Correction of age-associated defects in dendritic cells enables CD4+ T cells to eradicate tumors.

Defective host defenses later in life are associated with changes in immune cell activities, suggesting that age-specific considerations are needed in immunotherapy approaches. In this study, we found that PD-1 and CTLA4-based cancer immunotherapies are unable to eradicate tumors in elderly mice. This defect in anti-tumor activity correlated with two known age-associated immune defects: diminished abundance of systemic naive CD8+ T cells and weak migratory activities of dendritic cells (DCs). We identified a vaccine adjuvant, referred to as a DC hyperactivator, which corrects DC migratory defects in the elderly. Vaccines containing tumor antigens and DC hyperactivators induced T helper type 1 (TH1) CD4+ T cells with cytolytic activity that drive anti-tumor immunity in elderly mice. When administered early in life, DC hyperactivators were the only adjuvant identified that elicited anti-tumor CD4+ T cells that persisted into old age. These results raise the possibility of correcting age-associated immune defects through DC manipulation.

Control of gasdermin D oligomerization and pyroptosis by the Ragulator-Rag-mTORC1 pathway.

The process of pyroptosis is mediated by inflammasomes and a downstream effector known as gasdermin D (GSDMD). Upon cleavage by inflammasome-associated caspases, the N-terminal domain of GSDMD forms membrane pores that promote cytolysis. Numerous proteins promote GSDMD cleavage, but none are known to be required for pore formation after GSDMD cleavage. Herein, we report a forward genetic screen that identified the Ragulator-Rag complex as being necessary for GSDMD pore formation and pyroptosis in macrophages. Mechanistic analysis revealed that Ragulator-Rag is not required for GSDMD cleavage upon inflammasome activation but rather promotes GSDMD oligomerization in the plasma membrane. Defects in GSDMD oligomerization and pore formation can be rescued by mitochondrial poisons that stimulate reactive oxygen species (ROS) production, and ROS modulation impacts the ability of inflammasome pathways to promote pore formation downstream of GSDMD cleavage. These findings reveal an unexpected link between key regulators of immunity (inflammasome-GSDMD) and metabolism (Ragulator-Rag).

Gasdermin D permeabilization of mitochondrial inner and outer membranes accelerates and enhances pyroptosis.

Gasdermin D (GSDMD)-activated inflammatory cell death (pyroptosis) causes mitochondrial damage, but its underlying mechanism and functional consequences are largely unknown. Here, we show that the N-terminal pore-forming GSDMD fragment (GSDMD-NT) rapidly damaged both inner and outer mitochondrial membranes (OMMs) leading to reduced mitochondrial numbers, mitophagy, ROS, loss of transmembrane potential, attenuated oxidative phosphorylation (OXPHOS), and release of mitochondrial proteins and DNA from the matrix and intermembrane space. Mitochondrial damage occurred as soon as GSDMD was cleaved prior to plasma membrane damage. Mitochondrial damage was independent of the B-cell lymphoma 2 family and depended on GSDMD-NT binding to cardiolipin. Canonical and noncanonical inflammasome activation of mitochondrial damage, pyroptosis, and inflammatory cytokine release were suppressed by genetic ablation of cardiolipin synthase (Crls1) or the scramblase (Plscr3) that transfers cardiolipin to the OMM. Phospholipid scramblase-3 (PLSCR3) deficiency in a tumor compromised pyroptosis-triggered anti-tumor immunity. Thus, mitochondrial damage plays a critical role in pyroptosis.

Inflammasomes: Threat-Assessment Organelles of the Innate Immune System.

Inflammasomes are supramolecular organizing centers that operate to drive interleukin-1 (IL-1)-dependent inflammation. Depending on context, inflammatory caspases act upstream or downstream of inflammasome assembly, serving as the principal enzymes that control activities of these organelles. In this review, we discuss mechanisms of inflammasome assembly and signaling. We posit that upstream regulatory proteins, classically known as pattern-recognition receptors, operate to assess infectious and non-infectious threats to the host. Threat assessment is achieved through two general strategies: (1) direct binding of receptors to microbial or host-derived ligands or (2) indirect detection of changes in cellular homeostasis. Upon activation, these upstream regulatory factors seed the assembly of inflammasomes, leading to IL-1 family cytokine release from living (hyperactive) or dead (pyroptotic) cells. The molecular and physiological consequences of these distinct cell fate decisions are discussed.

Defying Death: The (W)hole Truth about the Fate of GSDMD Pores.

Pyroptosis is an inflammatory cell death response initiated by supramolecular organizing centers known as inflammasomes. In a recent issue of Science, Rühl et al. (2018) challenge the paradigm that inflammasome signaling necessitates pyroptosis by demonstrating that ESCRTIII-dependent membrane repair can delay or prevent gasdermin D-mediated cell death.

The Pore-Forming Protein Gasdermin D Regulates Interleukin-1 Secretion from Living Macrophages.

The interleukin-1 (IL-1) family cytokines are cytosolic proteins that exhibit inflammatory activity upon release into the extracellular space. These factors are released following various cell death processes, with pyroptosis being a common mechanism. Recently, it was recognized that phagocytes can achieve a state of hyperactivation, which is defined by their ability to secrete IL-1 while retaining viability, yet it is unclear how IL-1 can be secreted from living cells. Herein, we report that the pyroptosis regulator gasdermin D (GSDMD) was necessary for IL-1ß secretion from living macrophages that have been exposed to inflammasome activators, such as bacteria and their products or host-derived oxidized lipids. Cell- and liposome-based assays demonstrated that GSDMD pores were required for IL-1ß transport across an intact lipid bilayer. These findings identify a non-pyroptotic function for GSDMD, and raise the possibility that GSDMD pores represent conduits for the secretion of cytosolic cytokines under conditions of cell hyperactivation.

Gasdermin D pore-forming activity is redox-sensitive.

Reactive oxygen species (ROS) regulate the activities of inflammasomes, which are innate immune signaling organelles that induce pyroptosis. The mechanisms by which ROS control inflammasome activities are unclear and may be multifaceted. Herein, we report that the protein gasdermin D (GSDMD), which forms membrane pores upon cleavage by inflammasome-associated caspases, is a direct target of ROS. Exogenous and endogenous sources of ROS, and ROS-inducing stimuli that prime cells for pyroptosis induction, promote oligomerization of cleaved GSDMD, leading to membrane rupture and cell death. We find that ROS enhance GSDMD activities through oxidative modification of cysteine 192 (C192). Within macrophages, GSDMD mutants lacking C192 show impaired ability to form membrane pores and induce pyroptosis. Reciprocal mutagenesis studies reveal that C192 is the only cysteine within GSDMD that mediates ROS responsiveness. Cellular redox state is therefore a key determinant of GSDMD activities.

Biting the hand that feeds: Metabolic determinants of cell fate during infection.

Metabolic shifts can occur in cells of the innate immune system in response to microbial infection. Whether these metabolic shifts benefit host defense and propagation of an immune response appears to be context dependent. In an arms race, host-adapted microbes and mammalian cells vie for control of biosynthetic machinery, organelles, and metabolites. Herein, we discuss the intersection of host metabolism and cell-intrinsic immunity with implications for cell fate during infection. Sensation of microbial ligands in isolation results in host metabolic shifts that imbues normal innate immune function, such as cytokine secretion. However, living microbes have an arsenal of effectors and strategies to subvert cell-intrinsic immune responses by manipulating host metabolism. Consequently, host metabolism is monitored as an indicator of invasion or manipulation by a pathogen, primarily through the actions of guard proteins and inflammasome pathways. In this review, we frame initiation of cell-intrinsic immunity in the context of host metabolism to include a physiologic "Goldilocks zone" of allowable shifts with guard circuits monitoring wide perturbations away from this zone for the initiation of innate immune responses. Through comparison of studies with purified microbial ligands, dead microbes, and live pathogens we may begin to understand how shifts in metabolism determine the outcome of host-pathogen interactions.

Putting the p(hosphor) in pyroptosis.

A recent study in Science found Mycobacterium tuberculosis inhibits pyroptosis of the host cell by secreting a phosphatase (PtpB). PtpB targets the plasma membrane to dephosphorylate PI4P and PI(4,5)P2, inhibiting recruitment of the pore-forming gasdermin D N-terminal fragment. Pyroptosis inhibition contributes to virulence, as ptpB-deficient Mtb is attenuated in mice.

Diverse Control Mechanisms of the Interleukin-1 Cytokine Family.

The majority of interleukin-1 (IL-1) family cytokines lack amino terminal secretion signals or transmembrane domains for secretion along the conventional biosynthetic pathway. Yet, these factors must be translocated from the cytoplasm across the plasma membrane into the extracellular space in order to regulate inflammation. Recent work has identified an array of mechanisms by which IL-1 family cytokines can be released into the extracellular space, with supramolecular organizing centers known as inflammasomes serving as dominant drivers of this process. In this review, we discuss current knowledge of the mechanisms of IL-1 family cytokine synthesis, processing, and release from cells. Using this knowledge, we propose a model whereby host metabolic state dictates the route of IL-1ß secretion, with implications for microbial infection and sterile inflammation.

Glycine inhibits NINJ1 membrane clustering to suppress plasma membrane rupture in cell death.

First recognized more than 30 years ago, glycine protects cells against rupture from diverse types of injury. This robust and widely observed effect has been speculated to target a late downstream process common to multiple modes of tissue injury. The molecular target of glycine that mediates cytoprotection, however, remains elusive. Here, we show that glycine works at the level of NINJ1, a newly identified executioner of plasma membrane rupture in pyroptosis, necrosis, and post-apoptosis lysis. NINJ1 is thought to cluster within the plasma membrane to cause cell rupture. We demonstrate that the execution of pyroptotic cell rupture is similar for human and mouse NINJ1 and that NINJ1 knockout functionally and morphologically phenocopies glycine cytoprotection in macrophages undergoing lytic cell death. Next, we show that glycine prevents NINJ1 clustering by either direct or indirect mechanisms. In pyroptosis, glycine preserves cellular integrity but does not affect upstream inflammasome activities or accompanying energetic cell death. By positioning NINJ1 clustering as a glycine target, our data resolve a long-standing mechanism for glycine-mediated cytoprotection. This new understanding will inform the development of cell preservation strategies to counter pathologic lytic cell death.

HDAC6 mediates an aggresome-like mechanism for NLRP3 and pyrin inflammasome activation.

Inflammasomes are supramolecular complexes that play key roles in immune surveillance. This is accomplished by the activation of inflammatory caspases, which leads to the proteolytic maturation of interleukin 1ß (IL-1ß) and pyroptosis. Here, we show that nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3)- and pyrin-mediated inflammasome assembly, caspase activation, and IL-1ß conversion occur at the microtubule-organizing center (MTOC). Furthermore, the dynein adapter histone deacetylase 6 (HDAC6) is indispensable for the microtubule transport and assembly of these inflammasomes both in vitro and in mice. Because HDAC6 can transport ubiquitinated pathological aggregates to the MTOC for aggresome formation and autophagosomal degradation, its role in NLRP3 and pyrin inflammasome activation also provides an inherent mechanism for the down-regulation of these inflammasomes by autophagy. This work suggests an unexpected parallel between the formation of physiological and pathological aggregates.

How Inflammasomes Inform Adaptive Immunity.

An immune response consists of a finely orchestrated interplay between initial recognition of potential microbial threats by the innate immune system and subsequent licensed adaptive immune neutralization. The initial recognition integrates environmental cues derived from pathogen-associated molecular patterns and cell-intrinsic damage-associated molecular patterns to contextualize the insult and inform a tailored adaptive response via T and B lymphocytes. While there are much data to support the role of transcriptional responses downstream of pattern recognition receptors in informing the adaptive immune response, markedly less attention has been paid to the role of post-translational responses to pathogen-associated molecular pattern and damage-associated molecular pattern recognition by the innate immune system, and how this may influence adaptive immunity. A well-characterized post-translational consequence of pattern recognition receptor signaling is the assembly of a multimeric signaling platform, termed the inflammasome, by members of the nucleotide-binding oligomerization domain (Nod), leucine-rich repeat-containing receptors (NLRs), and pyrin and HIN domain (PYHIN) families. Inflammasomes assemble in response to cytosolic perturbations, such as mitochondrial dysfunction and aberrant ion fluxes in the case of the canonical NLRP3 inflammasome or the presence of bacterial lipopolysaccharides in the case of the non-canonical inflammasome. Assembly of the inflammasome allows for the cleavage and activation of inflammatory caspases. These activated inflammatory caspases in turn cleave pro-form inflammatory cytokines into their mature bioactive species and lead to unconventional protein secretion and lytic cell death. In this review, we discuss evidence for inflammasome-mediated instruction and contextualization of infectious and sterile agents to the adaptive immune system.

Coxiella burnetii Infection in a Community Operating a Large-Scale Cow and Goat Dairy, Missouri, 2013.

Coxiella burnetii is a zoonotic pathogen that causes Q fever in humans and is transmitted primarily from infected goats, sheep, or cows. Q fever typically presents as an acute febrile illness; however, individuals with certain predisposing conditions, including cardiac valvulopathy, are at risk for chronic Q fever, a serious manifestation that may present as endocarditis. In response to a cluster of Q fever cases detected by public health surveillance, we evaluated C. burnetii infection in a community that operates a large-scale cow and goat dairy. A case was defined as an individual linked to the community with a C. burnetii phase II IgG titer ≥ 128. Of 135 participants, 47 (35%) cases were identified. Contact with or close proximity to cows, goats, and their excreta was associated with being a case (relative risk 2.7, 95% confidence interval 1.3-5.3). Cases were also identified among individuals without cow or goat contact and could be related to windborne spread or tracking of C. burnetii on fomites within the community. A history of injection drug use was reported by 26/130 (20%) participants; follow-up for the presence of valvulopathy and monitoring for development of chronic Q fever may be especially important among this population.