Effect of praziquantel on the differential expression of mouse hepatic genes and parasite ATP binding cassette transporter gene family members during <i>Schistosoma mansoni</i> infection

<div><p>Schistosomiasis is a chronic parasitic disease caused by sexually dimorphic blood flukes of the genus <i>Schistosoma</i>. Praziquantel (PZQ) is the only drug widely available to treat the disease but does not kill juvenile parasites. Here we report the use of next generation sequencing to study the transcriptional effect of PZQ on murine hepatic inflammatory, immune and fibrotic responses to <i>Schistosoma mansoni</i> worms and eggs. An initial T helper cell 1 (Th1) response is induced against schistosomes in mice treated with drug vehicle (Vh) around the time egg laying begins, followed by a T helper cell 2 (Th2) response and the induction of genes whose action leads to granuloma formation and fibrosis. When PZQ is administered at this time, there is a significant reduction in egg burden yet the hepatic Th1, Th2 and fibrotic responses are still observed in the absence of granuloma formation suggesting some degree of gene regulation may be induced by antigens released from the dying adult worms. Quantitative real-time PCR was used to examine the relative expression of 16 juvenile and adult <i>S</i>. <i>mansoni</i> genes during infection and their response to Vh and PZQ treatment <i>in vivo</i>. While the response of stress genes in adult parasites suggests the worms were alive immediately following exposure to PZQ, they were unable to induce transcription of any of the 9 genes encoding ATP-binding cassette (ABC) transporters tested. In contrast, juvenile schistosomes were able to significantly induce the activities of ABCB, C and G family members, underscoring the possibility that these efflux systems play a major role in drug resistance.</p></div

The effects of PZQ on infected host liver.

<p>(A) Effect of treatment with Vh and PZQ on the number of parasites present in host livers at days 32, 35 and 46 post infection, n = 5 per treatment group. (B) Number of parasite eggs per gram of liver at days 32, 35, 39 and 46 post infection after treatment with Vh and PZQ (n = 4). (C) Weight of liver at days 32, 35, 39 and 46 post infection after treatment with Vh and PZQ (n = 4). Error bars represent mean with standard deviation. * p <0.05 and *** p < 0.001.</p

Canonical pathway analysis of T helper cell maturation and differentiation in PZQ treated infected mice.

<p>Signaling events in the T cell maturation and differentiation pathway at (A) 32, (B) 35, (C) 39 and (D) 46 days in infected PZQ (Sm_PZQ) treated mice. Increasing expression in infected mice relative to uninfected mice is indicated by deeper blue shading. None of the genes indicated were down-regulated. Non-expression and non-differential expression is indicated by a lack of shading.</p

Canonical pathway analysis of hepatic fibrosis and stellate cell activation in vehicle treated infected mice.

<p>Signaling events in the fibrotic and stellate cell activation pathway at (A) 32, (B) 35, (C) 39 and (D) 46 days post infection in infected Vh treated (Sm_Vh) mice. Increasing expression in infected mice relative to uninfected mice is indicated by deeper blue shading. Decreased expression is shown in dark red. Non-expression and non-differential expression is indicated by a lack of shading.</p

Temporal expression of mouse hepatic genes during infection with <i>S</i>. <i>mansoni</i>.

<p>Heat maps of (A) immune and (B) fibrotic gene markers depicting differentially expressed genes in infected Vh and PZQ treated mice relative to uninfected mice. Gray map sections represent genes not expressed at that time point or in that treatment group. Regions of blue and red indicate, relative to uninfected Vh treated controls, increased and decreased gene expression respectively. The color scale indicates log₂ fold change (FC) and the profile of each group is the average of three biological replicates. Gene names associated with the figure differ slightly from those used in the text. Relevant differences include: Ccl (rather than CCL as it appears in the main body of text), Cxc (CXC), Cxcl (CXCL), Il1b (IL1β), Tnfa (TNFα), Tgfb (TGFβ), Ifng (IFNγ), Cd (CD) and Timp (TIMP).</p

Quantitative real-time PCR analysis of <i>S</i>. <i>mansoni</i> gene expression.

<p>Results are normalized to <i>S</i>. <i>mansoni</i> GAPDH and fold change in gene expression in PZQ treated schistosomes is shown relative to Vh treated groups. (A) Schematic diagram explaining the source of <i>S</i>. <i>mansoni</i> RNA for qRT-PCR analysis. (DPI = days post infection). B-Q. Gene expression in juvenile schistosomes at days 25 and 28 is shown with shaded bars and adults at days 32 and 35 are shown hatched. (B-D) and (F-H) show the response of full ABC drug transporters while (E), and (I, J) are half transporters each with homology to a member of the human B, C and G families. (K-O) show the response of stress genes HSP70, ferritin and thioredoxin peroxidase (TPX-1, 2 & 3), (P) the response of calmodulin and (Q) response of voltage-gated Ca²⁺ channel β subunit respectively. Statistical differences in fold change between adult and juvenile <i>S</i>. <i>mansoni</i> were assessed using one-way ANOVA followed by Fisher’s least significant difference post hoc test for multiple comparisons. Error bars represent +1 standard error of the mean. * p <0.05, ** p <0.01, *** p <0.001.</p

Canonical pathway analysis of hepatic fibrosis and stellate cell activation in PZQ treated infected mice.

<p>Signaling events in the fibrotic and stellate cell activation pathway at (A) 32, (B) 35, (C) 39 and (D) 46 days post infection in infected PZQ (Sm_PZQ) treated mice. Increasing expression in infected mice relative to uninfected mice is indicated by deeper blue shading. Decreased expression is shown in dark red. Non-expression and non-differential expression is indicated by a lack of shading.</p