RIPK1 protects naive and regulatory T cells from TNFR1-induced apoptosis.

The T cell population size is stringently controlled before, during, and after immune responses, as improper cell death regulation can result in autoimmunity and immunodeficiency. RIPK1 is an important regulator of peripheral T cell survival and homeostasis. However, whether different peripheral T cell subsets show a differential requirement for RIPK1 and which programmed cell death pathway they engage in vivo remains unclear. In this study, we demonstrate that conditional ablation of Ripk1 in conventional T cells (Ripk1ΔCD4) causes peripheral T cell lymphopenia, as witnessed by a profound loss of naive CD4+, naive CD8+, and FoxP3+ regulatory T cells. Interestingly, peripheral naive CD8+ T cells in Ripk1ΔCD4 mice appear to undergo a selective pressure to retain RIPK1 expression following activation. Mixed bone marrow chimeras revealed a competitive survival disadvantage for naive, effector, and memory T cells lacking RIPK1. Additionally, tamoxifen-induced deletion of RIPK1 in CD4-expressing cells in adult life confirmed the importance of RIPK1 in post-thymic survival of CD4+ T cells. Ripk1K45A mice showed no change in peripheral T cell subsets, demonstrating that the T cell lymphopenia was due to the scaffold function of RIPK1 rather than to its kinase activity. Enhanced numbers of Ripk1ΔCD4 naive T cells expressed the proliferation marker Ki-67+ despite the peripheral lymphopenia and single-cell RNA sequencing revealed T cell-specific transcriptomic alterations that were reverted by additional caspase-8 deficiency. Furthermore, Ripk1ΔCD4Casp8 ΔCD4 and Ripk1ΔCD4Tnfr1-/- double-knockout mice rescued the peripheral T cell lymphopenia, revealing that RIPK1-deficient naive CD4+ and CD8+ cells and FoxP3+ regulatory T cells specifically die from TNF- and caspase-8-mediated apoptosis in vivo. Altogether, our findings emphasize the essential role of RIPK1 as a scaffold in maintaining the peripheral T cell compartment and preventing TNFR1-induced apoptosis.

Evolutionary origin of Hoxc13-dependent skin appendages in amphibians.

Cornified skin appendages, such as hair and nails, are major evolutionary innovations of terrestrial vertebrates. Human hair and nails consist largely of special intermediate filament proteins, known as hair keratins, which are expressed under the control of the transcription factor Hoxc13. Here, we show that the cornified claws of Xenopus frogs contain homologs of hair keratins and the genes encoding these keratins are flanked by promoters in which binding sites of Hoxc13 are conserved. Furthermore, these keratins and Hoxc13 are co-expressed in the claw-forming epithelium of frog toe tips. Upon deletion of hoxc13, the expression of hair keratin homologs is abolished and the development of cornified claws is abrogated in X. tropicalis. These results indicate that Hoxc13-dependent expression of hair keratin homologs evolved already in stem tetrapods, presumably as a mechanism for protecting toe tips, and that this ancestral genetic program was coopted to the growth of hair in mammals.

Functions of the RIP kinase family members in the skin.

The receptor interacting protein kinases (RIPK) are a family of serine/threonine kinases that are involved in the integration of various stress signals. In response to several extracellular and/or intracellular stimuli, RIP kinases engage signaling cascades leading to the activation of NF-κB and mitogen-activated protein kinases, cell death, inflammation, differentiation and Wnt signaling and can have kinase-dependent and kinase-independent functions. Although it was previously suggested that seven RIPKs are part of the RIPK family, phylogenetic analysis indicates that there are only five genuine RIPKs. RIPK1 and RIPK3 are mainly involved in controlling and executing necroptosis in keratinocytes, while RIPK4 controls proliferation and differentiation of keratinocytes and thereby can act as a tumor suppressor in skin. Therefore, in this review we summarize and discuss the functions of RIPKs in skin homeostasis as well as the signaling pathways involved.

ADAR1 prevents autoinflammation by suppressing spontaneous ZBP1 activation.

The RNA-editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) limits the accumulation of endogenous immunostimulatory double-stranded RNA (dsRNA)1. In humans, reduced ADAR1 activity causes the severe inflammatory disease Aicardi-Goutières syndrome (AGS)2. In mice, complete loss of ADAR1 activity is embryonically lethal3-6, and mutations similar to those found in patients with AGS cause autoinflammation7-12. Mechanistically, adenosine-to-inosine (A-to-I) base modification of endogenous dsRNA by ADAR1 prevents chronic overactivation of the dsRNA sensors MDA5 and PKR3,7-10,13,14. Here we show that ADAR1 also inhibits the spontaneous activation of the left-handed Z-nucleic acid sensor ZBP1. Activation of ZBP1 elicits caspase-8-dependent apoptosis and MLKL-mediated necroptosis of ADAR1-deficient cells. ZBP1 contributes to the embryonic lethality of Adar-knockout mice, and it drives early mortality and intestinal cell death in mice deficient in the expression of both ADAR and MAVS. The Z-nucleic-acid-binding Zα domain of ADAR1 is necessary to prevent ZBP1-mediated intestinal cell death and skin inflammation. The Zα domain of ADAR1 promotes A-to-I editing of endogenous Alu elements to prevent dsRNA formation through the pairing of inverted Alu repeats, which can otherwise induce ZBP1 activation. This shows that recognition of Alu duplex RNA by ZBP1 may contribute to the pathological features of AGS that result from the loss of ADAR1 function.

Point mutation I634A in the glucocorticoid receptor causes embryonic lethality by reduced ligand binding.

The glucocorticoid (GC) receptor (GR) is essential for normal development and in the initiation of inflammation. Healthy GRdim/dim mice with reduced dimerization propensity due to a point mutation (A465T) at the dimer interface of the GR DNA-binding domain (DBD) (here GRD/D) have previously helped to define the functions of GR monomers and dimers. Since GRD/D retains residual dimerization capacity, here we generated the dimer-nullifying double mutant GRD+L/D+L mice, featuring an additional mutation (I634A) in the ligand-binding domain (LBD) of GR. These mice are perinatally lethal, as are GRL/L mice (these mice have the I634A mutation but not the A465T mutation), displaying improper lung and skin formation. Using embryonic fibroblasts, high and low doses of dexamethasone (Dex), nuclear translocation assays, RNAseq, dimerization assays, and ligand-binding assays (and Kd values), we found that the lethal phenotype in these mice is due to insufficient ligand binding. These data suggest there is some correlation between GR dimerization potential and ligand affinity. We conclude that even a mutation as subtle as I634A, at a position not directly involved in ligand interactions sensu stricto, can still influence ligand binding and have a lethal outcome.

Plasma membrane perforation by GSDME during apoptosis-driven secondary necrosis.

Secondary necrosis has long been perceived as an uncontrolled process resulting in total lysis of the apoptotic cell. Recently, it was shown that progression of apoptosis to secondary necrosis is regulated by Gasdermin E (GSDME), which requires activation by caspase-3. Although the contribution of GSDME in this context has been attributed to its pore-forming capacity, little is known about the kinetics and size characteristics of this. Here we report on the membrane permeabilizing features of GSDME by monitoring the influx and efflux of dextrans of different sizes into/from anti-Fas-treated L929sAhFas cells undergoing apoptosis-driven secondary necrosis. We found that GSDME accelerates cell lysis measured by SYTOX Blue staining but does not affect the exposure of phosphatidylserine on the plasma membrane. Furthermore, loss of GSDME expression clearly hampered the influx of fluorescently labeled dextrans while the efflux happened independently of the presence or absence of GSDME expression. Importantly, both in- and efflux of dextrans were dependent on their molecular weight. Altogether, our results demonstrate that GSDME regulates the passage of compounds together with other plasma membrane destabilizing subroutines.

Characteristic ERK1/2 signaling dynamics distinguishes necroptosis from apoptosis.

ERK1/2 involvement in cell death remains unclear, although many studies have demonstrated the importance of ERK1/2 dynamics in determining cellular responses. To untangle how ERK1/2 contributes to two cell death programs, we investigated ERK1/2 signaling dynamics during hFasL-induced apoptosis and TNF-induced necroptosis in L929 cells. We observed that ERK1/2 inhibition sensitizes cells to apoptosis while delaying necroptosis. By monitoring ERK1/2 activity by live-cell imaging using an improved ERK1/2 biosensor (EKAR4.0), we reported differential ERK1/2 signaling dynamics between cell survival, apoptosis, and necroptosis. We also decrypted a temporally shifted amplitude- and frequency-modulated (AM/FM) ERK1/2 activity profile in necroptosis versus apoptosis. ERK1/2 inhibition, which disrupted ERK1/2 signaling dynamics, prevented TNF and IL-6 gene expression increase during TNF-induced necroptosis. Using an inducible cell line for activated MLKL, the final executioner of necroptosis, we showed ERK1/2 and its distinctive necroptotic ERK1/2 activity dynamics to be positioned downstream of MLKL.

Mouse Strain-Dependent Difference Toward the Staphylococcus aureus Allergen Serine Protease-Like Protein D Reveals a Novel Regulator of IL-33.

Staphylococcus aureus (S. aureus) can secrete a broad range of virulence factors, among which staphylococcal serine protease-like proteins (Spls) have been identified as bacterial allergens. The S. aureus allergen serine protease-like protein D (SplD) induces allergic asthma in C57BL/6J mice through the IL-33/ST2 signaling axis. Analysis of C57BL/6J, C57BL/6N, CBA, DBA/2, and BALB/c mice treated with intratracheal applications of SplD allowed us to identify a frameshift mutation in the serine (or cysteine) peptidase inhibitor, clade A, and member 3I (Serpina3i) causing a truncated form of SERPINA3I in BALB/c, CBA, and DBA/2 mice. IL-33 is a key mediator of SplD-induced immunity and can be processed by proteases leading to its activation or degradation. Full-length SERPINA3I inhibits IL-33 degradation in vivo in the lungs of SplD-treated BALB/c mice and in vitro by direct inhibition of mMCP-4. Collectively, our results establish SERPINA3I as a regulator of IL-33 in the lungs following exposure to the bacterial allergen SplD, and that the asthma phenotypes of mouse strains may be strongly influenced by the observed frameshift mutation in Serpina3i. The analysis of this protease-serpin interaction network might help to identify predictive biomarkers for type-2 biased airway disease in individuals colonized by S. aureus.

Sensing of endogenous nucleic acids by ZBP1 induces keratinocyte necroptosis and skin inflammation.

Aberrant detection of endogenous nucleic acids by the immune system can cause inflammatory disease. The scaffold function of the signaling kinase RIPK1 limits spontaneous activation of the nucleic acid sensor ZBP1. Consequently, loss of RIPK1 in keratinocytes induces ZBP1-dependent necroptosis and skin inflammation. Whether nucleic acid sensing is required to activate ZBP1 in RIPK1-deficient conditions and which immune pathways are associated with skin disease remained open questions. Using knock-in mice with disrupted ZBP1 nucleic acid-binding activity, we report that sensing of endogenous nucleic acids by ZBP1 is critical in driving skin pathology characterized by antiviral and IL-17 immune responses. Inducing ZBP1 expression by interferons triggers necroptosis in RIPK1-deficient keratinocytes, and epidermis-specific deletion of MLKL prevents disease, demonstrating that cell-intrinsic events cause inflammation. These findings indicate that dysregulated sensing of endogenous nucleic acid by ZBP1 can drive inflammation and may contribute to the pathogenesis of IL-17-driven inflammatory skin conditions such as psoriasis.

Inhibitors Targeting RIPK1/RIPK3: Old and New Drugs.

The scaffolding function of receptor-interacting protein kinase 1 (RIPK1) regulates prosurvival signaling and inflammatory gene expression, while its kinase activity mediates both apoptosis and necroptosis; the latter involving RIPK3 kinase activity. The mutual transition between the scaffold and kinase functions of RIPK1 is regulated by (de)ubiquitylation and (de)phosphorylation. RIPK1-mediated cell death leads to disruption of epithelial barriers and/or release of damage-associated molecular patterns (DAMPs), cytokines, and chemokines, propagating inflammatory and degenerative diseases. Many drug development programs have pursued targeting RIPK1, and to a lesser extent RIPK3 kinase activity. In this review, we classify existing and novel small-molecule drugs based on their pharmacodynamic (PD) type I, II, and III binding mode. Finally, we discuss their applicability and therapeutic potential in inflammatory and degenerative experimental disease models.

Immunodominant AH1 Antigen-Deficient Necroptotic, but Not Apoptotic, Murine Cancer Cells Induce Antitumor Protection.

Immunogenic cell death (ICD) occurs when a dying cell releases cytokines and damage-associated molecular patterns, acting as adjuvants, and expresses Ags that induce a specific antitumor immune response. ICD is studied mainly in the context of regulated cell death pathways, especially caspase-mediated apoptosis marked by endoplasmic reticulum stress and calreticulin exposure and, more recently, also in relation to receptor-interacting protein kinase-driven necroptosis, whereas unregulated cell death like accidental necrosis is nonimmunogenic. Importantly, the murine cancer cell lines used in ICD studies often express virally derived peptides that are recognized by the immune system as tumor-associated Ags. However, it is unknown how different cell death pathways may affect neoepitope cross-presentation and Ag recognition of cancer cells. We used a prophylactic tumor vaccination model and observed that both apoptotic and necroptotic colon carcinoma CT26 cells efficiently immunized mice against challenge with a breast cancer cell line that expresses the same immunodominant tumor Ag, AH1, but only necroptotic CT26 cells would mount an immune response against CT26-specific neoepitopes. By CRISPR/Cas9 genome editing, we knocked out AH1 and saw that only necroptotic CT26 cells were still able to protect mice against tumor challenge. Hence, in this study, we show that endogenous AH1 tumor Ag expression can mask the strength of immunogenicity induced by different cell death pathways and that upon knockout of AH1, necroptosis was more immunogenic than apoptosis in a prophylactic tumor vaccination model. This work highlights necroptosis as a possible preferred ICD form over apoptosis in the treatment of cancer.

Correction: A20 protects cells from TNF-induced apoptosis through linear ubiquitin-dependent and -independent mechanisms.

The original version of this article contained an error in the name of one of the co-authors (Wim Declercq). This has been corrected in the PDF and HTML versions.

Filaggrin Expression and Processing Deficiencies Impair Corneocyte Surface Texture and Stiffness in Mice.

Abundant corneocyte surface protrusions, observed in patients with atopic dermatitis with filaggrin loss-of-function mutations, are inversely associated with levels of natural moisturizing factors (NMFs) in the stratum corneum. To dissect the etiological role of NMFs and filaggrin deficiency in surface texture alterations, we examined mouse models with genetic deficiencies in the synthesis or degradation of filaggrin monomers for NMFs, cell stiffness (elastic modulus) and corneocyte surface protrusion density (dermal texture index). Five neonatal and adult mouse models carrying inactivating mutations of SASPase (Sasp-/-), filaggrin (Flgft/ft and Flg-/-), filaggrin-hornerin (FlgHrnr-/-), and bleomycin hydrolase (Blmh-/-) were investigated. Sasp-/- and Flg-/- were on the hairless mouse background. Atomic force microscopy was used to determine elastic modulus and dermal texture index. Corneocytes of each neonatal as well as hairless adult knockout mouse exhibited an increased number of protrusions and decreased elastic modulus. In these mice, NMFs were reduced except for Sasp-/-. Dermal texture index was inversely correlated with NMFs and elastic modulus. Our findings demonstrate that any filaggrin-NMF axis deficiency can affect corneocyte mechanical properties in mice and likely in humans. Differences in NMFs and corneocyte surface texture between neonatal and adult as well as hairless and hairy mice emphasize the need for carefully selecting the most appropriate animal models for studies.

Keratinocyte-specific ablation of Mcpip1 impairs skin integrity and promotes local and systemic inflammation.

MCPIP1 (Regnase-1, encoded by the ZC3H12A gene) regulates the mRNA stability of several inflammatory cytokines. Due to the critical role of this RNA endonuclease in the suppression of inflammation, Mcpip1 deficiency in mice leads to the development of postnatal multiorgan inflammation and premature death. Here, we generated mice with conditional deletion of Mcpip1 in the epidermis (Mcpip1EKO). Mcpip1 loss in keratinocytes resulted in the upregulated expression of transcripts encoding factors related to inflammation and keratinocyte differentiation, such as IL-36α/γ cytokines, S100a8/a9 antibacterial peptides, and Sprr2d/2h proteins. Upon aging, the Mcpip1EKO mice showed impaired skin integrity that led to the progressive development of spontaneous skin pathology and systemic inflammation. Furthermore, we found that the lack of epidermal Mcpip1 expression impaired the balance of keratinocyte proliferation and differentiation. Overall, we provide evidence that keratinocyte-specific Mcpip1 activity is crucial for the maintenance of skin integrity as well as for the prevention of excessive local and systemic inflammation. KEY MESSAGES: Loss of murine epidermal Mcpip1 upregulates transcripts related to inflammation and keratinocyte differentiation. Keratinocyte Mcpip1 function is essential to maintain the integrity of skin in adult mice. Ablation of Mcpip1 in mouse epidermis leads to the development of local and systemic inflammation.

Epithelial HMGB1 Delays Skin Wound Healing and Drives Tumor Initiation by Priming Neutrophils for NET Formation.

Regenerative responses predispose tissues to tumor formation by largely unknown mechanisms. High-mobility group box 1 (HMGB1) is a danger-associated molecular pattern contributing to inflammatory pathologies. We show that HMGB1 derived from keratinocytes, but not myeloid cells, delays cutaneous wound healing and drives tumor formation. In wounds of mice lacking HMGB1 selectively in keratinocytes, a marked reduction in neutrophil extracellular trap (NET) formation is observed. Pharmacological targeting of HMGB1 or NETs prevents skin tumorigenesis and accelerates wound regeneration. HMGB1-dependent NET formation and skin tumorigenesis is orchestrated by tumor necrosis factor (TNF) and requires RIPK1 kinase activity. NETs are present in the microenvironment of keratinocyte-derived tumors in mice and lesional and tumor skin of patients suffering from recessive dystrophic epidermolysis bullosa, a disease in which skin blistering predisposes to tumorigenesis. We conclude that tumorigenicity of the wound microenvironment depends on epithelial-derived HMGB1 regulating NET formation, thereby establishing a mechanism linking reparative inflammation to tumor initiation.

Blocking connexin43 hemichannels protects mice against tumour necrosis factor-induced inflammatory shock.

Upon intravenous injection of tumour necrosis factor (TNF) in mice, a systemic inflammatory response syndrome (SIRS) is initiated, characterized by an acute cytokine storm and induction of vascular hyperpermeability. Connexin43 hemichannels have been implicated in various pathological conditions, e.g. ischemia and inflammation, and can lead to detrimental cellular outcomes. Here, we explored whether targeting connexin43 hemichannels could alleviate TNF-induced endothelial barrier dysfunction and lethality in SIRS. Therefore, we verified whether administration of connexin43-targeting-peptides affected survival, body temperature and vascular permeability in vivo. In vitro, TNF-effects on connexin43 hemichannel function were investigated by single-channel studies and Ca2+-imaging. Blocking connexin43 hemichannels with TAT-Gap19 protected mice against TNF-induced mortality, hypothermia and vascular leakage, while enhancing connexin43 hemichannel function with TAT-CT9 provoked opposite sensitizing effects. In vitro patch-clamp studies revealed that TNF acutely activated connexin43 hemichannel opening in endothelial cells, which was promoted by CT9, and inhibited by Gap19 and intracellular Ca2+-buffering. In vivo experiments aimed at buffering intracellular Ca2+, and pharmacologically targeting Ca2+/calmodulin-dependent protein kinase-II, a known modulator of endothelial barrier integrity, demonstrated their involvement in permeability alterations. Our results demonstrate significant benefits of inhibiting connexin43 hemichannels to counteract TNF-induced SIRS-associated vascular permeability and lethality.

A20 protects cells from TNF-induced apoptosis through linear ubiquitin-dependent and -independent mechanisms.

The cytokine TNF promotes inflammation either directly by activating the MAPK and NF-κB signaling pathways, or indirectly by triggering cell death. A20 is a potent anti-inflammatory molecule, and mutations in the gene encoding A20 are associated with a wide panel of inflammatory pathologies, both in human and in the mouse. Binding of TNF to TNFR1 triggers the NF-κB-dependent expression of A20 as part of a negative feedback mechanism preventing sustained NF-κB activation. Apart from acting as an NF-κB inhibitor, A20 is also well-known for its ability to counteract the cytotoxic potential of TNF. However, the mechanism by which A20 mediates this function and the exact cell death modality that it represses have remained incompletely understood. In the present study, we provide in vitro and in vivo evidences that deletion of A20 induces RIPK1 kinase-dependent and -independent apoptosis upon single TNF stimulation. We show that constitutively expressed A20 is recruited to TNFR1 signaling complex (Complex I) via its seventh zinc finger (ZF7) domain, in a cIAP1/2-dependent manner, within minutes after TNF sensing. We demonstrate that Complex I-recruited A20 protects cells from apoptosis by stabilizing the linear (M1) ubiquitin network associated to Complex I, a process independent of its E3 ubiquitin ligase and deubiquitylase (DUB) activities and which is counteracted by the DUB CYLD, both in vitro and in vivo. In absence of linear ubiquitylation, A20 is still recruited to Complex I via its ZF4 and ZF7 domains, but this time protects the cells from death by deploying its DUB activity. Together, our results therefore demonstrate two distinct molecular mechanisms by which constitutively expressed A20 protect cells from TNF-induced apoptosis.

Delivery of Mixed-Lineage Kinase Domain-Like Protein by Vapor Nanobubble Photoporation Induces Necroptotic-Like Cell Death in Tumor Cells.

Modern molecular medicine demands techniques to efficiently deliver molecules directly into mammalian cells. As proteins are the final mediators of most cellular pathways, efficient intracellular protein delivery techniques are highly desired. In this respect, photoporation is a promising recent technique for the delivery of proteins directly into living cells. Here, we show the possibility to deliver a model saccharide (FD70) and a model protein (FITC-BSA) into murine B16 melanoma cells by using the vapor nanobubble photoporation technique with an efficiency of 62% and 38%, respectively. Next, we delivered the mixed-lineage kinase domain-like (MLKL) protein, the most terminal mediator of necroptosis currently known, and caspase-8 and -3 protein, which are important proteins in the initiation and execution of apoptosis. A significant drop in cell viability with 62%, 71% and 64% cell survival for MLKL, caspase-8 and caspase-3, respectively, was observed. Remarkably, maximal cell death induction was already observed within 1 h after protein delivery. Transduction of purified recombinant MLKL by photoporation resulted in rapid cell death characterized by cell swelling and cell membrane rupture, both hallmarks of necroptosis. As necroptosis has been identified as a type of cell death with immunogenic properties, this is of interest to anti-cancer immunotherapy. On the other hand, transduction of purified recombinant active caspase-3 or -8 into the tumor cells resulted in rapid cell death preceded by membrane blebbing, which is typical for apoptosis. Our results suggest that the type of cell death of tumor cells can be controlled by direct transduction of effector proteins that are involved in the executioner phase of apoptosis or necroptosis.

Synthesis and evaluation of novel benzotropolones as Atg4B inhibiting autophagy blockers.

Autophagy is an intracellular degradation/recycling pathway that provides nutrients and building blocks to cellular metabolism and keeps the cytoplasm clear of obsolete proteins and organelles. During recent years, dysregulated autophagy activity has been reported to be a characteristic of many different disease types, including cancer and neurodegenerative disorders. This has created a strong case for development of autophagy modulating compounds as potential treatments for these diseases. Inhibitors of autophagy have been proposed as a therapeutic intervention in, e.g., advanced cancer, and inhibiting the cysteine protease Atg4B has been put forward as a main strategy to block autophagy. We recently identified and demonstrated -both in vitro and in vivo - that compounds with a benzotropolone basic structure targeting Atg4B, can significantly slow down tumor growth and potentiate the effect of classical chemotherapy. In this study we report the synthesis and inhibition profile of new benzotropolone derivatives with additional structural modifications at 6 different positions. To obtain a solid inhibition profile, all compounds were evaluated on three levels, including two cell-based assays to confirm autophagy and intracellular Atg4B inhibition and an SDS-PAGE-based experiment to assess in vitro Atg4B affinity. Several molecules with a promising profile were identified.