Milena Sokolowska

Prostaglandin E2 Inhibits NLRP3 Inflammasome Activation through EP4 Receptor and Intracellular Cyclic AMP in Human Macrophages

PGE2 is a potent lipid mediator involved in maintaining homeostasis but also promotion of acute inflammation or immune suppression in chronic inflammation and cancer. Nucleotide-binding domain, leucine-rich repeat–containing protein (NLR)P3 inflammasome plays an important role in host defense. Uncontrolled activation of the NLRP3 inflammasome, owing to mutations in the NLRP3 gene, causes cryopyrin-associated periodic syndromes. In this study, we showed that NLRP3 inflammasome activation is inhibited by PGE2 in human primary monocyte-derived macrophages. This effect was mediated through PGE2 receptor subtype 4 (EP4) and an increase in intracellular cAMP, independently of protein kinase A or exchange protein directly activated by cAMP. A specific agonist of EP4 mimicked, whereas its antagonist or EP4 knockdown reversed, PGE2-mediated NLRP3 inhibition. PGE2 caused an increase in intracellular cAMP. Blockade of adenylate cyclase by its inhibitor reversed PGE2-mediated NLRP3 inhibition. Increase of intracellular cAMP by an activator of adenylate cyclase or an analog of cAMP, or a blockade of cAMP degradation by phosphodiesterase inhibitor decreased NLRP3 activation. Protein kinase A or exchange protein directly activated by cAMP agonists did not mimic, and their antagonists did not reverse, PGE2-mediated NLRP3 inhibition. Additionally, constitutive IL-1β secretion from LPS-primed PBMCs of cryopyrin-associated periodic fever syndromes patients was substantially reduced by high doses of PGE2. Moreover, blocking cytosolic phospholipase A2α by its inhibitor or small interfering RNA or inhibiting cyclooxygenase 2, resulting in inhibition of endogenous PGE2 production, caused an increase in NLRP3 inflammasome activation. Our results suggest that PGE2 might play a role in maintaining homeostasis during the resolution phase of inflammation and might serve as an autocrine and paracrine regulator.

The fish oil ingredient, docosahexaenoic acid, activates cytosolic phospholipase A2 via GPR120 receptor to produce prostaglandin E2 and plays an anti‐inflammatory role in macrophages

Docosahexaenoic acid (DHA) is one of the major ingredients of fish oil and has been reported to have anti‐inflammatory properties mediated through the GPR120 receptor. Whether cytosolic phospholipase A2 (cPLA2) and lipid mediators produced from cPLA2 activation are involved in the anti‐inflammatory role of DHA in macrophages has not been reported. We report here that DHA and the GPR120 agonist, GW9508, activate cPLA2 and cyclooxygenase 2 (COX‐2), and cause prostaglandin E2 (PGE2) release in a murine macrophage cell line RAW264.7 and in human primary monocyte‐derived macrophages. DHA and GW9508 activate cPLA2 via GPR120 receptor, G protein Gαq and scaffold protein β‐arrestin 2. Extracellular signal‐regulated kinase 1/2 activation is involved in DHA‐ and GW9508‐induced cPLA2 activation, but not p38 mitogen‐activated protein kinase. The anti‐inflammatory role of DHA and GW9508 is in part via activation of cPLA2, COX‐2 and production of PGE2 as a cPLA2 inhibitor or a COX‐2 inhibitor partially reverses the DHA‐ and GW9508‐induced inhibition of lipopolysaccharide‐induced interleukin‐6 secretion. The cPLA2 product arachidonic acid and PGE2 also play an anti‐inflammatory role. This effect of PGE2 is partially through inhibition of the nuclear factor‐κB signalling pathway and through the EP4 receptor of PGE2 because an EP4 inhibitor or knock‐down of EP4 partially reverses DHA inhibition of lipopolysaccharide‐induced interleukin‐6 secretion. Hence, DHA has an anti‐inflammatory effect partially through induction of PGE2.

Low molecular weight hyaluronan activates cytosolic phospholipase A2α and eicosanoid production in monocytes and macrophages

Background: Fragmented hyaluronan (a major extracellular matrix component) and eicosanoids (potent lipid mediators) are associated with chronic inflammatory diseases and cancer. Results: Fragmented hyaluronan stimulates lipid mediator production in human monocytes and macrophages and influences macrophage differentiation toward a distinct activation pattern. Conclusion: These findings reveal a novel link between hyaluronan-mediated inflammation and lipid metabolism. Significance: This link may provide new targets for disease therapeutics. Hyaluronan (HA) is the major glycosaminoglycan in the extracellular matrix. During inflammation, there is an increased breakdown of HA, resulting in the accumulation of low molecular weight (LMW) HA and activation of monocytes and macrophages. Eicosanoids, derived from the cytosolic phospholipase A2 group IVA (cPLA2α) activation, are potent lipid mediators also attributed to acute and chronic inflammation. The aim of this study was to determine the effect of LMW HA on cPLA2α activation, arachidonic acid (AA) release, and subsequent eicosanoid production and to examine the receptors and downstream mechanisms involved in these processes in monocytes and differently polarized macrophages. LMW HA was a potent stimulant of AA release in a time- and dose-dependent manner, induced cPLA2α, ERK1/2, p38, and JNK phosphorylation, as well as activated COX2 expression and prostaglandin (PG) E2 production in primary human monocytes, murine RAW 264.7, and wild-type bone marrow-derived macrophages. Specific cPLA2α inhibitor blocked HA-induced AA release and PGE2 production in all of these cells. Using CD44, TLR4, TLR2, MYD88, RHAMM or STAB2 siRNA-transfected macrophages and monocytes, we found that AA release, cPLA2α, ERK1/2, p38, and JNK phosphorylation, COX2 expression, and PGE2 production were activated by LMW HA through a TLR4/MYD88 pathway. Likewise, PGE2 production and COX2 expression were blocked in Tlr4−/− and Myd88−/− mice, but not in Cd44−/− mice, after LMW HA stimulation. Moreover, we demonstrated that LMW HA activated the M1 macrophage phenotype with the unique cPLA2α/COX2high and COX1/ALOX15/ALOX5/LTA4Hlow gene and PGE2/PGD2/15-HETEhigh and LXA4low eicosanoid profile. These findings reveal a novel link between HA-mediated inflammation and lipid metabolism.

Apocynin reduces reactive oxygen species concentrations in exhaled breath condensate in asthmatics

ABSTRACT Asthma is an inflammatory airway disease, and oxidative stress was proven to be involved in its pathogenesis. Apocynin effectively inhibits the main source of reactive oxygen species (ROS)—nicotinamide adenine dinucleotide phosphate (NADPH) oxidase—by blocking its activation. The aim of this study was to investigate the effect of inhaled apocynin on ROS and RNS (reactive nitrogen species) concentration in 14 nonsmoking mild asthmatics. Effects of nebulized apocynin (0.5 mg/mL) were assessed in exhaled breath condensate (EBC) after 30, 60, and 120 minutes, and safety parameters have been analyzed. Apocynin significantly decreased H2O2 concentration in EBC in comparison with placebo after 60 and 120 minutes. Moreover, apocynin significantly reduced NO−2 concentration 30 and 60 minutes after nebulization and caused a significant decrease of NO−3 concentration in EBC 60 and 120 minutes after administration, comparing with placebo. No adverse events have been observed throughout the study. This research confirmed anti-inflammatory properties of nebulized apocynin, which might be an effective and safe drug in bronchial asthma.

Allergen immunotherapy for allergic asthma: A systematic review and meta-analysis

To inform the development of the European Academy of Allergy and Clinical Immunologys (EAACI) Guidelines on Allergen Immunotherapy (AIT) for allergic asthma, we assessed the evidence on the effectiveness, cost‐effectiveness and safety of AIT.

Regulation of bronchial epithelial barrier integrity by type 2 cytokines and histone deacetylases in asthmatic patients

Background: Tight junctions (TJs) form a barrier on the apical side of neighboring epithelial cells in the bronchial mucosa. Changes in their integrity might play a role in asthma pathogenesis by enabling the paracellular influx of allergens, toxins, and microbes to the submucosal tissue. Objective: The regulation of bronchial epithelial TJs by TH2 cells and their cytokines and their involvement in epigenetic regulation of barrier function were investigated. Methods: The expression, regulation, and function of TJs were determined in air‐liquid interface (ALI) cultures of control and asthmatic primary human bronchial epithelial cells (HBECs) by means of analysis of transepithelial electrical resistance, paracellular flux, mRNA expression, Western blotting, and immunofluorescence staining. Results: HBECs from asthmatic patients showed a significantly low TJ integrity in ALI cultures compared with HBECs from healthy subjects. TH2 cell numbers and levels of their cytokines, IL‐4 and IL‐13, decreased barrier integrity in ALI cultures of HBECs from control subjects but not in HBECs from asthmatic patients. They induced a physical separation of the TJs of adjacent cells in immunofluorescence staining of the TJ molecules occludin and zonula occludens‐1. We observed that expression of histone deacetylases (HDACs) 1 and 9, and Silent information regulator genes (sirtuins [SIRTs]) 6 and 7 were significantly high in HBECs from asthmatic patients. IL‐4 and IL‐13 significantly increased the expression of HDACs and SIRTs. The role of HDAC activation on epithelial barrier leakiness was confirmed by HDAC inhibition, which improved barrier integrity through increased synthesis of TJ molecules in epithelium from asthmatic patients to the level seen in HBECs from control subjects. Conclusion: Our data demonstrate that barrier leakiness in asthmatic patients is induced by TH2 cells, IL‐4, and IL‐13 and HDAC activity. The inhibition of endogenous HDAC activity reconstitutes defective barrier by increasing TJ expression.

Apocynin decreases hydrogen peroxide and nirtate concentrations in exhaled breath in healthy subjects

The imbalance between reactive oxygen species (ROS) synthesis and antioxidants might be involved in the pathogenesis of many inflammatory diseases. NADPH oxidase, an enzyme responsible for ROS production, may represent an attractive therapeutic target to inhibit, for the treatment of these diseases. Apocynin is an inhibitor of activation of NADPH oxidase complex present in the inflammatory cells. In double blind, placebo-controlled, cross-over study, we investigated the effect of nebulized apocynin on ROS synthesis in 10 nonsmoking healthy volunteers. Apocynin (6ml of 0.5mg/ml) was administered by nebulization and its effects on H(2)O(2), NO(2)(-) and NO(3)(-) generation were assessed after 30, 60 and 120min by collecting exhaled breath condensate (EBC) samples using an EcoScreen analyzer. Additionally, respiratory parameters have been evaluated, utilizing spirometry and DLCO. We also analyzed peripheral blood differential counts and NO(2)(-) serum level, cough scale control and blood pressure as safety parameters. Apocynin caused reduction of H(2)O(2) concentration in EBC as compared to placebo, after 60min. of inhalation (0.18microM vs. 0.31microM, p<0.05) as well as after 120min. (0.2microM vs. 0.31microM, p<0.05). Similarly, apocynin significantly decreased concentration of NO(3)(-) as compared to placebo, after 60 and 120min. (6.8microM vs. 14.4microM and 6.5microM vs. 14.9microM respectively, p<0.05). Apocynin was well tolerated and no adverse events have been observed throughout the study. Thus, as apocynin significantly influence ROS concentration, it might have also antiinflammatory properties. As it is safe, it may have a potential to become a drug in airway inflammatory diseases treatment.

High-dose bee venom exposure induces similar tolerogenic B-cell responses in allergic patients and healthy beekeepers.

The involvement of B cells in allergen tolerance induction remains largely unexplored. This study investigates the role of B cells in this process, by comparing B‐cell responses in allergic patients before and during allergen immunotherapy (AIT) and naturally exposed healthy beekeepers before and during the beekeeping season.

Hydrogen peroxide and nitrite reduction in exhaled breath condensate of COPD patients

Chronic obstructive pulmonary disease (COPD) is predominantly the result of years of cigarette smoking. Increased oxidative stress in COPD derives from the increased burden of inhaled oxidants (cigarette smoke), air pollution and the increase in reactive oxygen and nitrogen species (ROS and RNS), generated by some inflammatory, immune, and structural airways cells. In view of the lack of therapy that might inhibit the progress of the disease, there is an urgent need for a successful therapeutic approach. Apocynin is a molecule inhibiting activation of NADPH oxidase - enzyme generating ROS and RNS precursor. Thus, our aim was to analyze apocynin influence on hydrogen peroxide and nitrite concentrations in EBC of COPD patients. Apocynin reduced concentration of H(2)O(2) in COPD patients 60 and 120 min after apocynin inhalation, in comparison to placebo (0.43 μM vs. 0.59 μM, and 0.4 μM vs. 0.59 μM respectively, p < 0.05). Moreover, apocynin decreased NO(2)(-) ions concentration in airways of COPD patients after apocynin nebulization (3.97 μM vs. 4.48 μM after 30 min, 3.82 μM vs. 4.48 μM after 60 min, and 3.76 μM vs. 4.48 μM after 30 min respectively, p < 0.05). No adverse effects have been observed. The results suggest that apocynin might be considered as anti-inflammatory agent, and, possibly used in therapy of COPD.

85‐kDa cytosolic phospholipase A2 group IVα gene promoter polymorphisms in patients with severe asthma: a gene expression and case–control study

Cytosolic phospholipase A2 (cPLA2) group IVα is a critical enzyme involved in the liberation of arachidonic acid from cellular membranes. cPLA2−/− mice have reduced allergen‐induced bronchoconstriction and bronchial hyperresponsiveness. The goal of this study was to investigate polymorphisms of the (CA)n and (T)n microsatellites and surrounding regions in the cPLA2α gene promoter. We analysed the cPLA2 promoter regions containing (CA)n and (T)n repeats in 87 patients with severe asthma and in 48 control subjects by bidirectional sequencing. Functional studies were performed utilizing reporter genes derived from subjects with varying numbers of these repeats, and on constructs with a series of deletions. We found that the (CA)n and (T)n regions are polymorphic and that constructs with CA or T repeats or CA and T repeats deleted revealed, respectively, a 41·8 ± 7%, 22·3 ± 5% and 100 ± 20% increase in reporter gene activity. A lower number of CA or T repeats caused higher cPLA2 promoter luciferase activity. The group of shorter alleles of the (CA)n microsatellite region (n = 12–18) (Pcor = 0·00006), and the group of shorter alleles of (T)n repeats region (n = 17–38) (Pcor = 0·0039) occurred significantly more often in patients with severe asthma. We also found novel SNPs in positions −292 C > G, −185 A > C, −180 T > C and −165 A > C. Two of them were associated with the severe asthma phenotype: −180T allele (Pcor = 0·03996) and −185 A allele (Pcor = 0·03966). These results demonstrate that (CA)n and (T)n repeats may have an influence on cPLA2 transcription which might play a role in severe asthma pathogenesis.