The integrin CD11b inhibits MSU-induced NLRP3 inflammasome activation in macrophages and protects mice against MSU-induced joint inflammation.

OBJECTIVE: In gout, monosodium urate crystals are taken up by macrophages, triggering the activation of the NLRP3 inflammasome and the maturation of IL-1ß. This study aimed to investigate the role of integrin CD11b in inflammasome activation in macrophages stimulated by MSU. METHODS: BMDM from WT and CD11b KO mice were stimulated in vitro with MSU crystals. Cellular supernatants were collected to assess the expression of the inflammatory cytokines by enzyme-linked immunosorbent assay and western blot methods. The role of integrin CD11b in MSU-induced gouty arthritis in vivo was investigated by intra-articular injection of MSU crystals. Real-time extracellular acidification rate and oxygen consumption rate of BMDMs were measured by Seahorse Extracellular Flux Analyzer. RESULTS: We demonstrate that CD11b-deficient mice developed exacerbated gouty arthritis with increased recruitment of leukocytes in the joint and higher IL-1ß levels in the sera. In macrophages, genetic deletion of CD11b induced a shift of macrophage metabolism from oxidative phosphorylation to glycolysis, thus decreasing the overall generation of intracellular ATP. Upon MSU stimulation, CD11b-deficient macrophages showed an exacerbated secretion of IL-1ß. Treating wild-type macrophages with a CD11b agonist, LA1, inhibited MSU-induced release of IL-1ß in vitro and attenuated the severity of experimental gouty arthritis. Importantly, LA1, was also effective in human cells as it inhibited MSU-induced release of IL-1ß by peripheral blood mononuclear cells from healthy donors. CONCLUSION: Our data identified the CD11b integrin as a principal cell membrane receptor that modulates NLRP3 inflammasome activation by MSU crystal in macrophages, which could be a potential therapeutic target to treat gouty arthritis in human patients.

HSP90ß controls NLRP3 autoactivation.

Gain-of-function mutations in NLRP3 are linked to cryopyrin-associated periodic syndromes (CAPS). Although NLRP3 autoinflammasome assembly triggers inflammatory cytokine release, its activation mechanisms are not fully understood. Our study used a functional genetic approach to identify regulators of NLRP3 inflammasome formation. We identified the HSP90ß-SGT1 chaperone complex as crucial for autoinflammasome activation in CAPS. A deficiency in HSP90ß, but not in HSP90α, impaired the formation of ASC specks without affecting the priming and expression of inflammasome components. Conversely, activating NLRP3 with stimuli such as nigericin or alum bypassed the need for SGT1 and HSP90ß, suggesting the existence of alternative inflammasome assembly pathways. The role of HSP90ß was further demonstrated in PBMCs derived from CAPS patients. In these samples, the pathological constitutive secretion of IL-1ß could be suppressed using a pharmacological inhibitor of HSP90ß. This finding underscores the potential of SGT1-HSP90ß modulation as a therapeutic strategy in CAPS while preserving NLRP3's physiological functions.

ER-trafficking triggers NRF1 ubiquitination to promote its proteolytic activation.

The transcription factor NRF1 resides in the endoplasmic reticulum (ER) and is constantly transported to the cytosol for proteasomal degradation. However, when the proteasome is defective, NRF1 escapes degradation and undergoes proteolytic cleavage by the protease DDI2, generating a transcriptionally active form that restores proteostasis, including proteasome function. The mechanisms that regulate NRF1 proteolytic activation and transcriptional potential remain poorly understood. This study demonstrates that the ER is a crucial regulator of NRF1 function by orchestrating its ubiquitination through the E3 ubiquitin ligase HRD1. We show that HRD1-mediated NRF1 ubiquitination is necessary for DDI2-mediated processing in cells. Furthermore, we found that deficiency in both RAD23A and RAD23B impaired DDI2-mediated NRF1 processing, indicating that these genes are essential components of the DDI2 proteolytic machinery. Our findings highlight the intricate mechanism by which the ER activates NRF1 to coordinate the transcriptional activity of an adaptation response in cells.

NLRP3 leucine-rich repeats control induced and spontaneous inflammasome activation in cryopyrin-associated periodic syndrome.

BACKGROUND: The cryopyrin-associated periodic syndromes (CAPS) comprise a group of rare autoinflammatory diseases caused by gain-of-function mutations in the NLRP3 gene. NLRP3 contains a leucine-rich repeats (LRR) domain with a highly conserved exonic organization that is subjected to extensive alternative splicing. Aberrant NLRP3 inflammasome assembly in CAPS causes chronic inflammation; however, the mechanisms regulating inflammasome function remain unclear. OBJECTIVE: We aimed to elucidate the mechanisms regulating NLRP3-mediated autoinflammation in human disease, characterizing the role of LRR in inflammasome activation. METHODS: We analyzed sequence read archive data to characterize the pattern of NLRP3 splicing in human monocytes and investigated the role of each LRR-coding exon in inflammasome regulation in genetically modified U937 cells representing CAPS and healthy conditions. RESULTS: We detected a range of NLRP3 splice variants in human primary cells and monocytic cell lines, including 2 yet-undescribed splice variants. We observe that lipopolysaccharides affect the abundance of certain splice variants, suggesting that they may regulate NLRP3 activation by affecting alternative splicing. We showed that exons 4, 5, 7, and 9 are essential for inflammasome function, both in the context of wild-type NLRP3 activation by the agonist molecule nigericin and in a model of CAPS-mediated NLRP3 inflammasome assembly. Moreover, the SGT1-NLRP3 interaction is decreased in nonfunctional variants, suggesting that alternative splicing may regulate the recruitment of proteins that facilitate inflammasome assembly. CONCLUSION: These findings demonstrate the contribution of the LRR domain in inflammasome function and suggest that navigating LRR exon usage within NLRP3 is sufficient to dampen inflammasome assembly in CAPS.

CDC42 regulates PYRIN inflammasome assembly.

The PYRIN inflammasome pathway is part of the innate immune response against invading pathogens. Unprovoked continuous activation of the PYRIN inflammasome drives autoinflammation and underlies several autoinflammatory diseases, including familial Mediterranean fever (FMF) syndrome. PYRIN inflammasome formation requires PYRIN dephosphorylation and oligomerization by molecular mechanisms that are poorly understood. Here, we use a functional genetics approach to find regulators of PYRIN inflammasome function. We identify the small Rho GTPase CDC42 to be essential for PYRIN activity and oligomerization of the inflammasome complex. While CDC42 catalytic activity enhances PYRIN phosphorylation, thereby inhibiting it, the inflammasome-supportive role of CDC42 is independent of its GDP/GTP binding or hydrolysis capacity and does not affect PYRIN dephosphorylation. These findings identify the dual role of CDC42 as a regulator of PYRIN and as a critical player required for PYRIN inflammasome assembly in health and disease.

The protease DDI2 regulates NRF1 activation in response to cadmium toxicity.

DNA-damage inducible 1 homolog 2 (DDI2) is a protease that activates the transcription factor NRF1. Cellular models have shown that this pathway contributes to cell-stress adaptation, for example, on proteasome inhibition. However, DDI2 physiological function is unknown. Ddi2 Knock-out (KO) mice were embryonic lethal. Therefore, we generated liver-specific Ddi2-KO animals and used comprehensive genetic analysis to identify the molecular pathways regulated by DDI2. Here, we demonstrate that DDI2 contributes to metallothionein (MT) expression in mouse and human hepatocytes at basal and upon cadmium (Cd) exposure. This transcriptional program is dependent on DDI2-mediated NRF1 proteolytic maturation. In contrast, NRF1 homolog NRF2 does not contribute to MT production. Mechanistically, we observed that Cd exposure inhibits proteasome activity, resulting in DDI2-mediated NRF1 proteolytic maturation. In line with these findings, DDI2 deficiency sensitizes cells to Cd toxicity. This study identifies a function for DDI2 that links proteasome homeostasis to heavy metal mediated toxicity.

The aspartyl protease DDI2 drives adaptation to proteasome inhibition in multiple myeloma.

Proteasome inhibitors, such as bortezomib, are first-line therapy against multiple myeloma (MM). Unfortunately, patients frequently become refractory to this treatment. The transcription factor NRF1 has been proposed to initiate an adaptation program that regulates proteasome levels. In the context of proteasome inhibition, the cytosolic protease DDI2 cleaves NRF1 to release an active fragment that translocates to the nucleus to promote the transcription of new proteasome subunits. However, the contribution of the DDI2-NRF1 pathway to bortezomib resistance is poorly understood. Here we show that upon prolonged bortezomib treatment, MM cells become resistant to proteasome inhibition by increasing the expression of DDI2 and consequently activation of NRF1. Furthermore, we found that many MM cells became more sensitive to proteasome impairment in the context of DDI2 deficiency. Mechanistically, we demonstrate that both the protease and the HDD domains of DDI2 are required to activate NRF1. Finally, we show that partial inhibition of the DDI2-protease domain with the antiviral drug nelfinavir increased bortezomib susceptibility in treated MM cells. Altogether, these findings define the DDI2-NRF1 pathway as an essential program contributing to proteasome inhibition responses and identifying DDI2 domains that could be targets of interest in bortezomib-treated MM patients.

Tumor-induced reshuffling of lipid composition on the endoplasmic reticulum membrane sustains macrophage survival and pro-tumorigenic activity.

Tumor-associated macrophages (TAMs) display pro-tumorigenic phenotypes for supporting tumor progression in response to microenvironmental cues imposed by tumor and stromal cells. However, the underlying mechanisms by which tumor cells instruct TAM behavior remain elusive. Here, we uncover that tumor-cell-derived glucosylceramide stimulated unconventional endoplasmic reticulum (ER) stress responses by inducing reshuffling of lipid composition and saturation on the ER membrane in macrophages, which induced IRE1-mediated spliced XBP1 production and STAT3 activation. The cooperation of spliced XBP1 and STAT3 reinforced the pro-tumorigenic phenotype and expression of immunosuppressive genes. Ablation of XBP1 expression with genetic manipulation or ameliorating ER stress responses by facilitating LPCAT3-mediated incorporation of unsaturated lipids to the phosphatidylcholine hampered pro-tumorigenic phenotype and survival in TAMs. Together, we uncover the unexpected roles of tumor-cell-produced lipids that simultaneously orchestrate macrophage polarization and survival in tumors via induction of ER stress responses and reveal therapeutic targets for sustaining host antitumor immunity.

Cell-autonomous inflammation of BRCA1-deficient ovarian cancers drives both tumor-intrinsic immunoreactivity and immune resistance via STING.

In this study, we investigate mechanisms leading to inflammation and immunoreactivity in ovarian tumors with homologous recombination deficiency (HRD). BRCA1 loss is found to lead to transcriptional reprogramming in tumor cells and cell-intrinsic inflammation involving type I interferon (IFN) and stimulator of IFN genes (STING). BRCA1-mutated (BRCA1mut) tumors are thus T cell inflamed at baseline. Genetic deletion or methylation of DNA-sensing/IFN genes or CCL5 chemokine is identified as a potential mechanism to attenuate T cell inflammation. Alternatively, in BRCA1mut cancers retaining inflammation, STING upregulates VEGF-A, mediating immune resistance and tumor progression. Tumor-intrinsic STING elimination reduces neoangiogenesis, increases CD8+ T cell infiltration, and reverts therapeutic resistance to dual immune checkpoint blockade (ICB). VEGF-A blockade phenocopies genetic STING loss and synergizes with ICB and/or poly(ADP-ribose) polymerase (PARP) inhibitors to control the outgrowth of Trp53-/-Brca1-/- but not Brca1+/+ ovarian tumors in vivo, offering rational combinatorial therapies for HRD cancers.

Detection of viruses by inflammasomes.

The innate immune system has evolved mechanisms to keep the viral infection under control and repair damaged tissues. Several pathways can identify the presence of pathogenic components, such as viral nucleic acids and viral proteins. Also, the innate immune system can detect cellular and tissue perturbations caused by infections. Inflammasomes are cellular pieces of machinery that can detect a pathogen's presence and its possible impact on cellular integrity. Thereby several inflammasomes, including the NLRP3 inflammasome and the AIM2 inflammasome, contribute to antiviral innate immunity. Inflammation driven by inflammasomes promotes immune defenses and initiate repair mechanisms. However, its overactivation may trigger acute inflammatory responses that may harm the host. This pathologic activation could contribute to the hyperinflammatory response observed in patients infected with viruses, including influenza, SARS, and possibly SARS-CoV2.

Different CFTR modulator combinations downregulate inflammation differently in cystic fibrosis.

Previously, we showed that serum and monocytes from patients with CF exhibit an enhanced NLRP3-inflammasome signature with increased IL-18, IL-1ß, caspase-1 activity and ASC speck release (Scambler et al. eLife 2019). Here we show that CFTR modulators down regulate this exaggerated proinflammatory response following LPS/ATP stimulation. In vitro application of ivacaftor/lumacaftor or ivacaftor/tezacaftor to CF monocytes showed a significant reduction in IL-18, whereas IL-1ß was only reduced with ivacaftor/tezacaftor. Thirteen adults starting ivacaftor/lumacaftor and eight starting ivacaftor/tezacaftor were assessed over three months. Serum IL-18 and TNF decreased significantly with treatments, but IL-1ß only declined following ivacaftor/tezacaftor. In (LPS/ATP-stimulated) PBMCs, IL-18/TNF/caspase-1 were all significantly decreased and IL-10 was increased with both combinations. Ivacaftor/tezacaftor alone showed a significant reduction in IL-1ß and pro-IL-1ß mRNA. This study demonstrates that these CFTR modulator combinations have potent anti-inflammatory properties, in addition to their ability to stimulate CFTR function, which could contribute to improved clinical outcomes.

Inflammasomes contributing to inflammation in arthritis.

Inflammasomes are intracellular multiprotein signaling platforms that initiate inflammatory responses in response to pathogens and cellular damage. Active inflammasomes induce the enzymatic activity of caspase-1, resulting in the induction of inflammatory cell death, pyroptosis, and the maturation and secretion of inflammatory cytokines IL-1ß and IL-18. Inflammasomes are activated in many inflammatory diseases, including autoinflammatory disorders and arthritis, and inflammasome-specific therapies are under development for the treatment of inflammatory conditions. In this review, we outline the different inflammasome platforms and recent findings contributing to our knowledge about inflammasome biology in health and disease. In particular, we discuss the role of the inflammasome in the pathogenesis of arthritic diseases, including rheumatoid arthritis, gout, ankylosing spondylitis, and juvenile idiopathic arthritis, and the potential of newly developed therapies that specifically target the inflammasome or its products for the treatment of inflammatory diseases.

ENaC-mediated sodium influx exacerbates NLRP3-dependent inflammation in cystic fibrosis.

Cystic Fibrosis (CF) is a monogenic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in defective CFTR-mediated chloride and bicarbonate transport, with dysregulation of epithelial sodium channels (ENaC). These changes alter fluid and electrolyte homeostasis and result in an exaggerated proinflammatory response driven, in part, by infection. We tested the hypothesis that NLRP3 inflammasome activation and ENaC upregulation drives exaggerated innate-immune responses in this multisystem disease. We identify an enhanced proinflammatory signature, as evidenced by increased levels of IL-18, IL-1ß, caspase-1 activity and ASC-speck release in monocytes, epithelia and serum with CF-associated mutations; these differences were reversed by pretreatment with NLRP3 inflammasome inhibitors and notably, inhibition of amiloride-sensitive sodium (Na+) channels. Overexpression of ß-ENaC, in the absence of CFTR dysfunction, increased NLRP3-mediated inflammation, indicating that dysregulated, ENaC-dependent signalling may drive exaggerated inflammatory responses in CF. These data support a role for sodium in modulating NLRP3 inflammasome activation.

Metabolic Reprograming of Cystic Fibrosis Macrophages via the IRE1α Arm of the Unfolded Protein Response Results in Exacerbated Inflammation.

Cystic Fibrosis (CF) is a recessive genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR mutations cause dysregulation of channel function with intracellular accumulation of misfolded proteins and endoplasmic reticulum (ER) stress, with activation of the IRE1α-XBP1 pathway that regulates a subset of unfolded protein response (UPR) genes. This pathway regulates a group of genes that control proinflammatory and metabolic responses in different immune cells; however, the metabolic state of immune cells and the role of this pathway in CF remain elusive. Our results indicate that only innate immune cells from CF patients present increased levels of ER stress, mainly affecting neutrophils, monocytes, and macrophages. An overactive IRE1α-XBP1 pathway reprograms CF M1 macrophages toward an increased metabolic state, with increased glycolytic rates and mitochondrial function, associated with exaggerated production of TNF and IL-6. This hyper-metabolic state, seen in CF macrophages, is reversed by inhibiting the RNase domain of IRE1α, thereby decreasing the increased glycolic rates, mitochondrial function and inflammation. Altogether, our results indicate that innate immune cells from CF patients are primarily affected by ER stress. Moreover, the IRE1α-XBP1 pathway of the UPR is responsible for the hyper-metabolic state seen in CF macrophages, which is associated with the exaggerated inflammatory response. Modulating ER stress, metabolism and inflammation, by targeting IRE1α, may improve the metabolic fitness of macrophages, and other immune cells in CF and other immune-related disorders.

A proximity-dependent biotinylation (BioID) approach flags the p62/sequestosome-1 protein as a caspase-1 substrate.

The inflammasome is a major component of the innate immune system, and its main function is to activate caspase-1, a cysteine protease that promotes inflammation by inducing interleukin-1ß (IL-1ß) maturation and release into the extracellular milieu. To prevent uncontrolled inflammation, this complex is highly regulated. When it is assembled, the inflammasome is insoluble, which has long precluded the analysis of its interactions with other proteins. Here we used the proximity-dependent biotinylation assay (BioID) to identify proteins associated with caspase-1 during inflammasome activation. Using the BioID in a cell-free system in which the inflammasome had been activated, we found that a caspase-1-biotin ligase fusion protein selectively labeled 111 candidates, including the p62/sequestosome-1 protein (p62). Using co-immunoprecipitation experiments, we demonstrated that p62 interacts with caspase-1. This interaction promoted caspase-1-mediated cleavage of p62 at Asp-329. Mechanistic and functional analyses revealed that caspase-1-mediated cleavage of p62 leads to loss of its interaction with the autophagosomal protein microtubule-associated protein 1 light chain 3 ß (LC3B). Strikingly, overexpression of a p62 N-terminal fragment generated upon caspase-1 cleavage decreased IL-1ß release, whereas overexpression of p62's C-terminal portion enhanced IL-1ß release, by regulating pro-IL1ß levels. Overall, the overexpression of both fragments together decreased IL-1ß release. Taken together, our results indicate that caspase-1-mediated p62 cleavage plays a complex role in balancing caspase-1-induced inflammation.

Hydrogen sulfide inhibits NLRP3 inflammasome activation and reduces cytokine production both in vitro and in a mouse model of inflammation.

A variety of stimuli, including monosodium urate (MSU) crystals, activate the NLRP3 inflammasome, and this activation involves several molecular mechanisms including xanthine oxidase (XO) up-regulation and mitochondrial dysfunction. Upon oligomerization of apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1 becomes active and cleaves the proinflammatory cytokine IL-1ß into its active secreted form. Hydrogen sulfide (H2S), a gasotransmitter mainly produced by cystathionine γ-lyase (CSE) in macrophages, could modulate inflammation. Here, we sought to investigate the effects of exogenous and endogenous H2S on NLRP3 inflammasome activation in vitro and in vivo Primed bone marrow-derived macrophages (BMDM) isolated from wildtype (wt) or CSE-deficient mice and human macrophages (THP1 cells and primary macrophages), were stimulated with MSU crystals in the presence or absence of a H2S donor, sodium thiosulfate (STS) or GYY4137 (GYY). In murine and human macrophages in vitro, both STS and GYY inhibited MSU crystal-induced IL-1ß secretion in a dose-dependent manner. Moreover, the H2S donors inhibited MSU crystal-induced XO/caspase-1 activities, mitochondrial reactive oxygen species (ROS) generation, and ASC oligomerization. Accordingly, IL-1ß secretion and XO/caspase-1 activities were higher in CSE-deficient BMDMs than in wt BMDMs. For in vivo studies, we experimentally induced peritonitis by intraperitoneal injection of MSU crystals into mice. GYY pretreatment ameliorated inflammation, evidenced by decreased IL-6/monocyte chemoattractant protein-1 (MCP-1) released into peritoneal lavages. Taken together, our results suggest that both exogenous (via H2S donors) and endogenous (via CSE) H2S production may represent approaches for managing, for example, acute gout or other inflammation conditions.

The AIM2 inflammasome: Sensor of pathogens and cellular perturbations.

Recognition of pathogens and altered self must be efficient and highly specific to orchestrate appropriate responses while limiting excessive inflammation and autoimmune reaction to normal self. AIM2 is a member of innate immune sensors that detects the presence of DNA, arguably the most conserved molecules in living organisms. However, AIM2 achieves specificity by detecting altered or mislocalized DNA molecules. It can detect damaged DNA, and the aberrant presence of DNA within the cytosolic compartment such as genomic DNA released into the cytosol upon loss of nuclear envelope integrity. AIM2 is also a key sensor of pathogens that detects the presence of foreign DNA accumulating in the cytosol during the life cycle of intracellular pathogens including viruses, bacteria, and parasites. AIM2 activation initiates the assembly of the inflammasome, an innate immune complex that leads to the activation of inflammatory caspases. This triggers the maturation and secretion of the cytokines IL-1ß and IL-18. It can also initiate pyroptosis, a proinflammatory form of cell death. The AIM2 inflammasome contributes to physiological responses and diseases. It is a key player in host defenses, but its deregulation can contribute immune-linked diseases, such as autoinflammatory and autoimmune pathologies. Moreover, AIM2 may play a role in cancer development. Recent studies have shown that the detection of self-DNA species by AIM2 is an important factor that contributes to diseases associated with perturbation of cellular homeostasis. Thus, in addition of being a sensor of pathogen associated molecular patterns (PAMPs), the AIM2 inflammasome is emerging as a key guardian of cellular integrity.

Inflammation initiated by stressed organelles.

Key cellular functions including those related to energy metabolism, organization of the genetic information or production of membrane-bound and secreted proteins are compartmentalized within organelles. Various stresses such as differentiation programs, viral and bacterial infections, perturbations in protein production, mechanical constraints, changes in the environment and nutriment accessibility can impact cellular homeostasis and organelle integrity. Perturbations of these cellular compartments trigger repair and adaptation programs aimed at restoring homeostasis. These events are often associated with low-grade inflammation also termed parainflammation. While the nature and mechanisms of danger signals released by irremediably damaged cells are well understood, how transiently stressed cells trigger inflammation is still poorly understood. Emerging studies highlighted new mechanisms by which stress pathways promote inflammation. Cytosolic innate immune pathways are engaged by signals stemming from perturbed organelles such as the mitochondria, the endoplasmic reticulum (ER) or the nuclear envelope (NE). These observations indicate that these pathways function as guardians of cellular homeostasis and may contribute to disease in pathologies characterized by perturbations of cellular homoeostasis. Mitochondria-stress, ER-stress or NE-stress are emerging as proinflammatory signals that contribute to human conditions and diseases.

Detection of ASC Oligomerization by Western Blotting.

The apoptosis-associated speck-like protein with a caspase-recruitment domain (ASC) adaptor protein bridges inflammasome sensors and caspase-1. Upon inflammasome activation, ASC nucleates in a prion-like manner into a large and single platform responsible for the recruitment and the activation of caspase-1. Active caspase-1 will in turn promote the proteolytic maturation of the pro-inflammatory cytokine IL-1ß. ASC oligomerization is a direct evidence for inflammasome activation and its detection allows a read-out independent of caspase-1 and IL-1ß. This protocol describes how to detect the oligomerization of ASC by Western blot.