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. 2023 Dec 8;16(1):450.
doi: 10.1186/s13071-023-06021-7.

Therapeutic effect of Echinococcus granulosus cyst fluid on bacterial sepsis in mice

Shuying Wang #  1   2   3 , Donghui Jiang #  4 , Feifei Huang  1   2 , Yayun Qian  1   2 , Meitao Qi  3 , Huihui Li  2 , Xiaoli Wang  2 , Zhi Wang  2 , Kaigui Wang  2 , Yin Wang  4 , Pengfei Du  4 , Bin Zhan  5 , Rui Zhou  1 , Liang Chu  6   7 , Xiaodi Yang  8
Affiliations

Therapeutic effect of Echinococcus granulosus cyst fluid on bacterial sepsis in mice

Shuying Wang et al. Parasit Vectors. .

Abstract

Background: The primary pathophysiological process of sepsis is to stimulate a massive release of inflammatory mediators to trigger systemic inflammatory response syndrome (SIRS), the major cause of multi-organ dysfunction and death. Like other helminths, Echinococcus granulosus induces host immunomodulation. We sought to determine whether E. granulosus cyst fluid (EgCF) displays a therapeutic effect on sepsis-induced inflammation and tissue damage in a mouse model.

Methods: The anti-inflammatory effects of EgCF were determined by in vitro culture with bone marrow-derived macrophages (BMDMs) and in vivo treatment of BALB/C mice with cecal ligation and puncture (CLP)-induced sepsis. The macrophage phenotypes were determined by flow cytometry, and the levels of cytokines in cell supernatants or in sera of mice were measured (ELISA). The therapeutic effect of EgCF on sepsis was evaluated by observing the survival rates of mice for 72 h after CLP, and the pathological injury to the liver, kidney, and lung was measured under a microscope. The expression of TLR-2/MyD88 in tissues was measured by western blot to determine whether TLR-2/MyD88 is involved in the sepsis-induced inflammatory signaling pathway.

Results: In vitro culture with BMDMs showed that EgCF promoted macrophage polarization to M2 type and inhibited lipopolysaccharide (LPS)-induced M1 macrophages. EgCF treatment provided significant therapeutic effects on CLP-induced sepsis in mice, with increased survival rates and alleviation of tissue injury. The EgCF conferred therapeutic efficacy was associated with upregulated anti-inflammatory cytokines (IL-10 and TGF-β) and reduced pro-inflammatory cytokines (TNF-α and INF-γ). Treatment with EgCF induced Arg-1-expressed M2, and inhibited iNOS-expressed M1 macrophages. The expression of TLR-2 and MyD88 in EgCF-treated mice was reduced.

Conclusions: The results demonstrated that EgCF confers a therapeutic effect on sepsis by inhibiting the production of pro-inflammatory cytokines and inducing regulatory cytokines. The anti-inflammatory effect of EgCF is carried out possibly through inducing macrophage polarization from pro-inflammatory M1 to regulatory M2 phenotype to reduce excessive inflammation of sepsis and subsequent multi-organ damage. The role of EgCF in regulating macrophage polarization may be achieved by inhibiting the TLR2/MyD88 signaling pathway.

Keywords: Cyst fluid; Echinococcus granulosus; Immunomodulation; Macrophage; Sepsis.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
SDS-PAGE analysis of EgCF. Total 40 μg of EgCF was separated by 12% polyacrylamide gel electrophoresis and stained with Coomassie blue
Fig. 2
Fig. 2
EgCF stimulated BMDM differentiation to M2 macrophages. a The mature BMDMs were obtained by incubating mouse bone marrow cells (adherent) with M-CSF for 7 days, defined as CD11b+F4/80+ subpopulations using FACS. b Gating strategy on flow cytometry to differentiate dead cells and adhere cells and BMDMs labeled with CD11b+F4/80+. c The isotype-matched immunoglobulins were used as a control for non-specific staining as a baseline. d, e EgCF enhanced M2 macrophages under LPS-stimulating conditions. BMDMs were incubated with EgCF (1 ug/ml), LPS (100 ng/ml), IL-4 (20 ng/ml), EgCF + LPS, or DMEM, respectively, for 24 h. The M1 (CD86) and M2 (CD206) markers were detected using FACS. The results were shown as the mean ± SEM for each group (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 3
Fig. 3
EgCF stimulated M2 type macrophage with regulatory cytokine secretions and inhabited LPS-induced M1 type cytokines. Mature BMDMs were incubated with EgCF (1 ug/ml), LPS (100 ng/ml), IL-4 (20 ng/ml), or EgCF + LPS for 24 h. a The levels of M1-related cytokines TNF-α and IFN-γ were measured in the supernatant by ELISA. b The levels of M2-related cytokines IL-10 and TGF-β were measured in the supernatant by ELISA. n = 6. The results are presented as mean ± SEM, *P < 0.05, ***P < 0.001
Fig. 4
Fig. 4
EgCF treatment improved the survival rate of mice with CLP-induced sepsis. After CLP or sham operation, mice were injected intraperitoneally with EgCF, DXM, or PBS. The survival rate was determined using Kaplan Meier method and compared by log-rank test. n = 10. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 5
Fig. 5
EgCF reduced liver, kidney, and lung injury caused by CLP-induced sepsis. a The histopathology results of liver, kidney, and lung stained with H&E. The tissue was obtained from mice 12 h after CLP and treated with EgCF, DXM, or PBS (arrows indicate inflammatory cell, shrunk glomerulus, and thickened interalveolar septum). b The pathological score comparison among different treated groups in liver, kidney, and lung tissue. The magnification × 200, scale bar = 100 µm. n = 6/group. The results are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 6
Fig. 6
The levels of ALT, AST, BUN, and Cr were reduced in the sera of mice treated with EgCF. The levels of ALT, AST, BUN, and Cr were measured in the sera from mice 12 h after CLP receiving different intervention. n = 6/group. The results are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
Fig. 7
Fig. 7
EgCF reduced the inflammatory cytokines (TNF-α, IFN-γ) (a) and induced regulatory cytokines IL-10 and TGF-β (b) in sera of mice with CLP-induced sepsis. The cytokine levels were measured by ELISA. n = 6/group. The results are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 8
Fig. 8
Treatment with EgCF inhibited iNOS mRNA expression and stimulated Arg-1 mRNA expression in the liver (a), kidney (b), and lung tissues (c) measured by quantitative PCR compared with housekeeper GAPDH. n = 6/group. The results are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 9
Fig. 9
EgCF suppressed the expression of TLR-2 and MyD88 in the liver (a), kidney (b), and lung (c) of mice with CLP-induced sepsis detected by western blot with specific antibody. The β-actin was used as a control. The density ratio of TLR-2/β-actin and MyD88/β-actin are shown on the right. The results are shown as the density mean ± SEM for each group. n = 3/group. The results are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001
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