Pathogenic NLRP3 mutants form constitutively active inflammasomes resulting in immune-metabolic limitation of IL-1ß production.

Cryopyrin-associated periodic syndrome (CAPS) is an autoinflammatory condition resulting from monoallelic NLRP3 variants that facilitate IL-1ß production. Although these are gain-of-function variants characterized by hypersensitivity to cell priming, patients with CAPS and animal models of the disease may present inflammatory flares without identifiable external triggers. Here we find that CAPS-associated NLRP3 variants are forming constitutively active inflammasome, which induce increased basal cleavage of gasdermin D, IL-18 release and pyroptosis, with a concurrent basal pro-inflammatory gene expression signature, including the induction of nuclear receptors 4 A. The constitutively active NLRP3-inflammasome of CAPS is responsive to the selective NLRP3 inhibitor MCC950 and its activation is regulated by deubiquitination. Despite their preactivated state, the CAPS inflammasomes are responsive to activation of the NF-κB pathway. NLRP3-inflammasomes with CAPS-associated variants affect the immunometabolism of the myeloid compartment, leading to disruptions in lipids and amino acid pathways and impaired glycolysis, limiting IL-1ß production. In summary, NLRP3 variants causing CAPS form a constitutively active inflammasome inducing pyroptosis and IL-18 release without cell priming, which enables the host's innate defence against pathogens while also limiting IL-1ß-dependent inflammatory episodes through immunometabolism modulation.

Measuring NLR Oligomerization III: Detection of NLRP3 and NLRC4 Complex by Bioluminescence Resonance Energy Transfer.

Bioluminescent resonance energy transfer (BRET) is a natural phenomenon resulting from a non-radiative energy transfer between a bioluminescent donor (Renilla luciferase) and a fluorescent protein acceptor. BRET signal is dependent on the distance and the orientation between the donor and the acceptor and could be used to study protein-protein interactions and conformational changes within proteins at real-time in living cells. This protocol describes the use of BRET technique to study NLRP3 oligomerization in living cells before and during NLRP3 inflammasome activation.

ASC oligomer favors caspase-1CARD domain recruitment after intracellular potassium efflux.

Signaling through the inflammasome is important for the inflammatory response. Low concentrations of intracellular K+ are associated with the specific oligomerization and activation of the NLRP3 inflammasome, a type of inflammasome involved in sterile inflammation. After NLRP3 oligomerization, ASC protein binds and forms oligomeric filaments that culminate in large protein complexes named ASC specks. ASC specks are also initiated from different inflammasome scaffolds, such as AIM2, NLRC4, or Pyrin. ASC oligomers recruit caspase-1 and then induce its activation through interactions between their respective caspase activation and recruitment domains (CARD). So far, ASC oligomerization and caspase-1 activation are K+-independent processes. Here, we found that when there is low intracellular K+, ASC oligomers change their structure independently of NLRP3 and make the ASCCARD domain more accessible for the recruitment of the pro-caspase-1CARD domain. Therefore, conditions that decrease intracellular K+ not only drive NLRP3 responses but also enhance the recruitment of the pro-caspase-1 CARD domain into the ASC specks.

BTK operates a phospho-tyrosine switch to regulate NLRP3 inflammasome activity.

Activity of the NLRP3 inflammasome, a critical mediator of inflammation, is controlled by accessory proteins, posttranslational modifications, cellular localization, and oligomerization. How these factors relate is unclear. We show that a well-established drug target, Bruton's tyrosine kinase (BTK), affects several levels of NLRP3 regulation. BTK directly interacts with NLRP3 in immune cells and phosphorylates four conserved tyrosine residues upon inflammasome activation, in vitro and in vivo. Furthermore, BTK promotes NLRP3 relocalization, oligomerization, ASC polymerization, and full inflammasome assembly, probably by charge neutralization, upon modification of a polybasic linker known to direct NLRP3 Golgi association and inflammasome nucleation. As NLRP3 tyrosine modification by BTK also positively regulates IL-1ß release, we propose BTK as a multifunctional positive regulator of NLRP3 regulation and BTK phosphorylation of NLRP3 as a novel and therapeutically tractable step in the control of inflammation.

Sensing low intracellular potassium by NLRP3 results in a stable open structure that promotes inflammasome activation.

The NLRP3 inflammasome is activated by a wide range of stimuli and drives diverse inflammatory diseases. The decrease of intracellular K+ concentration is a minimal upstream signal to most of the NLRP3 activation models. Here, we found that cellular K+ efflux induces a stable structural change in the inactive NLRP3, promoting an open conformation as a step preceding activation. This conformational change is facilitated by the specific NLRP3 FISNA domain and a unique flexible linker sequence between the PYD and FISNA domains. This linker also facilitates the ensemble of NLRP3PYD into a seed structure for ASC oligomerization. The introduction of the NLRP3 PYD-linker-FISNA sequence into NLRP6 resulted in a chimeric receptor able to be activated by K+ efflux­specific NLRP3 activators and promoted an in vivo inflammatory response to uric acid crystals. Our results establish that the amino-terminal sequence between PYD and NACHT domain of NLRP3 is key for inflammasome activation.

Addendum: MCC950 closes the active conformation of NLRP3 to an inactive state.

An Addendum to this paper has been published: https://doi.org/10.1038/s41589-021-00741-6.

Author Correction: Early endosome autoantigen 1 regulates IL-1ß release upon caspase-1 activation independently of gasdermin D membrane permeabilization.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Recent insights into the regulatory networks of NLRP3 inflammasome activation.

The NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome is a fascinating cellular machinery endowed with the capacity for rapid proteolytic processing of the pro-inflammatory cytokine IL-1ß and the cell death effector gasdermin D (GSDMD). Although its activity is essential to fight infection and support tissue homeostasis, the inflammasome complex, which consists of the danger sensor NLRP3, the adaptor apoptosis-associated speck-like protein containing a CARD (ASC; also known as PYCARD), caspase-1 and probably other regulatory proteins, also bears considerable potential for detrimental inflammation, as observed in human conditions such as gout, heart attack, stroke and Alzheimer's disease. Thus, multi-layered regulatory networks are required to ensure the fine balance between rapid responsiveness versus erroneous activation (sufficient and temporally restricted versus excessive and chronic activity) of the inflammasome. These involve multiple activation, secretion and cell death pathways, as well as modulation of the subcellular localization of NLRP3, and its structure and activity, owing to post-translational modification by other cellular proteins. Here, we discuss the exciting progress that has recently been made in deciphering the regulation of the NLRP3 inflammasome. Additionally, we highlight open questions and describe areas of research that warrant further exploration to obtain a more comprehensive molecular and cellular understanding of the NLRP3 inflammasome.

Isolation of functional mature peritoneal macrophages from healthy humans.

Macrophages play an important role in the inflammatory response. Their various biological functions are induced by different membrane receptors, including Toll-like receptors, which trigger several intracellular signaling cascades and activate the inflammasomes, which in turn elicit the release of inflammatory mediators such as cytokines. In this study, we present a novel method for the isolation of human mature peritoneal macrophages. This method can be easily implemented by gynecologists who routinely perform laparoscopy for sterilization by tubal ligation or surgically intervene in benign gynecological pathologies. Our method confirms that macrophages are the main peritoneal leukocyte subpopulation isolated from the human peritoneum in homeostasis. We showed that primary human peritoneal macrophages present phagocytic and oxidative activities, and respond to activation of the main proinflammatory pathways such as Toll-like receptors and inflammasomes, resulting in the secretion of different proinflammatory cytokines. Therefore, this method provides a useful tool for characterizing primary human macrophages as control cells for studies of molecular inflammatory pathways in steady-state conditions and for comparing them with those obtained from pathologies involving the peritoneal cavity. Furthermore, it will facilitate advances in the screening of anti-inflammatory compounds in the human system.

MCC950 closes the active conformation of NLRP3 to an inactive state.

NLRP3 (NOD-like receptor pyrin domain-containing protein 3) is an innate immune sensor that contributes to the development of different diseases, including monogenic autoinflammatory syndromes, gout, atherosclerosis, and Alzheimer's disease. The molecule sulfonylurea MCC950 is a NLRP3 inflammasome inhibitor with potential clinical utility. However, the mechanism of action of MCC950 remains unknown. Here, we characterize the mechanism of action of MCC950 in both wild-type and autoinflammatory-related NLRP3 mutants, and demonstrate that MCC950 closes the 'open' conformation of active NLRP3.

Early endosome autoantigen 1 regulates IL-1ß release upon caspase-1 activation independently of gasdermin D membrane permeabilization.

Unconventional protein secretion represents an important process of the inflammatory response. The release of the pro-inflammatory cytokine interleukin (IL)-1ß which burst during pyroptosis as a consequence of gasdermin D plasma membrane pore formation, can also occur through other unconventional secretion pathways dependent on caspase-1 activation. However, how caspase-1 mediates cytokine release independently of gasdermin D remains poorly understood. Here we show that following caspase-1 activation by different inflammasomes, caspase-1 cleaves early endosome autoantigen 1 (EEA1) protein at Asp127/132. Caspase-1 activation also results in the release of the endosomal EEA1 protein in a gasdermin D-independent manner. EEA1 knock-down results in adecreased release of caspase-1 and IL-1ß, but the pyroptotic release of other inflammasome components and lactate dehydrogenase was not affected. This study shows how caspase-1 control the release of EEA1 and IL-1ß in a pyroptotic-independent manner.

NLRP3 lacking the leucine-rich repeat domain can be fully activated via the canonical inflammasome pathway.

NLRP3 is a cytosolic sensor triggered by different pathogen- and self-derived signals that plays a central role in a variety of pathological conditions, including sterile inflammation. The leucine-rich repeat domain is present in several innate immune receptors, where it is frequently responsible for sensing danger signals and regulation of activation. Here we show by reconstitution of truncated and chimeric variants into Nlrp3-/- macrophages that the leucine-rich repeat domain is dispensable for activation and self-regulation of NLRP3 by several different triggers. The pyrin domain on the other hand is required to maintain NLRP3 in the inactive conformation. A fully responsive minimal NLRP3 truncation variant reconstitutes peritonitis in Nlrp3-/- mice. We demonstrate that in contrast to pathogen-activated NLRC4, the constitutively active NLRP3 molecule cannot engage wild-type NLRP3 molecules in a self-catalytic oligomerization. This lack of signal amplification is likely a protective mechanism to decrease sensitivity to endogenous triggers to impede autoinflammation.

Development of an Acrylate Derivative Targeting the NLRP3 Inflammasome for the Treatment of Inflammatory Bowel Disease.

Pharmacological inhibition of NLRP3 inflammasome activation may offer a new option in the treatment of inflammatory bowel disease. In this work, we report the design, synthesis, and biological screening of a series of acrylate derivatives as NLRP3 inhibitors. The in vitro determination of reactivity, cytotoxicity, NLRP3 ATPase inhibition, and antipyroptotic properties allowed the selection of 11 (INF39), a nontoxic, irreversible NLRP3 inhibitor able to decrease interleukin-1ß release from macrophages. Bioluminescence resonance energy transfer experiments proved that this compound was able to directly interfere with NLRP3 activation in cells. In vivo studies confirmed the ability of the selected lead to alleviate the effects of colitis induced by 2,4-dinitrobenzenesulfonic acid in rats after oral administration.

A novel CD14(high) CD16(high) subset of peritoneal macrophages from cirrhotic patients is associated to an increased response to LPS.

The aim of this study was to characterize monocyte-derived macrophages (M-DM) from blood and ascites of cirrhotic patients comparatively with those obtained from blood of healthy controls. The phenotypic profile based on CD14/CD16 expression was analyzed by flow cytometry. Cells were isolated and stimulated in vitro with LPS and heat killed Candida albicans. Phosphorylation of ERK, c-Jun, p38 MAPK, and PKB/Akt was analyzed by Western blotting. A novel CD14(high)CD16(high) M-DM subpopulation is present in ascites (∼33%). The CD14(++)CD16(+) intermediate subset is increased in the blood of cirrhotic patients (∼from 4% to 11%) and is predominant in ascites (49%), while the classical CD14(++)CD16(-) subpopulation is notably reduced in ascites (18%). Basal hyperactivation of ERK and JNK/c-Jun pathways observed in ascites M-DM correlates with CD14/CD16 high expressing subsets, while PI3K/PKB does it with the CD16 low expressing cells. In vitro LPS treatment highly increases ERK1/2, PKB/Akt and c-Jun phosphorylation, while that of p38 MAPK is decreased in M-DM from ascites compared to control blood M-DM. Stimulation of healthy blood M-DM with LPS and C. albicans induced higher phosphorylation levels of p38 than those from ascites. Regarding cytokines secretion, in vitro activated M-DM from ascites of cirrhotic patients produced significantly higher amounts of IL-6, IL-10 and TNF-α, and lower levels of IL-1ß and IL-12 than control blood M-DM. In conclusion, a new subpopulation of CD14(high)CD16(high) peritoneal M-DM has been identified in ascites of cirrhotic patients, which is very sensitive to LPS stimulation.

Role of MAP kinases and PI3K-Akt on the cytokine inflammatory profile of peritoneal macrophages from the ascites of cirrhotic patients.

AIMS: Several new approaches targeting inflammation associated with different diseases are in clinical development. OBJECTIVE: To explore the role played by MAPK and PI3K-Akt pathways on the release of cytokines in monocyte-derived macrophages (M-DM) obtained from the ascites of cirrhotic patients to identify novel targets for pharmaceutical intervention to prevent hepatic damage. METHODS: M-DM were isolated from the ascites of cirrhotic patients and stimulated in vitro with LPS and heat-killed Candida albicans in the presence or absence of the inhibitors for MEK1, p38 MAPK, JNK and PI3K. The MAPK phosphorylation levels were determined by Western Blot. Cell culture supernatants were assayed by ELISA for TNF-α, IL-6 and IL-10. RESULTS: The release of the pro-inflammatory cytokines IL-6 and TNF-α at baseline was more effectively reduced by the MAPK inhibitors, while the basal IL-10 anti-inflammatory cytokine secretion was only and strongly (90.3%) affected by the PI3K inhibitor. The incubation of peritoneal M-DM in the presence of LPS and C. albicans increased the release of IL-6, TNF-α and IL-10. LPS-induced pro-inflammatory cytokines secretion was more sensitive to MAPK inhibitors, whereas that induced by C. albicans was more susceptible to inhibition of PI3K. Finally, inhibition of PI3K almost completely suppressed the secretion of IL-10 in stimulated M-DM. CONCLUSIONS: These results demonstrate that pro-inflammatory cytokines release in M-DM from this clinical setting strongly depends on the MAPK signalling pathways, differs depending on the microbial stimulus added and confirms the prominent role of the PI3K-Akt pathway in the modulation of IL-10-mediated anti-inflammatory function.

The peritoneal macrophage inflammatory profile in cirrhosis depends on the alcoholic or hepatitis C viral etiology and is related to ERK phosphorylation.

BACKGROUND: The development of ascites in cirrhotic patients generally heralds a deterioration in their clinical status. A differential gene expression profile between alcohol- and hepatitis C virus (HCV)-related cirrhosis has been described from liver biopsies, especially those associated with innate immune responses. The aim of this work was to identify functional differences in the inflammatory profile of monocyte-derived macrophages from ascites in cirrhotic patients of different etiologies in an attempt to extrapolate studies from liver biopsies to immune cells in ascites. To this end 45 patients with cirrhosis and non-infected ascites, distributed according to disease etiology, HCV (n=15) or alcohol (n=30) were studied. Cytokines and the cell content in ascites were assessed by ELISA and flow cytometry, respectively. Cytokines and ERK phosphorylation in peritoneal monocyte-derived macrophages isolated and stimulated in vitro were also determined. RESULTS: A different pattern of leukocyte migration to the peritoneal cavity and differences in the primed status of macrophages in cirrhosis were observed depending on the viral or alcoholic etiology. Whereas no differences in peripheral blood cell subpopulations could be observed, T lymphocyte, monocyte and polymorphonuclear cell populations in ascites were more abundant in the HCV than the alcohol etiology. HCV-related cirrhosis etiology was associated with a decreased inflammatory profile in ascites compared with the alcoholic etiology. Higher levels of IL-10 and lower levels of IL-6 and IL-12 were observed in ascitic fluid from the HCV group. Isolated peritoneal monocyte-derived macrophages maintained their primed status in vitro throughout the 24 h culture period. The level of ERK1/2 phosphorylation was higher in ALC peritoneal macrophages at baseline than in HCV patients, although the addition of LPS induced a greater increase in ERK1/2 phosphorylation in HCV than in ALC patients. CONCLUSIONS: The macrophage inflammatory status is higher in ascites of alcohol-related cirrhotic patients than in HCV-related patients, which could be related with differences in bacterial translocation episodes or regulatory T cell populations. These findings should contribute to identifying potential prognostic and/or therapeutic targets for chronic liver diseases of different etiology.

Glycoconjugate expression on the cell wall of tps1/tps1 trehalose-deficient Candida albicans strain and implications for its interaction with macrophages.

The yeast Candida albicans has developed a variety of strategies to resist macrophage killing. In yeasts, accumulation of trehalose is one of the principal defense mechanisms under stress conditions. The gene-encoding trehalose-6-phosphate synthase (TPS1), which is responsible for trehalose synthesis, is induced in response to oxidative stress, as in phagolysosomes. Mutants unable to synthesize trehalose are sensitive to oxidative stress in vitro. In mice, the TPS1-deficient strain, tps1/tps1, displays a lower infection rate than its parental strain (CAI4). We have previously demonstrated the reduced binding capacity of tps1/tps1 and its lower resistance to macrophages. At the same time, its outer cell wall layer was seen to be altered. In this study, we show that depending on the culture conditions, the tps1/tps1 strain regulates the carbohydrate metabolism in a different way to CAI4, as reflected by the enhanced ß-mannosylation of cell wall components, especially at the level of the 120 kDa glycoprotein species, accessible at the cell surface of tps1/tps1 when cultured in liquid medium, but not on solid medium. This leads to changes in its surface properties, as revealed by decreased hydrophobicity, and the lower levels of ERK1/2 phosphorylation and tumor necrosis factor-α (TNF-α) production in macrophages, thus increasing the resistance to these cells. In contrast, in solid medium, in which over-glycosylation was less evident, tps1/tps1 showed similar macrophage interaction properties to CAI4, but was less resistant to killing, confirming the protective role of trehalose. Thus, the lack of trehalose is compensated by an over-glycosylation of the cell wall components in the tps1/tps1 mutant, which reduces susceptibility to killing.

Peritoneal macrophage priming in cirrhosis is related to ERK phosphorylation and IL-6 secretion.

BACKGROUND: Bacterial infections are common complications arising in patients with cirrhosis and ascites. Translocation of bacterial DNA is a dynamic process that is associated with an increased inflammatory response and a poor prognosis in this setting. The aim of this study was to study whether peritoneal macrophages remain in a chronic primed status to allow a rapid response to subsequent events of bacterial translocation. PATIENTS AND METHODS: Peritoneal monocyte-derived macrophages were isolated from 25 patients with cirrhosis and non-infected ascites and compared with donor's blood monocytes. Activation cell-surface markers were screened using flow-cytometry, and the phosphorylation state of ERK 1/2, p38 MAP Kinase, PKB/Akt and transcription factors c-Jun and p65 NFκB were evaluated using Western blot. Synthesis of tumour necrosis factor alpha, interleukin 6 (IL-6) and interleukin-10 (IL-10) at baseline and in response to bacterial stimuli was evaluated using ELISA. RESULTS: A high expression of CD54, CD86 and HLA-DR at baseline was displayed by peritoneal macrophages. Increased phosphorylated levels of ERK1/2, protein kinase B (PKB) and c-Jun, together with IL-6 production, were observed in peritoneal macrophages at baseline compared with donors' blood monocytes. A positive correlation was established between basal IL-6 levels and extracellular signal-regulated kinase (ERK) phosphorylation in peritoneal macrophages from patients with cirrhosis (r=0·9; P=0·005). Addition of lipopolysaccharide induced higher phosphorylation levels of all studied signalling intermediates than synthetic-oligodeoxydinucleotides, but similar end-stage p65 NFκB. CONCLUSIONS: A sustained immune response is present in ascitic fluid of cirrhotic patients, even in the temporal absence of bacterial antigens. This would facilitate a fast response, probably controlled by IL-6, against repeated bacterial-DNA translocation or in liver chronic inflammation.