title: Flagellin of Pseudomonas aeruginosa induces transforming growth factor beta 1 expression in normal bronchial epithelial cells through mitogen activated protein kinase cascades.
abstract: BACKGROUND
  Acute lung infection due to Pseudomonas aeruginosa (P. aeruginosa) is a serious problem, especially in patients with structural lung conditions or immune compromised hosts, leading to an overwhelming threat with a high risk of morbidity and mortality. As an outcome of infection, fibrosis can be linked with chronic lung diseases. But some fibrotic manifestations, such as an irreversible decrease of lung function and fibrous bands seen on chest imaging, have been found after an acute infection with P. aeruginosa. Fibrogenesis/remodeling resulting from acute lung infection by P. aeruginosa is rarely reported. This study was designed to explore the relation between fibrogenesis/remodeling and acute infection by P. aeruginosa in vitro. We used flagellin protein from P. aeruginosa, a key initiator of acute P. aeruginosa lung infection, to elucidate mechanisms by which acute lung infection with P. aeruginosa can cause fibrogenesis/remodeling.
  
  
  METHODS
  We studied the effect of flagellin from P. aeruginosa (flagellin for short) on the transforming growth factor beta 1 (TGF-β1) and interleukin-8 (IL-8) expression, and the possible involvement of the signaling pathway, tumor necrosis factor receptor-associated factor 6 (TRAF6)/mitogen activated protein kinase (MAPK) pathway. Flagellin was purified from the P. aeruginosa standard strain, PAO1. Normal bronchial epithelial cells BEAS-2B were challenged with different concentrations of flagellin, and cell viability assessment was performed by cell counting kit-8. BEAS-2B cells were incubated with flagellin with the specific MAPK inhibitors or TRAF6 siRNA. Cell lysates and the cultured supernatant were collected. The level of TGF-β1 and IL-8 were detected by enzyme-linked immunosorbant assay (ELISA). Western blotting was used to detect the protein levels of MAPK signal proteins p38, c-Jun NH(2)-terminal kinase (JNK) and extracellular regulated kinase (ERK).
  
  
  RESULTS
  Expression of TGF-β1 in BEAS-2B cells was elevated by flagellin vs. control groups ((104.3 ± 20.8) vs. (44.6 ± 4.4) pg/ml (P < 0.01)) and was ablated by either p38 or JNK inhibitors compared with flagellin treatment ((45.1 ± 18.8) vs. (104.3 ± 20.8) pg/ml and (48.1 ± 20.8) vs. (104.3 ± 20.8) pg/ml, respectively (P < 0.05)). Flagellin also elevated the expression of IL-8 in BEAS-2B cells vs. the control groups ((554.9 ± 57.7) vs. (51.4 ± 22.9) pg/ml (P < 0.01)), and p38 MAPK inhibitors weaken the expression by flagellin ((301.1 ± 155.1) vs. (554.9 ± 57.7) pg/ml (P < 0.05)). Western blotting revealed that all three MAPK proteins, p38, JNK and ERK were activated by flagellin challenge in an early phase, respectively in 15 minutes (P < 0.01), 30 minutes (P < 0.01) and 15 minutes (P < 0.01). TRAF6 siRNA which decreased expression of TRAF6, altered the activation of JNK, p38, and ERK following flagellin treatment, but its influence on the expression of TGF-β1 and IL-8 has no statistical significance.
  
  
  CONCLUSIONS
  Flagellin from P.aeruginosa PAO1 induces TGF-β1 expression in normal bronchial epithelial cells, BEAS-2B, through the MAPK signal cascade in vitro. It suggests that the fibrogenesis/remodeling process may be initiated from an early stage of acute lung infection due to P. aeruginosa.
authors.affiliation: Department of Respiratory Diseases, Peking University Fifth School of Clinical Medicine, Beijing Hospital, Beijing, China.
authors.forename: Jing-jing
authors.initials: JJ
authors.lastname: Yang
authors.forename: Dan-dan
authors.initials: DD
authors.lastname: Wang
authors.forename: Tie-ying
authors.initials: TY
authors.lastname: Sun
mesh_terms: D015153:Blotting, Western; D001980:Bronchi / Q000166:cytology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism*; D005408:Flagellin / Q000494:pharmacology*; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism*
chemical_list: D016209:Interleukin-8; D053773:Transforming Growth Factor beta1; D005408:Flagellin; D020928:Mitogen-Activated Protein Kinases