Safety and infectivity of female cercariae in Schistosoma-naïve, healthy participants: a controlled human Schistosoma mansoni infection study.

BACKGROUND: A controlled human infection model for schistosomiasis (CHI-S) can speed up vaccine development and provides insight into early immune responses following schistosome exposure. Recently, we established CHI-S model using single-sex male-only Schistosoma mansoni (Sm) cercariae in Schistosoma-naïve individuals. Given important differences in antigenic profile and human immune responses to schistosomes of different sex, we pioneered a single-sex female-only CHI-S model for future use in vaccine development. METHODS: We exposed 13 healthy, Schistosoma-naïve adult participants to 10 (n = 3) or 20 (n = 10) female cercariae and followed for 20 weeks, receiving treatment with praziquantel (PZQ) 60 mg/kg at week 8 and 12 after exposure. FINDINGS: The majority (11/13) participants reported rash and/or itch at the site of exposure, 5/13 had transient symptoms of acute schistosomiasis. Exposure to 20 cercariae led to detectable infection, defined as serum circulating anodic antigen levels >1.0 pg/mL, in 6/10 participants. Despite two rounds of PZQ treatment, 4/13 participants showed signs of persistent infection. Additional one- or three-day PZQ treatment (1 × 60 mg/kg and 3 × 60 mg/kg) or artemether did not result in cure, but over time three participants self-cured. Antibody, cellular, and cytokine responses peaked at week 4 post infection, with a mixed Th1, Th2, and regulatory profile. Cellular responses were (most) discriminative for symptoms. INTERPRETATION: Female-only infections exhibit similar clinical and immunological profiles as male-only infections but are more resistant to PZQ treatment. This limits future use of this model and may have important implications for disease control programs. FUNDING: European Union's Horizon 2020 (grant no. 81564).

Inflammatory and tolerogenic myeloid cells determine outcome following human allergen challenge.

Innate mononuclear phagocytic system (MPS) cells preserve mucosal immune homeostasis. We investigated their role at nasal mucosa following allergen challenge with house dust mite. We combined single-cell proteome and transcriptome profiling on nasal immune cells from nasal biopsies cells from 30 allergic rhinitis and 27 non-allergic subjects before and after repeated nasal allergen challenge. Biopsies of patients showed infiltrating inflammatory HLA-DRhi/CD14+ and CD16+ monocytes and proallergic transcriptional changes in resident CD1C+/CD1A+ conventional dendritic cells (cDC)2 following challenge. In contrast, non-allergic individuals displayed distinct innate MPS responses to allergen challenge: predominant infiltration of myeloid-derived suppressor cells (MDSC: HLA-DRlow/CD14+ monocytes) and cDC2 expressing inhibitory/tolerogenic transcripts. These divergent patterns were confirmed in ex vivo stimulated MPS nasal biopsy cells. Thus, we identified not only MPS cell clusters involved in airway allergic inflammation but also highlight novel roles for non-inflammatory innate MPS responses by MDSC to allergens in non-allergic individuals. Future therapies should address MDSC activity as treatment for inflammatory airway diseases.

Use of Toll-Like Receptor (TLR) Ligation to Characterize Human Regulatory B-Cells Subsets.

Toll-like receptors (TLRs) are pattern recognition receptors (PRRs), which constitute key components in the recognition of pathogens, thereby initiating innate immune responses and promoting adaptive immune responses. In B cells, TLR ligation is important for their activation and, together with CD40, for their differentiation. TLR ligands are also strong promoters of regulatory B (Breg)-cell development, by enhancing the production of IL-10 and their capacity to induce tolerance. In inflammatory diseases, such as autoimmunity or allergies, Breg-cell function is often impaired, while in chronic infections, such as with helminths, or cancer, Breg-cell function is boosted. Following pathogen exposure, B cells can respond directly by producing cytokines and/or IgM (innate response) and develop into various memory B (Bmem)-cell subsets with class-switched immunoglobulin receptors. Depending on the disease state or chronic infection conditions, various Breg subsets can be recognized as well. Currently, a large array of surface markers is known to distinguish between these large range of B-cell subsets. In recent years, the development of mass cytometers and spectral flow cytometry has allowed for high-dimensional detection of up to 48 markers, including both surface and intracellular/intranuclear markers. Therefore, this novel technology is highly suitable to provide a comprehensive overview of Bmem/Breg-cell subsets in different disease states and/or in clinical intervention trials. Here, we provide detailed instructions of the steps necessary to obtain high-quality data for high-dimensional analysis of multiple human Breg-cell subsets using various TLR ligands.

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.

Primary prevention of asthma: from risk and protective factors to targeted strategies for prevention.

Asthma is a complex disease that often starts in childhood. Genomic and environmental factors as well as aberrant immune maturation early in life can contribute to the onset of disease, with great disparity over time and geographical regions. Epidemiological studies have scrutinised environmental exposures and attempted to translate these exposures into prevention strategies. Some approaches for patients with asthma have been successful (eg, smoking ban, the Finnish Asthma Programme), and primary prevention of wheeze in pre-school children (age 0-5 years) by the supplementation of vitamin D or fish oil, or both, to pregnant women seems promising. Several recent prevention initiatives are based on strong asthma-protective environmental microbial exposures associated with traditional rural lifestyles. Preclinical studies with various bacterial lysates, bacterial and dietary metabolites, or helminthic compounds have yielded promising results that await translation into clinical practice. Given the immense societal and individual burden of asthma, there is an urgent need to further develop novel strategies to eradicate the disease.

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.

Innate and adaptive nasal mucosal immune responses following experimental human pneumococcal colonization.

Streptococcus pneumoniae (Spn) is a common cause of respiratory infection, but also frequently colonizes the nasopharynx in the absence of disease. We used mass cytometry to study immune cells from nasal biopsy samples collected following experimental human pneumococcal challenge in order to identify immunological mechanisms of control of Spn colonization. Using 37 markers, we characterized 293 nasal immune cell clusters, of which 7 were associated with Spn colonization. B cell and CD8+CD161+ T cell clusters were significantly lower in colonized than in non-colonized subjects. By following a second cohort before and after pneumococcal challenge we observed that B cells were depleted from the nasal mucosa upon Spn colonization. This associated with an expansion of Spn polysaccharide-specific and total plasmablasts in blood. Moreover, increased responses of blood mucosal associated invariant T (MAIT) cells against in vitro stimulation with pneumococcus prior to challenge associated with protection against establishment of Spn colonization and with increased mucosal MAIT cell populations. These results implicate MAIT cells in the protection against pneumococcal colonization and demonstrate that colonization affects mucosal and circulating B cell populations.

Extract of Helicobacter pylori Ameliorates Parameters of Airway Inflammation and Goblet Cell Hyperplasia following Repeated Allergen Exposure.

BACKGROUND: An inverse relation between Helicobacter pylori infection and asthma has been shown in epidemiological studies. Infection with H. pylori, or application of an extract of it before or after sensitization, inhibits allergic airway disease in mice. OBJECTIVES: The aim of this study was to investigate the effect of an extract of H. pylori on allergic airway disease induced by repeated allergen exposure in mice that were sensitized and challenged prior to extract application. METHOD: C57BL/6 mice were intranasally (i.n.) sensitized and challenged with house dust mite (HDM). After a minimum of 4 weeks, mice received the H. pylori extract intraperitoneally and were rechallenged i.n. with HDM. Allergen-specific antibodies were measured by ELISA. Cells present in the bronchoalveolar lavage fluid and dendritic cell (DC) subsets in the lung tissue were analyzed by flow cytometry. Tissue inflammation and goblet cell hyperplasia were assessed by histology. Cells of the mediastinal lymph node (mLN) were isolated and in vitro restimulated with HDM or H. pylori extract. RESULTS: Treatment with H. pylori extract before rechallenge reduced allergen-specific IgE, the DC numbers in the tissue, and goblet cell hyperplasia. Cells isolated from mLN of mice treated with the extract produced significantly more IL-10 and IL-17 after in vitro restimulation with HDM. mLN cells of H. pylori-treated mice that were re-exposed to the H. pylori extract produced significantly more interferon gamma. CONCLUSIONS: An extract of H. pylori is effective in reducing mucus production and various features of inflammation in HDM rechallenged mice.

Modulation of Host Immunity by Helminths: The Expanding Repertoire of Parasite Effector Molecules.

Helminths are extraordinarily successful parasites due to their ability to modulate the host immune response. They have evolved a spectrum of immunomodulatory molecules that are now beginning to be defined, heralding a molecular revolution in parasite immunology. These discoveries have the potential both to transform our understanding of parasite adaptation to the host and to develop possible therapies for immune-mediated disease. In this review we will summarize the current state of the art in parasite immunomodulation and discuss perspectives on future areas for research and discovery.

Therapeutic Application of an Extract of Helicobacter pylori Ameliorates the Development of Allergic Airway Disease.

Epidemiological and experimental studies have shown that exposure to the gastric bacterium Helicobacter pylori, especially in early life, prevents the development of asthma. Recent mouse studies have shown that this protective effect does not require live bacteria and that treatment with an extract of H. pylori in neonates prevents the development of airway inflammation and goblet cell metaplasia. In the current study, the effect of administration of an extract of H. pylori was assessed in a therapeutic study design with application of the extract just prior to allergen challenge. C57BL/6 mice were sensitized and challenged with OVA or house dust mite. Treatment with H. pylori extract just prior to the challenge significantly reduced airway inflammation, as assessed in bronchoalveolar lavage fluid and lung tissue, and reduced airway remodeling, as assessed by goblet cell quantification. These effects were apparent in the OVA model and in the house dust mite model. Injection of H. pylori extract reduced the processing of allergen by dendritic cells in the lungs and mediastinal lymph node. Bone marrow-derived dendritic cells exposed to H. pylori extract were affected with regard to their ability to process Ag. These data show that application of H. pylori extract after sensitization effectively inhibits allergic airway disease.

Schistosome Egg Migration: Mechanisms, Pathogenesis and Host Immune Responses.

Many parasitic worms possess complex and intriguing life cycles, and schistosomes are no exception. To exit the human body and progress to their successive snail host, Schistosoma mansoni eggs must migrate from the mesenteric vessels, across the intestinal wall and into the feces. This process is complex and not always successful. A vast proportion of eggs fail to leave their definite host, instead becoming lodged within intestinal or hepatic tissue, where they can evoke potentially life-threatening pathology. Thus, to maximize the likelihood of successful egg passage whilst minimizing host pathology, intriguing egg exit strategies have evolved. Notably, schistosomes actively exert counter-inflammatory influences on the host immune system, discreetly compromise endothelial and epithelial barriers, and modulate granuloma formation around transiting eggs, which is instrumental to their migration. In this review, we discuss new developments in our understanding of schistosome egg migration, with an emphasis on S. mansoni and the intestine, and outline the host-parasite interactions that are thought to make this process possible. In addition, we explore the potential immune implications of egg penetration and discuss the long-term consequences for the host of unsuccessful egg transit, such as fibrosis, co-infection and cancer development.

The effect of tiotropium in combination with olodaterol on house dust mite-induced allergic airway disease.

One of the major goals of asthma therapy is to maintain asthma control and prevent acute exacerbations. Long-acting bronchodilators are regularly used for the treatment of asthma patients and in clinical studies the anti-cholinergic tiotropium has recently been shown to reduce exacerbations in patients with asthma. So far it is unclear how tiotropium exerts this effect. For this purpose, we designed an allergen-driven rechallenge model of allergic airway inflammation in mice, to assess the effectiveness of tiotropium and the long-acting ß-2 adrenoceptor agonist olodaterol on allergen-induced exacerbations of airway disease. Female C57BL/6J mice were sensitized intranasally (i.n.) with 1 µg of house dust mite (HDM) extract followed by a challenge regime (5 consecutive days 10 µg HDM extract i.n.) after one week. Mice were exposed to a secondary challenge five weeks after sensitization and were treated i.n. with different concentrations of tiotropium or olodaterol (1, 10 and 100 µg/kg) or a combination thereof (10 µg/kg each) prior to and during the secondary challenge period. Three days after the last challenge, bronchoalveolar lavage (BAL) fluid and lung tissue were collected for flow cytometry and histologic analysis, respectively. Secondary challenge with HDM extract strongly induced allergic airway disease reflected by inflammatory cell infiltration and goblet cell metaplasia. Treatment with tiotropium, but not with olodaterol reduced tissue inflammation and goblet cell metaplasia in a dose-dependent manner. The combination of tiotropium and olodaterol was more effective in significantly reducing tissue inflammation compared to tiotropium treatment alone, and also led to a decrease in BAL cell counts. These data suggest that in a model of relapsing allergic airway disease tiotropium directly prevents exacerbations by reducing inflammation and mucus production in the airways. In addition, the combination of tiotropium and olodaterol exerts synergistic effects.

Schistosome egg antigens, including the glycoprotein IPSE/alpha-1, trigger the development of regulatory B cells.

Infection with the helminth Schistosoma (S.) mansoni drives the development of interleukin (IL)-10-producing regulatory B (Breg) cells in mice and man, which have the capacity to reduce experimental allergic airway inflammation and are thus of high therapeutic interest. However, both the involved antigen and cellular mechanisms that drive Breg cell development remain to be elucidated. Therefore, we investigated whether S. mansoni soluble egg antigens (SEA) directly interact with B cells to enhance their regulatory potential, or act indirectly on B cells via SEA-modulated macrophage subsets. Intraperitoneal injections of S. mansoni eggs or SEA significantly upregulated IL-10 and CD86 expression by marginal zone B cells. Both B cells as well as macrophages of the splenic marginal zone efficiently bound SEA in vivo, but macrophages were dispensable for Breg cell induction as shown by macrophage depletion with clodronate liposomes. SEA was internalized into acidic cell compartments of B cells and induced a 3-fold increase of IL-10, which was dependent on endosomal acidification and was further enhanced by CD40 ligation. IPSE/alpha-1, one of the major antigens in SEA, was also capable of inducing IL-10 in naïve B cells, which was reproduced by tobacco plant-derived recombinant IPSE. Other major schistosomal antigens, omega-1 and kappa-5, had no effect. SEA depleted of IPSE/alpha-1 was still able to induce Breg cells indicating that SEA contains more Breg cell-inducing components. Importantly, SEA- and IPSE-induced Breg cells triggered regulatory T cell development in vitro. SEA and recombinant IPSE/alpha-1 also induced IL-10 production in human CD1d+ B cells. In conclusion, the mechanism of S. mansoni-induced Breg cell development involves a direct targeting of B cells by SEA components such as the secretory glycoprotein IPSE/alpha-1.

Production and glyco-engineering of immunomodulatory helminth glycoproteins in plants.

Helminth parasites control host-immune responses by secreting immunomodulatory glycoproteins. Clinical trials and mouse model studies have demonstrated the potential of helminth-derived glycoproteins for the treatment of immune-related diseases, like allergies and autoimmune diseases. Studies are however hampered by the limited availability of native parasite-derived proteins. Moreover, recombinant protein production systems have thus far been unable to reconstitute helminth-like glycosylation essential for the functionality of some helminth glycoproteins. Here we exploited the flexibility of the N-glycosylation machinery of plants to reconstruct the helminth glycoproteins omega-1 and kappa-5, two major constituents of immunomodulatory Schistosoma mansoni soluble egg antigens. Fine-tuning transient co-expression of specific glycosyltransferases in Nicotiana benthamiana enabled the synthesis of Lewis X (LeX) and LDN/LDN-F glycan motifs as found on natural omega-1 and kappa-5, respectively. In vitro and in vivo evaluation of the introduction of native LeX motifs on plant-produced omega-1 confirmed that LeX on omega-1 contributes to the glycoprotein's Th2-inducing properties. These data indicate that mimicking the complex carbohydrate structures of helminths in plants is a promising strategy to allow targeted evaluation of therapeutic glycoproteins for the treatment of inflammatory disorders. In addition, our results offer perspectives for the development of effective anti-helminthic vaccines by reconstructing native parasite glycoprotein antigens.

LIM-only protein FHL2 regulates experimental pulmonary Schistosoma mansoni egg granuloma formation.

LIM-only protein FHL2 is associated with several immune and inflammatory diseases such as arthritis, influenza A virus infection, and lung inflammation. However, the role of FHL2 in macrophage differentiation and in the development of granuloma formation is unknown. Here, we show that expression of FHL2 is induced in mouse bone marrow derived macrophages (BMMs) following stimulation with M2 cytokines such as IL-4 and IL-10. FHL2-knockout (FHL2-KO) BMMs exhibit a proinflammatory M1 phenotype after LPS treatment and display a reduced anti-inflammatory M2 phenotype following IL-4 treatment. Furthermore, thioglycollate-induced migration of macrophages and B cells is enhanced in FHL2-KO mice. To evaluate the importance of FHL2 in the development of pulmonary granuloma formation, FHL2-KO mice were challenged with Schistosoma mansoni eggs. FHL2-KO mice show an enhanced number of granulomas and display decreased expression of Th2 markers and an exacerbated Th1 type of inflammation, characterized by enhanced expression of neutrophil markers and Th1 cytokines. Furthermore, the expression of barrier proteins is reduced in FHL2-KO lung compared to WT. Collectively, these data identify a previously unrecognized role for FHL2 in the pathogenesis of pulmonary granulomatous inflammation, partly through its effect on macrophage polarization, modulation of the Th1/Th2 balance and regulation of permeability in lung.

CD4+CD25hiFOXP3+ Regulatory T Cells and Cytokine Responses in Human Schistosomiasis before and after Treatment with Praziquantel.

BACKGROUND: Chronic schistosomiasis is associated with T cell hypo-responsiveness and immunoregulatory mechanisms, including induction of regulatory T cells (Tregs). However, little is known about Treg functional capacity during human Schistosoma haematobium infection. METHODOLOGY: CD4+CD25hiFOXP3+ cells were characterized by flow cytometry and their function assessed by analysing total and Treg-depleted PBMC responses to schistosomal adult worm antigen (AWA), soluable egg antigen (SEA) and Bacillus Calmette-Guérin (BCG) in S. haematobium-infected Gabonese children before and 6 weeks after anthelmintic treatment. Cytokines responses (IFN-γ, IL-5, IL-10, IL-13, IL-17 and TNF) were integrated using Principal Component Analysis (PCA). Proliferation was measured by CFSE. PRINCIPAL FINDINGS: S. haematobium infection was associated with increased Treg frequencies, which decreased post-treatment. Cytokine responses clustered into two principal components reflecting regulatory and Th2-polarized (PC1) and pro-inflammatory and Th1-polarized (PC2) cytokine responses; both components increased post-treatment. Treg depletion resulted in increased PC1 and PC2 at both time-points. Proliferation on the other hand, showed no significant difference from pre- to post-treatment. Treg depletion resulted mostly in increased proliferative responses at the pre-treatment time-point only. CONCLUSIONS: Schistosoma-associated CD4+CD25hiFOXP3+Tregs exert a suppressive effect on both proliferation and cytokine production. Although Treg frequency decreases after praziquantel treatment, their suppressive capacity remains unaltered when considering cytokine production whereas their influence on proliferation weakens with treatment.

E-cadherin expression in macrophages dampens their inflammatory responsiveness in vitro, but does not modulate M2-regulated pathologies in vivo.

IL-4/IL-13-induced alternatively activated macrophages (M(IL-4/IL-13), AAMs or M2) are known to express E-cadherin, enabling them to engage in heterotypic cellular interactions and IL-4-driven macrophage fusion in vitro. Here we show that E-cadherin overexpression in Raw 264.7 macrophages inhibits their inflammatory response to LPS stimulation, as demonstrated by a reduced secretion of inflammatory mediators like interleukin (IL)-6, tumor necrosis factor (TNF) and nitric oxide (NO). To study the function of E-cadherin in M(IL-4/IL-13) macrophages in vivo, we generated macrophage-specific E-cadherin-deficient C57BL/6 mice. Using this new tool, we analyzed immunological parameters during two typical AAM-associated Th2-driven diseases and assessed Th2-associated granuloma formation. Although E-cadherin is strongly induced in AAMs during Taenia crassiceps helminth infections and allergic airway inflammation, its deletion in macrophages does not affect the course of both Th2 cytokine-driven diseases. Moreover, macrophage E-cadherin expression is largely redundant for granuloma formation around Schistosoma mansoni ova. Overall, we conclude that E-cadherin is a valuable AAM marker which suppresses the inflammatory response when overexpressed. Yet E-cadherin deletion in macrophages does not affect M(LPS+IFNγ) and M(IL-4) polarization in vitro, nor in vivo macrophage function, at least in the conditions tested.

Microorganism-induced suppression of allergic airway disease: novel therapies on the horizon?

Allergic airway disease is a major global health burden, and novel treatment options are urgently needed. Numerous epidemiological and experimental studies suggest that certain helminths and bacteria protect against respiratory allergies. These microorganisms are strong regulators of the immune system, and various potential regulatory mechanisms by which they protect against allergic airway inflammation have been proposed. Whereas early studies addressed the beneficial effect of natural infections, the focus now shifts toward identifying the dominant protective molecules and exploring their efficacy in models of allergic airway disease. In this article, we will review the evidence for microbe-mediated protection from allergic airway disease, the potential modes of action involved and discuss advances as well as limitations in the translation of this knowledge into novel treatment strategies against allergic airway disease.