Airway epithelial NF-κB activation promotes Mycoplasma pneumoniae clearance in mice.

Respiratory infections including atypical bacteria Mycoplasma pneumoniae (Mp) contribute to the pathobiology of asthma and chronic obstructive pulmonary disease (COPD). Mp infection mainly targets airway epithelium and activates various signaling pathways such as nuclear factor κB (NF-κB). We have shown that short palate, lung, and nasal epithelium clone 1 (SPLUNC1) serves as a novel host defense protein and is up-regulated upon Mp infection through NF-κB activation in cultured human and mouse primary airway epithelial cells. However, the in vivo role of airway epithelial NF-κB activation in host defense against Mp infection has not been investigated. In the current study, we investigated the effects of in vivo airway epithelial NF-κB activation on lung Mp clearance and its association with airway epithelial SPLUNC1 expression.Non-antimicrobial tetracycline analog 9-t-butyl doxycycline (9-TB) was initially optimized in mouse primary tracheal epithelial cell culture, and then utilized to induce in vivo airway epithelial specific NF-κB activation in conditional NF-κB transgenic mice (CC10-(CA)IKKβ) with or without Mp infection. Lung Mp load and inflammation were evaluated, and airway epithelial SPLUNC1 protein was examined by immunohistochemistry. We found that 9-TB treatment in NF-κB transgene positive (Tg+), but not transgene negative (Tg-) mice significantly reduced lung Mp load. Moreover, 9-TB increased airway epithelial SPLUNC1 protein expression in NF-κB Tg+ mice.By using the non-antimicrobial 9-TB, our study demonstrates that in vivo airway epithelial NF-κB activation promotes lung bacterial clearance, which is accompanied by increased epithelial SPLUNC1 expression

Up-Regulation of MUC18 in Airway Epithelial Cells by IL-13: Implications in Bacterial Adherence

Airway bacterial infections are a major problem in lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. Increased Th2 cytokines, such as IL-13, are observed in lung diseases and may contribute to bacterial infections. How Th2 cytokines affect bacterial infection remains unknown. MUC18, an adhesion molecule shown to be involved in the pathogenesis of malignant melanoma, has been recently identified in airway epithelial cells of patients with COPD. We investigated MUC18 regulation by IL-13 and the role of MUC18 in bacterial adherence to epithelial cells. Human airway tissues, brushed bronchial epithelial cells from normal subjects and subjects with asthma, and epithelial cell lines (e.g., HEK293 cells) were used to study the regulation of MUC18 by IL-13 and the involvement of MUC18 in bacterial (e.g., Mycoplasma pneumoniae [Mp] and nontypeable Haemophilus influenzae [NTHi]) adherence to epithelial cells. Asthmatic bronchial epithelium expressed higher levels of MUC18 than normal bronchial epithelium. IL-13 increased MUC18 in cultured bronchial epithelial cells from normal subjects and particularly from subjects with asthma. IL-13–induced MUC18 expression may be modulated in part through transcription factor specificity protein 1. Overexpression of human MUC18 in HEK293 cells increased cell-associated Mp and NTHi levels. Moreover, MUC18 was shown to directly interact with Mp and NTHi. These results for the first time show that an allergic airway milieu (e.g., IL-13) increases MUC18 expression, which may contribute to increased bacterial infection/colonization in asthma and other lung diseases

9-TB treatment increases leukocytes in bronchoalveolar lavage (BAL) fluid of CC10-_CAIKKβ Tg+ mice with saline treatment.

<p>(<b>A</b>) – total leukocytes; (<b>B</b>) – neutrophils. N = 4–6 mice per group. Data are expressed as means ± SEM.</p

9-TB up-regulates airway epithelial SPLUNC1 protein in CC10-_CAIKKβ transgene positive (Tg+) mice.

<p>Lungs from saline-treated Tg+ mice were processed for SPLUNC1 immunohistochemistry. Representative SPLUNC1 staining in medium-size airways of Tg+ mice treated with vehicle solution (<b>A</b>) and 9-TB (<b>B</b>). Quantitative data of airway SPLUNC1 protein (<b>C</b>) are expressed as a percentage of stained area versus total (stained plus non-stained) airway epithelial area. N = 4 mice per group. Data are expressed as means ± SEM.</p

9-TB enhances NF-κB activation in saline-treated CC10-_CAIKKβ Tg+ mice.

<p>NF-κB activity in 9-TB- and saline-treated CC10-_CAIKKβ Tg+ mice was measured by using the NF-κB p65 ELISA in nuclear proteins extracted from mouse lungs (n = 4 mice per group). Data are expressed as means ± SEM.</p

9-TB reduces lung Mp load in CC10-_CAIKKβ transgene positive (Tg+) mice.

<p>Left lungs from Mp-infected mice (24 hours after infection) were homogenized and plated on PPLO-plates to count Mp CFUs. 9-TB significantly reduced lung Mp load in Tg+ mice, but had a minimal impact on Mp load in transgene negative (Tg–) mice. N = 4–6 mice per group. Data are expressed as means ± SEM.</p

9-TB treatment increases KC and IL-6 levels in CC10-_CAIKKβ transgene positive (Tg+), but not transgene negative (Tg–) mice with saline treatment.

<p>(<b>A</b>) – KC; (<b>B</b>) – IL-6. N = 4–6 mice per group. Data are expressed as means ± SEM.</p

Validation of the non-antimicrobial feature of a tetracycline analog 9-t-butyl doxycycline (9-TB).

<p>Tracheal epithelial cells from wild-type C57BL/6 mice were isolated and cultured under air-liquid interface (ALI) condition as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052969#s4" target="_blank">Materials and Methods</a> section. The effects of medium control, doxycycline (Dox, 0.5 µg/ml) or 9-TB (0.5 µg/ml) on Mp growth in the apical supernatants of epithelial cells were examined at 24 hour post infection. N = 3; CFUs = colony forming units. Data are expressed as means ± SEM.</p