IKKɛ induces STING non-IFN immune responses via a mechanism analogous to TBK1.

The cGAS-STING pathway responds to cytosolic DNA to elicit host immunity to infection. The activation of stimulator of interferon genes (STING) can trigger a number of critical cellular responses including inflammation, noncanonical autophagy, lipid metabolism, senescence, and cell death. STING-mediated immunity through the production of type I interferons (IFNs) and nuclear factor kappa B (NF-κB)-driven proinflammatory cytokines is primarily driven via the effector protein TBK1. We have previously found that IκBα kinase epsilon (IKKε), a homolog of TBK1, can also facilitate STING-NF-κB responses. Therefore, a thorough understanding of how IKKε participates in STING signaling is essential. Here, we used a combination of genetic and biochemical approaches to provide mechanistic details into how IKKε confers non-IFN (e.g., NF-κB and MAPK) STING responses in macrophages, including in the absence of TBK1. We demonstrate a conserved mechanism of STING binding between TBK1 and IKKε. These findings strengthen our understanding of cGAS-STING signaling and the preservation of host immunity in cases of TBK1-deficiency.

Termination of STING responses is mediated via ESCRT-dependent degradation.

cGAS-STING signalling is induced by detection of foreign or mislocalised host double-stranded (ds)DNA within the cytosol. STING acts as the major signalling hub, where it controls production of type I interferons and inflammatory cytokines. Basally, STING resides on the ER membrane. Following activation STING traffics to the Golgi to initiate downstream signalling and subsequently to endolysosomal compartments for degradation and termination of signalling. While STING is known to be degraded within lysosomes, the mechanisms controlling its delivery remain poorly defined. Here we utilised a proteomics-based approach to assess phosphorylation changes in primary murine macrophages following STING activation. This identified numerous phosphorylation events in proteins involved in intracellular and vesicular transport. We utilised high-temporal microscopy to track STING vesicular transport in live macrophages. We subsequently identified that the endosomal complexes required for transport (ESCRT) pathway detects ubiquitinated STING on vesicles, which facilitates the degradation of STING in murine macrophages. Disruption of ESCRT functionality greatly enhanced STING signalling and cytokine production, thus characterising a mechanism controlling effective termination of STING signalling.

Homeostatic apoptosis prevents competition-induced atrophy in follicular B cells.

While the intrinsic apoptosis pathway is thought to play a central role in shaping the B cell lineage, its precise role in mature B cell homeostasis remains elusive. Using mice in which mature B cells are unable to undergo apoptotic cell death, we show that apoptosis constrains follicular B (FoB) cell lifespan but plays no role in marginal zone B (MZB) cell homeostasis. In these mice, FoB cells accumulate abnormally. This intensifies intercellular competition for BAFF, resulting in a contraction of the MZB cell compartment, and reducing the growth, trafficking, and fitness of FoB cells. Diminished BAFF signaling dampens the non-canonical NF-κB pathway, undermining FoB cell growth despite the concurrent triggering of a protective p53 response. Thus, MZB and FoB cells exhibit a differential requirement for the intrinsic apoptosis pathway. Homeostatic apoptosis constrains the size of the FoB cell compartment, thereby preventing competition-induced FoB cell atrophy.

Germline heterozygous mutations in Nxf1 perturb RNA metabolism and trigger thrombocytopenia and lymphopenia in mice.

In eukaryotic cells, messenger RNA (mRNA) molecules are exported from the nucleus to the cytoplasm, where they are translated. The highly conserved protein nuclear RNA export factor1 (Nxf1) is an important mediator of this process. Although studies in yeast and in human cell lines have shed light on the biochemical mechanisms of Nxf1 function, its contribution to mammalian physiology is less clear. Several groups have identified recurrent NXF1 mutations in chronic lymphocytic leukemia (CLL), placing it alongside several RNA-metabolism factors (including SF3B1, XPO, RPS15) whose dysregulation is thought to contribute to CLL pathogenesis. We report here an allelic series of germline point mutations in murine Nxf1. Mice heterozygous for these loss-of-function Nxf1 mutations exhibit thrombocytopenia and lymphopenia, together with milder hematological defects. This is primarily caused by cell-intrinsic defects in the survival of platelets and peripheral lymphocytes, which are sensitized to intrinsic apoptosis. In contrast, Nxf1 mutations have almost no effect on red blood cell homeostasis. Comparative transcriptome analysis of platelets, lymphocytes, and erythrocytes from Nxf1-mutant mice shows that, in response to impaired Nxf1 function, the cytoplasmic representation of transcripts encoding regulators of RNA metabolism is altered in a unique, lineage-specific way. Thus, blood cell lineages exhibit differential requirements for Nxf1-mediated global mRNA export.

A small molecule interacts with VDAC2 to block mouse BAK-driven apoptosis.

Activating the intrinsic apoptosis pathway with small molecules is now a clinically validated approach to cancer therapy. In contrast, blocking apoptosis to prevent the death of healthy cells in disease settings has not been achieved. Caspases have been favored, but they act too late in apoptosis to provide long-term protection. The critical step in committing a cell to death is activation of BAK or BAX, pro-death BCL-2 proteins mediating mitochondrial damage. Apoptosis cannot proceed in their absence. Here we show that WEHI-9625, a novel tricyclic sulfone small molecule, binds to VDAC2 and promotes its ability to inhibit apoptosis driven by mouse BAK. In contrast to caspase inhibitors, WEHI-9625 blocks apoptosis before mitochondrial damage, preserving cellular function and long-term clonogenic potential. Our findings expand on the key role of VDAC2 in regulating apoptosis and demonstrate that blocking apoptosis at an early stage is both advantageous and pharmacologically tractable.

Intrinsic apoptosis circumvents the functional decline of circulating platelets but does not cause the storage lesion.

The circulating life span of blood platelets is regulated by the prosurvival protein BCL-XL It restrains the activity of BAK and BAX, the essential prodeath mediators of intrinsic apoptosis. Disabling the platelet intrinsic apoptotic pathway in mice by deleting BAK and BAX results in a doubling of platelet life span and concomitant thrombocytosis. Apoptotic platelets expose phosphatidylserine (PS) via a mechanism that is distinct from that driven by classical agonists. Whether there is any role for apoptotic PS in platelet function in vivo, however, is unclear. Apoptosis has also been associated with the platelet storage lesion (PSL), the constellation of biochemical deteriorations that occur during blood bank storage. In this study, we investigated the role of BAK/BAX-mediated apoptosis in hemostasis and thrombosis and in the development of the PSL. We show that although intrinsic apoptosis is rapidly induced during storage at 37°C, it is not detected when platelets are kept at the standard storage temperature of 22°C. Remarkably, loss of BAK and BAX did not prevent the development of the PSL at either temperature. BAK/BAX-deficient mice exhibited increased bleeding times and unstable thrombus formation. This phenotype was not caused by impaired PS exposure, but was associated with a defect in granule release from aged platelets. Strikingly, rejuvenation of BAK/BAX-deficient platelets in vivo completely rescued the observed hemostatic defects. Thus, apoptotic culling of old platelets from the bloodstream is essential to maintain a functional, hemostatically reactive platelet population. Inhibiting intrinsic apoptosis in blood banked platelets is unlikely to yield significant benefit.

BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis.

Mitochondrial apoptosis is mediated by BAK and BAX, two proteins that induce mitochondrial outer membrane permeabilization, leading to cytochrome c release and activation of apoptotic caspases. In the absence of active caspases, mitochondrial DNA (mtDNA) triggers the innate immune cGAS/STING pathway, causing dying cells to secrete type I interferon. How cGAS gains access to mtDNA remains unclear. We used live-cell lattice light-sheet microscopy to examine the mitochondrial network in mouse embryonic fibroblasts. We found that after BAK/BAX activation and cytochrome c loss, the mitochondrial network broke down and large BAK/BAX pores appeared in the outer membrane. These BAK/BAX macropores allowed the inner mitochondrial membrane to herniate into the cytosol, carrying with it mitochondrial matrix components, including the mitochondrial genome. Apoptotic caspases did not prevent herniation but dismantled the dying cell to suppress mtDNA-induced innate immune signaling.

Mutations in tropomyosin 4 underlie a rare form of human macrothrombocytopenia.

Platelets are anuclear cells that are essential for blood clotting. They are produced by large polyploid precursor cells called megakaryocytes. Previous genome-wide association studies in nearly 70,000 individuals indicated that single nucleotide variants (SNVs) in the gene encoding the actin cytoskeletal regulator tropomyosin 4 (TPM4) exert an effect on the count and volume of platelets. Platelet number and volume are independent risk factors for heart attack and stroke. Here, we have identified 2 unrelated families in the BRIDGE Bleeding and Platelet Disorders (BPD) collection who carry a TPM4 variant that causes truncation of the TPM4 protein and segregates with macrothrombocytopenia, a disorder characterized by low platelet count. N-Ethyl-N-nitrosourea-induced (ENU-induced) missense mutations in Tpm4 or targeted inactivation of the Tpm4 locus led to gene dosage-dependent macrothrombocytopenia in mice. All other blood cell counts in Tpm4-deficient mice were normal. Insufficient TPM4 expression in human and mouse megakaryocytes resulted in a defect in the terminal stages of platelet production and had a mild effect on platelet function. Together, our findings demonstrate a nonredundant role for TPM4 in platelet biogenesis in humans and mice and reveal that truncating variants in TPM4 cause a previously undescribed dominant Mendelian platelet disorder.

Physiological restraint of Bak by Bcl-xL is essential for cell survival.

Due to the myriad interactions between prosurvival and proapoptotic members of the Bcl-2 family of proteins, establishing the mechanisms that regulate the intrinsic apoptotic pathway has proven challenging. Mechanistic insights have primarily been gleaned from in vitro studies because genetic approaches in mammals that produce unambiguous data are difficult to design. Here we describe a mutation in mouse and human Bak that specifically disrupts its interaction with the prosurvival protein Bcl-xL Substitution of Glu75 in mBak (hBAK Q77) for leucine does not affect the three-dimensional structure of Bak or killing activity but reduces its affinity for Bcl-xL via loss of a single hydrogen bond. Using this mutant, we investigated the requirement for physical restraint of Bak by Bcl-xL in apoptotic regulation. In vitro, Bak(Q75L) cells were significantly more sensitive to various apoptotic stimuli. In vivo, loss of Bcl-xL binding to Bak led to significant defects in T-cell and blood platelet survival. Thus, we provide the first definitive in vivo evidence that prosurvival proteins maintain cellular viability by interacting with and inhibiting Bak.

IL-18 Production from the NLRP1 Inflammasome Prevents Obesity and Metabolic Syndrome.

Interleukin-18 (IL-18) is activated by Caspase-1 in inflammasome complexes and has anti-obesity effects; however, it is not known which inflammasome regulates this process. We found that mice lacking the NLRP1 inflammasome phenocopy mice lacking IL-18, with spontaneous obesity due to intrinsic lipid accumulation. This is exacerbated when the mice are fed a high-fat diet (HFD) or a high-protein diet, but not when mice are fed a HFD with low energy density (high fiber). Furthermore, mice with an activating mutation in NLRP1, and hence increased IL-18, have decreased adiposity and are resistant to diet-induced metabolic dysfunction. Feeding these mice a HFD further increased plasma IL-18 concentrations and strikingly resulted in loss of adipose tissue mass and fatal cachexia, which could be prevented by genetic deletion of IL-18. Thus, NLRP1 is an innate immune sensor that functions in the context of metabolic stress to produce IL-18, preventing obesity and metabolic syndrome.

Apoptotic caspases suppress mtDNA-induced STING-mediated type I IFN production.

Activated caspases are a hallmark of apoptosis induced by the intrinsic pathway, but they are dispensable for cell death and the apoptotic clearance of cells in vivo. This has led to the suggestion that caspases are activated not just to kill but to prevent dying cells from triggering a host immune response. Here, we show that the caspase cascade suppresses type I interferon (IFN) production by cells undergoing Bak/Bax-mediated apoptosis. Bak and Bax trigger the release of mitochondrial DNA. This is recognized by the cGAS/STING-dependent DNA sensing pathway, which initiates IFN production. Activated caspases attenuate this response. Pharmacological caspase inhibition or genetic deletion of caspase-9, Apaf-1, or caspase-3/7 causes dying cells to secrete IFN-ß. In vivo, this precipitates an elevation in IFN-ß levels and consequent hematopoietic stem cell dysfunction, which is corrected by loss of Bak and Bax. Thus, the apoptotic caspase cascade functions to render mitochondrial apoptosis immunologically silent.

Platelet production proceeds independently of the intrinsic and extrinsic apoptosis pathways.

BH3 mimetic drugs that target BCL-2 family pro-survival proteins to induce tumour cell apoptosis represent a new era in cancer therapy. Clinical trials of navitoclax (ABT-263, which targets BCL-2, BCL-XL and BCL-W) have shown great promise, but encountered dose-limiting thrombocytopenia. Recent work has demonstrated that this is due to the inhibition of BCL-XL, which is essential for platelet survival. These findings raise new questions about the established model of platelet shedding by megakaryocytes, which is thought to be an apoptotic process. Here we generate mice with megakaryocyte-specific deletions of the essential mediators of extrinsic (Caspase-8) and intrinsic (BAK/BAX) apoptosis. We show that megakaryocytes possess a Fas ligand-inducible extrinsic apoptosis pathway. However, Fas activation does not stimulate platelet production, rather, it triggers Caspase-8-mediated killing. Combined loss of Caspase-8/BAK/BAX does not impair thrombopoiesis, but can protect megakaryocytes from death in mice infected with lymphocytic choriomeningitis virus. Thus, apoptosis is dispensable for platelet biogenesis.

ABCA12 regulates ABCA1-dependent cholesterol efflux from macrophages and the development of atherosclerosis.

ABCA12 is involved in the transport of ceramides in skin, but it may play a wider role in lipid metabolism. We show that, in Abca12-deficient macrophages, cholesterol efflux failed to respond to activation with LXR agonists. Abca12 deficiency caused a reduction in the abundance of Abca1, Abcg1, and Lxrß. Overexpression of Lxrß reversed the effects. Mechanistically, Abca12 deficiency did not affect expression of genes involved in cholesterol metabolism. Instead, a physical association between Abca1, Abca12, and Lxrß proteins was established. Abca12 deficiency enhanced interaction between Abca1 and Lxrß and the degradation of Abca1. Overexpression of ABCA12 in HeLa-ABCA1 cells increased the abundance and stability of ABCA1. Abca12 deficiency caused an accumulation of cholesterol in macrophages and the formation of foam cells, impaired reverse cholesterol transport in vivo, and increased the development of atherosclerosis in irradiated Apoe(-/-) mice reconstituted with Apoe(-/-)Abca12(-/-) bone marrow. Thus, ABCA12 regulates the cellular cholesterol metabolism via an LXRß-dependent posttranscriptional mechanism.

Mcl-1 and Bcl-x(L) coordinately regulate megakaryocyte survival.

Mature megakaryocytes depend on the function of Bcl-x(L), a member of the Bcl-2 family of prosurvival proteins, to proceed safely through the process of platelet shedding. Despite this, loss of Bcl-x(L) does not prevent the growth and maturation of megakaryocytes, suggesting redundancy with other prosurvival proteins. We therefore generated mice with a megakaryocyte-specific deletion of Mcl-1, which is known to be expressed in megakaryocytes. Megakaryopoiesis, platelet production, and platelet lifespan were unperturbed in Mcl-1(Pf4Δ/Pf4Δ) animals. However, treatment with ABT-737, a BH3 mimetic compound that inhibits the prosurvival proteins Bcl-2, Bcl-x(L), and Bcl-w resulted in the complete ablation of megakaryocytes and platelets. Genetic deletion of both Mcl-1 and Bcl-x(L) in megakaryocytes resulted in preweaning lethality. Megakaryopoiesis in Bcl-x(Pf4Δ/Pf4Δ) Mcl-1(Pf4Δ/Pf4Δ) embryos was severely compromised, and these animals exhibited ectopic bleeding. Our studies indicate that the combination of Bcl-x(L) and Mcl-1 is essential for the viability of the megakaryocyte lineage.

Megakaryocytes possess a functional intrinsic apoptosis pathway that must be restrained to survive and produce platelets.

It is believed that megakaryocytes undergo a specialized form of apoptosis to shed platelets. Conversely, a range of pathophysiological insults, including chemotherapy, are thought to cause thrombocytopenia by inducing the apoptotic death of megakaryocytes and their progenitors. To resolve this paradox, we generated mice with hematopoietic- or megakaryocyte-specific deletions of the essential mediators of apoptosis, Bak and Bax. We found that platelet production was unperturbed. In stark contrast, deletion of the prosurvival protein Bcl-x(L) resulted in megakaryocyte apoptosis and a failure of platelet shedding. This could be rescued by deletion of Bak and Bax. We examined the effect on megakaryocytes of three agents that activate the intrinsic apoptosis pathway in other cell types: etoposide, staurosporine, and the BH3 mimetic ABT-737. All three triggered mitochondrial damage, caspase activation, and cell death. Deletion of Bak and Bax rendered megakaryocytes resistant to etoposide and ABT-737. In vivo, mice with a Bak(-/-) Bax(-/-) hematopoietic system were protected against thrombocytopenia induced by the chemotherapeutic agent carboplatin. Thus, megakaryocytes do not activate the intrinsic pathway to generate platelets; rather, the opposite is true: they must restrain it to survive and progress safely through proplatelet formation and platelet shedding.