Die Regulation des Zelltods ist ein kritischer Schritt in der Embryonalentwicklung, der Gewebehomöostase und bei der Abwehr eindringender Krankheitserreger. Hierbei spielt Caspase-8 (Casp8) eine entscheidende Rolle, indem das Enzym extrinsische Apoptose induziert. Außerdem hemmt Casp8 Nekroptose und sichert so die embryonale Entwicklung. Wir zeigen hier, dass eine weitere wichtige Funktion der enzymatische Aktivität von Casp8 die Hemmung der Pyroptose ist. Um die spezifische Rolle der enzymatischen Aktivität von Casp8 zu untersuchen, haben wir eine Mauslinie etabliert, welche eine Punktmutation in der Substratbindetasche von Casp8 (Mutation des katalytischen Cysteins 362 zu Serin) aufweist. Diese Mutation führt zum Verlust der enzymatischer Aktivität von Casp8 C362S. Die Expression von katalytisch inaktiver Casp8 C362S resultiert in eine embryonale Letalität bei E10,5 aufgrund von kardiovaskulären Defekten, ähnlich zu Casp8-/-Mäusen. Die Blockade von Nekroptose durch zusätzliche Deletion von MLKL verhinderte den kardiovaskulären Phänotyp, verursachte jedoch unerwartet die perinatale Letalität von Casp8 C362S/C362S Mäusen. Dies deutet darauf hin, dass der Verlust der enzymatischen Aktivität von Casp8 die perinatale Entwicklung durch zusätzliche, Nekroptose-unabhängigen Funktionen beeinträchtigt. Der spezifische Verlust der katalytischen Aktivität von Casp8 in Darmepithelzellen (IECs) führte zu einer Darmentzündung ähnlich zu Casp8 IEC-KO Mäusen. Eine zusätzliche Deletion von MLKL verschlimmerte die Entzündung des Darms und verursachte ein vorzeitiges Versterben von Casp8 C362S/IEC-/Mlkl-/-Mäusen durch die Induktion des pyroptischen Zelltods. In diesem Zusammenhang konnten Prozesse, welche charakteristisch für Pyroptose sind, wie die Bildung von ASC-Specks, die Aktivierung von Casp1 sowie die Produktion von IL-1β beobachtet werden. Unsere Zellkulturanalysen zeigen, dass katalytisch inaktive Casp8 mit ASC kolokalisiert, sowie ASC-Nukleation und Casp1-Aktivierung induziert. Dementsprechend verhinderte die Deletion von ASC oder Casp1 die Entzündung des Darms und den vorzeitigen Tod von Casp8 C362S/C362S /Mlkl-/-Mäusen. Diese Ergebnisse zeigen eine noch unbekannte und unerwartete Rolle für Casp8 als Proteingerüst eines Signalkomplexes, welcher unter Gegebenheiten bei denen Apoptose und Nekroptose blockiert sind, gebildet wird.

Conjugation of Met1-linked polyubiquitin (Met1-Ub) by the linear ubiquitin chain assembly complex (LUBAC) is an important regulatory modification in innate immune signaling. So far, only few Met1-Ub substrates have been described, and the regulatory mechanisms have remained elusive. We recently identified that the ovarian tumor (OTU) family deubiquitinase OTULIN specifically disassembles Met1-Ub. Here, we report that OTULIN is critical for limiting Met1-Ub accumulation after nucleotide-oligomerization domain-containing protein 2 (NOD2) stimulation, and that OTULIN depletion augments signaling downstream of NOD2. Affinity purification of Met1-Ub followed by quantitative proteomics uncovered RIPK2 as the predominant NOD2-regulated substrate. Accordingly, Met1-Ub on RIPK2 was largely inhibited by overexpressing OTULIN and was increased by OTULIN depletion. Intriguingly, OTULIN-depleted cells spontaneously accumulated Met1-Ub on LUBAC components, and NOD2 or TNFR1 stimulation led to extensive Met1-Ub accumulation on receptor complex components. We propose that OTULIN restricts Met1-Ub formation after immune receptor stimulation to prevent unwarranted proinflammatory signaling.

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Cell death plays an important role during pathogen infections. Here, we report that interferon-γ (IFNγ) sensitizes macrophages to Toll-like receptor (TLR)-induced death that requires macrophage-intrinsic death ligands and caspase-8 enzymatic activity, which trigger the mitochondrial apoptotic effectors, BAX and BAK. The pro-apoptotic caspase-8 substrate BID was dispensable for BAX and BAK activation. Instead, caspase-8 reduced pro-survival BCL-2 transcription and increased inducible nitric oxide synthase (iNOS), thus facilitating BAX and BAK signaling. IFNγ-primed, TLR-induced macrophage killing required iNOS, which licensed apoptotic caspase-8 activity and reduced the BAX and BAK inhibitors, A1 and MCL-1. The deletion of iNOS or caspase-8 limited SARS-CoV-2-induced disease in mice, while caspase-8 caused lethality independent of iNOS in a model of hemophagocytic lymphohistiocytosis. These findings reveal that iNOS selectively licenses programmed cell death, which may explain how nitric oxide impacts disease severity in SARS-CoV-2 infection and other iNOS-associated inflammatory conditions.

Corticosteroids are host-directed drugs with proven beneficial effect on survival of tuberculosis (TB) patients, but their precise mechanisms of action in this disease remain largely unknown. Here we show that corticosteroids such as dexamethasone inhibit necrotic cell death of cells infected with Mycobacterium tuberculosis (Mtb) by facilitating mitogen-activated protein kinase phosphatase 1 (MKP-1)-dependent dephosphorylation of p38 MAPK. Characterization of infected mixed lineage kinase domain-like (MLKL) and tumor necrosis factor receptor 1 (TNFR1) knockout cells show that the underlying mechanism is independent from TNFα-signaling and necroptosis. Our results link corticosteroid function and p38 MAPK inhibition to abrogation of necrotic cell death mediated by mitochondrial membrane permeability transition, and open new avenues for research on novel host-directed therapies (HDT).

BackgroundImmune infiltration is implicated in the development of acquired resistance to anti-angiogenic cancer therapy. We therefore investigated the correlation between neutrophil infiltration in metastasis of colorectal cancer (CRC) patients and survival after treatment with bevacizumab. Our study identifies CD177+ tumour neutrophil infiltration as an adverse prognostic factor for bevacizumab treatment. We further demonstrate that a novel anti-VEGF/anti-Ang2 compound (BI-880) can overcome resistance to VEGF inhibition in experimental tumour models.MethodsA total of 85 metastatic CRC patients were stratified into cohorts that had either received chemotherapy alone (n = 39) or combined with bevacizumab (n = 46). Tumour CD177+ neutrophil infiltration was correlated to clinical outcome. The impact of neutrophil infiltration on anti-VEGF or anti-VEGF/anti-Ang2 therapy was studied in both xenograft and syngeneic tumour models by immunohistochemistry.ResultsThe survival of bevacizumab-treated CRC patients in the presence of CD177+ infiltrates was significantly reduced compared to patients harbouring CD177− metastases. BI-880 treatment reduced the development of hypoxia associated with bevacizumab treatment and improved vascular normalisation in xenografts. Furthermore, neutrophil depletion or BI-880 treatment restored treatment sensitivity in a syngeneic tumour model of anti-VEGF resistance.ConclusionsOur findings implicate CD177 as a biomarker for bevacizumab and suggest VEGF/Ang2 inhibition as a strategy to overcome neutrophil associated resistance to anti-angiogenic treatment.

The vasculature represents a highly plastic compartment, capable of switching from a quiescent to an active proliferative state during angiogenesis. Metabolic reprogramming in endothelial cells (ECs) thereby is crucial to cover the increasing cellular energy demand under growth conditions. Here we assess the impact of mitochondrial bioenergetics on neovascularisation, by deleting cox10 gene encoding an assembly factor of cytochrome c oxidase (COX) specifically in mouse ECs, providing a model for vasculature-restricted respiratory deficiency. We show that EC-specific cox10 ablation results in deficient vascular development causing embryonic lethality. In adult mice induction of EC-specific cox10 gene deletion produces no overt phenotype. However, the angiogenic capacity of COX-deficient ECs is severely compromised under energetically demanding conditions, as revealed by significantly delayed wound-healing and impaired tumour growth. We provide genetic evidence for a requirement of mitochondrial respiration in vascular endothelial cells for neoangiogenesis during development, tissue repair and cancer.

The X-linked inhibitor of apoptosis protein (XIAP) is a potent caspase inhibitor, best known for its anti-apoptotic function in cancer. During apoptosis, XIAP is antagonized by SMAC, which is released from the mitochondria upon caspase-mediated activation of BID. Recent studies suggest that XIAP is involved in immune signaling. Here, we explore XIAP as an important mediator of an immune response against the enteroinvasive bacterium Shigella flexneri, both in vitro and in vivo. Our data demonstrate for the first time that Shigella evades the XIAP-mediated immune response by inducing the BID-dependent release of SMAC from the mitochondria. Unlike apoptotic stimuli, Shigella activates the calpain-dependent cleavage of BID to trigger the release of SMAC, which antagonizes the inflammatory action of XIAP without inducing apoptosis. Our results demonstrate how the cellular death machinery can be subverted by an invasive pathogen to ensure bacterial colonization.