Pyroptosis and polarization of macrophages in septic acute lung injury induced by lipopolysaccharide in mice

Abstract

Background: Pyroptosis and polarization are significant contributors to the onset and development of many diseases. At present, the relationship between pyroptosis and polarization in acute lung injury (ALI) caused by sepsis remains unclear.

Methods: The ALI model for sepsis was created in mice and categorized into the blank control, lipopolysaccharide (LPS) group, LPS + low-dose Belnacasan group, LPS + high-dose Belnacasan group, LPS + low-dose Wedelolactone group, LPS + high-dose Wedelolactone group, and positive control group. The wet-dry specific gravity was evaluated to compare pulmonary edema. Hematoxylin-eosin, Masson, and terminal deoxynucleotidyl transferase dUTP nick end labeling staining techniques were conducted to observe and contrast the pathological changes in lung tissue. ELISA was utilized to identify M1 and M2 macrophages and correlated inflammatory factors. Immunohistochemical staining and flow cytometry were employed to identify markers of M1 and M2 macrophages in lung tissue. Propidium iodide staining, together with flow cytometry, was utilized to observe the degree and positive rate of pyroptosis of alveolar macrophages. Western blot analysis was conducted to detect the expression levels of Caspase 1, Caspase 11, GSDMD, and IL-18 in the lung tissues of each group. The real-time quantitative polymerase chain reaction method was used to ascertain relative expression levels of NLRP3, Caspase 1, Caspase 11, GSDMD, IL-18, iNOS, and Arg-1 in lung tissues of all groups.

Results: In mice with sepsis-induced ALI, both classical and nonclassical pathways of pyroptosis are observed. Inhibiting pyroptosis has been found to ameliorate lung injury, pulmonary edema, and inflammation induced by LPS. Notably, the expression of NLRP3, Caspase 1, Caspase 11, GSDMD, IL-1β, IL-18, TGF-β, CD86, CD206, iNOS, and Arg-1 were all altered in this process. Additionally, alveolar macrophages were polarized along with pyroptosis in mice with ALI caused by sepsis.

Conclusion: Pyroptosis of alveolar macrophages in the context of ALI in mice infected with sepsis has been linked to the polarization of alveolar macrophages toward type M1.

Keywords: acute lung injury; alveolar macrophage; polarization; pyroptosis; sepsis.

FIGURE 1

Pyroptosis inhibition alleviated lung pathological injury in  lipopolysaccharide (LPS)‐induced mice. (A) hematoxylin–eosin staining judged pathological alternations in lung tissues (×200), N = 3. (B) Comparison of lung tissue pathological score. (C, D) TUNEL assay detected the pyroptosis of lung cells in mice (×400), N = 6. *p < .05 versus control, ^# p < .05 versus LPS.

FIGURE 2

W/D ratio was measured to observe the change of pulmonary edema, N = 3. *p < .05 versus control, ^# p < .05 versus lipopolysaccharide.

FIGURE 3

ELISA examined (A) IL‐1β and (B) IL‐18 activities in the mouse serum, N = 5. *p < .005 versus control, ^# p < .005 versus lipopolysaccharide.

FIGURE 4

In mice with acute lung injury caused by sepsis, both classical and nonclassical pathways of pyroptosis occurred. (A) The cell graph after propidium iodide staining was observed by microscope (×200), N = 3. (B, C) Flow cytometric profiles of pyroptosis cells, N = 5. (D) Analysis of messenger RNA (mRNA) levels of NLRP3, Caspase 1, Caspase 11, GSDMD, and IL‐1β in lung. (E, F) The protein levels of Caspase 1, Caspase 1 P20, and Caspase 1 P10 in lung were measured via western blot. (G, H) The protein levels of GSDMD and GSDMD‐N in lung were measured via western blot. (I–K) The protein levels of Caspase 11 and IL‐18 in lung were measured via western blot. *p < .05 versus control, ^# p < .05 versus lipopolysaccharide.

FIGURE 5

Inhibition of pyroptosis can affect the polarization of alveolar macrophages. (A) Representative images and quantitative analysis of M2 rate were assessed using flow cytometry. (B) Representative images and quantitative analysis of M1 rate were assessed using flow cytometry. (C) The immunohistochemical results of F4/80, CD86, and CD206 in the lungs (×200), N = 3. (D) The concentrations of IL‐1β in alveolar lavage fluid were examined with ELISA kits, N = 3. (E) The concentrations of TGF‐β in alveolar lavage fluid were examined with ELISA kits, N = 5. (F) Analysis of messenger RNA (mRNA) levels of iNOS and Arg‐1 in lung, N = 5. *p < .05 versus control, ^# p < .05 versus lipopolysaccharide.