Role of TIPE2 in macrophage pyroptosis in mice

Abstract

Objective To evaluate the role of tumor necrosis factor alpha-inducible protein 8 like-2 (TIPE2) in macrophage pyroptosis in mice. Methods Mouse macrophages J774A.1 were seeded in 6-cm culture dishes (5 ml/dish) at the density of 2×105 cells/ml and divided into 4 groups (n=18 each) using a random number table method: blank vector control group (C group), blank vector plus lipopolysaccharide (LPS)/ATP group (C+ LPS/ATP group), TIPE2 overexpression group (T group) and TIPE2 overexpression plus LPS/ATP group (T+ LPS/ATP group). Cells were infected with lentivirus without TIPE2 in C and C+ LPS/ATP groups.TIPE2 overexpression stable cell line was constructed in T group and T+ LPS/ATP group.LPS 1.0 μg/ml was added and cells were incubated for 5 h, and then ATP 5.0 mmol/L was added and cells were incubated for 1 h in C+ LPS/ATP group and T+ LPS/ATP group.Cells were collected for detection of the expression of TIPE2, NOD-like receptor protein 3 (NLRP3), interleukin-1beta (IL-1β) and interleukin-8 (IL-18) by Western blot.Flow cytometry was used to detect the pyroptotic cells, and the percentage of pyroptotic cells was calculated.The concentrations of tumor necrosis factor-alpha (TNF-α), IL-6, IL-1β and IL-18 in cell culture media were determined by enzyme-linked immunosorbent assay. Results Compared with group C, the expression of TIPE2 was significantly down-regulated, the expression of NLRP3, IL-1β and IL-18 was up-regulated, and the percentage of pyroptotic cells and concentrations of TNF-α, IL-6, IL-1β and IL-18 in cell culture media were increased in group C+ LPS/ATP (P<0.05). Compared with group T, the expression of TIPE2 was significantly down-regulated, the expression of NLRP3, IL-1β and IL-18 was up-regulated, and the percentage of pyroptotic cells and concentrations of TNF-α, IL-6, IL-1β and IL-18 in cell culture media were increased in group T+ LPS/ATP (P<0.05). Compared with group C+ LPS/ATP, the expression of TIPE2 was significantly up-regulated, the expression of NLRP3, IL-1β and IL-18 was down-regulated, and the percentage of pyroptotic cells and concentrations of TNF-α, IL-6, IL-1β and IL-18 in cell culture media were decreased in group T+ LPS/ATP (P<0.05). Conclusion TIPE2 can inhibit macrophage pyroptosis, and the mechanism may be related to inhibiting activation of NLRP3 inflammasome in mice. Key words: Cell death; TIPE2