although current vaccine strategies recognize the value of humoral and cellular immunity, the precise mechanisms that act at the placental interface remain elusive. Immunity resulting from natural infection appears to limit rather than prevent reactivation of latent viruses and susceptibility to re-infection, leaving a challenge for universal vaccination to improve upon natural immunity levels. Despite these hurdles, early phase clinical trials have achieved primary end points in CMV seronegative subjects. Efficacy studies must be expanded to mixed populations of CMV-naive and naturally infected subjects to understand the overall efficacy and potential. Together with CMV vaccine candidates currently in clinical development, additional promising preclinical strategies continue to come forward; however, these face limitations due to the insufficient understanding of host defense mechanisms that prevent transmission, as well as the age-old challenges of reaching the appropriate threshold of immunogenicity, efficacy, durability and potency. This review focuses on the current understanding of natural and CMV vaccine-induced protective immunity.

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 (RIPK1) kinase is a key regulator of TNF-induced NF-κB activation, apoptosis, and necroptosis through its kinase and scaffolding activities. Dissecting the balance of RIP1 kinase activity and scaffolding function in vivo during development and TNF-dependent inflammation has been hampered by the perinatal lethality of RIP1-deficient mice. In this study, we generated RIP1 kinase–dead (Ripk1K45A) mice and showed they are viable and healthy, indicating that the kinase activity of RIP1, but not its scaffolding function, is dispensable for viability and homeostasis. After validating that the Ripk1K45A mice were specifically protected against necroptotic stimuli in vitro and in vivo, we crossed them with SHARPIN-deficient cpdm mice, which develop severe skin and multiorgan inflammation that has been hypothesized to be mediated by TNF-dependent apoptosis and/or necroptosis. Remarkably, crossing Ripk1K45A mice with the cpdm strain protected against all cpdm-related pathology. Together, these data suggest that RIP1 kinase represents an attractive therapeutic target for TNF-driven inflammatory diseases.

Tegument proteins pp150 and pUL96 function at a late step in cytomegalovirus (CMV) maturation. Here, we show that pp150 interacts directly with pUL96; however, the N-terminal region of pp150 and the C-terminal region of pUL96, which are critical for these proteins to function, are not required for this interaction. Moreover, the largely dispensable C-terminal region of pp150 is critical for pp150-pUL96 interaction. To further study the role of pUL96, several point and clustered mutations were engineered into the CMV Towne bacterial artificial chromosome (Towne-BAC) genome, replacing the conserved negatively charged C-terminal residues of pUL96. Although individual point mutations (E 122 A, D 124 A, and D 125 A) reduced virus growth slightly, the clustered mutations of 122 EVDDAV 127 significantly reduced virus growth, produced small syncytial plaque phenotypes, and impacted a late stage of virus maturation. When the UL96 C-terminal alanine conversion mutant (B6-BAC) virus was serially passaged in cell culture, it gained a plaque size comparable to that of Towne-BAC, displayed an altered restriction fragment length pattern, and replicated with increased growth kinetics. Whole-genome sequencing of this passaged virus (UL96P10) and the similarly passaged Towne-BAC virus revealed major differences only in the RNA4.9 and UL96 regions. When one of the mutations in the UL96 coding region was engineered into the B6-BAC virus, it significantly increased the plaque size and rescued the virus growth rate. Thus, accumulation of compensatory mutations only in UL96 in this revertant and the specific involvement of functionally dispensable regions of pp150 in the pUL96-pp150 interaction point toward a role for pUL96 in virus maturation that does not depend upon pp150. IMPORTANCE Human cytomegalovirus causes significant medical problems in newborns, as well as in people with low immunity. In this study, we investigated the functions of two essential virus proteins, pp150 and pUL96, and determined the impact of their mutual interaction on virus replication. These studies provide valuable information that is critical for the development of targeted antiviral therapies.

Summary 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.

A multidisciplinary meeting addressed priorities related to development of vaccines against cytomegalovirus (CMV), the cause of congenital CMV (cCMV) disease and of serious disease in the immunocompromised. Participants discussed optimal uses of a CMV vaccine, aspects of clinical study design, and the value of additional research. A universal childhood CMV vaccine could potentially rapidly reduce cCMV disease, as infected children are sources of viral transmission to seronegative and seropositive mothers. A vaccine administered to adolescents or adult women could also reduce cCMV disease by making them immune prior to pregnancy. Clinical trials of CMV vaccines in women should evaluate protection against cCMV infection, an essential precursor of cCMV disease, which is a more practical and acceptable endpoint for assessing vaccine effects on maternal-fetal transmission. Clinical trials of vaccines to evaluate prevention of CMV disease in stem cell transplant recipients could use CMV viremia at a level triggering pre-emptive antiviral therapy as an endpoint, because widespread use of pre-emptive and prophylactic antivirals has rendered CMV-induced disease too rare to be a practical endpoint for clinical trials. In solid organ transplant patients, CMV-associated disease is sufficiently common for use as a primary endpoint. Additional research to advance CMV vaccine development should include identifying factors that predict fetal loss due to CMV, determining age-specific incidence and transmission rates, defining the mechanism and relative contributions of maternal reactivation and re-infection to cCMV disease, developing assays that can distinguish between reactivation and re-infection in seropositive vaccinees, further defining predictors of sequelae from cCMV infection, and identifying clinically relevant immune response parameters to CMV (including developing validated assays that could assess CMV antibody avidity) that could lead to the establishment of immune correlates of protection.

The identification of noncanonical (caspase-1–independent) pathways for IL-1β production has unveiled an intricate interplay between inflammatory and death-inducing signaling platforms. We found a heretofore unappreciated role for caspase-8 as a major pathway for IL-1β processing and release in murine bone marrow–derived dendritic cells (BMDC) costimulated with TLR4 agonists and proapoptotic chemotherapeutic agents such as doxorubicin (Dox) or staurosporine (STS). The ability of Dox to stimulate release of mature (17-kDa) IL-1β was nearly equivalent in wild-type (WT) BMDC, Casp1−/−Casp11−/− BMDC, WT BMDC treated with the caspase-1 inhibitor YVAD, and BMDC lacking the inflammasome regulators ASC, NLRP3, or NLRC4. Notably, Dox-induced production of mature IL-1β was temporally correlated with caspase-8 activation in WT cells and greatly suppressed in Casp8−/−Rip3−/− or Trif−/− BMDC, as well as in WT BMDC treated with the caspase-8 inhibitor, IETD. Similarly, STS stimulated robust IL-1β processing and release in Casp1−/−Casp11−/− BMDC that was IETD sensitive. These data suggest that TLR4 induces assembly of caspase-8–based signaling complexes that become licensed as IL-1β–converting enzymes in response to Dox and STS. The responses were temporally correlated with downregulation of cellular inhibitor of apoptosis protein 1, suggesting suppressive roles for this and likely other inhibitor of apoptosis proteins on the stability and/or proteolytic activity of the caspase-8 platforms. Thus, proapoptotic chemotherapeutic agents stimulate the caspase-8–mediated processing and release of IL-1β, implicating direct effects of such drugs on a noncanonical inflammatory cascade that may modulate immune responses in tumor microenvironments.