Concomitant hepatocyte apoptosis and regeneration is a hallmark of chronic liver diseases (CLDs) predisposing to hepatocellular carcinoma (HCC). Here, we mechanistically link caspase-8-dependent apoptosis to HCC development via proliferation- and replication-associated DNA damage. Proliferation-associated replication stress, DNA damage, and genetic instability are detectable in CLDs before any neoplastic changes occur. Accumulated levels of hepatocyte apoptosis determine and predict subsequent hepatocarcinogenesis. Proliferation-associated DNA damage is sensed by a complex comprising caspase-8, FADD, c-FLIP, and a kinase-dependent function of RIPK1. This platform requires a non-apoptotic function of caspase-8, but no caspase-3 or caspase-8 cleavage. It may represent a DNA damage-sensing mechanism in hepatocytes that can act via JNK and subsequent phosphorylation of the histone variant H2AX.

Caspase-8, the initiator caspase of the death receptor pathway of apoptosis, its adapter molecule, FADD, required for caspase-8 activation, and cFLIPL, a caspase-8-like protein that lacks a catalytic site and blocks caspase-8-mediated apoptosis, are each essential for embryonic development. Animals deficient in any of these genes present with E10.5 embryonic lethality. Recent studies have shown that development in caspase-8-deficient mice is rescued by ablation of RIPK3, a kinase that promotes a form of programmed, necrotic cell death. Here, we show that FADD, RIPK3 double-knockout mice develop normally but that the lethal effects of cFLIP deletion are not rescued by RIPK3 deficiency. Remarkably, in mice lacking FADD, cFLIP, and RIPK3, embryonic development is normal. This can be explained by the convergence of two cell processes: the enzymatic activity of the FADD-caspase-8-cFLIPL complex blocks RIPK3-dependent signaling (including necrosis), whereas cFLIPL blocks RIPK3-independent apoptosis promoted by the FADD-caspase-8 complex.

Expression of enzymatically inactive caspase-8, or deletion of caspase-8 from basal epidermal keratinocytes, triggers chronic skin inflammation in mice. Unlike similar inflammation resulting from arrest of nuclear factor kappaB activation in the epidermal cells, the effect induced by caspase-8 deficiency did not depend on TNF, IL-1, dermal macrophage function, or expression of the toll-like receptor adapter proteins MyD88 or TRIF. Both interferon regulatory factor (IRF) 3 and TANK-binding kinase were constitutively phosphorylated in the caspase-8-deficient epidermis, and knockdown of IRF3 in the epidermis-derived cells from these mice abolished the expression of up-regulated genes. Temporal and spatial analyses of the alterations in gene expression that result from caspase-8 deficiency reveal that the changes are initiated before birth, around the time that cornification develops, and occur mainly in the suprabasal layer. Finally, we found that caspase-8-deficient keratinocytes display an enhanced response to gene activation by transfected DNA. Our findings suggest that an enhanced response to endogenous activators of IRF3 in the epidermis, presumably generated in association with keratinocyte differentiation, contributes to the skin inflammatory process triggered by caspase-8 deficiency.

Activation of the pseudokinase mixed lineage kinase domain-like (MLKL) upon its phosphorylation by the protein kinase RIPK3 triggers necroptosis, a form of programmed cell death in which rupture of cellular membranes yields release of intracellular components. We report that MLKL also associated with endosomes and controlled the transport of endocytosed proteins, thereby enhancing degradation of receptors and ligands, modulating their induced signaling and facilitating the generation of extracellular vesicles. This role was exerted on two quantitative grades: a constitutive one independent of RIPK3, and an enhanced one, triggered by RIPK3, where the association of MLKL with the endosomes was enhanced, and it was found to bind endosomal sorting complexes required for transport (ESCRT) proteins and the flotillins and to be excluded, together with them, from cells within vesicles. We suggest that release of phosphorylated MLKL within extracellular vesicles serves as a mechanism for self-restricting the necroptotic activity of this protein.

The NF-kappaB-inducing kinase (NIK) induces proteolytic processing of NF-kappaB2/p100 and, hence, the generation of NF-kappaB dimers such as p52:RelB but was suggested not to signal for the processing of IkappaB. Here, we show that although the induction of IkappaB degradation in lymphocytes by TNF is independent of NIK, its induction by CD70, CD40 ligand, and BLyS/BAFF, which all also induce NF-kappaB2/p100 processing, does depend on NIK function. Both CD70 and TNF induce recruitment of the IKK kinase complex to their receptors. In the case of CD70, but not TNF, this process is associated with NIK recruitment and is followed by prolonged receptor association of just IKK1 and NIK. Recruitment of the IKK complex to CD27, but not that of NIK, depends on NIK kinase function. Our findings indicate that NIK participates in a unique set of proximal signaling events initiated by specific inducers, which activate both canonical and noncanonical NF-kappaB dimers.

B cell homeostasis is regulated by multiple signaling processes, including nuclear factor-kappaB (NF-kappaB), BAFF-, and B cell receptor signaling. Conditional disruption of genes involved in these pathways has shed light on the mechanisms governing signaling from the cell surface to the nucleus. We describe a novel mouse strain that expresses solely and excessively a naturally occurring splice variant of CYLD (CYLD(ex7/8) mice), which is a deubiquitinating enzyme that is integral to NF-kappaB signaling. This shorter CYLD protein lacks the TRAF2 and NEMO binding sites present in full-length CYLD. A dramatic expansion of mature B lymphocyte populations in all peripheral lymphoid organs occurs in this strain. The B lymphocytes themselves exhibit prolonged survival and manifest a variety of signaling disarrangements that do not occur in mice with a complete deletion of CYLD. Although both the full-length and the mutant CYLD are able to interact with Bcl-3, a predominant nuclear accumulation of Bcl-3 occurs in the CYLD mutant B cells. More dramatic, however, is the accumulation of the NF-kappaB proteins p100 and RelB in CYLD(ex7/8) B cells, which, presumably in combination with nuclear Bcl-3, results in increased levels of Bcl-2 expression. These findings suggest that CYLD can both positively and negatively regulate signal transduction and homeostasis of B cells in vivo, depending on the expression of CYLD splice variants.

Excessive responses to pattern-recognition receptors are prevented by regulatory mechanisms that affect the amounts and activities of the downstream signaling proteins. We report that activation of the transcription factor IRF3 by the ribonucleic acid sensor RIG-I was restricted by caspase-8-mediated cleavage of the RIP1 protein, which resulted in conversion of RIP1 from a signaling enhancer to a signaling inhibitor. The proteins RIP1 and caspase-8 were recruited to the RIG-I complex after viral infection and served antagonistic regulatory roles. Conjugation of ubiquitin chains to RIP1 facilitated assembly of the RIG-I complex, resulting in enhanced phosphorylation of IRF3. However, the ubiquitination of RIP1 also rendered it susceptible to caspase-8-mediated cleavage that yielded an inhibitory RIP1 fragment. The dependence of RIP1 cleavage on the same molecular change as that facilitating RIG-I signaling allows for RIG-I signaling to be restricted in its duration without compromising its initial activation.

Tumor necrosis factor (TNF) is a potent cytokine exerting critical functions in the activation and regulation of immune and inflammatory responses. Due to its pleiotropic activities, the amplitude and duration of TNF function must be tightly regulated. One of the mechanisms that may have evolved to modulate TNF function is the proteolytic cleavage of its cell surface receptors. In humans, mutations affecting shedding of the p55TNF receptor (R) have been linked with the development of the TNFR-associated periodic syndromes, disorders characterized by recurrent fever attacks and localized inflammation. Here we show that knock-in mice expressing a mutated nonsheddable p55TNFR develop Toll-like receptor-dependent innate immune hyperreactivity, which renders their immune system more efficient at controlling intracellular bacterial infections. Notably, gain of function for antibacterial host defenses ensues at the cost of disbalanced inflammatory reactions that lead to pathology. Mutant mice exhibit spontaneous hepatitis, enhanced susceptibility to endotoxic shock, exacerbated TNF-dependent arthritis, and experimental autoimmune encephalomyelitis. These results introduce a new concept for receptor shedding as a mechanism setting up thresholds of cytokine function to balance resistance and susceptibility to disease. Assessment of p55TNFR shedding may thus be of prognostic value in infectious, inflammatory, and autoimmune diseases.

Deletion of the Casp8 gene in epithelial tissues of mice results in severe inflammatory pathologies. Its ubiquitous deletion, or its specific deletion in endothelial cells, results in intrauterine death associated with capillary damage. These pathologies are all preventable by co-deletion of Casp8 and the genes encoding either the RIPK1 or the RIPK3 protein kinase. Since activation of RIPK3 in Caspase-8-deficient cells can trigger necroptotic cell death, and since RIPK1 can activate RIPK3, it is widely assumed that the inflammatory states resulting from Caspase-8 deficiency occur as a consequence of RIPK3-induced necroptosis. Here, we report that although on a Ripk3-null background Casp8 deletion in mice does not result in outright pathological changes, it triggers enhanced expression of a variety of inflammatory genes in utero, which gradually subsides after birth. Deletion of Ripk1, or even of only one of its two alleles, obliterates this activation. Resembling the embryonic pathology observed in RIPK3-expressing cells, the activation of inflammatory genes observed on a Ripk3-null background seems to be initiated in endothelial cells. Analysis of endothelial cells isolated from livers of Caspase-8-deficient embryos revealed neither an increase in the amount of RIPK1 in these cells after Casp8 deletion, nor triggering of RIPK1 phosphorylation. These findings indicate that the triggering of inflammation by Casp8 deletion in mice occurs, in part, independently of necroptosis or other functions of RIPK3, and rather reflects enhanced RIPK1-dependent signaling for activation of inflammatory genes.

Phosphorylation of the pseudokinase mixed lineage kinase domain-like protein (MLKL) by the protein kinase RIPK3 targets MLKL to the cell membrane, where it triggers necroptotic cell death. We report that conjugation of K63-linked polyubiquitin chains to distinct lysine residues in the N-terminal HeLo domain of phosphorylated MLKL (facilitated by the ubiquitin ligase ITCH that binds MLKL via a WW domain) targets MLKL instead to endosomes. This results in the release of phosphorylated MLKL within extracellular vesicles. It also prompts enhanced endosomal trafficking of intracellular bacteria such as Listeria monocytogenes and Yersinia enterocolitica to the lysosomes, resulting in decreased bacterial yield. Thus, MLKL can be directed by specific covalent modifications to differing subcellular sites, whence it signals either for cell death or for non-deadly defense mechanisms.

The global emergence of novel pathogenic viruses presents an important challenge for research, as high biosafety levels are required to process samples. While inactivation of infectious agents facilitates the use of less stringent safety conditions, its effect on other biological entities of interest present in the sample is generally unknown. Here, we analyzed the effect of five inactivation methods (heat, ethanol, formaldehyde, psoralen, and TRIzol) on microbiome composition and diversity in samples collected from four different body sites (gut, nasal, oral, and skin) and compared them against untreated samples from the same tissues. We performed 16S rRNA gene sequencing and estimated abundance and diversity of bacterial taxa present in all samples. Nasal and skin samples were the most affected by inactivation, with ethanol and TRIzol inducing the largest changes in composition, and heat, formaldehyde, TRIzol, and psoralen inducing the largest changes in diversity. Oral and stool microbiomes were more robust to inactivation, with no significant changes in diversity and only moderate changes in composition. Firmicutes was the taxonomic group least affected by inactivation, while Bacteroidetes had a notable enrichment in nasal samples and moderate enrichment in fecal and oral samples. Actinobacteria were more notably depleted in fecal and skin samples, and Proteobacteria exhibited a more variable behavior depending on sample type and inactivation method. Overall, our results demonstrate that inactivation methods can alter the microbiome in a tissue-specific manner and that careful consideration should be given to the choice of method based on the sample type under study. IMPORTANCE Understanding how viral infections impact and are modulated by the microbiome is an important problem in basic research but is also of high clinical relevance under the current pandemic. To facilitate the study of interactions between microbial communities and pathogenic viruses under safe conditions, the infectious agent is generally inactivated prior to processing samples. The effect of this inactivation process in the microbiome is, however, unknown. Further, it is unclear whether biases introduced by inactivation methods are dependent on the sample type under study. Estimating the magnitude and nature of the changes induced by different methods in samples collected from various body sites thus provides important information for current and future studies that require inactivation of pathogenic agents.