Sex-Specific Modulation of the Host Transcriptome in the Spleen of Schistosoma mansoni-Infected Mice

Abstract

Background: Schistosomiasis is a severe parasitic disease that is primarily driven by the host's immune response to schistosome eggs trapped in tissue and by the granulomatous inflammatory and fibrotic reaction they cause. Despite significant progress in understanding the complex immunological processes involved in the relationship between schistosomes and their host, neither an effective vaccine against the infection nor anti-fibrotic drugs currently exists, making the search for new targets for schistosome drugs and vaccine candidates even more important. In order to identify new molecular targets for defense against or elimination of the parasite, we investigate herein the interplay between the host and male or female schistosomes, clearly separating this from the action of the parasite eggs.

Methods: For this purpose, we infected 6-8-week-old female NMRI mice with 100 male (M), female (F), or both (MF) S. mansoni cercariae and performed a comparative transcriptomic and flow cytometric analysis of their spleens.

Results: Principal component analysis of a total of 22,207 transcripts showed a clear clustering of the experimental groups. We identified a total of 1,293 genes in group M, 512 genes in group F, and 4,062 genes in group MF that were differentially expressed compared to naive controls. The highest percentage of regulated genes (2,972; 65.9%) was found in group MF alone, but there was a large overlap between groups M and MF (798; 17.7%) and a small overlap between groups F and MF (91; 2.0%). Only 4.5% of genes (201) were revealed to be regulated in all experimental groups (M/F/MF). In addition, we were able to show that both worm sexes trigger immune responses in an egg-independent manner (non-polarized Th1 and Th2 response), with female worms exerting less regulatory influence than males.

Conclusion: Our data show that adult schistosomes trigger sex-specific, egg-independent immune responses. The lists of genes regulated by adult female or male worms presented here may be useful in deciphering host-parasite interactions to identify targets for schistosome elimination.

Keywords: Schistosoma mansoni; host–parasite interaction, immunomodulation; transcriptome analysis in spleens; unisexual infection.

Figure 1

Unisexual infection with female and male Schistosoma mansoni leads to significant enlargement of the spleen. Quantification of (A) specific weight and (B) total cell counts in the spleens of infected (M, F, and MF) and control mice (naive) over time (4 and 8 wk p.i., n = 6–8). Data are presented as mean ± SEM. p values < 0.05 were considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001; M, infected with male cercariae; F, infected with female cercariae; MF, infected with male and female cercariae.

Figure 2

Transcriptomic analysis of spleens of mice uni- and bisexually infected with Schistosoma mansoni. Transcriptomic analysis of spleens reveals different gene expression profiles after infection with S. mansoni (8 wk p.i.). (A) Principal component analysis, (B) bar chart, and (C) Venn diagram represent an overview of transcriptomic analysis of spleens (n = 2–3). Inclusion of all genes showing altered gene expression compared to controls (fold change >2 or à2, LIMMA p value < 0.05, and FDR q value < 0.05); M, infected with male cercariae; F, infected with female cercariae; MF, infected with male and female cercariae.

Figure 3

Volcano plot and heatmap of differentially expressed genes in spleen tissue of mice unisexually and bisexually infected with Schistosoma mansoni. (A) Volcano plots (upper panel) visualizing differentially expressed genes of infected (unisexual and bisexual) mice compared to healthy controls (fold change >2 or ≤2, LIMMA p value < 0.05“. and „Heat map of highly regulated genes related to the host immune response in splenic tissue of infected (M, F, MF)“ into „Heat map of highly regulated genes related to the host immune response in splenic tissue of infected (M, male cercariae; F, female cercariae; MF, male and female cercariae) and FDR q value <0.05) plotted against the significance level (negative log10 p value) and showing significantly (p < 0.05) increased (green) and decreased (red) genes in the spleen. Highly regulated genes are encircled in black. Gene ontology analysis (lower panel) displaying enrichments of biological processes of up- and downregulated genes after infection with S. mansoni (8 wk p.i.) compared to naive mice. The ten most enriched categories of biological processes associated with the regulated genes are presented. (B) Heat map of highly regulated genes related to the host immune response in splenic tissue of infected (M, F, MF) compared to control (naive) mice (8 wk p.i., n = 2–3). The color scale on the top illustrates the log2 fold change values shown in the heat map. Upregulated genes are highlighted in green whereas downregulated genes are marked red. The baseline data are shown in Supplementary Table 1 .

Figure 4

Validation of microarray data by RT-qPCR of six representative, highly regulated genes. Relative gene expression of tlr5, cd209a, clec4g, ccl24, epb4.2, and cdc20 in spleens of Schistosoma mansoni-infected mice (M, F, MF) was determined by real-time PCR (8 wk p.i., n = 2–3). Real-time PCR data are expressed as fold-change and presented as bar graphs, while the corresponding microarray data are visualized as heat maps below the graphs (upregulation/green, downregulation/red, or unchanged/black). Real-time PCR data are presented as mean ± SEM. p values < 0.05 were considered statistically signi!cant. *p < 0.05; M, infected with male cercariae; F, infected with female cercariae; MF, infected with male and female cercariae.

Figure 5

Infection with male Schistosoma mansoni leads to an increase in splenic CD86⁺ dendritic cells and a decrease in splenic CD4⁺ T cells. (A) Total numbers and (B) percentages of eosinophils, neutrophils, macrophages (MOs), dendritic cells (DCs), B cells, T cells, Th cells, and cytotoxic T cells as well as the proportion of CD86 on dendritic cells were analyzed by flow cytometry in homogenized spleen tissue of infected (M, F, and MF) and control (naive) mice (8 wk p.i., n = 4–8). Data are presented as mean ± SEM. p values < 0.05 were considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; M, infected with male cercariae; F, infected with female cercariae; MF, infected with male and female cercariae.

Figure 6

Unisexual infection with Schistosoma mansoni leads to a non-polarized Th1/Th2 immune response. Splenocytes were isolated from spleens of unisexually (M, F) and bisexually infected (MF) mice as well as healthy controls. Isolated splenocytes were stimulated with 10 μg/ml soluble worm antigen preparation containing eggs (SWAP). Supernatants were collected after 72 h and amounts of INF-γ, IL-13, IL-4, and IL-10 were quantified using ELISA (n = 5–8). Data are presented as mean ± SEM. p values <0.05 were considered statistically significant. *p < 0.05, ** p< 0.01, ***p < 0.001. M, infected with male cercariae; F, infected with female cercariae; MF, infected with male and female cercariae.