Reduced IL-1β Secretion Research Articles

Intestinal mesenchymal cells regulate immune responses and promote epithelial regeneration in vitro and in dextran sulfate sodium-induced experimental colitis in mice.

Disruption of the intestinal mucosal tolerance, that is, the immunological unresponsiveness to innocuous food antigens and the commensal microbiota, in the colon is associated with several chronic diseases including inflammatory bowel disease (IBD). Understanding the mechanisms responsible for intestinal mucosal tolerance has potential translational value for its therapy and management. Human intestinal mesenchymal cells (iMCs) play important roles in colonic mucosal tolerance, but further studies on their tissue regenerative and immunomodulatory capacities are necessary in order to fully understand their function in health and disease. In this study, we have isolated and analysed the capacity of human iMCs to promote wound healing and modulate immune responses in vitro and in vivo, using the dextran sulfate sodium (DSS)-induced colitis model. Cultured iMCs were CD45- CD73+ CD90+ CD105+ and accelerated the wound closure in a normal colon mucosa (NCM) 356 human epithelial cell wound healing assay. Furthermore, iMCs blocked the LPS-mediated induction of TNF-α in THP-1 macrophages and inhibited the proliferation of peripheral blood mononuclear cells, partly through the induction of indoleamine-2,3-dioxygenase. In DSS colitic mice, iMCs administration reduced the disease activity index and ameliorated intestinal tissue damage and permeability. Furthermore, iMCs reduced intestinal inflammation, evidenced by a decreased mRNA expression of pro-inflammatory cytokines, reduced IL-1β secretion by intestinal explants and inhibited colonic iNOS protein expression. Our data show that human iMCs isolated from the noninflamed intestine possess tissue-regenerative and immunomodulatory capacities that could potentially be harnessed/restored in order to reduce IBD severity.

Curcumin suppresses neuroinflammation to protect neurons by preventing NLRP3 inflammasome activation

Introduction Nucleotide-binding and oligomerization domain like receptors protein 3 (NLRP3) inflammasome-mediated interleukin (IL)-1β secretion plays an important role in the progression of Alzheimer’s disease (AD). Curcumin has been shown to improve cognitive impairment and learning ability of AD mice by reducing IL-1β secretion. However, its exact mechanism of action remains unclear. In the present study, we explored the relationship between the neuroprotective effect of curcumin and activation of the NLRP3 inflammasome pathway. Methods BV2 cells were primed with 500 ng/mL lipopolysaccharide (LPS) for 4 h and subsequently treated with 50 μM Aβ25-35 for 24 h or pretreated with 2.5–10 μM curcumin for 4 h and exposed to 50 μM Aβ25-35 for 24 h. The effects of curcumin and Aβ25-35 were assessed by the CCK8 assay. ELISA was used for the detection of IL-1β, IL-6, and tumor necrosis factor (TNF)-α levels in the supernatant of the cell culture medium. The viability of SH-SY5Y cells, which were incubated with conditioned medium (CM) was assessed using the CCK8 assay. The percentage of apoptotic SH-SY5Y cells incubated with CM was assessed using Annexin V-FITC/PI staining flow cytometry analysis. The expression levels of NLRP3, caspase-1 and IL-1β were observed by western blot and immunofluorescence staining analyses; the mRNA levels of nlrp3, caspase-1 and IL-1β were analyzed using qRT-PCR. Results Low (2.5 μM), medium (5 μM), and high (10 μM) concentrations of curcumin and 50 μM Aβ25-35 were used to perform the experiments in the present study. Curcumin attenuated the IL-1β, IL-6, and TNF-α release and increased SH-SY5Y cell activity, while decreasing the apoptotic percentage of SH-SY5Y cells using Aβ25-35 for cell stimulation ( p < 0.05). Furthermore, curcumin inhibited the expression of NLRP3, caspase-1 and IL-1β and nlrp3 in BV-2 cells ( p < 0.05), However, curcumin did not affect the expression levels of caspase-1 and IL-1β ( p > 0.05) Conclusion Overall, the data indicated that curcumin is a promising neuroprotective agent for suppressing neuroinflammation by inhibiting the NLRP3 inflammasome pathway.

Multi-walled carbon nanotubes induce IL-1β secretion by activating hemichannels-mediated ATP release in THP-1 macrophages

Multi-walled carbon nanotubes (MWCNTs) are known to induce pulmonary inflammatory effects through stimulating pro-inflammatory cytokine secretion from alveolar macrophages. Despite extensive studies on MWCNTs’ pro-inflammatory reactivity, the understanding of molecular mechanisms involved is still incomplete. In this study, we investigated hemichannel’s involvement in MWCNTs-induced macrophage IL-1β release. Our results showed that the unmodified and COOH MWCNTs could induce ATP release and ATP-P2X7R axis-dependent IL-1β secretion from THP-1 macrophages. By using various inhibitors, we confirmed that the MWCNTs-induced ATP release was primarily through hemichannels. EtBr dye uptake assay detected significant hemichannels opening in MWCNTs exposed THP-1 macrophages. Inhibition of hemichannels by CBX, 43Gap27, or 10Panx1 pretreatment results in decreased ATP and IL-1β release. The addition of ATP restored the reduced IL-1β secretion level from hemichannel inhibition. We also confirmed with five other types of MWCNTs that the induction of hemichannels by MWCNTs strongly correlates with their capacity to induce IL-1β secretion. Taken together, we conclude that hemichannels-mediated ATP release and subsequent NLRP3 inflammasome activation through P2X7R may be one mechanism by which MWCNTs induce macrophage IL-1β secretion. Our findings may provide a novel molecular mechanism for MWCNTs induced IL-1β secretion.

Direct Binding to NLRP3 Pyrin Domain as a Novel Strategy to Prevent NLRP3-Driven Inflammation and Gouty Arthritis.

The NLRP3 inflammasome is closely linked to the pathophysiology of a wide range of inflammatory diseases. This study was undertaken to identify small molecules that directly bind to NLRP3 in order to develop pharmacologic interventions for NLRP3-related diseases. A structure-based virtual screening analysis was performed with ~62,800 compounds to select efficient NLRP3 inhibitors. The production of caspase 1-p10 and interleukin-1β (IL-1β) was measured by immunoblotting and enzyme-linked immunosorbent assay to examine NLRP3 inflammasome activation. Two gouty arthritis models and an air pouch inflammation model induced by monosodium urate monohydrate (MSU) crystal injection were used for in vivo experiments. Primary synovial fluid cells from gout patients were used to determine the relevance of NLRP3 inflammasome inhibition in human gout. Beta-carotene (provitamin A) suppressed the NLRP3 inflammasome activation induced by various activators, including MSU crystals, in mouse bone marrow-derived primary macrophages (P < 0.05). Surface plasmon resonance analysis demonstrated the direct binding of β-carotene to the pyrin domain (PYD) of NLRP3 (KD = 3.41 × 10-6 ). Molecular modeling and mutation assays revealed the interaction mode between β-carotene and the NLRP3 PYD. Inflammatory symptoms induced by MSU crystals were attenuated by oral administration of β-carotene in gouty arthritis mouse models (P < 0.05), correlating with its suppressive effects on the NLRP3 inflammasome in inflamed tissues. Furthermore, β-carotene reduced IL-1β secretion from human synovial fluid cells isolated from gout patients (P < 0.05), showing its inhibitory efficacy in human gout. Our results present β-carotene as a selective and direct inhibitor of NLRP3, and the binding of β-carotene to NLRP3 PYD as a novel pharmacologic strategy to combat NLRP3 inflammasome-driven diseases, including gouty arthritis.

Therapeutic hypothermia rescues severe acute lung injury (ALI) by IL-1β-induced neutrophil extracellular traps (NETs) formation through down-regulation of Gasdermin D

Abstract ARDS is a serious ALI defined by the acute onset of non-cardiogenic pulmonary edema and hypoxemia. We have previously published that NLRP3 inflammasome is required to develop hypoxemia in LPS plus mechanical ventilator (MV) two-hit-induced ALI. We initially observed that, despite equal neutrophil infiltration, severe alveolar leakage and hypoxemia were not observed in Il1r1KO mice, confirming that NLRP3 inflammasome is critical in this two-hit induced hypoxemia. However, this hypoxemia was significantly attenuated in neutrophil-depleted mice. Furthermore, we found that NETs formations were significantly attenuated in Il1r1KO mice. These data demonstrated that IL-1R signaling plays a regulating role in neutrophil functionality in the alveoli, and not their infiltration. We next examined whether NETs formation contributed to develop hypoxemia in two-hit ALI. by Pad4 inhibitor and DNase I. Both treatments improved the development of hypoxemia. In the course of experiments, we serendipitously found that body temperature is a critical factor regulating severe hypoxemia. Therapeutic hypothermia at 32°C during MV rescued development of hypoxemia with significantly attenuated alveolar leakage, reduced IL-1β secretion, and less NETs formation. 32°C condition inhibited IL-1β secretion by Mϕ as well as NETs induction. Unexpectedly, we observed similar Caspase-1 activities in LPS-primed Mϕ stimulated with ATP or nigericin at 32°C while IL-1βsecretion was significantly reduced. We found decreased GSDMD expression and unsecreted mature IL-1β at 32 °C treatment. Overall our data suggests that hypothermia suppresses ALI development by inhibiting IL-1β secretion and NETs formation through down-regulation of GSDMD.

Dehydrocostus lactone inhibits NLRP3 inflammasome activation by blocking ASC oligomerization and prevents LPS-mediated inflammation in vivo

Uncontrolled activation of NLRP3 inflammasome initiates a series of human inflammatory diseases. Targeting NLRP3 inflammasome has attracted considerable attention in developing potential therapeutic interventions. Here, we reported that dehydrocostus lactone (DCL), a main component of Saussurea lappa from the traditional Chinese medicine, inhibited NLRP3 inflammasome-mediated caspase-1 activation and subsequent interleukin (IL)-1β production in primary mouse macrophages and human peripheral blood mononuclear cells and exerted an inhibitory effect on NLRP3-driven inflammation. Mechanistically, DCL significantly blocked the ASC oligomerization, which is essential for the assembly of activated inflammasome. Importantly, in vivo experiments showed that DCL reduced IL-1β secretion and peritoneal neutrophils recruitment in LPS-mediated inflammation mouse model, which is demonstrated to be NLRP3 dependent. These results suggest that DCL is a potent pharmacological inhibitor of NLRP3 inflammasome and may be developed as a therapeutic drug for treating NLRP3-associated diseases.

Regulation of interleukin-1 beta secretion from macrophages via modulation of potassium ion (K+ ) channel activity.

A causal relationship exists between macrophage cholesterol levels and inflammation, for example, Interleukin-1β (IL-1β) secretion. A decrease in intracellular K+ is essential for inflammasome activation/IL-1β secretion and, herein, we examined the hypothesis that cellular cholesterol affects K+ -channel activity and K+ -efflux using mouse peritoneal macrophages (MPMs) and human/THP1 macrophages. An increase in cellular cholesterol led to a significant increase in K+ currents (>350% in both MPM and THP1). Enhancing cholesterol efflux returned K+ currents back to basal levels with corresponding increase in intracellular K+ (11.2-14.5%) and reduced IL-1β secretion (32-62%). These data demonstrate a novel mechanism by which cellular cholesterol modulates inflammation/inflammasome via regulation of K+ -channel activity and intracellular K+ levels. Attenuation of IL-1β secretion by Nateglinide/Repaglinide further suggests involvement of Kir6 channels.

Abstract 578: Macrophage Cholesterol Levels Modulate Ion Channel Activity: Effect(s) on Ion Flux Dependent Inflammatory Events

Accumulation of cholesterol loaded macrophage foam cells is the hallmark of atherosclerotic lesions where high cellular cholesterol content not only contributes to the plaque volume but is also responsible for the inflammatory milieu. Causal relationship exist between macrophage cholesterol content and inflammatory status involving TLR4, NFκB or cholesterol crystal mediated activation of inflammasome and IL1β secretion. Since K+ efflux is central to inflammasome activation, in this study we examined the hypothesis that cellular cholesterol content directly affects K+ channel activity as well as K+ efflux. K+ currents were monitored in mouse peritoneal macrophages (MPMs) from chow or western diet (WD) fed LDLR-/- or LDLR-/-CEHTg mice. WD feeding led to a significant increase in the K+ currents in MPMs from LDLR-/- mice (A, red). In contrast, WD feeding did not affect K+ currents in MPMs from LDLR-/-CEHTg mice; these MPMs have reduced cellular cholesterol accumulation due to increased efflux. Consistently, cholesterol loading with AcLDL led to an increase in K+ currents in THP1 macrophages (B, red) and this increase was attenuated following cholesterol efflux. The physiological effect of changes in K+ channel activity or K+ efflux on inflammasome activation in MPMs by different activators (K+ ionophore Nigericin, N or ALUM crystals) was examined. Increase in extracellular K+ (+KCl) or inhibition of K+ efflux by glyburide significantly reduced IL1β secretion (C&amp;D). Consistently, cellular K+ levels after LPS+N or LPS+ALUM treatment were significantly reduced (E). While in MPMs only K ATP channel inhibitors reduced IL1β secretion (F), in human THP1 macrophages inhibition of K ATP as well as Na/K pump inhibitors significantly reduced IL1β secretion (G). These data demonstrate a novel mechanism for cholesterol mediated regulation of inflammatory pathways by regulation of K+ channel activity and also illustrate species specific differences in K+ channels involved

Autophagy Mediates Interleukin-1β Secretion in Human Neutrophils.

Interleukin-1β (IL-1β), a major pro-inflammatory cytokine, is a leaderless cytosolic protein whose secretion does not follow the classical endoplasmic reticulum-to-Golgi pathway, and for which a canonical mechanism of secretion remains to be established. Neutrophils are essential players against bacterial and fungi infections. These cells are rapidly and massively recruited from the circulation into infected tissues and, beyond of displaying an impressive arsenal of toxic weapons effective to kill pathogens, are also an important source of IL-1β in infectious conditions. Here, we analyzed if an unconventional secretory autophagy mechanism is involved in the exportation of IL-1β by these cells. Our findings indicated that inhibition of autophagy with 3-methyladenine and Wortmannin markedly reduced IL-1β secretion induced by LPS + ATP, as did the disruption of the autophagic flux with Bafilomycin A1 and E64d. These compounds did not noticeable affect neutrophil viability ruling out that the effects on IL-1β secretion were due to cell death. Furthermore, VPS34IN-1, a specific autophagy inhibitor, was still able to reduce IL-1β secretion when added after it was synthesized. Moreover, siRNA-mediated knockdown of ATG5 markedly reduced IL-1β secretion in neutrophil-differentiated PLB985 cells. Upon LPS + ATP stimulation, IL-1β was incorporated to an autophagic compartment, as was revealed by its colocalization with LC3B by confocal microscopy. Overlapping of IL-1β-LC3B in a vesicular compartment peaked before IL-1β increased in culture supernatants. On the other hand, stimulation of autophagy by cell starvation augmented the colocalization of IL-1β and LC3B and then promoted neutrophil IL-1β secretion. In addition, specific ELISAs indicated that although both IL-1β and pro-IL-1β are released to culture supernatants upon neutrophil stimulation, autophagy only promotes IL-1β secretion. Furthermore, the serine proteases inhibitor AEBSF reduced IL-1β secretion. Moreover, IL-1β could be also found colocalizing with elastase, suggesting both some vesicles containing IL-1β intersect azurophil granules content and that serine proteases also regulate IL-1β secretion. Altogether, our findings indicate that an unconventional autophagy-mediated secretory pathway mediates IL-1β secretion in human neutrophils.

Reduced NLRP3 Gene Expression Limits the IL-1β Cleavage via Inflammasome in Monocytes from Severely Injured Trauma Patients.

Objective Traumatic injury or severe surgery leads to a profound immune response with a diminished functionality of monocytes and subsequently their IL-1β release. IL-1β plays an important role in host immunity and protection against infections. Its biological activation via IL-1β-precursor processing requires the transcription of inflammasome components and their activation. Deregulated activity of NOD-like receptor inflammasomes (NLR) like NLRP3 that leads to the maturation of IL-1β has been described in various diseases. While the role of other inflammasomes has been studied in monocytes, nothing is known about NLRP3 inflammasome after a traumatic injury. Here, the role of the NLRP3 inflammasome in impaired monocyte functionality after a traumatic injury was analyzed. Measurements and Main Results Ex vivo-in vitro stimulation of isolated CD14+ monocytes with lipopolysaccharide (LPS) showed a significantly higher IL-1β secretion in healthy volunteers (HV) compared to trauma patients (TP) after admission. Reduced IL-1β secretion was paralleled by significantly lowered gene expression of NLRP3 in monocytes from TP compared to those of HV. Transfection of monocytes with NLRP3-encoding plasmid recovered the functionality of monocytes from TP regarding the IL-1β secretion. Conclusions This study demonstrates that CD14+ monocytes from TP are significantly diminished in their function and that the presence of NLRP3 components is necessary in recovering the ability of monocytes to produce active IL-1β. This recovery of the NLRP3 inflammasome in monocytes may imply a new target for treatment and therapy of immune suppression after severe injury.

Anti-inflammatory activity of Echium amoenum extract on macrophages mediated by inhibition of inflammatory mediators and cytokines expression.

Echium amoenum (Boraginaceae) is an important remedy used for various illnesses. In this study, we investigated the anti-inflammatory effects of E. amoenum in the J774.1A macrophage cell line. We prepared ethyl acetate, dichloromethane and hexane extracts from E. amoenum flowers and examined their possible cytotoxic effects using MTT assay. Lipopolysaccharide (LPS)-stimulated macrophages were treated with the extracts after which we measured nitric oxide (NO) production by Griess method. Inducible NO synthase (iNOS), cyclooxygenase-2 (COX2), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 gene expressions were examined by real time-PCR. IL-1β and IL-6 cytokine levels were measured by enzyme-linked immunosorbent assay (ELISA). The hexane extract with a half maximal inhibitory concentration (IC50) of 39.8 μg/mL most effectively reduced NO production. Real time-PCR analysis indicated reduced levels of iNOS ((0.05 ± 0.006 relative fold change (RFC)) and COX2 (0.06 ± 0.002 RFC) gene expressions with the 100 μg/mL hexane extract (P < 0.001). IL1-β, TNF-α, and IL-6 gene expression levels decreased at all concentrations of the extract (less than ≈ 0.28 RFC). Treatment of LPS-stimulated cells with 100 μg/mL of the extract reduced IL-1β secretion to 27.9 ± 0.21 pg/mL and IL-6 to 555 ± 166 pg/mL. In conclusion, E. amoenum hexane extract showed the greatest reduction in macrophage NO secretion compared to other extracts. This extract could modulate the inflammatory mode of the macrophages by causing reductions in iNOS and COX2 enzymes as well as IL-1β, IL-6, and TNF-α cytokine levels. The results of this study have shown the anti-inflammatory effects of this plant. Further studies regarding its therapeutic potential in inflammatory disorders are recommended.

Purified type III secretion system needle proteins are novel TLR agonists that induce NF-κB signaling and stimulate NLRP3-dependent IL-1β production by innate cells in vitro

Abstract Purified type III secretion (T3S) system needle proteins from a number of Gram-negative bacteria have been identified as protective antigens as well as strong adjuvants in vivo. These proteins have been shown to act as TLR2 and TLR4 agonists, producing robust pro-inflammatory cytokine release in a MyD88-dependant manner from both human and mouse cells in vitro. Additionally, it has been established that TLR agonists alone can stimulate IL-1β through the NLRP3 inflammasome. Interestingly, we have demonstrated that T3S needle proteins induce NF-κB signaling downstream of both TLR2 and TLR4. Given the vaccine potential of T3S needle proteins, it is necessary to further understand the signaling mechanisms underlying their adjuvant properties. Here we characterized the innate signaling pathways engaged by T3S needle proteins. We show T3S needle proteins induce NF-κB signaling and pro-inflammatory cytokine release from mouse macrophages more strongly through TLR4 as compared to TLR2. Utilizing primary cells from inflammasome component knockout mice, we demonstrate that IL-1β secretion from T3S needle protein stimulated cells was dependent on the NLRP3 inflammasome, as well as caspase-1. Interestingly, pre-treatment with cytochalasin D reduced IL-1β secretion and resulted in the accumulation of intracellular pro-IL-1β after T3S needle protein stimulation; suggesting that internalization of T3S needle proteins is critical for inflammasome activation and IL-1β secretion. These findings further identify critical innate signaling pathways engaged by T3S needle proteins. T3S needle proteins have tremendous vaccine potential due to their ability to act as protective antigens and their innate immunity stimulating properties.

Mangiferin suppresses endoplasmic reticulum stress in perivascular adipose tissue and prevents insulin resistance in the endothelium.

Mangiferin is a naturally occurring glucosylxanthone with beneficial effects on glucose and lipid homeostasis. This study investigates the potential therapeutic effect of Mangiferin in perivascular adipose tissue (PVAT) and whether it contributes to regulating insulin action in the endothelium. Palmitate challenge evoked ROS-associated endoplasmic reticulum stress (ER stress) and NLRP3 inflammasome activation in PVAT. The conditioned medium from PA-stimulated PVAT was prepared to induce endothelial insulin resistance, and improved endothelium-dependent vasodilation in response to insulin was detected in vitro and in vivo. Mangiferin treatment enhanced LKB1-dependent AMPK activity and suppressed ER stress with downregulation of TXNIP induction, leading to the inhibition of NLRP3 inflammasome activation evidenced by attenuated NLRP3 and cleaved caspase-1 expression as well as reduced IL-1β secretion. Moreover, Mangiferin restored insulin-mediated Akt and eNOS phosphorylations with increased NO production, immunohistochemistry examination of adipocytes, and endothelial tissue in high-fat diet-fed mice also showed that oral administration of Mangiferin inhibited ER stress and NLRP3 induction in PVAT, and then effectively prevented insulin resistance in the vessel endothelium. Taken together, these results revealed that Mangiferin suppressed ER stress-associated NLRP3 inflammasome activation in PVAT through regulation of AMPK activity, which prevented endothelial insulin resistance. These findings suggested that the amelioration of PVAT dysfunction may be a therapeutic strategy for the prevention of endothelial insulin resistance.

Polydatin attenuates potassium oxonate-induced hyperuricemia and kidney inflammation by inhibiting NF-κB/NLRP3 inflammasome activation via the AMPK/SIRT1 pathway.

This study was designed to investigate the effects of polydatin (PLD) on potassium oxonate-induced hyperuricemic rats. Hyperuricemic rats were treated with potassium oxonate (250 mg kg-1) intragastrically for 7 days, and polydatin (25, 50 mg kg-1) or allopurinol (5 mg kg-1) was administered to the rats 1 h after the potassium oxonate exposure. Polydatin administration decreased the levels of uric acid and creatinine in serum and urine, leading to inhibition of pro-inflammatory cytokine production in serum and kidney. Western blot analyses illustrated that polydatin down-regulated the translocation of NF-κB p65, the degradation of IκBα, and the protein levels of inflammasome components (NLRP3, ASC, and caspase-1), which led to reduced IL-1β secretion. Notably, polydatin treatment activated AMP kinase (AMPK) protein and increased SIRT1 expression. Taken together, polydatin might be a promising agent for gouty treatment to inhibit renal NF-κB/NLRP3 inflammasome activation via the AMPK/SIRT1 pathway.

Hemin and Cobalt Protoporphyrin Inhibit NLRP3 Inflammasome Activation by Enhancing Autophagy: A Novel Mechanism of Inflammasome Regulation

Inflammasomes are intracellular protein platforms, which, upon activation, produce the highly proinflammatory cytokines interleukin (IL)-1β and IL-18. Heme, hemin and their degradation products possess significant immunomodulatory functions. Here, we studied whether hemin regulates inflammasome function in macrophages. Both hemin and its derivative, cobalt protoporphyrin (CoPP), significantly reduced IL-1β secretion by cultured human primary macrophages, the human monocytic leukemia cell line and also mouse bone marrow-derived and peritoneal macrophages. Intraperitoneal administration of CoPP to mice prior to urate crystal-induced peritonitis alleviated IL-1β secretion to the peritoneal cavity. In cultured macrophages, hemin and CoPP inhibited NLRP3 inflammasome assembly by reducing the amount of intracellular apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC). The reduction of ASC was associated with enhanced autophagosome formation and autophagic flux. Inhibition of autophagy prevented the CoPP-induced depletion of ASC, implying that the depletion was caused by increased autophagy. Our data indicate that hemin functions as an endogenous negative regulator of the NLRP3 inflammasome. The inhibition is mediated via enhanced autophagy that results in increased degradation of ASC. This regulatory mechanism may provide a novel approach for the treatment of inflammasome-related diseases.

Enzymatically Inactive Procaspase 1 stabilizes the ASC Pyroptosome and Supports Pyroptosome Spreading during Cell Division

Caspase-1 is a key player during the initiation of pro-inflammatory innate immune responses, activating pro-IL-1β in so-called inflammasomes. A subset of patients with recurrent febrile episodes and systemic inflammation of unknown origin harbor mutations in CASP1 encoding caspase-1. CASP1 variants result in reduced enzymatic activity of caspase-1 and impaired IL-1β secretion. The apparent paradox of reduced IL-1β secretion but systemic inflammation led to the hypothesis that CASP1 mutations may result in variable protein interaction clusters, thus activating alternative signaling pathways. To test this hypothesis, we established and characterized an in vitro system of transduced immortalized murine macrophages expressing either WT or enzymatically inactive (p.C284A) procaspase-1 fusion reporter proteins. Macrophages with variant p.C284A caspase-1 did not secrete IL-1β and exhibited reduced inflammatory cell death, referred to as pyroptosis. Caspase-1 and apoptosis-associated speck-like protein containing a CARD (ASC) formed cytosolic macromolecular complexes (so-called pyroptosomes) that were significantly increased in number and size in cells carrying the p.C284A caspase-1 variant compared with WT caspase-1. Furthermore, enzymatically inactive caspase-1 interacted with ASC longer and with increased intensity compared with WT caspase-1. Applying live cell imaging, we documented for the first time that pyroptosomes containing enzymatically inactive variant p.C284A caspase-1 spread during cell division. In conclusion, variant p.C284A caspase-1 stabilizes pyroptosome formation, potentially enhancing inflammation by two IL-1β-independent mechanisms: pyroptosomes convey an enhanced inflammatory stimulus through the recruitment of additional proteins (such as RIP2, receptor interacting protein kinase 2), which is further amplified through pyroptosome and cell division.

Disease activity accounts for long-term efficacy of IL-1 blockers in pyogenic sterile arthritis pyoderma gangrenosum and severe acne syndrome.

To provide a rationale for anti-IL-1 treatment in pyogenic sterile arthritis, pyoderma gangrenosum and acne (PAPA) by defining whether IL-1β secretion is enhanced; requires NLRP3; and correlates with proline-serine-threonine phosphatase-interacting protein 1 mutations, disease activity and/or the clinical picture in PAPA. Monocytes were isolated from 13 patients and 35 healthy donors and studied at baseline and following activation. Secretion pattern of IL-1β, IL-1α, IL-1Ra, IL-6, IL-18 and TNF-α was assessed in supernatants by ELISA. The NLRP3 requirement for IL-1β secretion was investigated by silencing technique in PAPA and healthy donor monocytes. Long-term follow-up (mean 26 months, range 4-38) was performed in five patients enrolled in an anti-IL-1 regimen. IL-1β secretion in PAPA is increased, requires NLRP3 and correlates with disease activity. Patients with a history of osteoarticular flares release more IL-1β, IL-6 and TNF-α compared with those with predominant cutaneous recurrences. Monocytes from patients in anti-IL-1 treatment dramatically reduced IL-1β secretion after ex vivo activation, and long-term follow-up demonstrated decreased frequency of flares and normalization of acute phase reactants in all the patients. A straightforward correlation between genotype and IL-1β signalling was not observed suggesting that factors other than mutation itself may play a role in regulating IL-1β secretion and response to treatment in PAPA. PAPA patients with active lesions display increased NLRP3-mediated IL-1β secretion, and long-term efficacy of IL-1 blockade was demonstrated. Even if other mechanisms related to the complex proline-serine-threonine phosphatase-interacting protein 1 protein networking might play additional roles, this study further supports the potential of IL-1 blockade as an effective therapeutic strategy in PAPA syndrome.

Lysosomal storage and impaired autophagy lead to inflammasome activation in Gaucher macrophages.

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, is characterized by the presence of glucosylcer-amide macrophages, the accumulation of glucosylceramide in lysosomes and the secretion of inflammatory cytokines. However, the connection between this lysosomal storage and inflammation is not clear. Studying macrophages derived from peripheral monocytes from patients with type 1 Gaucher disease with genotype N370S/N370S, we confirmed an increased secretion of interleukins IL-1β and IL-6. In addition, we found that activation of the inflammasome, a multiprotein complex that activates caspase-1, led to the maturation of IL-1β in Gaucher macrophages. We show that inflammasome activation in these cells is the result of impaired autophagy. Treatment with the small-molecule glucocerebrosidase chaperone NCGC758 reversed these defects, inducing autophagy and reducing IL-1β secretion, confirming the role of the deficiency of lysosomal glucocerebrosidase in these processes. We found that in Gaucher macrophages elevated levels of the autophagic adaptor p62 prevented the delivery of inflammasomes to autophagosomes. This increase in p62 led to activation of p65-NF-kB in the nucleus, promoting the expression of inflammatory cytokines and the secretion of IL-1β. This newly elucidated mechanism ties lysosomal dysfunction to inflammasome activation, and may contribute to the massive organomegaly, bone involvement and increased susceptibility to certain malignancies seen in Gaucher disease. Moreover, this link between lysosomal storage, impaired autophagy, and inflammation may have implications relevant to both Parkinson disease and the aging process. Defects in these basic cellular processes may also provide new therapeutic targets.

Generation of inducible immortalized bone marrow derived cell lines expressing mutant procaspase-1 C284A on a caspase-1 knock-out background

Caspase-1, belonging to the family of cystein proteases, is well-known for its involvement in IL-1β and IL-18 maturation. Although, caspase-1 is one of the best described caspase-family-members, open questions concerning autoinflammatory diseases, caused by procaspase-1 variants (e.g. ICE-Fever), remain. ICE-Fever patients suffer from chronic febrile episodes, although procaspase-1 variants show reduced enzymatic activity leading to limited IL-1β secretion. The paradox of reduced IL-1β secretion but increased inflammation led to the hypothesis, that CASP1-variants enhance alternative signaling pathways. However, the role of procaspase-1 variants and their interaction partners during IL-1β maturation or NF-κB activation is still poorly understood.

Enhanced activity of NLRP3 inflammasome in peripheral blood cells of patients with active rheumatoid arthritis

IntroductionInterleukin-1β (IL-1β) is a major inflammatory cytokine, produced predominantly by innate immune cells through NLRP3-inflammasome activation. Both intrinsic and extrinsic danger signals may activate NLRP3. Genetic variations in NLRP3-inflammasome components have been reported to influence rheumatoid arthritis (RA) susceptibility and severity. We sought to assess the activity of NLRP3-inflammasome in patients with active RA compared to healthy individuals.MethodIntracellular protein expression of NLRP3, ASC, pro- and active caspase-1, pro- and active IL-1β was assessed by immunoblotting both at baseline and upon inflammasome activation. NLRP3 function (IL-1β secretion) was assessed upon priming of TLR2 (Pam(3)CysSK(4), TLR3 (poly(I:C)) or TLR4 (LPS) and ATP sequential treatment. We used caspase inhibitors (casp-1, 3/7 and 8) to assess their contribution to IL-1β maturation. All experiments were performed in whole blood cells.ResultsActive RA patients (n = 11) expressed higher basal intracellular levels of NLRP3 (p < 0.008), ASC (p < 0.003), active caspase-1 (p < 0.02) and pro-IL-1β (p < 0.001). Upon priming with TLR4 (LPS) and ATP, RA-derived cell extracts (n = 7) displayed increased expression of NLRP3 (p < 0.01) and active caspase-1 (p < 0.001). Secreted IL-1β in culture supernatants from whole blood cells activated with TLR4 (LPS) or TLR3 agonist (poly(I:C)) plus ATP was higher in RA patients (n = 20) versus controls (n = 18) (p < 0.02 for both). Caspase-1 inhibition significantly reduced IL-1β secretion induced by all stimuli, whereas caspase-8 inhibition affected only TLR4 and TLR3 cell priming.ConclusionPatients with active RA have increased expression of NLRP3 and NLRP3-mediated IL-1β secretion in whole blood cells upon stimulation via TLR3 and TLR4 but not TLR2. In these patients, IL-1β secretion seems to be predominately driven by caspase-1 and caspase-8. Targeting NLRP3 or downstream caspases may be of benefit in suppressing IL-1β production in RA.