Differential expression of chemokine and matrix re-modelling genes is associated with contrasting schistosome-induced hepatopathology in murine models

Abstract

The pathological outcomes of schistosomiasis are largely dependent on the molecular and cellular mechanisms of the host immune response. In this study, we investigated the contribution of variations in host gene expression to the contrasting hepatic pathology observed between two inbred mouse strains following Schistosoma japonicum infection. Whole genome microarray analysis was employed in conjunction with histological and immunohistochemical analysis to define and compare the hepatic gene expression profiles and cellular composition associated with the hepatopathology observed in S. japonicum-infected BALB/c and CBA mice. We show that the transcriptional profiles differ significantly between the two mouse strains with high statistical confidence. We identified specific genes correlating with the more severe pathology associated with CBA mice, as well as genes which may confer the milder degree of pathology associated with BALB/c mice. In BALB/c mice, neutrophil genes exhibited striking increases in expression, which coincided with the significantly greater accumulation of neutrophils at granulomatous regions seen in histological sections of hepatic tissue. In contrast, up-regulated expression of the eosinophil chemokine CCL24 in CBA mice paralleled the cellular influx of eosinophils to the hepatic granulomas. Additionally, there was greater down-regulation of genes involved in metabolic processes in CBA mice, reflecting the more pronounced hepatic damage in these mice. Profibrotic genes showed similar levels of expression in both mouse strains, as did genes associated with Th1 and Th2 responses. However, imbalances in expression of matrix metalloproteinases (e.g. MMP12, MMP13) and tissue inhibitors of metalloproteinases (TIMP1) may contribute to the contrasting pathology observed in the two strains. Overall, these results provide a more complete picture of the molecular and cellular mechanisms which govern the pathological outcome of hepatic schistosomiasis. This improved understanding of the immunopathogenesis in the murine model schistosomiasis provides the basis for a better appreciation of the complexities associated with chronic human schistosomiasis.

Figure 1. Parasitological and histological comparisons between BALB/c and CBA mice.

Worm abundance (A) and hepatic egg burden (B) did not differ significantly between strains; egg clustering (C) was significantly greater in CBA mice compared to BALB/c mice at 7 weeks p.i, yet the mean number of eggs per cluster (D) did not differ significantly between strains. Granuloma area (E) was significantly greater in CBA mice compared to BALB/c mice, as was hepatic fibrosis at 7 weeks p.i (F). Statistical significance between strains was determined using 2-Way ANOVA with Bonferroni post hoc tests. ▴ = BALB/c replicates, ▪ = CBA replicates, *** = p<0.001, * = p<0.05, ns = no significant difference. Error bars represent mean with standard error of the mean (SEM).

Figure 2. Histological staining highlights differences in tissue damage between BALB/c and CBA mice.

Granulomatous pathology was more severe in CBA mice despite a similar egg burden (A and B; Haematoxylin and Eosin x40). Collagen deposition (red) was also greater in CBA mice (C and D; Sirius Red x40). All images were derived from mice at 7 weeks p.i. and were taken from murine livers with similar egg burdens. A and C; B and D were taken from sections of the same mouse. Scale bar equals 400 µm.

Figure 3. Comparison of cell infiltration in CBA mice and BALB/c mice.

Hepatic neutrophil infiltration (A) was markedly higher in BALB/c mice compared to CBA mice. Conversely, eosinophil numbers (B) were significantly greater in CBA mice compared to BALB/c mice. Statistical significance between strains was determined using 2-Way ANOVA with Bonferroni post hoc tests. ▴ = BALB/c replicates, ▪ = CBA replicates, *** = p<0.001, ** = p<0.01, * = p<0.05, ns = no significant difference. Error bars represent mean with standard error of the mean (SEM).

Figure 4. Cellular infiltration differs between BALB/c and CBA mice.

Large areas of neutrophil accumulation (Leder stain; cells with pink cytoplasm indicated by arrows) adjacent to eggs were more common in BALB/c mice (A) compared with relatively scarce neutrophil accumulation in the granulomas of CBA mice (B). Conversely, large areas of eosinophil accumulation (Giemsa stain; cells with pink cytoplasms indicated by arrows) were more frequent in the granulomas of CBA mice (D) compared with those in BALB/c mice (C). All images were derived from mice at 7 weeks p.i. and were taken from murine livers with similar egg burdens and represent granulomas containing a similar number of eggs and at a similar stage of development. Scale bar = 50 µm (X400).

Figure 5. Venn diagrams to illustrate the overlap between genes showing significantly altered expression.

A and B are derived from the list of 2,937 genes which were significantly changing over time in BALB/c mice (2-Way ANOVA, p<0.05). A shows the overlapping expression of genes within this 2,937 gene list that were significantly up-regulated at either 4, 7 or 9 weeks p.i. B demonstrates the overlap of expression for genes which were down-regulated at either 4, 7 or 9 weeks p.i. Similarly, C and D correspond to the 2,938 genes showing differential expression over time in CBA mice (2-Way ANOVA, p<0.05), where C represents up-regulated genes and D represents down-regulated genes. The majority of these genes show overlapping expression between 7 and 9 weeks p.i. E and F represent the 687 genes which were differentially expressed between strains (2-Way ANOVA, p<0.05). E depicts the overlapping expression of genes which were more highly expressed in BALB/c mice at either 4, 7 or 9 weeks p.i. F demonstrates the overlapping time-points at which genes transcribed at much higher levels in CBA mice were expressed.

Figure 6. Real-time PCR validates microarray results.

Microarray fold-changes relative to uninfected controls for each strain and time-point are presented above the individual bars. Real-time PCR data are presented in the column graphs as copy number. PCR copy number provides an absolute comparison between all groups; microarray fold-change provides a relative comparison between all groups, as these data are normalised to respective control groups.