Molecular mechanisms and regulation of inflammasome activation and signaling: sensing of pathogens and damage molecular patterns

Background:Mitochondrial function is implicated as a target of environmental toxicants and found in disease or injury models, contributing to acute and chronic inflammation. One mechanism by which mitochondrial damage can propagate inflammation is via activation of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing receptor (NLRP)3 inflammasome, a protein complex that processes mature interleukin . plays an important role in the innate immune response and dysregulation is associated with autoinflammatory disorders.Objective:The objective was to evaluate whether mitochondrial toxicants recruit inflammasome activation and processing.Method:Murine macrophages (RAW 264.7) exposed to tri-organotins (triethyltin bromide (TETBr), trimethyltin hydroxide (TMTOH), triphenyltin hydroxide (TPTOH), bis(tributyltin)oxide) [Bis(TBT)Ox] were examined for pro-inflammatory cytokine induction. TMTOH and TETBr were examined in RAW 264.7 and bone marrow-derived macrophages for mitochondrial bioenergetics, reactive oxygen species (ROS) production, and inflammasome activation via visualization of aggregate formation, caspase-1 flow cytometry, enzyme-linked immunosorbent assay and Western blots, and microRNA (miRNA) and mRNA arrays.Results:TETBr and TMTOH induced inflammasome aggregate formation and release in lipopolysaccharide (LPS)-primed macrophages. Mitochondrial bioenergetics and mitochondrial ROS were suppressed. Il1a and Il1b induction with LPS or challenge was diminished. Differential miRNA and mRNA profiles were observed. Lower miR-151-3p targeted cyclic adenosine monophosphate (cAMP)-mediated and AMP-activated protein kinase signaling pathways; higher miR-6909-5p, miR-7044-5p, and miR-7686-5p targeted Wnt beta-catenin signaling, retinoic acid receptor activation, apoptosis, signal transducer and activator of transcription 3, IL-22, IL-12, and IL-10 signaling. Functional enrichment analysis identified apoptosis and cell survival canonical pathways.Conclusion:Select mitotoxic tri-organotins disrupted murine macrophage transcriptional response to LPS, yet triggered inflammasome activation. The differential response pattern suggested unique functional changes in the inflammatory response that may translate to suppressed host defense or prolong inflammation. We posit a framework to examine immune cell effects of environmental mitotoxic compounds for adverse health outcomes. https://doi.org/10.1289/EHP8314

The NLRP11 protein is only expressed in primates and participates in the activation of the canonical NLRP3 and non-canonical NLRP3 inflammasome activation after infection with gram-negative bacteria. Here, we generated a series of defined NLRP11 deletion mutants to further analyze the role of NLRP11 in NLRP3 inflammasome activation. Like the complete NLRP11 deletion mutant (NLRP11-/-), the NLRP11 mutant lacking the NAIP, C2TA, HET-E, and TP1 (NACHT) and leucine-rich repeat (LRR) domains (NLRP11∆N_LRR) showed reduced activation of the canonical NLRP3 inflammasome, whereas a pyrin domain mutant (NLRP11∆PYD) had no effect on NLRP3 activation. The NLRP11-/- and NLRP11∆N_LRR mutants, but not the NLRP11∆PYD mutant, also displayed reduced activation of caspase-4 during infection with the intracytosolic, gram-negative pathogen Shigella flexneri. We found that the human-adapted, acid-fast pathogen Mycobacterium tuberculosis and the opportunistic pathogen Mycobacterium kansasii both activate the non-canonical NLRP11 inflammasome in a caspase-4/caspase-5-dependent pathway. In conclusion, we show that NLRP11 functions in the non-canonical caspase-4/caspase-5 inflammasome activation pathway and the canonical NLRP3 inflammasome pathway and that NLRP11 is required for full recognition of mycobacteria by each of these pathways. Our work extends the spectrum of bacterial pathogen recognition by the non-canonical NLRP11-caspase4/caspase-5 pathway beyond gram-negative bacteria.IMPORTANCEThe activation of inflammasome complexes plays a crucial role in intracellular pathogen detection. NLRP11 and caspase-4 are essential for recognizing lipopolysaccharide (LPS), a molecule found in gram-negative bacteria such as the human pathogens Shigella spp., which activate both canonical NLRP3 and non-canonical inflammasome pathways. Through a series of deletion mutants, we demonstrate that the NACHT and LRR domains of NLRP11, but not its pyrin domain, are critical for detection of S. flexneri. Notably, our research reveals that the acid-fast bacterium M. tuberculosis is also detected by NLRP11 and caspase-4, despite not producing LPS. These findings significantly expand the range of pathogens recognized by NLRP11 and caspase-4 to now include acid-fast bacteria that do not contain LPS and underscore the versatility of these innate immune components in pathogen detection.

Inflammasomes are essential components of the innate immune system and its defense against infections, whereas the dysregulation of inflammasome activation has a detrimental effect on human health. The activation of inflammasomes is subjected to tight regulation to maintain immune homeostasis, yet the underlying mechanism remains elusive. Here, we identify USP3 as a direct deubiquitinating enzyme (DUB) for ASC, the central adapter mediating the assembly and activation of most inflammasomes. USP3 removes the K48-linked ubiquitination on ASC and strengthens its stability by blocking proteasomal degradation. Additionally, USP3 promotes inflammasome activation, and this function was confirmed in mouse models of aluminum (Alum)-induced peritonitis, F. novicida infection and flagellin-induced pneumonia in vivo. Our work unveils that USP3 functions as a key regulator of ASC ubiquitination and maintains the physiological role of ASC in mediating inflammasome activation, and we propose a new mechanism by which the ubiquitination of ASC regulates inflammasome activation.

Atheroprone flow promotes inflammation in endothelial cells, and this process is critical for pathogenesis of many chronic inflammatory conditions such as coronary and carotid artery atherosclerosis, as well as abdominal aortic aneurysm. Signal mediators activated by atheroprone (disturbed) flow that have been described include nuclear factor κB and protein kinase ζ, which is very different from atheroprotective (steady laminar) flow.1 In this issue of Circulation , an article from Xiao et al2 shows the critical role of sterol regulatory element binding protein 2 (SREBP2) on atheroprone flow–mediated Nod-like receptor protein 3 (NLRP3) inflammasome activation. In particular, they showed that atheroprone flow induced both mature form of SREBP2 (SREBP2-N) and SREBP2 mRNA induction, which transcriptionally increase NADPH oxidase 2 (Nox2) and NLRP3 expression, thereby leading to interleukin 1β (IL-1β) expression and endothelial inflammation (Figure 1). In this editorial, we briefly review the NLRP3 inflammasome and SREBP activation system, which play a key role in modulating atheroprone flow–mediated endothelial cell inflammation. We also discuss the following important considerations for the future: the role of local NLRP3 and IL-1β expression, mechanisms for two different types of flow (atheroprone flow versus atheroprotective flow) on SREBP2 activation, and other NLRP3 activators including thioredoxin-interacting protein (TXNIP). Figure 1. Scheme for sterol regulatory element binding protein 2 (SREBP2)-mediated Nod-like receptor protein 3 (NLRP3) inflammasome activation. IL indicates interleukin; Nox, NADPH oxidase; ROS, reactive oxygen species; and TXNIP, thioredoxin-interacting protein. Article see p 632 The inflammasome is a protein complex that serves as a platform for the maturation of caspase-1 subsequent activation, leading to proteolytic maturation and secretion of IL-1β and IL-18 (Figure 8 in Xiao et al2). Three essential components of inflammasome are a sensor protein, the adapter protein ASC, and the inflammatory protease caspase-1. As a sensor protein, Nod-like receptor (NLR) family (NLRP1, NLRP3, and …

The activation of NLRP3 inflammasome and its function in anti-infection immunity of largemouth bass (Micropterus salmoides).

Autophagy is a key regulator of cellular homeostasis that can be activated by pathogen-associated molecules and recently has been shown to influence IL-1β secretion by macrophages. However, the mechanisms behind this are unclear. Here, we describe a novel role for autophagy in regulating the production of IL-1β in antigen-presenting cells. After treatment of macrophages with Toll-like receptor ligands, pro-IL-1β was specifically sequestered into autophagosomes, whereas further activation of autophagy with rapamycin induced the degradation of pro-IL-1β and blocked secretion of the mature cytokine. Inhibition of autophagy promoted the processing and secretion of IL-1β by antigen-presenting cells in an NLRP3- and TRIF-dependent manner. This effect was reduced by inhibition of reactive oxygen species but was independent of NOX2. Induction of autophagy in mice in vivo with rapamycin reduced serum levels of IL-1β in response to challenge with LPS. These data demonstrate that autophagy controls the production of IL-1β through at least two separate mechanisms: by targeting pro-IL-1β for lysosomal degradation and by regulating activation of the NLRP3 inflammasome.

Activation of the inflammasome leads to caspase-1 activation and maturation of pro-inflammatory cytokines, IL-1β and IL-18. The inflammasome plays a pivotal role in host defense against microbial pathogens. However, its uncontrolled activation is associated with inflammatory disorders, suggesting that regulation of inflammasome activation is important to prevent detrimental effects. In this study, we found that endogenous and exogenous NO inhibits the NLRP3 inflammasome. iNOS was involved in the regulation of NLRP3 inflammasome activation by either IFN-β pretreatment or long-term LPS stimulation. Furthermore, the NO donor SNAP markedly inhibited NLRP3 inflammasome activation, while the AIM2 and NLRC4 inflammasomes were only partially inhibited by SNAP. An increase in mitochondrial reactive oxygen species induced by ATP was only modestly affected by SNAP treatment. S-nitrosylation of NLRP3 was detected in macrophages treated with SNAP, and this modification may account for the NO-mediated mechanism controlling inflammasome activation. Taken together, these results revealed a novel role of NO in regulating the NLRP3 inflammasome.

The Nlrp3 inflammasomes are essential for protection against several infections and autoimmune diseases. Despite the plethora of research focused on understanding the roles of NLRP3 in various disease settings, the mechanisms controlling its activation remain enigmatic. Herein, we show through genetic studies that FADD or caspase-8 is required for both transcriptional priming and posttranslational activation of the canonical and non-canonical Nlrp3 inflammasomes. FADD and caspase-8 were required for caspase-1 activation during canonical Nlrp3 inflammasome activation by both soluble (LPS+ATP) and particulate (Silica) ligands. We showed that FADD and caspase-8 also regulate Nlrp3 expression. Furthermore, both FADD and caspase-8 were critical for caspase-11 and caspase-1 activation during non-canonical Nlrp3 inflammasome activation by C. rodentium and E. coli. These requirements of FADD and caspase-8 for caspase-1 activation were specific to the Nlrp3 inflammasome since the activation of Nlrc4 inflammasome by S. typhimurium was not hampered by deletion of FADD or caspase-8. In vivo LPS challenge or C. rodentium infection of FADD or caspase-8-deficient mice resulted in impaired IL-1β production compared to their littermate controls. Thus, our results demonstrate that FADD and caspase-8 are critical mediators of the canonical and non-canonical Nlrp3 inflammasomes.

Rationale Inflammasome assembly and activation is a complex process regulated, in part, by posttranslational modification of inflammasome proteins. ASC is a universal adaptor in virtually all inflammasome platforms and its modification is critical in regulation of inflammasome function. In particular, PTK inhibitor AG126 greatly inhibits inflammasome activation. Methods We generated stable THP-1 cells with either overexpression of ASC (YFP-ASC) or knock down of ASC (siRNA and CRISPR) followed by ASC knock in. To test the role of ASC phosphorylation in inflammasome assembly and activity, we mutated the highly conserved tyrosine 146 to Y146A. Cells were stimulated with LPS or live bacteria and inflammasome activity was measured by fluorescent microscopy, ELISA, and immunoblotting. Results Upon inflammasome activation ASC is released from the cell together with IL-1β, IL-18 and other inflammasome proteins. Inhibition of select tyrosine kinases severely reduces inflammasome activation, pyroptosis and ASC release. Elimination of the potential phosphorylation site Y146 dramatically changes visible ASC complex formation – from a single round speck to a filamentous structure. Cells with the Y146A ASC mutation show significantly abrogated inflammasome activation as compared to a wild type. Screening of a PTK inhibitor library (Selleckchem) linked several tyrosine kinases to regulating ASC phosphorylation and inflammasome activity. Conclusion ASC phosphorylation at Y146 by tyrosine kinases is an important regulator of inflammasome activity and pyroptosis.

MicroRNA-133a-1 regulates inflammasome activation through uncoupling protein-2

The assembly and activation of the inflammasomes are tightly regulated by post‐translational modifications, including ubiquitin. Deubiquitinases (DUBs) counteract the addition of ubiquitin and are essential regulators of immune signalling pathways, including those acting on the inflammasome. How DUBs control the assembly and activation of inflammasomes is unclear. Here, we show that the DUBs USP7 and USP47 regulate inflammasome activation in macrophages. Chemical inhibition of USP7 and USP47 blocks inflammasome formation, independently of transcription, by preventing ASC oligomerisation and speck formation. We also provide evidence that the ubiquitination status of NLRP3 itself is altered by inhibition of USP7 and USP47. Interestingly, we found that the activity of USP7 and USP47 increased in response to inflammasome activators. Using CRISPR/Cas9 in the macrophage cell line THP‐1, we show that inflammasome activation is reduced when both USP7 and USP47 are knocked down. Altogether, these data reveal a new post‐transcriptional role for USP47 and USP7 in inflammation by regulating inflammasome activation and the release of the pro‐inflammatory cytokines IL‐1β and IL‐18, and implicate dual USP7 and USP47 inhibitors as potential therapeutic agents for inflammatory disease.

Guanylate-binding proteins (GBPs) are a subfamily of interferon-inducible proteins that undertake distinct roles in the the context of bacteria, virus, chlamydia and parasites infections. These proteins exert a notable influence on the progression and outcomes of infectious diseases. Within the realm of host cell-autonomous immunity against pathogens, GBPs have been identified as the regulators of pyroptosis through canonical and noncanonical inflammasome activation pathways. In this review, we summarize the structure and evolution of GBP family members, the canonical and noncanonical inflammasome activation pathways, the roles of GBPs in regulating inflammasome activation, and the mechanisms of GBPs affecting infections induced by different pathogens. We hope to provide new basic research clues for the pathogenesis and diagnosis and treatment of infectious diseases.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta, leading to hallmark motor symptoms such as bradykinesia, tremors, and rigidity. Emerging evidence suggests that the dysregulation or aberrant expression of long noncoding RNAs (lncRNAs) plays a critical role in the pathogenesis of PD by activating the inflammasome, either directly or via oxidative stress. Aberrant lncRNA expression has been linked to alterations in genes related to oxidative stress, causing an imbalance between reactive oxygen species (ROS) and antioxidant defenses. This imbalance contributes to mitochondrial dysfunction and neuronal damage. The NLRP3 inflammasome is a multiprotein complex comprising a sensor protein (eg, NLRP3), an adaptor protein (ASC), and an effector protein (caspase‐1). Its activation involves priming via NF‐κB signaling and is triggered by ROS, mitochondrial dysfunction, death‐associated molecular patterns, or extracellular ATP. Once activated, the inflammasome promotes the cleavage and maturation of the proinflammatory cytokines IL‐1β and IL‐18, amplifying neuroinflammation and leading to neurodegeneration in PD. Crosstalk between dysregulated lncRNAs, ROS production, and inflammasome activation creates a vicious cycle of neuroinflammation and neurodegeneration, exacerbating PD progression. This review explores the molecular mechanisms linking lncRNA dysregulation to inflammasome activation in PD, either directly or through oxidative stress. It also highlights key lncRNAs involved in these processes. Furthermore, potential therapeutic strategies targeting these pathways, such as antioxidants, lncRNA modulators, and inflammasome inhibitors, offer promising avenues to mitigate neuroinflammation and slow neurodegeneration in PD.

The NLRP11 protein is only expressed in primates and participates in the activation of the canonical NLRP3 and non-canonical NLRP3 inflammasome activation after infection with gram-negative bacteria. Here, we generated a series of defined NLRP11 deletion mutants to further analyze the role of NLRP11 in NLRP3 inflammasome activation. Like the complete NLRP11 deletion mutant (NLRP11−/−), the NLRP11 mutant lacking the NACHT and LRR domains (NLRP11ΔN_LRR) showed reduced activation of the canonical NLRP3 inflammasome, whereas a pyrin domain mutant (NLRP11ΔPYD) had no effect on NLRP3 activation. The NLRP11−/− and NLRP11ΔN_LRR mutants but not the NLRP11ΔPYD mutant also displayed reduced activation of caspase-4 during infection with the intracytosolic, gram-negative pathogen Shigella flexneri. We found that the human adapted, acid-fast pathogen Mycobacterium tuberculosis and the opportunistic pathogen M. kansasii both activate the non-canonical NLRP11 inflammasome in a caspase-4/5-dependent pathway. In conclusion, we show that NLRP11 functions in the non-canonical caspase-4/5 inflammasome activation pathway and the canonical NRLP3 inflammasome pathway, and that NLRP11 is required for full recognition of mycobacteria by each of these pathways. Our work extends the spectrum of bacterial pathogen recognition by the non-canonical NLRP11-caspase4/5 pathway beyond gram-negative bacteria.

The NLRP3 (NOD-like receptor family, pyrin domain containing 3) inflammasome is a multiprotein complex that orchestrates innate immune responses to infection and cell stress through activation of caspase-1 and maturation of inflammatory cytokines pro-interleukin-1β (pro-IL-1β) and pro-IL-18. Activation of the inflammasome during infection can be protective, but unregulated NLRP3 inflammasome activation in response to non-pathogenic endogenous or exogenous stimuli can lead to unintended pathology. NLRP3 associates with mitochondria and mitochondrial molecules, and activation of the NLRP3 inflammasome in response to diverse stimuli requires cation flux, mitochondrial Ca(2+) uptake, and mitochondrial reactive oxygen species accumulation. It remains uncertain whether NLRP3 surveys mitochondrial integrity and senses mitochondrial damage, or whether mitochondria simply serve as a physical platform for inflammasome assembly. The structure of the active, caspase-1-processing NLRP3 inflammasome also requires further clarification, but recent studies describing the prion-like properties of ASC have advanced the understanding of how inflammasome assembly and caspase-1 activation occur while raising new questions regarding the propagation and resolution of NLRP3 inflammasome activation. Here, we review the mechanisms and pathways regulating NLRP3 inflammasome activation, discuss emerging concepts in NLRP3 complex organization, and expose the knowledge gaps hindering a comprehensive understanding of NLRP3 activation.