Therapeutic effect of Schistosoma japonicum cystatin on bacterial sepsis in mice

Abstract

Background: Sepsis is a life-threatening complication of an infection and remains one of the leading causes of mortality in surgical patients. Bacteremia induces excessive inflammatory responses that result in multiple organ damage. Chronic helminth infection and helminth-derived materials have been found to immunomodulate host immune system to reduce inflammation against some allergic or inflammatory diseases. Schistosoma japonicum cystatin (Sj-Cys) is a cysteine protease inhibitor that induces regulatory T-cells and a potential immunomodulatory. The effect of Sj-Cys on reducing sepsis inflammation and mortality was investigated.

Methods: Sepsis was induced in BALB/c mice using cecal ligation and puncture (CLP), followed by intraperitoneal injection of different doses (10, 25 or 50 μg) of recombinant Sj-Cys (rSj-Cys). The therapeutic effect of rSj-Cys on sepsis was evaluated by observing the survival rates of mice for 96 h after CLP and the pathological injury of liver, kidney and lung by measuring the levels of alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN) and creatinine (Cr) in sera and the tissue sections pathology, and the expression of MyD88 in liver, kidney and lung tissues. The immunological mechanism was investigated by examining pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) and IL-10 and TGF-β1 in mice sera and in culture of macrophages stimulated by lipopolysaccharides (LPS).

Results: rSj-Cys treatment provided significant therapeutic effects on CLP-induced sepsis in mice demonstrated with increased survival rates, alleviated overall disease severity and tissue injury of liver, kidney and lung. The rSj-Cys conferred therapeutic efficacy was associated with upregualted IL-10 and TGF-β1 cytokines and reduced pro-inflammatory cytokines TNF-α, IL-6, IL-1β. MyD88 expression in liver, kidney and lung tissues of rSj-Cys-treated mice was reduced. In vitro assay with macrophages also showed that rSj-Cys inhibited the release of pro-inflammatory cytokines and mediator nitric oxide (NO) after being stimulated by lipopolysaccharide (LPS).

Conclusions: The results suggest the anti-inflammatory potential of rSj-Cys as a promising therapeutic agent on sepsis. The immunological mechanism underlying its therapeutic effect may involve the downregulation of pro-inflammatory cytokines and upregulation of IL-10 and TGF-β1 cytokines possibly via downregulation of the TLR adaptor-transducer MyD88 pathway. The findings suggest rSj-Cys is a potential therapeutic agent for sepsis and other inflammatory diseases.

Keywords: Cecal ligation and puncture; Cystatin; Immunomodulation; Schistosoma japonicum; Sepsis.

Fig. 1

SDS-PAGE of purified rSj-Cys. Total 2 μg of purified rSj-Cys was separated by 12% polyacrylamide gel electrophoresis

Fig. 2

rSj-Cys reduces the releasing of pro-inflammatory cytokines and nitrous oxide from murine peritoneal exudate cells stimulated with LPS. The adherent peritoneal exudate cells were cultured and stimulated with LPS in the presence or absence of rSj-Cys. The levels of TNF-α, IL-6, IL-1β and NO were measured in culture supernatants collected after 24 h incubation. The results are shown as the mean ± SEM for each group. *P < 0.05, ***P < 0.001

Fig. 3

rSj-Cys treatment reduced mortality of mice with sepsis induced by CLP. After CLP surgery, mice were injected intraperitoneally with different doses of rSj-Cys. Mice with sham surgery and treated with PBS were used as control. The survival rate was determined using Kaplan-Meier method and compared by log-rank test (n = 10 mice per group). ***P < 0.001

Fig. 4

rSj-Cys reduced the inflammatory cytokines (TNF-α, IL-6, IL-1β) and induced IL-10 and TGF-β1 releasing in mice with CLP-induced sepsis. The levels of these cytokines in sera of mice were measured by ELISA 12 h after the surgery. The results are shown as the mean ± SEM for each group (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

rSj-Cys reduced liver injury caused by CLP-induced sepsis. a The serum levels of ALT and AST were reduced in mice treated with rSj-Cys. b Representative liver sections showing reduced hepatocytes swelling and inflammatory cell infiltration in rSj-Cys treated mice (×200; Scale-bars: 100 μm) (red arrow: hepatocellular necrosis; black arrow: inflammatory cell) and the improved liver injury score in rSj-Cys treated mice (c). The results are shown as the mean ± SEM for each group (n = 6). *P < 0.05, ***P < 0.001

Fig. 6

rSj-Cys reduced kidney injury caused by CLP-induced sepsis. a The serum levels of BUN and Cr were reduced in mice treated with rSj-Cys. b Representative kidney sections showing reduced renal tissue disrupture and inflammatory cell infiltration in rSj-Cys treated mice (×200; Scale-bars: 100 μm) (arrows indicate shrunk glomerulus) and the improved kidney injury score (c). The results are shown as the mean ± SEM for each group (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

rSj-Cys reduced lung injury caused by CLP-induced sepsis. a Representative lung tissue sections showing reduced alveolar structural disruption and reduced inflammatory cell infiltration in rSj-Cys treated mice (×200; Scale-bars: 100 μm) (arrows indicate interalveolar septum thickened). b The improved lung injury score based on the tissue injury. The results are shown as the means ± SEM for each group (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 8

rSj-Cys suppressed the expression of MyD88 in liver (a), kidney (b) and lung (c) of mice with CLP-induced sepsis detected by western blot. The β-actin was detected as control. The density ratio of MyD88/β-actin is shown on the right. The results are shown as the density mean ± SEM for each group (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001