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

Abstract

Background/objective: 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.

Methodology/main results: 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.

Conclusion: 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.

Figure 1. Validation of the non-antimicrobial feature of a tetracycline analog 9-t-butyl doxycycline (9-TB).

Tracheal epithelial cells from wild-type C57BL/6 mice were isolated and cultured under air-liquid interface (ALI) condition as described in the Materials and Methods 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.

Figure 2. 9-TB enhances NF-κB activation in saline-treated CC10-_CAIKKβ Tg+ mice.

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.

Figure 3. 9-TB treatment increases leukocytes in bronchoalveolar lavage (BAL) fluid of CC10-_CAIKKβ Tg+ mice with saline treatment.

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

Figure 4. 9-TB treatment increases KC and IL-6 levels in CC10-_CAIKKβ transgene positive (Tg+), but not transgene negative (Tg–) mice with saline treatment.

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

Figure 5. 9-TB reduces lung Mp load in CC10-_CAIKKβ transgene positive (Tg+) mice.

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.

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

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 (A) and 9-TB (B). Quantitative data of airway SPLUNC1 protein (C) 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.