Dehydroepiandrosterone exacerbates nigericin-induced abnormal autophagy and pyroptosis via GPER activation in LPS-primed macrophages

As a widely acknowledged FDA-approved dietary supplement or over-the-counter medicines, dehydroepiandrosterone (DHEA) exerts anti-inflammatory and immunomodulatory function. Pyroptosis is an important form of programmed cell death (PCD), and which acts a key role in the body’s anti-infection and inflammatory responses. But the effects and mechanisms of DHEA on pyroptosis remain unclear. Here, we found that DHEA inhibited the NLRP3 inflammasome components expression by blocking inflammatory signals in lipopolysaccharide (LPS)-primed macrophages, and prevented the bacterial toxin nigericin (Nig)-induced NLRP3 inflammasome assembly. However, DHEA exacerbated NLRP3-independent cell death in Nig-treated inflammatory macrophages. During this process, DHEA induced the abnormal autophagy, which reflected as the blocking of autophagic flux and the accumulation of autophagy receptor p62 (SQSTM1) protein. In addition, DHEA caused a burst of reactive oxygen species (ROS) and activated extracellular signal-regulated kinase (ERK) phosphorylation in LPS plus Nig-stimulated macrophages but not in LPS-treated macrophages. Mechanistically, the present study certified that the activation of G protein-coupled estrogen receptor (GPER) signal mediated the cell death induced by DHEA in Nig-stimulated inflammatory macrophages, as GPER specific inhibitor G15 alleviated the abnormal autophagy and ultimately prevented the gasdermin D (GSDMD)-mediated pyroptosis induced by DHEA. Collectively, DHEA can exacerbate Nig-induced abnormal autophagy and pyroptosis via activation of GPER in LPS-primed macrophages, which prompts us the potential application value of DHEA in anti-infection or anti-tumor immunity.Subject terms: Cell death and immune response, Immune cell death     

ASC, Ipaf and Cryopyrin/Nalp3: bona fide intracellular adapters of the caspase-1 inflammasome

The adapter molecules ASC, Ipaf and Cryopyrin/Nalp3 have each been proposed to regulate caspase-1 within a multi-protein complex called the "inflammasome". Activation of caspase-1 leads to the cleavage and activation of pro-inflammatory cytokines such as interleukin (IL)-1beta and IL-18. The analysis of mice deficient in ASC, Ipaf and Cryopyrin/Nalp3 has revealed that the inflammasome is a dynamic entity that is assembled from different adapters in a stimulus-dependent manner.     

AIM2/ASC triggers caspase-8-dependent apoptosis in Francisella-infected caspase-1-deficient macrophages

The inflammasome is a signalling platform leading to caspase-1 activation. Caspase-1 causes pyroptosis, a necrotic-like cell death. AIM2 is an inflammasome sensor for cytosolic DNA. The adaptor molecule ASC mediates AIM2-dependent caspase-1 activation. To date, no function besides caspase-1 activation has been ascribed to the AIM2/ASC complex. Here, by comparing the effect of gene inactivation at different levels of the inflammasome pathway, we uncovered a novel cell death pathway activated in an AIM2/ASC-dependent manner. Francisella tularensis, the agent of tularaemia, triggers AIM2/ASC-dependent caspase-3-mediated apoptosis in caspase-1-deficient macrophages. We further show that AIM2 engagement leads to ASC-dependent, caspase-1-independent activation of caspase-8 and caspase-9 and that caspase-1-independent death is reverted upon caspase-8 inhibition. Caspase-8 interacts with ASC and active caspase-8 specifically colocalizes with the AIM2/ASC speck thus identifying the AIM2/ASC complex as a novel caspase-8 activation platform. Furthermore, we demonstrate that caspase-1-independent apoptosis requires the activation of caspase-9 and of the intrinsic pathway in a typical type II cell manner. Finally, we identify the AIM2/ASC-dependent caspase-1-independent pathway as an innate immune mechanism able to restrict bacterial replication in vitro and control IFN-γ levels in vivo in Casp1(KO) mice. This work underscores the crosstalk between inflammasome components and the apoptotic machinery and highlights the versatility of the pathway, which can switch from pyroptosis to apoptosis.     

Cryopyrin and pyrin activate caspase-1, but not NF-kappaB, via ASC oligomerization

Mutations in cryopyrin and pyrin proteins are responsible for several autoinflammatory disorders in humans, suggesting that these proteins play important roles in regulating inflammation. Using a HEK293 cell-based reconstitution system that stably expresses ASC and procaspase-1 we demonstrated that neither cryopyrin nor pyrin or their corresponding disease-associated mutants could significantly activate NF-kappaB in this system. However, both cryopyrin and two disease-associated cryopyrin mutants induced ASC oligomerization and ASC-dependent caspase-1 activation, with the disease-associated mutants being more potent than the wild-type (WT) cryopyrin, because of increased self-oligomerization. Contrary to the proposed anti-inflammatory activity of WT pyrin, our results demonstrated that pyrin, like cryopyrin, can also assemble an inflammasome complex with ASC and procaspase-1 leading to ASC oligomerization, caspase-1 activation and interleukin-1beta processing. Thus, we propose that pyrin could function as a proinflammatory molecule.     

The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation

Pyroptosis is a caspase-1-dependent inflammatory form of cell death. The adapter protein ASC binds directly to caspase-1 and is critical for caspase-1 activation in response to a broad range of stimuli. To elucidate the mechanism of activation of caspase-1 by ASC and its exact role in macrophage pyroptosis, we performed time-lapse confocal bioimaging analysis on human THP-1 macrophages stably expressing an ASC-GFP fusion protein. We show that stimulation of these cells with several proinflammatory stimuli trigger the formation of a large supramolecular assembly of ASC, termed here pyroptosome. Only one distinct pyroptosome in each stimulated cell is formed, which rapidly recruits and activates caspase-1 resulting in pyroptosis and the release of the intracellular proinflammatory cytokines. The pyroptosome is largely composed of oligomerized ASC dimers. Dimerization of ASC is driven by subphysiological concentrations of potassium as in vitro incubation of purified recombinant ASC in the presence of subphysiological concentrations of potassium induces the assembly of a functional pyroptosome. Furthermore, stimulation of potassium efflux in THP-1 cells with potassium-depleting agents induces formation of the pyroptosome, while increasing potassium concentrations in the culture medium or pharmacological inhibition of this efflux inhibits its assembly. Our results establish that macrophage pyroptosis is mediated by a unique pyroptosome, distinct from the inflammasome.     

Identification of Ipaf, a human caspase-1-activating protein related to Apaf-1

Procaspase-9 contains an NH2-terminal caspase-associated recruitment domain (CARD), which is essential for direct association with Apaf-1 and activation. Procaspase-1 also contains an NH2-terminal CARD domain, suggesting that its mechanism of activation, like that of procaspase-9, involves association with an Apaf-1-related molecule. Here we describe the identification of a human Apaf-1-related protein, named Ipaf that contains an NH2-terminal CARD domain, a central nucleotide-binding domain, and a COOH-terminal regulatory leucine-rich repeat domain (LRR). Ipaf associates directly and specifically with the CARD domain of procaspase-1 through CARD-CARD interaction. A constitutively active Ipaf lacking its COOH-terminal LRR domain can induce autocatalytic processing and activation of procaspase-1 and caspase-1-dependent apoptosis in transfected cells. Our results suggest that Ipaf is a specific and direct activator of procaspase-1 and could be involved in activation of caspase-1 in response to pro-inflammatory and apoptotic stimuli.     

Macrophage activation redirects yersinia-infected host cell death from apoptosis to caspase-1-dependent pyroptosis

Infection of macrophages by Yersinia species results in YopJ-dependent apoptosis, and naïve macrophages are highly susceptible to this form of cell death. Previous studies have demonstrated that macrophages activated with lipopolysaccharide (LPS) prior to infection are resistant to YopJ-dependent cell death; we found this simultaneously renders macrophages susceptible to killing by YopJ⁻ Yersinia pseudotuberculosis (Yptb). YopJ⁻ Yptb-induced macrophage death was dependent on caspase-1 activation, resulting in rapid permeability to small molecules, followed by membrane breakdown and DNA damage, and accompanied by cleavage and release of proinflammatory interleukin-18. Induction of caspase-1-dependent death, or pyroptosis, required the bacterial type III translocon but none of its known translocated proteins. Wild-type Yptb infection also triggered pyroptosis: YopJ-dependent activation of proapoptotic caspase-3 was significantly delayed in activated macrophages and resulted in caspase-1-dependent pyroptosis. The transition to susceptibility was not limited to LPS activation; it was also seen in macrophages activated with other Toll-like receptor (TLR) ligands and intact nonviable bacteria. Yptb infection triggered macrophage activation and activation of caspase-1 in vivo. Y. pestis infection of activated macrophages also stimulated caspase-1 activation. These results indicate that host signaling triggered by TLR and other activating ligands during the course of Yersinia infection redirects both the mechanism of host cell death and the downstream consequences of death by shifting from noninflammatory apoptosis to inflammatory pyroptosis.     

Critical roles of ASC inflammasomes in caspase-1 activation and host innate resistance to Streptococcus pneumoniae infection

Streptococcus pneumoniae is a Gram-positive, extracellular bacterium that is responsible for significant mortality and morbidity worldwide. Pneumolysin (PLY), a cytolysin produced by all clinical isolates of the pneumococcus, is one of the most important virulence factors of this pathogen. We have previously reported that PLY is an essential factor for activation of caspase-1 and consequent secretion of IL-1β and IL-18 in macrophages infected with S. pneumoniae. However, the host molecular factors involved in caspase-1 activation are still unclear. To further elucidate the mechanism of caspase-1 activation in macrophages infected with S. pneumoniae, we examined the involvement of inflammasomes in inducing this cellular response. Our study revealed that apoptosis-associated specklike protein containing a caspase recruitment domain (ASC), an adaptor protein for inflammasome receptors such as nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) and absent in melanoma 2 (AIM2), is essentially required for the induction of caspase-1 activation by S. pneumoniae. Caspase-1 activation was partially impaired in NLRP3(-/-) macrophages, whereas knockdown and knockout of AIM2 resulted in a clear decrease in caspase-1 activation in response to S. pneumoniae. These results suggest that ASC inflammasomes, including AIM2 and NLRP3, are critical for caspase-1 activation induced by S. pneumoniae. Furthermore, ASC(-/-) mice were more susceptible than wild-type mice to S. pneumoniae, with impaired secretion of IL-1β and IL-18 into the bronchoalveolar lavage after intranasal infection, suggesting that ASC inflammasomes contribute to the protection of host from infection with PLY-producing S. pneumoniae.     

New insights into the regulation of apoptosis,necroptosis, and pyroptosis by receptor interacting protein kinase 1 and caspase-8

Necroptosis and pyroptosis are inflammatory forms of regulated necrotic cell death as opposed to apoptosis that is generally considered immunologically silent. Recent studies revealed unexpected links in the pathways regulating and executing cell death in response to activation of signaling cascades inducing apoptosis, necroptosis, and pyroptosis. Emerging evidence suggests that receptor interacting protein kinase 1 and caspase-8 control the cross-talk between apoptosis, necroptosis, and pyroptosis and determine the type of cell death induced in response to activation of cell death signaling.     

Sestrin 1 ameliorates cardiac hypertrophy via autophagy activation

Cardiac hypertrophy is one of the major risk factors of cardiovascular morbidity and mortality. Autophagy is acknowledged to be an important mechanism regulating cardiac hypertrophy. Sestrin 1, a downstream target gene of p53, has been proven to regulate autophagy. However, the role of Sestrin 1 in cardiac hypertrophy remains unknown. Our study showed that Sestrin 1 mRNA and protein expression declined in pressure overload cardiac hypertrophy and phenylephrine (PE)‐induced cardiac hypertrophy. Knockdown of Sestrin 1 by RNAi deteriorated PE‐induced cardiac hypertrophy, whereas the overexpression of Sestrin 1 by adenovirus transfection blunted hypertrophy. We discovered that knockdown of Sestrin 1 resulted in impaired autophagy while overexpression of Sestrin 1 resulted in increased autophagy without affecting lysosomal function. In addition, the antihypertrophic effect of Sestrin 1 overexpression was eliminated by autophagy blockade. Importantly, Sestrin 1 targets at the AMPK/mTORC1/autophagy pathway to inhibit cardiac hypertrophy by interaction with AMPK which is responsible for autophagy regulation. Taken together, our data indicate that Sestrin 1 regulates AMPK/mTORC1/autophagy axis to attenuate cardiac hypertrophy.     

Anti-GBM glomerulonephritis involves IL-1 but is independent of NLRP3/ASC inflammasome-mediated activation of caspase-1

IL-1β and IL-18 are proinflammatory cytokines that contribute to renal immune complex disease, but whether IL-1β and IL-18 are mediators of intrinsic glomerular inflammation is unknown. In contrast to other cytokines the secretion of IL-1β and IL-18 requires a second stimulus that activates the inflammasome-ASC-caspase-1 pathway to cleave pro-IL-1β and -IL-18 into their mature and secretable forms. As the NLRP3 inflammasome and caspase-1 were shown to contribute to postischemic and postobstructive tubulointerstitial inflammation, we hypothesized a similar role for NLRP3, ASC, and caspase-1 in glomerular immunopathology. This concept was supported by the finding that lack of IL-1R1 reduced antiserum-induced focal segmental necrosis, crescent formation, and tubular atrophy when compared to wildtype mice. Lack of IL-18 reduced tubular atrophy only. However, NLRP3-, ASC- or caspase-1-deficiency had no significant effect on renal histopathology or proteinuria of serum nephritis. In vitro studies with mouse glomeruli or mesangial cells, glomerular endothelial cells, and podocytes did not reveal any pro-IL-1β induction upon LPS stimulation and no caspase-1 activation after an additional exposure to the NLRP3 agonist ATP. Only renal dendritic cells, which reside mainly in the tubulointerstitium, expressed pro-IL-1β and were able to activate the NLRP3-caspase-1 axis and secrete mature IL-1β. Together, the NLRP3-ASC-caspase-1 axis does not contribute to intrinsic glomerular inflammation via glomerular parenchymal cells as these cannot produce IL-1β during sterile inflammation.     

Differential regulation of caspase-1 activation via NLRP3/NLRC4 inflammasomes mediated by aerolysin and type III secretion system during Aeromonas veronii infection

Aeromonas spp. are Gram-negative bacteria that cause serious infectious disease in humans. Such bacteria have been shown to induce apoptosis in infected macrophages, yet the host responses triggered by macrophage death are largely unknown. In this study, we demonstrate that the infection of mouse bone marrow-derived macrophages with Aeromonas veronii biotype sobria triggers activation of caspase-1 with the ensuing release of IL-1β and pyroptosis. Caspase-1 activation in response to A. veronii infection requires the adaptor apoptosis-associated speck-like protein containing a caspase recruitment domain and both the NLRP3 and NLRC4 inflammasomes. Furthermore, caspase-1 activation requires aerolysin and a functional type III secretion system in A. veronii. Aerolysin-inducing caspase-1 activation is mediated through the NLRP3 inflammasome, with aerolysin-mediated cell death being largely dependent on the NLRP3 inflammasome. In contrast, the type III secretion system activates both the NLRP3 and NLRC4 inflammasomes. Inflammasome-mediated caspase-1 activation is also involved in host defenses against systemic A. veronii infection in mice. Our results indicated that multiple factors from both the bacteria and the host play a role in eliciting caspase-1 activation during A. veronii infection.     

Inflammasome regulation by adaptor isoforms,ASC and ASCb,via differential self-assembly

ASC is an essential adaptor of the inflammasome, a micrometer-size multiprotein complex that processes proinflammatory cytokines. Inflammasome formation depends on ASC self-association into large assemblies via homotypic interactions of its two death domains, PYD and CARD. ASCb, an alternative splicing isoform, activates the inflammasome to a lesser extent compared with ASC. Thus, it has been postulated that adaptor isoforms differentially regulate inflammasome function. At the amino acid level, ASC and ASCb differ only in the length of the linker connecting the two death domains. To understand inflammasome regulation at the molecular level, we investigated the self-association properties of ASC and ASCb using real-time NMR, dynamic light scattering (DLS), size-exclusion chromatography, and transmission electron microscopy (TEM). The NMR data indicate that ASC self-association is faster than that of ASCb; a kinetic model for this oligomerization results in differing values for both the reaction order and the rate constants. Furthermore, DLS analysis indicates that ASC self-associates into more compact macrostructures compared with ASCb. Finally, TEM data show that ASCb has a reduced tendency to form densely packed filaments relative to ASC. Overall, these differences can only be explained by an effect of the linker length, as the NMR results show structural equivalence of the PYD and CARD in both proteins. The effect of linker length was corroborated by molecular docking with the procaspase-1 CARD domain. Altogether, our results indicate that ASC’s faster and less polydisperse polymerization is more efficient, plausibly explaining inflammasome activation differences by ASC isoforms at the molecular level.     

Regulating caspase-1 during infection: roles of NLRs, AIM2, and ASC

Pathogens are detected by a variety of innate immune sensors in host cells leading to rapid induction of cell autonomous responses. Proinflammatory cytokine secretion and a specialized form of inflammatory cell death called pyroptosis are induced during infection through activation of caspase-1. Pathogen-induced caspase-1 activation is regulated in large part by a vast array of cystosolic sensor proteins, including NLRs and AIM2, and an adaptor protein called ASC. Together, these proteins cooperate in forming caspase-1 activation platforms and, more importantly, direct caspase-1 toward cytokine secretion or cell death.     

Intracytosolic Listeria monocytogenes induces cell death through caspase-1 activation in murine macrophages

Listeria monocytogenes induces apoptosis in vitro and in vivo in a variety of cell types. However, the mechanism of cell death in L. monocytogenes -infected macrophages was initially reported to be distinct from apoptosis. Here, we studied the mechanism of L. monocytogenes -induced cell death using sensitive fluorescent techniques. We found that caspase-1 activation preceded cell death of macrophages infected with L. monocytogenes , using fluorogenic substrates. Caspase-1 activation was diminished after infection with wild-type L. monocytogenes when cells were treated with NH₄Cl, or if they were infected with a listeriolysin mutant that cannot escape from the phagolysosome. Mitochondrial membrane integrity was preserved during the infection. A particular mechanism of cell death, recently termed 'pyroptosis', is associated with infection by intracellular microorganisms, and has an inherent pro-inflammatory character, due to involvement of caspase-1 activation with consequent IL-1β and IL-18 production. Cell death through caspase-1 activation would constitute a defence mechanism of macrophages which induces cell death to eliminate the bacteria's intracytosolic niche and recruits early host's defences through the secretion of inflammatory cytokines.     

NRF2 activation by 2-methoxycinnamaldehyde attenuates inflammatory responses in macrophages via enhancing autophagy flux

A well-controlled inflammatory response is crucial for the recovery from injury and maintenance of tissue homeostasis. The anti-inflammatory response of 2-methoxycinnamaldehyde (2-MCA), a natural compound derived from cinnamon, has been studied; however, the underlying mechanism on macrophage has not been fully elucidated. In this study, LPS-stimulated production of TNF-α and NO was reduced by 2-MCA in macrophages. 2-MCA significantly activated the NRF2 pathway, and expression levels of autophagy-associated proteins in macrophages, including LC3 and P62, were enhanced via NRF2 activation regardless of LPS treatment, suggesting the occurrence of 2-MCA-mediated autophagy. Moreover, evaluation of autophagy flux using luciferase-conjugated LC3 revealed that incremental LC3 and P62 levels are coupled to enhanced autophagy flux. Finally, reduced expression levels of TNF-α and NOS2 by 2-MCA were reversed by autophagy inhibitors, such as bafilomycin A1 and NH4Cl, in LPS-stimulated macrophages. In conclusion, 2-MCA enhances autophagy flux in macrophages via NRF2 activation and consequently reduces LPS-induced inflammation.     

Role of AMPK in atherosclerosis via autophagy regulation

Atherosclerosis is characterized by the accumulation of lipids and deposition of fibrous elements in the vascular wall, which is the primary cause of cardiovascular diseases. Adenosine monophosphate-activated protein kinase (AMPK) is a metabolic sensor of energy metabolism that regulates multiple physiological processes, including lipid and glucose metabolism and the normalization of energy imbalances. Overwhelming evidence indicates that AMPK activation markedly attenuates atherosclerosis development. Autophagy inhibits cell apoptosis and inflammation and promotes cholesterol efflux and efferocytosis. Physiological autophagy is essential for maintaining normal cardiovascular function. Increasing evidence demonstrates that autophagy occurs in developing atherosclerotic plaques. Emerging evidence indicates that AMPK regulates autophagy via a downstream signaling pathway. The complex relationship between AMPK and autophagy has attracted the attention of many researchers because of this close relationship to atherosclerosis development. This review demonstrates the role of AMPK and autophagy in atherosclerosis. An improved understanding of this interrelationship will create novel preventive and therapeutic strategies for atherosclerosis.