Early Induction of Human Regulatory Dermal Antigen Presenting Cells by Skin-Penetrating Schistosoma Mansoni Cercariae

Following initial invasion of Schistosoma mansoni cercariae, schistosomula reside in the skin for several days during which they can interact with the dermal immune system. While murine experiments have indicated that exposure to radiation-attenuated (RA) cercariae can generate protective immunity which is initiated in the skin stage, contrasting non-attenuated cercariae, such data is missing for the human model. Since murine skin does not form a reliable marker for immune responses in human skin, we used human skin explants to study the interaction with non-attenuated and RA cercariae with dermal innate antigen presenting cells (APCs) and the subsequent immunological responses. We exposed human skin explants to cercariae and visualized their invasion in real time (initial 30 min) using novel imaging technologies. Subsequently, we studied dermal immune responses and found an enhanced production of regulatory cytokine interleukin (IL)-10, pro-inflammatory cytokine IL-6 and macrophage inflammatory protein (MIP)-1α within 3 days of exposure. Analysis of dermal dendritic cells (DDCs) for their phenotype revealed an increased expression of immune modulators programmed death ligand (PD-L) 1 and 2, and increased IL-10 production. Ex vivo primed DDCs suppress Th1 polarization of naïve T-cells and increase T-cell IL-10 production, indicating their regulatory potential. These immune responses were absent or decreased after exposure to RA parasites. Using transwells, we show that direct contact between APCs and cercariae is required to induce their regulatory phenotype. To the best of our knowledge this is the first study that attempts to provide insight in the human dermal S. mansoni cercariae invasion and subsequent immune responses comparing non-attenuated with RA parasites. We reveal that cercariae induce a predominantly regulatory immune response whereas RA cercariae fail to achieve this. This initial understanding of the dermal immune suppressive capacity of S. mansoni cercariae in humans provides a first step toward the development of an effective schistosome vaccine

Plasmodium sporozoites induce regulatory macrophages.

Professional antigen-presenting cells (APCs), like macrophages (Mϕs) and dendritic cells (DCs), are central players in the induction of natural and vaccine-induced immunity to malaria, yet very little is known about the interaction of SPZ with human APCs. Intradermal delivery of whole-sporozoite vaccines reduces their effectivity, possibly due to dermal immunoregulatory effects. Therefore, understanding these interactions could prove pivotal to malaria vaccination. We investigated human APC responses to recombinant circumsporozoite protein (recCSP), SPZ and anti-CSP opsonized SPZ both in monocyte derived MoDCs and MoMϕs. Both MoDCs and MoMϕs readily took up recCSP but did not change phenotype or function upon doing so. SPZ are preferentially phagocytosed by MoMϕs instead of DCs and phagocytosis greatly increased after opsonization. Subsequently MoMϕs show increased surface marker expression of activation markers as well as tolerogenic markers such as Programmed Death-Ligand 1 (PD-L1). Additionally they show reduced motility, produce interleukin 10 and suppressed interferon gamma (IFNγ) production by antigen specific CD8+ T cells. Importantly, we investigated phenotypic responses to SPZ in primary dermal APCs isolated from human skin explants, which respond similarly to their monocyte-derived counterparts. These findings are a first step in enhancing our understanding of pre-erythrocytic natural immunity and the pitfalls of intradermal vaccination-induced immunity

Dectin-1/2–induced autocrine PGE₂ signaling licenses dendritic cells to prime Th2 responses

<div><p>The molecular mechanisms through which dendritic cells (DCs) prime T helper 2 (Th2) responses, including those elicited by parasitic helminths, remain incompletely understood. Here, we report that soluble egg antigen (SEA) from <i>Schistosoma mansoni</i>, which is well known to drive potent Th2 responses, triggers DCs to produce prostaglandin E2 (PGE₂), which subsequently—in an autocrine manner—induces OX40 ligand (OX40L) expression to license these DCs to drive Th2 responses. Mechanistically, SEA was found to promote PGE₂ synthesis through Dectin-1 and Dectin-2, and via a downstream signaling cascade involving spleen tyrosine kinase (Syk), extracellular signal-regulated kinase (ERK), cytosolic phospholipase A₂ (cPLA₂), and cyclooxygenase 1 and 2 (COX-1 and COX-2). In addition, this pathway was activated independently of the actions of omega-1 (ω-1), a previously described Th2-priming glycoprotein present in SEA. These findings were supported by in vivo murine data showing that ω-1–independent Th2 priming by SEA was mediated by Dectin-2 and Syk signaling in DCs. Finally, we found that Dectin-2^−/−, and to a lesser extent Dectin-1^−/− mice, displayed impaired Th2 responses and reduced egg-driven granuloma formation following <i>S</i>. <i>mansoni</i> infection, highlighting the physiological importance of this pathway in Th2 polarization during a helminth infection. In summary, we identified a novel pathway in DCs involving Dectin-1/2-Syk-PGE₂-OX40L through which Th2 immune responses are induced.</p></div

SEA stimulates PGE₂ secretion and primes Th2 responses independently of ω-1 in human moDCs.

<p>(A) PGE₂ concentration in supernatants from moDC cultures after stimulation with indicated reagents. Concentrations are determined based on an internal standard. Data represent mean ± SEM of 4 independent experiments. (B) moDCs stimulated with indicated lipids (concentration of 2.5 ng/mL for LXA₄, PGE₂, and PGD₂; 12.5 μg/mL for 5-HETE, 8-HETE, and 11-HETE; 25 μg/mL 9-HODE and 13-HODE) were analyzed for Th2 polarizing potential as described in Materials and methods. The ratio of percentage of IL-4⁺ over percentage of IFN-γ⁺ T cells based on intracellular cytokine staining was calculated relative to the control condition. (C) moDCs were pulsed with indicated stimuli and subsequently cocultured with a CD40L-expressing cell line. Supernatants were collected after 24 h, and IL-12p70 concentration was determined by ELISA. (D) T-cell polarization was determined as in panel B. Top and bottom panels show representative flow cytometry plots of intracellular staining of CD4⁺ T cells for indicated cytokines, and the ratio of IL-4 over IFN-γ ratio of these plots based on 4 experiments. Numbers in plots represent frequencies of cells in indicated quadrants. (E) PGE₂ levels as determined by LC-MS/MS in supernatants of moDCs stimulated with indicated stimuli. Data represent mean ± SEM of 3 independent experiments. (A) Statistical significance of different time points per condition compared to baseline (0 h) time point. “*” and “#”: <i>P</i> < 0.05; “**”: <i>P</i> < 0.01. (A) based on two-way ANOVA test or (B–D) for significantly different with the LPS control (*) or between-test conditions (#) based on paired analysis (paired Student <i>t</i> test). Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005504#pbio.2005504.s009" target="_blank">S1 Data</a>. CD4, cluster of differentiation 4; HETE, Hydroxyeicosatetraenoic acid; HODE, Hydroxyoctadecadienoic acid; IFN-γ, interferon γ; IL-4, interleukin 4; LC-MS/MS, liquid chromatography tandem mass spectrometry; LPS, lipopolysaccharide; LXA₄, lipoxin A₄; moDC, monocyte-derived DC; PGE₂, prostaglandin E2; SEA, soluble egg antigen; Th2, T helper 2.</p

The helminth glycoprotein omega‐1 improves metabolic homeostasis in obese mice through type 2 immunity‐independent inhibition of food intake

Type 2 immunity plays an essential role in the maintenance of metabolic homeostasis and its disruption during obesity promotes meta‐inflammation and insulin resistance. Infection with the helminth parasite Schistosoma mansoni and treatment with its soluble egg antigens (SEA) induce a type 2 immune response in metabolic organs and improve insulin sensitivity and glucose tolerance in obese mice, yet, a causal relationship remains unproven. Here, we investigated the effects and underlying mechanisms of the T2 ribonuclease omega‐1 (ω1), one of the major S mansoni immunomodulatory glycoproteins, on metabolic homeostasis. We show that treatment of obese mice with plant‐produced recombinant ω1, harboring similar glycan motifs as present on the native molecule, decreased body fat mass, and improved systemic insulin sensitivity and glucose tolerance in a time‐ and dose‐dependent manner. This effect was associated with an increase in white adipose tissue (WAT) type 2 T helper cells, eosinophils, and alternatively activated macrophages, without affecting type 2 innate lymphoid cells. In contrast to SEA, the metabolic effects of ω1 were still observed in obese STAT6‐deficient mice with impaired type 2 immunity, indicating that its metabolic effects are independent of the type 2 immune response. Instead, we found that ω1 inhibited food intake, without affecting locomotor activity, WAT thermogenic capacity or whole‐body energy expenditure, an effect also occurring in leptin receptor‐deficient obese and hyperphagic db/db mice. Altogether, we demonstrate that while the helminth glycoprotein ω1 can induce type 2 immunity, it improves whole‐body metabolic homeostasis in obese mice by inhibiting food intake via a STAT6‐independent mechanism

SEA promotes PGE₂ synthesis and drives Th2 polarization via signaling through Dectin-1 and Dectin-2 in human moDCs.

<p>(A) cPLA₂ activity 8 h after stimulation. Zymosan was taken along as a positive control for cPLA₂ activation. (B) Protein expression of COX-1 and COX-2 were assessed by western blot. β-actin was used as housekeeping protein. One of 3 experiments is shown. (C) Following 1 h pre-incubation with specific inhibitors for cPLA₂ (Pyr.) or COX-1 and COX-2 (SC and ind., respectively), moDCs were stimulated for 12 h with LPS plus SEAΔα-1/ω-1, and supernatants were collected for PGE₂ determination by LC-MS/MS. (D) At the indicated time points after stimulation with depicted stimuli, phosphorylation of ERK was determined by flow cytometry. A representative flow cytometry plot of intracellular staining for phospho-ERK is shown on the left. (E) PGE₂ levels were determined as in panel C. U0216 was used as inhibitor of ERK. (F) moDCs were treated 45 min with indicated blocking antibodies or isotype controls after which the cells were incubated with PF-647–labeled SEA. Antigen binding/uptake was analyzed by flow cytometry and plotted as relative differences. A representative flow cytometry plot of SEA uptake is depicted on the left. (G) PGE₂ levels were assessed as in panel C, following pre-incubation with blocking antibodies as described in panel F. (H) Syk and (I, J) ERK phosphorylation were determined as described in panel D following pre-incubation with blocking antibodies as described in panel F or panel J with Syk inhibitor R406. Representative flow cytometry plots of Syk (panel H) and ERK (panel I, J) phosphorylation is shown on the left. (K) PGE₂ levels were assessed as in panel C. (L, M) moDCs were pre-incubated with indicated blocking antibodies, followed by 48 h stimulation with LPS plus SEAΔα-1/ω-1, after which OX40L expression was determined by flow cytometry. Data are based on geometric mean florescence. (M) Cells described in panel L were used for T-cell polarization assay as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005504#pbio.2005504.g002" target="_blank">Fig 2A</a>. Data represent mean ± SEM of 2 (panel D, H, I) or at least 3 independent experiments (panel A, C–G, J–M) and are shown relative to control conditions, which are set to 1 (panel A, C–E, G–M) or 100% (F). “*” and “#”: <i>P</i> < 0.05; “**” and “##”: <i>P</i> < 0.01; “***” and “###”: <i>P</i> < 0.001 for significant differences with the control (*) or between-test condition (#) based on paired analysis (paired Student <i>t</i> test). Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005504#pbio.2005504.s009" target="_blank">S1 Data</a>. COX, cyclooxygenase; cPLA₂, cytosolic phospholipase A₂; DC-SIGN, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin; ERK, extracellular signal-regulated kinase; ind., Indometacin; LC-MS/MS, liquid chromatography tandem mass spectrometry; LPS, lipopolysaccharide; moDC, monocyte-derived DC; MR, mannose receptor; OX40L, OX40 ligand; PGE₂, prostaglandin E₂; Pyr., Pyrrophenone; SC, SC236; SEA, soluble egg antigen; Syk, spleen tyrosine kinase; Th2, T helper 2.</p

Dectin-2 signaling is required for induction of a Th2 response during <i>S</i>. <i>mansoni</i> infection.

<p>WT and Dectin-2^−/− mice were infected with <i>S</i>. <i>mansoni</i>. After 8 wk of infection, cells from spleens (A) or mLNs (B) were restimulated with SEA or anti-CD3/CD28 for 72 h, and cytokine levels were analyzed in supernatants by Luminex or ELISA. Bars represent mean ± SEM of combined data of at least 2 or 3 independent experiments with 5 to 10 mice per group. (C) Granuloma sizes around eggs trapped in the liver of 8-wk–infected mice were assessed in Masson blue–stained liver sections. Data are based on 10 mice per group. Number of worms (D) and liver and intestinal eggs (E) in mice infected with <i>S</i>. <i>mansoni</i> for 8 wk. (F) IL-1β protein levels in livers of mice infected with <i>S</i>. <i>mansoni</i> for 8 wk. **<i>P</i> < 0.01 and ***<i>P</i> < 0.001 for significant differences relative to the control mice based on unpaired analysis (unpaired Student <i>t</i> test). Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005504#pbio.2005504.s009" target="_blank">S1 Data</a>. CD3, cluster of differentiation 3; IL-1β, interleukin 1β; mLN, mesenteric lymph node; SEA, soluble egg antigen; Th2, T helper 2; WT, wild-type.</p

Proposed model of <i>S</i>. <i>mansoni</i>–driven Th2 polarization.

<p><i>S</i>. <i>mansoni</i> egg antigens, but not ω-1, interact with Dectin-1 and Dectin-2 expressed by DCs to promote 2 intracellular pathways in moDCs in an Syk-dependent manner: ERK-cPLA₂-COX and ROS activity that culminate in PGE₂ and PGE₂ isomer synthesis, respectively. Both PGE₂ and its isomers can then bind to EP2 and EP4 in an autocrine loop to trigger OX40L expression, which endows the DCs with the capacity to prime a Th2 response. ω-1, omega-1; COX, cyclooxygenase; cPLA₂, cytosolic phospholipase A₂; DC, dendritic cell; EP2, prostaglandin E₂ receptor 2; ERK, extracellular signal-regulated kinase; moDC, monocyte-derived DC; OX40L, OX40 ligand; PGE₂, prostaglandin E₂; ROS, reactive oxygen species; Syk, spleen tyrosine kinase; Th2, T helper 2.</p

ω-1–independent Th2 polarization by SEA is dependent on PGE₂ synthesis by moDCs.

<p>(A–C) T-cell polarization assay as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005504#pbio.2005504.g001" target="_blank">Fig 1B</a>. (A) Neutralizing anti-PGE₂ antibody was added during stimulation of moDCs with indicated reagents or (B) during DC–T cell coculture. (C) EP2 and EP4 receptor inhibitors (EP2-I and EP4-I) were added during stimulation of moDCs with indicated stimuli. (A–C) Left: representative flow cytometry plots are shown of intracellular staining of CD4⁺ T cells for IL-4 and IFN-γ. Numbers in plots represent frequencies of cells in indicated quadrants. Right: these data were used to calculate the fold change in frequency of IL-4⁺ and IFN-γ⁺ T cells polarized by moDCs stimulated with indicated stimuli relative to the cytokine production by T cells polarized by LPS-stimulated moDCs, for which the values were set to 1. Bars represent mean ± SEM of at least 4 independent experiments. Significance was calculated based on the ratio of IL-4 over IFN-γ between conditions. *<i>P</i> < 0.05 and **<i>P</i> < 0.01 for significantly different from control conditions based on paired analysis (paired Student <i>t</i> test). Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005504#pbio.2005504.s009" target="_blank">S1 Data</a>. ω-1, omega-1; CD4, cluster of differentiation 4; EP2, prostaglandin E₂ receptor 2; IL-4, interleukin 4; IFNγ, interferon γ; LPS, lipopolysaccharide; moDC, monocyte-derived DC; PGE₂, prostaglandin E₂; SEA, soluble egg antigen; Th2, T helper 2.</p