IL-1beta Secretion Research Articles

Presence of NLRP3 in myeloid cells increases sensitivity to membrane damage (157.3)

Abstract Pore forming toxins, such as tetanolysin O (TLO), as well as the potassium ionophore nigericin, stimulate the NLRP3 inflammasome. We and others have shown that NLRP3 inflammasome induced processing and secretion of mature IL-1 beta is dependent on the activity of caspase-1 as well as potassium efflux. Unexpectedly, we now observe that NLRP3 knockout macrophages are more resistant to membrane damage compared to wild type macrophages exposed to nigericin or TLO, as assessed by a dye uptake assay. We also were able to increase sensitivity to membrane damage in myeloid cells deficient in ASC, an adaptor protein required for formation of the NLRP3 inflammasome, by over-expression of ASC using retroviral transduction. Through the use of both pharmacological inhibitors and caspase-1 knockout mice, we observed this NLRP3 and ASC dependent effect on membrane integrity is independent of the activity of caspase-1, unlike the secretion of mature IL-1 beta. However, the loss of membrane integrity is dependent on potassium efflux, as is mature IL-1 beta secretion. These data suggest a mechanism through which the NLRP3 inflammasome is able to influence the membrane integrity of myeloid cells in a caspase-1 independent manner, distinguishing the loss of membrane integrity from IL-1 beta processing and secretion.

Granuloma Formation and Host Defense in Chronic Mycobacterium tuberculosis Infection Requires PYCARD/ASC but Not NLRP3 or Caspase-1

The NLR gene family mediates host immunity to various acute pathogenic stimuli, but its role in chronic infection is not known. This paper addressed the role of NLRP3 (NALP3), its adaptor protein PYCARD (ASC), and caspase-1 during infection with Mycobacterium tuberculosis (Mtb). Mtb infection of macrophages in culture induced IL-1β secretion, and this requires the inflammasome components PYCARD, caspase-1, and NLRP3. However, in vivo Mtb aerosol infection of Nlrp3−/−, Casp-1−/−, and WT mice showed no differences in pulmonary IL-1β production, bacterial burden, or long-term survival. In contrast, a significant role was observed for Pycard in host protection during chronic Mtb infection, as shown by an abrupt decrease in survival of Pycard−/− mice. Decreased survival of Pycard−/− animals was associated with defective granuloma formation. These data demonstrate that PYCARD exerts a novel inflammasome-independent role during chronic Mtb infection by containing the bacteria in granulomas.

Staphylococcus aureus Stimulates Neutrophil Targeting Chemokine Expression in Keratinocytes through an Autocrine IL-1α Signaling Loop

Staphylococcus aureus is a significant human pathogen that can colonize the skin. Neutrophils are well known to be involved in clearance of the bacterium. This study focused on exploring the role that human keratinocytes have as first responders to bacterial challenges. IL-1alpha and IL-1beta increased mRNA production and protein secretion of the neutrophil chemotactic CXCL1, CXCL2, and IL-8 in keratinocytes. S. aureus and the bacterial cell wall components lipoteichoic acid (LTA) and peptidoglycan (PGN) induced similar expression profiles in a Toll-like receptor (TLR)-2-dependent manner. Interestingly, the S. aureus-induced mRNA levels peaked at later time points than those induced by IL-1. The S. aureus-activated chemokine production was preceded by significant IL-1alpha and IL-1beta secretion. Expression of IL-1alpha was significantly higher than that of IL-1beta. Inhibition of IL-1RI using neutralizing antibodies revealed that S. aureus-derived LTA and PGN-induced chemokine expression requires IL-1RI engagement. Surprisingly, we further found that chemokine secretion is dependent upon endocrine IL-1alpha, but not IL-1beta, signaling. Our data show that the innate immune response of keratinocytes is regulated differently than those of other cell types. This may represent a fail-safe system that protects the host against genetic variation and immune evasion mechanisms developed by pathogens.

Temporal Interleukin-1β Secretion from Primary Human Peripheral Blood Monocytes by P2X7-independent and P2X7-dependent Mechanisms

The processing and regulated secretion of IL-1β are critical points of control of the biological activity of this important pro-inflammatory cytokine. IL-1β is produced by both monocytes and macrophages, but the rate and mechanism of release differ according to the differentiation status and the origin of these cells. We aimed to study the control of processing and release in human blood monocytes and human monocyte-derived macrophages. Toll-like receptor (TLR)-induced IL-1β production and release were investigated for dependence upon caspase-1, P2X7 receptor activation, and loss of membrane asymmetry associated with microvesicle shedding. TLR agonists induced P2X7 receptor-dependent IL-1β release in both monocytes and macrophages; however, only monocytes also showed P2X7 receptor-independent release of mature IL-1β. Furthermore, in monocytes ATP-mediated PS exposure could be activated independently of IL-1β production. Release of IL-1β from monocytes showed selectivity for specific TLR agonists and was accelerated by P2X7 receptor activation. Human monocytes released more IL-1β/cell than macrophages. These data have important implications for inflammatory diseases that involve monocyte activation and IL-1 release.

SIGNR1 ligation on murine peritoneal macrophages induces IL-12 production through NFκB activation

We have previously shown that murine resident peritoneal macrophages (PEMs) are activated in response to uptake of oligomannose-coated liposomes (OMLs), leading to production of interleukin (IL)-12. To understand the mechanism of activation of PEMs by OMLs, in the present study we investigated the role of a mannose-binding C-type lectin receptor, SIGNR1, in production of proinflammatory cytokines by PEMs, in which SIGNR1 acts as a physiological receptor for OMLs. Engagement of SIGNR1 on PEMs with an anti-SIGNR1-specific rat IgM antibody, ERTR9, induced production of IL-12 and tumor necrosis factor (TNF)-alpha from PEMs, while secretion of IL-6 and IL-1beta was not detected with the same treatment. The level of phosphorylated IkappaB kinase in PEMs also increased in response to ERTR9 treatment of the cells. Treatment of PEMs with a specific nuclear factor kappa-B (NFkappaB) inhibitor, BAY11-7082, reduced ERTR9-dependent IL-12 production. Intraperitoneal treatment with BAY11-7082 also led to reduction of subsequent OML-induced IL-12 production from PEMs. These results indicate that SIGNR1-mediated intercellular signaling may induce production of cytokines such as IL-12 through NFkappaB activation.

Upregulation of interleukin‐1 by Epstein–Barr virus latent membrane protein 1 and its possible role in nasopharyngeal carcinoma cell growth

Nasopharyngeal carcinoma (NPC) is associated with Epstein-Barr virus (EBV) infection. We previously found that interleukin (IL)-1alpha and IL-1beta significantly increased in NPC tissues. This study investigated what EBV-encoded proteins were involved in such IL-1 production. IL-1alpha and IL-1beta messenger ribonucleic acids (mRNAs) were detected in the EBV latent membrane protein 1 (LMP1) transfectant (LMP135) only by reverse transcriptase-polymerase chain reaction (RT-PCR). LMP1-mediated IL-1alpha and IL-1beta production could be enhanced by tumor necrosis factor alpha (TNF-alpha), determined by enzyme-linked immunosorbent assay (ELISA). Moreover, IL-1alpha and IL-1beta mRNAs and proteins were increased in a dose-dependent manner in epithelial cells transiently transfected by an LMP1 plasmid. Besides, immortalized human epidermal keratinocyte (RHEK-1) epithelial cells could be enhanced to proliferate by IL-1alpha and IL-1beta determined by water-soluble tetrazolium salt (WST-1) assay. EBV LMP1 is capable of upregulating IL-1alpha and IL-1beta secretions from epithelial cells and positively modulated by TNF-alpha. This may consequently contribute to tumor growth in patients with NPC.

Involvement of the AIM2, NLRC4, and NLRP3 Inflammasomes in Caspase-1 Activation by Listeria monocytogenes

Infection with Listeria monocytogenes can cause meningitis and septicemia in newborn, elderly, or immunocompromised individuals. Pregnant women are particularly susceptible to Listeria, leading to a potentially fatal infection. Cytosolic Listeria activates the proinflammatory caspase-1 and induces the processing and secretion of interleukins IL-1beta and IL-18 as well as caspase-1-dependent pyroptosis. This study elucidates the role of various inflammasome components of host macrophages in the proinflammatory response to infection with Listeria. Here, we have used macrophages from AIM2-, NLRC4-, NLRP3-, and ASC-deficient mice to demonstrate that AIM2, NLRC4, and NLRP3 inflammasomes as well as the adaptor protein ASC all contribute to activation of caspase-1 in Listeria-infected macrophages. We show that Listeria DNA, which escapes into the cytosol of infected macrophages, triggers AIM2 oligomerization, caspase-1 activation, and pyroptosis. Interestingly, we found that flagellin-deficient Listeria, like Francisella tularensis, is recognized primarily by the AIM2 inflammasome, as no caspase-1 activation or cell death was observed in AIM2-deficient macrophages infected with this Listeria mutant. We further show that prior priming of NLRC4-deficient macrophages with LPS is sufficient for Listeria-induced caspase-1 activation in these macrophages, suggesting that TLR4 signaling is important for activation of the AIM2 and NLRP3 inflammasomes by Listeria in the absence of NLRC4. Taken together, our results indicate that Listeria infection is sensed by multiple inflammasomes that collectively orchestrate a robust caspase-1 activation and proinflammatory response.

Effect of TLR4 engagement on TLR3-induced proinflammatory signaling in dendritic cells through IL-10, STAT3, and p38-dependent pathways and on antimelanoma CD8+ T-cell priming.

8568 Background: Toll-like receptor agonists have shown promise as adjuvants for immune therapy, but optimal combinations or pathways through which they signal have yet to be identified. Methods: DCs were stimulated with TLR3, TLR4 and TLR7/8 agonists and their combinations. Upon evaluation of phenotype and cytokine production these DCs were then used to prime anti-tumor CD8+ T cell responses. Results: DCs stimulated with the TLR3 agonist PolyI:C displayed the strongest inflammatory responses (heightened secretion of IL-12p70, TNF-α, IP-10, and IL-1beta) and induced the highest frequency of polyfunctional antigen-specific cells towards melanoma antigen Mart-1/MelanA. While the addition of TLR7/8 agonists improved these responses, the inclusion of LPS substantially suppressed pro-inflammatory cytokine production in vitro and in vivo. Inhibition was dependent upon the rapid, LPS-dependent induction of IL-10, and could be reversed by blockade of STAT-3, p38 or antibodies to IL-10 and its receptor. Conclusions: Our findings identify combinations of TLR agonists that can enhance or limit immune responses directed towards tumor antigens based on their relative ability to induce immune suppressive pathways that involve IL-10. No significant financial relationships to disclose.

Inflammation and Fibrosis duringChlamydia pneumoniaeInfection Is Regulated by IL-1 and the NLRP3/ASC Inflammasome

Chlamydia pneumoniae is a common respiratory pathogen associated with atypical pneumonia, and it has been suggested as a trigger or promoter of several chronic inflammatory conditions, such as asthma and atherosclerosis. The beta form of IL-1 (IL-1beta) is a proinflammatory cytokine released by many cell types and is an important mediator of inflammation during infection. IL-1beta production is a tightly controlled process that includes regulation at multiple levels and typically requires two distinct signals for activation and release. In this study, we investigated the ability of C. pneumoniae to induce IL-1beta secretion. We found that C. pneumoniae was unique among the other Chlamydia species tested in its ability to potently induce secretion of mature IL-1beta from unprimed bone marrow-derived macrophages during a productive infection. TLR2 was required for induction of pro-IL-1beta, whereas the NLRP3/ASC was required for caspase-1 activation and pro-IL-1beta cleavage to produce mature IL-1beta. Caspase-1 cleavage was independent of endogenous ATP release, but required potassium flux, lysosomal acidification, and cathepsin B release. We further investigated the role of IL-1 in host defense against C. pneumoniae-induced pneumonia using mice deficient in the type I IL-1R. Although the IL-1R(-/-) mice developed an inflammatory infiltrate, the number of infiltrating neutrophils was lower, whereas there was evidence of increased infiltrating fibroblasts and mesenchymal cells and more lung fibrosis. We conclude that C. pneumoniae directly activates the NLRP3/ASC inflammasome, leading to the release of biologically active IL-1beta, and that concurrent IL-1 signaling is required for optimal host defense against acute bacterial pneumonia.

Interleukin (IL)‐31 induces pro‐inflammatory cytokines in human monocytes and macrophages following stimulation with staphylococcal exotoxins

IL-31 is a cytokine expressed by T cells following activation with cytokines or staphylococcal exotoxins. A major function of IL-31 in atopic dermatitis (AD) is the induction of pruritus in the skin via the IL-31 receptor on sensory nerve cells. However, the regulation of the IL-31 receptor and pro-inflammatory functions of IL-31 in human monocytes and monocyte-derived cells are yet to be studied in detail. To investigate the regulation and function of IL-31 receptors in resting and activated human monocytes, macrophages and dendritic cells. Human monocytes, macrophages and dendritic cells were stimulated with staphylococcal exotoxins (SEB, alpha-toxin) or cytokines (IFN-gamma, IL-13). IL-31RA expression and regulation were then investigated at both the mRNA and the protein level. Subsequently, functional effects of IL-31 stimulation on cytokine secretion were measured at the protein level. Staphylococcal exotoxins significantly up-regulated IL-31RA expression on monocytes and macrophages but not on dendritic cells at both the mRNA and the protein level. IL-31 enhanced the secretion of IL-1beta, IL-6 and IL-18 and up-regulated CD86 expression. In patients with AD, functional IL-31RA was also detected following stimulation of PBMC with IFN-gamma. However, this was not observed in healthy individuals. IL-31 induces pro-inflammatory effects in activated human monocytes and macrophages. This may have implications for cutaneous inflammation in eczema where an over-expression of IL-31 has been described previously. Moreover, our findings provide a new link between staphylococcal colonization and the worsening of inflammation via IL-31. Further therapeutic considerations may include IL-31 as a target in AD.

Rationale and timeliness for IL‐1β‐targeted therapy to reduce allogeneic organ injury at procurement and to diminish risk of rejection after transplantation

Ischemia-reperfusion injury (IRI) involving allograft transplantation and procured organs may in part be induced by stimulation of a newly described innate pro-inflammatory immune system (i.e., NALP-3-inflammasome), which can cause secretion of IL-1beta and subsequent neutrophilic inflammation. Ischemia and/or hypoxia/anoxia can induce anaerobic metabolism with metabolic acidosis and subsequent development of danger signals known to stimulate IL-1beta secretion from the NALP-3 inflammasome. Observations from IRI studies and hereditary auto-inflammatory syndromes with NALP-3 inflammasome mutations suggest that IL-1beta secretion can induce robust neutrophilic inflammation that is responsive to IL-1beta targeted therapy. Based on these observations and data from transplantation studies, it may be timely to consider commercially available IL-1beta targeted biologic therapy to improve allograft tolerance and viability of procured organs.

Chemotherapeutic Agents Targeting the Tubulin Cytoskeleton Modify LPS-induced Cytokine Secretion by Dendritic Cells and Increase Antigen Presentation

Recent studies support the idea that certain chemotherapeutic agents do not act only by cytotoxicity, but also have immunomodulatory activity. Because of their role in mitosis chemotherapeutic agents targeting microtubules are widely used, and this study determined the outcome of disturbing tubulin cytoskeleton dynamics on human dendritic cell function. Dendritic cells (DCs) play a major role in the generation of the adaptive immune response owing to their capacity for antigen presentation leading to T lymphocyte activation and differentiation and there is compelling evidence for their contribution to the antitumoral immune response. Two agents that target the tubulin cytoskeleton were used, taxol and colchicine, and a brief pretreatment with either increased antigen presentation by DCs independently of significant phenotypic change, cell death, or cytokine production. Although taxol and colchicine use different mechanisms to disrupt microtubules, NF-kappabeta was activated by either. We therefore determined whether the cytokine secretion profile in response to lipopolysaccharide (LPS) was modified. LPS stimulation of DCs induced IL-10, IL-12p70, TNFalpha and IL-1beta secretion, and taxol pretreatment modified this response by down-regulating IL-1beta secretion whereas colchicine induced a proinflammatory cytokine profile with reduced IL-10 and increased IL-12p70 and TNFalpha secretion. Taken together, these data reveal new immunomodulatory strategies of microtubule disrupting agents in dendritic cells, that of modifying the cytokine response to LPS and that of increasing T lymphocyte activation without induction of inflammatory cytokine secretion by dendritic cells.

The effect of lipopolysaccharide on gene expression of TLR4 and MD-2 in rat alveolar macrophage and its secretion of inflammation cytokines

To investigate the effect of lipopolysaccharide (LPS) on gene expression of Toll-Like receptor 4 (TLR4) and myeloid differentiation protein-2 (MD-2) in rat alveolar macrophage NR8383 cell and its secretion of inflammation cytokines with or without small interference RNA target MD-2 (MD2-siRNA) treatment. NR8383 cell was cultured with F-12K medium and stimulated with LPS (0.01 approximately 10 mg/L) for 2 h or stimulated with 1 microg/ml LPS for 2-24 h. MD2-siRNA oligo was transfected into NR8383 cell by Lipofectamine 2000. The expression of TLR4 mRNA and MD-2 mRNA in cell were detected by semi-quantitative revels transcription polymerase (RT-PCR). The contents of TNF-alpha, IL-6 and IL-1beta in the cell cultured supernatant were tested by ELISA. One way ANOVA was used for difference comparison during groups and Pearson correlation was used for correlation analysis. (1) The mRNA expression of TLR4 and MD-2 in control NR8383 cell were 0.52+/-0.05 and 0.44+/-0.09, respectively. There was no obviously change after 0.01 mg/L LPS stimulation. The increase occurred in a dose dependent manner from 0.1 mg/L to 10 mg/L. The highest TLR4 and MD-2 mRNA expression were 0.72+/-0.06 and 0.65+/-0.10 (F=17.255 and 6.045, P<0.01). The changes of TNF-alpha, IL-6 and IL-1beta contents in cell cultured supernatant were similar with that of TLR4 and MD-2 gene expression. TNF-alpha, IL-6 and IL-1beta content were (25.8+/-3.4) ng/L, (62.4+/-4.7) ng/L and (31.6+/-1.7) ng/L in control group, while the corresponding contents were (58.9+/-5.3) ng/L, (96.5+/-3.9) ng/L and (55.4+/-5.4) ng/L after 10 mg/L LPS simulation (F=29.55, 54. 47 and 31.45, P<0. 01). (2) When stimulated with 1 microg/ml LPS, the mRNA expressions of TLR4 and MD-2 in NR8383 cell were increased from hour 2. The highest mRNA expressions of TLR4 and MD-2 occurred at hour 6, and then decreased slowly from hour 8. The mRNA expressions at hour 24 were still higher than those in cells without LPS stimulation (F=5.279 and 4.106, P<0.01). The changes of TNF-alpha, IL-6 and IL-1beta content in cell cultured supernatant were also similar with that of gene expression (F=10.64, 11.23 and 17.58, P<0. 01). The secretion peaks of TNF-alpha and IL-1beta occurred from hour 6 to hour 8, and the secretion peak of IL-6 occurred from hour 8 to hour12. (3) The mRNA expression of MD-2 was related with that of TLR4 positively (r=0.513, P<0.01). (4) The interfere efficiency of MD-2 siRNA was 67%. There was no obvious increase of TNF-alpha, Il-1beta and IL-6 in MD-2 siRNA treated group after LPS stimulation. Higher dose LPS can up-regulate the gene expression of TLR4 and MD-2 in rat alveolar macrophage NR8383 cell with a long time and promote the secretion of TNF-alpha, IL-6 and IL-1beta. MD-2 siRNA can inhibit NR8383 cell secrete TNF-alpha, IL-6 and IL-1beta induced by LPS.

Listeria monocytogenes Triggers AIM2-Mediated Pyroptosis upon Infrequent Bacteriolysis in the Macrophage Cytosol

A host defense strategy against pathogens is the induction of cell death, thereby eliminating the pathogen's intracellular niche. Pyroptosis, one such form of cell death, is dependent on inflammasome activation. In a genetic screen to identify Listeria monocytogenes mutants that induced altered levels of host cell death, we identified a mutation in lmo2473 that caused hyperstimulation of IL-1beta secretion and pyroptosis following bacteriolysis in the macrophage cytosol. In addition, strains engineered to lyse in the cytosol by expression of both bacteriophage holin and lysin or induced to lyse by treatment with ampicillin stimulated pyroptosis. Pyroptosis was independent of the Nlrp3 and Nlrc4 inflammasome receptors but dependent on the inflammasome adaptor ASC and the cytosolic DNA sensor AIM2. Importantly, wild-type L. monocytogenes were also found to lyse, albeit at low levels, and trigger AIM2-dependent pyroptosis. These data suggested that pyroptosis is triggered by bacterial DNA released during cytosolic lysis.

Natural Selection of Human Embryos: Decidualizing Endometrial Stromal Cells Serve as Sensors of Embryo Quality upon Implantation

BackgroundPregnancy is widely viewed as dependent upon an intimate dialogue, mediated by locally secreted factors between a developmentally competent embryo and a receptive endometrium. Reproductive success in humans is however limited, largely because of the high prevalence of chromosomally abnormal preimplantation embryos. Moreover, the transient period of endometrial receptivity in humans uniquely coincides with differentiation of endometrial stromal cells (ESCs) into highly specialized decidual cells, which in the absence of pregnancy invariably triggers menstruation. The role of cyclic decidualization of the endometrium in the implantation process and the nature of the decidual cytokines and growth factors that mediate the crosstalk with the embryo are unknown.Methodology/Principal FindingsWe employed a human co-culture model, consisting of decidualizing ESCs and single hatched blastocysts, to identify the soluble factors involved in implantation. Over the 3-day co-culture period, approximately 75% of embryos arrested whereas the remainder showed normal development. The levels of 14 implantation factors secreted by the stromal cells were determined by multiplex immunoassay. Surprisingly, the presence of a developing embryo had no significant effect on decidual secretions, apart from a modest reduction in IL-5 levels. In contrast, arresting embryos triggered a strong response, characterized by selective inhibition of IL-1β, -6, -10, -17, -18, eotaxin, and HB-EGF secretion. Co-cultures were repeated with undifferentiated ESCs but none of the secreted cytokines were affected by the presence of a developing or arresting embryo.ConclusionsHuman ESCs become biosensors of embryo quality upon differentiation into decidual cells. In view of the high incidence of gross chromosomal errors in human preimplantation embryos, cyclic decidualization followed by menstrual shedding may represent a mechanism of natural embryo selection that limits maternal investment in developmentally impaired pregnancies.

Clostridium difficile Toxin–Induced Inflammation and Intestinal Injury Are Mediated by the Inflammasome

Clostridium difficile-associated disease (CDAD) is the leading cause of nosocomial diarrhea in the United States. C difficile toxins TcdA and TcdB breach the intestinal barrier and trigger mucosal inflammation and intestinal damage. The inflammasome is an intracellular danger sensor of the innate immune system. In the present study, we hypothesize that TcdA and TcdB trigger inflammasome-dependent interleukin (IL)-1beta production, which contributes to the pathogenesis of CDAD. Macrophages exposed to TcdA and TcdB were assessed for IL-1beta production, an indication of inflammasome activation. Macrophages deficient in components of the inflammasome were also assessed. Truncated/mutated forms of TcdB were assessed for their ability to activate the inflammasome. The role of inflammasome signaling in vivo was assessed in ASC-deficient and IL-1 receptor antagonist-treated mice. TcdA and TcdB triggered inflammasome activation and IL-1beta secretion in macrophages and human mucosal biopsy specimens. Deletion of Nlrp3 decreased, whereas deletion of ASC completely abolished, toxin-induced IL-1beta release. TcdB-induced IL-1beta release required recognition of the full-length toxin but not its enzymatic function. In vivo, deletion of ASC significantly reduced toxin-induced inflammation and damage, an effect that was mimicked by pretreatment with the IL-1 receptor antagonist anakinra. TcdA and TcdB trigger IL-1beta release by activating an ASC-containing inflammasome, a response that contributes to toxin-induced inflammation and damage in vivo. Pretreating mice with the IL-1 receptor antagonist anakinra afforded the same level of protection that was observed in ASC-/- mice. These data suggest that targeting inflammasome or IL-1beta signaling may represent new therapeutic targets in the treatment of CDAD.

Atrovirinone inhibits proinflammatory mediator synthesis through disruption of NF-κB nuclear translocation and MAPK phosphorylation in the murine monocytic macrophage RAW 264.7

In a previous communication we showed that atrovirinone, a 1,4-benzoquinone isolated from the roots of Garcinia atroviridis, was able to inhibit several major proinflammatory mediators of inflammation. In this report we show that atrovirinone inhibits NO and PGE(2) synthesis through inhibition of iNOS and COX-2 expression. We also show that atrovirinone inhibits the secretion of IL-1beta and IL-6 in a dose dependent fashion whereas the secretion of IL-10, the anti-inflammatory cytokine, was enhanced. Subsequently we determined that the inhibition of proinflammatory cytokine synthesis and inducible enzyme expression was due to a dose-dependent inhibition of phosphorylation of p38 and ERK1/2. We also showed that atrovirinone prevented phosphorylation of I-kappaBalpha, which resulted in a reduction of p65NF-kappaB nuclear translocation as demonstrated by expression analysis. We conclude that atrovirinone is a potential anti-inflammatory drug lead that targets both the MAPK and NF-kappaB pathway.

The Role of Potassium in Inflammasome Activation by Bacteria

Many Gram-negative bacteria possess a type III secretion system (TTSS¶) that can activate the NLRC4 inflammasome, process caspase-1 and lead to secretion of mature IL-1β. This is dependent on the presence of intracellular flagellin. Previous reports have suggested that this activation is independent of extracellular K+ and not accompanied by leakage of K+ from the cell, in contrast to activation of the NLRP3 inflammasome. However, non-flagellated strains of Pseudomonas aeruginosa are able to activate NLRC4, suggesting that formation of a pore in the cell membrane by the TTSS apparatus may be sufficient for inflammasome activation. Thus, we set out to determine if extracellular K+ influenced P. aeruginosa inflammasome activation. We found that raising extracellular K+ prevented TTSS NLRC4 activation by the non-flagellated P. aeruginosa strain PA103ΔUΔT at concentrations above 90 mm, higher than those reported to inhibit NLRP3 activation. Infection was accompanied by efflux of K+ from a minority of cells as determined using the K+-sensitive fluorophore PBFI, but no formation of a leaky pore. We obtained exactly the same results following infection with Salmonella typhimurium, previously described as independent of extracellular K+. The inhibitory effect of raised extracellular K+ on NLRC4 activation thus reflects a requirement for a decrease in intracellular K+ for this inflammasome component as well as that described for NLRP3.

NLRP3 inflammasome participates in lysosomal destabilization in macrophages exposed to the bacterial toxin tetanolysin O (136.30)

Abstract The NLRP3 inflammasome is a multiprotein complex that includes the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD), which binds with capase-1 via CARD-CARD interactions and is involved with capase-1 activation. Our lab has previously shown that exposure of murine bone marrow derived macrophages (BMDM) with the cholesterol dependent cytolysin tetanolysin O (TLO), produced by Clostridium tetani, activates the NLRP3 inflammasome and induces IL-1beta secretion. Furthermore, we have shown that TLO treatment of BMDM results in the loss of acidity in lysosomes. In this study, our objective was to determine if the effects of TLO on the lysosome in BMDM are dependent upon NLRP3 and whether the inflammasome plays a role in lysosomal destabilization. Our data show that in NLRP3-deficient macrophages lysosomal permeabilization is diminished following toxin exposure relative to equivalently treated wild type cells. The results to be presented further our understanding of how NLRP3 functions in macrophages exposed to stressful stimuli such as bacterial toxins.

Two haplotypes of the P2X7receptor containing the Ala‐348 to Thr polymorphism exhibit a gain‐of‐function effect and enhanced interleukin‐1β secretion

The P2X(7) receptor is an ATP-gated cation channel expressed in immune cells and plays a role in proinflammatory cytokine release from monocytes and macrophages. This study investigated the coinheritance of 12 functionally relevant single nucleotide polymorphisms (SNPs) in the human P2X(7) gene (P2RX7), and the functional effect of each singly and in combination was assessed by measurements of ATP-induced currents and ethidium(+) uptake. Genotyping of 3430 Caucasian subjects identified 4 common haplotypes in addition to the common (wild-type) P2X(7)-1. Two haplotypes (denoted P2X(7)-2 and P2X(7)-4) contained various combinations of gain-of-function SNPs. P2X(7)-4 was identified uniquely by the Gln-460 to Arg polymorphism (rs2230912). When expressed in HEK-293 cells, recombinant P2X(7)-2, and P2X(7)-4 haplotypes displayed a 3-fold and 5-fold increase, respectively, in receptor function compared to the wild-type P2X(7)-1. Both P2X(7) haplotypes contained the Ala-348>Thr polymorphism (rs1718119), and this mutation was critical for the gain-of-function effect. Peripheral blood monocytes and erythrocytes from subjects homozygous for gain-of-function P2X(7) haplotypes exhibited increased ATP-induced ethidium(+) uptake and (86)Rb(+) efflux, respectively, and this correlated with increased IL-1beta secretion from LPS-primed monocytes. Inheritance of these P2X(7) haplotypes predisposing to increased proinflammatory cytokine secretion may be important in genetic association studies of inflammatory, infectious, and psychiatric disorders.