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.

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/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. 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.

Highlights • Necroptosis and apoptosis are alternate programmed cell death pathways. • Necroptosis, like apoptosis, provides cell autonomous host defense. • Herpesviruses block necroptosis by encoding RHIM signaling competitors. • Herpesvirus suppression of caspase 8 unleashes necroptosis. Abstract Herpesviruses suppress cell death to assure sustained infection in their natural hosts. Murine cytomegalovirus (MCMV) encodes suppressors of apoptosis as well as M45-encoded viral inhibitor of RIP activation (vIRA) to block RIP homotypic interaction motif (RHIM)-signaling and recruitment of RIP3 (also called RIPK3), to prevent necroptosis. MCMV and human cytomegalovirus encode a viral inhibitor of caspase (Casp)8 activation to block apoptosis, an activity that unleashes necroptosis. Herpes simplex virus (HSV)1 and HSV2 incorporate both RHIM and Casp8 suppression strategies within UL39-encoded ICP6 and ICP10, respectively, which are herpesvirus-conserved homologs of MCMV M45. Both HSV proteins sensitize human cells to necroptosis by blocking Casp8 activity while preventing RHIM-dependent RIP3 activation and death. In mouse cells, HSV1 ICP6 interacts with RIP3 and, surprisingly, drives necroptosis. Thus, herpesviruses have illuminated the contribution of necoptosis to host defense in the natural host as well as its potential to restrict cross-species infections in nonnatural hosts.

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(-/-)Ripk3(K51A/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(-/-)Ripk3(K51A/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)gamma 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 IFN gamma 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.

Viral infection activates cytokine expression and triggers cell death, the modulation of which is important for successful pathogenesis. Necroptosis is a form of programmed necrosis dependent on two related RIP homotypic interaction motif (RHIM)-containing signaling adaptors, receptor-interacting protein kinases (RIP) 1 and 3. We find that murine cytomegalovirus infection induces RIP3-dependent necrosis. Whereas RIP3 kinase activity and RHIM-dependent interactions control virus-associated necrosis, virus-induced death proceeds independently of RIP1 and is therefore distinct from TNF alpha-dependent necroptosis. Viral M45-encoded inhibitor of RIP activation (vIRA) targets RIP3 during infection and disrupts RIP3-RIP1 interactions characteristic of TNF alpha-induced necroptosis, thereby suppressing both death pathways. Importantly, attenuation of vIRA mutant virus in wild-type mice is normalized in RIP3-deficient mice. Thus, vIRA function validates necrosis as central to host defense against viral infections and highlights the benefit of multiple virus-encoded cell-death suppressors that inhibit not only apoptotic, but also necrotic mechanisms of virus clearance.

Human cytomegalovirus-encoded UL91 is a betagamma gene that is essential for viral replication. Here we show that the 111-amino-acid (aa) UL91 protein controls accumulation of true-late (gamma 2) viral transcripts. The primate betaherpesvirus conserved N-terminal region from aa 1 to 71 is sufficient to fully reconstitute function. Evaluation of viral DNA, RNA, and antigen revealed that UL91 protein is expressed with leaky-late (gamma 1) kinetics, localizes in the nucleus without influencing viral DNA synthesis, and must be present from 48 h postinfection to support full expression of late viral transcripts and proteins. In the absence of UL91, viral capsid assembly in the nucleus of infected cells is significantly reduced, and mature, cytoplasmic virions fail to form. Taken together, the evidence shows that UL91 regulates late viral gene expression by a mechanism that is apparently conserved in beta-herpesviruses and gammaherpesviruses.

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(=3D)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.

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 cas pase-8 or caspase-11. Both proteases had to be present to support tumor necrosis factor- and interferon-beta-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.

Background Osteoporosis is a complex disease with a strong genetic contribution. A recently published genome-wide association study (GWAS) for estimated bone mineral density (eBMD) identified 1103 independent genome-wide significant association signals. Most of these variants are non-coding, suggesting that regulatory effects may drive many of the associations. To identify genes with a role in osteoporosis, we integrate the eBMD GWAS association results with those from our previous osteoclast expression quantitative trait locus (eQTL) dataset. Results We identify sixty-nine significant cis-eQTL effects for eBMD GWAS variants after correction for multiple testing. We detect co-localisation of eBMD GWAS and osteoclast eQTL association signals for 21 of the 69 loci, implicating a number of genes including CCR5, ZBTB38, CPE, GNA12, RIPK3, IQGAP1 and FLCN. Summary-data-based Mendelian Randomisation analysis of the eBMD GWAS and osteoclast eQTL datasets identifies significant associations for 53 genes, with TULP4 presenting as a strong candidate for pleiotropic effects on eBMD and gene expression in osteoclasts. By performing analysis using the GARFIELD software, we demonstrate significant enrichment of osteoporosis risk variants among high-confidence osteoclast eQTL across multiple GWAS P value thresholds. Mice lacking one of the genes of interest, the apoptosis/necroptosis gene RIPK3, show disturbed bone micro-architecture and increased osteoclast number, highlighting a new biological pathway relevant to osteoporosis. Conclusion We utilise a unique osteoclast eQTL dataset to identify a number of potential effector genes for osteoporosis risk variants, which will help focus functional studies in this area.

Interleukin 1 beta (IL-1 beta) is critical for the in vivo survival, expansion and effector function of IL-17-producing helper T (T(H)17) cells during autoimmune responses, including experimental autoimmune encephalomyelitis (EAE). However, the spatiotemporal role and cellular source of IL-1 beta during EAE pathogenesis are poorly defined. In the present study, we uncovered a T cell-intrinsic inflammasome that drives IL-1 beta production during T(H)17-mediated EAE pathogenesis. Activation of T cell antigen receptors induced expression of pro-IL-1 beta, whereas ATP stimulation triggered T cell production of IL-1 beta via ASC-NLRP3-dependent caspase-8 activation. IL-1R was detected on T(H)17 cells but not on type 1 helper T (T(H)1) cells, and ATP-treated T(H)17 cells showed enhanced survival compared with ATP-treated T(H)1 cells, suggesting autocrine action of T(H)17-derived IL-1 beta. Together these data reveal a critical role for IL-1 beta produced by a T(H)17 cell-intrinsic ASC-NLRP3-caspase-8 inflammasome during inflammation of the central nervous system.

Pathogens specifically target both the caspase 8-dependent apoptotic cell death pathway and the necrotic cell death pathway that is dependent on receptor-interacting protein 1 (RIP1; also known as RIPK1) and RIP3 (also known as RIPK3). The fundamental co-regulation of these two cell death pathways emerged when the midgestational death of mice deficient in FAS-associated death domain protein (FADD) or caspase 8 was reversed by elimination of RIP1 or RIP3, indicating a far more entwined relationship than previously appreciated. Thus, mammals require caspase 8 activity during embryogenesis to suppress the kinases RIP1 and RIP3 as part of the dialogue between two distinct cell death processes that together fulfil reinforcing roles in the host defence against intracellular pathogens such as herpesviruses.

Background - Asymptomatic cytomegalovirus (CMV) replication is frequent after cardiac transplantation in recipients with pretransplantation CMV infection. How subclinical viral replication influences cardiac allograft disease remains poorly understood, as does the importance of T-cell immunity in controlling such replication.Methods and Results - Thirty-nine cardiac recipients who were pretransplantation CMV antibody positive were longitudinally studied for circulating CMV-specific CD4 and CD8 T-cell responses, CMV viral load in blood neutrophils, and allograft rejection during the first posttransplantation year. Nineteen of these recipients were also analyzed for changes of coronary artery intimal, lumen, and whole-vessel area. All recipients received early prophylactic therapy with ganciclovir. No recipients developed overt CMV disease. Those with detectable levels of CMV-specific CD4 T cells in the first month after transplantation were significantly protected from high mean and peak posttransplantation viral load (P < 0.05), acute rejection (P < 0.005), and loss of allograft coronary artery lumen (P < 0.05) and of whole-vessel area (P < 0.05) compared with those who lacked this immune response. The losses of lumen and vessel area were both significantly correlated with the time after transplantation at which a CD4 T-cell response was first detected (P < 0.05) and with the cumulative graft rejection score (P < 0.05).Conclusions - The early control of subclinical CMV replication after transplantation by T-cell immunity may limit cardiac allograft rejection and vascular disease. Interventions to increase T-cell immunity might be clinically useful in limiting these adverse viral effects.

Background-Treatment of acute rejection (AR) in heart transplantation relies on histopathological grading of endomyocardial biopsies according to International Society for Heart and Lung Transplantation guidelines. Intragraft gene expression profiling may be a way to complement histological evaluation. Methods and Results-Transcriptional profiling was performed on 26 endomyocardial biopsies, and expression patterns were compared with the 1990 International Society for Heart and Lung Transplantation AR grades. Importantly, transcriptional profiles from settings with an equivalent AR grade appeared the same. In addition, grade 0 profiles could not be distinguished from 1A profiles, and grade 3A profiles could not be distinguished from 3B profiles. Comparing the AR groupings (0 + 1A, 1B, and 3A + 3B), 0 + 1A showed more striking differences from 1B than from 3A + 3B. When these findings were extrapolated to the 2005 revised guidelines, the combination of 1A and 1B into a single category (1R) appears to have brought together endomyocardial biopsies with different underlying processes that are not evident from histological evaluation. Grade 1B was associated with upregulated immune response genes, as 1 categorical distinction from grade 1A. Although grade 1B was distinct from the clinically relevant AR grades 3A and 3B, all of these grades shared a small number of overlapping pathways consistent with common physiological underpinnings. Conclusion-The gene expression similarities and differences identified here in different AR settings have the potential to revise the clinical perspective on acute graft rejection, pending the results of larger studies. (Circulation. 2011; 123: 2236-2243.)

The endosomal sorting complex required for transport (ESCRT) machinery controls the incorporation of cargo into intraluminal vesicles of multivesicular bodies. This machinery is used during envelopment of many RNA viruses and some DNA viruses, including herpes simplex virus type 1. Other viruses mature independent of ESCRT components, instead relying on the intrinsic behavior of viral matrix and envelope proteins to drive envelopment. Human cytomegalovirus (HCMV) maturation has been reported to proceed independent of ESCRT components (A. Fraile-Ramos et al. Cell. Microbiol. 9: 2955-2967, 2007). A virus complementation assay was used to evaluate the role of dominant-negative (DN) form of a key ESCRT ATPase, vacuolar protein sorting-4 (Vps4(DN)) in HCMV replication. Vps4(DN) specifically inhibited viral replication, whereas wild-type-Vps4 had no effect. In addition, a DN form of charged multivesicular body protein 1 (CHMP1(DN)) was found to inhibit HCMV. In contrast, DN tumor susceptibility gene-101 (Tsg101(DN)) did not impact viral replication despite the presence of a PTAP motif within pp150/ppUL32, an essential tegument protein involved in the last steps of viral maturation and release. Either Vps4(DN) or CHMP1(DN) blocked viral replication at a step after the accumulation of late viral proteins, suggesting that both are involved in maturation. Both Vps4A and CHMP1A localized in the vicinity of viral cytoplasmic assembly compartments, sites of viral maturation that develop in CMV-infected cells. Thus, ESCRT machinery is involved in the final steps of HCMV replication.

Cytomegalovirus replication depends upon a betaherpesvirus-conserved 150-kDa tegument phosphoprotein (pp150; encoded by UL32) that supports the final steps in virion maturation at cytoplasmic assembly compartments. Amino acid substitutions were introduced into conserved region 1 (CR1) and CR2 of pp150, affecting a region that may interact with nucleocapsids. Two independent CR2 point mutants (N201A and G207A) failed to support viral replication in evaluations by a transient complementation assay or after reconstruction into recombinant viruses. An assembly compartment-like cytoplasmic inclusion developed in UL32 mutant virus-infected cells that was similar to that of wild-type virus-infected cells. The cellular localization of the trans-Golgi marker Golgin-97 suggested differences in the organization of the assembly compartment compared to that of wild-type virus-infected cells. Replication-defective CR2 point mutants exhibited the same phenotype as that of a virus carrying a complete deletion of the UL32 open reading frame in these assays. Electron micrographs of fibroblasts at 3 or 5 days postinfection with a deletion mutant (Delta UL32) grown on UL32-complementing cells showed a similar number and morphology of capsids in the nucleus, but the cytoplasmic region associated with virion assembly appeared highly vesiculated and contained few recognizable nucleocapsids or complete virus particles. These data demonstrate that the principle role of pp150 is to retain nucleocapsid organization through secondary envelopment at the assembly compartment.