title: Autophagy mechanisms in sputum and peripheral blood cells of patients with severe asthma: a new therapeutic target.
abstract: BACKGROUND
  Autophagy and genetic predisposition have been suggested to potentially play roles in the development of asthma. However, little is known about the role of autophagy in the pathogenesis of severe asthma.
  
  
  OBJECTIVE
  We compared autophagy in the sputum granulocytes, peripheral blood cells (PBCs) and peripheral blood eosinophils (PBEs) between patients with severe asthma and those with non-severe asthma and investigated the functional effects of autophagy.
  
  
  METHODS
  We enrolled 36 patients with severe asthma, 14 with non-severe asthma and 23 normal healthy controls in this study. Sputum granulocytes, PBCs and PBEs were isolated from each subject. Autophagy was evaluated based on the expression of microtubule-associated protein light chain 3 (LC3) by Western blot, confocal microscopy, transmission electron microscopy and flow cytometry. IL-8 levels were measured by ELISA. To induce autophagy, HL-60 cells, human primary small airway epithelial cells (SAECs) and A549 cells were treated with IL-5, IL-1β and TNF-α. To inhibit autophagy, PI3K inhibitors (LY29400 and 3-methyladenine [3-MA]) and hydroxychloroquine (HCQ) were used. Knockdown of ATG5 and Beclin-1 was performed in A549 cells, and the therapeutic effects of dexamethasone were evaluated.
  
  
  RESULTS
  Higher autophagy levels were noted in sputum granulocytes, PBCs and PBEs from patients with severe asthma than from patients with non-severe asthma and healthy controls (P < 0.05 for all). IL-5 increased autophagy levels in both PBCs and PBEs (P < 0.05). 3-MA attenuated the increased expression of LC3-II and eosinophil cationic protein in HL-60 cells induced by IL-5 (P = 0.034 for both). Dexamethasone did not affect autophagy levels in PBEs. IL-1β increased LC3-II expression and IL-8 production (P < 0.01) in SAECs, and this was attenuated by LY294002, 3-MA, HCQ and knockdown of ATG5 and Beclin-1 (in A549 cells) (P < 0.01).
  
  
  CONCLUSIONS
  Autophagy could play a role in the pathogenesis of severe asthma. Autophagy modulation may be a novel therapeutic target for conventional therapy-resistant severe asthma.
authors.affiliation: Department of Allergy and Clinical Immunology, Ajou University School of Medicine, Suwon, South Korea.
authors.forename: G-Y
authors.initials: GY
authors.lastname: Ban
authors.affiliation: Department of Allergy and Clinical Immunology, Ajou University School of Medicine, Suwon, South Korea.|Department of Biomedical Sciences, The Graduate School, Ajou University, Suwon, South Korea.
authors.forename: D L
authors.initials: DL
authors.lastname: Pham
authors.affiliation: Department of Allergy and Clinical Immunology, Ajou University School of Medicine, Suwon, South Korea.
authors.forename: T H K
authors.initials: TH
authors.lastname: Trinh
authors.affiliation: Department of Allergy and Clinical Immunology, Ajou University School of Medicine, Suwon, South Korea.
authors.forename: S-I
authors.initials: SI
authors.lastname: Lee
authors.affiliation: Department of Allergy and Clinical Immunology, Ajou University School of Medicine, Suwon, South Korea.
authors.forename: D-H
authors.initials: DH
authors.lastname: Suh
authors.affiliation: Department of Allergy and Clinical Immunology, Ajou University School of Medicine, Suwon, South Korea.
authors.forename: E-M
authors.initials: EM
authors.lastname: Yang
authors.affiliation: Department of Allergy and Clinical Immunology, Ajou University School of Medicine, Suwon, South Korea.
authors.forename: Y-M
authors.initials: YM
authors.lastname: Ye
authors.affiliation: Department of Allergy and Clinical Immunology, Ajou University School of Medicine, Suwon, South Korea.
authors.forename: Y S
authors.initials: YS
authors.lastname: Shin
authors.affiliation: Department of Biomedical Sciences, The Graduate School, Ajou University, Suwon, South Korea.|Department of Microbiology, Ajou University School of Medicine, Suwon, South Korea.
authors.forename: Y-J
authors.initials: YJ
authors.lastname: Chwae
authors.affiliation: Department of Allergy and Clinical Immunology, Ajou University School of Medicine, Suwon, South Korea.|Department of Biomedical Sciences, The Graduate School, Ajou University, Suwon, South Korea.
authors.forename: H-S
authors.initials: HS
authors.lastname: Park
mesh_terms: D000328:Adult; D051017:Apoptosis Regulatory Proteins / Q000235:genetics; D001249:Asthma / Q000175:diagnosis / Q000209:etiology* / Q000378:metabolism* / Q000628:therapy; D001343:Autophagy; D000071187:Autophagy-Related Protein 5; D000071186:Beclin-1; D016022:Case-Control Studies; D002460:Cell Line; D016207:Cytokines; D005260:Female; D005541:Forced Expiratory Volume; D055785:Gene Knockdown Techniques; D006098:Granulocytes / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D007958:Leukocyte Count; D007962:Leukocytes / Q000276:immunology* / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D008869:Microtubule-Associated Proteins / Q000235:genetics; D008875:Middle Aged; D010588:Phagosomes / Q000378:metabolism; D012720:Severity of Illness Index; D013183:Sputum / Q000166:cytology* / Q000276:immunology*; D055815:Young Adult
chemical_list: C502778:ATG5 protein, human; D051017:Apoptosis Regulatory Proteins; D000071187:Autophagy-Related Protein 5; C491997:BECN1 protein, human; D000071186:Beclin-1; D016207:Cytokines; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D007073:Immunoglobulin E
keywords: autophagy; eosinophils; epithelial cells; severe asthma; treatment