Nontypeable Haemophilus influenzae infection differently modulates E-cadherin expression under changing environmental oxygen concentrations

Einleitung: Ongoing airway remodeling is characteristic for chronic obstructive pulmonary disease (COPD) progression. The adherens junctions protein E-cadherin was shown to be reduced in COPD patients, which is implicated with decreased barrier tightness in lung epithelial cells. Nontypeable Haemophilus influenzae (NTHi) infection has been frequently detected during exacerbation of COPD but the contribution to E-cadherin reduction in alveolar epithelial cells is still largely unknown. In later stages of the disease, chronic bronchitis and emphysema cause airflow limitations that result in reduced oxygen availability in lung tissue. We therefore investigated whether NTHi contributes to loss of E-cadherin under normoxic or hypoxic conditions.

Methoden: A549 cells were infected with NTHi for 24h under normoxia (20% O₂) or hypoxia (2% O₂). To analyze the influence of NTHi on alveolar epithelial cells, cell morphology as well as E-cadherin was analyzed by microscopy, qRT-PCR and western blot analysis. The expression of transforming growth factor-β (TGF-β) and fibroblast growth factor 2 (FGF2) was analyzed by qRT-PCR. The protein amount of the E-cadherin repressors Slug and Snail as well as hypoxia-inducible factor-1α (HIF-1α) were analyzed using western blot analysis. The role of HIF-1α and mTOR was analyzed by HIF-1α knock-down using stealth siRNA and rapamycin, respectively.

Ergebnisse: Within 24h NTHi infection led to decreased E-cadherin protein expression and spindle-shaped cell morphology under normoxia and hypoxia. This mechanism was mediated via enhanced expression of Slug and FGF2 in a mTOR-mediated manner, which resulted in decreased E-cadherin mRNA expression under normoxia. In contrast, TGF-β and Snail were not expressed during infection and thus not involved in E-cadherin downregulation. Interestingly, NTHi infection under hypoxia resulted in decreased protein but not mRNA expression levels of E-cadherin. Thereby, HIF-1α stabilization in NTHi infected cells under hypoxia contributed to the unaffected E-cadherin mRNA expression.

Diskussion: We conclude that infection with NTHi under normoxia and hypoxia leads to loss of E-cadherin protein expression through different mechanisms. Thus, NTHi infection might reduce epithelial integrity which could result in higher sensitivity to harmful inhaled substances causing exacerbation.