Highly regulated programs for airway epithelial cell proliferation and differentiation during development and repair are often disrupted in disease. These processes have been studied in mouse models; however, it is difficult to isolate and identify epithelial cell-specific responses in vivo. To investigate these processes in vitro, we characterized a model for primary culture of mouse tracheal epithelial cells. Small numbers of cells seeded at low density (7.5 × 104 cells/cm2) rapidly proliferated and became polarized. Subsequently, supplemented media and air-liquid interface conditions resulted in development of highly differentiated epithelia composed of ciliated and nonciliated cells with gene expression characteristic of native airways. Genetically altered or injured mouse tracheal epithelial cells also reflected in vivo patterns of airway epithelial cell gene expression. Passage of cells resulted in continued proliferation but limited differentiation after the first passage, suggesting that transit-amplifying cell populations were present but with independent programs for proliferation and differentiation. This approach provides a high-fidelity in vitro model for evaluation of gene regulation and expression in mouse airway epithelial cells.

To understand the pathogenesis of chronic inflammatory disease, we analyzed an experimental mouse model of a chronic lung disease that resembles asthma and chronic obstructive pulmonary disease (COPD) in humans. In this model, chronic lung disease develops after infection with a common type of respiratory virus is cleared to trace levels of noninfectious virus. Unexpectedly, the chronic inflammatory disease arises independently of an adaptive immune response and is driven by IL-13 produced by macrophages stimulated by CD1d-dependent TCR-invariant NKT cells. This innate immune axis is also activated in the lungs of humans with chronic airway disease due to asthma or COPD. These findings provide new insight into the pathogenesis of chronic inflammatory disease with the discovery that the transition from respiratory viral infection into chronic lung disease requires persistent activation of a novel NKT cell-macrophage innate immune axis. It has been widely speculated that infections are linked to the development of chronic inflammatory diseases. Although the connection between infection and chronic disease is uncertain, it likely depends on an aberrant immune response. In particular, it is believed that the innate immune system mediates the acute response to an infectious agent 1 , while an atypical adaptive immune response may cause chronic inflammation 2. Furthermore, infectioninduced alterations in the adaptive immune response that produce T cell or antibody-mediated

Chronic obstructive lung disease is characterized by persistent abnormalities in epithelial and immune cell function that are driven, at least in part, by infection. Analysis of parainfluenza virus infection in mice revealed an unexpected role for innate immune cells in IL-13-dependent chronic lung disease, but the upstream driver for the immune axis in this model and in humans with similar disease was undefined. We demonstrate here that lung levels of IL-33 are selectively increased in postviral mice with chronic obstructive lung disease and in humans with very severe chronic obstructive pulmonary disease (COPD). In the mouse model, IL-33/IL-33 receptor signaling was required for Il13 and mucin gene expression, and Il33 gene expression was localized to a virus-induced subset of airway serous cells and a constitutive subset of alveolar type 2 cells that are both linked conventionally to progenitor function. In humans with COPD, IL33 gene expression was also associated with IL13 and mucin gene expression, and IL33 induction was traceable to a subset of airway basal cells with increased capacities for pluripotency and ATP-regulated release of IL-33. Together, these findings provide a paradigm for the role of the innate immune system in chronic disease based on the influence of long-term epithelial progenitor cells programmed for excess IL-33 production.

Epithelial hyperplasia and metaplasia are common features of inflammatory and neoplastic disease, but the basis for the altered epithelial phenotype is often uncertain. Here we show that long-term ciliated cell hyperplasia coincides with mucous (goblet) cell metaplasia after respiratory viral clearance in mouse airways. This chronic switch in epithelial behavior exhibits genetic susceptibility and depends on persistent activation of EGFR signaling to PI3K that prevents apoptosis of ciliated cells and on IL-13 signaling that promotes transdifferentiation of ciliated to goblet cells. Thus, EGFR blockade (using an irreversible EGFR kinase inhibitor designated EKB-569) prevents virus-induced increases in ciliated and goblet cells whereas IL-13 blockade (using s-IL-13Rα2-Fc) exacerbates ciliated cell hyperplasia but still inhibits goblet cell metaplasia. The distinct effects of EGFR and IL-13 inhibitors after viral reprogramming suggest that these combined therapeutic strategies may also correct epithelial architecture in the setting of airway inflammatory disorders characterized by a similar pattern of chronic EGFR activation, IL-13 expression, and ciliated-to-goblet cell metaplasia. IntroductionEpithelial cell hyperplasia and metaplasia are common consequences of inflammation and may be associated with protective as well as pathogenic outcomes. In the lung, airway epithelial remodeling can be life threatening, since mucous cell metaplasia is the foundation for hypersecretion that can obstruct the airway lumen. Despite the critical nature of this process, little is known about how mucous cell metaplasia develops in the setting of acute or chronic inflammatory disease. Particularly, little is known about the mechanism for what is likely the most common cause of mucous cell metaplasia in the lung, i.e., respiratory viral infection, since previous work has focused on bacterial, allergic, and carcinogenic stimuli. Perhaps because of the paucity of mechanistic information, no effective and specific pharmacologic treatment is currently available to treat epithelial cell metaplasia in general or mucous cell metaplasia in particular.In this context, recent work on mucous cell metaplasia has often focused on signaling pathways initiated by activation of the IL-13 receptor (IL-13R) and EGFR (also designated ErbB1 and HER1). The experimental role of IL-13R was established when a decoy receptor for IL-13 (soluble IL-13Rα2-Fc) was found to inhibit allergen-induced mucous (goblet) cell formation in mice (1, 2). These reports have been followed by evidence that IL-13 can directly drive mucin gene expression in airway epithelial cells cultured under physiologic conditions and in vivo (3-6). Moreover, IL-13 is often overexpressed in the setting of mucous cell metaplasia in asthma

Programs that direct cellular differentiation are dependent on the strict temporal expression of regulatory factors that can be provided by Rho GTPases. Ciliogenesis is a complex sequence of events involving the generation and docking of basal bodies at the apical membrane, followed by ciliary axoneme generation. Although a cilia proteome has been assembled, programs that direct ciliated cell differentiation are not well established, particularly in mammalian systems. Using mouse primary culture airway epithelial cells, we identified a critical stage of ciliogenesis requiring the temporal establishment of an apical web-like structure of actin for basal body docking and subsequent axoneme growth. Apical web formation and basal body docking were prevented by interruption of actin remodeling and were dependent on RhoA activation. Additional evidence for this program was provided by analysis of Foxj1-null mice that failed to dock basal bodies and lacked apical actin. Foxj1 expression coincided with actin web formation, activated RhoA and RhoB, and persisted despite RhoA inhibition, suggesting that Foxj1 promoted RhoA during ciliogenesis. Apical ezrin localization was also dependent on Foxj1, actin remodeling, and RhoA, but was not critical for ciliogenesis. Thus, temporal Foxj1 and RhoA activity are essential regulatory events for cytoskeletal remodeling during mammalian ciliogenesis.