Sex-determining region Y-box 2-positive alveolar cells are responsive to bleomycin-induced lung injury
The pulmonary alveolar epithelium serves as a critical barrier, playing a pivotal role in lung gas exchange and host defense. This epithelium is primarily composed of alveolar type I (AT1) and type II (AT2) cells, which are tightly connected to maintain the integrity of the alveolar structure. In response to various injuries, several types of stem and progenitor cells are capable of repairing the adult alveolar epithelium. For instance, bronchioalveolar stem cells (BASCs) have been identified as cells that can differentiate into both alveolar and airway cells. Additionally, a subset of alveolar cells expressing the laminin receptor a6b4, but not surfactant protein C (SPC), has been shown to regenerate into AT2 cells following bleomycin-induced lung injury. Furthermore, rare keratin 5-positive basal cells have been found to give rise to alveolar epithelial lineages after influenza infection.
Sex-determining region Y-box 2 (Sox2) is a transcription factor known for its role in maintaining and regulating self-renewal and pluripotency in embryonic stem cells. However, the functional roles of Sox2 in the repair of alveolar epithelium injuries remain largely unexplored. This study aimed to investigate whether alveolar cells expressing Sox2 are involved in the repair of alveolar epithelium following injury.
To explore this, mouse lung cells were isolated through elastase digestion and subjected to fluorescence-activated cell sorting (FACS) using a transgenic surfactant protein C (SFTPC)-green fluorescent protein (GFP) reporter mouse line. This approach allowed for the analysis of SPC expression in alveolar cells. Flow cytometry was performed using primary antibodies against CD31-biotin, CD34-biotin, CD45-biotin, CD24-phycoerythrin (PE), anti-epithelial cellular adhesion molecule-PE-cyanine 7, and Sca-1-allophycocyanin. A secondary antibody against streptavidin was used, and 7-amino-actinomycin D was added to label dead cells. The flow cytometry analysis was conducted using an FACS Aria III sorter.
Gene expression was measured using LightCycler real-time polymerase chain reaction (PCR). The primers used were specific for b-actin and Sox2. The relative messenger RNA (mRNA) expression level of the target genes was calculated using the comparative Ct (threshold cycle) method, normalized to b-actin mRNA in the same sample. The specific DCt was calculated as follows: DCt = (Ctb-actin) – (Cttarget), and the relative expression was defined as 2^ -DCt. To detect the location of Sox2-positive cells, Sox2-GFP reporter transgenic mice and immunofluorescence analysis were employed. Primary antibodies included chicken polyclonal anti-GFP antibody, Sox2, and pro-SPC. Secondary antibodies included Rabbit anti-Chicken immunoglobulin of yolk (Ig Y)-fluorescein isothiocyanate, Donkey anti-Rat Alexa Fluor 488, and Donkey anti-Rabbit Alexa Fluor 594. Stained sections were imaged using a Zeiss LSM710 confocal microscope.
The study then investigated whether Sox2-positive alveolar cells are involved in alveolar epithelial repair using a bleomycin-induced lung injury model. Mice were anesthetized and intratracheally injected with 2.5 U/kg bleomycin, while control animals received phosphate-buffered saline alone. At designated time points after bleomycin injection, the mice were euthanized, and their lung tissues were harvested for immunofluorescence staining or flow cytometry analysis. All procedures were conducted in strict adherence to the protocol approved by the Haihe Hospital Animal Care and Use Committee.
Statistical analysis was performed using SPSS 17.0 software. Measurement data conforming to the normal distribution were expressed as mean ± standard deviation. Comparisons between two groups were performed using Student’s t-test, with P < 0.05 indicating statistical significance.
The study utilized a transgenic SFTPC-GFP reporter mouse line to analyze SPC expression in alveolar cells through an FACS-based strategy. Dot plot analysis of the alveolar cell population revealed subsets of SPChi (AT2 cells expressing high levels of SPC) and SPClow cells (alveolar cells expressing low levels of SPC) with different GFP fluorescence intensities. Quantitative real-time PCR analysis of these fractionated lung epithelial cells indicated that the level of Sox2 mRNA was lower in SPClow alveolar cells than in Club cells but significantly higher than in AT2 cells. Immunofluorescence analysis of lung tissues from SFTPC-GFP mice for GFP and Sox2 expression revealed that SPClow alveolar cells were Sox2-positive and localized in the alveolar region. The Sox2-GFP reporter transgenic mice further validated the presence of Sox2-positive cells within the alveolar space.
The study then determined whether Sox2-positive alveolar cells are involved in alveolar epithelial injuries. Bleomycin was administered to mice by intratracheal instillation. Observations indicated that Sox2+SPClow cells resided within the alveolar space near the bronchioalveolar duct junction (BADJ). Immunofluorescence staining of lung sections showed that the percentage of Sox2+SPClow cells in total SPC+ alveolar cells increased on days 14 and 21 after bleomycin-induced injury.
In summary, the study identified an alveolar cell population expressing a low level of SPC in mouse lung tissue. These SPClow alveolar cells express Sox2 at a lower level than airway progenitor Club cells but at a higher level than AT2 cells. Immunofluorescent staining of lungs from Sox2-GFP mice confirmed the presence of Sox2-expressing cells in the alveolar space. Importantly, Sox2+SPClow alveolar cells were responsive to bleomycin-induced lung injury, with their abundance peaking at day 14 post-injury, suggesting significant alveolar repair activity at this time. This Sox2+SPClow alveolar cell population appears distinct from previously identified epithelial stem and progenitor cells involved in alveolar epithelium repair.
The molecular basis for the function of epithelial stem and progenitor cells in the lung remains largely unknown. The study demonstrated that the level of Sox2 expression varies among AT2, Sox2+SPClow, and Club cells. Sox2 is a transcription factor expressed exclusively in tracheal epithelium and conducting airway epithelium in the lung. The functional roles of Sox2 in regulating alveolar Sox2+SPClow cells remain to be fully understood. While Sox2+SPClow alveolar cells are responsive to bleomycin-induced lung injury, further research is needed to elucidate how these cells contribute to alveolar epithelial regeneration.
This study was supported by grants from the National Natural Science Foundation of China, the Natural Science Foundation of Tianjin, and the Science and Technology Fund of Tianjin Haihe Hospital.
doi.org/10.1097/CM9.0000000000001086
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