Pannexin‐1‐dependent caspase‐1 activation and secretion of IL‐1β is regulated by zinc

Inflammatory processes induced by IL‐1β are critical for host defence responses, but are also implicated in disease. Zinc deficiency is a common consequence of, or contributor to, human inflammatory disease. However, the molecular mechanisms through which zinc contributes to inflammatory disease remain largely unknown. We report here that zinc metabolism regulates caspase‐1 activation and IL‐1β secretion. One of the endogenous mediators of IL‐1β secretion is adenosine triphosphate, acting via the P2X7‐receptor and caspase‐1 activation in cells primed with an inflammatory stimulus such as LPS. We show that this process is selectively abolished by a brief pre‐treatment with the zinc chelator N,N,N′,N′‐tetrakis‐(2‐pyridylmethyl) ethylene diamine (TPEN). These effects on IL‐1β secretion were independent of rapid changes in free zinc within the cell, not a direct effect on caspase‐1 activity, and upstream of caspase‐1 activation. TPEN did however inhibit the activity of pannexin‐1, a hemi‐channel critical for adenosine triphosphate and nigericin‐induced IL‐1β release. These data provide new insights into the mechanisms of caspase‐1 activation and how zinc metabolism contributes to inflammatory mechanisms.     

Caspase‐4 mediates non‐canonical activation of the NLRP3 inflammasome in human myeloid cells

Inflammasome activation culminates in activation of caspase‐1, which leads to the maturation and subsequent release of cytokines of the interleukin 1 (IL‐1) family and results in a particular form of cell death known as pyroptosis. In addition, in the murine system, a so‐called non‐canonical inflammasome involving caspase‐11 has been described that directly responds to cytosolic LPS. Here, we show that the human monocytic cell line THP1 activates the inflammasome in response to cytosolic LPS in a TLR4‐independent fashion. This response is mediated by caspase‐4 and accompanied by caspase‐1 activation, pyroptosis, and IL‐1β maturation. In addition to caspase‐4, efficient IL‐1β conversion upon intracellular LPS delivery relies on potassium efflux, NLRP3, ASC, and caspase‐1, indicating that although caspase‐4 activation alone is sufficient to induce pyroptosis, this process depends on the NLRP3 inflammasome activation to drive IL‐1β maturation. Altogether, this study provides evidence for the presence of a non‐canonical inflammasome in humans and its dependence on caspase‐4.     

NADPH oxidase derived reactive oxygen species are involved in human neutrophil IL‐1β secretion but not in inflammasome activation

Neutrophils are essential players in acute inflammatory responses. Upon stimulation, neutrophils activate NADPH oxidase, generating an array of reactive oxygen species (ROS). Interleukin‐1 beta (IL‐1β) is a major proinflammatory cytokine synthesized as a precursor that has to be proteolytically processed to become biologically active. The role of ROS in IL‐1β processing is still controversial and has not been previously studied in neutrophils. We report here that IL‐1β processing in human neutrophils is dependent on caspase‐1 and on the serine proteases elastase and/or proteinase 3. NADPH oxidase deficient neutrophils activated caspase‐1 and did not exhibit differences in NALP3 expression, indicating that ROS are neither required for inflammasome activation nor for its priming, as has been reported for macrophages. Strikingly, ROS exerted opposite effects on the processing and secretion of IL‐1β; whereas ROS negatively controlled caspase‐1 activity, as reported in mononuclear phagocytes, ROS were found to be necessary for the exportation of mature IL‐1β out of the cell, a role never previously described. The complex ROS‐mediated regulation of neutrophil IL‐1β secretion might constitute a physiological mechanism to control IL‐1β‐dependent inflammatory processes where neutrophils play a crucial role.     

Complement C5a potentiates uric acid crystal‐induced IL‐1β production

Anaphylatoxin C5a released upon complement activation is associated with both acute and chronic inflammations such as gout. The pathogenesis of gout was identified as uric acid crystal deposition in the joints that activates inflammasome, leading to IL‐1β release. However, little is known about the interaction between complement activation and monosodium urate/uric acid (MSU) crystal‐induced inflammasome activation or IL‐1β production. Here, we report that MSU crystal‐induced proinflammatory cytokines/chemokines in human whole blood is predominantly regulated by C5a through its interaction with C5a receptor. C5a induces pro‐IL‐1β and IL‐1β production in human primary monocytes, and potentiates MSU or cholesterol crystals in IL‐1β production. This potentiation is caspase‐1 dependent and requires intracellular Ca²⁺ mobilization, K⁺ efflux, and cathepsin B activity. Our results provide insight into the role of C5a as an endogenous priming signal that is required for the initiation of uric acid crystal‐induced IL‐1β production. C5a could potentially be a therapeutic target together with IL‐1β antagonists for the treatment of complement‐dependent and inflammasome‐associated diseases.     

Unconventional protein secretion by gasdermin pores

Unconventional protein secretion (UPS) allows the release of specific leaderless proteins independently of the classical endoplasmic reticulum (ER)-Golgi secretory pathway. While it remains one of the least understood mechanisms in cell biology, UPS plays an essential role in immunity as it controls the release of the IL-1 family of cytokines, which coordinate host defense and inflammatory responses. The unconventional secretion of IL-1β and IL-18, the two most prominent members of the IL-1 family, is initiated by inflammasome complexes – cytosolic signaling platforms that are assembled in response to infectious or noxious stimuli. Inflammasomes activate inflammatory caspases that proteolytically mature IL-1β/− 18, but also induce pyroptosis, a lytic form of cell death. Pyroptosis is caused by gasdermin-D (GSDMD), a member of the gasdermin protein family, which is activated by caspase cleavage and forms large β-barrel plasma membrane pores. This pore-forming activity is shared with other family members that are activated during infection or upon treatment with chemotherapy drugs. While the induction of cell death was assumed to be the main function of gasdermin pores, accumulating evidence suggests that they have also non-lytic functions, such as in the release of cytokines and alarmins, or in regulating ion fluxes. This has raised the possibility that gasdermin pores are one of the main mediators of UPS. Here, I summarize and discuss new insights into gasdermin activation and pore formation, how gasdermin pores achieve selective cargo release, and how gasdermin pore formation and ninjurin-1-driven plasma membrane rupture are executed and regulated.     

Caspase‐11 non‐canonical inflammasome: a critical sensor of intracellular lipopolysaccharide in macrophage‐mediated inflammatory responses

Inflammatory responses mediated by macrophages are part of the innate immune system, whose role is to protect against invading pathogens. Lipopolysaccharide (LPS) found in the outer membrane of Gram‐negative bacteria stimulates an inflammatory response by macrophages. During the inflammatory response, extracellular LPS is recognized by Toll‐like receptor 4, one of the pattern recognition receptors that activates inflammatory signalling pathways and leads to the production of inflammatory mediators. The innate immune response is also triggered by intracellular inflammasomes, and inflammasome activation induces pyroptosis and the secretion of pro‐inflammatory cytokines such as interleukin‐1β (IL‐1β) and IL‐18 by macrophages. Cysteine‐aspartic protease (caspase)‐11 and the human orthologues caspase‐4/caspase‐5 were recently identified as components of the ‘non‐canonical inflammasome’ that senses intracellular LPS derived from Gram‐negative bacteria during macrophage‐mediated inflammatory responses. Direct recognition of intracellular LPS facilitates the rapid oligomerization of caspase‐11/4/5, which results in pyroptosis and the secretion of IL‐1β and IL‐18. LPS is released into the cytoplasm from Gram‐negative bacterium‐containing vacuoles by small interferon‐inducible guanylate‐binding proteins encoded on chromosome 3 (GBP^chr3)‐mediated lysis of the vacuoles. In vivo studies have clearly shown that caspase‐11^−/− mice are more resistant to endotoxic septic shock by excessive LPS challenge. Given the evidence, activation of caspase‐11 non‐canonical inflammasomes by intracellular LPS is distinct from canonical inflammasome activation and provides a new paradigm in macrophage‐mediated inflammatory responses.     

Potassium efflux fires the canon: Potassium efflux as a common trigger for canonical and noncanonical NLRP3 pathways

Murine caspase‐11 and its human orthologues, caspase‐4 and caspase‐5, activate an inflammatory response following cytoplasmic recognition of cell wall constituents from Gram‐negative bacteria, such as LPS. This inflammatory response involves pyroptotic cell death and the concomitant release of IL‐1α, as well as the production of IL‐1β and IL‐18 through the noncanonical NLR family, pyrin domain containing 3 (NLRP3) pathway. This commentary discusses three papers in this issue of the European Journal of Immunology that advance our understanding of the roles of caspase‐11, ‐4, and ‐5 in the noncanonical pathway. By utilizing the new gene editing technique, clustered regularly interspaced short palindromic repeats (CRISPR), as well as sensitive cell imaging techniques, these papers establish that cytoplasmic LPS‐dependent IL‐1β production requires the NLRP3 inflammasome and that its activation is dependent on K⁺ efflux, whereas IL‐1α release and pyroptotic cell death pathways are NLRP3‐independent. These findings expand on previous research implicating K⁺ efflux as the principal trigger for NLRP3 activation and suggest that canonical and noncanonical NLRP3 pathways are not as dissimilar as first thought.     

Histones trigger sterile inflammation by activating the NLRP3 inflammasome

Sterile cell death mediated inflammation is linked to several pathological disorders and involves danger recognition of intracellular molecules released by necrotic cells that activate different groups of innate pattern recognition receptors. Toll‐like receptors directly interact with their extrinsic or intrinsic agonists and induce multiple proinflammatory mediators. In contrast, the NLRP3 inflammasome is rather thought to represent a downstream element integrating various indirect stimuli into proteolytic cleavage of interleukin (IL)–1β and IL‐18. Here, we report that histones released from necrotic cells induce IL‐1β secretion in an NLRP3–ASC‐caspase‐1‐dependent manner. Genetic deletion of NLRP3 in mice significantly attenuated histone‐induced IL‐1β production and neutrophil recruitment. Furthermore, necrotic cells induced neutrophil recruitment, which was significantly reduced by histone‐neutralizing antibodies or depleting extracellular histones via enzymatic degradation. These results identify cytosolic uptake of necrotic cell‐derived histones as a triggering mechanism of sterile inflammation, which involves NLRP3 inflammasome activation and IL‐1β secretion via oxidative stress.     

NLRP3 inflammasome activation downstream of cytoplasmic LPS recognition by both caspase‐4 and caspase‐5

Humans encode two inflammatory caspases that detect cytoplasmic LPS, caspase‐4 and caspase‐5. When activated, these trigger pyroptotic cell death and caspase‐1‐dependent IL‐1β production; however the mechanism underlying this process is not yet confirmed. We now show that a specific NLRP3 inhibitor, MCC950, prevents caspase‐4/5‐dependent IL‐1β production elicited by transfected LPS. Given that both caspase‐4 and caspase‐5 can detect cytoplasmic LPS, it is possible that these proteins exhibit some degree of redundancy. Therefore, we generated human monocytic cell lines in which caspase‐4 and caspase‐5 were genetically deleted either individually or together. We found that the deletion of caspase‐4 suppressed cell death and IL‐1β production following transfection of LPS into the cytoplasm, or in response to infection with Salmonella typhimurium. Although deletion of caspase‐5 did not confer protection against transfected LPS, cell death and IL‐1β production were reduced after infection with Salmonella. Furthermore, double deletion of caspase‐4 and caspase‐5 had a synergistic effect in the context of Salmonella infection. Our results identify the NLRP3 inflammasome as the specific platform for IL‐1β maturation, downstream of cytoplasmic LPS detection by caspase‐4/5. We also show that both caspase‐4 and caspase‐5 are functionally important for appropriate responses to intracellular Gram‐negative bacteria.     

Caspase‐11: The driving factor for noncanonical inflammasomes

Inflammasomes are large multiprotein platforms that mediate the processing of caspase‐1, which in turn promotes the maturation and release of IL‐1β and IL‐18 in response to microbial and danger signals. While the canonical pathway of inflammasome activation has been known for some time, a novel mechanism of noncanonical inflammasome activation mediated by caspase‐11 was more recently identified. This pathway engages caspase‐11 to trigger both caspase‐1‐dependent and ‐independent production of the inflammatory cytokines IL‐1β, IL‐18, and IL‐1α, as well as to promote pyroptosis, a form of genetically programmed cell death that is associated with the release of such cytokines. In this review, we gather together studies on both the mechanisms and implications of caspase‐11‐mediated noncanonical inflammasome activation, and discuss the emerging importance of this pathway in regulating host defense against intracellular bacterial pathogens.     

Porphyromonas gingivalis‐induced inflammatory responses in THP1 cells are altered by native and modified low‐density lipoproteins in a strain‐dependent manner

Strong epidemiological evidence supports an association between cardiovascular and periodontal disease and furthermore, the periodontopathogen Porphyromonas gingivalis has been identified in blood and from atheromatous plaques. Blood exposed to P. gingivalis shows an increased protein modification of low‐density lipoprotein (LDL). In this study, we investigate the inflammatory responses of THP1 cells incubated with P. gingivalis and the effects of native or modified LDL on these responses. Reactive oxygen species (ROS) and IL‐1β were observed in THP1 cells following infection with P. gingivalis ATCC33277 and W50. Caspase 1 activity was quantified in THP1 cells and correlated with IL‐1β accumulation. Oxidized LDL (oxLDL) induced IL‐1β release and CD36 expression on THP1 cells. Modified LDL co‐stimulated with ATCC33277 exhibited regulatory effects on caspase 1 activity, IL‐1β release and CD36 expression in THP1 cells, whereas W50 induced more modest responses in THP1 cells. In summary, we show that P. gingivalis is capable of inducing pro‐inflammatory responses in THP1 cells, and native and modified LDL could alter these responses in a dose‐ and strain‐dependent manner. Strain‐dependent differences in THP1 cell responses could be due to the effect of P. gingivalis proteases, presence or absence of capsule and proteolytic transformation of native and modified LDL.     

Pro‐inflammatory effects of extracellular Hsp70 on NCI‐H292 human bronchial epithelial cell line

Extracellular Hsp70 (eHsp70) exerts its biological actions via Toll‐like receptors 2 and 4, and is increased in sera of chronic obstructive pulmonary disease (COPD) patients. The aim of this study was to explore the pro‐inflammatory effects and cytotoxicity of eHsp70 alone and in combination with bacterial components lipoteichoic acid (LTA) and lipopolysaccharide (LPS) on NCI‐H292 airway epithelial cells. NCI‐H292 cells were treated with recombinant human Hsp70 protein (rhHsp70), LPS, LTA and their combinations for 4, 12, 24 and 48 hours. IL‐6, IL‐8 and TNF‐α levels were measured by an ELISA method. Cell viability was determined by the MTS method, and caspase‐3/7, caspase‐8 and caspase‐9 assays. rhHsp70 induced secretion of IL‐6 and IL‐8 in a concentration‐ and time‐dependent manner, with the highest secretion at 24 hours. rhHsp70 combined with LTA had antagonistic and with LPS synergistic effect on IL‐6 secretion, while the interactions between rhHsp70 and LPS or LTA on IL‐8 were synergistic. TNF‐α was not detected in the applied conditions. rhHsp70, LPS or LTA did not affect cell viability, and rhHsp70 even suppressed caspase‐3/7 activities. We suggest that pro‐inflammatory effects of eHsp70, together with other damaging molecules and/or COPD risk factors, might contribute to the aggravation of chronic inflammation in human bronchial epithelium.     

The CDK domain of p21 is a suppressor of IL‐1β‐mediated inflammation in activated macrophages

Significant morbidity and mortality can be attributed to inflammatory diseases; therefore, a greater understanding of the mechanisms involved in the progression of inflammation is crucial. Here, we demonstrate that p21^(WAF1/CIP1), an established suppressor of cell cycle progression, is a inhibitor of IL‐1β synthesis in macrophages. Mice deficient in p21 (p21^?/?) display increased susceptibility to endotoxic shock, which is associated with increased serum levels of IL‐1β. Administration of IL‐1 receptor antagonist reduces LPS‐induced lethality in p21^?/? mice. Analysis of isolated macrophages, which are one of the central producers of IL‐1β, reveals that deficiency for p21 led to more IL‐1β mRNA and pro‐protein synthesis following TLR ligation. The increase in IL‐1β pro‐protein is associated with elevated secretion of active IL‐1β by p21^?/? macrophages. siRNA‐mediated knockdown of p21 in human macrophages results in increased IL‐1β secretion as well. A peptide mapping strategy shows that the cyclin‐dependent‐kinase (CDK)‐binding domain of p21 is sufficient to reduce the secretion of IL‐1β by p21^?/? macrophages. These data suggest a novel role for p21 and specifically for the CDK‐binding domain of p21^(WAF1/CIP1) in inhibiting inflammation.     

Impaired NLRP3 inflammasome activity during fetal development regulates IL‐1β production in human monocytes

Interleukin‐1β (IL‐1β) production is impaired in cord blood monocytes. However, the mechanism underlying this developmental attenuation remains unclear. Here, we analyzed the extent of variability within the Toll‐like receptor (TLR)/NLRP3 inflammasome pathways in human neonates. We show that immature low CD14 expressing/CD16^pos monocytes predominate before 33 weeks of gestation, and that these cells lack production of the pro‐IL‐1β precursor protein upon LPS stimulation. In contrast, high levels of pro‐IL‐1β are produced within high CD14 expressing monocytes, although these cells are unable to secrete mature IL‐1β. The lack of secreted IL‐1β in these monocytes parallels a reduction of NLRP3 induction following TLR stimulation resulting in a lack of caspase‐1 activity before 29 weeks of gestation, whereas expression of the apoptosis‐associated speck‐like protein containing a CARD and function of the P2×7 receptor are preserved. Our analyses also reveal a strong inhibitory effect of placental infection on LPS/ATP‐induced caspase‐1 activity in cord blood monocytes. Lastly, secretion of IL‐1β in preterm neonates is restored to adult levels during the neonatal period, indicating rapid maturation of these responses after birth. Collectively, our data highlight important developmental mechanisms regulating IL‐1β responses early in gestation, in part due to a downregulation of TLR‐mediated NLRP3 expression. Such mechanisms may serve to limit potentially damaging inflammatory responses in a developing fetus.     

Nuclear retention of IL‐1α by necrotic cells: A mechanism to dampen sterile inflammation

Sterile inflammation is a host response to tissue injury that is mediated by damage‐associated molecular patterns released from dead cells. Sterile inflammation worsens damage in a number of injury paradigms. The pro‐inflammatory cytokine IL‐1α is reported to be a damage‐associated molecular pattern released from dead cells, and it is known to exacerbate brain injury caused by stroke. In the brain, IL‐1α is produced by microglia, the resident brain macrophages. We found that IL‐1α is actively trafficked to the nuclei of microglia, and hence tested the hypothesis that trafficking of IL‐1α to the nucleus would inhibit its release following necrotic cell death, limiting sterile inflammation. Microglia subjected to oxygen‐glucose deprivation died via necrosis. Under these conditions, microglia expressing nuclear IL‐1α released significantly less IL‐1α than microglia with predominantly cytosolic IL‐1α. The remaining IL‐1α was immobilized in the nuclei of the dead cells. Thus, nuclear retention of IL‐1α may serve to limit inflammation following cell death.     

Soluble HLA‐I‐mediated secretion of TGF‐β1 by human NK cells and consequent down‐regulation of anti‐tumor cytolytic activity

Soluble HLA class I (sHLA‐I) molecules can regulate survival of NK cells and their anti‐tumor killing activity. Herein, we have analysed whether interaction of sHLA‐I with CD8 and/or different isoforms of killer Ig‐like receptors (KIR) induced secretion of transforming growth factor (TGF)‐β1. CD8⁺KIR^? NK cell clones secreted TGF‐β1 upon the interaction of sHLA‐I with CD8 molecule. sHLA‐Cw4 or sHLA‐Cw3 alleles engaging inhibitory isoforms of KIR, namely KIR2DL1 or KIR2DL2, strongly downregulated TGF‐β1 production elicited through CD8. On the other hand, sHLA‐Cw4 or sHLA‐Cw3 alleles induced secretion of TGF‐β1 by ligation of stimulatory KIR2DS1 or KIR2DS2 isoforms. TGF‐β1 strongly reduced NK cell‐mediated tumor cell lysis and production of pro‐inflammatory cytokines such as TNF‐α and IFN‐γ. Also, TGF‐β1 inhibited NK cell cytolysis induced by the engagement of stimulatory receptors including NKG2D, DNAM1, 2B4, CD69, NKp30, NKp44 and NKp46. The IL‐2‐dependent surface upregulation of some of these receptors was prevented by TGF‐β1. Furthermore, TGF‐β1 hampered IL‐2‐induced NK cell proliferation but not IL‐2‐mediated rescue from apoptosis of NK cells. Depletion of TGF‐β1 restored all the NK cell‐mediated functional activities analysed. Taken together these findings suggest that sHLA‐I antigens may downregulate the NK cell‐mediated innate response by inducing TGF‐β1 release.     

Caspase‐11 activates a canonical NLRP3 inflammasome by promoting K+ efflux

Recognition of microbe‐associated molecular patterns or endogenous danger signals by a subset of cytosolic PRRs results in the assembly of multiprotein signaling complexes, the so‐called inflammasomes. Canonical inflammasomes are assembled by NOD‐like receptor (NLR) or PYHIN family members and activate caspase‐1, which promotes the induction of pyroptosis and the release of mature interleukin‐1β/‐18. Recently, a noncanonical inflammasome pathway was discovered that results in caspase‐11 activation in response to bacterial lipopolysaccharide (LPS) in the cytosol. Interestingly, caspase‐11 induces pyroptosis by itself, but requires NLRP3, the inflammasome adapter ASC, and caspase‐1 to promote cytokine secretion. Here, we have studied the mechanism by which caspase‐11 controls IL‐1β secretion. Investigating NLRP3/ASC complex formation, we find that caspase‐11 functions upstream of a canonical NLRP3 inflammasome. The activation of NLRP3 by caspase‐11 during LPS transfection is a cell‐intrinsic process and is independent of the release of danger signals. Furthermore, we show that active caspase‐11 leads to a drop of intracellular potassium levels, which is necessary to activate NLRP3. Our study, therefore, sheds new light on the mechanism of noncanonical inflammasome signaling.     

IL‐1β, IL‐18, and eicosanoids promote neutrophil recruitment to pore‐induced intracellular traps following pyroptosis

Inflammasomes activate caspase‐1, initiating a lytic form of programmed cell death termed pyroptosis, which is an important innate immune defense mechanism against intracellular infections. We recently demonstrated in a mouse infection model of pyroptosis that instead of releasing bacteria into the extracellular space, bacteria remain trapped within the pyroptotic cell corpse, termed the pore‐induced intracellular trap (PIT). This trapping mediates efferocytosis of the PIT and associated bacteria by neutrophils; bacteria are subsequently killed via neutrophil ROS. Using this pyroptosis model, we now show that the pro‐inflammatory cytokines IL‐1β and IL‐18 and inflammatory lipid mediators termed eicosanoids are required for effective clearance of bacteria downstream of pyroptosis. We further show that IL‐1β, IL‐18, and eicosanoids affect this in part by mediating neutrophil recruitment to the PIT. This is in addition to our prior findings that complement is also important to attract neutrophils. Thus, the PIT initiates a robust and coordinated innate immune response involving multiple mediators that attract neutrophils to efferocytose the PIT and its entrapped bacteria.