Joshua D. Webster

Activity of Protein Kinase RIPK3 Determines Whether Cells Die by Necroptosis or Apoptosis

Life and Cell Death Trying to protect animals from one form of cell death may lead to death by another. Two protein kinases, known as RIPK1 and RIPK3 promote signaling that leads to cell death by necroptosis. However, Newton et al. (p. 1357, published online 20 February; see the Perspective by Zhang and Chan) found that inhibition of RIPK3 was not always beneficial. Instead, mice expressing a form of RIPK3 with no catalytic activity died from increased apoptotic cell death, but animals lacking the RIPK3 protein entirely, did not die perhaps because RIPK3 restrains apoptosis mediated by caspase-8 by an independent mechanism. A particular protein kinase functions at a critical control point that determines whether—and how—cells die. [Also see Perspective by Zhang and Chan] Receptor-interacting protein kinase 1 (RIPK1) and RIPK3 trigger pro-inflammatory cell death termed “necroptosis.” Studies with RIPK3-deficient mice or the RIPK1 inhibitor necrostatin-1 suggest that necroptosis exacerbates pathology in many disease models. We engineered mice expressing catalytically inactive RIPK3 D161N or RIPK1 D138N to determine the need for the active kinase in the whole animal. Unexpectedly, RIPK3 D161N promoted lethal RIPK1- and caspase-8–dependent apoptosis. In contrast, mice expressing RIPK1 D138N were viable and, like RIPK3-deficient mice, resistant to tumor necrosis factor (TNF)–induced hypothermia. Cells expressing RIPK1 D138N were resistant to TNF-induced necroptosis, whereas TNF-induced signaling pathways promoting gene transcription were unperturbed. Our data indicate that the kinase activity of RIPK3 is essential for necroptosis but also governs whether a cell activates caspase-8 and dies by apoptosis.

RIPK3 deficiency or catalytically inactive RIPK1 provides greater benefit than MLKL deficiency in mouse models of inflammation and tissue injury

Necroptosis is a caspase-independent form of cell death that is triggered by activation of the receptor interacting serine/threonine kinase 3 (RIPK3) and phosphorylation of its pseudokinase substrate mixed lineage kinase-like (MLKL), which then translocates to membranes and promotes cell lysis. Activation of RIPK3 is regulated by the kinase RIPK1. Here we analyze the contribution of RIPK1, RIPK3, or MLKL to several mouse disease models. Loss of RIPK3 had no effect on lipopolysaccharide-induced sepsis, dextran sodium sulfate-induced colitis, cerulein-induced pancreatitis, hypoxia-induced cerebral edema, or the major cerebral artery occlusion stroke model. However, kidney ischemia–reperfusion injury, myocardial infarction, and systemic inflammation associated with A20 deficiency or high-dose tumor necrosis factor (TNF) were ameliorated by RIPK3 deficiency. Catalytically inactive RIPK1 was also beneficial in the kidney ischemia–reperfusion injury model, the high-dose TNF model, and in A20−/− mice. Interestingly, MLKL deficiency offered less protection in the kidney ischemia–reperfusion injury model and no benefit in A20−/− mice, consistent with necroptosis-independent functions for RIPK1 and RIPK3. Combined loss of RIPK3 (or MLKL) and caspase-8 largely prevented the cytokine storm, hypothermia, and morbidity induced by TNF, suggesting that the triggering event in this model is a combination of apoptosis and necroptosis. Tissue-specific RIPK3 deletion identified intestinal epithelial cells as the major target organ. Together these data emphasize that MLKL deficiency rather than RIPK1 inactivation or RIPK3 deficiency must be examined to implicate a role for necroptosis in disease.

RIPK1 inhibits ZBP1-driven necroptosis during development

Receptor-interacting protein kinase 1 (RIPK1) promotes cell survival—mice lacking RIPK1 die perinatally, exhibiting aberrant caspase-8-dependent apoptosis and mixed lineage kinase-like (MLKL)-dependent necroptosis. However, mice expressing catalytically inactive RIPK1 are viable, and an ill-defined pro-survival function for the RIPK1 scaffold has therefore been proposed. Here we show that the RIP homotypic interaction motif (RHIM) in RIPK1 prevents the RHIM-containing adaptor protein ZBP1 (Z-DNA binding protein 1; also known as DAI or DLM1) from activating RIPK3 upstream of MLKL. Ripk1RHIM/RHIM mice that expressed mutant RIPK1 with critical RHIM residues IQIG mutated to AAAA died around birth and exhibited RIPK3 autophosphorylation on Thr231 and Ser232, which is a hallmark of necroptosis, in the skin and thymus. Blocking necroptosis with catalytically inactive RIPK3(D161N), RHIM mutant RIPK3, RIPK3 deficiency, or MLKL deficiency prevented lethality in Ripk1RHIM/RHIM mice. Loss of ZBP1, which engages RIPK3 in response to certain viruses but previously had no defined role in development, also prevented perinatal lethality in Ripk1RHIM/RHIM mice. Consistent with the RHIM of RIPK1 functioning as a brake that prevents ZBP1 from engaging the RIPK3 RHIM, ZBP1 interacted with RIPK3 in Ripk1RHIM/RHIMMlkl−/− macrophages, but not in wild-type, Mlkl−/− or Ripk1RHIM/RHIMRipk3RHIM/RHIM macrophages. Collectively, these findings indicate that the RHIM of RIPK1 is critical for preventing ZBP1/RIPK3/MLKL-dependent necroptosis during development.

Whole-Slide Imaging and Automated Image Analysis Considerations and Opportunities in the Practice of Pathology

Digital pathology, the practice of pathology using digitized images of pathologic specimens, has been transformed in recent years by the development of whole-slide imaging systems, which allow for the evaluation and interpretation of digital images of entire histologic sections. Applications of whole-slide imaging include rapid transmission of pathologic data for consultations and collaborations, standardization and distribution of pathologic materials for education, tissue specimen archiving, and image analysis of histologic specimens. Histologic image analysis allows for the acquisition of objective measurements of histomorphologic, histochemical, and immunohistochemical properties of tissue sections, increasing both the quantity and quality of data obtained from histologic assessments. Currently, numerous histologic image analysis software solutions are commercially available. Choosing the appropriate solution is dependent on considerations of the investigative question, computer programming and image analysis expertise, and cost. However, all studies using histologic image analysis require careful consideration of preanalytical variables, such as tissue collection, fixation, and processing, and experimental design, including sample selection, controls, reference standards, and the variables being measured. The fields of digital pathology and histologic image analysis are continuing to evolve, and their potential impact on pathology is still growing. These methodologies will increasingly transform the practice of pathology, allowing it to mature toward a quantitative science. However, this maturation requires pathologists to be at the forefront of the process, ensuring their appropriate application and the validity of their results. Therefore, histologic image analysis and the field of pathology should co-evolve, creating a symbiotic relationship that results in high-quality reproducible, objective data.

Deubiquitinase DUBA is a post-translational brake on interleukin-17 production in T cells

T-helper type 17 (TH17) cells that produce the cytokines interleukin-17A (IL-17A) and IL-17F are implicated in the pathogenesis of several autoimmune diseases. The differentiation of TH17 cells is regulated by transcription factors such as RORγt, but post-translational mechanisms preventing the rampant production of pro-inflammatory IL-17A have received less attention. Here we show that the deubiquitylating enzyme DUBA is a negative regulator of IL-17A production in T cells. Mice with DUBA-deficient T cells developed exacerbated inflammation in the small intestine after challenge with anti-CD3 antibodies. DUBA interacted with the ubiquitin ligase UBR5, which suppressed DUBA abundance in naive T cells. DUBA accumulated in activated T cells and stabilized UBR5, which then ubiquitylated RORγt in response to TGF-β signalling. Our data identify DUBA as a cell-intrinsic suppressor of IL-17 production.

Regulation of proximal T cell receptor signaling and tolerance induction by deubiquitinase Usp9X.

Naik et al. show that the deubiquitinating enzyme Usp9X is a regulator of T cell activation and its loss induces the development of a lupuslike autoimmune disease in mice.

Coordinated ubiquitination and phosphorylation of RIP1 regulates necroptotic cell death

Proper regulation of cell death signaling is crucial for the maintenance of homeostasis and prevention of disease. A caspase-independent regulated form of cell death called necroptosis is rapidly emerging as an important mediator of a number of human pathologies including inflammatory bowel disease and ischemia–reperfusion organ injury. Activation of necroptotic signaling through TNF signaling or organ injury leads to the activation of kinases receptor-interacting protein kinases 1 and 3 (RIP1 and RIP3) and culminates in inflammatory cell death. We found that, in addition to phosphorylation, necroptotic cell death is regulated by ubiquitination of RIP1 in the necrosome. Necroptotic RIP1 ubiquitination requires RIP1 kinase activity, but not necroptotic mediators RIP3 and MLKL (mixed lineage kinase-like). Using immunoaffinity enrichment and mass spectrometry, we profiled numerous ubiquitination events on RIP1 that are triggered during necroptotic signaling. Mutation of a necroptosis-related ubiquitination site on RIP1 reduced necroptotic cell death and RIP1 ubiquitination and phosphorylation, and disrupted the assembly of RIP1 and RIP3 in the necrosome, suggesting that necroptotic RIP1 ubiquitination is important for maintaining RIP1 kinase activity in the necrosome complex. We also observed RIP1 ubiquitination in injured kidneys consistent with a physiological role of RIP1 ubiquitination in ischemia–reperfusion disease. Taken together, these data reveal that coordinated and interdependent RIP1 phosphorylation and ubiquitination within the necroptotic complex regulate necroptotic signaling and cell death.

Canine cutaneous mast cell tumors: A combined clinical and pathologic approach to diagnosis, prognosis, and treatment selection.

In view of the varied biologic behavior and the costs of treatment for canine cutaneous mast cell tumors, development of appropriate treatment plans for individual affected dogs can be difficult, but decisions regarding treatment should be made using a systematic, evidence-based approach. This manuscript reviews the current state of diagnostics and prognostication of canine cutaneous mast cell tumors, and suggests a combined approach based on clinical and pathologic assessment for decision making regarding treatment choices. The current state of histologic grading, evaluation of proliferation indices, evaluation of mutations in the c-kit gene and KIT expression, evaluation of excision and clinical staging are examined. On the basis of the current understanding of prognostication and treatment response, algorithms for selection of local and systemic therapy are presented.

β-Cell Insulin Secretion Requires the Ubiquitin Ligase COP1

A variety of signals finely tune insulin secretion by pancreatic β cells to prevent both hyper-and hypoglycemic states. Here, we show that post-translational regulation of the transcription factors ETV1, ETV4, and ETV5 by the ubiquitin ligase COP1 (also called RFWD2) in β cells is critical for insulin secretion. Mice lacking COP1 in β cells developed diabetes due to insulin granule docking defects that were fully rescued by genetic deletion of Etv1, Etv4, and Etv5. Genes regulated by ETV1, ETV4, or ETV5 in the absence of mouse COP1 were enriched in human diabetes-associated genes, suggesting that they also influence human β-cell pathophysiology. In normal β cells, ETV4 was stabilized upon membrane depolarization and limited insulin secretion under hyperglycemic conditions. Collectively, our data reveal that ETVs negatively regulate insulin secretion for the maintenance of normoglycemia.

Repression of Stress-Induced LINE-1 Expression Protects Cancer Cell Subpopulations from Lethal Drug Exposure

Maintenance of phenotypic heterogeneity within cell populations is an evolutionarily conserved mechanism that underlies population survival upon stressful exposures. We show that the genomes of a cancer cell subpopulation that survives treatment with otherwise lethal drugs, the drug-tolerant persisters (DTPs), exhibit a repressed chromatin state characterized by increased methylation of histone H3 lysines 9 and 27 (H3K9 and H3K27). We also show that survival of DTPs is, in part, maintained by regulators of H3K9me3-mediated heterochromatin formation and that the observed increase in H3K9me3 in DTPs is most prominent over long interspersed repeat element 1 (LINE-1). Disruption of the repressive chromatin over LINE-1 elements in DTPs results in DTP ablation, which is partially rescued by reducing LINE-1 expression or function.