Supplementary Material for: Antibacterial Defense of Human Airway Epithelial Cells from Chronic Obstructive Pulmonary Disease Patients Induced by Acute Exposure to Nontypeable Haemophilus influenzae: Modulation by Cigarette Smoke

Antimicrobial proteins and peptides (AMPs) are a central component of the antibacterial activity of airway epithelial cells. It has been proposed that a decrease in antibacterial lung defense contributes to an increased susceptibility to microbial infection in smokers and patients with chronic obstructive pulmonary disease (COPD). However, whether reduced AMP expression in the epithelium contributes to this lower defense is largely unknown. We investigated the bacterial killing activity and expression of AMPs by air-liquid interface-cultured primary bronchial epithelial cells from COPD patients and non-COPD (ex-)smokers that were stimulated with nontypeable <i>Haemophilus influenzae </i>(NTHi). In addition, the effect of cigarette smoke on AMP expression and the activation of signaling pathways was determined. COPD cell cultures displayed reduced antibacterial activity, whereas smoke exposure suppressed the NTHi-induced expression of AMPs and further increased IL-8 expression in COPD and non-COPD cultures. Moreover, smoke exposure impaired NTHi-induced activation of NF-κB, but not MAP-kinase signaling. Our findings demonstrate that the antibacterial activity of cultured airway epithelial cells induced by acute bacterial exposure was reduced in COPD and suppressed by cigarette smoke, whereas inflammatory responses persisted. These findings help to explain the imbalance between protective antibacterial and destructive inflammatory innate immune responses in COPD.

抗菌蛋白与多肽（Antimicrobial proteins and peptides，AMPs）是气道上皮细胞抗菌活性的核心组成部分。有研究提出，肺部抗菌防御功能下降会导致吸烟者及慢性阻塞性肺疾病（chronic obstructive pulmonary disease，COPD）患者更易发生微生物感染。然而，上皮细胞中AMPs表达下调是否参与了这一防御功能减弱的过程，目前尚不清楚。本研究针对COPD患者及非COPD的既往吸烟者，采用经非分型流感嗜血杆菌（nontypeable Haemophilus influenzae，NTHi）刺激的气液界面培养原代支气管上皮细胞，检测其细菌杀伤活性及AMPs的表达水平。此外，本研究还探究了香烟烟雾对AMPs表达及信号通路激活的影响。研究结果显示，COPD组细胞培养物表现出抗菌活性减弱；而香烟烟雾暴露可抑制NTHi诱导的AMPs表达，并进一步升高COPD组与非COPD组培养物中IL-8的表达水平。此外，香烟烟雾暴露会削弱NTHi诱导的核因子κB（NF-κB）激活，但对丝裂原活化蛋白激酶（MAP-kinase）信号通路无明显影响。本研究证实，急性细菌暴露诱导的培养气道上皮细胞抗菌活性在COPD患者中减弱，并可被香烟烟雾进一步抑制，而炎症反应则持续存在。上述研究结果有助于阐释慢性阻塞性肺疾病中保护性抗菌固有免疫应答与破坏性炎症固有免疫应答之间的失衡机制。