Death Is Coming and the Clot Thickens, as Pyroptosis Feeds the Fire.

Pyroptotic cell death during endotoxemia causes death via unknown mechanisms. In this issue of Immunity, Wu et al. (2019) show that T3SS rod proteins or LPS induces inflammasome activation, macrophage pyroptosis, and accompanying tissue factor release, directly connecting inflammation to coagulation.

The Coagulation and Immune Systems Are Directly Linked through the Activation of Interleukin-1α by Thrombin.

Ancient organisms have a combined coagulation and immune system, and although links between inflammation and hemostasis exist in mammals, they are indirect and slower to act. Here we investigated direct links between mammalian immune and coagulation systems by examining cytokine proproteins for potential thrombin protease consensus sites. We found that interleukin (IL)-1α is directly activated by thrombin. Thrombin cleaved pro-IL-1α at a site perfectly conserved across disparate species, indicating functional importance. Surface pro-IL-1α on macrophages and activated platelets was cleaved and activated by thrombin, while tissue factor, a potent thrombin activator, colocalized with pro-IL-1α in the epidermis. Mice bearing a mutation in the IL-1α thrombin cleavage site (R114Q) exhibited defects in efficient wound healing and rapid thrombopoiesis after acute platelet loss. Thrombin-cleaved IL-1α was detected in humans during sepsis, pointing to the relevance of this pathway for normal physiology and the pathogenesis of inflammatory and thrombotic diseases.

Intracellular interleukin-1 receptor 2 binding prevents cleavage and activity of interleukin-1α, controlling necrosis-induced sterile inflammation.

Necrosis can induce profound inflammation or be clinically silent. However, the mechanisms underlying such tissue specificity are unknown. Interleukin-1α (IL-1α) is a key danger signal released upon necrosis that exerts effects on both innate and adaptive immunity and is considered to be constitutively active. In contrast, we have shown that necrosis-induced IL-1α activity is tightly controlled in a cell type-specific manner. Most cell types examined expressed a cytosolic IL-1 receptor 2 (IL-1R2) whose binding to pro-IL-1α inhibited its cytokine activity. In cell types exhibiting a silent necrotic phenotype, IL-1R2 remained associated with pro-IL-1α. Cell types possessing inflammatory necrotic phenotypes either lacked IL-1R2 or had activated caspase-1 before necrosis, which degraded and dissociated IL-1R2 from pro-IL-1α. Full IL-1α activity required cleavage by calpain after necrosis, which increased its affinity for IL-1 receptor 1. Thus, we report a cell type-dependent process that fundamentally governs IL-1α activity postnecrosis and the mechanism allowing conditional release of this blockade.

Cell surface IL-1α trafficking is specifically inhibited by interferon-γ, and associates with the membrane via IL-1R2 and GPI anchors.

IL-1 is a powerful cytokine that drives inflammation and modulates adaptive immunity. Both IL-1α and IL-1ß are translated as proforms that require cleavage for full cytokine activity and release, while IL-1α is reported to occur as an alternative plasma membrane-associated form on many cell types. However, the existence of cell surface IL-1α (csIL-1α) is contested, how IL-1α tethers to the membrane is unknown, and signaling pathways controlling trafficking are not specified. Using a robust and fully validated system, we show that macrophages present bona fide csIL-1α after ligation of TLRs. Pro-IL-1α tethers to the plasma membrane in part through IL-1R2 or via association with a glycosylphosphatidylinositol-anchored protein, and can be cleaved, activated, and released by proteases. csIL-1α requires de novo protein synthesis and its trafficking to the plasma membrane is exquisitely sensitive to inhibition by IFN-γ, independent of expression level. We also reveal how prior csIL-1α detection could occur through inadvertent cell permeabilisation, and that senescent cells do not drive the senescent-associated secretory phenotype via csIL-1α, but rather via soluble IL-1α. We believe these data are important for determining the local or systemic context in which IL-1α can contribute to disease and/or physiological processes.

Interleukin-1α Activity in Necrotic Endothelial Cells Is Controlled by Caspase-1 Cleavage of Interleukin-1 Receptor-2: IMPLICATIONS FOR ALLOGRAFT REJECTION.

Inflammation is a key instigator of the immune responses that drive atherosclerosis and allograft rejection. IL-1α, a powerful cytokine that activates both innate and adaptive immunity, induces vessel inflammation after release from necrotic vascular smooth muscle cells (VSMCs). Similarly, IL-1α released from endothelial cells (ECs) damaged during transplant drives allograft rejection. However, IL-1α requires cleavage for full cytokine activity, and what controls cleavage in necrotic ECs is currently unknown. We find that ECs have very low levels of IL-1α activity upon necrosis. However, TNFα or IL-1 induces significant levels of active IL-1α in EC necrotic lysates without alteration in protein levels. Increased activity requires cleavage of IL-1α by calpain to the more active mature form. Immunofluorescence and proximity ligation assays show that IL-1α associates with interleukin-1 receptor-2, and this association is decreased by TNFα or IL-1 and requires caspase activity. Thus, TNFα or IL-1 treatment of ECs leads to caspase proteolytic activity that cleaves interleukin-1 receptor-2, allowing IL-1α dissociation and subsequent processing by calpain. Importantly, ECs could be primed by IL-1α from adjacent damaged VSMCs, and necrotic ECs could activate neighboring normal ECs and VSMCs, causing them to release inflammatory cytokines and up-regulate adhesion molecules, thus amplifying inflammation. These data unravel the molecular mechanisms and interplay between damaged ECs and VSMCs that lead to activation of IL-1α and, thus, initiation of adaptive responses that cause graft rejection.

Senescent Vascular Smooth Muscle Cells Drive Inflammation Through an Interleukin-1α-Dependent Senescence-Associated Secretory Phenotype.

OBJECTIVE: Vascular smooth muscle cells (VSMCs) that become senescent are both present within atherosclerotic plaques and thought to be important to the disease process. However, senescent VSMCs are generally considered to only contribute through inaction, with failure to proliferate resulting in VSMC- and collagen-poor unstable fibrous caps. Whether senescent VSMCs can actively contribute to atherogenic processes, such as inflammation, is unknown. APPROACH AND RESULTS: We find that senescent human VSMCs develop a proinflammatory state known as a senescence-associated secretory phenotype. Senescent human VSMCs release high levels of multiple cytokines and chemokines driven by secreted interleukin-1α acting in an autocrine manner. Consequently, the VSMC senescence-associated secretory phenotype promotes chemotaxis of mononuclear cells in vitro and in vivo. In addition, senescent VSMCs release active matrix metalloproteinase-9, secrete less collagen, upregulate multiple inflammasome components, and prime adjacent endothelial cells and VSMCs to a proadhesive and proinflammatory state. Importantly, maintaining the senescence-associated secretory phenotype places a large metabolic burden on senescent VSMCs, such that they can be selectively killed by inhibiting glucose utilization. CONCLUSIONS: Senescent VSMCs may actively contribute toward the chronic inflammation associated with atherosclerosis through the interleukin-1α-driven senescence-associated secretory phenotype and the priming of adjacent cells to a proatherosclerotic state. These data also suggest that inhibition of this potentially important source of chronic inflammation in atherosclerosis requires blockade of interleukin-1α and not interleukin-1ß.

Mitochondrial DNA damage can promote atherosclerosis independently of reactive oxygen species through effects on smooth muscle cells and monocytes and correlates with higher-risk plaques in humans.

BACKGROUND: Mitochondrial DNA (mtDNA) damage occurs in both circulating cells and the vessel wall in human atherosclerosis. However, it is unclear whether mtDNA damage directly promotes atherogenesis or is a consequence of tissue damage, which cell types are involved, and whether its effects are mediated only through reactive oxygen species. METHODS AND RESULTS: mtDNA damage occurred early in the vessel wall in apolipoprotein E-null (ApoE(-/-)) mice, before significant atherosclerosis developed. mtDNA defects were also identified in circulating monocytes and liver and were associated with mitochondrial dysfunction. To determine whether mtDNA damage directly promotes atherosclerosis, we studied ApoE(-/-) mice deficient for mitochondrial polymerase-γ proofreading activity (polG(-/-)/ApoE(-/-)). polG(-/-)/ApoE(-/-) mice showed extensive mtDNA damage and defects in oxidative phosphorylation but no increase in reactive oxygen species. polG(-/-)/ApoE(-/-) mice showed increased atherosclerosis, associated with impaired proliferation and apoptosis of vascular smooth muscle cells, and hyperlipidemia. Transplantation with polG(-/-)/ApoE(-/-) bone marrow increased the features of plaque vulnerability, and polG(-/-)/ApoE(-/-) monocytes showed increased apoptosis and inflammatory cytokine release. To examine mtDNA damage in human atherosclerosis, we assessed mtDNA adducts in plaques and in leukocytes from patients who had undergone virtual histology intravascular ultrasound characterization of coronary plaques. Human atherosclerotic plaques showed increased mtDNA damage compared with normal vessels; in contrast, leukocyte mtDNA damage was associated with higher-risk plaques but not plaque burden. CONCLUSIONS: We show that mtDNA damage in vessel wall and circulating cells is widespread and causative and indicates higher risk in atherosclerosis. Protection against mtDNA damage and improvement of mitochondrial function are potential areas for new therapeutics.

Apoptosis of vascular smooth muscle cells induces features of plaque vulnerability in atherosclerosis.

Vascular smooth muscle cell (VSMC) apoptosis occurs in many arterial diseases, including aneurysm formation, angioplasty restenosis and atherosclerosis. Although VSMC apoptosis promotes vessel remodeling, coagulation and inflammation, its precise contribution to these diseases is unknown, given that apoptosis frequently accompanies vessel injury or alterations to flow. To study the direct consequences of VSMC apoptosis, we generated transgenic mice expressing the human diphtheria toxin receptor (hDTR, encoded by HBEGF) from a minimal Tagln (also known as SM22alpha) promoter. Despite apoptosis inducing loss of 50-70% of VSMCs, normal arteries showed no inflammation, reactive proliferation, thrombosis, remodeling or aneurysm formation. In contrast, VSMC apoptosis in atherosclerotic plaques of SM22alpha-hDTR Apoe-/- mice induced marked thinning of fibrous cap, loss of collagen and matrix, accumulation of cell debris and intense intimal inflammation. We conclude that VSMC apoptosis is 'silent' in normal arteries, which have a large capacity to withstand cell loss. In contrast, VSMC apoptosis alone is sufficient to induce features of plaque vulnerability in atherosclerosis. SM22alpha-hDTR Apoe-/- mice may represent an important new model to test agents proposed to stabilize atherosclerotic plaques.

Thrombin-activated interleukin-1α drives atherogenesis, but also promotes vascular smooth muscle cell proliferation and collagen production.

AIMS: Atherosclerosis is driven by multiple processes across multiple body systems. For example, the innate immune system drives both atherogenesis and plaque rupture via inflammation, while coronary artery-occluding thrombi formed by the coagulation system cause myocardial infarction and death. However, the interplay between these systems during atherogenesis is understudied. We recently showed that coagulation and immunity are fundamentally linked by the activation of interleukin-1α (IL-1α) by thrombin, and generated a novel knock-in mouse in which thrombin cannot activate endogenous IL-1α [IL-1α thrombin mutant (IL-1αTM)]. METHODS AND RESULTS: Here, we show significantly reduced atherosclerotic plaque formation in IL-1αTM/Apoe-/- mice compared with Apoe-/- and reduced T-cell infiltration. However, IL-1αTM/Apoe-/- plaques have reduced vascular smooth muscle cells, collagen, and fibrous caps, indicative of a more unstable phenotype. Interestingly, the reduced atherogenesis seen with thrombin inhibition was absent in IL-1αTM/Apoe-/- mice, suggesting that thrombin inhibitors can affect atherosclerosis via reduced IL-1α activation. Finally, bone marrow chimeras show that thrombin-activated IL-1α is derived from both vessel wall and myeloid cells. CONCLUSIONS: Together, we reveal that the atherogenic effect of ongoing coagulation is, in part, mediated via thrombin cleavage of IL-1α. This not only highlights the importance of interplay between systems during disease and the potential for therapeutically targeting IL-1α and/or thrombin, but also forewarns that IL-1 may have a role in plaque stabilization.

Vascular smooth muscle cell apoptosis induces interleukin-1-directed inflammation: effects of hyperlipidemia-mediated inhibition of phagocytosis.

RATIONALE: Atherosclerosis is characterized by lipid accumulation in the vessel wall, inflammation, and both macrophage and vascular smooth muscle cell (VSMC) apoptosis. However, whereas VSMC apoptosis in mice with established atherosclerotic plaques or hyperlipidemia increases serum levels of the proatherogenic cytokines monocyte chemotactic protein (MCP)-1, tumor necrosis factor alpha, and interleukin (IL)-6, the link between hyperlipidemia, apoptosis and inflammation, and the mechanisms by which apoptotic cells promote inflammation in atherosclerosis are unknown. OBJECTIVE: To determine whether hyperlipidemia affects apoptotic cell clearance, and identify the molecular pathways downstream of VSMC apoptosis that may promote inflammation. METHODS AND RESULTS: We find that human VSMCs are potent and efficient phagocytes of apoptotic human VSMCs, but phagocytosis is significantly reduced by oxidized low-density lipoprotein in vitro or hyperlipidemia in vivo. Necrotic human aortic VSMCs release IL-1alpha, which induces IL-6 and MCP-1 production from viable human VSMCs in vitro. In contrast, secondary necrotic VSMCs release both IL-1alpha and caspase-activated IL-1beta, augmenting IL-6 and MCP-1 production. Conditionally inducing VSMC apoptosis in situ in hyperlipidemic SM22alpha-hDTR/ApoE(-/-) mice to levels seen in human plaques increases serum MCP-1, tumor necrosis factor alpha, and IL-6, which is prevented by blocking IL-1. CONCLUSIONS: We conclude that VSMC necrosis releases IL-1alpha, whereas secondary necrosis of apoptotic VSMCs releases both IL-1alpha and beta. IL-1 from necrotic VSMCs induces the surrounding viable VSMCs to produce proinflammatory cytokines. Thus, failed clearance of apoptotic VSMCs caused by hyperlipidemia in vivo may promote the increased serum cytokines and chronic inflammation associated with atherosclerosis.

Cell death, damage-associated molecular patterns, and sterile inflammation in cardiovascular disease.

Cell death and inflammation are ancient processes of fundamental biological importance in both normal physiology and pathology. This is evidenced by the profound conservation of mediators, with ancestral homologues identified from plants to humans, and the number of diseases driven by aberrant control of either process. Apoptosis is the most well-studied cell death, but many forms exist, including autophagy, necrosis, pyroptosis, paraptosis, and the obscure dark cell death. Cell death occurs throughout the cardiovascular system, from initial shaping of the heart and vasculature during development to involvement in pathologies, including atherosclerosis, aneurysm, cardiomyopathy, restenosis, and vascular graft rejection. However, determining whether cell death primarily drives pathology or is a secondary bystander effect is difficult. Inflammation, the primary response of innate immunity, is considered essential in initiating and driving vascular diseases. Cell death and inflammation are inextricably linked with their effectors modulating the other process. Indeed, an evolutionary link between cell death and inflammation occurs at caspase-1 (which activates interleukin-1ß), which can induce death by pyroptosis, and is a member of the caspase family vital for apoptosis. This review examines cell death in vascular disease, how it can induce inflammation, and finally the emergence of inflammasomes in vascular pathology.

Smooth muscle cell apoptosis promotes vessel remodeling and repair via activation of cell migration, proliferation, and collagen synthesis.

OBJECTIVE: Although vascular smooth muscle cell (VSMC) apoptosis occurs after vessel injury and during remodeling, the direct role of VSMC death in determining final vessel structure is unclear. We sought to determine the role of VSMC apoptosis in vessel remodeling, medial repair, and neointima formation and to identify the mediators involved. METHODS AND RESULTS: The left common carotid artery was ligated in SM22α-human diphtheria toxin receptor mice, in which diphtheria toxin treatment selectively induces VSMC apoptosis. Apoptosis induced from day 7 to day 14 after ligation significantly increased neointimal and medial areas, cell proliferation, migration, and vessel size. Neointima formation depended on VSMCs, as VSMC depletion before ligation significantly reduced neointimal area and cellularity. In culture, conditioned media from apoptotic VSMCs promoted VSMC migration, proliferation, and collagen synthesis. Interleukin-6 (IL-6) secretion increased 5-fold and IL-1α 1.5-fold after apoptosis, whereas IL-6 inhibition negated the effect of apoptotic VSMC supernatants on VSMC migration, proliferation, and matrix synthesis. CONCLUSION: Signaling from apoptotic VSMCs directly promotes vessel remodeling, medial repair, and neointima formation after flow reduction. Although lumen size appears to depend on flow, VSMC apoptosis is an important determinant of vessel, medial, and neointimal size after flow reduction.

Chronic apoptosis of vascular smooth muscle cells accelerates atherosclerosis and promotes calcification and medial degeneration.

Vascular smooth muscle cell (VSMC) accumulation is implicated in plaque development. In contrast, VSMC apoptosis is implicated in plaque rupture, coagulation, vessel remodeling, medial atrophy, aneurysm formation, and calcification. Although VSMC apoptosis accompanies multiple pathologies, there is little proof of direct causality, particularly with the low levels of VSMC apoptosis seen in vivo. Using a mouse model of inducible VSMC-specific apoptosis, we demonstrate that low-level VSMC apoptosis during either atherogenesis or within established plaques of apolipoprotein (Apo)E(-/-) mice accelerates plaque growth by two-fold, associated with features of plaque vulnerability including a thin fibrous cap and expanded necrotic core. Chronic VSMC apoptosis induced development of calcified plaques in younger animals and promoted calcification within established plaques. In addition, VSMC apoptosis induced medial expansion, associated with increased elastic lamina breaks, and abnormal matrix deposition reminiscent of cystic medial necrosis in humans. VSMC apoptosis prevented outward remodeling associated with atherosclerosis resulting in marked vessel stenosis. We conclude that VSMC apoptosis is sufficient to accelerate atherosclerosis, promote plaque calcification and medial degeneration, prevent expansive remodeling, and promote stenosis in atherosclerosis.

Alternative Pathways of IL-1 Activation, and Its Role in Health and Disease.

Cytokines activate or inhibit immune cell behavior and are thus integral to all immune responses. IL-1α and IL-1ß are powerful apical cytokines that instigate multiple downstream processes to affect both innate and adaptive immunity. Multiple studies show that IL-1ß is typically activated in macrophages after inflammasome sensing of infection or danger, leading to caspase-1 processing of IL-1ß and its release. However, many alternative mechanisms activate IL-1α and IL-1ß in atypical cell types, and IL-1 function is also important for homeostatic processes that maintain a physiological state. This review focuses on the less studied, yet arguably more interesting biology of IL-1. We detail the production by, and effects of IL-1 on specific innate and adaptive immune cells, report how IL-1 is required for barrier function at multiple sites, and discuss how perturbation of IL-1 pathways can drive disease. Thus, although IL-1 is primarily studied for driving inflammation after release from macrophages, it is clear that it has a multifaceted role that extends far beyond this, with various unconventional effects of IL-1 vital for health. However, much is still unknown, and a detailed understanding of cell-type and context-dependent actions of IL-1 is required to truly understand this enigmatic cytokine, and safely deploy therapeutics for the betterment of human health.

Temporal inhibition of autophagy reveals segmental reversal of ageing with increased cancer risk.

Autophagy is an important cellular degradation pathway with a central role in metabolism as well as basic quality control, two processes inextricably linked to ageing. A decrease in autophagy is associated with increasing age, yet it is unknown if this is causal in the ageing process, and whether autophagy restoration can counteract these ageing effects. Here we demonstrate that systemic autophagy inhibition induces the premature acquisition of age-associated phenotypes and pathologies in mammals. Remarkably, autophagy restoration provides a near complete recovery of morbidity and a significant extension of lifespan; however, at the molecular level this rescue appears incomplete. Importantly autophagy-restored mice still succumb earlier due to an increase in spontaneous tumour formation. Thus, our data suggest that chronic autophagy inhibition confers an irreversible increase in cancer risk and uncovers a biphasic role of autophagy in cancer development being both tumour suppressive and oncogenic, sequentially.

Compartmentalized megakaryocyte death generates functional platelets committed to caspase-independent death.

Caspase-directed apoptosis usually fragments cells, releasing nonfunctional, prothrombogenic, membrane-bound apoptotic bodies marked for rapid engulfment by macrophages. Blood platelets are functional anucleate cells generated by specialized fragmentation of their progenitors, megakaryocytes (MKs), but committed to a constitutive caspase-independent death. Constitutive formation of the proplatelet-bearing MK was recently reported to be caspase-dependent, apparently involving mitochondrial release of cytochrome c, a known pro-apoptogenic factor. We extend those studies and report that activation of caspases in MKs, either constitutively or after Fas ligation, yields platelets that are functionally responsive and evade immediate phagocytic clearance, and retain mitochondrial transmembrane potential until constitutive platelet death ensues. Furthermore, the exclusion from the platelet progeny of caspase-9 present in the progenitor accounts for failure of mitochondrial release of cytochrome c to activate caspase-3 during platelet death. Thus, progenitor cell death by apoptosis can result in birth of multiple functional anucleate daughter cells.

Platelet Isolation and Activation Assays.

Platelets regulate hemostasis and are the key determinants of pathogenic thrombosis following atherosclerotic plaque rupture. Platelets circulate in an inactive state, but become activated in response to damage to the endothelium, which exposes thrombogenic material such as collagen to the blood flow. Activation results in a number of responses, including secretion of soluble bioactive molecules via the release of alpha and dense granules, activation of membrane adhesion receptors, release of microparticles, and externalization of phosphatidylserine. These processes facilitate firm adhesion to sites of injury and the recruitment and activation of other platelets and leukocytes, resulting in aggregation and thrombus formation. Platelet activation drives the hemostatic response, and also contributes to pathogenic thrombus formation. Thus, quantification of platelet-associated responses is key to many pathophysiologically relevant processes. Here we describe protocols for isolating, counting, and activating platelets, and for the rapid quantification of cell surface proteins using flow cytometry.

Vascular smooth muscle cells in atherosclerosis.

Vascular smooth muscle cells (VSMCs) are a major cell type present at all stages of an atherosclerotic plaque. According to the 'response to injury' and 'vulnerable plaque' hypotheses, contractile VSMCs recruited from the media undergo phenotypic conversion to proliferative synthetic cells that generate extracellular matrix to form the fibrous cap and hence stabilize plaques. However, lineage-tracing studies have highlighted flaws in the interpretation of former studies, revealing that these studies had underestimated both the content and functions of VSMCs in plaques and have thus challenged our view on the role of VSMCs in atherosclerosis. VSMCs are more plastic than previously recognized and can adopt alternative phenotypes, including phenotypes resembling foam cells, macrophages, mesenchymal stem cells and osteochondrogenic cells, which could contribute both positively and negatively to disease progression. In this Review, we present the evidence for VSMC plasticity and summarize the roles of VSMCs and VSMC-derived cells in atherosclerotic plaque development and progression. Correct attribution and spatiotemporal resolution of clinically beneficial and detrimental processes will underpin the success of any therapeutic intervention aimed at VSMCs and their derivatives.

IL-1α cleavage by inflammatory caspases of the noncanonical inflammasome controls the senescence-associated secretory phenotype.

Interleukin-1 alpha (IL-1α) is a powerful cytokine that modulates immunity, and requires canonical cleavage by calpain for full activity. Mature IL-1α is produced after inflammasome activation and during cell senescence, but the protease cleaving IL-1α in these contexts is unknown. We show IL-1α is activated by caspase-5 or caspase-11 cleavage at a conserved site. Caspase-5 drives cleaved IL-1α release after human macrophage inflammasome activation, while IL-1α secretion from murine macrophages only requires caspase-11, with IL-1ß release needing caspase-11 and caspase-1. Importantly, senescent human cells require caspase-5 for the IL-1α-dependent senescence-associated secretory phenotype (SASP) in vitro, while senescent mouse hepatocytes need caspase-11 for the SASP-driven immune surveillance of senescent cells in vivo. Together, we identify IL-1α as a novel substrate of noncanonical inflammatory caspases and finally provide a mechanism for how IL-1α is activated during senescence. Thus, targeting caspase-5 may reduce inflammation and limit the deleterious effects of accumulated senescent cells during disease and Aging.