Multicellular Parasites Research Articles

Modulation of neutrophil NETosis: interplay between infectious agents and underlying host physiology.

The ability of neutrophils and other leucocyte members of the innate immune system to expel their DNA into the extracellular environment in a controlled manner in order to trap and kill pathogenic microorganisms lead to a paradigm shift in our understanding of host microbe interactions. Surprisingly, the neutrophil extracellular trap (NET) cast by neutrophils is very wide and extends to the entrapment of viruses as well as multicellular eukaryotic parasites. Not unexpectedly, it has emerged that pathogenic microorganisms can employ a wide array of strategies to avoid ensnarement, including expression of DNAse enzymes that destroy the lattice backbone of NETs. Alternatively, they may use molecular mimicry to avoid detection or trigger events leading to the expression of immune modulatory cytokines such as IL-10, which dampen the NETotic response of neutrophils. In addition, the host microenvironment may contribute to the innate immune response by the production of lectin-like molecules that bind to bacteria and promote their entrapment on NETs. An example of this is the production of surfactant protein D by the lung epithelium. In addition, pregnancy provides a different challenge, as the mother needs to mount an effective response against pathogens, without harming her unborn child. An examination of these decoy and host response mechanisms may open the path for new therapies to treat pathologies mediated by overt NETosis.

Modulation of Signal Strength Switches Notch from an Inducer of T Cells to an Inducer of ILC2

Innate lymphoid cells (ILCs) are emerging key players of the immune system with close lineage relationship to T cells. ILC2 play an important role in protective immunity against multicellular parasites, but are also involved in the pathogenesis of type 2 immune diseases. Here, we have studied the developmental requirements for human ILC2. We report that ILC2 are present in the thymus of young human donors, possibly reflecting local differentiation. Furthermore, we show that uncommitted lineage−CD34+CD1a−human thymic progenitors have the capacity to develop into ILC2 in vitro under the influence of Notch signaling, either by stimulation with the Notch ligand Delta like 1 (Dll1) or by expression of the active intracellular domain of NOTCH1 (NICD1). The capacity of NICD1 to mobilize the ILC2 differentiation program was sufficiently potent to override commitment to the T cell lineage in CD34+CD1a+ progenitors and force them into the ILC2 lineage. As Notch is an important factor also for T cell development, these results raise the question how one and the same signaling pathway can elicit such distinct developmental outcomes from the same precursors. We provide evidence that Notch signal strength is a critical determinant in this decision: by tuning signal amplitude, Notch can be converted from a T cell inducer (low signal strength) to an ILC2 inducer (high signal strength). Thus, this study enhances our understanding of human ILC2 development and identifies a mechanism determining specificity of Notch signal output during T cell and ILC2 differentiation.

Hookworm virulence factors: making the most of the host

Hookworm disease from Necator americanus and Ancylostoma duodenale affects approximately 700 million people, with N. americanus being the predominant species. Unlike other pathogens (e.g., bacterial infections), where “virulence” is described in regards to acute pathogenesis and case-fatality, hookworms are well-evolved, multicellular parasites that establish long-term infections in their human hosts with a subtle and chronic, but insidious, pathogenesis, usually in the form of iron deficiency anemia from parasite blood feeding that, over time, has devastating effects on the human host especially when it involves children or women of child bearing years. As such, many of the typical terms for “virulence factors” used in other reviews in this special edition cannot be applied to hookworm (e.g., “colonization”, “invasion”, “or “toxicity”); rather the virulence of hookworm infection comes in terms of their ability to maintain a chronic blood-feeding infection in the lumen of relatively healthy human hosts, an infection that is usually measured in years but can sometimes be measured in decades. In the current manuscript, we describe the routes of invasion hookworms take into their human hosts and the means by which they modulate the human immune system to maintain this long-term parasitism. Little data on hookworm infection comes from actual human infections; instead, much of the data is derived from observations of laboratory animal models, in which hookworms fail to establish this distinctive “chronic infection,” either due to physiological or immunological responses of these animal models. Hence, the mode and effects of chronic immunity must be extrapolated from this very different sort of infection to humans. Herein, we aim to synthesize immunological information from both types of models in the context of immune regulation and protection in order to identify future research focuses for the development of new treatment alternatives (i.e. drugs and vaccines).

Chemosensory behaviors of parasites

Many multicellular parasites seek out hosts by following trails of host-emitted chemicals. Host seeking is a characteristic of endoparasites such as parasitic worms as well as of ectoparasites such as mosquitoes and ticks. For host location, many of these parasites use CO(2), a respiration byproduct, in combination with host-specific chemicals. Recent work has begun to elucidate the behavioral responses of parasites to CO(2) and other host chemicals, and to unravel the mechanisms of these responses. Here we discuss recent findings that have greatly advanced our understanding of the chemosensory behaviors of host-seeking parasites. We focus primarily on well-studied parasites such as nematodes and insects, but also note broadly relevant findings in a few less well studied parasites.

The world within: living with our microbial guests and guides

and throughoutour lives, humans serve as reservoirs forextremely complex and dynamic communitiesof microorganisms of varying origins. These microbesinclude archaea, bacteria, viruses, bacteriophage, andeukaryotes (uni- and multicellular parasites). Althoughconstituting a potential threat to health and well-beingthrough parasitism, these microbial communities haveco-evolved over millions of years with the healthyhuman host to provide a range of beneficial, and oftenessential, services. Although still rudimentary, ourunderstanding of the beneficial roles played by thehuman microbiome has grown appreciably in recentyears,as high-throughput,culture-independenttechnol-ogyandrelatedmoleculartechnologieshavebeenadap-ted to complement the role of traditional microbiologiccultures to facilitate the study of the human ecosystem.Most studies identify individuals within complex popu-lations by focusing on molecular characterization ofDNA from bacterial 16S ribosomal genes, a sequencethat distinguishes each organism by its phylum, genus,and even species, depending on the length of the se-quence. By studying communities in their native habi-tats, rather than in liquid broth or on Petri plates, wehave gained significant insight into the dynamic, multi-factorial interactions that occur among host, pathogen,commensal community, and environment. Undernormal, healthy circumstances, these interactions occuracross both the integument and mucosal surfaces(eg, airways, intestinal, reproductive tracts, mouth),the surfaces of the human body exposed to the environ-ment that are the primary sites of microbial residence.As thoughtfully reviewed in this issue of TranslationalResearch, the authors consider the microbial ecologyof the intestine,

Host resistance to a gastrointestinal parasite regulated by a SNP modulating SMAD3 binding to a TGFβ-regulated transcriptional enhancer of Mina, encoding an immunoregulatory member of the Jmjc protein family (70.9)

Abstract In a hunt for Th2-bias regulatory loci in mice, we have used interval-specific congenic mapping to positionally clone a gene called Mina, encoding an immunoregulatory member of the JmjC protein family. Th2-bias is a genetic trait defined as the propensity of naïve T helper cells to develop in vitro under neutral conditions into T helper 2 (Th2) effector cells. Th2 cells play a critical role in host protection from multicellular parasites such as intestinal helminths and when disregulated contribute to allergic diseases such as asthma. The Th2-bias trait is known to vary across different inbred mouse strains and correlates inversely with the relative abundance of intracellular Mina protein. Adding further support for a causal link between Mina protein level and Th2-bias, Mina was shown to act as a dose-dependent transcriptional corepressor of the gene encoding interleukin-4 (IL4), a key regulator of Th2 development. We have now identified a SNP that explains differential Mina expression level in high and low Th2-biased strains such as BALB/c and C57BL/6. Most interestingly, this SNP acts by modulating SMAD3 binding to a TGFβ-regulated intronic Mina enhancer. We are currently tying this into its physiological role in pulmonary inflammation and intestinal helminth infection using Mina conditional KO mice, which exhibit dramatic phenotypes in both models.

Exploiting structural biology in the fight against parasitic diseases

Despite spectacular advances in structural biology over the past half-century, only approximately 2% of the structures in the Protein Data Bank are from eukaryotic parasites and less than 0.5% are from multicellular parasites. Even when only major human pathogens are considered, 3D structures of parasites are vastly underrepresented. Yet approximately one-third of the global burden of human disease comes from parasites. It is time to divert greater effort and resources in structural biology to benefit the fight against parasitic diseases. Using as leverage recent technological and methodological advances, a concerted effort to determine macromolecular structures from parasite pathogens would provide invaluable mechanistic insights on vital processes of the parasites and would suggest novel strategies for inhibiting infection.

Redox-Active Antiparasitic Drugs

Parasitic diseases affect hundreds of millions of people worldwide and represent major health problems. Treatment is becoming extremely difficult due to the emergence of drug resistance, the absence of effective vaccines, and the spread of insecticide-resistant vectors. Thus, identification of affordable and readily available drugs against resistant parasites is of global demand. Susceptibility of many parasites to oxidative stress is a well-known phenomenon. Therefore, generation of reactive oxygen species (ROS) or inhibition of endogenous antioxidant enzymes would be a novel therapeutic approach to develop antiparasitic drugs. This article highlights the unique metabolic pathways along with redox enzymes of unicellular (Plasmodium falciparum, Trypanosoma cruzi, Trypanosoma brucei, Leishmania donovani, Entamoeba histolytica, and Trichomonas vaginalis) and multicellular parasites (Schistosoma mansoni), which could be utilized to promote ROS-mediated toxicity. Enzymes involved in various vital redox reactions could be potential targets for drug development. The identification of redox-active antiparasitic drugs along with their mode of action will help researchers around the world in designing novel drugs in the future.

An essential role for TH2-type responses in limiting acute tissue damage during experimental helminth infection

Helminths induce potent Th2-type immune responses that can mediate worm expulsion but the importance of this response in controlling acute tissue damage caused by migrating multi-cellular parasites through vital tissues remains uncertain. We used a helminth infection model where parasitic nematode larvae migrate transiently through the lung causing damage resulting in hemorrhage and inflammation. Our findings showed initial elevations in IL-17 contributed to inflammation and lung damage while subsequent IL-4R signaling controlled IL-17 elevations, enhanced expression of insulin-like growth factor 1 and IL-10 and stimulated development of M2 cells, each of which contributed to rapid resolution of tissue damage. These studies indicate an essential role for the Th2-type immune response in mediating acute wound healing during helminth infection.

TLR signaling in mast cells: common and unique features

In addition to the well known role of mast cells in immunity to multi-cellular parasites and in the pathogenesis of allergy and asthma, the importance of mast cells in the immune defense against bacteria and viruses is increasingly being recognized. Their location in the skin, gut, and airways puts mast cells in an ideal location to encounter and respond to pathogens, and in order to perform this function, these cells express a variety of pattern recognition receptors, including Toll-like receptors (TLRs). Mast cells respond to TLR ligands by secreting cytokines, chemokines, and lipid mediators, and some studies have found that TLR ligands can also cause degranulation, although this finding is contentious. In addition, stimulation via TLR ligands can synergize with signaling via the FcεRI, potentially enhancing the response of the cells to antigen in vivo. A great deal is now known about TLR signaling pathways. Some features of these pathways are cell type-specific, however, and work is under way to fully elucidate the TLR signaling cascades in the mast cell. Already, some interesting differences have been identified. This review aims to address what is known about the responses of mast cells to TLR ligands and the signaling pathways involved. Given the location of mast cells at sites exposed to the environment, the response of these cells to TLR ligands must be carefully regulated. The known mechanisms behind this regulation are also reviewed here.

Hematopoietic Stem Cell Gene Therapy as a Treatment for Autoimmune Diseases

A key function of the immune system is to protect us from foreign pathogens such as viruses, bacteria, fungi and multicellular parasites. However, it is also important in many other aspects of human health such as cancer surveillance, tissue transplantation, allergy and autoimmune disease. Autoimmunity can be defined as a chronic immune response that targets self-antigens leading to tissue pathology and clinical disease. Autoimmune diseases, as a group of diseases that include type 1 diabetes, multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus, have no effective cures, and treatment is often based on long-term broad-spectrum immunosuppressive regimes. While a number of strategies aimed at providing disease specific treatments are being explored, one avenue of study involves the use of hematopoietic stem cells to promote tolerance. In this manuscript, we will review the literature in this area but in particular examine the relatively new experimental field of gene therapy and hematopoietic stem cell transplantation as a molecular therapeutic strategy to combat autoimmune disease.

Immunotherapy of allergic diseases by bacterial products

Allergic diseases are characterized by excessive immunoglobulin E (IgE) production, mast cell degranulation, tissue eosinophilia and mucus hypersecretion. These responses also take place during infection with multicellular parasites, and are linked to the production of a characteristic set of cytokines by T helper (Th)2 cells. By contrast, many other microbial infections are associated with cytokine production by Th1 or Th17 cells. There have been extensive efforts to subvert the Th2 responses of allergic diseases by treatment with microbial products.

Identification of Schistosoma mansoni microRNAs

BackgroundMicroRNAs (miRNAs) constitute a class of single-stranded RNAs which play a crucial role in regulating development and controlling gene expression by targeting mRNAs and triggering either translation repression or messenger RNA (mRNA) degradation. miRNAs are widespread in eukaryotes and to date over 14,000 miRNAs have been identified by computational and experimental approaches. Several miRNAs are highly conserved across species. In Schistosoma, the full set of miRNAs and their expression patterns during development remain poorly understood. Here we report on the development and implementation of a homology-based detection strategy to search for miRNA genes in Schistosoma mansoni. In addition, we report results on the experimental detection of miRNAs by means of cDNA cloning and sequencing of size-fractionated RNA samples.ResultsHomology search using the high-throughput pipeline was performed with all known miRNAs in miRBase. A total of 6,211 mature miRNAs were used as reference sequences and 110 unique S. mansoni sequences were returned by BLASTn analysis. The existing mature miRNAs that produced these hits are reported, as well as the locations of the homologous sequences in the S. mansoni genome. All BLAST hits aligned with at least 95% of the miRNA sequence, resulting in alignment lengths of 19-24 nt. Following several filtering steps, 15 potential miRNA candidates were identified using this approach. By sequencing small RNA cDNA libraries from adult worm pairs, we identified 211 novel miRNA candidates in the S. mansoni genome. Northern blot analysis was used to detect the expression of the 30 most frequent sequenced miRNAs and to compare the expression level of these miRNAs between the lung stage schistosomula and adult worm stages. Expression of 11 novel miRNAs was confirmed by northern blot analysis and some presented a stage-regulated expression pattern. Three miRNAs previously identified from S. japonicum were also present in S. mansoni.ConclusionEvidence for the presence of miRNAs in S. mansoni is presented. The number of miRNAs detected by homology-based computational methods in S. mansoni is limited due to the lack of close relatives in the miRNA repository. In spite of this, the computational approach described here can likely be applied to the identification of pre-miRNA hairpins in other organisms. Construction and analysis of a small RNA library led to the experimental identification of 14 novel miRNAs from S. mansoni through a combination of molecular cloning, DNA sequencing and expression studies. Our results significantly expand the set of known miRNAs in multicellular parasites and provide a basis for understanding the structural and functional evolution of miRNAs in these metazoan parasites.

RNAi silencing of calcium-regulated heat-stable protein of 24 kDa in Schistosoma japonicum affects parasite growth

The calcium-regulated heat-stable protein of 24 kDa (CRHSP-24) is a major calcineurin phosphoprotein that functions in multiple signal transduction pathways in cell metabolism. Schistosomes are multicellular parasites that infect 200 million people worldwide, even though treatment has been available for two decades. To determine the function of schistosome CRHSP-24 (SjCRHSP-24), we successfully knocked down SjCRHSP-24 in Schistosoma japonicum by RNA interference (RNAi). By establishing controls for measuring off-target RNAi effects, we found that different double-stranded (dsRNA) sequences had different levels of effectiveness. While all tested dsRNAs reduced CRHSP-24 transcript levels, the S2 dsRNA consistently reduced CRHSP-24 expression to >95% of the control. Knockdown of the SjCRHSP-24 gene significantly affected the morphology and vitality of S. japonicum.

Integrated Cytokine and Metabolic Analysis of Pathological Responses to Parasite Exposure in Rodents

Parasitic infections cause a myriad of responses in their mammalian hosts, on immune as well as on metabolic level. A multiplex panel of cytokines and metabolites derived from four parasite-rodent models, namely, Plasmodium berghei−mouse, Trypanosoma brucei brucei−mouse, Schistosoma mansoni−mouse, and Fasciola hepatica−rat were statistically coanalyzed. 1H NMR spectroscopy and multivariate statistical analysis were used to characterize the urine and plasma metabolite profiles in infected and noninfected animals. Each parasite generated a unique metabolic signature in the host. Plasma cytokine concentrations were obtained using the ‘Meso Scale Discovery’ multi cytokine assay platform. Multivariate data integration methods were subsequently used to elucidate the component of the metabolic signature which is associated with inflammation and to determine specific metabolic correlates with parasite-induced changes in plasma cytokine levels. For example, the relative levels of acetyl glycoproteins extracted from the plasma metabolite profile in the P. berghei-infected mice were statistically correlated with IFN-γ, whereas the same cytokine was anticorrelated with glucose levels. Both the metabolic and the cytokine data showed a similar spatial distribution in principal component analysis scores plots constructed for the combined murine data, with samples from all infected animals clustering according to the parasite species and whereby the protozoan infections (P. berghei and T. b. brucei) grouped separately from the helminth infection (S. mansoni). For S. mansoni, the main infection-responsive cytokines were IL-4 and IL-5, which covaried with lactate, choline, and d-3-hydroxybutyrate. This study demonstrates that the inherently differential immune response to single- and multicellular parasites not only manifests in the cytokine expression, but also consequently imprints on the metabolic signature, and calls for in-depth analysis to further explore direct links between immune features and biochemical pathways.

Ryanodine receptor calcium channels and their partners as drug targets

Ryanodine receptors (RyRs) are high conductance intracellular cation channels that release calcium ions from stores such as the endoplasmic reticulum and sarcoplasmic reticulum. Although RyRs are expressed in many cell types, their roles have only been extensively characterised in tissues in which they are abundant: RyR1 is essential for excitation–contraction coupling in skeletal muscle; whereas RyR2 is required for the analogous signal transduction pathway in heart. Defects in RyR1 cause malignant hyperthermia and a spectrum of myopathies in skeletal muscle; whereas RyR2 dysregulation can result in fatal cardiac arrhythmias and is involved in heart failure. Altered RyR gating has been implicated in a range of other diseases, including epilepsy, neurodegeneration, pain and cancer. RyRs interact with a range of toxic substances, providing insights into their functional and structural properties. Consequently, these channel complexes represent potential therapeutic targets for treatment of numerous diseases. Furthermore, strategies for combating multicellular parasites and agricultural pests could exploit pharmacological differences between their RyRs and those of vertebrates. However, available pharmacological tools for manipulation of RyR gating are generally unsuitable for clinical, veterinary or agricultural use, owing to their lack of selectivity, inappropriate solubility in the aqueous or lipid environment, or generation of side-effects. The expression, subcellular distribution and gating of RyRs is modified by a wide variety of cellular proteins, some of which are expressed in a developmentally or tissue-restricted manner. This commentary examines the possibility of manipulating the expression and function of such proteins in order develop new drugs acting on RyR channel complexes.

Mass Spectrometric Identification of Aberrantly Glycosylated Human Apolipoprotein C-III Peptides in Urine from Schistosoma mansoni-infected Individuals

Schistosomiasis is a parasitic infection caused by Schistosoma flatworms, prime examples of multicellular parasites that live in the mammalian host for many years. Glycoconjugates derived from the parasite have been shown to play an important role in many aspects of schistosomiasis, and some of them are present in the circulation of the host. The aim of this study was to identify novel glycoconjugates related to schistosomiasis in urine of Schistosoma mansoni-infected individuals using a combination of glycopeptide separation techniques and in-depth mass spectrometric analysis. Surprisingly, we characterized a heterogeneous population of novel aberrantly O-glycosylated peptides derived from the C terminus of human apolipoprotein C-III (apoC-III) in urine of S. mansoni-infected individuals that were not detected in urine of non-infected controls. The glycan composition of these glycopeptides is completely different from what has been described previously for apoC-III. Most importantly, they lack sialylation and display a high degree of fucosylation. This study exemplifies the potential of mass spectrometry for the identification and characterization of O-glycopeptides without prior knowledge of either the glycan or the peptide sequence. Furthermore, our results indicate for the first time that as a result of S. mansoni infection the glycosylation of a host protein is altered.

Histochemistry and functional organization of the dorsal skin of Ancistrus dolichopterus (Siluriformes: Loricariidae)

The structural organization and histo-cytochemical features of dorsal skin of Ancistrus dolichopterus (acari bodo) are the main focus of this work. The epidermis, dermis and subcutis are the principal layers of the skin. The epidermis mainly consists of epithelial and mucous cells. Interspersed between them are lymphocytes, pigment cells, eosinophilic granular cells (EGC), and the taste buds as sensory structures. The high number of EGCs is implicated in general and specific immunological defense from pathogenic bacteria and multicellular parasites. The epithelial cells and mucous cells contain glycoproteins with oxidizable vicinal diols, carboxyl groups and O-sulphate esters and their high secretory activity is correlated with the bottom dwelling habit of this species. A thick stratum laxum contains overlapping osteoderms bearing denticles, and the stratum compactum make the integument thicker to help the fish in negative buoyancy for maneuvering near the bottom and protection. The entire body surface is covered by conical, backwardly directed denticles. These are composed of a dentine cone, surrounding a pulp cavity with the top covered by mineralized cap, and are the true homologues of teeth. These structures provide effective protection from abrasion and enemies. These structural peculiarities and histochemical features indicate additional physiological role of the skin of A. dolichopterus.

Regulation of pathogenesis and immunity in helminth infections

Helminths are multicellular eukaryotic parasites that infect over one quarter of the world’s population. Through coevolution with the human immune system, these organisms have learned to exploit immunoregulatory pathways, resulting in asymptomatic tolerance of infections in many individuals. When infections and the resulting immune responses become dysregulated, however, acute and chronic pathologies often develop. A recent international meeting focused on how these parasites modulate host immunity and how control of parasitic and immunopathological disease might be achieved.

Old and rising stars in the lymphoid liver

Old and rising stars in the lymphoid liver