Knocking 'em Dead: Pore-Forming Proteins in Immune Defense.

Immune cells use a variety of membrane-disrupting proteins [complement, perforin, perforin-2, granulysin, gasdermins, mixed lineage kinase domain-like pseudokinase (MLKL)] to induce different kinds of death of microbes and host cells, some of which cause inflammation. After activation by proteolytic cleavage or phosphorylation, these proteins oligomerize, bind to membrane lipids, and disrupt membrane integrity. These membrane disruptors play a critical role in both innate and adaptive immunity. Here we review our current knowledge of the functions, specificity, activation, and regulation of membrane-disrupting immune proteins and what is known about the mechanisms behind membrane damage, the structure of the pores they form, how the cells expressing these lethal proteins are protected, and how cells targeted for destruction can sometimes escape death by repairing membrane damage.

The Formation and Function of Granulomas.

Granulomas are organized aggregates of macrophages, often with characteristic morphological changes, and other immune cells. These evolutionarily ancient structures form in response to persistent particulate stimuli-infectious or noninfectious-that individual macrophages cannot eradicate. Granulomas evolved as protective responses to destroy or sequester particles but are frequently pathological in the context of foreign bodies, infections, and inflammatory diseases. We summarize recent findings that suggest that the granulomatous response unfolds in a stepwise program characterized by a series of macrophage activations and transformations that in turn recruit additional cells and produce structural changes. We explore why different granulomas vary and the reasons that granulomas are protective and pathogenic. Understanding the mechanisms and role of granuloma formation may uncover new therapies for the multitude of granulomatous diseases that constitute serious medical problems while enhancing the protective function of granulomas in infections.

mTOR-regulated mitochondrial metabolism limits mycobacterium-induced cytotoxicity.

Necrosis of macrophages in the granuloma, the hallmark immunological structure of tuberculosis, is a major pathogenic event that increases host susceptibility. Through a zebrafish forward genetic screen, we identified the mTOR kinase, a master regulator of metabolism, as an early host resistance factor in tuberculosis. We found that mTOR complex 1 protects macrophages from mycobacterium-induced death by enabling infection-induced increases in mitochondrial energy metabolism fueled by glycolysis. These metabolic adaptations are required to prevent mitochondrial damage and death caused by the secreted mycobacterial virulence determinant ESAT-6. Thus, the host can effectively counter this early critical mycobacterial virulence mechanism simply by regulating energy metabolism, thereby allowing pathogen-specific immune mechanisms time to develop. Our findings may explain why Mycobacterium tuberculosis, albeit humanity's most lethal pathogen, is successful in only a minority of infected individuals.

TNF Induces Pathogenic Programmed Macrophage Necrosis in Tuberculosis through a Mitochondrial-Lysosomal-Endoplasmic Reticulum Circuit.

Necrosis of infected macrophages constitutes a critical pathogenetic event in tuberculosis by releasing mycobacteria into the growth-permissive extracellular environment. In zebrafish infected with Mycobacterium marinum or Mycobacterium tuberculosis, excess tumor necrosis factor triggers programmed necrosis of infected macrophages through the production of mitochondrial reactive oxygen species (ROS) and the participation of cyclophilin D, a component of the mitochondrial permeability transition pore. Here, we show that this necrosis pathway is not mitochondrion-intrinsic but results from an inter-organellar circuit initiating and culminating in the mitochondrion. Mitochondrial ROS induce production of lysosomal ceramide that ultimately activates the cytosolic protein BAX. BAX promotes calcium flow from the endoplasmic reticulum into the mitochondrion through ryanodine receptors, and the resultant mitochondrial calcium overload triggers cyclophilin-D-mediated necrosis. We identify ryanodine receptors and plasma membrane L-type calcium channels as druggable targets to intercept mitochondrial calcium overload and necrosis of mycobacterium-infected zebrafish and human macrophages.

Cell-autonomous regulation of complement C3 by factor H limits macrophage efferocytosis and exacerbates atherosclerosis.

Complement factor H (CFH) negatively regulates consumption of complement component 3 (C3), thereby restricting complement activation. Genetic variants in CFH predispose to chronic inflammatory disease. Here, we examined the impact of CFH on atherosclerosis development. In a mouse model of atherosclerosis, CFH deficiency limited plaque necrosis in a C3-dependent manner. Deletion of CFH in monocyte-derived inflammatory macrophages propagated uncontrolled cell-autonomous C3 consumption without downstream C5 activation and heightened efferocytotic capacity. Among leukocytes, Cfh expression was restricted to monocytes and macrophages, increased during inflammation, and coincided with the accumulation of intracellular C3. Macrophage-derived CFH was sufficient to dampen resolution of inflammation, and hematopoietic deletion of CFH in atherosclerosis-prone mice promoted lesional efferocytosis and reduced plaque size. Furthermore, we identified monocyte-derived inflammatory macrophages expressing C3 and CFH in human atherosclerotic plaques. Our findings reveal a regulatory axis wherein CFH controls intracellular C3 levels of macrophages in a cell-autonomous manner, evidencing the importance of on-site complement regulation in the pathogenesis of inflammatory diseases.

Unconventional Ways to Live and Die: Cell Death and Survival in Development, Homeostasis, and Disease.

Balancing cell death and survival is essential for normal development and homeostasis and for preventing diseases, especially cancer. Conventional cell death pathways include apoptosis, a form of programmed cell death controlled by a well-defined biochemical pathway, and necrosis, the lysis of acutely injured cells. New types of regulated cell death include necroptosis, pyroptosis, ferroptosis, phagoptosis, and entosis. Autophagy can promote survival or can cause death. Newly described processes of anastasis and resuscitation show that, remarkably, cells can recover from the brink of apoptosis or necroptosis. Important new work shows that epithelia achieve homeostasis by extruding excess cells, which then die by anoikis due to loss of survival signals. This mechanically regulated process both maintains barrier function as cells die and matches rates of proliferation and death. In this review, we describe these unconventional ways in which cells have evolved to die or survive, as well as the contributions that these processes make to homeostasis and cancer.

Directional mast cell degranulation of tumor necrosis factor into blood vessels primes neutrophil extravasation.

Tissue resident mast cells (MCs) rapidly initiate neutrophil infiltration upon inflammatory insult, yet the molecular mechanism is still unknown. Here, we demonstrated that MC-derived tumor necrosis factor (TNF) was crucial for neutrophil extravasation to sites of contact hypersensitivity-induced skin inflammation by promoting intraluminal crawling. MC-derived TNF directly primed circulating neutrophils via TNF receptor-1 (TNFR1) while being dispensable for endothelial cell activation. The MC-derived TNF was infused into the bloodstream by directional degranulation of perivascular MCs that were part of the vascular unit with access to the vessel lumen. Consistently, intravenous administration of MC granules boosted neutrophil extravasation. Pronounced and rapid intravascular MC degranulation was also observed upon IgE crosslinking or LPs challenge indicating a universal MC potential. Consequently, the directional MC degranulation of pro-inflammatory mediators into the bloodstream may represent an important target for therapeutic approaches aimed at dampening cytokine storm syndromes or shock symptoms, or intentionally pushing immune defense.

Six distinct NFκB signaling codons convey discrete information to distinguish stimuli and enable appropriate macrophage responses.

Macrophages initiate inflammatory responses via the transcription factor NFκB. The temporal pattern of NFκB activity determines which genes are expressed and thus, the type of response that ensues. Here, we examined how information about the stimulus is encoded in the dynamics of NFκB activity. We generated an mVenus-RelA reporter mouse line to enable high-throughput live-cell analysis of primary macrophages responding to host- and pathogen-derived stimuli. An information-theoretic workflow identified six dynamical features-termed signaling codons-that convey stimulus information to the nucleus. In particular, oscillatory trajectories were a hallmark of responses to cytokine but not pathogen-derived stimuli. Single-cell imaging and RNA sequencing of macrophages from a mouse model of Sjögren's syndrome revealed inappropriate responses to stimuli, suggestive of confusion of two NFκB signaling codons. Thus, the dynamics of NFκB signaling classify immune threats through six signaling codons, and signal confusion based on defective codon deployment may underlie the etiology of some inflammatory diseases.

LC3-associated phagocytosis of neutrophils triggers tumor ferroptotic cell death in glioblastoma.

Necrosis in solid tumors is commonly associated with poor prognostic but how these lesions expand remains unclear. Studies have found that neutrophils associate with and contribute to necrosis development in glioblastoma by inducing tumor cell ferroptosis through transferring myeloperoxidase-containing granules. However, the mechanism of neutrophilic granule transfer remains elusive. We performed an unbiased small molecule screen and found that statins inhibit neutrophil-induced tumor cell death by blocking the neutrophilic granule transfer. Further, we identified a novel process wherein neutrophils are engulfed by tumor cells before releasing myeloperoxidase-containing contents into tumor cells. This neutrophil engulfment is initiated by integrin-mediated adhesion, and further mediated by LC3-associated phagocytosis (LAP), which can be blocked by inhibiting the Vps34-UVRAG-RUBCN-containing PI3K complex. Myeloperoxidase inhibition or Vps34 depletion resulted in reduced necrosis formation and prolonged mouse survival in an orthotopic glioblastoma mouse model. Thus, our study unveils a critical role for LAP-mediated neutrophil internalization in facilitating the transfer of neutrophilic granules, which in turn triggers tumor cell death and necrosis expansion. Targeting this process holds promise for improving glioblastoma prognosis.

Plasma Cells Are Obligate Effectors of Enhanced Myelopoiesis in Aging Bone Marrow.

Aging results in increased myelopoiesis, which is linked to the increased incidence of myeloid leukemias and production of myeloid-derived suppressor cells. Here, we examined the contribution of plasma cells (PCs) to age-related increases in myelopoiesis, as PCs exhibit immune regulatory function and sequester in bone marrow (BM). PC number was increased in old BM, and they exhibited high expression of genes encoding inflammatory cytokines and pathogen sensors. Antibody-mediated depletion of PCs from old mice reduced the number of myeloid-biased hematopoietic stem cells and mature myeloid cells to levels in young animals, but lymphopoiesis was not rejuvenated, indicating that redundant mechanisms inhibit that process. PCs also regulated the production of inflammatory factors from BM stromal cells, and disruption of the PC-stromal cell circuitry with inhibitors of the cytokines IL-1 and TNF-α attenuated myelopoiesis in old mice. Thus, the age-related increase in myelopoiesis is driven by an inflammatory network orchestrated by PCs.

Regulated cell death: signaling and mechanisms.

Cell turnover is a fundamental feature in metazoans. Cells can die passively, as a consequence of severe damage to their structural integrity, or actively, owing to a more confined biological disruption such as DNA damage. Passive cell death is uncontrolled and often harmful to the organism. In contrast, active cell death is tightly regulated and serves to support the organism's life. Apoptosis-the primary form of regulated cell death-is relatively well defined. Necroptosis-an alternative, distinct kind of regulated cell death discovered more recently-is less well understood. Apoptosis and necroptosis can be triggered either from within the cell or by extracellular stimuli. Certain signaling components, including several death ligands and receptors, can regulate both processes. Whereas apoptosis is triggered and executed via intracellular proteases called caspases, necroptosis is suppressed by caspase activity. Here we highlight current understanding of the key signaling mechanisms that control regulated cell death.

A variety of death modes of neutrophils and their role in the etiology of autoimmune diseases.

Neutrophils are important in the context of innate immunity and actively contribute to the progression of diverse autoimmune disorders. Distinct death mechanisms of neutrophils may exhibit specific and pivotal roles in autoimmune diseases and disease pathogenesis through the orchestration of immune homeostasis, the facilitation of autoantibody production, the induction of tissue and organ damage, and the incitement of pathological alterations. In recent years, more studies have provided in-depth examination of various neutrophil death modes, revealing nuances that challenge conventional understanding and underscoring their potential clinical utility in diagnosis and treatment. This review explores the multifaceted processes and characteristics of neutrophil death, with a focus on tailored investigations within various autoimmune diseases. It also highlights the potential interplay between neutrophil death and the landscape of autoimmune disorders. The review encapsulates the pertinent pathways implicated in various neutrophil death mechanisms across diverse autoimmune diseases while also charts possible avenues for future research.

Development of a hepatic cryoinjury model to study liver regeneration.

The liver is a remarkable organ that can regenerate in response to injury. Depending on the extent of injury, the liver can undergo compensatory hyperplasia or fibrosis. Despite decades of research, the molecular mechanisms underlying these processes are poorly understood. Here, we developed a new model to study liver regeneration based on cryoinjury. To visualise liver regeneration at cellular resolution, we adapted the CUBIC tissue-clearing approach. Hepatic cryoinjury induced a localised necrotic and apoptotic lesion characterised by inflammation and infiltration of innate immune cells. After this initial phase, we observed fibrosis, which resolved as regeneration re-established homeostasis in 30 days. Importantly, this approach enables the comparison of healthy and injured parenchyma within an individual animal, providing unique advantages to previous models. In summary, the hepatic cryoinjury model provides a fast and reproducible method for studying the cellular and molecular pathways underpinning fibrosis and liver regeneration.

Caspase-8 Collaborates with Caspase-11 to Drive Tissue Damage and Execution of Endotoxic Shock.

The execution of shock following high dose E. coli lipopolysaccharide (LPS) or bacterial sepsis in mice required pro-apoptotic caspase-8 in addition to pro-pyroptotic caspase-11 and gasdermin D. Hematopoietic cells produced MyD88- and TRIF-dependent inflammatory cytokines sufficient to initiate shock without any contribution from caspase-8 or caspase-11. Both proteases had to be present to support tumor necrosis factor- and interferon-ß-dependent tissue injury first observed in the small intestine and later in spleen and thymus. Caspase-11 enhanced the activation of caspase-8 and extrinsic cell death machinery within the lower small intestine. Neither caspase-8 nor caspase-11 was individually sufficient for shock. Both caspases collaborated to amplify inflammatory signals associated with tissue damage. Therefore, combined pyroptotic and apoptotic signaling mediated endotoxemia independently of RIPK1 kinase activity and RIPK3 function. These observations bring to light the relevance of tissue compartmentalization to disease processes in vivo where cytokines act in parallel to execute diverse cell death pathways.

Plasma membrane repair empowers the necrotic survivors as innate immune modulators.

The plasma membrane is crucial to the survival of animal cells, and damage to it can be lethal, often resulting in necrosis. However, cells possess multiple mechanisms for repairing the membrane, which allows them to maintain their integrity to some extent, and sometimes even survive. Interestingly, cells that survive a near-necrosis experience can recognize sub-lethal membrane damage and use it as a signal to secrete chemokines and cytokines, which activate the immune response. This review will present evidence of necrotic cell survival in both in vitro and in vivo systems, including in C. elegans, mouse models, and humans. We will also summarize the various membrane repair mechanisms cells use to maintain membrane integrity. Finally, we will propose a mathematical model to illustrate how near-death experiences can transform dying cells into innate immune modulators for their microenvironment. By utilizing their membrane repair activity, the biological effects of cell death can extend beyond the mere elimination of the cells.

Fundamental Mechanisms of Regulated Cell Death and Implications for Heart Disease.

Twelve regulated cell death programs have been described. We review in detail the basic biology of nine including death receptor-mediated apoptosis, death receptor-mediated necrosis (necroptosis), mitochondrial-mediated apoptosis, mitochondrial-mediated necrosis, autophagy-dependent cell death, ferroptosis, pyroptosis, parthanatos, and immunogenic cell death. This is followed by a dissection of the roles of these cell death programs in the major cardiac syndromes: myocardial infarction and heart failure. The most important conclusion relevant to heart disease is that regulated forms of cardiomyocyte death play important roles in both myocardial infarction with reperfusion (ischemia/reperfusion) and heart failure. While a role for apoptosis in ischemia/reperfusion cannot be excluded, regulated forms of necrosis, through both death receptor and mitochondrial pathways, are critical. Ferroptosis and parthanatos are also likely important in ischemia/reperfusion, although it is unclear if these entities are functioning as independent death programs or as amplification mechanisms for necrotic cell death. Pyroptosis may also contribute to ischemia/reperfusion injury, but potentially through effects in non-cardiomyocytes. Cardiomyocyte loss through apoptosis and necrosis is also an important component in the pathogenesis of heart failure and is mediated by both death receptor and mitochondrial signaling. Roles for immunogenic cell death in cardiac disease remain to be defined but merit study in this era of immune checkpoint cancer therapy. Biology-based approaches to inhibit cell death in the various cardiac syndromes are also discussed.

Tyrosine Kinase SYK Licenses MyD88 Adaptor Protein to Instigate IL-1α-Mediated Inflammatory Disease.

Mice carrying a hypomorphic point mutation in the Ptpn6 gene (Ptpn6spin mice) develop an inflammatory skin disease that resembles neutrophilic dermatosis in humans. Here, we demonstrated that interleukin-1α (IL-1α) signaling through IL-1R and MyD88 in both stromal and immune cells drive inflammation in Ptpn6spin mice. We further identified SYK as a critical kinase that phosphorylates MyD88, promoted MyD88-dependent signaling and mediates dermatosis in Ptpn6spin mice. Our studies further demonstrated that SHP1 encoded by Ptpn6 binds and suppresses SYK activation to inhibit MyD88 phosphorylation. Downstream of SHP1 and SYK-dependent counterregulation of MyD88 tyrosine phosphorylation, we have demonstrated that the scaffolding function of receptor interacting protein kinase 1 (RIPK1) and tumor growth factor-ß activated kinase 1 (TAK1)-mediating signaling were required to spur inflammatory disease. Overall, these studies identify SHP1 and SYK crosstalk as a critical regulator of MyD88 post-translational modifications and IL-1-driven inflammation.

Inhibition of the mPTP and Lipid Peroxidation Is Additively Protective Against I/R Injury.

BACKGROUND: During myocardial ischemia/reperfusion (I/R) injury, high levels of matrix Ca2+ and reactive oxygen species (ROS) induce the opening of the mitochondrial permeability transition pore (mPTP), which causes mitochondrial dysfunction and ultimately necrotic death. However, the mechanisms of how these triggers individually or cooperatively open the pore have yet to be determined. METHODS: Here, we use a combination of isolated mitochondrial assays and in vivo I/R surgery in mice. We challenged isolated liver and heart mitochondria with Ca2+, ROS, and Fe2+ to induce mitochondrial swelling. Using inhibitors of the mPTP (cyclosporine A or ADP) lipid peroxidation (ferrostatin-1, MitoQ), we determined how the triggers elicit mitochondrial damage. Additionally, we used the combination of inhibitors during I/R injury in mice to determine if dual inhibition of these pathways is additivity protective. RESULTS: In the absence of Ca2+, we determined that ROS fails to trigger mPTP opening. Instead, high levels of ROS induce mitochondrial dysfunction and rupture independently of the mPTP through lipid peroxidation. As expected, Ca2+ in the absence of ROS induces mPTP-dependent mitochondrial swelling. Subtoxic levels of ROS and Ca2+ synergize to induce mPTP opening. Furthermore, this synergistic form of Ca2+- and ROS-induced mPTP opening persists in the absence of CypD (cyclophilin D), suggesting the existence of a CypD-independent mechanism for ROS sensitization of the mPTP. These ex vivo findings suggest that mitochondrial dysfunction may be achieved by multiple means during I/R injury. We determined that dual inhibition of the mPTP and lipid peroxidation is significantly more protective against I/R injury than individually targeting either pathway alone. CONCLUSIONS: In the present study, we have investigated the relationship between Ca2+ and ROS, and how they individually or synergistically induce mitochondrial swelling. Our findings suggest that Ca2+ mediates mitochondrial damage through the opening of the mPTP, although ROS mediates its damaging effects through lipid peroxidation. However, subtoxic levels both Ca2+ and ROS can induce mPTP-mediated mitochondrial damage. Targeting both of these triggers to preserve mitochondria viability unveils a highly effective therapeutic approach for mitigating I/R injury.

MLKL Requires the Inositol Phosphate Code to Execute Necroptosis.

Necroptosis is an important form of lytic cell death triggered by injury and infection, but whether mixed lineage kinase domain-like (MLKL) is sufficient to execute this pathway is unknown. In a genetic selection for human cell mutants defective for MLKL-dependent necroptosis, we identified mutations in IPMK and ITPK1, which encode inositol phosphate (IP) kinases that regulate the IP code of soluble molecules. We show that IP kinases are essential for necroptosis triggered by death receptor activation, herpesvirus infection, or a pro-necrotic MLKL mutant. In IP kinase mutant cells, MLKL failed to oligomerize and localize to membranes despite proper receptor-interacting protein kinase-3 (RIPK3)-dependent phosphorylation. We demonstrate that necroptosis requires IP-specific kinase activity and that a highly phosphorylated product, but not a lowly phosphorylated precursor, potently displaces the MLKL auto-inhibitory brace region. These observations reveal control of MLKL-mediated necroptosis by a metabolite and identify a key molecular mechanism underlying regulated cell death.

The mitochondrial ATP synthase is a negative regulator of the mitochondrial permeability transition pore.

The mitochondrial permeability transition pore (mPTP) is a channel in the inner mitochondrial membrane whose sustained opening in response to elevated mitochondrial matrix Ca2+ concentrations triggers necrotic cell death. The molecular identity of mPTP is unknown. One proposed candidate is the mitochondrial ATP synthase, whose canonical function is to generate most ATP in multicellular organisms. Here, we present mitochondrial, cellular, and in vivo evidence that, rather than serving as mPTP, the mitochondrial ATP synthase inhibits this pore. Our studies confirm previous work showing persistence of mPTP in HAP1 cell lines lacking an assembled mitochondrial ATP synthase. Unexpectedly, however, we observe that Ca2+-induced pore opening is markedly sensitized by loss of the mitochondrial ATP synthase. Further, mPTP opening in cells lacking the mitochondrial ATP synthase is desensitized by pharmacological inhibition and genetic depletion of the mitochondrial cis-trans prolyl isomerase cyclophilin D as in wild-type cells, indicating that cyclophilin D can modulate mPTP through substrates other than subunits in the assembled mitochondrial ATP synthase. Mitoplast patch clamping studies showed that mPTP channel conductance was unaffected by loss of the mitochondrial ATP synthase but still blocked by cyclophilin D inhibition. Cardiac mitochondria from mice whose heart muscle cells we engineered deficient in the mitochondrial ATP synthase also demonstrate sensitization of Ca2+-induced mPTP opening and desensitization by cyclophilin D inhibition. Further, these mice exhibit strikingly larger myocardial infarctions when challenged with ischemia/reperfusion in vivo. We conclude that the mitochondrial ATP synthase does not function as mPTP and instead negatively regulates this pore.