Jani Lappalainen

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.

Heat Shock Proteins in Diabetes and Wound Healing

The heat shock proteins (HSPs), originally identified as heat-inducible gene products, are a highly conserved family of proteins that respond to a wide variety of stress. Although HSPs are among the most abundant intracellular proteins, they are expressed at low levels under normal physiological conditions, and show marked induction in response to various stressors. HSPs function primarily as molecular chaperones, facilitating the folding of other cellular proteins, preventing protein aggregation, or targeting improperly folded proteins to specific pathways for degradation. By modulating inflammation, wound debris clearance, cell proliferation, migration and collagen synthesis, HSPs are essential for normal wound healing of the skin. In this review, our goal is to discuss the role and clinical implications of HSP with respect to skin wound healing and diabetes. The numerous defects in the function of HSPs associated with diabetes could contribute to the commonly observed complications and delayed wound healing in diabetics. Several physical, pharmacological and genetic approaches may be considered to address HSP-directed therapies both in the laboratory and in the clinics.

Increased Expression of Elastolytic Cathepsins S, K, and V and Their Inhibitor Cystatin C in Stenotic Aortic Valves

Objective—To investigate the possible role of elastolytic cathepsins S, K, and V and their endogenous inhibitor cystatin C in adverse extracellular matrix remodeling of stenotic aortic valves. Methods and Results—Stenotic aortic valves were collected at valve replacement surgery and control valves at cardiac transplantations. The expression of cathepsins S, K, and V and cystatin C was studied by conventional and real-time polymerase chain reaction and by immunohistochemistry. Total cathepsin activity in the aortic valves was quantified by a fluorometric microassay. When compared with control valves, stenotic valves showed increased mRNA expression of cathepsins S, K, and V (P<0.05 for each) and a higher total cathepsin activity (P<0.001). In stenotic valves, cystatin C mRNA was increased (P<0.05), and cystatin C protein was found particularly in areas with infiltrates of inflammatory cells. Both cathepsin S and cystatin C were present in bony areas of the valves, whereas cathepsin V localized to endothelial cells in areas rich of neovascularization. Incubation of thin sections of aortic valves with cathepsins S, K, and V resulted in severe disruption of elastin fibers, and this cathepsin effect could be blocked by adding cystatin C to the incubation system. Conclusions—Stenotic aortic valves show increased expression and activity of elastolytic cathepsins S, K, and V. These cathepsins may accelerate the destruction of aortic valvular extracellular matrix, so promoting the progression of aortic stenosis.

Exercise-induced oxidative stress and muscle stress protein responses in trotters

Acute exercise induces oxidative stress and heat shock protein (HSP) expression. Information on the protection of stress proteins against oxidant insult and muscle damage during moderate exercise is scanty. We aimed to show how a single bout of moderate exercise affects the markers of oxidative stress and heat shock factor-1 (HSF1; the transcriptional regulator of HSP synthesis), and HSP70, HSP90 and glucose-regulated protein (GRP75) expression in horses. Eight clinically normal and regularly trained standardbred trotters were treadmill-exercised for 45xa0min at moderate intensity. Blood samples were collected prior to and immediately after exercise and at 4 and 24xa0h of recovery. Muscle biopsy samples from the middle gluteal muscle were taken before exercise and after 4xa0h of recovery. Acute exercise did not activate HSF1 or induce expression of HSP70, HSP90 or GRP75 in skeletal muscle. One bout of acute exercise increased protein oxidation, which was measured by protein carbonyls in plasma and muscle, but it did not effect 4-hydroxynonenal protein adducts, which are markers of lipid peroxidation. Furthermore, mild muscle damage was observed 4xa0h after exercise. Our results showed that horses are susceptible to oxidative stress. One bout of exercise at moderate intensity and duration did not induce HSP responses despite the increased protein oxidation and tissue inflammation in equine muscle.

Diabetes impairs exercise training-associated thioredoxin response and glutathione status in rat brain

Regular exercise plays an important preventive and therapeutic role in oxidative stress-associated diseases such as diabetes and its complications. Thiol antioxidants including thioredoxin (TRX) and glutathione (GSH) have a crucial role in controlling cellular redox status. In this study, the effects of 8 wk of exercise training on brain TRX and GSH systems, and antioxidant enzymes were tested in rats with or without streptozotocin-induced diabetes. We found that in untrained animals, the levels of TRX-1 (TRX1) protein and activity, and thioredoxin-interacting protein (TXNip) were similar in diabetic and nondiabetic animals. Exercise training, however, increased TRX1 protein in nondiabetic animals without affecting TXNip levels, whereas diabetes inhibited the effect of training on TRX1 protein and also increased TXNip mRNA. In addition, the proportion of oxidized glutathione (GSSG) to total GSH was increased in animals with diabetes, indicating altered redox status and possibly increased oxidative stress. Glutathione peroxidase-1 (GPX1) levels were not affected by diabetes or exercise training, although diabetes increased total GPX activity. Both diabetes and exercise training decreased glutathione reductase (GRD) activity and cytosolic superoxide dismutase (Cu,Zn-SOD) levels. Nevertheless, diabetes or training had no effect on Cu,Zn-SOD mRNA, Mn-SOD protein, total SOD activity, or catalase mRNA, protein, or activity. Our findings suggest that exercise training increases TRX1 levels in brain without a concomitant rise in TXNip, and that experimental diabetes is associated with an incomplete TRX response to training. Increased oxidative stress may be both a cause and a consequence of perturbed antioxidant defenses in the diabetic brain.

Dietary antioxidants for the athlete

Physical exercise induces oxidative stress and tissue damage. Although a basal level of reactive oxygen species (ROS) is required to drive redox signaling and numerous physiologic processes, excess ROS during exercise may have adverse implications on health and performance. Antioxidant nutrients may be helpful in that regard. Caution should be exercised against excess antioxidant supplements, however. This article presents a digest for sports practitioners. The following three recommendations are made: 1) it is important to determine the individual antioxidant need of each athlete performing a specific sport; 2) multinutrient preparations, as opposed to megadoses of any single form of nutrient, seem to be a more prudent path to choose; and 3) for outcomes of antioxidant supplementation, performance should not be the only criteria. Overall well being of the athlete, faster recovery, and minimization of injury time could all be affected by antioxidant therapy.

OxLDL-IgG immune complexes induce expression and secretion of proatherogenic cytokines by cultured human mast cells

OBJECTIVEnHuman atherosclerotic lesions contain mast cells and immunoglobulin G immune complexes containing oxidized low-density lipoproteins (oxLDL-IgG ICs). Here we studied whether such oxLDL-IgG ICs can activate human mast cells and induce them to express and secrete pro-inflammatory cytokines that are potentially capable of inducing and amplifying atherogenic processes.nnnMETHODS AND RESULTSnIncubation of cultured human mast cells in the presence of oxLDL-IgG ICs led to a significant dose-dependent upregulation of the expression and secretion of tumor necrosis factor-alpha (TNF-a) and interleukin-8 (IL-8), and the chemotactic cytokine monocyte chemoattractant protein-1 (MCP-1). The secretory responses were dose-dependent and associated with moderate release of histamine and tryptase, which are preformed mast cell mediators contained in the cytoplasmic secretory granules of the cells. Also native LDL-IgG ICs induced similar pro-inflammatory cytokine response, suggesting that ICs per se are important for the IgG IC-induced mast cell activation.nnnCONCLUSIONnMast cells in atherosclerotic lesions which also contain oxLDL-IgG ICs may become activated by the ICs and secrete many pro-inflammatory cytokines. Our results suggest that intimal mast cells act as a cellular link between oxLDL-IgG ICs and the inflammatory response in atherosclerosis.

Human mast cells arise from a common circulating progenitor

BACKGROUNDnHuman tissue mast cells (MCs) have the potential to express several neutral granule proteases, which are the most precise markers of the phenotypic heterogeneity of MCs. However, the full extent of such heterogeneity is limited by the fact that MCs containing either tryptase only or tryptase and chymase have long been considered to be the sole MC phenotypes. Moreover, the potential developmental relationship between human MCs of different protease phenotypes has remained a matter of dispute.nnnOBJECTIVEnWe attempted to define how human MCs with different protease phenotypes relate to their circulating progenitors.nnnMETHODSnMCs were generated from human peripheral blood-derived CD34(+) progenitors in the presence of kit ligand (KITLG) and the cytokines IL-3, IL-9, and IL-6 under serum-free conditions, or by KITLG alone in the presence or absence of serum. The expression of chymase, carboxypeptidase A3, cathepsin G, granzyme B, and the tryptases derived from the TPSAB1, TPSB2, TPSD1, and TPSG1/PRSS31 genes were determined weekly at the mRNA and/or protein levels.nnnRESULTSnIncubation of CD34(+) progenitors in the presence of KITLG and the cytokines IL-3, IL-9, and IL-6 promoted the development of a single population of MCs with a uniform tryptase(+), chymase(+), CPA3(+), cathepsin G(+), and granzyme B(+) phenotype. Interestingly, the presence of KITLG alone was sufficient to induce the expression of all the above proteases.nnnCONCLUSIONnAll circulating human MC progenitors have the potential to differentiate into MCs expressing the complete panel of neutral granule proteases, implying that human MCs originate from a common MC-committed progenitor.

Expression of antizyme inhibitor 2 in mast cells and role of polyamines as selective regulators of serotonin secretion.

Background Upon IgE-mediated activation, mast cells (MC) exocytose their cytoplasmic secretory granules and release a variety of bioactive substances that trigger inflammatory responses. Polyamines mediate numerous cellular and physiological functions. We report here that MCs express antizyme inhibitor 2 (AZIN2), an activator of polyamine biosynthesis, previously reported to be exclusively expressed in the brain and testis. We have investigated the intracellular localization of AZIN2 both in resting and activated MCs. In addition, we have examined the functional role of polyamines, downstream effectors of AZIN2, as potential regulators of MC activity. Methodology/Principal Findings Immunostainings show that AZIN2 is expressed in primary and neoplastic human and rodent MCs. We demonstrate that AZIN2 localizes in the Vamp-8 positive, serotonin-containing subset of MC granules, but not in tryptase-containing granules, as revealed by double immunofluorescence stainings. Furthermore, activation of MCs induces rapid upregulation of AZIN2 expression and its redistribution, suggesting a role for AZIN2 in secretory granule exocytosis. We also demonstrate that release of serotonin from activated MCs is polyamine-dependent whereas release of histamine and β-hexosaminidase is not, indicating a granule subtype-specific function for polyamines. Conclusions/Significance The study reports for the first time the expression of AZIN2 outside the brain and testis, and demonstrates the intracellular localization of endogenous AZIN2 in MCs. The granule subtype-specific expression and its induction after MC activation suggest a role for AZIN2 as a local, in situ regulator of polyamine biosynthesis in association with serotonin-containing granules of MCs. Furthermore, our data indicates a novel function for polyamines as selective regulators of serotonin release from MCs.