Experimental Research On The Role Of IRF5-regulated Alveolar Macrophage Polarization In Severe Acute Pancreatitis Associated Lung Injury

Objective:(1) To establish the lung injury mouse model of severe acute pancreatitis (SAP) and determine the most serious time of lung injury.(2) To elucidate the relationship between alveolar macrophages polarization and SAP associated lung injury.(3) To investigate the role of interferon regulatory factor 5 (IRF5) in the regulation of alveolar macrophage polarization.(4) To investigate whether lentivirus-mediated IRF5 interference (IRF5 shRNA)has protective effect on SAP associated lung injury in vivo.Methods:(1) Development of mouse lung injury model and evaluation of lung injury:Development of mouse model of SAP associated lung injury by intraperitoneal injection of frogin and lipopolysaccharide. The activity of myeloperoxidase (MPO),the concentration of lipopolysaccharide malondialdehyde (MDA), the levels of TNF-?and IL-1? in the lung tissue were measured to confirm the degree of lung injury and to determine the most serious time point of lung injury.(2) To confirm the relationship between alveolar macrophage polarization and lung injury: The flow cytometry were applied to detect the content of macrophages and other inflammatory cells in alveolar lavage fluid of SAP mouse models. Futhermore,alveolar macrophages were sorted as Ml and M2 macrophages. Real-time quantitative PCR was used to detect the levels of macrophage associated inflammatory factors such as TNF-? and IL-1?. The relationship between polarized alveolar macrophages(M1, M2) infiltration and lung injury was analyzed by immunohistochemical staining and laser confocal immunofluorescence staining..(3) To study the role of IRF5 in alveolar macrophage polarization: The alveolar M1 and M2 macrophages were sorted by flow cytometry. Real-time quantitative PCR was used to detect the expression of IRF5 in M1 and M2 macrophages. The alveolar Ml macrophages were transfected by IRF5 siRNA. Real-time quantitative PCR and Western blotting were used to detect the expression of markers of M1 and M2 macrophages to identify the polarized state of alveolar macrophages.(4) To study the protective effect of IRF5 shRNA on SAP associated lung injury:Lentivirus-mediated IRF5 interference were prepared and the most significant sequence of interference was identified as the interference fragment of the subsequent IRF5 gene. The IRF5 shRNA were injected into the mice by tail vein. After 48 h, the level of serum amylase, the lung tissue W/D, the activity of MPO, the concentration of MDA, the levels of TNF-? and IL-1? in lung tissue were measured. The lung histopathology scores, immunohistochemical staining and laser confocal immunofluorescence staining were used to evaluate the severity of lung injury.Results:(1) The mouse model of SAP associated lung injury was successfully developed.The most serious time of acute lung injury was determined at 48 h after SAP model established.(2) SAP injury degree was related with the activation of alveolar macrophages. In the early stage of lung injury (24h after SAP), M1 macrophages was in a dominant position. In the later stage (96h after SAP), the number of M2 macrophages significantly increased.(3) The expression of IRF5 in alveolar Ml macrophages was significantly higher than that in alveolar M2 macrophages (P < 0.01). After IRF5 siRNA transfecting with alveolar M1 macrophages, the expression of TNF-?, IL-12 and iNOS mRNA were significantly lower than that of untreated M1 macrophages (P < 0.01). The relative expression of IL-10 and Arg-1 mRNA were significantly higher than that of untreated M2 macrophages (P < 0.01), and there was no significant difference between treated M1 macrophages and untreated M2 macrophages (P > 0.05). After IRF5 siRNA transfecting with alveolar M1 macrophages, alveolar Ml macrophages shifted to M2 state.(4) Compared with saline group and Scramble shRNA group, the lung injury of IRF5 shRNA group was significantly reduced. After IRF5 shRNA infecting, alveolar macrophages were gradually decreased, M2 macrophages were significantly increased.Alveolar macrophages were transformed from M1 macrophages to M2 macrophages.Conclusions: IRF5 can be used as an important marker for the identification of alveolar M1 and M2 macrophages. IRF5 was a key molecule to regulate macrophage polarization. IRF5 shRNA had a significant immunoprotective effect on SAP lung injury.