R indicate that use of 9-TB is an excellent approach to activate airway epithelial NF-kB for studying the impact of in vivo NF-kB activation on various epithelial functions following bacterial infection. Our previous study suggests that SPLUNC1 was inducible upon Mp infection in cultured airway epithelial cells largely through NF-kB pathway [5]. Our current study has extended our previous work by revealing in vivo airway epithelial SPLUNC1 upregulation following NF-kB activation. Future studies are warranted to explicitly define the contribution of airway epithelial SPLUNC1 up-regulation to bacterial clearance in 9-TB-treated NF-kB transgenic mice. This could be achieved by applying a mouse SPLUNC1 neutralizing antibody prior to bacterial infection. There are several limitations to our present study. First, it focused on an acute (e.g., day 1 post infection) infection model. Although our acute mouse model is highly relevant to acute exacerbations of lung diseases, chronic bacterial infection modelFigure 6. 9-TB up-regulates airway epithelial SPLUNC1 protein in CC10-CAIKKb transgene positive (Tg+) mice. Lungs from salinetreated 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 6 SEM. doi:10.1371/journal.pone.0052969.gAirway NF-kB Activation and Bacterial Infectionwill be needed in the future to study the role of airway epithelial NF-kB pathway in disease progression. Second, as NF-kB activation in mouse airway epithelium activates an array of host defence molecules (e.g., KC and IL-6), the enhanced airway epithelial SPLUNC1 expression in 9-TB-treated NF-kB transgenic mice is expected to serve only as one of the mechanisms involved in enhanced lung Mp clearance. To explicitly demonstrate the role of SPLUNC1 in airway epithelial cell NF-kB-mediated lung defense against Mp, future studies are warranted to breed SPLUNC1 knockout mice or their wild-type littermates with NF-kB transgenic mice, and infect the new strains of mice with Mp. Moreover, as other mediators (e.g., KC and IL-6) induced by NF-kB activation have been shown to promote Mp clearance [19], the contribution of those additional mediators will be considered in our future studies by using knockout mice or neutralizing purchase CPI-455 antibodies. Addtionally, we may need to examine other antimicrobial substances (e.g., lactotransferrin and b defensin 2) that can also be increased following NF-kB activation. Third, although the canonial NF-kB pathway is predominatly activated in our CC10-CAIKKb mouse model [20,21,22], IKKb activation may have NF-kB-independent effects. For example, IKKb activation can phosphorylate adaptor protein DOK1, and subsequently inhibit MAP kinase signaling [23]. Because MAP kinases are involved in inflammatory cytokine production, and even SPLUNC1 induction [24] CX-5461 web during bacterial infection, it is likely that IKKb activation may serve as a negative regulatory mechanism to prevent excessive activation of canonical NF-kB pathway. The balance of IKKb-induced NF-kB activation and MAP kinase inhibition during mycoplasma infection warrants future studies to better understand the functions of IKKbmediated signaling in airway epithelial cells. Lastly, in th.R indicate that use of 9-TB is an excellent approach to activate airway epithelial NF-kB for studying the impact of in vivo NF-kB activation on various epithelial functions following bacterial infection. Our previous study suggests that SPLUNC1 was inducible upon Mp infection in cultured airway epithelial cells largely through NF-kB pathway [5]. Our current study has extended our previous work by revealing in vivo airway epithelial SPLUNC1 upregulation following NF-kB activation. Future studies are warranted to explicitly define the contribution of airway epithelial SPLUNC1 up-regulation to bacterial clearance in 9-TB-treated NF-kB transgenic mice. This could be achieved by applying a mouse SPLUNC1 neutralizing antibody prior to bacterial infection. There are several limitations to our present study. First, it focused on an acute (e.g., day 1 post infection) infection model. Although our acute mouse model is highly relevant to acute exacerbations of lung diseases, chronic bacterial infection modelFigure 6. 9-TB up-regulates airway epithelial SPLUNC1 protein in CC10-CAIKKb transgene positive (Tg+) mice. Lungs from salinetreated 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 6 SEM. doi:10.1371/journal.pone.0052969.gAirway NF-kB Activation and Bacterial Infectionwill be needed in the future to study the role of airway epithelial NF-kB pathway in disease progression. Second, as NF-kB activation in mouse airway epithelium activates an array of host defence molecules (e.g., KC and IL-6), the enhanced airway epithelial SPLUNC1 expression in 9-TB-treated NF-kB transgenic mice is expected to serve only as one of the mechanisms involved in enhanced lung Mp clearance. To explicitly demonstrate the role of SPLUNC1 in airway epithelial cell NF-kB-mediated lung defense against Mp, future studies are warranted to breed SPLUNC1 knockout mice or their wild-type littermates with NF-kB transgenic mice, and infect the new strains of mice with Mp. Moreover, as other mediators (e.g., KC and IL-6) induced by NF-kB activation have been shown to promote Mp clearance [19], the contribution of those additional mediators will be considered in our future studies by using knockout mice or neutralizing antibodies. Addtionally, we may need to examine other antimicrobial substances (e.g., lactotransferrin and b defensin 2) that can also be increased following NF-kB activation. Third, although the canonial NF-kB pathway is predominatly activated in our CC10-CAIKKb mouse model [20,21,22], IKKb activation may have NF-kB-independent effects. For example, IKKb activation can phosphorylate adaptor protein DOK1, and subsequently inhibit MAP kinase signaling [23]. Because MAP kinases are involved in inflammatory cytokine production, and even SPLUNC1 induction [24] during bacterial infection, it is likely that IKKb activation may serve as a negative regulatory mechanism to prevent excessive activation of canonical NF-kB pathway. The balance of IKKb-induced NF-kB activation and MAP kinase inhibition during mycoplasma infection warrants future studies to better understand the functions of IKKbmediated signaling in airway epithelial cells. Lastly, in th.
