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Abstract Background Cerebral ischemia is a devastating condition in which the outcome is heavily influenced by inflammatory processes, which can augment primary injury caused by reduced blood supply. The cytokines interleukin-1α (IL-1α) and IL-1β are key contributors to ischemic brain injury. However, there is very little evidence that IL-1 expression occurs at the protein level early enough (within hours) to influence brain damage after stroke. In order to determine this we investigated the temporal and spatial profiles of IL-1α and IL-1β expression after cerebral ischemia. Findings We report here that in mice, as early as 4 h after reperfusion following ischemia induced by occlusion of the middle cerebral artery, IL-1α, but not IL-1β, is expressed by microglia-like cells in the ischemic hemisphere, which parallels an upregulation of IL-1α mRNA. 24 h after ischemia IL-1α expression is closely associated with areas of focal blood brain barrier breakdown and neuronal death, mostly near the penumbra surrounding the infarct. The sub-cellular distribution of IL-1α in injured areas is not uniform suggesting that it is regulated. Conclusions The early expression of IL-1α in areas of focal neuronal injury suggests that it is the major form of IL-1 contributing to inflammation early after cerebral ischemia. This adds to the growing body of evidence that IL-1α is a key mediator of the sterile inflammatory response.

Inflammasomes are immune complexes whose activation leads to the release of pro-inflammatory cytokines IL-18 and IL-1β. Type I IFNs play a role in fighting infection and stimulate the expression of IFN-stimulated genes (ISGs) involved in inflammation. Despite the importance of these cytokines in inflammation, the regulation of inflammasomes by type I IFNs remains poorly understood. Here, we analysed RNA-sequencing data from patients with monogenic interferonopathies and found an up-regulation of several inflammasome-related genes. To investigate the effect of type I IFN on the inflammasome, we treated human monocyte-derived macrophages with IFN-α and observed an increase in CASP1 and GSDMD mRNA levels over time, whereas IL1B and NLRP3 were not directly correlated to IFN-α exposure time. IFN-α treatment reduced the release of mature IL-1β and IL-18, but not caspase-1, in response to ATP-mediated NLRP3 inflammasome activation, suggesting regulation occurs at cytokine expression levels and not the inflammasome itself. However, more studies are required to investigate how regulation by IFN-α occurs and impacts NLRP3 and other inflammasomes at both transcriptional and post-translational levels., (© 2024 Díaz-Pino et al.)

Posttranslational modifications are crucial in determining the functions of proteins in the cell. Modification of the NLRP3 inflammasome by the ubiquitin system has recently emerged as a new level of regulation of the inflammasome complex. Here we describe a method to detect poly-ubiquitination of NRLP3 using two different approaches: (i) detection with a ubiquitin antibody or (ii) using TUBEs (Tandem Ubiquitin Binding entities). This approach can be used to detect ubiquitination of other NLRs or other proteins., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

IL-37 is an anti-inflammatory member of the IL-1 family that dampens inflammation associated with many noncommunicable diseases. However, mechanisms of IL-37 regulation remain understudied. We aimed to investigate the enzymatic cleavage of IL-37 that potentiates extracellular signalling, as well as pathways of IL-37 secretion. In human monocytes, mature IL-37 (mIL-37) was released following canonical NLRP3 inflammasome activation. The release of IL-37 was blocked by inhibiting plasma membrane permeability and in gasdermin-D-deficient THP-1 cells. While the cleavage of IL-37 was found to be constitutive, the release of mIL-37 was blocked in NLRP3-deficient THP-1 cells and by NLRP3 inhibitor MCC950 in THP-1s and primary human monocytes. IL-37 secretion also occurred after 18-h exposure to LPS, independently of the alternative NLRP3 inflammasome. This LPS-dependent IL-37 secretion required plasma membrane permeability, but not conventional protein secretion apparatus. Mutagenesis of the suggested caspase-1 cleavage site (D20) or the proposed alternative cleavage site (V46) did not completely block IL-37 processing. Therefore, we propose a novel pathway in which IL-37 is cleaved by caspase-1-independent mechanisms and released following canonical and alternative NLRP3 inflammasome triggers by differential pathways., (© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Non-steroidal anti-inflammatory drugs (NSAIDs) can damage the small intestine, mainly through an involvement of enteric bacteria. This study examined the pathophysiology of NSAID-associated intestinal lesions in a rat model of diclofenac-enteropathy and evaluated the effect of rifaximin on small bowel damage. Enteropathy was induced in 40-week old male rats by intragastric diclofenac (4 mg/kg BID, 14 days). Rifaximin (delayed release formulation) was administered (50 mg/kg BID) 1 h before the NSAID. At the end of treatments, parameters dealing with ileal damage, inflammation, barrier integrity, microbiota composition, and TLR-NF-κB-inflammasome pathway were evaluated. In addition, the modulating effect of rifaximin on NLRP3 inflammasome was tested in an in vitro cell system. Diclofenac induced intestinal damage and inflammation, triggering an increase in tissue concentrations of tumor necrosis factor and interleukin-1β, higher expression of TLR-2 and TLR-4, MyD88, NF-κB and activation of caspase-1. In addition, the NSAID decreased ileal occludin expression and provoked a shift of bacterial phyla toward an increase in Proteobacteria and Bacteroidetes abundance. All these changes were counterbalanced by rifaximin co-administration. This drug was also capable of increasing the proportion of Lactobacilli, a genus depleted by the NSAID. In LPS-primed THP-1 cells stimulated by nigericin (a model to study the NLRP3 inflammasome), rifaximin reduced IL-1β production in a concentration-dependent fashion, this effect being associated with inhibition of the up-stream caspase-1 activation. In conclusion, diclofenac induced ileal mucosal lesions, driving inflammatory pathways and microbiota changes. In conclusion, rifaximin prevents diclofenac-induced enteropathy through both anti-bacterial and anti-inflammatory activities.

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., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

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-1β 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.

Posttranslational modifications are crucial in determining the functions of proteins in the cell. Modification of the NLRP3 inflammasome by the ubiquitin system has recently emerged as a new level of regulation of the inflammasome complex. Here, we describe a method to detect polyubiquitination of NRLP3 using two different approaches: (1) detection with an ubiquin antibody or (2) using TUBE (Tandem Ubiquitin Binding entities). This approach can be used to detect ubiquitination of other NLR or other components of the inflammasome.

Inflammasomes are molecular complexes that initiate innate immune response. They are mainly expressed by immune cells; however, molecular manipulations in these cells remain very difficult. Here, we describe a simple protocol to overexpress and activate functional NRLP3 inflammasomes in HEK293 cells.

Apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC) is a key adaptor molecule required for the inflammatory processes. ASC acts by bridging NLRP proteins, such as NLRP3, with procaspase-1 within the inflammasome complex, which subsequently results in the activation of caspase-1 and the secretion of IL-1β and IL-18. In response to bacterial infection, ASC also forms specks by self-oligomerization to activate caspase-1 and induce pyroptosis. Hitherto, the role of these specks in NLRP3 inflammasome activation in response to danger signals, such as a hypotonic environment, largely has been unexplored. In this article, we report that, under hypotonic conditions and independently of NLRP3, ASC was able to form specks that did not activate caspase-1. These specks were not associated with pyroptosis and were controlled by transient receptor potential vanilloid 2 channel-mediated signaling. However, interaction with NLRP3 enhanced ASC speck formation, leading to fully functional inflammasomes and caspase-1 activation. This study reveals that the ASC speck can present different oligomerization assemblies and represents an essential step in the activation of functional NLRP3 inflammasomes., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Members of the nucleotide binding and oligomerization domain-like receptors (NLRs) and the PYD and CARD domain containing adaptor protein (PYCARD) assemble into multi-protein platforms, termed inflammasomes, to mediate in the activation of caspase-1 and the subsequent secretion of IL-1β and IL-18, and the induction of pyroptotic cell death. While the recognition site for caspase-1 is well conserved in mammals, most of the non-mammalian IL-1β genes cloned so far lack this conserved site. We report here that stimulation or infection of seabream macrophages (MØ) led to the caspase-1-independent processing and release of IL-1β. In addition, several classical activators of the NLRP3 inflammasome failed to activate caspase-1 and to induce the processing and release of IL-1β. Furthermore, the processing of IL-1β in seabream MØ is not prevented by caspase-1 or pan-caspase inhibitors, and recombinant seabream caspase-1 failed to process IL-1β. However, the pharmacological inhibition of caspase-1 impaired Salmonella enterica sv. Typhimurium-induced cell death. These results suggest a role for the inflammasome and caspase-1 in the regulation of pyroptotic cell death in fish and support the idea that its use as a molecular platform for the processing of pro-inflammatory cytokines arose after the divergence of fish and tetrapods.

Cell volume regulation is a primitive response to alterations in environmental osmolarity. The NLRP3 inflammasome is a multiprotein complex that senses pathogen- and danger-associated signals. Here, we report that, from fish to mammals, the basic mechanisms of cell swelling and regulatory volume decrease (RVD) are sensed via the NLRP3 inflammasome. We found that a decrease in extracellular osmolarity induced a K(+)-dependent conformational change of the preassembled NLRP3-inactive inflammasome during cell swelling, followed by activation of the NLRP3 inflammasome and caspase-1, which was controlled by transient receptor potential channels during RVD. Both mechanisms were necessary for interleukin-1β processing. Increased extracellular osmolarity prevented caspase-1 activation by different known NLRP3 activators. Collectively, our data identify cell volume regulation as a basic conserved homeostatic mechanism associated with the formation of the NLRP3 inflammasome and reveal a mechanism for NLRP3 inflammasome activation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Introduction: The inflammasomes have emerged as key mediators of inflammation and immunity, yet clinical application of this knowledge has been limited by a lack of specific and drug-like antagonists. Recent studies using inflammasome knockout mice have shown that different inflammasomes control immunity in different pathologies. Drug-like antagonists acting up- or down-stream of the inflammasome pathway have been successfully used in clinics as important therapeutics to treat different inflammatory diseases., Areas Covered: The current literature has been reviewed on the role of inflammasomes in inflammatory disease, focusing on potential therapeutic applications of selective inflammasome antagonists as anti-inflammatory agents. Particular emphasis has been placed on the potential role of the different inflammasomes in common inflammatory diseases. The latest clinical developments for drugs targeting inflammasome pathways are covered., Expert Opinion: Recent studies using inflammasome knockout mice suggest its importance as a potential therapeutic target for the treatment of inflammatory disease. However, efficacious antagonists for the inflammasome for use in clinical studies are still at an early stage of development. Developing selective inflammasome antagonists is a challenge that if met, offers promise for the treatment of chronic inflammatory diseases. Major developments in this area will include the identification of reliable high-throughput screening methods for compounds directly targeting inflammasome assembly.

The control of biochemical fluxes is distributed, and to perturb complex intracellular networks effectively it is often necessary to modulate several steps simultaneously. However, the number of possible permutations leads to a combinatorial explosion in the number of experiments that would have to be performed in a complete analysis. We used a multiobjective evolutionary algorithm to optimize reagent combinations from a dynamic chemical library of 33 compounds with established or predicted targets in the regulatory network controlling IL-1β expression. The evolutionary algorithm converged on excellent solutions within 11 generations, during which we studied just 550 combinations out of the potential search space of ~9 billion. The top five reagents with the greatest contribution to combinatorial effects throughout the evolutionary algorithm were then optimized pairwise. A p38 MAPK inhibitor together with either an inhibitor of IκB kinase or a chelator of poorly liganded iron yielded synergistic inhibition of macrophage IL-1β expression. Evolutionary searches provide a powerful and general approach to the discovery of new combinations of pharmacological agents with therapeutic indices potentially greater than those of single drugs.

Caspases are a family of cysteine proteases that fulfil critical roles in mammalian apoptosis and in the proteolytic activation of cytokines. In humans, the caspase family includes 13 members whose functions seem to correlate with their phylogenetic relationship. They are classified into two main groups, the cell death (apoptotic) and the inflammatory caspases. Caspase-1 is the best characterized inflammatory caspase and is responsible for the processing of interleukin-1beta (IL-1beta), IL-18 and IL-33. Despite the importance of caspase-1 in inflammation, no information is available on the presence and activity of this enzyme in fish. In this study, we cloned a caspase-1-like gene from the bony fish gilthead seabream (Sparus aurata L.) which shows a conserved N-terminal caspase-recruitment domain (CARD) and a C-terminal caspase catalytic domain. The seabream caspase-1 gene was expressed in 1 day post-hatching larvae and its mRNA levels increased throughout development. In adult fish, caspase-1 was found to be constitutively expressed in all immune tissues analyzed and, unexpectedly, infection of fish and stimulation of professional phagocytes in vitro decreased its mRNA levels. It was also demonstrated that the recombinant seabream caspase-1 ectopically expressed in HEK293 cells was able to cleave a caspase-1 specific substrate, this activity being enhanced upon activation of the rat P2X7 receptor with BzATP. Finally, seabream fibroblast cell line SAF-1 and primary leukocytes showed endogenous caspase-1 activity, which was almost completely inhibited by a caspase-1 specific inhibitor.

Interleukin-1beta (IL-1beta) is the prototypic pro-inflammatory cytokine. All the biological effects of IL-1beta are mediated through interaction with type 1 IL-1 receptor (IL-1RI), whereas another receptor, called type 2 IL-1R (IL-1RII), lacks an intracellular signalling domain and acts as a decoy receptor that down-regulates responses to IL-1beta. Although both receptors are present in bony fish, their expression and biological role in the regulation of IL-1beta activity in non-mammalian vertebrates remain to be established. In this study, a homologue of mammalian IL-1RII was isolated and characterized in the gilthead seabream (Sparus aurata). The seabream IL-1RII harboured two Ig-like domains in its extracellular region and a short cytoplasmic tail lacking a signalling domain. The seabream IL-1RII cDNA showed an unexpectedly long 3'UTR compared with that from other species and contained three ATTTA instability motifs, which seem to be responsible for its relatively short half-life (less than 2h). The expression of seabream IL-1RII was dramatically up-regulated after infection with Vibrio anguillarum in all the immune tissues examined and was even more strongly induced than the IL-1beta gene in the head kidney, spleen and liver. Strikingly, the mRNA levels of IL-1RII were 15-fold higher than those of IL-1beta in the liver, suggesting a role for this organ in the neutralization of IL-1beta leaking into the systemic circulation from the sites of inflammation. In vitro, bacterial DNA and flagellin increased the mRNA levels of IL-1RII in macrophages, while only flagellin was able to weakly induce its expression in acidophilic granulocytes. Finally, the seabream IL-1RII was localized in the plasma membrane when expressed in HEK293 cells and was able to bind IL-1beta.

Phagocytic cells form the cellular arm of the innate immune system. A primary role of these cells is an ability to discriminate large number of potential pathogens from self, using a restricted number of receptors. In the gilthead seabream, acidophilic granulocytes and macrophages have been described as the professional phagocytes of this species. However, no direct functional comparisons between these two phagocytic lineages exist for the seabream or for other teleost species. Therefore, purified fractions of acidophilic granulocytes and macrophages were used to characterize the ability of these cells to recognize and respond to different pathogen-associated molecular patterns (PAMPs) and the data obtained were then correlated with the expression of several pattern-recognition receptors (PRRs). The time course of the respiratory burst of acidophilic granulocytes stimulated with different PAMPs showed that muramyldipeptide (MDP), the ligand for NOD2 in mammals, induced maximal activation earlier than several ligands for toll-like receptors (TLRs), including bacterial DNA, flagellin and lipopolysaccharide (LPS). In addition, all these PAMPs strongly increased the phagocytic and bactericidal activities of acidophilic granulocytes, while other PAMPs, including poly I:C, Pam3CSK(4) and zymosan, failed to do so. The stimulation of acidophilic granulocytes and macrophages by PAMPs also resulted in the up-regulation of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), cyclooxygenase-2 (COX-2) and TLRs, although the kinetics and expression profiles observed for each cell type differed. These results suggest different roles for professional phagocytes of fish in the recognition and elimination of pathogens and in the regulation of adaptive immune responses.

Mammalian interleukin-1beta (IL-1beta) is produced as a biologically inactive precursor molecule, which is proteolytically cleaved to an active form by IL-1beta-converting enzyme (ICE) after the activation of P2X(7) receptor by extracellular ATP. The mechanism of IL-1beta release in non-mammalian vertebrates is largely unknown, although most of the IL-1beta gene sequences lack a conserved ICE recognition site. Here we have cloned the P2X(7) receptor from the bony fish seabream and compared agonist and antagonist profiles at this and other non-mammalian P2X(7) receptors expressed in HEK cells, as well in seabream SAF-1 cells expressing endogenous P2X(7) receptors. We used this information to further investigate the mechanisms of IL-1beta release induced by mammalian and fish P2X(7) receptors. Despite phosphatidylserine externalization and cell permeabilization in seabream leukocytes after the addition of high BzATP concentrations, IL-1beta remained unprocessed within the cell. However, activation of rat P2X(7) receptors ectopically expressed in HEK293 together with human ICE led to the specific secretion of unprocessed seabream IL-1beta. In contrast, neither seabream nor zebrafish P2X(7) receptors induced the secretion of mammalian or fish IL-1beta when expressed in HEK293, while a chimeric receptor harboring the ATP-binding domain of seabream P2X(7) and the intracellular region of its rat counterpart did so. These findings indicate that P2X(7) receptor-mediated activation of ICE and release of IL-1beta result from different downstream signaling pathways and suggest that although the mechanisms involved in IL-1beta secretion are conserved throughout evolution, distinct inflammatory signals have been selected for the secretion of this cytokine in different vertebrates.

We report the molecular cloning of the colony-stimulating factor-1 receptor gene from the bony fish gilthead seabream (sbCSF-1R). The deduced sbCSF-1R shows a predicted signal sequence, a transmembrane domain and a tyrosine kinase domain, all in conserved positions. A transcript showing a premature stop codon that predicted the removal of 84 C-terminal amino acids was also found. RT-PCR expression studies demonstrate that, although the sbCSF-1R transcripts are found in different immune tissues, including gill, liver, spleen, blood, peritoneal exudate, thymus and head-kidney (HK), their expression is confined to the monocyte/macrophage lineage. Furthermore, the expression of sbCSF-1R might be modulated by the activation stage of the macrophages, since both the infection of fish and the in vitro activation of leukocytes resulted in the down-regulation of gene expression. These data indicate that the CSF-1R may be used as a specific probe for cells of the monocyte/macrophage lineage in the gilthead seabream, an immunological tractable fish model. In addition, the functional characterisation of the CSF-1R and its ligand may shed light into the mechanisms of proliferation and the pathways of differentiation of macrophages in bony fish.