Airway Epithelial Progenitors Are Region Specific and Show Differential Responses to Bleomycin-Induced Lung Injury

Mechanisms that regulate regional epithelial cell diversity and pathologic remodeling in airways are poorly understood. We hypothesized that regional differences in cell composition and injury-related tissue remodeling result from the type and composition of local progenitors. We used surface markers and the spatial expression pattern of an SFTPC-GFP transgene to subset epithelial progenitors by airway region. Green fluorescent protein (GFP) expression ranged from undetectable to high in a proximal-to-distal gradient. GFPhi cells were subdivided by CD24 staining into alveolar (CD24neg) and conducting airway (CD24low) populations. This allowed for the segregation of three types of progenitors displaying distinct clonal behavior in vitro. GFPneg and GFPlow progenitors both yielded lumen containing colonies but displayed transcriptomes reflective of pseudostratified and distal conducting airways, respectively. CD24lowGFPhi progenitors were present in an overlapping distribution with GFPlow progenitors in distal airways, yet expressed lower levels of Sox2 and expanded in culture to yield undifferentiated self-renewing progeny. Colony-forming ability was reduced for each progenitor cell type after in vivo bleomycin exposure, but only CD24lowGFPhi progenitors showed robust expansion during tissue remodeling. These data reveal intrinsic differences in the properties of regional progenitors and suggest that their unique responses to tissue damage drive local tissue remodeling. Disclosure of potential conflicts of interest is found at the end of this article

Severe lung fibrosis requires an invasive fibroblast phenotype regulated by hyaluronan and CD44

Hyaluronan synthase 2 and CD44 are required for severe lung fibrosis in response to bleomycin

Apical Secretion of FSTL1 in the Respiratory Epithelium for Normal Lung Development.

Follistatin-like 1 (FSTL1) is a secreted bone morphogenetic protein (BMP) antagonist, and it plays a crucial role in normal lung development. Deletion of Fstl1 leads to postnatal death in mice due to respiratory failure. To further explore the role of FSTL1 in mouse lung development, we created a transgene SFTPC-Fstl1 allele mouse displaying significant epithelial overexpression of Fstl1 in all stages of lung development. However, epithelial overexpression of Fstl1 did not alter lung morphogenesis, epithelial differentiation and lung function. Moreover, we found that FSTL1 function was blocked by the epithelial polarization, which was reflected by the remarkable apical secretion of FSTL1 and the basolateral BMP signaling. Taken together, this study demonstrates that tightly spatial interaction of FSTL1 and BMP signaling plays an essential role in lung development

Apical secretion of FSTL1 and basolateral location of BMPRII in polarized lung epithelial cells.

<p>(<b>A</b>) FSTL1 levels in BAL and serum from adult <i>SFTPC-Fstl1</i> mice (P60) as determined by western blotting analysis. (<b>B-C</b>) Primary AECs <b>(B)</b> isolated from E18.5 <i>SFTPC-Fstl1</i> transgenic mice and MLE-12 cells <b>(C)</b> transfected with <i>pcDNA3</i>.<i>1-Fstl1</i> (<i>pc-Fstl1</i>). FSTL1 level in conditioned medium from the apical (AP) or basolateral (BL) side was determined by western blotting. (<b>D</b>) Primary mouse AECs from E18.5 lungs were stained with an anti-FLAG antibody (Red). X-Y (horizontal) and X-Z (vertical) sections are shown in the top and bottom panels, respectively. The yellow line shows the Y position of X-Z section. Scale bars, 7.5 μm.</p

Characterization of <i>SFTPC-Fstl1</i> transgenic mouse lungs.

<p>(<b>A</b>) qRT-PCR analysis of <i>Fstl1</i> mRNA expression in E18.5 lungs from three lines of transgenic mice. n = 3. ***, <i>P</i> < 0.001. Data represent the mean ± SEM in triplicates. (<b>B</b>) Western blotting analysis of FSTL1 expression in E18.5 lungs. β-actin was used as a loading control. (<b>C</b>) Immunohistochemical staining for epithelial expression of FSTL1 in transgenic (Tg) lungs from E12.5 to P60. P60 WT staining was used as control. Scale bars, 50 μm. (<b>D</b>) Co-localization of FSTL1 and SP-C in distal region of E18.5 transgenic lungs. Scale bars, 25 μm. (<b>E</b>) Expression of differentiation markers for lung epithelial cells and microvascular network development in E18.5 lungs. Scale bars, 100 μm.</p