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The anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) plays an important role in survival and differentiation of leukocytes, more specifically of neutrophils. Here, we investigated the impact of myeloid Mcl-1 deletion in atherosclerosis. Western type diet fed LDL receptor-deficient mice were transplanted with either wild-type (WT) or LysMCre Mcl-1 fl/fl (Mcl-1 -/- ) bone marrow. Mcl-1 myeloid deletion resulted in enhanced apoptosis and lipid accumulation in atherosclerotic plaques. In vitro, Mcl-1 deficient macrophages also showed increased lipid accumulation, resulting in increased sensitivity to lipid-induced cell death. However, plaque size, necrotic core and macrophage content were similar in Mcl-1 -/- compared to WT mice, most likely due to decreased circulating and plaque-residing neutrophils. Interestingly, Mcl-1 -/- peritoneal foam cells formed up to 45% more multinucleated giant cells (MGCs) in vitro compared to WT, which concurred with an increased MGC presence in atherosclerotic lesions of Mcl-1 -/- mice. Moreover, analysis of human unstable atherosclerotic lesions also revealed a significant inverse correlation between MGC lesion content and Mcl-1 gene expression, coinciding with the mouse data. Taken together, these findings suggest that myeloid Mcl-1 deletion leads to a more apoptotic, lipid and MGC-enriched phenotype. These potentially pro-atherogenic effects are however counteracted by neutropenia in circulation and plaque.

Apoptosis can be induced by either death receptors on the plasma membrane (extrinsic pathway) or the damage of the genome and/or cellular organelles (intrinsic pathway). Previous studies suggest that cellular caspase 8 (FLICE)-like inhibitory protein (c-FLIP) promotes cell survival in death receptor-induced apoptosis pathway in T lymphocytes. Independent of death receptor signaling, mitochondria sense apoptotic stimuli and mediate the activation of effector caspases. Whether c-FLIP regulates mitochondrion-dependent apoptotic signals remains unknown. In this study, c-FLIP gene was deleted in mature T lymphocytes in vitro, and the role of c-FLIP protein in intrinsic apoptosis pathway was studied. In resting T cells treated with the intrinsic apoptosis inducer, c-FLIP suppressed cytochrome c release from mitochondria. Bim-deletion rescued the enhanced apoptosis in c-FLIP-deficient T cells, whereas inhibition of caspase 8 did not. Different from activated T cells, there was no necroptosis or increase in reactive oxygen species in c-FLIP-deficient resting T cells. These data suggest that c-FLIP is a negative regulator of intrinsic apoptosis pathway in T lymphocytes., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Understanding the signals that regulate eosinophil survival and death is critical to developing new treatments for asthma, atopy, and gastrointestinal disease. Previous studies suggest that TNF-α stimulation protects eosinophils from apoptosis, and this TNF-α-mediated protection is mediated by the upregulation of an unknown protein by NF-κB. Here, we show for the first time that eosinophils express the caspase 8-inhibitory protein c-FLIP, and c-FLIP expression is upregulated upon TNF-α stimulation. Considering that c-FLIP expression is regulated by NF-κB, we hypothesized that c-FLIP might serve as the "molecular switch" that converts TNFRI activation to a pro-survival signal in eosinophils. Indeed, we found that one c-FLIP isoform, c-FLIPL, is required for mouse eosinophil survival in the presence of TNF-α both in vitro and in vivo. Importantly, our results suggest c-FLIP as a potential therapeutic target for the treatment of eosinophil-mediated disease.

Background & Aims: The intestinal epithelium generates a barrier that protects mammals from potentially harmful intestinal contents, such as pathogenic bacteria. Dysregulation of epithelial cell death has been implicated in barrier dysfunction and in the pathogenesis of intestinal inflammation. We investigated mechanisms of cell-death regulation in the intestinal epithelium of mice., Methods: Conditional knockout mice (either inducible or permanent) with deletion of cellular FLICE-inhibitory protein (cFlip) or caspase-8 in the intestinal epithelium were analyzed by histology and high-resolution endoscopy. We assessed the effects of cFlip or caspase-8 deficiency on intestinal homeostasis., Results: Expression of cFlip in the intestinal epithelium was required for constitutive activation of caspase-8 under steady-state conditions. Intestinal expression of cFlip was required for development; disruption of the gene encoding cFlip from the intestinal epithelium (cFlip(fl/fl) VillinCre(+) mice) resulted in embryonic lethality. When cFlip was deleted from the intestinal epithelium of adult mice (cFlip(iΔIEC) mice), the animals died within a few days from severe tissue destruction, epithelial cell death, and intestinal inflammation. Death of cFlip-depleted intestinal epithelial cells was regulated extrinsically and required the presence of death receptor ligands, such as tumor necrosis factor-α and CD95 ligand, but was independent of receptor-interacting protein 3. cFlip deficiency was associated with strong up-regulation of caspase-8 and caspase-3 activity and excessive apoptosis in intestinal crypts., Conclusions: cFlip is required for intestinal tissue homeostasis in mice. It controls the level of activation of caspase-8 to promote survival of intestinal epithelial cells., (Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.)

FADD, caspase-8, and cFLIP regulate the outcome of cell death signaling. Mice that constitutively lack these molecules die at an early embryonic age, whereas tissue-specific constitutive deletion of FADD or caspase-8 results in inflammatory skin disease caused by increased necroptosis. The function of cFLIP in the skin in vivo is unknown. In contrast to tissue-specific caspase-8 knockout, we show that mice constitutively lacking cFLIP in the epidermis die around embryonic days 10 and 11. When cFLIP expression was abrogated in adult skin of cFLIPfl/fl-K14CreERtam mice, severe inflammation of the skin with concomitant caspase activation and apoptotic, but not necroptotic, cell death developed. Apoptosis was dependent of autocrine tumor necrosis factor production triggered by loss of cFLIP. In addition, epidermal cFLIP protein was lost in patients with severe drug reactions associated with epidermal apoptosis. Our data demonstrate the importance of cFLIP for the integrity of the epidermis and for silencing of spontaneous skin inflammation.

Although extensive studies have demonstrated the functional impairment of antigen-specific CD4(+) and CD8(+) T-cells during chronic hepatitis C virus (HCV) infection, the functional status of global CD4(+) and CD8(+) T-cells remains unclear. In this report, we recruited 42 long-term (~20 years) treatment-naïve chronic HCV (CHC) patients and 15 healthy donors (HDs) to investigate differences in global CD4(+) and CD8(+) T-cells function. We show that CD4(+) and CD8(+) T-cells from CHC patients underwent increased apoptosis after TCR stimulation. Furthermore, IFN-γ, IL-9 and IP-10 were elevated in CHC patients' plasma and promoted activation-induced T-cells death. Global CD4(+) and CD8(+) T-cells also showed unique transcriptional profiles in the expression of apoptosis-related genes. We identified BCL2, PMAIP1, and CASP1 in CD4(+) T-cells and IER3 and BCL2A1 in CD8(+) T-cells from CHC patients as HCV-specific gene signatures. Importantly, the gene expression patterns of CD4(+) and CD8(+) T-cells from CHC patients differ from those in CD4(+) and CD8(+) T-cells from human immunodeficiency virus type 1 (HIV-1) or hepatitis B virus (HBV) infected individuals. Our results indicate that chronic HCV infection causes a systemic change in cytokine levels that primes T-cells for activation-induced apoptosis. Furthermore, HCV infection programs unique apoptosis-related gene expression profiles in CD4(+) and CD8(+) T-cells, leading to their enhanced activation-induced apoptosis. These results provide novel insights to the pathogenesis of chronic HCV infection.

Necroptosis, a caspase-independent, receptor (TNFRSF)-interacting serine-threonine kinase 1 (RIPK1)/RIPK3-dependent necrotic cell death, occurs in cells when apoptosis is blocked. A high level of macroautophagy (herein referred to as autophagy) is usually detected in necroptotic cells, although it is still controversial as to whether excessive autophagy leads to cell death or is cytoprotective. In a recently published paper, we show that the anti-apoptotic protein CFLAR (CASP8 and FADD-like apoptosis regulator) long isoform (CFLARL) plays a critical role in all three fundamental intracellular processes: autophagy, necroptosis, and apoptosis in T lymphocytes. CFLARL-deficient T cells suffer from severe cell death upon T cell receptor stimulation, in which both apoptosis and necroptosis are involved. Autophagy is enhanced in both naïve and activated CFLARL-deficient T cells and plays a cytoprotective function. Here, we summarize our findings and discuss the future direction in the study of the interplay of autophagy, apoptosis and necroptosis in T lymphocytes.

As a catalytically inactive homolog of caspase-8, a proapoptotic initiator caspase, c-FLIP blocks apoptosis by binding to and inhibiting caspase-8. The transcription factor nuclear factor κB (NF-κB) plays a pivotal role in maintaining the homeostasis of the intestine and the liver by preventing death receptor-induced apoptosis, and c-FLIP plays a role in the NF-κB-dependent protection of cells from death receptor signaling. Because c-Flip-deficient mice die in utero, we generated conditional c-Flip-deficient mice to investigate the contribution of c-FLIP to homeostasis of the intestine and the liver at developmental and postnatal stages. Intestinal epithelial cell (IEC)- or hepatocyte-specific deletion of c-Flip resulted in perinatal lethality as a result of the enhanced apoptosis and programmed necrosis of the IECs and the hepatocytes. Deficiency in the gene encoding tumor necrosis factor-α (TNF-α) receptor 1 (Tnfr1) partially rescued perinatal lethality and the development of colitis in IEC-specific c-Flip-deficient mice but did not rescue perinatal lethality in hepatocyte-specific c-Flip-deficient mice. Moreover, adult mice with interferon (IFN)-inducible deficiency in c-Flip died from hepatitis soon after depletion of c-FLIP. Pretreatment of IFN-inducible c-Flip-deficient mice with a mixture of neutralizing antibodies against TNF-α, Fas ligand (FasL), and TNF-related apoptosis-inducing ligand (TRAIL) prevented hepatitis. Together, these results suggest that c-FLIP controls the homeostasis of IECs and hepatocytes by preventing cell death induced by TNF-α, FasL, and TRAIL.

Recent advances in the understanding of the molecular processes contributing to autophagy have provided insight into the relationship between autophagy and apoptosis. In contrast to the concept of "autophagic cell death," accumulating evidence suggests that autophagy serves a largely cytoprotective role in physiologically relevant conditions. The cytoprotective function of autophagy is mediated in many circumstances by negative modulation of apoptosis. Apoptotic signaling, in turn, serves to inhibit autophagy. While the mechanisms mediating the complex counter-regulation of apoptosis and autophagy are not yet fully understood, important points of crosstalk include the interactions between Beclin-1 and Bcl-2/Bcl-xL and between FADD and Atg5, caspase- and calpain-mediated cleavage of autophagy-related proteins, and autophagic degradation of caspases. Continued investigation of these and other means of crosstalk between apoptosis and autophagy is necessary to elucidate the mechanisms controlling the balance between survival and death both under normal conditions and in diseases including cancer.

During the development and normal function of T lymphocytes, the cells are subject to several checkpoints at which they must "decide" to live or die. At these critical times and during homeostasis, the molecules that regulate the classical apoptotic pathways and survival pathways such as autophagy have critical roles in controlling this decision. Our laboratory has focused on the roles of apoptotic and autophagic proteins in T lymphocyte development and function. Using genetic models in mice and in vitro analyses of T cell functions, we have outlined critical roles for the Bcl-2 family (regulators of the intrinsic pathway of apoptosis), c-FLIP (an anti-apoptotic protein in the extrinsic pathway of apoptosis), and autophagy in T lymphocytes.

Although c-FLIP has been identified as an important player in the extrinsic (death receptor-induced) apoptosis pathway, its endogenous function in mature T lymphocytes remains undefined. c-FLIP may inhibit or promote T cell death as previous data demonstrate that the c-FLIP(L) isoform can promote or inhibit caspase 8 activation while the c-FLIP(S) isoform promotes or inhibits T cell death when overexpressed. Although the c-FLIP(R) isoform inhibits cell death in cell lines, its function in T cells remains unknown. To investigate the function of c-FLIP in mature T cells, we have generated several genetic mouse models with c-FLIP or its individual isoforms deleted in mature T cells. Surprisingly, we found that c-FLIP protects mature T cells not only from apoptosis induced by the death receptors Fas and TNFR but also from TCR-mediated and spontaneous apoptosis. Thus, c-FLIP plays an essential role in protecting mature T cells from a death signal induced through the TCR itself and is required for naive T cell survival. Our results demonstrate that c-FLIP functions beyond the extrinsic death pathway.

Apoptosis plays an essential role in T cell biology. Thymocytes expressing nonfunctional or autoreactive TCRs are eliminated by apoptosis during development. Apoptosis also leads to the deletion of expanded effector T cells during immune responses. The dysregulation of apoptosis in the immune system results in autoimmunity, tumorogenesis and immunodeficiency. Two major pathways lead to apoptosis: the intrinsic cell death pathway controlled by Bcl-2 family members and the extrinsic cell death pathway controlled by death receptor signaling. These two pathways work together to regulate T lymphocyte development and function.

Apoptosis-related genes play important roles in thymocyte maturation. We show that cellular FLICE-like inhibitory protein (c-FLIP), a procaspase-8-like apoptotic regulator, plays an essential role in the efficient development of mature T lymphocytes. Mice conditionally lacking c-FLIP in T lymphocytes display severe defects in the development of mature T cells, as indicated by a dramatically reduced number of CD4+ and CD8+ T cells in the spleen and lymph nodes of mutant mice. The impaired T lymphocyte maturation in c-FLIP conditional knockout mice occurs at the single-positive thymocyte stage and may be caused by enhanced apoptosis in vivo. Moreover, although c-FLIP has been implicated in T cell receptor signaling through nuclear factor (NF)-kappaB and Erk pathways, activation of NF-kappaB and Erk in c-FLIP-deficient thymocytes appears largely intact. Collectively, our data suggest that the primary role of c-FLIP in thymocyte maturation is to protect cells from apoptosis.

The antiapoptotic protein Bcl-x(L) is induced in activated T lymphocytes upon costimulation through CD28, 4-1BB, and OX40. Bcl-x(L) is also highly enriched in memory T lymphocytes. Based on this body of evidence, it was thought that Bcl-x(L) plays an essential role in the generation of effector and memory T lymphocytes. We report that mice with a conditional deletion of Bcl-x in T lymphocytes develop a normal CD8(+) T cell response to Listeria monocytogenes infection. Furthermore, Bcl-x conditional knockout mice exhibit normal T-dependent humoral immune responses. These results indicate that Bcl-x is dispensable for the generation of effector and memory T lymphocytes and suggest that costimulation of T lymphocytes promotes their survival through a Bcl-x(L) independent mechanism.

Bcl2l1 (Bcl-XL) belongs to the Bcl-2 family, Bcl2 and Bcl2-XL are major anti-apoptotic proteins, and the apoptosis of osteoblasts is a key event for bone homeostasis. As the functions of Bcl2l1 in osteoblasts and bone homeostasis remain unclear, we generated osteoblast-specific Bcl2l1-deficient (Bcl2l1fl/flCre) mice using 2.3-kb Col1a1 Cre. Trabecular bone volume and the trabecular number were lower in Bcl2l1fl/flCre mice of both sexes than in Bcl2l1fl/fl mice. In bone histomorphometric analysis, osteoclast parameters were increased in Bcl2l1fl/flCre mice, whereas osteoblast parameters and the bone formation rate were similar to those in Bcl2l1fl/fl mice. TUNEL-positive osteoblastic cells and serum TRAP5b levels were increased in Bcl2l1fl/flCre mice. The deletion of Bcl2l1 in osteoblasts induced Tnfsf11 expression, whereas the overexpression of Bcl-XL had no effect. In a co-culture of Bcl2l1-deficient primary osteoblasts and wild-type bone-marrow-derived monocyte/macrophage lineage cells, the numbers of multinucleated TRAP-positive cells and resorption pits increased. Furthermore, serum deprivation or the deletion of Bcl2l1 in primary osteoblasts increased apoptosis and ATP levels in the medium. Therefore, the reduction in trabecular bone in Bcl2l1fl/flCre mice may be due to enhanced bone resorption through osteoblast apoptosis and the release of ATP from apoptotic osteoblasts, and Bcl2l1 may inhibit bone resorption by preventing osteoblast apoptosis. [ABSTRACT FROM AUTHOR]

Abstract: Apoptosis is a mechanism that regulates hepatic tissue homeostasis and contributes to both acute and chronic injury in liver disease. The apoptotic signaling cascade involves activation of the death-inducing signaling complex (DISC) and subsequent recruitment of proteins containing death effector domains (DED), which regulate downstream effector molecules. Prominent among these are the Fas-associated death domain (FADD) and the cellular caspase 8-like inhibitory protein (cFLIP), and alterations in these proteins can lead to severe disruption of physiological processes, including acute liver failure or hepatocellular carcinoma. Their role in cell signaling events independent of the DISC remains undetermined. Oxidative stress can cause cell injury from direct effects on molecules or by activating intracellular signaling pathways including the mitogen-activated protein kinases (MAPKs). In this context, prolonged activation of the cJun N-terminal kinase (JNK)/AP-1/cJun signaling pathway promotes hepatocellular apoptosis, whereas activation of the extracellular signal-regulated kinase (Erk) exerts protection. We investigated the roles of FADD and cFLIP in acute oxidant stress induced by the superoxide generator menadione in hepatocytes. Menadione resulted in dose-dependent predominantly necrotic cell death. Hepatocytes expressing a truncated, dominant-negative FADD protein were partially protected, whereas cFLIP-deficient hepatocytes displayed increased cell death from menadione. In parallel, Erk phosphorylation was enhanced in hepatocytes expressing dnFADD and decreased in cFLIP-deficient hepatocytes. Hepatocyte injury was accompanied by increased release of proapoptotic factors and increased JNK/cJun activation. Thus, FADD and cFLIP contribute to the regulation of cell death from acute oxidant stress in hepatocytes involving MAPK signaling. This implies that DED-containing proteins are involved in the regulation of cellular survival beyond their role in cell death receptor–ligand-mediated apoptosis. [Copyright &y& Elsevier]

Background & Aims: Apoptosis is crucially involved in acute and chronic liver injury, including viral, cholestatic, toxic, and metabolic liver disease. Additionally, dysregulation of apoptosis signaling pathways has been implicated in hepatocarcinogenesis. The most prominent members of the apoptosis-mediating tumor necrosis factor receptor superfamily are the TNF-R1 (CD120a) and the CD95 (Apo-1/Fas) receptor. Although extensively studied, the intracellular signaling events in hepatocytes are only incompletely understood. Methods: To examine the role of the caspase-8 homolog cellular FLICE-inhibitory protein (c-FLIP) in liver injury, we generated mice with hepatocyte specific deletion of c-FLIP. Three models of acute liver injury were employed: the agonistic anti-CD95 antibody Jo2, d-galactosamine and LPS (GalN/LPS), and concanavalin A. Results: Conditional ablation of c-FLIP in hepatocytes augmented liver injury and cell death in all three models of liver injury. CD95- and GalN/LPS-induced liver injury was ameliorated by a pancaspase inhibitor, while ConA-induced injury was unaffected by caspase inhibition. Augmented activation of the MAPK JNK was observed in parallel to liver injury in c-FLIP knockout mice in all injury models; however, inhibition of JNK only affected TNF- and ConA-mediated injury. Conclusions: In summary, c-FLIP is a central regulator of cell death in hepatocytes, involving increased activation of caspases and the MAPK JNK. [ABSTRACT FROM AUTHOR]