An ex vivo model for studying hepatic schistosomiasis and the effect of released protein from dying eggs

Abstract

Background: We report the use of an ex vivo precision cut liver slice (PCLS) mouse model for studying hepatic schistosomiasis. In this system, liver tissue is unfixed, unfrozen, and alive for maintenance in culture and subsequent molecular analysis.

Methods and findings: Using thick naive mouse liver tissue and sterile culture conditions, the addition of soluble egg antigen (SEA) derived from Schistosoma japonicum eggs, followed 4, 24 and 48 hrs time points. Tissue was collected for transcriptional analysis and supernatants collected to quantitate liver enzymes, cytokines and chemokines. No significant hepatotoxicity was demonstrated by supernatant liver enzymes due to the presence of SEA. A proinflammatory response was observed both at the transcriptional level and at the protein level by cytokine and chemokine bead assay. Key genes observed elevated transcription in response to the addition of SEA included: IL1-α and IL1-β, IL6, all associated with inflammation. The recruitment of antigen presenting cells was reflected in increases in transcription of CD40, CCL4 and CSF1. Indications of tissue remodeling were seen in elevated gene expression of various Matrix MetalloProteinases (MMP3, 9, 10, 13) and delayed increases in TIMP1. Collagen deposition was significantly reduced in the presence of SEA as shown in COL1A1 expression by qPCR after 24 hrs culture. Cytokine and chemokine analysis of the culture supernatants confirmed the elevation of proteins including IL6, CCL3, CCL4 and CXCL5.

Conclusions: This ex vivo model system for the synchronised delivery of parasite antigen to liver tissue provides an insight into the early phase of hepatic schistosomiasis, corresponding with the release of soluble proteins from dying schistosome eggs.

Fig 1. Malondialdehyde (MDA) assay of culture media containing liver slice tissue exposed or not to soluble egg antigen (SEA).

While MDA concentrations in media increased over the 48 hour time course, indicating hepatotoxicity, there was no statistical difference between treatment groups at any time point (p-value>0.05).

Fig 2. Correlation of 13,746 genes identified after quality control filtering.

Colouring ranging from r² = 0.6 for black to r² = 1 red. Individual samples are grouped by time point and correlation between biological replicates varied between r² = 0.965–0.985. Correlation between individual time points with pre-cut (whole) tissue resulted in r² = 0.97 for time point 0 (after cutting and 2 hours rest), 0.88 after 4 hours, 0.73 after 24 hours and 0.57 after 48 hours.

Fig 3. Real time validation of a subset of the 13,746 genes.

Genes were identified both as differentially expressed and unchanged due to the presence of soluble egg antigen (SEA) from Schistosoma japonicum. Relative gene expression was normalised to the house keeping gene HPRT. Time points refer to pre-cut intact tissue (PC) and tissue slices in culture before (0) and after (4, 24 and 48 hours) the addition of soluble egg antigen (SEA). P values = *≤0.05, **≤0.01, ****≤0.0001.

Fig 4. Cytokine and chemokine levels were determined in liver section culture supernatants.

Using Legendplex multi-analyte flow assay kits, IL-6, CXCL1, CCL2, CCL3, CCL4, CXCL5 and CCL22 were detected. Levels of significance were calculated using two-way ANOVA with Sidaks multiple comparison test. P values = *≤0.05, **≤0.01, ****≤0.0001, NS = not significant >0.05.