Recombinant Adenovirus AD-IL-12 Reverses The Phenotype And Function Of M2 Macrophages

Objective:To investigate whether recombinant adenovirus Ad-IL-12 could reverse the phenotype and function of M2 macrophages, and related signaling molecule changes.Methods:M2 macrophages, which were established by the exogenous cytokines, were transfected with adenovirus carrying the desired gene IL-12. About 60h later, the changes of a series of markers related to macrophage polarization (some pro-and anti-inflammatory factors) were detected by PCR, the alterations of cell surface markers were assessed by FCM. The functions of M2 macrophage were indirectly detected by some biological functions of hepatic carcinoma cell lines HepG2 treated with conditioned medium collected from three different treated groups. The proliferation of HepG2 was detected by CCK-8 assay, and the apoptosis and cell cycle were detected by FCM. Finally, the changes of some signaling molecules following the alteration of macrophage phenotype and function were detected with western blot.Results:The model of M2 macrophage was successfully established. The fluorescence microscope images and the expression of IL-12p35, p40 proved that recombinant adenovirus Ad-IL-12 was successfully transfected and the IL-12 gene was effectively expressed. About 60h later, proinflammatory factors (IL-23, IL-1β, et al.) were upregulated, while the anti-inflammatory factors (IL-10, TGF-β) were downregulated in M2 macrophages of overexpression of IL-12 compared with the blank group and empty vector group. The difference was statistically significant (P< 0.05). FCM showed that cell surface marker CD86 of M1 macrophage was increased while CD206 of M2 macrophage surface marker was decreased in Ad-IL-12 group. The hepatic carcinoma cell lines HepG2 was treated with conditioned medium collecting from three different treated groups for 72 h. CCK-8 assay proved that the proliferation of HepG2 was significantly inhibited from the third day in Ad-IL-12 group compared with blank group and empty vector group, while the difference between the last two groups was not statistical. FCM showed that the early apoptosis of HepG2 was significantly increased in Ad-IL-12 group (rate of apoptosis was 11.33%) compared with in blank group and empty vector group (rate of apoptosis was 2.91% and 3.01%, respectively), the cell cycle was arrested in G0/G1 phase (65.88% compared with 44.63% and 48.42%). The expression of protein p-stat3, wnt2, p-catenin, c-myc and NF-κ BP50 were downregulated while protein p-statl and NF-κ BP65 were both significantly upregulated in Ad-IL-12 group compared with control groups through the western blot.Conclutions:Through transfecting recombinant adenovirus Ad-IL-12 into the established M2 macrophages, we have found that recombinant adenovirus Ad-IL-12 could reverse the phenotype and function of M2 macrophages to M1, and following this process, some signaling molecules are changed.