Interleukin-1 receptor-associated kinase M regulates lipopolysaccharide-induced respiratory burst in peritoneal macrophages of mouse through inhibiting p22phox

Abstract

Objective To investigate whether interleukin-1 receptor-associated kinase M (IRAK-M) regulates p22phox-mediated lipopolysaccharide (LPS)-induced macrophage respiratory burst. Methods Mouse peritoneal macrophages were isolated and cultured in votro. The cells were divided into endotoxin tolerance (ET) group and control group (NC group). The ET group was stimulated with 10 μg/L lipopolysaccharide (LPS) for 2 h, then with 100 μg/L LPS re-stimulation. The NC group was not pretreated, directly stimulated by 100 μg/L LPS. Supernatant was collected 3 h later, TNF-α and IL-6 levels were detected by enzyme-linked immunosorbent assay (ELISA). H2O2 and O2- levels were also detected. Western blot was used to detect the expression levels of IRAK-M and p22phox proteins. IRAK-M small interfering RNA (siRNA) was used to interfere with IRAK-M. Groups are set as follows: NC-control group, the interfering with IRAK-M+LPS stimulation group (NC-si-IRAK-M group), the interfering with endotoxin tolerance group (ET-control group), and the interfering with endotoxin tolerance group (ET-si-IRAK-M group). The interference efficiency was detected by polymerase chain reaction (PCR). The protein expression levels of IRAK-M and p22phox were also measured. After successful IRAK-M interference, p22phox was inhibited by apocynin. The groups were: dimethyl sulfoxide (DMSO)+endotoxin tolerance group (control-placebo group), apocynin+endotoxin tolerance group (control-apocynin group), DMSO+interference IRAK-M+endotoxin tolerance group (si-IRAK-M-placebo group) and interfering with IRAK-M+apocynin+endotoxin tolerance group (si-IRAK-M-apocynin group). The supernatant was taken for detection of TNF-α, IL-6, H2O2 and O2- levels. Results Comparing with ET-control group, after interference with IRAK-M, the protein content of ET-si-IRAK-M was significantly decreased. In contrast, the protein content of p22phox was significantly increased (P<0.05); after interfering with IRAK-M and inhibiting p22phox, the levels of TNF-α, IL-6 and H2O2 O2- in the IRAK-M-apocynin group were significantly lower than the levels of these proteins in si-IRAK-M-placebo group (P<0.05). Compared with NC group, TNF-α, IL-6, H2O2 and O2- levels were decreased in ET group. The ET group has low expression of p22phox protein and high expression of IRAK-M protein (P<0.05). Compared with the ET-control group, the expression of IRAK-M in the ET-si-IRAK-M group was significantly decreased while the expression of p22phox was significantly increased after interference IRAK-M(P<0.05). Compared with the si-IRAK-M-placebo group, the levels of TNF-α, IL-6 and H2O2, O2-in the si-IRAK-M-apocynin group were significantly decreased after interfering with IRAK-M and inhibiting p22phox(P<0.05). Conclusions IRAK-M exerts a negative regulatory effect on endotoxin-induced respiratory outbursts of macrophages in mice by inhibiting p22phox. Key words: Interleukin-1 receptor-associated kinase M; p22phox; Immunosuppression; Macrophage; Respiratory burst; Oxidative stress; Sepsis