RIPK1 blocks early postnatal lethality mediated by caspase-8 and RIPK3.

Receptor-interacting protein kinase (RIPK)-1 is involved in RIPK3-dependent and -independent signaling pathways leading to cell death and/or inflammation. Genetic ablation of ripk1 causes postnatal lethality, which was not prevented by deletion of ripk3, caspase-8, or fadd. However, animals that lack RIPK1, RIPK3, and either caspase-8 or FADD survived weaning and matured normally. RIPK1 functions in vitro to limit caspase-8-dependent, TNFR-induced apoptosis, and animals lacking RIPK1, RIPK3, and TNFR1 survive to adulthood. The role of RIPK3 in promoting lethality in ripk1(-/-) mice suggests that RIPK3 activation is inhibited by RIPK1 postbirth. Whereas TNFR-induced RIPK3-dependent necroptosis requires RIPK1, cells lacking RIPK1 were sensitized to necroptosis triggered by poly I:C or interferons. Disruption of TLR (TRIF) or type I interferon (IFNAR) signaling delayed lethality in ripk1(-/-)tnfr1(-/-) mice. These results clarify the complex roles for RIPK1 in postnatal life and provide insights into the regulation of FADD-caspase-8 and RIPK3-MLKL signaling by RIPK1.

Apoptosis-Inducing-Factor-Dependent Mitochondrial Function Is Required for T Cell but Not B Cell Function.

The role of apoptosis inducing factor (AIF) in promoting cell death versus survival remains controversial. We report that the loss of AIF in fibroblasts led to mitochondrial electron transport chain defects and loss of proliferation that could be restored by ectopic expression of the yeast NADH dehydrogenase Ndi1. Aif-deficiency in T cells led to decreased peripheral T cell numbers and defective homeostatic proliferation, but thymic T cell development was unaffected. In contrast, Aif-deficient B cells developed and functioned normally. The difference in the dependency of T cells versus B cells on AIF for function and survival correlated with their metabolic requirements. Ectopic Ndi1 expression rescued homeostatic proliferation of Aif-deficient T cells. Despite its reported roles in cell death, fibroblasts, thymocytes and B cells lacking AIF underwent normal death. These studies suggest that the primary role of AIF relates to complex I function, with differential effects on T and B cells.

The Pseudokinase MLKL and the Kinase RIPK3 Have Distinct Roles in Autoimmune Disease Caused by Loss of Death-Receptor-Induced Apoptosis.

The kinases RIPK1 and RIPK3 and the pseudo-kinase MLKL have been identified as key regulators of the necroptotic cell death pathway, although a role for MLKL within the whole animal has not yet been established. Here, we have shown that MLKL deficiency rescued the embryonic lethality caused by loss of Caspase-8 or FADD. Casp8(-/-)Mlkl(-/-) and Fadd(-/-)Mlkl(-/-) mice were viable and fertile but rapidly developed severe lymphadenopathy, systemic autoimmune disease, and thrombocytopenia. These morbidities occurred more rapidly and with increased severity in Casp8(-/-)Mlkl(-/-) and Fadd(-/-)Mlkl(-/-) mice compared to Casp8(-/-)Ripk3(-/-) or Fadd(-/-)Ripk3(-/-) mice, respectively. These results demonstrate that MLKL is an essential effector of aberrant necroptosis in embryos caused by loss of Caspase-8 or FADD. Furthermore, they suggest that RIPK3 and/or MLKL may exert functions independently of necroptosis. It appears that non-necroptotic functions of RIPK3 contribute to the lymphadenopathy, autoimmunity, and excess cytokine production that occur when FADD or Caspase-8-mediated apoptosis is abrogated.

Myeloid-derived suppressor activity is mediated by monocytic lineages maintained by continuous inhibition of extrinsic and intrinsic death pathways.

Nonresolving inflammation expands a heterogeneous population of myeloid suppressor cells capable of inhibiting T cell function. This heterogeneity has confounded the functional dissection of individual myeloid subpopulations and presents an obstacle for antitumor immunity and immunotherapy. Using genetic manipulation of cell death pathways, we found the monocytic suppressor-cell subset, but not the granulocytic subset, requires continuous c-FLIP expression to prevent caspase-8-dependent, RIPK3-independent cell death. Development of the granulocyte subset requires MCL-1-mediated control of the intrinsic mitochondrial death pathway. Monocytic suppressors tolerate the absence of MCL-1 provided cytokines increase expression of the MCL-1-related protein A1. Monocytic suppressors mediate T cell suppression, whereas their granulocytic counterparts lack suppressive function. The loss of the granulocytic subset via conditional MCL-1 deletion did not alter tumor incidence implicating the monocytic compartment as the functionally immunosuppressive subset in vivo. Thus, death pathway modulation defines the development, survival, and function of myeloid suppressor cells.

ZBP1/DAI Drives RIPK3-Mediated Cell Death Induced by IFNs in the Absence of RIPK1.

Receptor-interacting protein kinase 1 (RIPK1) regulates cell fate and proinflammatory signaling downstream of multiple innate immune pathways, including those initiated by TNF-α, TLR ligands, and IFNs. Genetic ablation of Ripk1 results in perinatal lethality arising from both RIPK3-mediated necroptosis and FADD/caspase-8-driven apoptosis. IFNs are thought to contribute to the lethality of Ripk1-deficient mice by activating inopportune cell death during parturition, but how IFNs activate cell death in the absence of RIPK1 is not understood. In this study, we show that Z-form nucleic acid binding protein 1 (ZBP1; also known as DAI) drives IFN-stimulated cell death in settings of RIPK1 deficiency. IFN-activated Jak/STAT signaling induces robust expression of ZBP1, which complexes with RIPK3 in the absence of RIPK1 to trigger RIPK3-driven pathways of caspase-8-mediated apoptosis and MLKL-driven necroptosis. In vivo, deletion of either Zbp1 or core IFN signaling components prolong viability of Ripk1-/- mice for up to 3 mo beyond parturition. Together, these studies implicate ZBP1 as the dominant activator of IFN-driven RIPK3 activation and perinatal lethality in the absence of RIPK1.

Design and evaluation of an open-source, conformable skin-cooling system for body magnetic resonance guided focused ultrasound treatments.

PURPOSE: Magnetic resonance guided focused ultrasound (MRgFUS) treatment of tumors uses inter-sonication delays to allow heat to dissipate from the skin and other near-field tissues. Despite inter-sonication delays, treatment of tumors close to the skin risks skin burns. This work has designed and evaluated an open-source, conformable, skin-cooling system for body MRgFUS treatments to reduce skin burns and enable ablation closer to the skin. METHODS: A MR-compatible skin cooling system is described that features a conformable skin-cooling pad assembly with feedback control allowing continuous flow and pressure maintenance during the procedure. System performance was evaluated with hydrophone, phantom and in vivo porcine studies. Sonications were performed 10 and 5 mm from the skin surface under both control and forced convective skin-cooling conditions. 3D MR temperature imaging was acquired in real time and the accumulated thermal dose volume was measured. Gross analysis of the skin post-sonication was further performed. Device conformability was demonstrated at several body locations. RESULTS: Hydrophone studies demonstrated no beam aberration, but a 5-12% reduction of the peak pressure due to the presence of the skin-cooling pad assembly in the acoustic near field. Phantom evaluation demonstrated there is no MR temperature imaging precision reduction or any other artifacts present due to the coolant flow during MRgFUS sonication. The porcine studies demonstrated skin burns were reduced in size or eliminated when compared to the control condition. CONCLUSION: An open-source design of an MRgFUS active skin cooling system demonstrates device conformability with a reduction of skin burns while ablating superficial tissues.

The transcription factor Myc controls metabolic reprogramming upon T lymphocyte activation.

To fulfill the bioenergetic and biosynthetic demand of proliferation, T cells reprogram their metabolic pathways from fatty acid ß-oxidation and pyruvate oxidation via the TCA cycle to the glycolytic, pentose-phosphate, and glutaminolytic pathways. Two of the top-ranked candidate transcription factors potentially responsible for the activation-induced T cell metabolic transcriptome, HIF1α and Myc, were induced upon T cell activation, but only the acute deletion of Myc markedly inhibited activation-induced glycolysis and glutaminolysis in T cells. Glutamine deprivation compromised activation-induced T cell growth and proliferation, and this was partially replaced by nucleotides and polyamines, implicating glutamine as an important source for biosynthetic precursors in active T cells. Metabolic tracer analysis revealed a Myc-dependent metabolic pathway linking glutaminolysis to the biosynthesis of polyamines. Therefore, a Myc-dependent global metabolic transcriptome drives metabolic reprogramming in activated, primary T lymphocytes. This may represent a general mechanism for metabolic reprogramming under patho-physiological conditions.

Gold(I)-NHC-catalysed synthesis of benzofurans via migratory cyclization of 2-alkynylaryl ethers.

Homogeneous cationic gold(i) catalysis emerged as a preferred avenue for the activation of alkenes and alkynes towards reactions with weak nucleophiles, especially in cyclization reactions. Here we report an intramolecular carboalkoxylation reaction of electron-rich benzyl ethers of 2-ethynylaryl phenols catalysed by a digold(i)-NHC complex. The reaction proceeds efficiently with low catalyst loading and the resulting 2,3-disubstituted benzofurans form in moderate to good yields. Based on the results of a cross-over experiment, spectroscopic data, and DFT calculations, we propose a mechanism that accounts for the observed chemo- and regioselectivity.

Effect of k-space-weighted image contrast and ultrasound focus size on the accuracy of proton resonance frequency thermometry.

PURPOSE: To construct a predictive model that describes how the duration and symmetry of a k-space-weighted image contrast (KWIC) window affects the temporal resolution of differently sized ultrasound foci when using a pseudo-golden angle stack-of-stars acquisition. METHODS: We performed a modulation analysis of proton resonance frequency temperature measurements to create the temporal modulation transfer function for KWIC windows of different symmetry and temporal duration. We reconstructed simulated ultrasound heating trajectories and stack-of-stars k-space data as well as experimental phantom data using the same trajectories. Images were reconstructed using symmetric and asymmetric KWIC windows of 3 different temporal durations. Simulated results were compared against temporal modulation transfer function predictions, experimental results, and the original simulated temperatures. RESULTS: The temporal modulation transfer function shows that temporal resolution with KWIC reconstructions depend on the object size. The KWIC window duration affected SNR and severity of undersampling artifacts. Accuracy and response delay improved as the KWIC window duration decreased or the size of the heated region within the KWIC plane increased. Precision worsened as the window duration decreased. Using a symmetric window eliminated the response delay to heated region size but introduced a large reconstruction delay. CONCLUSION: The accuracy and precision of proton resonance frequency temperature measurements from a stack-of-stars acquisition using a sliding KWIC window reconstruction are dependent on the size of the KWIC window and the size and shape of the heated region. The temporal modulation transfer function of KWIC reconstructions for any object size can predict the temporal response to changes in signal being acquired, such as temperature and contrast enhancement.

RIPK-dependent necrosis and its regulation by caspases: a mystery in five acts.

Caspase-8, FADD, and FLIP orchestrate apoptosis in response to death receptor ligation. Mysteriously however, these proteins are also required for normal embryonic development and immune cell proliferation, an observation that has led to their implication in several nonapoptotic processes. While many scenarios have been proposed, recent genetic and biochemical evidence points to unregulated signaling by the receptor-interacting protein kinases-1 (RIPK1) and RIPK3 as the lethal defect in caspase-8-, FADD-, and FLIP-deficient animals and tissues. The RIPKs are known killers, being responsible for a nonapoptotic form of cell death with features similar to necrosis. However, the mechanism by which caspase-8, FADD, and FLIP prevent runaway RIPK activation is unknown, and the signals that trigger these events during development and immune cell activation remain at large. In this review, we will lay out the evidence as it now stands, reinterpreting earlier observations in light of new clues and considering where the investigation might lead.

A unified model of mammalian BCL-2 protein family interactions at the mitochondria.

During apoptosis, the BCL-2 protein family controls mitochondrial outer membrane permeabilization (MOMP), but the dynamics of this regulation remain controversial. We employed chimeric proteins composed of exogenous BH3 domains inserted into a tBID backbone that can activate the proapoptotic effectors BAX and BAK to permeabilize membranes without being universally sequestered by all antiapoptotic BCL-2 proteins. We thus identified two "modes" whereby prosurvival BCL-2 proteins can block MOMP, by sequestering direct-activator BH3-only proteins ("MODE 1") or by binding active BAX and BAK ("MODE 2"). Notably, we found that MODE 1 sequestration is less efficient and more easily derepressed to promote MOMP than MODE 2. Further, MODE 2 sequestration prevents mitochondrial fusion. We provide a unified model of BCL-2 family function that helps to explain otherwise paradoxical observations relating to MOMP, apoptosis, and mitochondrial dynamics.

Synthesis of 2-substituted benzo[b]thiophenes via gold(i)-NHC-catalyzed cyclization of 2-alkynyl thioanisoles.

Benzo[b]thiophene heterocycles are important components of many important small molecule pharmaceuticals and drug candidates as well as organic semiconducting materials. Many methods have been developed for the construction of a benzo[b]thiophene core via cyclization reaction of alkynes. Although few catalytic reactions were disclosed, most methods rely on stoichiometric activation of alkynes. Here we report an efficient method for the synthesis of 2-substituted benzo[b]thiophenes from 2-alkynyl thioanisoles catalyzed by a gold(i)-IPr hydroxide that is applicable to a wide range of substrates with diverse electronic and steric properties. Additionally, we demonstrate experimentally that the acid additive and its conjugate base are essential to catalyst turnover.

Cell-Extrinsic TNF Collaborates with TRIF Signaling To Promote Yersinia-Induced Apoptosis.

Innate immune responses that are crucial for control of infection are often targeted by microbial pathogens. Blockade of NF-κB and MAPK signaling by the Yersinia virulence factor YopJ inhibits cytokine production by innate immune cells but also triggers cell death. This cell death requires RIPK1 kinase activity and caspase-8, which are engaged by TLR4 and the adaptor protein TRIF. Nevertheless, TLR4- and TRIF-deficient cells undergo significant apoptosis, implicating TLR4/TRIF-independent pathways in the death of Yersinia-infected cells. In this article, we report a key role for TNF/TNFR1 in Yersinia-induced cell death of murine macrophages, which occurs despite the blockade of NF-κB and MAPK signaling imposed by Yersinia on infected cells. Intriguingly, direct analysis of YopJ injection revealed a heterogeneous population of injection-high and injection-low cells, and demonstrated that TNF expression came from the injection-low population. Moreover, TNF production by this subpopulation was necessary for maximal apoptosis in the population of highly injected cells, and TNFR-deficient mice displayed enhanced susceptibility to Yersinia infection. These data demonstrate an important role for collaboration between TNF and pattern recognition receptor signals in promoting maximal apoptosis during bacterial infection, and demonstrate that heterogeneity in virulence factor injection and cellular responses play an important role in promoting anti-Yersinia immune defense.

Validation of hybrid angular spectrum acoustic and thermal modelling in phantoms.

In focused ultrasound (FUS) thermal ablation of diseased tissue, acoustic beam and thermal simulations enable treatment planning and optimization. In this study, a treatment-planning methodology that uses the hybrid angular spectrum (HAS) method and the Pennes' bioheat equation (PBHE) is experimentally validated in homogeneous tissue-mimicking phantoms. Simulated three-dimensional temperature profiles are compared to volumetric MR thermometry imaging (MRTI) of FUS sonications in the phantoms, whose acoustic and thermal properties are independently measured. Additionally, Monte Carlo (MC) uncertainty analysis is performed to quantify the effect of tissue property uncertainties on simulation results. The mean error between simulated and experimental spatiotemporal peak temperature rise was +0.33°C (+6.9%). Despite this error, the experimental temperature rise fell within the expected uncertainty of the simulation, as determined by the MC analysis. The average errors of the simulated transverse and longitudinal full width half maximum (FWHM) of the profiles were -1.9% and 7.5%, respectively. A linear regression and local sensitivity analysis revealed that simulated temperature amplitude is more sensitive to uncertainties in simulation inputs than in the profile width and shape. Acoustic power, acoustic attenuation and thermal conductivity had the greatest impact on peak temperature rise uncertainty; thermal conductivity and volumetric heat capacity had the greatest impact on FWHM uncertainty. This study validates that using the HAS and PBHE method can adequately predict temperature profiles from single sonications in homogeneous media. Further, it informs the need to accurately measure or predict patient-specific properties for improved treatment planning of ablative FUS surgeries.

Thermal diffusivity and perfusion constants from in vivo MR-guided focussed ultrasound treatments: a feasibility study.

PURPOSE: This study investigates the feasibility of non-invasively determining thermal diffusivity (α) and the Pennes perfusion parameter (w) from pre-clinical and clinical magnetic resonance-guided focussed ultrasound (MRgFUS) temperature data. MATERIALS AND METHODS: Pre-clinical MRgFUS experiments were performed in rabbit muscle (N = 3, 28 sonications) using three-dimensional MR thermometry. Eight sonications were made in a clinical QA phantom with two-dimensional thermometry. Retrospective property determination was performed on clinical uterine fibroid (N = 8, 9 sonications) and desmoid tumour (N = 4, 7 sonications) data. The property determination method fits an analytical solution to MRgFUS temperatures in the coronal MR plane, including all temperatures acquired during heating and one cooling image. When possible, additional cooling data were acquired for property determination. RESULTS: Rabbit α and w from Heating Data (α = 0.164 mm2s-1, w = 7.9 kg m-3 s-1) and Heating and Cooling Data (α = 0.146 mm2s-1, w = 3.3 kg m-3 s-1) were within the range of gold-standard invasive measurements, with >50% reduction in variability by including cooling data. QA phantom property determination with cooling data yielded properties within 3% of expected values (α = 0.144 mm2s-1, w = 0.0 kg m-3 s-1), a difference that was not statistically significant (p = 0.053). Uterine fibroid (Heating Data: α = 0.212 mm2s-1, w = 11.0 kg m-3 s-1) and desmoid tumour (Heating & Cooling Data: α = 0.245 mm2s-1, w = 4.7 kg m-3 s-1) properties are feasible but lack independent verification. CONCLUSIONS: Thermal diffusivity and the Pennes perfusion parameter can be obtained from in vivo data and with clinical MRgFUS protocols. Property values are consistently improved by including cooling data. The utility of this property determination method will increase as clinical protocols implement improved temperature imaging.

Experimental assessment of phase aberration correction for breast MRgFUS therapy.

PURPOSE: This study validates that phase aberrations in breast magnetic resonance-guided focussed ultrasound (MRgFUS) therapies can be corrected in a clinically relevant time frame to generate more intense, smaller and more spatially accurate foci. MATERIALS AND METHODS: Hybrid angular spectrum (HAS) ultrasound calculations in an magnetic resonance imaging (MRI)-based tissue model, were used to compute phase aberration corrections for improved experimental MRgFUS heating in four heterogeneous breast-mimicking phantoms (n = 18 total locations). Magnetic resonance(MR) temperature imaging was used to evaluate the maximum temperature rise, focus volume and focus accuracy for uncorrected and phase aberration-corrected sonications. Thermal simulations assessed the effectiveness of the phase aberration correction implementation. RESULTS: In 13 of 18 locations, the maximum temperature rise increased by an average of 30%, focus volume was reduced by 40% and focus accuracy improved from 4.6 to 3.6 mm. Mixed results were observed in five of the 18 locations, with focus accuracy improving from 6.1 to 2.5 mm and the maximum temperature rise decreasing by 8% and focus volume increasing by 10%. Overall, the study demonstrated significant improvements (p < 0.005) in maximum temperature rise, focus volume and focus accuracy. Simulations predicted greater improvements than observed experimentally, suggesting potential for improvement in implementing the technique. The complete phase aberration correction procedure, including model generation, segmentation and phase aberration computations, required less than 45 min per sonication location. CONCLUSION: The significant improvements demonstrated in this study i.e., focus intensity, size and accuracy from phase aberration correction have the potential to improve the efficacy, time-efficiency and safety of breast MRgFUS therapies.

Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis.

Caspase-8 has two opposing biological functions--it promotes cell death by triggering the extrinsic pathway of apoptosis, but also has a survival activity, as it is required for embryonic development, T-lymphocyte activation, and resistance to necrosis induced by tumour necrosis factor-α (TNF-α) and related family ligands. Here we show that development of caspase-8-deficient mice is completely rescued by ablation of receptor interacting protein kinase-3 (RIPK3). Adult animals lacking both caspase-8 and RIPK3 display a progressive lymphoaccumulative disease resembling that seen with defects in CD95 or CD95-ligand (also known as FAS and FASLG, respectively), and resist the lethal effects of CD95 ligation in vivo. We have found that caspase-8 prevents RIPK3-dependent necrosis without inducing apoptosis by functioning in a proteolytically active complex with FLICE-like inhibitory protein long (FLIP(L), also known as CFLAR), and this complex is required for the protective function.

Non-apoptotic role of BID in inflammation and innate immunity.

Innate immunity is a fundamental defence response that depends on evolutionarily conserved pattern recognition receptors for sensing infections or danger signals. Nucleotide-binding and oligomerization domain (NOD) proteins are cytosolic pattern-recognition receptors of paramount importance in the intestine, and their dysregulation is associated with inflammatory bowel disease. They sense peptidoglycans from commensal microorganisms and pathogens and coordinate signalling events that culminate in the induction of inflammation and anti-microbial responses. However, the signalling mechanisms involved in this process are not fully understood. Here, using genome-wide RNA interference, we identify candidate genes that modulate the NOD1 inflammatory response in intestinal epithelial cells. Our results reveal a significant crosstalk between innate immunity and apoptosis and identify BID, a BCL2 family protein, as a critical component of the inflammatory response. Colonocytes depleted of BID or macrophages from Bid(-/-) mice are markedly defective in cytokine production in response to NOD activation. Furthermore, Bid(-/-) mice are unresponsive to local or systemic exposure to NOD agonists or their protective effect in experimental colitis. Mechanistically, BID interacts with NOD1, NOD2 and the IκB kinase (IKK) complex, impacting NF-κB and extracellular signal-regulated kinase (ERK) signalling. Our results define a novel role of BID in inflammation and immunity independent of its apoptotic function, furthering the mounting evidence of evolutionary conservation between the mechanisms of apoptosis and immunity.

Characterization of cytoplasmic caspase-2 activation by induced proximity.

Caspase-2 is an initiator caspase activated in response to heat shock and other stressors that induce apoptosis. Activation of caspase-2 requires induced proximity resulting after recruitment to caspase-2 activation complexes such as the PIDDosome. We have adapted bimolecular fluorescence complementation (BiFC) to measure caspase-2 induced proximity in real time in single cells. Nonfluorescent fragments of the fluorescent protein Venus that can associate to reform the fluorescent complex were fused to caspase-2, allowing visualization and kinetic measurements of caspase-2 induced proximity after heat shock and other stresses. This revealed that the caspase-2 activation platform occurred in the cytosol and not in the nucleus in response to heat shock, DNA damage, cytoskeletal disruption, and other treatments. Activation, as measured by this approach, in response to heat shock was RAIDD dependent and upstream of mitochondrial outer-membrane permeabilization. Furthermore, we identify Hsp90alpha as a key negative regulator of heat shock-induced caspase-2 activation.

Developmental checkpoints guarded by regulated necrosis.

The process of embryonic development is highly regulated through the symbiotic control of differentiation and programmed cell death pathways, which together sculpt tissues and organs. The importance of programmed necrotic (RIPK-dependent necroptosis) cell death during development has recently been recognized as important and has largely been characterized using genetically engineered animals. Suppression of necroptosis appears to be essential for murine development and occurs at three distinct checkpoints, E10.5, E16.5, and P1. These distinct time points have helped delineate the molecular pathways and regulation of necroptosis. The embryonic lethality at E10.5 seen in knockouts of caspase-8, FADD, or FLIP (cflar), components of the extrinsic apoptosis pathway, resulted in pallid embryos that did not exhibit the expected cellular expansions. This was the first suggestion that these factors play an important role in the inhibition of necroptotic cell death. The embryonic lethality at E16.5 highlighted the importance of TNF engaging necroptosis in vivo, since elimination of TNFR1 from casp8 (-/-), fadd (-/-), or cflar (-/-), ripk3 (-/-) embryos delayed embryonic lethality from E10.5 until E16.5. The P1 checkpoint demonstrates the dual role of RIPK1 in both the induction and inhibition of necroptosis, depending on the upstream signal. This review summarizes the role of necroptosis in development and the genetic evidence that helped detail the molecular mechanisms of this novel pathway of programmed cell death.