Co-ordinated gene expression in the liver and spleen during Schistosoma japonicum infection regulates cell migration

Abstract

Determining the molecular events induced in the spleen during schistosome infection is an essential step in better understanding the immunopathogenesis of schistosomiasis and the mechanisms by which schistosomes modulate the host immune response. The present study defines the transcriptional and cellular events occurring in the murine spleen during the progression of Schistosoma japonicum infection. Additionally, we compared and contrasted these results with those we have previously reported for the liver. Microarray analysis combined with flow cytometry and histochemistry demonstrated that transcriptional changes occurring in the spleen were closely related to changes in cellular composition. Additionally, the presence of alternatively activated macrophages, as indicated by up-regulation of Chi3l3 and Chi3l4 and expansion of F4/80(+) macrophages, together with enhanced expression of the immunoregulatory genes ANXA1 and CAMP suggests the spleen may be an important site for the control of S. japonicum-induced immune responses. The most striking difference between the transcriptional profiles of the infected liver and spleen was the contrasting expression of chemokines and cell adhesion molecules. Lymphocyte chemokines, including the homeostatic chemokines CXCL13, CCL19 and CCL21, were significantly down-regulated in the spleen but up-regulated in the liver. Eosinophil (CCL11, CCL24), neutrophil (CXCL1) and monocyte (CXCL14, CCL12) chemokines and the cell adhesion molecules VCAM1, NCAM1, PECAM1 were up-regulated in the liver but unchanged in the spleen. Chemokines up-regulated in both organs were expressed at significantly higher levels in the liver. Co-ordinated expression of these genes probably contributes to the development of a chemotactic signalling gradient that promotes recruitment of effector cells to the liver, thereby facilitating the development of hepatic granulomas and fibrosis. Together these data provide, for the first time, a comprehensive overview of the molecular events occurring in the spleen during schistosomiasis and will substantially further our understanding of the local and systemic mechanisms driving the immunopathogenesis of this disease.

Figure 1. Splenic pathology induced by schistosome infection.

Upper panel: Spleen weight increased over time following S. japonicum infection reaching approximately 4.5 times that of controls at 7 weeks p.i. The graph represents mean spleen weight of mice pooled for microarray analysis ±1SD (n = 4 per group). Lower panel: Schistosome induced splenomegaly was associated with increasing congestion of the red pulp and loss of definition between the red (lighter staining) and white pulp (darker staining) over time A: Control; B: 4 weeks p.i.; C: 6 weeks p.i.; D:7 weeks p.i. (Haematoxylin and eosin ×40; Bar equals 100µm.).

Figure 2. Splenomegaly is associated with accumulation of neutrophils, eosinophils and F4/80⁺ macrophages in the red pulp.

There was a significant increases in the number of neutrophils (A–C: Leder stain for neutrophils (pink stain, arrowed) ×200 in uninfected control mice (B) and at 7 weeks p.i. (C)), eosinophils (D–F, Giemsa stain ×400 in uninfected control mice (E) and at 7 weeks p.i. (F)) and F4/80⁺ macrophages (G–I: F4/80 staining in uninfected control mice (H) and at 7 weeks p.i (I). (red-brown) ×40) in the splenic red pulp from as early as 6 weeks p.i. Values represent mean cells/high power field (eosinophils and neutrophils) or percent positive staining (F4/80) ±1SD of mice pooled for microarray analysis (n = 4 per group). Bar = 100µm.

Figure 3. Flow cytometry revealed dynamic changes in lymphocyte populations of the spleen over time.

A: Flow cytometry demonstrated a significant increase in the total number of CD4⁺ and CD8⁺ T-cells and CD19⁺ B-cells in the spleen at 4 weeks p.i. followed by return of these cells to baseline levels by 6 weeks p.i. B: The ratio of T- and B-cells to total live cells decreased significantly over time. Values represent means ±1SD. N = 5 per group except for 6 weeks p.i. where one mouse harboured no adult worms and was excluded from all further analyses.

Figure 4. Hierarchical clustering and prominent gene ontologies of genes differentially expressed in the spleen.

Four distinct clusters representing genes that were significantly down-regulated (cluster 1); up-regulated earlier (cluster 2), consistently up-regulated (cluster 3) and up-regulated later (cluster 4) were identified by hierarchical clustering analysis. Prominent biological processes and molecular functions (gene ontologies) associated with genes 2-fold or greater up- or down-regulated in each of these clusters and genes associated with these ontologies are listed in the boxed text. Data are represented in heat map form where green represents down-regulated gene expression, red represents up-regulated expression, with relatively unchanged expression coloured black.

Figure 5. Contrasting expression of chemokines in the liver and spleen by microarray analysis.

Comparison of spleen and liver transcriptional profiles identified a number of chemokines with contrasting expression. A: Chemokines up-regulated in the liver and down-regulated in the spleen during schistosome infection. B: Chemokines up-regulated in the liver and unchanged or below levels of detection in the spleen. C: Chemokines up-regulated in both organs showed greater expression in the liver. Graphs represent average fold change relative to uninfected tissue by microarray analysis. Dotted lines represent a ±2 fold cut-off for biological significance. Expression values for the liver are derived from our previous study of the transcriptional profile of the S. japonicum infected liver .