Amanda Gonçalves

MLKL Compromises Plasma Membrane Integrity by Binding to Phosphatidylinositol Phosphates

Although mixed lineage kinase domain-like (MLKL) protein has emerged as a specific and crucial protein for necroptosis induction, how MLKL transduces the death signal remains poorly understood. Here, we demonstrate that the full four-helical bundle domain (4HBD) in the N-terminal region of MLKL is required and sufficient to induce its oligomerization and trigger cell death. Moreover, we found that a patch of positively charged amino acids on the surface of the 4HBD binds to phosphatidylinositol phosphates (PIPs) and allows recruitment of MLKL to the plasma membrane. Importantly, we found that recombinant MLKL, but not a mutant lacking these positive charges, induces leakage of PIP-containing liposomes as potently as BAX, supporting a model in which MLKL induces necroptosis by directly permeabilizing the plasma membrane. Accordingly, we found that inhibiting the formation of PI(5)P and PI(4,5)P2 specifically inhibits tumor necrosis factor (TNF)-mediated necroptosis but not apoptosis.

RIPK1 ensures intestinal homeostasis by protecting the epithelium against apoptosis

Receptor interacting protein kinase 1 (RIPK1) has an essential role in the signalling triggered by death receptors and pattern recognition receptors. RIPK1 is believed to function as a node driving NF-κB-mediated cell survival and inflammation as well as caspase-8 (CASP8)-dependent apoptotic or RIPK3/MLKL-dependent necroptotic cell death. The physiological relevance of this dual function has remained elusive because of the perinatal death of RIPK1 full knockout mice. To circumvent this problem, we generated RIPK1 conditional knockout mice, and show that mice lacking RIPK1 in intestinal epithelial cells (IECs) spontaneously develop severe intestinal inflammation associated with IEC apoptosis leading to early death. This early lethality was rescued by antibiotic treatment, MYD88 deficiency or tumour-necrosis factor (TNF) receptor 1 deficiency, demonstrating the importance of commensal bacteria and TNF in the IEC Ripk1 knockout phenotype. CASP8 deficiency, but not RIPK3 deficiency, rescued the inflammatory phenotype completely, indicating the indispensable role of RIPK1 in suppressing CASP8-dependent apoptosis but not RIPK3-dependent necroptosis in the intestine. RIPK1 kinase-dead knock-in mice did not exhibit any sign of inflammation, suggesting that RIPK1-mediated protection resides in its kinase-independent platform function. Depletion of RIPK1 in intestinal organoid cultures sensitized them to TNF-induced apoptosis, confirming the in vivo observations. Unexpectedly, TNF-mediated NF-κB activation remained intact in these organoids. Our results demonstrate that RIPK1 is essential for survival of IECs, ensuring epithelial homeostasis by protecting the epithelium from CASP8-mediated IEC apoptosis independently of its kinase activity and NF-κB activation.

Depletion of RIPK3 or MLKL blocks TNF-driven necroptosis and switches towards a delayed RIPK1 kinase-dependent apoptosis

In human cells, the RIPK1–RIPK3–MLKL–PGAM5–Drp1 axis drives tumor necrosis factor (TNF)-induced necroptosis through mitochondrial fission, but whether this pathway is conserved among mammals is not known. To answer this question, we analyzed the presence and functionality of the reported necroptotic axis in mice. As in humans, knockdown of receptor-interacting kinase-3 (RIPK3) or mixed lineage kinase domain like (MLKL) blocks TNF-induced necroptosis in L929 fibrosarcoma cells. However, repression of either of these proteins did not protect the cells from death, but instead induced a switch from TNF-induced necroptosis to receptor-interacting kinase-1 (RIPK1) kinase-dependent apoptosis. In addition, although mitochondrial fission also occurs during TNF-induced necroptosis in L929 cells, we found that knockdown of phosphoglycerate mutase 5 (PGAM5) and dynamin 1 like protein (Drp1) did not markedly protect the cells from TNF-induced necroptosis. Depletion of Pink1, a reported interactor of both PGAM5 and Drp1, did not affect TNF-induced necroptosis. These results indicate that in these murine cells mitochondrial fission and Pink1 dependent processes, including Pink-Parkin dependent mitophagy, apparently do not promote necroptosis. Our data demonstrate that the core components of the necrosome (RIPK1, RIPK3 and MLKL) are crucial to induce TNF-dependent necroptosis both in human and in mouse cells, but the associated mechanisms may differ between the two species or cell types.

The IL-33/ST2 axis is crucial in type 2 airway responses induced by Staphylococcus aureus–derived serine protease–like protein D

Background: Chronic airway inflammatory diseases, such as chronic rhinosinusitis with nasal polyps and asthma, show increased nasal Staphylococcus aureus colonization. Staphylococcus aureus–derived serine protease–like protein (Spl) D and other closely related proteases secreted by S aureus have recently been identified as inducers of allergic asthma in human subjects and mice, but their mechanism of action is largely unknown. Objective: We investigated the role of recombinant SplD in driving TH2‐biased responses and IgE formation in a murine model of allergic asthma. Methods: Allergic asthma was induced in C57BL/6 J wild‐type mice, Toll‐like receptor (TLR) 4 knockout (Tlr4−/−) mice, and recombination‐activating gene (Rag2) knockout (Rag2−/−) mice by means of repeated intratracheal applications of SplD. Inflammatory parameters in the airways were assessed by means of flow cytometry, ELISA, Luminex, and immunohistochemistry. Serum SplD‐specific IgE levels were analyzed by using ELISA. Results: We observed that repeated intratracheal exposure to SplD led to IL‐33 and eotaxin production, eosinophilia, bronchial hyperreactivity, and goblet cell hyperplasia in the airways. Blocking IL‐33 activity with a soluble ST2 receptor significantly decreased the numbers of eosinophils, IL‐13+ type 2 innate lymphoid cells and IL‐13+CD4+ T cells and IL‐5 and IL‐13 production by lymph node cells but had no effect on IgE production. SplD‐induced airway inflammation and IgE production were largely dependent on the presence of the functional adaptive immune system and independent of TLR4 signaling. Conclusion: The S aureus–derived protein SplD is a potent allergen of S aureus and induces a TH2‐biased inflammatory response in the airways in an IL‐33–dependent but TRL4‐independent manner. The soluble ST2 receptor could be an efficient strategy to interfere with SplD‐induced TH2 inflammation but does not prevent the allergic sensitization. Graphical abstract Figure. No caption available.

Caspase-1 Engagement and TLR-Induced c-FLIP Expression Suppress ASC/Caspase-8-Dependent Apoptosis by Inflammasome Sensors NLRP1b and NLRC4

Summary The caspase activation and recruitment domain (CARD)-based inflammasome sensors NLRP1b and NLRC4 induce caspase-1-dependent pyroptosis independent of the inflammasome adaptor ASC. Here, we show that NLRP1b and NLRC4 trigger caspase-8-mediated apoptosis as an alternative cell death program in caspase-1−/− macrophages and intestinal epithelial organoids (IECs). The caspase-8 adaptor FADD was recruited to ASC specks, which served as cytosolic platforms for caspase-8 activation and NLRP1b/NLRC4-induced apoptosis. We further found that caspase-1 protease activity dominated over scaffolding functions in suppressing caspase-8 activation and induction of apoptosis of macrophages and IECs. Moreover, TLR-induced c-FLIP expression inhibited caspase-8-mediated apoptosis downstream of ASC speck assembly, but did not affect pyroptosis induction by NLRP1b and NLRC4. Moreover, unlike during pyroptosis, NLRP1b- and NLRC4-elicited apoptosis retained alarmins and the inflammasome-matured cytokines interleukin 1β (IL-1β) and IL-18 intracellularly. This work identifies critical mechanisms regulating apoptosis induction by the inflammasome sensors NLRP1b and NLRC4 and suggests converting pyroptosis into apoptosis as a paradigm for suppressing inflammation.

Noninvasive Whole-Body Imaging of Phosphatidylethanolamine as a Cell Death Marker Using 99mTc-Duramycin During TNF-Induced SIRS

Systemic inflammatory response syndrome (SIRS) is an inflammatory state affecting the whole body. It is associated with the presence of pro- and antiinflammatory cytokines in serum, including tumor necrosis factor (TNF). TNF has multiple effects and leads to cytokine production, leukocyte infiltration, and blood pressure reduction and coagulation, thereby contributing to tissue damage and organ failure. A sterile mouse model of sepsis, TNF-induced SIRS, was used to visualize the temporal and spatial distribution of damage in susceptible tissues during SIRS. For this, a radiopharmaceutical agent, 99mTc-duramycin, that binds to exposed phosphatidylethanolamine on dying cells was longitudinally visualized using SPECT/CT imaging. Methods: C57BL/6J mice were challenged with intravenous injections of murine TNF or vehicle, and necrostatin-1 was used to interfere with cell death. Two hours after vehicle or TNF treatment, mice received 99mTc-duramycin intravenously (35.44 ± 3.80 MBq). Static whole-body 99mTc-duramycin SPECT/CT imaging was performed 2, 4, and 6 h after tracer injection. Tracer uptake in different organs was quantified by volume-of-interest analysis using PMOD software and expressed as SUVmean. After the last scan, ex vivo biodistribution was performed to validate the SPECT imaging data. Lastly, terminal deoxynucleotidyl-transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was performed to correlate the obtained results to cell death. Results: An increased 99mTc-duramycin uptake was detected in mice injected with TNF, when compared with control mice, in lungs (0.55 ± 0.1 vs. 0.34 ± 0.05), intestine (0.75 ± 0.13 vs. 0.56 ± 0.1), and liver (1.03 ± 0.14 vs. 0.64 ± 0.04) 4 h after TNF and remained significantly elevated until 8 h after TNF. The imaging results were consistent with ex vivo γ-counting results. Significantly increased levels of tissue damage were detected via TUNEL staining in the lungs and intestine of mice injected with TNF. Interestingly, necrostatin-1 pretreatment conferred protection against lethal SIRS and reduced the 99mTc-duramycin uptake in the lungs 8 h after TNF (SUV, 0.32 ± 0.1 vs. 0.51 ± 0.15). Conclusion: This study demonstrated that noninvasive 99mTc-duramycin SPECT imaging can be used to characterize temporal and spatial kinetics of injury and cell death in susceptible tissues during TNF-induced SIRS, making it useful for global, whole-body assessment of tissue damage during diseases associated with inflammation and injury.

Oral delivery of Escherichia coli persistently infected with M2e-displaying bacteriophages partially protects against influenza A virus

&NA; We describe a novel live oral vaccine type. Conceptually, this vaccine is based on a non‐lytic, recombinant filamentous bacteriophage that displays an antigen of interest. To provide proof of concept we used the amino‐terminal part of a conserved influenza A virus epitope, i.e. matrix protein 2 ectodomain (M2e) residues 2 to 16, as the antigen of interest. Rather than using the phages as purified virus‐like particles as a vaccine, these phages were delivered to intestinal Peyers patches as a live bacterium‐phage combination that comprises Escherichia coli cells that conditionally express invasin derived from Yersinia pseudotuberculosis. Invasin‐expressing E. coli cells were internalized by mammalian Hep‐2 cells in vitro and adhered to mouse intestinal microfold (M) cells ex vivo. Invasin‐expressing E. coli cells were permissive for recombinant filamentous bacteriophage f88 that displays M2e and became persistently infected. Oral administration of the live engineered E. coli‐invasin‐phage combination to mice induced M2e‐specific serum IgG antibodies. Mice that had been immunized with invasin‐expressing E. coli cells that carried M2e2‐16 displaying fd phages seroconverted to M2e and showed partial protection against challenge with influenza A virus. Oral delivery of a live vaccine comprising a bacterial host that is targeted to Peyers patches and is persistently infected with an antigen‐displaying phage, can thus be exploited as an oral vaccine. Graphical abstract Figure. No caption available.

A20 critically controls microglia activation and inhibits inflammasome-dependent neuroinflammation

Microglia, the mononuclear phagocytes of the central nervous system (CNS), are important for the maintenance of CNS homeostasis, but also critically contribute to CNS pathology. Here we demonstrate that the nuclear factor kappa B (NF-κB) regulatory protein A20 is crucial in regulating microglia activation during CNS homeostasis and pathology. In mice, deletion of A20 in microglia increases microglial cell number and affects microglial regulation of neuronal synaptic function. Administration of a sublethal dose of lipopolysaccharide induces massive microglia activation, neuroinflammation, and lethality in mice with microglia-confined A20 deficiency. Microglia A20 deficiency also exacerbates multiple sclerosis (MS)-like disease, due to hyperactivation of the Nlrp3 inflammasome leading to enhanced interleukin-1β secretion and CNS inflammation. Finally, we confirm a Nlrp3 inflammasome signature and IL-1β expression in brain and cerebrospinal fluid from MS patients. Collectively, these data reveal a critical role for A20 in the control of microglia activation and neuroinflammation.As resident macrophages of the brain, microglia are important for neuroinflammatory responses. This work shows that nuclear factor kappa B regulatory protein A20 is important for microglia activation and regulation during inflammation of the central nervous system.

Localization and Expression of Nuclear Factor of Activated T-Cells 5 in Myoblasts Exposed to Pro-inflammatory Cytokines or Hyperosmolar Stress and in Biopsies from Myositis Patients

Aims: Regeneration in skeletal muscle relies on regulated myoblast migration and differentiation, in which the transcription factor nuclear factor of activated T-cells 5 (NFAT5) participates. Impaired muscle regeneration and chronic inflammation are prevalent in myositis. Little is known about the impact of inflammation on NFAT5 localization and expression in this group of diseases. The goal of this study was to investigate NFAT5 physiology in unaffected myoblasts exposed to cytokine or hyperosmolar stress and in myositis. Methods: NFAT5 intracellular localization and expression were studied in vitro using a cell culture model of myositis. Myoblasts were exposed to DMEM solutions enriched with pro-inflammatory cytokines IFN-γ with IL-1β or hyperosmolar DMEM obtained by NaCl supplementation. NFAT5 localization was visualized using immunohistochemistry (IHC) and Western blotting (WB) in fractionated cell lysates. NFAT5 expression was assessed by WB and RT-qPCR. In vivo localization and expression of NFAT5 were studied in muscle biopsies of patients diagnosed with polymyositis (n = 6), dermatomyositis (n = 10), inclusion body myositis (n = 11) and were compared to NFAT5 localization and expression in non-myopathic controls (n = 13). Muscle biopsies were studied by means of quantitative IHC and WB of total protein extracts. Results: In unaffected myoblasts, hyperosmolar stress ensues in NFAT5 nuclear translocation and increased NFAT5 mRNA and protein expression. In contrast, pro-inflammatory cytokines did not lead to NFAT5 nuclear translocation nor increased expression. Cytokines IL-1β with IFN-γ induced colocalization of NFAT5 with histone deacetylase 6 (HDAC6), involved in cell motility. In muscle biopsies from dermatomyositis and polymyositis patients, NFAT5 colocalized with HDAC6, while in IBM, this was often absent. Conclusions: Our data suggest impaired NFAT5 localization and expression in unaffected myoblasts in response to inflammation. This disturbed myogenic NFAT5 physiology could possibly explain deleterious effects on muscle regeneration in myositis.

Monitoring Ubiquitin-Coated Bacteria via Confocal Microscopy

Salmonella is a gram-negative facultative intracellular pathogen that is capable of infecting a variety of hosts. Inside host cells, most Salmonella bacteria reside and replicate within Salmonella-containing vacuoles. They use virulence proteins to manipulate the host cell machinery for their own benefit and hijack the host cytoskeleton to travel toward the perinuclear area. However, a fraction of bacteria escapes into the cytosol where they get decorated with a dense layer of polyubiquitin, which labels the bacteria for clearance by autophagy. More specifically, autophagy receptor proteins recognize the ubiquitinated bacteria and deliver them to autophagosomes, which subsequently fuse to lysosomes. Here, we describe methods used to infect HeLa cells with Salmonella bacteria and to detect their ubiquitination via immunofluorescence and laser scanning confocal microscopy.