In our study, we treated highly differentiated cultures of primary human tracheobronchial epithelial (TBE) cells with a panel of cytokines (interleu- kin-1␣, 1␤, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, and tumor necrosis factor ␣). We found that IL-6 and IL-17 could stimulate the mucin genes, MUC5B and MUC5AC. The Th2 cytokines IL-4, IL-9, and IL-13 did not stimulate MUC5AC or MUC5B in our experiments. A similar stimulation of MUC5B/Muc5b expression by IL-6 and IL-17 was demonstrated in primary monkey and mouse TBE cells. Further investigation of MUC5B expression demonstrated that IL-17's effect is at least partly mediated through IL-6 by a JAK2-dependent autocrine/paracrine loop. Finally, evidence is presented to show that both IL-6 and IL-17 mediate MUC5B expression through the ERK signaling pathway.

Using microarray gene expression analysis, we first observed a profound elevation of human β-defensin-2 (hBD-2) message in IL-17-treated primary human airway epithelial cells. Further comparison of this stimulation with a panel of cytokines (IL-1α, 1β, 2–13, and 15–18; IFN-γ; GM-CSF; and TNF-α) demonstrated that IL-17 was the most potent cytokine to induce hBD-2 message (>75-fold). IL-17-induced stimulation of hBD-2 was time and dose dependent, and this stimulation also occurred at the protein level. Further studies demonstrated that hBD-2 stimulation was attenuated by IL-17R-specific Ab, but not by IL-1R antagonist or the neutralizing anti-IL-6 Ab. This suggests an IL-17R-mediated signaling pathway rather than an IL-17-induced IL-1αβ and/or IL-6 autocrine/paracrine loop. hBD-2 stimulation was sensitive to the inhibition of the JAK pathway, and to the inhibitors that affect NF-κB translocation and the DNA-binding activity of its p65 NF-κB subunit. Transient transfection of airway epithelial cells with an hBD-2 promoter-luciferase reporter gene expression construct demonstrated that IL-17 stimulated promoter-reporter gene activity, suggesting a transcriptional mechanism for hBD-2 induction. These results support an IL-17R-mediated signaling pathway involving JAK and NF-κB in the transcriptional stimulation of hBD-2 gene expression in airway epithelium. Because IL-17 has been identified in a number of airway diseases, especially diseases related to microbial infection, these findings provide a new insight into how IL-17 may play an important link between innate and adaptive immunity, thereby combating infection locally within the airway epithelium.

Partially reduced metabolites of molecular oxygen, superoxide (O ÅÀ2 ) and hydrogen peroxide (H 2 O 2 ), are detected in respiratory tract lining fluid, and it is assumed that these are key components of innate immunity. Whether these reactive oxygen species (ROS) are produced specifically by the respiratory epithelium in response to infection, or are a non-specific by-product of oxidant-producing inflammatory cells is not well characterized. Increasing evidence supports the hypothesis that the dual function NAD(P)H oxidases/peroxidases, Duox1 and Duox2, are important sources of regulated H 2 O 2 production in respiratory tract epithelium. However, no studies to date have characterized the regulation of Duox gene expression. Accordingly, we examined Duox1 and Duox2 mRNA expression by real-time PCR in primary respiratory tract epithelial cultures after treatment with multiple cytokines. Herein, we determined that Duox1 expression was increased several-fold by treatment with the Th2 cytokines IL-4 and IL-13, whereas Duox2 expression was highly induced following treatment with the Th1 cytokine IFN-c. Duox2 expression was also elevated by polyinosine-polycytidylic acid (poly(I:C)) and rhinovirus infection. Diphenyleneiodonium (DPI)-inhibitable apical H 2 O 2 production was similarly increased by the addition of Th1 or Th2 cytokines. These results demonstrate for the first time the regulation of Duox expression by immunomodulatory Th1 and Th2 cytokines, and suggest a mechanism by which ROS production can be regulated in the respiratory tract as part of the host defense response.

Mucins are important components that exert a variety of functions in cell-cell interaction, epidermal growth factor receptor signaling, and airways protection. In the conducting airways of the lungs, mucins are the major contributor to the viscoelastic property of mucous secretion, which is the major barrier to trapping inhaled microbial organism, particulates, and oxidative pollutants. The homeostasis of mucin production is an important feature in conducting airways for the maintenance of mucociliary function. Aberrant mucin secretion and accumulation in airway lumen are clinical hallmarks associated with various lung diseases, such as asthma, chronic obstructive pulmonary disease, cystic fibrosis, emphysema, and lung cancer. Among 20 known mucin genes identified, 11 of them have been verified at either the mRNA and/or protein level in airways. The regulation of mucin genes is complicated, as are the mediators and signaling pathways. This review summarizes the current view on the mediators, the signaling pathways, and the transcriptional units that are involved in the regulation of airway mucin gene expression. In addition, we also point out essential features of epigenetic mechanisms for the regulation of these genes.

Mucin over-production is one of the hallmarks of chronic airway diseases such as chronic obstructive pulmonary disease, asthma, and cystic fibrosis. NF-κB activation in airway epithelial cells has been shown to play a positive inflammatory role in chronic airway diseases; however, the role of NF-κB in mucin gene expression is unresolved. In this study, we have shown that the proinflammatory cytokines, IL-1β and IL-17A, both of which utilize the NF-κB pathway, are potent inducers of mucin (MUC)5AC mRNA and protein synthesis by both well-differentiated primary normal human bronchial epithelial cells and the human bronchial epithelial cell line, HBE1. MUC5AC induction by these cytokines was both time- and dose-dependent and occurred at the level of promoter activation, as measured by a reporter gene assay. These effects were attenuated by the small molecule inhibitor NF-κB inhibitor III, as well as p65 small-interfering RNA, suggesting that the regulation of MUC5AC expression by these cytokines is via an NF-κB-based transcriptional mechanism. Further investigation of the promoter region identified a putative NF-κB binding site at position-3594/-3582 in the promoter of MUC5AC as critical for the regulation of MUC5AC expression by both IL-1β and IL-17A. Chromatin immunoprecipitation analysis confirmed enhanced binding of the NF-κB subunit p50 to this region following cytokine stimulation. We conclude that an NF-κB-based transcriptional mechanism is involved in MUC5AC regulation by IL-1β and IL-17A in the airway epithelium. This is the first demonstration of the participation of NF-κB and its specific binding site in cytokine-mediated airway MUC5AC expression.