Echinococcus granulosus cyst fluid suppresses inflammatory responses by inhibiting TRAF6 signalling in macrophages

Abstract

Echinococcus granulosus sensu lato has complex defence mechanisms that protect it from the anti-parasitic immune response for long periods. Echinococcus granulosus cyst fluid (EgCF) is involved in the immune escape. Nevertheless, whether and how EgCF modulates the inflammatory response in macrophages remains poorly understood. Here, real-time polymerase chain reaction and enzyme-linked immunosorbent assay revealed that EgCF could markedly attenuate the lipopolysaccharide (LPS)-induced production of pro-inflammatory factors including tumour necrosis factor-α, interleukin (IL)-12 and IL-6 but increase the expression of IL-10 at mRNA and protein levels in mouse peritoneal macrophages and RAW 264.7 cells. Mechanically, western blotting and immunofluorescence assay showed that EgCF abolished the activation of nuclear factor (NF)-κB p65, p38 mitogen-activated protein kinase (MAPK) and ERK1/2 signalling pathways by LPS stimulation in mouse macrophages. EgCF's anti-inflammatory role was at least partly contributed by promoting proteasomal degradation of the critical adaptor TRAF6. Moreover, the EgCF-promoted anti-inflammatory response and TRAF6 proteasomal degradation were conserved in human THP-1 macrophages. These findings collectively reveal a novel mechanism by which EgCF suppresses inflammatory responses by inhibiting TRAF6 and the downstream activation of NF-κB and MAPK signalling in both human and mouse macrophages, providing new insights into the molecular mechanisms underlying the E. granulosus-induced immune evasion.

Keywords: Cystic echinococcosis; EgCF; TRAF6; inflammation; macrophages.

Graphical abstract

Fig. 1.

EgCF inhibits LPS-induced inflammatory response in mouse peritoneal macrophages. Mouse peritoneal macrophages were treated with EgCF (2.15 mg mL⁻¹) and LPS (1 μg mL⁻¹) for the indicated time points. The mRNA expression levels of TNF-α (A), IL-12 (B), IL-6 (C) and IL-10 (D) were analysed by real-time PCR. Mouse peritoneal macrophages were incubated with LPS in the presence or absence of EgCF for 24 h. Secretion of TNF-α (E), IL-6 (F) and IL-10 (G) in the cell culture supernatants was measured by ELISA.

Fig. 2.

EgCF suppresses LPS-induced inflammatory response in RAW 264.7 cells. RAW 264.7 cells were treated with EgCF (2.15 mg mL⁻¹) and LPS (1 μg mL⁻¹) for the periods indicated. The mRNA expression levels of IL-6 (A), TNF-α (B) and IL-10 (C) were analysed by real-time PCR. RAW 264.7 were incubated with LPS in the presence or absence of EgCF for 24 h. Secretion of IL-6 (D) and IL-10 (E) in cell culture supernatants was measured by ELISA.

Fig. 3.

EgCF inhibits LPS-induced NF-κB and MAPK signalling activation in mouse macrophages. RAW 264.7 cells (A) and mouse peritoneal macrophages (B) were treated with EgCF and LPS for the indicated time points. Activation status of NF-κB p65, p38 MAPK and ERK1/2 signalling was detected via western blotting. PM, peritoneal macrophages. (C) Mouse peritoneal macrophages were incubated with LPS in the presence or absence of EgCF for 4 h. Cells were subjected to immunofluorescence assay for NF-κB p65 nuclear translocation, and the rates of peritoneal macrophages with nuclear p65 staining were quantified.

Fig. 4.

EgCF promotes the proteasomal degradation of TRAF6 in mouse macrophages. RAW 264.7 cells (A) and mouse peritoneal macrophages (B) were treated with EgCF and LPS for the periods indicated. TRAF6 protein levels were evaluated by western blotting. PM, peritoneal macrophages. (C) The protein expression of TRAF6 was checked by western blotting in RAW 264.7 cells treated with EgCF and LPS in the presence of CHX (50 μg mL⁻¹) for 2 or 4 h. (D) RAW 264.7 cells were treated with EgCF and LPS in the presence of MG132 (20 μm) to block the proteasomal degradation for 4 h. Western blotting was used to analyse TRAF6 protein levels.

Fig. 5.

EgCF inhibits LPS-induced inflammatory response by promoting proteasomal degradation of TRAF6 in human macrophages. Relative mRNA expression levels of IL-6 (A) and TNF-α (B) in THP-1 cells treated with EgCF and LPS for the indicated time points were measured by real-time PCR. (C) The protein levels TRAF6 and the activation status of NF-κB p65, p38 MAPK and ERK1/2 signalling in THP-1 cells stimulated with LPS and EgCF for the periods indicated were checked by western blotting. (D) THP-1 cells were treated with EgCF and LPS in the presence of CHX (50 μg mL⁻¹) for 2 or 4 h. Western blotting was used to analyse TRAF6 protein expression. (E) The protein levels of TRAF6 were evaluated by western blotting in THP-1 cells treated with EgCF and LPS in the presence of MG132 (20 μm) to block the proteasomal degradation for 4 h.

Fig. 6.

Working model for EgCF suppressing LPS-induced inflammatory response in macrophages. EgCF promotes the proteasomal degradation of TRAF6 and subsequently impairs the LPS-induced activation of NF-κB and MAPKs signalling pathways and thus the inflammatory response.