A Praziquantel Treatment Study of Immune and Transcriptome Profiles in Schistosoma haematobium-Infected Gabonese Schoolchildren.

BACKGROUND: Although Schistosoma haematobium infection has been reported to be associated with alterations in immune function, in particular immune hyporesponsiveness, there have been only few studies that have used the approach of removing infection by drug treatment to establish this and to understand the underlying molecular mechanisms. METHODS: Schistosoma haematobium-infected schoolchildren were studied before and after praziquantel treatment and compared with uninfected controls. Cellular responses were characterized by cytokine production and flow cytometry, and in a subset of children RNA sequencing (RNA-Seq) transcriptome profiling was performed. RESULTS: Removal of S haematobium infection resulted in increased schistosome-specific cytokine responses that were negatively associated with CD4+CD25+FOXP3+ T-cells and accompanied by increased frequency of effector memory T-cells. Innate responses to Toll like receptor (TLR) ligation decreased with treatment and showed positive association with CD4+CD25+FOXP3+ T-cells. At the transcriptome level, schistosome infection was associated with enrichment in cell adhesion, whereas parasite removal was associated with a more quiescent profile. Further analysis indicated that alteration in cellular energy metabolism was associated with S haematobium infection and that the early growth response genes 2 and 3 (EGR 2 and EGR3), transcription factors that negatively regulate T-cell activation, may play a role in adaptive immune hyporesponsiveness. CONCLUSIONS: Using a longitudinal study design, we found contrasting effects of schistosome infection on innate and adaptive immune responses. Whereas the innate immune system appears more activated, the adaptive immunity is in a hyporesponsive state reflected in alterations in CD4+CD25+FOXP3+ T-cells, cellular metabolism, and transcription factors involved in anergy

Enhanced Pro-Inflammatory Cytokine Responses following Toll-Like-Receptor Ligation in Schistosoma haematobium-Infected Schoolchildren from Rural Gabon

BACKGROUND: Schistosoma infection is thought to lead to down-regulation of the host's immune response. This has been shown for adaptive immune responses, but the effect on innate immunity, that initiates and shapes the adaptive response, has not been extensively studied. In a first study to characterize these responses, we investigated the effect of Schistosoma haematobium infection on cytokine responses of Gabonese schoolchildren to a number of Toll-like receptor (TLR) ligands. METHODOLOGY: Peripheral blood mononuclear cells (PBMCs) were collected from S. haematobium-infected and uninfected schoolchildren from the rural area of Zile in Gabon. PBMCs were incubated for 24 h and 72 h with various TLR ligands, as well as schistosomal egg antigen (SEA) and adult worm antigen (AWA). Pro-inflammatory TNF-alpha and anti-inflammatory/regulatory IL-10 cytokine concentrations were determined in culture supernatants. PRINCIPAL FINDINGS: Infected children produced higher adaptive IL-10 responses than uninfected children against schistosomal antigens (72 h incubation). On the other hand, infected children had higher TNF-alpha responses than uninfected children and significantly higher TNF-alpha to IL-10 ratios in response to FSL-1 and Pam3, ligands of TLR2/6 and TLR2/1 respectively. A similar trend was observed for the TLR4 ligand LPS while Poly(I:C) (Mda5/TLR3 ligand) did not induce substantial cytokine responses (24 h incubation). CONCLUSIONS: This pilot study shows that Schistosoma-infected children develop a more pro-inflammatory TLR2-mediated response in the face of a more anti-inflammatory adaptive immune response. This suggests that S. haematobium infection does not suppress the host's innate immune system in the context of single TLR ligation

Cytokine Responses to Schistosoma mansoni and Schistosoma haematobium in Relation to Infection in a Co-endemic Focus in Northern Senegal

Background In Africa, many areas are co-endemic for the two major Schistosoma species, S. mansoni and S. haematobium. Epidemiological studies have suggested that host immunological factors may play an important role in co-endemic areas. As yet, little is known about differences in host immune responses and possible immunological interactions between S. mansoni and S. haematobium in humans. The aim of this study was to analyze host cytokine responses to antigens from either species in a population from a co-endemic focus, and relate these to S. mansoni and S. haematobium infection. Methodology Whole blood cytokine responses were investigated in a population in the north of Senegal (n = 200). Blood was stimulated for 72 h with schistosomal egg and adult worm antigens of either Schistosoma species. IL-10, IL-5, IFN-γ, TNF-α, and IL-2 production was determined in culture supernatants. A multivariate (i.e. multi-response) approach was used to allow a joint analysis of all cytokines in relation to Schistosoma infection. Principal Findings Schistosoma haematobium egg and worm antigens induced higher cytokine production, suggesting that S. haematobium may be more immunogenic than S. mansoni. However, both infections were strongly associated with similar, modified Th2 cytokine profiles. Conclusions/Significance This study is the first to compare S. mansoni and S. haematobium cytokine responses in one population residing in a co-endemic area. These findings are in line with previous epidemiological studies that also suggested S. haematobium egg and worm stages to be more immunogenic than those of S. mansoni.status: publishe

Cytokine Responses to <i>Schistosoma mansoni</i> and <i>Schistosoma haematobium</i> in Relation to Infection in a Co-endemic Focus in Northern Senegal

<div><p>Background</p><p>In Africa, many areas are co-endemic for the two major <i>Schistosoma</i> species, <i>S. mansoni</i> and <i>S. haematobium</i>. Epidemiological studies have suggested that host immunological factors may play an important role in co-endemic areas. As yet, little is known about differences in host immune responses and possible immunological interactions between <i>S. mansoni</i> and <i>S. haematobium</i> in humans. The aim of this study was to analyze host cytokine responses to antigens from either species in a population from a co-endemic focus, and relate these to <i>S. mansoni</i> and <i>S. haematobium</i> infection.</p><p>Methodology</p><p>Whole blood cytokine responses were investigated in a population in the north of Senegal (n = 200). Blood was stimulated for 72 h with schistosomal egg and adult worm antigens of either <i>Schistosoma</i> species. IL-10, IL-5, IFN-γ, TNF-α, and IL-2 production was determined in culture supernatants. A multivariate (i.e. multi-response) approach was used to allow a joint analysis of all cytokines in relation to <i>Schistosoma</i> infection.</p><p>Principal Findings</p><p><i>Schistosoma haematobium</i> egg and worm antigens induced higher cytokine production, suggesting that <i>S. haematobium</i> may be more immunogenic than <i>S. mansoni</i>. However, both infections were strongly associated with similar, modified Th2 cytokine profiles.</p><p>Conclusions/Significance</p><p>This study is the first to compare <i>S. mansoni</i> and <i>S. haematobium</i> cytokine responses in one population residing in a co-endemic area. These findings are in line with previous epidemiological studies that also suggested <i>S. haematobium</i> egg and worm stages to be more immunogenic than those of <i>S. mansoni</i>.</p></div

Levels of <i>Schistosoma</i>-induced cytokine responses in 72 h whole blood cultures (n = 200).

<p>Blood samples from one individual were divided into five and stimulated with <i>Schistosoma</i> antigens (SEAm, SEAh, AWAm, or AWAh), and with medium only (negative control; see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003080#s2" target="_blank">Materials and Methods</a>).</p>a<p>Crude cytokine levels are reported. IQR: Interquartile range (Tukey's hinges).</p>b<p>Wilcoxon Signed Rank test comparing <i>S. mansoni</i>- and <i>S. haematobium</i>-induced cytokine levels within individuals (either for SEA or AWA).</p>c<p><i>Schistosoma</i> egg antigen.</p>d<p>Adult worm antigen.</p

Variation in <i>Schistosoma</i> antigen-induced cytokine responses in relation to <i>Schistosoma</i> infection intensity.

<p>Each three-dimensional (3D) nMDS ordination is represented in two 2D planes (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003080#pntd.0003080.s001" target="_blank">Supporting Information S1</a>). Left and right panels represent the 1^st and 2^nd, and 2^nd and 3^rd dimensions, respectively. <b>Panels A</b> and <b>B</b> show the <i>S. mansoni</i> egg antigen (SEAm)-induced cytokine profile, <b>Panels C</b> and <b>D</b> that of <i>S. haematobium</i> SEA(h), <b>Panels E</b> and <b>F</b> that of <i>S. mansoni</i> adult worm antigens (AWAm), and <b>Panels G</b> and <b>H</b> show <i>S. haematobium</i> AWA(h)-induced cytokine profiles. Green dots represent individuals. Distances between dots approximate the rank order of dissimilarities in cytokine profiles between the respective individuals with stress values (i.e. discrepancies) of 0.051 for SEAm, 0.041 for SEAh, 0.058 for AWAm, and 0.061 for AWAh. Red arrows indicate linear gradients of normalized net cytokine responses on which the nMDS is based. Green dot sizes are proportional to individual values of normalized infection intensity of <i>S. mansoni</i> (for simplicity dots were only labelled with <i>S. mansoni</i> (not <i>S. haematobium</i>) infection intensity). Black arrows indicate linear gradients of post hoc fitted normalized infection intensity of <i>S. mansoni</i> (‘Sm’) and <i>S. haematobium</i> (‘Sh’). The length of the arrows is proportional to the goodness of fit onto the cytokine profile within one 2D plane, but lengths cannot be compared between cytokine and infection intensity arrows. Arrows are only depicted if their fit was significant at the level of <i>p</i> = 0.05 in 3D ordinations (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003080#pntd-0003080-t004" target="_blank">Table 4</a>), as well as in the respective 2D planes. In Panel H, the arrows of IL-5 response and <i>S. mansoni</i> infection intensity are overlapping and their labels are therefore illegible. ^aThe biological a posteriori interpretation of nMDS1 (left x-axis) and nMDS2 (y-axis) were added between brackets on the axis labels, but nMDS3 (right x-axis) could not be interpreted.</p

Distribution of <i>Schistosoma</i> infection in the study population.

<p>Distribution of <i>Schistosoma</i> infection in the study population.</p

Association between <i>Schistosoma</i> infection and <i>Schistosoma</i> antigen-induced cytokine profiles.

<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003080#pntd-0003080-g001" target="_blank">Figure 1</a> shows the fit of infection intensity and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003080#pntd-0003080-g002" target="_blank">Figure 2</a> that of infection status (uninfected, single <i>S. mansoni</i>, single <i>S. haematobium</i>, versus mixed infections) onto each of the four <i>Schistosoma</i> antigen-induced cytokine profiles (either SEAm, SEAh, AWAm or AWAh), obtained by the ‘metaMDS’ and ‘envfit’ functions (see also <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003080#pntd.0003080.s001" target="_blank">Supporting Information S1</a>) <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003080#pntd.0003080-R1" target="_blank">[12]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003080#pntd.0003080-Oksanen1" target="_blank">[13]</a>. Here, the goodness of these fits, i.e. squared correlation coefficients (R²), are shown. The statistical significance was assessed using permutation tests (n = 999), and presented <i>p</i>-values are approximations.</p

Cytokine production in response to schistosomal products in PBMC cultures.

<p>PBMCs were stimulated with schistosomal egg antigen (SEA) or adult worm antigen (AWA). White and grey boxes correspond to <i>S. haematobium</i>-free and infected children respectively with the whiskers indicating minimal and maximal concentrations. * <i>p</i><0.05; ** <i>p</i><0.01. <b>Panel A</b>: Cytokine production did not differ between groups at 24 h (mainly innate response) but the adaptive IL-10 response after 72 h of incubation to both schistosomal products was significantly higher in infected children than in <i>S. haematobium</i>-free children. Furthermore, only infected children produced a significant innate TNF-α response to schistosomal products at 24 h. These plots are not adjusted for spontaneous cytokine production. <b>Panel B</b>: After 24 h (predominantly innate), most cytokine ratios induced by schistosomal products were more anti-inflammatory than the ratios induced by medium alone. When the adaptive response had developed after 72 h however, only infected children produced significant anti-inflammatory cytokine balances while pro-inflammatory indices were lower in infected children than in uninfected children (AWA; <i>p</i><0.05).</p

Cytokine production in response to TLR ligands in PBMC cultures.

<p>White and grey boxes correspond to <i>S. haematobium</i>-free and infected children respectively with the whiskers indicating minimal and maximal concentrations. * <i>p</i><0.05; ** <i>p</i><0.01. <b>Panel A</b>: At 24 h, the ‘innate’ time-point, infected children produced significantly more TNF-α in response to the TLR2/1 ligand Pam3CSK4 (Pam3) compared with uninfected children. TLR-mediated IL-10 production did not significantly deviate between infection groups. When innate cytokine responses faded at 72 h, similar trends were observed. These plots were not adjusted for spontaneous cytokine production. <b>Panel B</b>: At both time points, pro-inflammatory indices (i.e. cytokine ratio induced by one of the stimuli relative to the spontaneously produced ratio) induced by the TLR2 ligands, Pam3 and FSL-1, were significantly higher in infected <i>versus</i> uninfected children.</p