Human TBK1 deficiency leads to autoinflammation driven by TNF-induced cell death.

TANK binding kinase 1 (TBK1) regulates IFN-I, NF-κB, and TNF-induced RIPK1-dependent cell death (RCD). In mice, biallelic loss of TBK1 is embryonically lethal. We discovered four humans, ages 32, 26, 7, and 8 from three unrelated consanguineous families with homozygous loss-of-function mutations in TBK1. All four patients suffer from chronic and systemic autoinflammation, but not severe viral infections. We demonstrate that TBK1 loss results in hypomorphic but sufficient IFN-I induction via RIG-I/MDA5, while the system retains near intact IL-6 induction through NF-κB. Autoinflammation is driven by TNF-induced RCD as patient-derived fibroblasts experienced higher rates of necroptosis in vitro, and CC3 was elevated in peripheral blood ex vivo. Treatment with anti-TNF dampened the baseline circulating inflammatory profile and ameliorated the clinical condition in vivo. These findings highlight the plasticity of the IFN-I response and underscore a cardinal role for TBK1 in the regulation of RCD.

Immune dysregulation in SHARPIN-deficient mice is dependent on CYLD-mediated cell death.

SHARPIN, together with RNF31/HOIP and RBCK1/HOIL1, form the linear ubiquitin chain assembly complex (LUBAC) E3 ligase that catalyzes M1-linked polyubiquitination. Mutations in RNF31/HOIP and RBCK/HOIL1 in humans and Sharpin in mice lead to autoinflammation and immunodeficiency, but the mechanism underlying the immune dysregulation remains unclear. We now show that the phenotype of the Sharpincpdm/cpdm mice is dependent on CYLD, a deubiquitinase previously shown to mediate removal of K63-linked polyubiquitin chains. Dermatitis, disrupted splenic architecture, and loss of Peyer's patches in the Sharpincpdm/cpdm mice were fully reversed in Sharpincpdm/cpdm Cyld-/- mice. We observed enhanced association of RIPK1 with the death-signaling Complex II following TNF stimulation in Sharpincpdm/cpdm cells, a finding dependent on CYLD since we observed reversal in Sharpincpdm/cpdm Cyld-/- cells. Enhanced RIPK1 recruitment to Complex II in Sharpincpdm/cpdm cells correlated with impaired phosphorylation of CYLD at serine 418, a modification reported to inhibit its enzymatic activity. The dermatitis in the Sharpincpdm/cpdm mice was also ameliorated by the conditional deletion of Cyld using LysM-cre or Cx3cr1-cre indicating that CYLD-dependent death of myeloid cells is inflammatory. Our studies reveal that under physiological conditions, TNF- and RIPK1-dependent cell death is suppressed by the linear ubiquitin-dependent inhibition of CYLD. The Sharpincpdm/cpdm phenotype illustrates the pathological consequences when CYLD inhibition fails.

Interleukin-1ß-induced IRAK1 ubiquitination is required for TH-GM-CSF cell differentiation in T cell-mediated inflammation.

Accumulating evidence suggests granulocyte macrophage-colony stimulating factor (GM-CSF) can function as an inflammatory mediator, but whether GM-CSF-producing CD4+ T cells (TH-GM-CSF) are a distinct T helper cell subset is lacking. Herein we demonstrate that interleukin (IL)-1ß exclusively drives differentiation of naïve CD4+ T cells into TH-GM-CSF cells via inducing ubiquitination of IL-1 receptor-associated kinase 1 (IRAK1) and subsequent activation of the transcription factor NF-kappaB (NF-κB), independent of RAR-related orphan receptor gamma (RORγt) required for TH17 differentiation. In vivo, TH-GM-CSF cells are present in murine Citrobacter Rodentium infections and mediate colitis following adoptive transfer of CD4+ T cells into Rag1-/- mice via GM-CSF-induced macrophage activation. The TH-GM-CSF cell phenotype is stable and distinct from the TH17 genetic program, but IL-1ß can convert pre-formed TH17 cells into TH-GM-CSF cells, thereby accounting for previously reported associations between IL-17 and GM-CSF. Together, our results newly identify IL-1ß/NF-κB-dependent TH-GM-CSF cells as a unique T helper cell subset and highlight the importance of CD4+ T cell-derived GM-CSF induced macrophage activation as a previously undescribed T cell effector mechanism.

More to Life than NF-κB in TNFR1 Signaling.

TNF is a master proinflammatory cytokine whose pathogenic role in inflammatory disorders has long been attributed to induction of proinflammatory mediators. TNF also activates cell survival and death pathways, and recent studies demonstrated that TNF also causes inflammation by inducing cell death. The default response of most cells to TNF is survival and NF-κB-mediated upregulation of prosurvival molecules is a well-documented protective mechanism downstream of TNFR1. Recent studies revealed the existence of an NF-κB-independent cell death checkpoint that restricts cell demise by inactivating RIPK1. Disruption of this checkpoint leads to RIPK1 kinase-dependent death and causes inflammation in vivo. These revelations bring complexity to the control of TNF-induced cell death, and suggest clinical benefit of RIPK1 inhibitors in TNF-driven human inflammatory disorders.

Cloaked in ubiquitin, a killer hides in plain sight: the molecular regulation of RIPK1.

In the past decade, studies have shown how instrumental programmed cell death (PCD) can be in innate and adaptive immune responses. PCD can be a means to maintain homeostasis, prevent or promote microbial pathogenesis, and drive autoimmune disease and inflammation. The molecular machinery regulating these cell death programs has been examined in detail, although there is still much to be explored. A master regulator of programmed cell death and innate immunity is receptor-interacting protein kinase 1 (RIPK1), which has been implicated in orchestrating various pathologies via the induction of apoptosis, necroptosis, and nuclear factor-κB-driven inflammation. These and other roles for RIPK1 have been reviewed elsewhere. In a reflection of the ability of tumor necrosis factor (TNF) to induce either survival or death response, this molecule in the TNF pathway can transduce either a survival or a death signal. The intrinsic killing capacity of RIPK1 is usually kept in check by the chains of ubiquitin, enabling it to serve in a prosurvival capacity. In this review, the intricate regulatory mechanisms responsible for restraining RIPK1 from killing are discussed primarily in the context of the TNF signaling pathway and how, when these mechanisms are disrupted, RIPK1 is free to unveil its program of cellular demise.

Tumor necrosis factor-driven cell death in donor organ as a barrier to immunological tolerance.

PURPOSE OF REVIEW: Regulated cell death (RCD) is likely to play a role in organ rejection but it is unclear how it may be invoked. A well-known trigger of regulated cell death is tumor necrosis factor-alpha (TNF), which activates both caspase-dependent apoptosis and caspase-independent necroptosis. TNF is best known as a pro-inflammatory cytokine because it activates NFκB and MAPK signaling to induce expression of pro-inflammatory genes. RECENT FINDINGS: Emerging data from animal models now suggest that TNF-induced cell death can also be inflammatory. Therefore, the role of cellular demise in regulating immunity should be considered. In transplantation, TNF could have a role in cellular injury or death from ischemia reperfusion (IR) injury and this may dictate organ survival. The default response to TNF in most cells is survival, rather than death, because of the presence of cell death checkpoints. However, cells succumb to TNF-driven death when these checkpoints are disrupted, and sensitivity to death likely reflects a reduction in molecules that fortify these checkpoints. We propose that a cell's propensity to die in response to TNF may underlie allograft rejection. SUMMARY: Genetic, epigenetic, and posttranslational control of death checkpoint regulators in donor tissues may determine graft survival. Therapeutically, drugs that prevent donor cell demise could be useful in preventing organ rejection.

Differential induction of plasma cells by isoforms of human TACI.

Subjects with common variable immune deficiency may have mutations in transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI). Unlike the murine gene, human TACI undergoes alternative messenger (m)RNA splicing to produce isoforms with 1 or 2 ligand-binding domains. Because both isoforms are found in human B cells, we compared their functions in transduced murine B and human pre-B cells. Although murine cells and pre-B cells transduced with the long TACI isoform retained surface CD19 and immunoglobulin G, cells transduced with the short TACI isoform completely lost these B-cell characteristics. Expression of the short TACI isoform produced intense nuclear factor κB activation, nuclear p65 translocation, and colocalization with myeloid differentiation factor 88 and calcium-modulating cyclophilin ligand. The short TACI-transduced cells became larger and CD138 positive, demonstrated upregulated BLIMP1 and XBP1 mRNA, and acquired the morphology of plasma cells. In contrast, cells bearing the long isoform had significantly less BLIMP1 and XBP1 mRNA and, for human pre-B cells, remained CD138 negative. Although human B cells express both isoforms, the short isoform predominates in CD27(+) B cells, toll-like receptor 9-activated peripheral B cells, and splenic marginal zone B cells. Although the transcriptional controls for alternative splicing of isoforms remain unknown, differential signals via isoforms may control plasma-cell generation in humans.

Tumor suppressor cylindromatosis (CYLD) controls HIV transcription in an NF-κB-dependent manner.

UNLABELLED: Characterizing the cellular factors that play a role in the HIV replication cycle is fundamental to fully understanding mechanisms of viral replication and pathogenesis. Whole-genome small interfering RNA (siRNA) screens have identified positive and negative regulators of HIV replication, providing starting points for investigating new cellular factors. We report here that silencing of the deubiquitinase cylindromatosis protein (CYLD), increases HIV infection by enhancing HIV long terminal repeat (LTR)-driven transcription via the NF-κB pathway. CYLD is highly expressed in CD4(+) T lymphocytes, monocyte-derived macrophages, and dendritic cells. We found that CYLD silencing increases HIV replication in T cell lines. We confirmed the positive role of CYLD silencing in HIV infection in primary human CD4(+) T cells, in which CYLD protein was partially processed upon activation. Lastly, Jurkat T cells latently infected with HIV (JLat cells) were more responsive to phorbol 12-myristate 13-acetate (PMA) reactivation in the absence of CYLD, indicating that CYLD activity could play a role in HIV reactivation from latency. In summary, we show that CYLD acts as a potent negative regulator of HIV mRNA expression by specifically inhibiting NF-κB-driven transcription. These findings suggest a function for this protein in modulating productive viral replication as well as in viral reactivation. IMPORTANCE: HIV transcription is regulated by a number of host cell factors. Here we report that silencing of the lysine 63 deubiquitinase CYLD increases HIV transcription in an NF-κB-dependent manner. We show that CYLD is expressed in HIV target cells and that its silencing increases HIV infection in transformed T cell lines as well as primary CD4(+) T cells. Similarly, reactivation of latent provirus was facilitated in the absence of CYLD. These data suggest that CYLD, which is highly expressed in CD4(+) T cells, can control HIV transcription in productive infection as well as during reactivation from latency.

TBK1 and IKKε protect target cells from IFNγ-mediated T cell killing via an inflammatory apoptotic mechanism.

Cytotoxic T cells produce interferon gamma (IFNγ), which plays a critical role in anti-microbial and anti-tumor responses. However, it is not clear whether T cell-derived IFNγ directly kills infected and tumor target cells, and how this may be regulated. Here, we report that target cell expression of the kinases TBK1 and IKKε regulate IFNγ cytotoxicity by suppressing the ability of T cell-derived IFNγ to kill target cells. In tumor targets lacking TBK1 and IKKε, IFNγ induces expression of TNFR1 and the Z-nucleic acid sensor, ZBP1, to trigger RIPK1-dependent apoptosis, largely in a target cell-autonomous manner. Unexpectedly, IFNγ, which is not known to signal to NFκB, induces hyperactivation of NFκB in TBK1 and IKKε double-deficient cells. TBK1 and IKKε suppress IKKα/ß activity and in their absence, IFNγ induces elevated NFκB-dependent expression of inflammatory chemokines and cytokines. Apoptosis is thought to be non-inflammatory, but our observations demonstrate that IFNγ can induce an inflammatory form of apoptosis, and this is suppressed by TBK1 and IKKε. The two kinases provide a critical connection between innate and adaptive immunological responses by regulating three key responses: (1) phosphorylation of IRF3/7 to induce type I IFN; (2) inhibition of RIPK1-dependent death; and (3) inhibition of NFκB-dependent inflammation. We propose that these kinases evolved these functions such that their inhibition by pathogens attempting to block type I IFN expression would enable IFNγ to trigger apoptosis accompanied by an alternative inflammatory response. Our findings show that loss of TBK1 and IKKε in target cells sensitizes them to inflammatory apoptosis induced by T cell-derived IFNγ.

SIRPα maintains macrophage homeostasis by interacting with PTK2B kinase in Mycobacterium tuberculosis infection and through autophagy and necroptosis.

BACKGROUND: To determine whether SIRPα can be a diagnostic marker of pulmonary tuberculosis (PTB) and the molecular mechanism of SIRPα regulating macrophages to kill Mycobacterium tuberculosis (MTB). METHODS: Meta-analysis combined with subsequent qRT-PCR, western-blotting and flow cytometry assay were used to detect SIRPα expression in PTB patients. Cell-based assays were used to explore the regulation of macrophage function by SIRPα. SIRPα-/- and wide type macrophages transplanted C57BL/6J mice were used to determine the function of SIRPα on MTB infection in vivo. FINDINGS: SIRPα levels are closely correlated with the treatment outcomes among PTB patients. Cell-based assay demonstrated that MTB significantly induces the expression of SIRPα on macrophages. SIRPα deficiency enhances the killing ability of macrophages against MTB through processes that involve enhanced autophagy and reduced necroptosis of macrophages. Mechanistically, SIRPα forms a direct interaction with PTK2B through its intracellular C-terminal domain, thus inhibiting PTK2B activation in macrophages. Necroptosis inhibition due to SIRPα deficiency requires PTK2B activity. The transfer of SIRPα-deficient bone marrow-derived macrophages (BMDMs) into wild type mice resulted in a drop of bacterial load in the lungs but an enhancement of inflammatory lung damage, and the combination of ulinastatin and SIRPα-/-→WT treatment could decrease the inflammation and maintain the bactericidal capacity. INTERPRETATION: Our data define SIRPα a novel biomarker for tuberculosis infection and underlying mechanisms for maintaining macrophage homeostasis. FUNDING: This work was financially supported by the Chinese National Natural Science Foundation project (No.81401635). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

IFN-γ+ cytotoxic CD4+ T lymphocytes are involved in the pathogenesis of colitis induced by IL-23 and the food colorant Red 40.

The food colorant Red 40 is an environmental risk factor for colitis development in mice with increased expression of interleukin (IL)-23. This immune response is mediated by CD4+ T cells, but mechanistic insights into how these CD4+ T cells trigger and perpetuate colitis have remained elusive. Here, using single-cell transcriptomic analysis, we found that several CD4+ T-cell subsets are present in the intestines of colitic mice, including an interferon (IFN)-γ-producing subset. In vivo challenge of primed mice with Red 40 promoted rapid activation of CD4+ T cells and caused marked intestinal epithelial cell (IEC) apoptosis that was attenuated by depletion of CD4+ cells and blockade of IFN-γ. Ex vivo experiments showed that intestinal CD4+ T cells from colitic mice directly promoted apoptosis of IECs and intestinal enteroids. CD4+ T cell-mediated cytotoxicity was contact-dependent and required FasL, which promoted caspase-dependent cell death in target IECs. Genetic ablation of IFN-γ constrained IL-23- and Red 40-induced colitis development, and blockade of IFN-γ inhibited epithelial cell death in vivo. These results advance the understanding of the mechanisms regulating colitis development caused by IL-23 and food colorants and identify IFN-γ+ cytotoxic CD4+ T cells as a new potential therapeutic target for colitis.

Induction of diabetes in aged C57B6 mice results in severe nephropathy: an association with oxidative stress, endoplasmic reticulum stress, and inflammation.

Kidney aging is a slowly progressive process that is postulated to be accelerated by intervening diseases, such as diabetes, due in part to the addition of excessive stress and inflammation from the intervening disease to the underlying aging process. This hypothesis was tested by inducing diabetes with streptozotocin in 18-month-old, aging mice. After 4 months of diabetes, these mice developed severe albuminuria, elevated creatinine levels, and renal lesions including extensive apoptotic cell death, glomerulosclerosis, afferent and efferent hyalinosis, and tubulointerstitial inflammation and fibrosis. These symptoms were associated with elevated oxidative stress. The presence of endoplasmic reticulum (ER) stress in 22-month-old diabetic kidneys resulted in up-regulation of C/EBP homologous protein (CHOP), which may play a role in increasing kidney lesions because CHOP-deficient proximal tubular cells were resistant to ER stress-induced cell death, and CHOP-deficient mice were protected from diabetic nephropathy. Moreover, CHOP-deficient mice did not develop albuminuria as they aged. Inflammation, another key component of progressive diabetic nephropathy, was prominent in 22-month-old diabetic kidneys. The expression of tumor-necrosis factor-alpha in 22-month-old diabetic kidneys may play a role in inflammation, ER stress, and apoptosis. Thus, diabetes may accelerate the underlying kidney aging process present in old mice.

T cell-derived tumor necrosis factor induces cytotoxicity by activating RIPK1-dependent target cell death.

TNF ligation of TNF receptor 1 (TNFR1) promotes either inflammation and cell survival by (a) inhibiting RIPK1's death-signaling function and activating NF-κB or (b) causing RIPK1 to associate with the death-inducing signaling complex to initiate apoptosis or necroptosis. The cellular source of TNF that results in RIPK1-dependent cell death remains unclear. To address this, we employed in vitro systems and murine models of T cell-dependent transplant or tumor rejection in which target cell susceptibility to RIPK1-dependent cell death could be genetically altered. We show that TNF released by T cells is necessary and sufficient to activate RIPK1-dependent cell death in target cells and thereby mediate target cell cytolysis independently of T cell frequency. Activation of the RIPK1-dependent cell death program in target cells by T cell-derived TNF accelerates murine cardiac allograft rejection and synergizes with anti-PD1 administration to destroy checkpoint blockade-resistant murine melanoma. Together, the findings uncover a distinct immunological role for TNF released by cytotoxic effector T cells following cognate interactions with their antigenic targets. Manipulating T cell TNF and/or target cell susceptibility to RIPK1-dependent cell death can be exploited to either mitigate or augment T cell-dependent destruction of allografts and malignancies to improve outcomes.

Ubiquitination of RIP1 regulates an NF-kappaB-independent cell-death switch in TNF signaling.

TNF receptor 1 (TNFR1) can trigger opposing responses within the same cell: a prosurvival response or a cell-death pathway [1, 2]. Cell survival requires NF-kappaB-mediated transcription of prosurvival genes [3-9]; apoptosis occurs if NF-kappaB signaling is blocked [5, 7-9]. Hence, activation of NF-kappaB acts as a cell-death switch during TNF signaling. This study demonstrates that the pathway includes another cell-death switch that is independent of NF-kappaB. We show that lysine 63-linked ubiquitination of RIP1 on lysine 377 inhibits TNF-induced apoptosis first through an NF-kappaB-independent mechanism and, subsequently, through an NF-kappaB-dependent mechanism. In contrast, in the absence of ubiquitination, RIP1 serves as a proapoptotic signaling molecule by engaging CASPASE-8. Therefore, RIP1 is a dual-function molecule that can be either prosurvival or prodeath depending on its ubiquitination state, and this serves as an NF-kappaB-independent cell-death switch early in TNF signaling. These results provide an explanation for the conflicting reports on the role of RIP1 in cell death; this role was previously suggested to be both prosurvival and prodeath [10-12]. Because TRAF2 is the E3 ligase for RIP1 [13], these observations provide an explanation for the NF-kappaB-independent antiapoptotic function previously described for TRAF2 [14-16].

The tumour suppressor CYLD is a negative regulator of RIG-I-mediated antiviral response.

On detecting viral RNAs, the RNA helicase retinoic acid-inducible gene I (RIG-I) activates the interferon regulatory factor 3 (IRF3) signalling pathway to induce type I interferon (IFN) gene transcription. How this antiviral signalling pathway might be negatively regulated is poorly understood. Microarray and bioinformatic analysis indicated that the expression of RIG-I and that of the tumour suppressor CYLD (cylindromatosis), a deubiquitinating enzyme that removes Lys 63-linked polyubiquitin chains, are closely correlated, suggesting a functional association between the two molecules. Ectopic expression of CYLD inhibits the IRF3 signalling pathway and IFN production triggered by RIG-I; conversely, CYLD knockdown enhances the response. CYLD removes polyubiquitin chains from RIG-I as well as from TANK binding kinase 1 (TBK1), the kinase that phosphorylates IRF3, coincident with an inhibition of the IRF3 signalling pathway. Furthermore, CYLD protein level is reduced in the presence of tumour necrosis factor and viral infection, concomitant with enhanced IFN production. These findings show that CYLD is a negative regulator of RIG-I-mediated innate antiviral response.

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.

Reversal of CYLD phosphorylation as a novel therapeutic approach for adult T-cell leukemia/lymphoma (ATLL).

Adult T-cell leukemia/lymphoma (ATLL) is a malignancy of mature T cells associated with chronic infection by human T-cell lymphotropic virus type-1 (HTLV-1). ATLL patients with aggressive subtypes have dismal outcomes. We demonstrate that ATLL cells co-opt an early checkpoint within the tumor necrosis factor receptor 1 (TNFR1) pathway, resulting in survival advantage. This early checkpoint revolves around an interaction between the deubiquitinase CYLD and its target RIPK1. The status of RIPK1 K63-ubiquitination determines cell fate by creating either a prosurvival signal (ubiquitinated RIPK1) or a death signal (deubiquitinated RIPK1). In primary ATLL samples and in cell line models, an increased baseline level of CYLD phosphorylation was observed. We therefore tested the hypothesis that this modification of CYLD, which has been reported to inhibit its deubiquitinating function, leads to increased RIPK1 ubiquitination and thus provides a prosurvival signal to ATLL cells. CYLD phosphorylation can be pharmacologically reversed by IKK inhibitors, specifically by TBK1/IKKε and IKKß inhibitors (MRT67307 and TPCA). Both of the IKK sub-families can phosphorylate CYLD, and the combination of MRT67307 and TPCA have a marked effect in reducing CYLD phosphorylation and triggering cell death. ATLL cells overexpressing a kinase-inactive TBK1 (TBK1-K38A) demonstrate lower CYLD phosphorylation and subsequently reduced proliferation. IKK blockade reactivates CYLD, as evidenced by the reduction in RIPK1 ubiquitination, which leads to the association of RIPK1 with the death-inducing signaling complex (DISC) to trigger cell death. In the absence of CYLD, RIPK1 ubiquitination remains elevated following IKK blockade and it does not associate with the DISC. SMAC mimetics can similarly disrupt CYLD phosphorylation and lead to ATLL cell death through reduction of RIPK1 ubiquitination, which is CYLD dependent. These results identify CYLD as a crucial regulator of ATLL survival and point to its role as a potential novel target for pharmacologic modification in this disease.

Ripoptocide - A Spark for Inflammation.

The clinical success of biologics that inhibit TNF (Tumor Necrosis Factor) in inflammatory bowel diseases (IBD), psoriasis and rheumatoid arthritis (RA) has clearly established a pathogenic role for this cytokine in these inflammatory disorders. TNF binding to its receptors activates NFκB and MAPK signaling, inducing the expression of downstream pro-inflammatory genes. This is thought to be the primary mechanism by which TNF elicits inflammation. TNF is also a well-known trigger of caspase-dependent apoptosis or caspase-independent necroptosis. Whether cell death has any role in TNF-mediated inflammation has been less clear. Emerging data from animal models now suggest that cellular demise caused by TNF may indeed provoke inflammation. The default response of most cells to TNF stimulation is survival, rather than death, due to the presence of two sequential cell death checkpoints. The early checkpoint is transcription-independent involving the non-degradative ubiquitination of RIPK1 to prevent RIPK1 from becoming a death-signaling molecule. The later checkpoint requires the induction of pro-survival genes by NFκB-mediated transcription. When the early checkpoint is disrupted, RIPK1 initiates cell death and we suggest the term ripoptocide to describe this manner of death (encompassing both apoptosis and necroptosis). The sensitivity of a cell to ripoptocide is determined by the balance between regulatory molecules that enforce and those that disassemble the early checkpoint. As there is evidence suggesting that ripoptocide is inflammatory, individuals may develop inflammation due to ripoptocide brought about by genetic, epigenetic or post-translational alteration of these checkpoint regulators. For these individuals, drugs that reinforce the early checkpoint and inhibit ripoptocide could be useful in ameliorating inflammation.

Single-Cell and Population-Level Analyses Using Real-Time Kinetic Labeling Couples Proliferation and Cell Death Mechanisms.

Quantifying cytostatic and cytotoxic outcomes are integral components of characterizing perturbagens used as research tools and in drug discovery pipelines. Furthermore, data-rich acquisition, coupled with robust methods for analysis, is required to properly assess the function and impact of these perturbagens. Here, we present a detailed and versatile method for single-cell and population-level analyses using real-time kinetic labeling (SPARKL). SPARKL integrates high-content live-cell imaging with automated detection and analysis of fluorescent reporters of cell death. We outline several examples of zero-handling, non-disruptive protocols for detailing cell death mechanisms and proliferation profiles. Additionally, we suggest several methods for mathematically analyzing these data to best utilize the collected kinetic data. Compared to traditional methods of detection and analysis, SPARKL is more sensitive, accurate, and high throughput while substantially eliminating sample processing and providing richer data.

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.