Drug-Induced Exposure of Schistosoma mansoni Antigens SmCD59a and SmKK7

BACKGROUND: Schistosomiasis is a serious health problem especially in developing countries and affects more than 243 million people. Only few anthelmintic drugs are available up to now. A major obstacle for drug treatment is the different developmental stages and the varying host compartments during worm development. Anthelmintic drugs have been tested mainly on adult schistosomes or freshly transformed cercariae. Knowledge concerning the larval stages is lacking. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we used in vitro-grown schistosomula (aged between 2 to 14 days) to investigate drug effects of the three anthelmintics praziquantel, artemether, and oxamniquine. Further, we analyzed the antibody accessibility of two exemplary schistosome antigens SmCD59a and SmKK7, before and after drug treatment. Our results demonstrated that praziquantel applied at a concentration of 1 μM inhibited development of all life stages. Application of 10 μM praziquantel led to dramatic morphological changes of all schistosomula. Artemether at 1 and 10 μM had differential effects depending on whether it was applied to 2-day as compared to 7- and 14-day schistosomula. While 2-day schistosomula were not killed but inhibited from further development, severe morphological damage was seen in 7- and 14-day schistosomula. Oxamniquine (1 and 10 μM) led to severe morphological impairment in all life stages. Analyzing the accessibility of the antigens SmCD59a and SmKK7 before drug treatment showed no antibody binding on living intact schistosomula. However, when schistosomula were treated with anthelmintics, both antigens became exposed on the larvae. Oxamniquine turned out to be most effective in promoting antibody binding to all schistosomula stages. CONCLUSION: This study has revealed marked differences in anthelmintic drug effects against larvae. Drug treatment increases surface antigen presentation and renders larvae accessible to antibody attack

Schistosoma mansoni Egg-Released IPSE/alpha-1 Dampens Inflammatory Cytokine Responses via Basophil Interleukin (IL)-4 and IL-13

Schistosomes control inflammation in their hosts via highly effective mechanisms such as induction of Tregs, Bregs, and alternatively activated macrophages (AAMs). Notably, IPSE/alpha-1, the major secretory product from Schistosoma mansoni eggs, triggers basophils to release interleukin (IL)-4 and IL-13. Both cytokines are essential for AAM induction, suggesting an important role for IPSE/alpha-1 in inflammation control. Here, we show by in vitro co-culture experiments that IPSE/alpha-1-induced basophil IL-4/IL-13 inhibited pro-inflammatory cytokine release from human LPS-activated monocytes. This effect was cell/cell contact-independent but dependent on IL-4, since it was abrogated in the presence of anti-IL-4 antibodies. Importantly, the IPSE/alpha-1-induced IL-4/IL-13 release from basophils was amplified in the presence of LPS. Moreover, monocytes co-cultured in the presence of LPS with IPSE/alpha-1-stimulated basophils adopted an AAM-like phenotype as assessed by elevated expression of CD206 and CD209. The putative in vivo relevance of these findings was supported by immunohistological staining of S. mansoni-infected murine tissue revealing close physical contact between IPSE/alpha-1 and basophils in schistosome egg granulomas. Taken together, we found that IPSE/alpha-1 dampens inflammatory cytokine responses by triggering basophil IL-4/IL-13, in particular in the context of TLR activation, thereby turning inflammatory monocytes into anti-inflammatory AAMs. This might represent a mechanism used by schistosomes to control inflammation in the host

Development of <i>in vitro</i>-cultured schistosomula.

<p><b><i>S</i>. <i>mansoni</i> larvae were cultured <i>in vitro</i> in IMDM cell culture medium supplied with human PBMC, serum and whole blood.</b> Images were taken at day 2, 7, 14 and 21 after transformation. By day 7 the gut (filled with black hemozoin) had developed; by day 14 the typical divided gut became clearly visible, and by day 21 oral and ventral suckers appeared. PMBC (P), s (schistosomula), g (gut), os (oral sucker), vs (ventral sucker).</p

Differential unmasking of the surface antigen SmCD59a by treatment with PZQ, ART and OXA.

<p>Drugs were added either at day 7 or day 14 and analyzed 2 days after administration of PZQ and ART and 6 days after OXA application. For the analysis of antigen accessibility, schistosomula were incubated with anti-SmCD59a antiserum. Antibody binding was detected with a fluorophore-labeled anti-rat IgG (IF). Corresponding lower images show the bright field images (BF). Scale bars = 100 μm.</p

Percentage of unmasked schistosomula after treatment with different OXA doses.

<p>14-day schistosomula were treated with 10, 1 or 0.1 μM OXA. 6 days after drug application schistosomula were incubated with anti-SmCD59a antiserum. Antibody binding was detected with a fluorophore-labeled anti-rat IgG and positive larvae were counted under an inverted fluorescence microscope. Data from three independent experiments are shown.</p

Differential unmasking of the antigen SmKK7 by treatment with PZQ and OXA.

<p>Drugs were added at day 2 and analyzed 2 days after administration of PZQ and 6 days after OXA application. For the analysis of antigen accessibility, schistosomula were incubated with anti-SmKK7 antiserum. Antibody binding was detected with a fluorophore-labeled anti-rat IgG (IF). Corresponding lower images show the bright field images (BF). Scale bars = 100 μm.</p

3D projection of schistosomula reveals differential localization of SmCD59a and SmKK7.

<p>Formalin-fixed and permeabilized schistosomula were analyzed at day 14 for SmCD59a localization and at day 7 for SmKK7 localization. Schistosomula were incubated with rat anti-SmCD59a antiserum, rat anti-SmKK7 antiserum, a control sample from uninfected rats (NRS) and a control sample from infected rats (RIS). Binding of IgG antibodies were detected with fluorophore-labeled anti-rat IgG. Immunofluorescence was visualized by projection overlay of a confocal z-stack. Corresponding panels show the bright field image for control of larval development.</p

SmCD59a and SmKK7 on living, intact schistosomula are not accessible by anti-SmCD59a and anti-SmKK7 antisera.

<p>Binding of rat anti-SmCD59a antiserum (upper panel), rat anti-SmKK7 antiserum (median panel) and serum from infected rat (RIS; lower panel) to schistosomula at day 2, day 7 and day 14 after transformation. Antibody binding was detected with a fluorophore-labeled anti-rat IgG (IF). Corresponding lower images show the bright field images for control larval development (BF). Anti-SmCD59a and anti-SmKK7 antisera do not recognize the antigens on the surface of the live schistosomula, while the RIS shows strong binding, strongest on day 2. Scale bars = 100 μm.</p

Schistosomicidal effects of PZQ, ART and OXA on 14-day-schistosomula.

<p>Magnification of the <i>in vitro</i>-cultured schistosomula treated with PZQ, ART, and OXA at 10 μM. Drugs were added at day 14 and schistosomula were analyzed at day 21 (late treatment). 0.1% DMSO in medium (solvent of the drugs; used as negative control) had no effect on schistosomula development (representatives out of triplicate wells are shown; see also <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003593#pntd.0003593.g002" target="_blank">Fig. 2</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003593#pntd.0003593.s002" target="_blank">S2</a>–<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003593#pntd.0003593.s005" target="_blank">S5</a> Videos).</p

Immunohistochemical analysis of SmCD59a and SmKK7 expression in schistosomula.

<p>Formalin-fixed and permeabilized schistosomula were analyzed by immunofluorescence microscopy at day 2, 7 and 14 following transformation. Schistosomula were incubated with rat anti-SmCD59a antiserum and rat anti-SmKK7 antiserum, binding of IgG antibodies were detected with fluorophore-labeled anti-rat IgG. The IF marked panels show the SmCD59a and SmKK7 fluorescence signal. Corresponding lower panels show the bright field image for control of the larval development (BF). Scale bars = 100 μm.</p