P21 facilitates macrophage chemotaxis by promoting CCL7 in the lung epithelial cell lines treated with radiation and bleomycin

Background: Interstitial lung diseases (ILDs) can be induced and even exacerbated by radiotherapy in thoracic Cancer patients. The roles of immune responses underlying the development of these severe lung injuries are still obscure and need to be investigated.

Methods: A severe lung damage murine model was established by delivering 16 Gy X-rays to the chest of mice that had been pre-treated with bleomycin (BLM) and thus hold ILDs. Bioinformatic analyses were performed on the GEO datasets of radiation-induced lung injury (RILI) and BLM-induced pulmonary fibrosis (BIPF), and RNA-sequencing data of the severely damaged lung tissues. The screened differentially expressed genes (DEGs) were verified in lung epithelial cell lines by qRT-PCR assay. The injured lung tissue pathology was analyzed with H&E and Masson's staining, and immunohistochemistry staining. The macrophage chemotaxis and activity promoted by the stressed epithelial cells were determined by using a cell co-culture system. The expressions of p21 in MLE-12 and Beas-2B cells were detected by qRT-PCR, western blot, and immunofluorescence. The concentration of CCL7 in cell supernatant was measured by ELISA assay. In some experiments, Beas-2B cells were transfected with p21-siRNA or CCL7-siRNA before irradiation and/or BLM treatment.

Results: After the treatment of irradiation and/or BLM, the inflammatory and immune responses, chemokine-mediated signaling pathways were steadily activated in the severely injured lung, and p21 was screened out by the bioinformatic analysis and further verified to be upregulated in both mouse and human lung epithelial cell lines. The expression of P21 was positively correlated with macrophage infiltration in the injured lung tissues. Co-culturing with stressed Beas-2B cells or its conditioned medium containing CCL7 protein, U937 macrophages were actively polarized to M1-phase and their migration ability was obviously increased along with the damage degree of Beas-2B cells. Furthermore, knockdown p21 reduced CCL7 expression in Beas-2B cells and then decreased the chemotaxis of co-cultured macrophages.

Conclusions: P21 promoted CCL7 release from the severely injured lung epithelial cell lines and contributed to the macrophage chemotaxis in vitro, which provides new insights for better understanding the inflammatory responses in lung injury.

Bleomycin-induced pulmonary fibrosis; CCL7; Macrophage chemotaxis; Radiation-induced lung injury; p21.