Radiation Induces Pulmonary Fibrosis by Promoting the Fibrogenic Differentiation of Alveolar Stem Cells

Abstract

The lung is a radiosensitive organ, which imposes limits on the therapeutic dose in thoracic radiotherapy. Irradiated alveolar epithelial cells promote radiation-related pneumonitis and fibrosis. However, the role of lung stem cells (LSCs) in the development of radiation-induced lung injury is still unclear. In this study, we found that both LSCs and LSC-derived type II alveolar epithelial cells (AECII) can repair radiation-induced DNA double-strand breaks, but the irradiated LSCs underwent growth arrest and cell differentiation faster than the irradiated AECII cells. Moreover, radiation drove LSCs to fibrosis as shown with the elevated levels of markers for epithelial-mesenchymal transition and myofibroblast (α-smooth muscle actin (α-SMA)) differentiation in in vitro and ex vivo studies. Increased gene expressions of connective tissue growth factor and α-SMA were found in both irradiated LSCs and alveolar cells, suggesting that radiation could induce the fibrogenic differentiation of LSCs. Irradiated LSCs showed an increase in the expression of surfactant protein C (SP-C), the AECII cell marker, and α-SMA, and irradiated AECII cells expressed SP-C and α-SMA. These results indicated that radiation induced LSCs to differentiate into myofibroblasts and AECII cells; then, AECII cells differentiated further into either myofibroblasts or type I alveolar epithelial cells (AECI). In conclusion, our results revealed that LSCs are sensitive to radiation-induced cell damage and may be involved in radiation-induced lung fibrosis.

Figure 1

The lung cell differentiation and the effects of radiation exposure on LSCs and alveolar cells. (a) Workflow and time line of in vitro lung cell differentiation experiments. LSCs were isolated from neonatal ICR mice and then sequentially differentiated into alveolar cells by culture with MCDB-201 medium. LSCs, AECII, and AECI cells were examined through immunostaining with anti-CD157, anti-pro-SP-C, and anti-T1α antibodies. Scale bars, 100 μm. (b) Cell numbers of irradiated LSCs and alveolar cells were examined at day 3 postirradiation. The results are represented as mean ± SD (n = 3, ^∗p < 0.05) relative to initial cell number. (c) Immunostaining of γ-H2AX expression (green, left panels) and nuclear morphology (blue, right panels) in LSCs and alveolar cells that received 8 Gy at 1 h and 16 h. (d) The lung cells repair radiation-induced DNA double-strand breaks as determined by nuclear foci formation of γ-H2AX. The nuclear γ-H2AX foci per cell are means ± SD from LSCs and AECII cells (n > 100) or AECI cells (n > 75) relative to the initial sample. (e) The cell morphology of irradiated lung cells at day 3 postirradiation. Scale bars, 100 μm. (f) The cell differentiation of irradiated lung stem cells was examined at day 3 postirradiation by immunostaining with specific protein markers CD157, SP-C, or T1α. Scale bars, 100 μm.

Figure 2

Fibrotic response of LSCs after exposure to irradiation. Total RNA was extracted from irradiated LSCs exposed to 2 or 8 Gy of X-rays followed by incubation under normal conditions for 7 days. The relative levels of the indicated mRNA for Oct-4, Nanog, and SOX-2 (a) as well as E-cadherin, N-cadherin, and α-SMA (b) were measured by real-time PCR. (c, d) The represented α-SMA expression in irradiated LSCs exposed to 2 or 8 Gy of X-ray followed by incubation under normal conditions for 1–7 days. Scale bars, 100 μm. (e) Irradiated LSCs exposed to 8 Gy of X-ray followed by incubation under normal conditions for 7 days. Cells were double stained with α-SMA (green) and FSP1 (red) antibodies and then stained with DAPI (blue). Scale bars, 100 μm. (f) LSCs were double stained with pro-SP-C (red) and α-SMA (green) antibodies and then stained with DAPI (blue). The white arrows indicate SP-C⁺/α-SMA⁺ cells. Scale bars, 100 μm.

Figure 3

The fibrogenic differentiation of LSCs from irradiated mice. (a) Workflow of ex vivo lung cell irradiation experiments. The cell growth and differentiation of LSCs isolated from irradiated neonatal mice were analyzed following incubation for 14 days and compared with those isolated from nontreated mice. Scale bars, 100 μm. (b) LSCs were isolated from control neonatal mice or mice exposed to the indicated dosage of radiation. Cell morphology and density were examined after seeding 1 × 10⁵ isolated LSCs within 5 days. Scale bars, 100 μm. (c) The populations of α-SMA⁺ (green) and SP-C⁺ (red) cells were costained in isolated LSCs and were analyzed, followed by incubation for 14 days. The nuclei of cells were stained blue with DAPI. Scale bars, 70 μm.

Figure 4

Radiation exposure promotes fibrogenic signaling in LSCs and AECII cells. (a) The mRNA was extracted from LSCs after treatment with or without 8 Gy X-ray followed by incubation under normal conditions for 7 days. Relative gene expression was examined by the Fibrosis PCR Array. (b) The mRNA was extracted from LSCs, AECII, and AECI cells exposed to 2 or 8 Gy. The relative levels of the indicated mRNAs were measured by real-time PCR. The data were normalized to GAPDH and expressed relative to nonradiated control cells. (c) LSCs were treated with CTGF (50 ng/ml) or TGF-β (5 ng/ml) for 3 days, and the populations of α-SMA⁺ and SP-C⁺ cells were analyzed. Scale bars, 100 μm.

Figure 5

Nintedanib attenuates radiation-induced pulmonary cell fibrogenic differentiation. (a, b) LSCs and LSC-differentiated alveolar cells were exposed to 4 Gy followed by treatment with or without nintedanib (1 μM) and then incubated for 3 days. The levels of SP-C (red) and α-SMA (green) protein were determined by coimmunostaining. The arrowheads indicate SP-C⁺/α-SMA⁺ cells. The nuclei of cells were stained blue with DAPI. Scale bars, 75 μm.

Figure 6

Model of LSC differentiation in alveolar development and irradiated tissue. LSCs (blue): lung stem cells; AECII (yellow): type II alveolar epithelial cells; AECI (green): type I alveolar epithelial cells; RT: radiation.