Roles of Neutrophil Activation in Asthma

Abstract

Background: Neutrophilic inflammation has been implicated in allergic asthma as well as occupational asthma, which is usually associated with severe phenotype. Autophagy plays crucial roles in innate as well as adaptive immune responses by contributing to the proliferation, activation and biological function of various immune cells such as macrophages, neutrophils, mast cells, T and B cells. Autophagy could regulate neutrophil survival and formation of neutrophil extracellular DNA trap (NET), which is an extracellular release of decondensed chromatin from neutrophils triggered by various stimuli including cytokines [e.g. intetleukin(IL)-8 or tumor necrosis factor (TNF)-α], phorbol 12-myristate 13-acetate (PMA) or microbial proteins. In allergic asthma, autophagy and NET were detected in asthmatic airways and inflammatory cells; however, their roles in pathogenesis of asthma have not yet been identified. S100A8 and S100A9 are Ca2+- and Zn2+-binding proteins of the S100 family, which could be produced by lipopolysaccharide-stimulated granulocytes and provoke an innate immune-mediated airway inflammation. S100A9 protein is known to be involved in uncontrolled neutrophilic asthma. The role of S100A8 and S100A9 in baker’s asthma pathogenesis has not been investigated.

Objectives: This thesis has investigated: 1) associations of neutrophilic inflammation in asthma with genetic variants of autophagy related gene (ATG) 5 and ATG7; 2) levels of autophagy and NET production from peripheral blood neutrophils (PBNs) of severe asthma (SA) and non-severe asthma (NSA) patients, as well as the roles of NET in airway inflammation; 3) involvement of serum S100A8 and S100A9 in neutrophilic inflammation of occupational asthma induced by wheat flour

Methods: In the first study, we recruited 408 asthma patients and 201 normal controls (NC) to a genetic association study of ATG5 and ATG7 variants with neutrophilic airway inflammation. ATG5 (-769T>C, -335G>A, and 8830C>T) and ATG7 (-100A>G and 25108G>C) polymorphisms were genotyped using the SNaPshot method. The transcriptional activities of ATG5 -769T>C and -335G>A variants were investigated by luciferase reporter assays. In the second study, 68 asthma patients (including 30 SA and 38 NSA patients) were recruited to investigate the production levels of neutrophilic autophagy and NET from PBNs. Isolated PBNs were treated with interleukin (IL)-8 (100 ng/ml). Autophagy (LC3 expression) and NET production levels were evaluated by Western bloting, immunofluorescent microscopy and PicoGreen assay. Cell survival and tight junction protein (occludin and claudin-1) expression levels in airway epithelial cells (AECs) were evaluated by CCK8 assay and Western blot analysis. Peripheral blood eosinophils (PBEs) were isolated from peripheral blood of human donors using MACS system. Secretion levels of IL-8, eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) were measured by ELISA. In baker’s asthma cohort, we recruited 381 bakery workers and 100 unexposed NCs. Skin prick tests for bakery allergens were performed to determine atopic status of study subjects. Serum levels of S100A8, S100A9, myeloperoxidase (MPO), tumor necrosis factor (TNF)-α, and IL-8 were measured using ELISA. Predictive values of serum S100A8 and S100A9 in bakery workers were estimated by receiver-operating characteristic (ROC) curves. Polymorphisms of toll like receptor 4 gene (TLR4) -2027A>G and -1608T>C, were genotyped using the SnaPshot method.

Results: In asthma patients, the GA/AA genotypes at ATG5 -335G>A SNP were significantly associated with higher neutrophil counts in sputum (P<0.05); the CC/TT genotype at ATG5 8830C>T was significantly associated with lower FEV1% predicted value (P<0.05). Promoter activity assay revealed that DNA fragments containing ATG5 -769T and -335G alleles exhibited significantly higher promoter activities compared to those with -769C and -335A in both human AECs (A549, P<0.01) and human mast cells (HMC-1, P<0.001). GG and CC genotype at ATG7 -100A>G and 25108G>C were significantly associated with high serum levels of IL-8 (P<0.001 and P<0.00, respectively).

In the second study, naïve and IL-8 treated neutrophils of SA patients expressed higher autophagy and NET production levels than NSA patients (P<0.001 for both experiments). IL-8 increased autophagy and NET levels in the PBNs of SA, but not in the NSA group. NET levels correlated with autophagy levels in PBNs (P <0.001). Levels of IL-8 induced NET production negatively correlated with FEV1/FVC (r=-0.700, P=0.016). NET dose-dependently induced cell death, cell detatchment, degradation of tight-junction proteins and IL-8 production from AECs dose dependently. NET-induced ECP and EDN releases were higher in PBEs of SA patients compared to NSA patients.

In baker’s asthma study, the bakery workers had higher serum levels of S100A8 and S100A9 compared to the NCs (P<0.001); however, no significant differences were noted according to work-related symptoms. The area under the ROC curve of serum S100A8 was 0.886 for occupational exposure (P< 0.001). The CC genotype at TLR4 -1608T>C was significantly associated with a higher serum S100A8 level (P=0.025). Serum S100A8 and S100A9 levels were correlated with serum levels of MPO (r=0.396 and 0.189, respectively), TNF-α (r=0.536 and 0.280, respectively), and IL-8 (r=0.540 and 0.205, respectively; P<0.001 for all).

Conclusions: This thesis demonstrated the role of neutrophils in the airway inflammatory mechanism of adult asthma. Neutrophilic airway inflammation in asthma could be regulated by genetic polymorphisms of ATG5 and ATG7. Neutrophilic autophagy and NET could enhance asthma severity by damaging airway epithelium and triggering inflammatory responses of AECs and eosinophils. Modulating neutrophil autophagy and NET production may be a new therapeutic strategy for SA treatment. In baker’s asthma, S100A8 and S100A9 could be involved in neutrophilic inflammation of airway under the regulation of TLR4 polymorphisms. Additionally, serum S100A8 level could be a potential biomarker for predicting occupational exposure to wheat flour in bakery workers.