Jazalle McClendon

Neutrophil transmigration triggers repair of the lung epithelium via β-catenin signaling

Injury to the epithelium is integral to the pathogenesis of many inflammatory lung diseases, and epithelial repair is a critical determinant of clinical outcome. However, the signaling pathways regulating such repair are incompletely understood. We used in vitro and in vivo models to define these pathways. Human neutrophils were induced to transmigrate across monolayers of human lung epithelial cells in the physiological basolateral-to-apical direction. This allowed study of the neutrophil contribution not only to the initial epithelial injury, but also to its repair, as manifested by restoration of transepithelial resistance and reepithelialization of the denuded epithelium. Microarray analysis of epithelial gene expression revealed that neutrophil transmigration activated β-catenin signaling, and this was verified by real-time PCR, nuclear translocation of β-catenin, and TOPFlash reporter activity. Leukocyte elastase, likely via cleavage of E-cadherin, was required for activation of β-catenin signaling in response to neutrophil transmigration. Knockdown of β-catenin using shRNA delayed epithelial repair. In mice treated with intratracheal LPS or keratinocyte chemokine, neutrophil emigration resulted in activation of β-catenin signaling in alveolar type II epithelial cells, as demonstrated by cyclin D1 expression and/or reporter activity in TOPGAL mice. Attenuation of β-catenin signaling by IQ-1 inhibited alveolar type II epithelial cell proliferation in response to neutrophil migration induced by intratracheal keratinocyte chemokine. We conclude that β-catenin signaling is activated in lung epithelial cells during neutrophil transmigration, likely via elastase-mediated cleavage of E-cadherin, and regulates epithelial repair. This pathway represents a potential therapeutic target to accelerate physiological recovery in inflammatory lung diseases.

Unbiased quantitation of alveolar type II to alveolar type i cell transdifferentiation during repair after lung injury in mice

Abstract The alveolar epithelium consists of squamous alveolar type (AT) I and cuboidal ATII cells. ATI cells cover 95‐98% of the alveolar surface, thereby playing a critical role in barrier integrity, and are extremely thin, thus permitting efficient gas exchange. During lung injury, ATI cells die, resulting in increased epithelial permeability. ATII cells re‐epithelialize the alveolar surface via proliferation and transdifferentiation into ATI cells. Transdifferentiation is characterized by down‐regulation of ATII cell markers, up‐regulation of ATI cell markers, and cell spreading, resulting in a change in morphology from cuboidal to squamous, thus restoring normal alveolar architecture and function. The mechanisms underlying ATII to ATI cell transdifferentiation have not been well studied in vivo. A prerequisite for mechanistic investigation is a rigorous, unbiased method to quantitate this process. Here, we used SPCCreERT2;mTmG mice, in which ATII cells and their progeny express green fluorescent protein (GFP), and applied stereologic techniques to measure transdifferentiation during repair after injury induced by LPS. Transdifferentiation was quantitated as the percent of alveolar surface area covered by ATII‐derived (GFP+) cells expressing ATI, but not ATII, cell markers. Using this methodology, the time course and magnitude of transdifferentiation during repair was determined. We found that ATI cell loss and epithelial permeability occurred by Day 4, and ATII to ATI cell transdifferentiation began by Day 7 and continued until Day 16. Notably, transdifferentiation and barrier restoration are temporally correlated. This methodology can be applied to investigate the molecular mechanisms underlying transdifferentiation, ultimately revealing novel therapeutic targets to accelerate repair after lung injury.

Flow Cytometry Underestimates and Planimetry Overestimates Alveolar Epithelial Type 2 Cell Expansion after Lung Injury

High-profile investigations have focused on alveolar regeneration after lung injury, notably expansion of the alveolar epithelial type 2 cell (AEC2) population (1–4). Investigations of AEC2 expansion, particularly mechanistic investigations using pharmacologic or genetic manipulation, require an accurate, unbiased method of quantitation to ensure valid comparison between experimental groups. AEC2 expansion is typically assessed by flow cytometry of cells recoverable from a lung digest (1–3, 5–7) or by counting cell profiles in arbitrary fields of immunostained lung sections (planimetry) (1, 2, 4, 7). AEC2 expansion is usually measured as the percentage of AEC2s that are actively cycling, as determined by incorporation of nucleoside analogs (1–7) or AEC2 number as a percentage of a larger population, for example, total lung cells (1, 2, 5); rarely is absolute AEC2 number assessed (1, 7). These methods have not been validated and have important theoretical limitations. Flow cytometric analysis may be limited by incomplete and variable recovery of cells. Planimetric analysis may be biased by tissue inflation and shrinkage, cell loss during sectioning, the overrepresentation of larger cells in two-dimensional sections, and nonrandom sampling, which is particularly problematic for lung injury with a patchy distribution (8–10). Evaluating AEC2 expansion as the percentage of actively cycling cells is limited by the duration of the pulse and ascertains S phase, not cell division. Expressing the number of actively cycling cells as a percentage of total AEC2s, or total AEC2 number as a percentage of a larger population, may be confounded by changes in the denominator. Epithelial regeneration requires increased absolute AEC2 numbers to replace cells lost during injury. Stereology is an unbiased approach recommended by the American Thoracic Society for the quantitation of cell number (10) but is rarely used to assess AEC2 expansion. Here, we employed stereology as the “gold standard” method for quantifying AEC2 expansion during repair after lung injury. We measured the absolute number of AEC2s, the key parameter for alveolar regeneration, and the absolute number and percentage of actively cycling AEC2s. The results were compared with flow cytometric and planimetric analyses.

Isolation of Rat and Mouse Alveolar Type II Epithelial Cells

The gas exchange surface of the lungs is lined by an epithelium consisting of alveolar type (AT) I and ATII cells. ATII cells function to produce surfactant, play a role in host defense and fluid and ion transport, and serve as progenitors. ATI cells are important for gas exchange and fluid and ion transport. Our understanding of the biology of these cells depends on the investigation of isolated cells. Here, we present methods for the isolation of mouse and rat ATII cells.