Kari K. Eklund

Cholesterol Crystals Activate the NLRP3 Inflammasome in Human Macrophages: A Novel Link between Cholesterol Metabolism and Inflammation

Background Chronic inflammation of the arterial wall is a key element in the pathogenesis of atherosclerosis, yet the factors that trigger and sustain the inflammation remain elusive. Inflammasomes are cytoplasmic caspase-1-activating protein complexes that promote maturation and secretion of the proinflammatory cytokines interleukin(IL)-1β and IL-18. The most intensively studied inflammasome, NLRP3 inflammasome, is activated by diverse substances, including crystalline and particulate materials. As cholesterol crystals are abundant in atherosclerotic lesions, and IL-1β has been linked to atherogenesis, we explored the possibility that cholesterol crystals promote inflammation by activating the inflammasome pathway. Principal Findings Here we show that human macrophages avidly phagocytose cholesterol crystals and store the ingested cholesterol as cholesteryl esters. Importantly, cholesterol crystals induced dose-dependent secretion of mature IL-1β from human monocytes and macrophages. The cholesterol crystal-induced secretion of IL-1β was caspase-1-dependent, suggesting the involvement of an inflammasome-mediated pathway. Silencing of the NLRP3 receptor, the crucial component in NLRP3 inflammasome, completely abolished crystal-induced IL-1β secretion, thus identifying NLRP3 inflammasome as the cholesterol crystal-responsive element in macrophages. The crystals were shown to induce leakage of the lysosomal protease cathepsin B into the cytoplasm and inhibition of this enzyme reduced cholesterol crystal-induced IL-1β secretion, suggesting that NLRP3 inflammasome activation occurred via lysosomal destabilization. Conclusions The cholesterol crystal-induced inflammasome activation in macrophages may represent an important link between cholesterol metabolism and inflammation in atherosclerotic lesions.

Serum Amyloid A Activates the NLRP3 Inflammasome via P2X7 Receptor and a Cathepsin B-Sensitive Pathway

Serum amyloid A (SAA) is an acute-phase protein, the serum levels of which can increase up to 1000-fold during inflammation. SAA has a pathogenic role in amyloid A-type amyloidosis, and increased serum levels of SAA correlate with the risk for cardiovascular diseases. IL-1β is a key proinflammatory cytokine, and its secretion is strictly controlled by the inflammasomes. We studied the role of SAA in the regulation of IL-1β production and activation of the inflammasome cascade in human and mouse macrophages, as well as in THP-1 cells. SAA could provide a signal for the induction of pro–IL-1β expression and for inflammasome activation, resulting in secretion of mature IL-1β. Blocking TLR2 and TLR4 attenuated SAA-induced expression of IL1B, whereas inhibition of caspase-1 and the ATP receptor P2X7 abrogated the release of mature IL-1β. NLRP3 inflammasome consists of the NLRP3 receptor and the adaptor protein apoptosis-associated speck-like protein containing CARD (a caspase-recruitment domain) (ASC). SAA-mediated IL-1β secretion was markedly reduced in ASC−/− macrophages, and silencing NLRP3 decreased IL-1β secretion, confirming NLRP3 as the SAA-responsive inflammasome. Inflammasome activation was dependent on cathepsin B activity, but it was not associated with lysosomal destabilization. SAA also induced secretion of cathepsin B and ASC. In conclusion, SAA can induce the expression of pro–IL-1β and activation of the NLRP3 inflammasome via P2X7 receptor and a cathepsin B-sensitive pathway. Thus, during systemic inflammation, SAA may promote the production of IL-1β in tissues. Furthermore, the SAA-induced secretion of active cathepsin B may lead to extracellular processing of SAA and, thus, potentially to the development of amyloid A amyloidosis.

Extracellular Acidosis Is a Novel Danger Signal Alerting Innate Immunity via the NLRP3 Inflammasome

Background: Local acidosis has been demonstrated in ischemic tissues and at inflammatory sites. Results: Acidic extracellular pH triggers NLRP3 inflammasome activation and interleukin-1β secretion in human macrophages. Conclusion: Acidic pH represents a novel danger signal alerting the innate immunity. Significance: Local acidosis may promote inflammation at ischemic and inflammatory sites. Local extracellular acidification has been demonstrated at sites of ischemia and inflammation. IL-1β is one of the key proinflammatory cytokines, and thus, its synthesis and secretion are tightly regulated. The NLRP3 (nucleotide-binding domain leucine-rich repeat containing family, pyrin domain containing 3) inflammasome complex, assembled in response to microbial components or endogenous danger signals, triggers caspase-1-mediated maturation and secretion of IL-1β. In this study, we explored whether acidic environment is sensed by immune cells as an inflammasome-activating danger signal. Human macrophages were exposed to custom cell culture media at pH 7.5–6.0. Acidic medium triggered pH-dependent secretion of IL-1β and activation of caspase-1 via a mechanism involving potassium efflux from the cells. Acidic extracellular pH caused rapid intracellular acidification, and the IL-1β-inducing effect of acidic medium could be mimicked by acidifying the cytosol with bafilomycin A1, a proton pump inhibitor. Knocking down the mRNA expression of NLRP3 receptor abolished IL-1β secretion at acidic pH. Remarkably, alkaline extracellular pH strongly inhibited the IL-1β response to several known NLRP3 activators, demonstrating bipartite regulatory potential of pH on the activity of this inflammasome. The data suggest that acidic environment represents a novel endogenous danger signal alerting the innate immunity. Low pH may thus contribute to inflammation in acidosis-associated pathologies such as atherosclerosis and post-ischemic inflammatory responses.

Mast cells in the pathogenesis of rheumatic diseases and as potential targets for anti-rheumatic therapy

Summary:  Increasing evidence suggests that mast cells (MCs), in addition to acute allergic reactions, are involved in the pathogenesis of chronic inflammatory diseases and in particular in rheumatoid arthritis (RA). MCs reside in connective tissues and in synovial tissue of joints. They produce an array of proinflammatory mediators, tissue destructive proteases, and cytokines, most prominently tumor necrosis factor‐α, which is one of the key cytokines in the pathogenesis of RA. MCs may also participate in the development of secondary or amyloid A amyloidosis, as the partial degradation of the serum amyloid A (SAA) protein by MCs leads to the generation of a highly amyloidogenic N‐terminal fragment of SAA. MCs may contribute to the pathogenesis of connective tissue diseases, scleroderma, vasculitic syndromes, and systemic lupus erythematosus, although the data available are limited. Inhibition of the most important growth factor receptor of human MCs, c‐Kit, by the selective tyrosine kinase inhibitor imatinib mesylate, induces apoptosis of synovial tissue MCs. As MCs are long‐lived cells, induction of their apoptosis could be a feasible approach to inhibit their functions. Preliminary findings suggest that a drug that inhibits c‐Kit could have anti‐rheumatic activity in the treatment of patients with RA and spondyloarthropathies.

Treatment of rheumatoid arthritis with imatinib mesylate: clinical improvement in three refractory cases

BACKGROUND. Imatinib mesylate is an inhibitor of a few tyrosine kinases including KIT, which is an important growth factor receptor of mast cells. AIM. To study the efficacy and safety of imatinib in the treatment of rheumatoid arthritis. METHOD. Three patients with severe rheumatoid were treated with imatinib for 12 weeks. The number of tender and swollen joints, patient-assessed disease activity and pain as assessed by a visual analogue scale, a health assessment questionnaire (HAQ) score, serum C-reactive protein (CRP) and blood erythrocyte sedimentation rate (ESR) were used as the primary outcome measures. RESULTS. All outcome measures improved. The swollen joint count decreased in all patients, and the tender joint count in two of the three patients. The patients reported less pain and disease activity, and the HAQ scores improved. Serum CRP and blood ESR improved in two patients. One patient interrupted therapy due to a rash. CONCLUSIONS. Imatinib mesylate may have considerable anti-rheumatic efficacy. The mechanism of action is not known, but one possible target for the action of imatinib is inhibition of the KIT receptor on mast cells.

Cost–utility of different treatment strategies after the failure of tumour necrosis factor inhibitor in rheumatoid arthritis in the Finnish setting

Objective. To evaluate the cost–utility of different treatment strategies in severe RA after TNF-inhibitor failure. Methods. The cost-effectiveness of treatment strategies was compared in a group of hypothetical Finnish RA patients. Initially, the patients received either best supportive care (BSC) or one of the following treatments before BSC: adalimumab (ADAL), abatacept (ABAT), etanercept (ETAN), infliximab (INFL) or rituximab (RTX). Further treatments were added to the most cost-effective strategy in a stepwise manner. The analysis was performed on an Excel-based Markov state transition model using the probabilistic approach. The clinical outcomes related to treatments were estimated from published clinical trials. The gained quality-adjusted life-years (QALYs) were estimated based on Health Utilities Index (HUI-3) and disease severity scores (HAQ). The resource use and costs were obtained from the Finnish treatment practice, one published study, the Finnish Unit Cost list and Finnish Medicine Tariffs. Results. Treatment with RTX was more effective and less costly than treatment with ADAL, ABAT or ETAN after TNF-inhibitor failure. An additional QALY gained with RTX costs 30 248 euros compared with BSC. The incremental cost-effectiveness ratios (ICERs) are 50 941, 50 372, 36 121 and 67 003 euros per QALY gained for adding ADAL, ETAN, INFL and ABAT to the RTX strategy, respectively. According to the cost-effectiveness acceptability frontier (CEAF), only BSC or treatments with RTX or RTX followed by INFL should be considered after TNF-inhibitor failure, if willingness to pay is between 0 and 50 000 euros per QALY gained. Conclusions. Treatment with RTX is a cost-effective treatment strategy in RA patients in Finland.

Ethanol induces apoptosis in human mast cells.

AIMS Alcohol abuse is associated with increased frequency of infections attributed to ethanol-induced immune suppression. The precise mechanism of immune suppression is however not known. Mast cells (MC) belong to the innate immune system and they have been implicated in the first line of immune defence against bacteria and parasites. Therefore we studied the effects of ethanol and its first metabolite acetaldehyde on mast cell viability, proliferation and apoptosis. MAIN METHODS Human mast cell line (HMC)-1 cells, mouse bone marrow derived mast cells (mBMMC) and human peripheral blood derived mast cells (HuMC) were used. Effects of ethanol and acetaldehyde on mast cell proliferation were determined by assessing incorporation of [(3)H]thymidine into cellular DNA and by trypan blue exclusion. Apoptosis was assessed by measuring apoptotic nucleosomes and caspase-3, -8 and -9 activities using ELISA and by using Tunel assay. The expression of anti- and proapoptotic proteins Bcl-2 and Bax was analyzed by RT-PCR and western blot, respectively. KEY FINDINGS Ethanol, but not acetaldehyde inhibited dose-dependently the proliferation and viability HMC-1 and mBMMC cells. The decreased viability was caused by apoptotic cell death of the MC. Significant apoptosis of HMC-1 cells was observed in the presence of 43mM (2.5 per thousand) ethanol. Induction of apoptosis was associated with clearly increased caspase-3 activity and moderately increased caspase-8 and 9 activities. Ethanol also shifted the Bcl-2/Bax balance towards apoptosis. SIGNIFICANCE The ethanol-induced reduction of MC viability could contribute to immunosuppression associated with ethanol abuse.

Selective activation of mast cells in rheumatoid synovial tissue results in production of TNF-α, IL-1β and IL-1Ra

Abstract.Objectives and Design:To study the consequences of mast cell activation in human synovial tissue.Methods:Synovial tissue was obtained from 18 RA patients and mast cells was selectively activated in synovial tissue explant cultures. Expression of TNF-α, IL-1β and IL-1Ra were determined and tissue distribution of IL-1β was studied.Results:Compared to untreated synovia, selective activation of synovial mast cells increased significantly the production of TNF-α (0.49 ± 0.88 vs. 4.56 ± 3.18 pg/mg wet tissue, p < 0.001) and IL-1β (0.058 ± 0.032 vs. 2.55 ± 1.98 pg/mg wet tissue, p = 0.013). The expression of TNF-α and IL-1β mRNA increased significantly (19-fold (p = 0.009) and 13-fold (p = 0.031), respectively). Mast cell activation induced IL-1β expression in particular in nearby CD68 positive synovial macrophages. Secretion of IL-1Ra was also increased but to a lesser degree than that of IL-1β.Conclusions:Synovial mast cells produce proinflammmatory cytokines and may thus contribute to the inflammation in RA.

Monovalent cation dependent phase behaviour of dipalmitoylphosphatidylglycerol

Abstract The phase behaviour of dipalmitoylphosphatidylglycerol (DPPG) was studied as a function of [NaCl] using differential scanning calorimetry and the effects of monovalent cations, Na+, K+, Li+ and Cs+ were compared at 300 mM cation concentration. Main phase transition, pretransition, and subtransition temperatures and enthalpies were found to depend on the concentration of NaCl and the monovalent cation species. Compared to the main phase transition both pretransition and subtransition were more sensitive and also exerted greater specificity with respect to the different cations. Considerable hysteresis (δTm) in the main transition temperature (Tm) upon heating and cooling was evident in the absence of salt. Upon increasing [NaCl], a decrease in δTm was observed. Subtransition and pretransition temperatures and enthalpies increased upon increasing [NaCl] from 50 mM to 1 M. The appearance of subtransition was considerably faster in the presence of high NaCl concentrations. No pretransition was observed in the absence of salt or in the presence of 300 mM LiCl. Pretransition temperatures of DPPG in the presence of 300 mM NaCl, KCl, and CsCl were 34.0, 34.3 and 30.4°C, respectively. The subtransition temperatures after 5 days of incubation at 6°C with 300 mM NaCl, KCl, CsCl were 29.3, 22.6 and 23.6°C, respectively. The highest subtransition temperature of 32.6°C was observed in the presence of 300 mM LiCl but compared to other cations the appearance of subtransition in the presence of Li+ required considerably longer incubation times.

Ethanol Inhibits Activation of NLRP3 and AIM2 Inflammasomes in Human Macrophages–A Novel Anti-Inflammatory Action of Alcohol

Objective In the pathogenesis of coronary atherosclerosis, local macrophage-driven inflammation and secretion of proinflammatory cytokines, interleukin-1β (IL-1β) in particular, are recognized as key factors. Moderate alcohol consumption is associated with a reduced risk of coronary artery disease mortality. Here we examined in cultured human macrophages whether ethanol modulates the intracellular processes involved in the secretion of IL-1β. Results Ethanol decreased dose-dependently the production of mature IL-1β induced by activators of the NLRP3 inflammasome, i.e. ATP, cholesterol crystals, serum amyloid A and nigericin. Ethanol had no significant effect on the expression of NLRP3 or IL1B mRNA in LPS-primed macrophages. Moreover, secretion of IL-1β was decreased in parallel with reduction of caspase-1 activation, demonstrating that ethanol inhibits inflammasome activation instead of synthesis of pro-IL-1β. Acetaldehyde, a highly reactive metabolite of ethanol, had no effect on the ATP-induced IL-1β secretion. Ethanol also attenuated the secretion of IL-1β triggered by synthetic double-stranded DNA, an activator of the AIM2 inflammasome. Ethanol conferred the inhibitory functions by attenuating the disruption of lysosomal integrity and ensuing leakage of the lysosomal protease cathepsin B and by reducing oligomerization of ASC. Conclusion Ethanol-induced inhibition of the NLRP3 inflammasome activation in macrophages may represent a biological pathway underlying the protective effect of moderate alcohol consumption on coronary heart disease.