Purpose Neutrophils are considered key effector cells in the pathogenic mechanisms of airway inflammation in asthma. This study assessed the activation status of neutrophils in adult asthmatics, and the therapeutic potential of FTY720, a synthetic sphingosine-1-phosphate analog, on activated neutrophils using an in vitro stimulation model. Methods We isolated peripheral blood neutrophils (PBNs) from 59 asthmatic patients (including 20 aspirin-exacerbated respiratory disease [AERD] and 39 aspirin-tolerant asthma [ATA] groups). PBNs were stimulated with N -formyl-methionyl-leucyl-phenylalanine (fMLP) or lipopolysaccharide (LPS) and their activation status was determined based on reactive oxygen species (ROS) production, cell surface expression of CD11b, interleukin (IL)-8 and matrix metallopeptidase (MMP)-9 release. PBNs were primed with FTY720 to evaluate its anti-inflammatory action. Results In vitro PBN stimulation with fMLP or LPS induced a significant increase in ROS/CD11b/IL-8/MMP-9 levels ( P < 0.05 for all). In asthmatics, fMLP-induced ROS level was significantly correlated with values of forced expiratory volume in 1 second/forced vital capacity (r = −0.278; P = 0.036), maximal mid-expiratory flow (r = −0.309; P = 0.019) and PC20 methacholine (r = −0.302; P = 0.029). In addition, ROS levels were significantly higher in patients with AERD and in those with severe asthma than in those with ATA or non-severe asthma ( P < 0.05 for all). FTY720 treatment could suppress ROS/CD11b levels, and LPS-induced IL-8 and MMP-9 levels ( P < 0.05 for all). Responders to FTY720 treatment had significantly higher neutrophil counts in sputum ( P = 0.004). Conclusions Our findings suggest a useful in vitro PBN stimulation model for evaluating the neutrophil functional status and the therapeutic potentials of neutrophil-targeting candidates in asthmatics.
Among the 56 ESBL-producing isolates, 43 isolates (76.8%) were resistant to fluoroquinolones, but all isolates were susceptible to carbapenems and amikacin. The polymerase chain reaction sequencing results showed that the dominant CTX-M genotype was CTX-M-15 (19/46, 41.3%) in the ESBL-producing E. coli isolates. By contrast, CTX-M-14 and CTX-M-3 were the major genotypes found in Klebsiella spp. Phylogenetic analysis revealed that 21 ESBL-producing E. coli isolates belonged to group D (21/46, 45.6%), followed by group A (13/46, 28.3%), group B2 (11/46, 23.9%), and group B1 (1/46, 2.2%). Only four E. coli isolates (4/46, 8.7%) belonged to the ST131 clone. PFGE showed that the ESBL-producing Enterobacteriaceae were genetically unrelated. The conjugation assay showed that two plasmids harboring CTX-M-15 in E. coli isolates were genetic unrelated, whereas seven plasmids harboring CTX-M-14 (5/7 and 2/7) and four plasmids harboring CTX-M-55 (4/4) showed genetic relatedness, indicating the dissemination of resistance plasmids in this area.
The ATP-binding cassette subfamily C member 4 gene encodes a transmembrane protein involved in the export of proinflammatory molecules, including leukotriene, prostaglandin, and sphingosine-1-phosphate across the plasma membrane. Those metabolites play important roles in asthma. We investigated the potential associations between ABCC4 gene polymorphisms and asthma phenotype. In total, 270 asthma patients and 120 normal healthy controls were enrolled for a genetic association study. Two polymorphisms (−1508A>G and −642C>G) in the ABCC4 promoter were genotyped. The functional variability of the promoter polymorphisms was analyzed by luciferase reporter assay. Inflammatory cytokine levels were measured by enzyme-linked immunosorbent assay. Serum and urinary eicosanoid metabolites, sphingosine-1-phosphate, were evaluated by quadrupole time-of-flight mass spectrometry. Asthma patients carrying the G allele at −1508A>G had significantly higher serum levels of periostin, myeloperoxidase, and urinary levels of 15-hydroxyeicosatetraenoic acid and sphingosine-1-phosphate (P = 0.016, P = 0.027, P = 0.032, and P = 0.010, resp.) compared with noncarrier asthma patients. Luciferase activity was significantly enhanced in human epithelial A549 cells harboring a construct containing the −1508G allele (P < 0.01 for each) compared with a construct containing the −1508A allele. A functional polymorphism in the ABCC4 promoter, −1508A>G, may increase extracellular 15-hydroxyeicosatetraenoic acid, sphingosine-1-phosphate, and periostin levels, contributing to airway inflammation in asthmatics.
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