E bacteriostatic, but not bactericidal for Mp [4]. Therefore, understanding the host defense mechanisms against Mp infection would offer more effective therapies to treat chronic lung diseases. Mp infection is known to predominantly target airway epithelium, leading to epithelial damage and inflammatory cytokine production. Airway epithelium, the first line of hostdefense against environmental hazards, utilizes various signaling pathways to Anlotinib modulate host defense against bacteria [5,6,7]. For example, airway epithelial nuclear transcription factor kB (NF-kB) can be activated following Mp infection [5], which promotes the production of chemokines involved in leukocyte recruitment and activation. Thus, studying the role of NF-kB in airway epithelial cell responses to bacterial infection is critical to find better strategies to eliminate bacteria from airways of asthma and COPD patients. Several groups of investigators have generated doxycycline (Dox)-inducible NF-kB transgenic mice to study the role of airway epithelial NF-kB activation in airway allergic inflammation [8,9]. So far, the role of airway epithelial NF-kB signaling in lung bacterial infection and clearance remains poorly understood. Although Chen et al has demonstrated the feasibility of Pseudomonas aeruginosa (Pa) infection in Dox-inducible NF-kB transgenic mice [10], that study is limited for its broad application because PaAirway NF-kB Activation and Bacterial Infectionis resistant to Dox [11]. Indeed, Pa is about 266 times more resistant to the bactericidal effect of Dox than other strains of bacteria (e.g. Mp) that are highly relevant to some of the most prominent lung diseases including asthma and COPD [12,13,14]. To overcome the antimicrobial activity of Dox, in the present study, we utilized non-antimicrobial tetracycline MedChemExpress H 4065 analog tetracycline analog 9-t-butyl doxycycline (9-TB) in conditional NF-kB transgenic mice that were infected with Mp. 9-TB is a novel tetracycline analog that has been used in cell culture and animal studies [15,16]. The primary goal of our study was to test if in vivo airway epithelial NF-kB activation was critical to lung defense against Mp. Our secondary goal is to reveal the potential mechanisms by which in vivo airway epithelial NF-kB activation enhances host defense against Mp. Our previous publications have shown that short palate, lung, and nasal epithelium clone 1 (SPLUNC1), a member of the PLUNC family that is localized in large airway epithelium, exerts antimicrobial activity against Mp. Moreover, SPLUNC1 was induced in cultured human and mouse primary airway epithelial cells upon Mp infection largely through the activation of NF-kB pathway [5,17,18]. Therefore, in the present study, we examined mouse (in 
vivo) airway epithelial SPLUNC1 expression following NF-kB activation to provide a potential mechanism for NF-kB-mediated host defense against bacterial infection.Lung NF-kB Activation in 9-TB-treated CC10-CAIKKb Tg+ (NF-kB Tg+) MiceTo address if 9-TB increases NF-kB activation, we measured whole lung NF-kB activation levels in CC10-CAIKKb Tg+ mice with or without administration of 9-TB (please refer to “Material and Method” section for details on CC10-CAIKKb transgenic mouse strain). At 24 hr after the last 9-TB treatment, 9-TB treated-mice, as compared to vehicle control mice, demonstrated increased NFkB activation (Figure 2).Increased Lung Leukocytes and Cytokines in 9-TB-treated NF-kB Tg+ MiceNF-kB activation regulates the product.E bacteriostatic, but not bactericidal for Mp [4]. Therefore, understanding the host defense mechanisms against Mp infection would offer more effective therapies to treat chronic lung diseases. Mp infection is known to predominantly target airway epithelium, leading to epithelial damage and inflammatory cytokine production. Airway epithelium, the first line of hostdefense against environmental hazards, utilizes various signaling pathways to modulate host defense against bacteria [5,6,7]. For example, airway epithelial nuclear transcription factor kB (NF-kB) can be activated following Mp infection [5], which promotes the production of chemokines involved in leukocyte recruitment and activation. Thus, studying the role of NF-kB in airway epithelial cell responses to bacterial infection is critical to find better strategies to eliminate bacteria from airways of asthma and COPD patients. Several groups of investigators have generated 
doxycycline (Dox)-inducible NF-kB transgenic mice to study the role of airway epithelial NF-kB activation in airway allergic inflammation [8,9]. So far, the role of airway epithelial NF-kB signaling in lung bacterial infection and clearance remains poorly understood. Although Chen et al has demonstrated the feasibility of Pseudomonas aeruginosa (Pa) infection in Dox-inducible NF-kB transgenic mice [10], that study is limited for its broad application because PaAirway NF-kB Activation and Bacterial Infectionis resistant to Dox [11]. Indeed, Pa is about 266 times more resistant to the bactericidal effect of Dox than other strains of bacteria (e.g. Mp) that are highly relevant to some of the most prominent lung diseases including asthma and COPD [12,13,14]. To overcome the antimicrobial activity of Dox, in the present study, we utilized non-antimicrobial tetracycline analog tetracycline analog 9-t-butyl doxycycline (9-TB) in conditional NF-kB transgenic mice that were infected with Mp. 9-TB is a novel tetracycline analog that has been used in cell culture and animal studies [15,16]. The primary goal of our study was to test if in vivo airway epithelial NF-kB activation was critical to lung defense against Mp. Our secondary goal is to reveal the potential mechanisms by which in vivo airway epithelial NF-kB activation enhances host defense against Mp. Our previous publications have shown that short palate, lung, and nasal epithelium clone 1 (SPLUNC1), a member of the PLUNC family that is localized in large airway epithelium, exerts antimicrobial activity against Mp. Moreover, SPLUNC1 was induced in cultured human and mouse primary airway epithelial cells upon Mp infection largely through the activation of NF-kB pathway [5,17,18]. Therefore, in the present study, we examined mouse (in vivo) airway epithelial SPLUNC1 expression following NF-kB activation to provide a potential mechanism for NF-kB-mediated host defense against bacterial infection.Lung NF-kB Activation in 9-TB-treated CC10-CAIKKb Tg+ (NF-kB Tg+) MiceTo address if 9-TB increases NF-kB activation, we measured whole lung NF-kB activation levels in CC10-CAIKKb Tg+ mice with or without administration of 9-TB (please refer to “Material and Method” section for details on CC10-CAIKKb transgenic mouse strain). At 24 hr after the last 9-TB treatment, 9-TB treated-mice, as compared to vehicle control mice, demonstrated increased NFkB activation (Figure 2).Increased Lung Leukocytes and Cytokines in 9-TB-treated NF-kB Tg+ MiceNF-kB activation regulates the product.