Emerging organoid-immune co-culture models for cancer research: from oncoimmunology to personalized immunotherapies.

In the past decade, treatments targeting the immune system have revolutionized the cancer treatment field. Therapies such as immune checkpoint inhibitors have been approved as first-line treatment in a variety of solid tumors such as melanoma and non-small cell lung cancer while other therapies, for instance, chimeric antigen receptor (CAR) lymphocyte transfer therapies, are still in development. Although promising results are obtained in a small subset of patients, overall clinical efficacy of most immunotherapeutics is limited due to intertumoral heterogeneity and therapy resistance. Therefore, prediction of patient-specific responses would be of great value for efficient use of costly immunotherapeutic drugs as well as better outcomes. Because many immunotherapeutics operate by enhancing the interaction and/or recognition of malignant target cells by T cells, in vitro cultures using the combination of these cells derived from the same patient hold great promise to predict drug efficacy in a personalized fashion. The use of two-dimensional cancer cell lines for such cultures is unreliable due to altered phenotypical behavior of cells when compared with the in vivo situation. Three-dimensional tumor-derived organoids, better mimic in vivo tissue and are deemed a more realistic approach to study the complex tumor-immune interactions. In this review, we present an overview of the development of patient-specific tumor organoid-immune co-culture models to study the tumor-specific immune interactions and their possible therapeutic infringement. We also discuss applications of these models which advance personalized therapy efficacy and understanding the tumor microenvironment such as: (1) Screening for efficacy of immune checkpoint inhibition and CAR therapy screening in a personalized manner. (2) Generation of tumor reactive lymphocytes for adoptive cell transfer therapies. (3) Studying tumor-immune interactions to detect cell-specific roles in tumor progression and remission. Overall, these onco-immune co-cultures might hold a promising future toward developing patient-specific therapeutic approaches as well as increase our understanding of tumor-immune interactions.

Modelling immune cytotoxicity for cholangiocarcinoma with tumour-derived organoids and effector T cells.

BACKGROUND: Immunotherapy with immune checkpoint inhibitors (ICIs) is being explored to improve cholangiocarcinoma (CCA) therapy. However, it remains difficult to predict which ICI will be effective for individual patients. Therefore, the aim of this study is to develop a co-culture method with patient-derived CCA organoids and immune cells, which could represent anti-cancer immunity in vitro. METHODS: CCA organoids were co-cultured with peripheral blood mononuclear cells or T cells. Flow cytometry, time-lapse confocal imaging for apoptosis, and quantification of cytokeratin 19 fragment (CYFRA) release were applied to analyse organoid and immune cell behaviour. CCA organoids were also cultured in immune cell-conditioned media to analyse the effect of soluble factors. RESULTS: The co-culture system demonstrated an effective anti-tumour organoid immune response by a decrease in live organoid cells and an increase in apoptosis and CYFRA release. Interpatient heterogeneity was observed. The cytotoxic effects could be mediated by direct cell-cell contact and by release of soluble factors, although soluble factors only decreased viability in one organoid line. CONCLUSIONS: In this proof-of-concept study, a novel CCA organoid and immune cell co-culture method was established. This can be the first step towards personalised immunotherapy for CCA by predicting which ICIs are most effective for individual patients.

Neutrophils and Neutrophil Extracellular Traps Drive Necroinflammation in COVID-19.

The COVID-19 pandemic is progressing worldwide with an alarming death toll. There is an urgent need for novel therapeutic strategies to combat potentially fatal complications. Distinctive clinical features of severe COVID-19 include acute respiratory distress syndrome, neutrophilia, and cytokine storm, along with severe inflammatory response syndrome or sepsis. Here, we propose the putative role of enhanced neutrophil infiltration and the release of neutrophil extracellular traps, complement activation and vascular thrombosis during necroinflammation in COVID-19. Furthermore, we discuss how neutrophilic inflammation contributes to the higher mortality of COVID-19 in patients with underlying co-morbidities such as diabetes and cardiovascular diseases. This perspective highlights neutrophils as a putative target for the immunopathologic complications of severely ill COVID-19 patients. Development of the novel therapeutic strategies targeting neutrophils may help reduce the overall disease fatality rate of COVID-19.

Neutrophil Extracellular Traps (NETs) Take the Central Stage in Driving Autoimmune Responses.

Following fifteen years of research, neutrophil extracellular traps (NETs) are widely reported in a large range of inflammatory infectious and non-infectious diseases. Cumulating evidences from in vitro, in vivo and clinical diagnostics suggest that NETs may play a crucial role in inflammation and autoimmunity in a variety of autoimmune diseases, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV). Most likely, NETs contribute to breaking self-tolerance in autoimmune diseases in several ways. During this review, we discuss the current knowledge on how NETs could drive autoimmune responses. NETs can break self-tolerance by being a source of autoantigens for autoantibodies found in autoimmune diseases, such as anti-citrullinated protein antibodies (ACPAs) in RA, anti-dsDNA in SLE and anti-myeloperoxidase and anti-protein 3 in AAV. Moreover, NET components could accelerate the inflammatory response by mediating complement activation, acting as danger-associated molecular patterns (DAMPs) and inflammasome activators, for example. NETs also can activate other immune cells, such as B cells, antigen-presenting cells and T cells. Additionally, impaired clearance of NETs in autoimmune diseases prolongs the presence of active NETs and their components and, in this way, accelerate immune responses. NETs have not only been implicated as drivers of inflammation, but also are linked to resolution of inflammation. Therefore, NETs may be central regulators of inflammation and autoimmunity, serve as biomarkers, as well as promising targets for future therapeutics of inflammatory autoimmune diseases.

Mitochondria Permeability Transition versus Necroptosis in Oxalate-Induced AKI.

BACKGROUND: Serum oxalate levels suddenly increase with certain dietary exposures or ethylene glycol poisoning and are a well known cause of AKI. Established contributors to oxalate crystal-induced renal necroinflammation include the NACHT, LRR and PYD domains-containing protein-3 (NLRP3) inflammasome and mixed lineage kinase domain-like (MLKL) protein-dependent tubule necroptosis. These studies examined the role of a novel form of necrosis triggered by altered mitochondrial function. METHODS: To better understand the molecular pathophysiology of oxalate-induced AIK, we conducted in vitro studies in mouse and human kidney cells and in vivo studies in mice, including wild-type mice and knockout mice deficient in peptidylprolyl isomerase F (Ppif) or deficient in both Ppif and Mlkl. RESULTS: Crystals of calcium oxalate, monosodium urate, or calcium pyrophosphate dihydrate, as well as silica microparticles, triggered cell necrosis involving PPIF-dependent mitochondrial permeability transition. This process involves crystal phagocytosis, lysosomal cathepsin leakage, and increased release of reactive oxygen species. Mice with acute oxalosis displayed calcium oxalate crystals inside distal tubular epithelial cells associated with mitochondrial changes characteristic of mitochondrial permeability transition. Mice lacking Ppif or Mlkl or given an inhibitor of mitochondrial permeability transition displayed attenuated oxalate-induced AKI. Dual genetic deletion of Ppif and Mlkl or pharmaceutical inhibition of necroptosis was partially redundant, implying interlinked roles of these two pathways of regulated necrosis in acute oxalosis. Similarly, inhibition of mitochondrial permeability transition suppressed crystal-induced cell death in primary human tubular epithelial cells. PPIF and phosphorylated MLKL localized to injured tubules in diagnostic human kidney biopsies of oxalosis-related AKI. CONCLUSIONS: Mitochondrial permeability transition-related regulated necrosis and necroptosis both contribute to oxalate-induced AKI, identifying PPIF as a potential molecular target for renoprotective intervention.

To NET or not to NET:current opinions and state of the science regarding the formation of neutrophil extracellular traps.

Since the discovery and definition of neutrophil extracellular traps (NETs) 14 years ago, numerous characteristics and physiological functions of NETs have been uncovered. Nowadays, the field continues to expand and novel mechanisms that orchestrate formation of NETs, their previously unknown properties, and novel implications in disease continue to emerge. The abundance of available data has also led to some confusion in the NET research community due to contradictory results and divergent scientific concepts, such as pro- and anti-inflammatory roles in pathologic conditions, demarcation from other forms of cell death, or the origin of the DNA that forms the NET scaffold. Here, we present prevailing concepts and state of the science in NET-related research and elaborate on open questions and areas of dispute.

CXCL12 blockade preferentially regenerates lost podocytes in cortical nephrons by targeting an intrinsic podocyte-progenitor feedback mechanism.

Insufficient podocyte regeneration after injury is a central pathomechanism of glomerulosclerosis and chronic kidney disease. Podocytes constitutively secrete the chemokine CXCL12, which is known to regulate homing and activation of stem cells; hence we hypothesized a similar effect of CXCL12 on podocyte progenitors. CXCL12 blockade increased podocyte numbers and attenuated proteinuria in mice with Adriamycin-induced nephropathy. Similar studies in lineage-tracing mice revealed enhanced de novo podocyte formation from parietal epithelial cells in the setting of CXCL12 blockade. Super-resolution microscopy documented full integration of these progenitor-derived podocytes into the glomerular filtration barrier, interdigitating with tertiary foot processes of neighboring podocytes. Quantitative 3D analysis revealed that conventional 2D analysis underestimated the numbers of progenitor-derived podocytes. The 3D analysis also demonstrated differences between juxtamedullary and cortical nephrons in both progenitor endowment and Adriamycin-induced podocyte loss, with more robust podocyte regeneration in cortical nephrons with CXCL12 blockade. Finally, we found that delayed CXCL12 inhibition still had protective effects. In vitro studies found that CXCL12 inhibition uncoupled Notch signaling in podocyte progenitors. These data suggest that CXCL12-driven podocyte-progenitor feedback maintains progenitor quiescence during homeostasis, but also limits their intrinsic capacity to regenerate lost podocytes, especially in cortical nephrons. CXCL12 inhibition could be an innovative therapeutic strategy in glomerular disorders.

Activated platelets induce MLKL-driven neutrophil necroptosis and release of neutrophil extracellular traps in venous thrombosis.

Venous thromboembolic (VTE) disease, often manifesting as deep vein thrombosis or pulmonary embolism, involves clot formation consisting of blood cells and platelets locked in plasma protein and chromatin networks. The latter derives from neutrophil extracellular traps released by dying neutrophils; however, the molecular mechanisms of neutrophil death in VTE remains unknown. We speculated that mixed lineage kinase-like (MLKL)-driven neutrophil necroptosis contributes to VTE. Indeed, human inferior venous cava thrombus material stained positive for phosphorylated MLKL, the activated version of MLKL that executes necroptotic cell death. In mice, MLKL immunostaining showed co-localization of MLKL with citrullinated histone H3, a marker of neutrophil extracellular trap (NET) formation. These data provide indirect support for a role of MLKL-mediated necroptosis. As a functional proof, both the stabilizer of receptor-interacting protein kinase-1 (RIPK1) and necroptosis inhibitor necrostatin-1s as well as genetic deficiency of MLKL partially prevented clot formation upon inferior vena cava ligation in mice. In both experiments terminal deoxynucleotidyl transferase dUTP nick-end labeling, RIPK3, and citrullinated histone H3+ areas were markedly reduced within the remnant thrombus. In vitro, thrombin-activated platelets induced cell death and NET formation in human neutrophils, which was inhibited by necrostatin-1s treatment. Necrostatin-1s and necrosulfonamide also inhibited neutrophil-platelet aggregate formation induced by tumor necrosis factor-α but had no effect on platelet activation itself. We conclude that in VTE, activated platelets, and possibly other triggers, induce neutrophil necroptosis, a process contributing to clot formation by releasing chromatin in the extracellular space.

Author Correction: Particles of different sizes and shapes induce neutrophil necroptosis followed by the release of neutrophil extracellular trap-like chromatin.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

The macrophage phenotype and inflammasome component NLRP3 contributes to nephrocalcinosis-related chronic kidney disease independent from IL-1-mediated tissue injury.

Primary/secondary hyperoxalurias involve nephrocalcinosis-related chronic kidney disease (CKD) leading to end-stage kidney disease. Mechanistically, intrarenal calcium oxalate crystal deposition is thought to elicit inflammation, tubular injury and atrophy, involving the NLRP3 inflammasome. Here, we found that mice deficient in NLRP3 and ASC adaptor protein failed to develop nephrocalcinosis, compromising conclusions on nephrocalcinosis-related CKD. In contrast, hyperoxaluric wild-type mice developed profound nephrocalcinosis. NLRP3 inhibition using the ß-hydroxybutyrate precursor 1,3-butanediol protected such mice from nephrocalcinosis-related CKD. Interestingly, the IL-1 inhibitor anakinra had no such effect, suggesting IL-1-independent functions of NLRP3. NLRP3 inhibition using 1,3-butanediol treatment induced a shift of infiltrating renal macrophages from pro-inflammatory (CD45+F4/80+CD11b+CX3CR1+CD206-) and pro-fibrotic (CD45+F4/80+CD11b+CX3CR1+CD206+TGFß+) to an anti-inflammatory (CD45+F4/80+CD11b+CD206+TGFß-) phenotype, and prevented renal fibrosis. Finally, in vitro studies with primary murine fibroblasts confirmed the non-redundant role of NLRP3 in the TGF-ß signaling pathway for fibroblast activation and proliferation independent of the NLRP3 inflammasome complex formation. Thus, nephrocalcinosis-related CKD involves NLRP3 but not necessarily via intrarenal IL-1 release but rather via other biological functions including TGFR signaling and macrophage polarization. Hence, NLRP3 may be a promising therapeutic target in hyperoxaluria and nephrocalcinosis.

Particles of different sizes and shapes induce neutrophil necroptosis followed by the release of neutrophil extracellular trap-like chromatin.

The human body is exposed to a wide range of particles of industrial, environmental or internal origin such as asbestos, alum, silica or crystals of urate, calcium phosphate, calcium oxalate, cystine or cholesterol. Phagocytic clearance of such particles involves neutrophils and macrophages. Here we report that neutrophils encountering such particles of diverse sizes and shapes undergo necrotic cell death, a process associated with the formation of neutrophil extracellular trap (NET)-like extracellular DNA. In human neutrophils receptor-interacting protein kinase (RIPK)-1 inhibition with necrostatin-1s or mixed lineage kinase domain-like (MLKL) inhibition with necrosulfonamide abrogated cell death and associated-neutrophil extracellular DNA release induced by all of the aforementioned particles. Similar results were obtained with Mlkl-deficient mice neutrophils for all particles in vitro. Furthermore, Mlkl-deficient mice lacked tophus formation upon injection of MSU crystals into subcutaneous air pouches. These findings imply that nano- or microparticle-induced neutrophil extracellular DNA release is the consequence of neutrophil necroptosis, a regulated form of cell necrosis defined by RIPK1-RIPK3-MLKL signaling. Interestingly, this finding was consistent across different particle sizes and shapes. The RIPK1-RIPK3-MLKL signaling pathway may represent a potential therapeutic target in nano- or microparticle-related diseases (crystallopathies).

Phagocytosis of environmental or metabolic crystalline particles induces cytotoxicity by triggering necroptosis across a broad range of particle size and shape.

In crystallopathies, crystals or crystalline particles of environmental and metabolic origin deposit within tissues, induce inflammation, injury and cell death and eventually lead to organ-failure. The NLRP3-inflammasome is involved in mediating crystalline particles-induced inflammation, but pathways leading to cell death are still unknown. Here, we have used broad range of intrinsic and extrinsic crystal- or crystalline particle-sizes and shapes, e.g. calcium phosphate, silica, titanium dioxide, cholesterol, calcium oxalate, and monosodium urate. As kidney is commonly affected by crystallopathies, we used human and murine renal tubular cells as a model system. We showed that all of the analysed crystalline particles induce caspase-independent cell death. Deficiency of MLKL, siRNA knockdown of RIPK3, or inhibitors of necroptosis signaling e.g. RIPK-1 inhibitor necrostatin-1s, RIPK3 inhibitor dabrafenib, and MLKL inhibitor necrosulfonamide, partially protected tubular cells from crystalline particles cytotoxicity. Furthermore, we identify phagocytosis of crystalline particles as an upstream event in their cytotoxicity since a phagocytosis inhibitor, cytochalasin D, prevented their cytotoxicity. Taken together, our data confirmed the involvement of necroptosis as one of the pathways leading to cell death in crystallopathies. Our data identified RIPK-1, RIPK3, and MLKL as molecular targets to limit tissue injury and organ failure in crystallopathies.

Molecular Pathophysiology of Gout.

Three contradictory clinical presentations of gout have puzzled clinicians and basic scientists for some time: first, the crescendo of sterile inflammation in acute gouty arthritis; second, its spontaneous resolution, despite monosodium urate (MSU) crystal persistence in the synovium; and third, immune anergy to MSU crystal masses observed in tophaceous or visceral gout. Here, we provide an update on the molecular pathophysiology of these gout manifestations, namely, how MSU crystals can trigger the auto-amplification loop of necroinflammation underlying the crescendo of acute gouty arthritis. We also discuss new findings, such as how aggregating neutrophil extracellular traps (NETs) might drive the resolution of arthritis and how these structures, together with granuloma formation, might support immune anergy, but yet promote tissue damage and remodeling during tophaceous gout.

Histones and Neutrophil Extracellular Traps Enhance Tubular Necrosis and Remote Organ Injury in Ischemic AKI.

Severe AKI is often associated with multiorgan dysfunction, but the mechanisms of this remote tissue injury are unknown. We hypothesized that renal necroinflammation releases cytotoxic molecules that may cause remote organ damage. In hypoxia-induced tubular epithelial cell necrosis in vitro, histone secretion from ischemic tubular cells primed neutrophils to form neutrophil extracellular traps. These traps induced tubular epithelial cell death and stimulated neutrophil extracellular trap formation in fresh neutrophils. In vivo, ischemia-reperfusion injury in the mouse kidney induced tubular necrosis, which preceded the expansion of localized and circulating neutrophil extracellular traps and the increased expression of inflammatory and injury-related genes. Pretreatment with inhibitors of neutrophil extracellular trap formation reduced kidney injury. Dual inhibition of neutrophil trap formation and tubular cell necrosis had an additive protective effect. Moreover, pretreatment with antihistone IgG suppressed ischemia-induced neutrophil extracellular trap formation and renal injury. Renal ischemic injury also increased the levels of circulating histones, and we detected neutrophil infiltration and TUNEL-positive cells in the lungs, liver, brain, and heart along with neutrophil extracellular trap accumulation in the lungs. Inhibition of neutrophil extracellular trap formation or of circulating histones reduced these effects as well. These data suggest that tubular necrosis and neutrophil extracellular trap formation accelerate kidney damage and remote organ dysfunction through cytokine and histone release and identify novel molecular targets to limit renal necroinflammation and multiorgan failure.

Neutrophil extracellular traps in tissue pathology.

Neutrophil extracellular traps (NETs) are innate immune systems against invading pathogens. NETs are characterized as released DNA mixed with cytoplasmic antimicrobial proteins such as myeloperoxidase, proteinase3 and neutrophil elastase. While NETs are thought to have an important role in host defense, recent work has suggested that NETs contribute to tissue injury in non-infectious disease states. Uncontrolled NET formation in autoimmune diseases, metabolic disorders, cancers and thrombotic diseases can exacerbate a disease or even be a major initiator of tissue injury. But spotting NETs in tissues is not easy. Here we review the available histopathological evidence on the presence of NETs in a variety of diseases. We discuss technical difficulties and potential sources of misinterpretation while trying to detect NETs in tissue samples.

Hyperoxaluria Requires TNF Receptors to Initiate Crystal Adhesion and Kidney Stone Disease.

Intrarenal crystals trigger inflammation and renal cell necroptosis, processes that involve TNF receptor (TNFR) signaling. Here, we tested the hypothesis that TNFRs also have a direct role in tubular crystal deposition and progression of hyperoxaluria-related CKD. Immunohistochemical analysis revealed upregulated tubular expression of TNFR1 and TNFR2 in human and murine kidneys with calcium oxalate (CaOx) nephrocalcinosis-related CKD compared with controls. Western blot and mRNA expression analyses in mice yielded consistent data. When fed an oxalate-rich diet, wild-type mice developed progressive CKD, whereas Tnfr1-, Tnfr2-, and Tnfr1/2-deficient mice did not. Despite identical levels of hyperoxaluria, Tnfr1-, Tnfr2-, and Tnfr1/2-deficient mice also lacked the intrarenal CaOx deposition and tubular damage observed in wild-type mice. Inhibition of TNFR signaling prevented the induced expression of the crystal adhesion molecules, CD44 and annexin II, in tubular epithelial cells in vitro and in vivo, and treatment with the small molecule TNFR inhibitor R-7050 partially protected hyperoxaluric mice from nephrocalcinosis and CKD. We conclude that TNFR signaling is essential for CaOx crystal adhesion to the luminal membrane of renal tubules as a fundamental initiating mechanism of oxalate nephropathy. Furthermore, therapeutic blockade of TNFR might delay progressive forms of nephrocalcinosis in oxalate nephropathy, such as primary hyperoxaluria.

Regulated necrosis-related molecule mRNA expression in humans and mice and in murine acute tissue injury and systemic autoimmunity leading to progressive organ damage, and progressive fibrosis.

The species-specific, as well as organ-specific expression of regulated necrosis (RN)-related molecules, is not known. We determined the expression levels of tumour necrosis factor receptor-1 (TNFR1), receptor activated protein kinase (RIPK)1, RIPK3, mixed lineage kinase domain-like (MLKL), CASP8, Fas-associated protein with death domain (FADD), cellular inhibitor of apoptosis protein (CIAP)1, CIAP2, glutathione peroxidase-4 (GPX4), cyclophilin D (CYPD), CASP1, NLRP3 and poly(ADP-ribose) polymerase-1 (PARP1) in human and mouse solid organs. We observed significant differences in expression of these molecules between human and mice. In addition, we characterized their expression profiles in acute as well as persistent tissue injury and chronic tissue remodelling using acute and chronic kidney injury models. We observed that the degree and pattern of induction of RN-related molecules were highly dependent on the trigger and disease pathogenesis. Furthermore, we studied their expression patterns in mice with lupus-like systemic autoimmunity, which revealed that the expression of MLKL, GPX4 and PARP1 significantly increased in the spleen along disease progression and CASP1, RIPK1, RIPK3 and CYPD were higher at the earlier stages but were significantly decreased in the later stages. In contrast, in the kidney, the expression of genes involved in pyroptosis, e.g. NLRP3 and CASP1 were significantly increased and TNFR1, RIPK1, RIPK3, CIAP1/2 and GPX4 were significantly decreased along the progression of lupus nephritis (LN). Thus, the organ- and species-specific expression of RN-related molecules should be considered during designing experiments, interpreting the results as well as extrapolating the conclusions from one species or organ to another species or organ respectively.

How Kidney Cell Death Induces Renal Necroinflammation.

The nephrons of the kidney are independent functional units harboring cells of a low turnover during homeostasis. As such, physiological renal cell death is a rather rare event and dead cells are flushed away rapidly with the urinary flow. Renal cell necrosis occurs in acute kidney injuries such as thrombotic microangiopathies, necrotizing glomerulonephritis, or tubular necrosis. All of these are associated with intense intrarenal inflammation, which contributes to further renal cell loss, an autoamplifying process referred to as necroinflammation. But how does renal cell necrosis trigger inflammation? Here, we discuss the role of danger-associated molecular patterns (DAMPs), mitochondrial (mito)-DAMPs, and alarmins, as well as their respective pattern recognition receptors. The capacity of DAMPs and alarmins to trigger cytokine and chemokine release initiates the recruitment of leukocytes into the kidney that further amplify necroinflammation. Infiltrating neutrophils often undergo neutrophil extracellular trap formation associated with neutrophil death or necroptosis, which implies a release of histones, which act not only as DAMPs but also elicit direct cytotoxic effects on renal cells, namely endothelial cells. Proinflammatory macrophages and eventually cytotoxic T cells further drive kidney cell death and inflammation. Dissecting the molecular mechanisms of necroinflammation may help to identify the best therapeutic targets to limit nephron loss in kidney injury.

Murine Double Minute-2 Inhibition Ameliorates Established Crescentic Glomerulonephritis.

Rapidly progressive glomerulonephritis is characterized by glomerular necroinflammation and crescent formation. Its treatment includes unspecific and toxic agents; therefore, the identification of novel therapeutic targets is required. The E3-ubiquitin ligase murine double minute (MDM)-2 is a nonredundant element of NF-κB signaling and the negative regulator of tumor suppressor gene TP53-mediated cell cycle arrest and cell death. We hypothesized that the MDM2 would drive crescentic glomerulonephritis by NF-κB-dependent glomerular inflammation and by p53-dependent parietal epithelial cell hyperproliferation. Indeed, the pre-emptive MDM2 blockade by nutlin-3a ameliorated all aspects of crescentic glomerulonephritis. MDM2 inhibition had identical protective effects in Trp53-deficient mice, with the exception of crescent formation, which was not influenced by nutlin-3a treatment. In vitro experiments confirmed the contribution of MDM2 for induction of NF-κB-dependent cytokines in murine glomerular endothelial cells and for p53-dependent parietal epithelial cell proliferation. To evaluate MDM2 blockade as a potential therapeutic intervention in rapidly progressive glomerulonephritis, we treated mice with established glomerulonephritis with nutlin-3a. Delayed onset of nutlin-3a treatment was equally protective as the pre-emptive treatment in abrogating crescentic glomerulonephritis. Together, the pathogenic effects of MDM2 are twofold, that is, p53-independent NF-κB activation increasing intraglomerular inflammation and p53-dependent parietal epithelial cell hyperplasia and crescent formation. We therefore propose MDM2 blockade as a potential novel therapeutic strategy in rapidly progressive glomerulonephritis.

Matters of life and death. How neutrophils die or survive along NET release and is "NETosis" = necroptosis?

Neutrophil extracellular trap (NET) formation is a hallmark of many disorders that involve neutrophil recruitment, tissue damage, and inflammation. As NET formation is often associated with neutrophil death, the term "NETosis" has become popular. Upon discovery that neutrophils may survive NET release, apparent misnomers, such as "vital NETosis," have been proposed. Meanwhile, it has become obvious that certain stimuli can trigger neutrophil necroptosis, a process associated with NET-like chromatin release. Here, we discuss the relationship between NET release and neutrophil death in view highlighting that many assays used in the field do not properly distinguish between the two. An updated nomenclature is needed replacing the term "NETosis" to meet the growing variety of settings leading to chromatin release with and without neutrophil death. Dissecting which triggers of NET release involve which signaling pathway will help to define drugable molecular targets that inhibit NET release and/or neutrophil necrosis in specific disorders.