The Impact of Intestinal Inflammation on Nematode's Excretory-Secretory Proteome.

Parasitic nematodes and their products are promising candidates for therapeutics against inflammatory bowel diseases (IBD). Two species of nematodes, the hookworm Necator americanus and the whipworm Trichuis suis, are being used in clinical treatment trials of IBD referred to as "helminth therapy". Heligmosomoides polygyrus is a well-known model for human hookworm infections. Excretory-secretory (ES) products of H. polygyrus L4 stage that developed during colitis show a different immunomodulatory effect compared to the ES of H. polgyrus from healthy mice. The aim of the study was to evaluate excretory-secretory proteins produced by H. polygyrus L4 stage males and females that developed in the colitic milieu. Mass spectrometry was used to identify proteins. Blast2GO was used to investigate the functions of the discovered proteins. A total of 387 proteins were identified in the ES of H. polygyrus L4 males (HpC males), and 330 proteins were identified in the ES of L4 females that developed in the colitic milieu (HpC females). In contrast, only 200 proteins were identified in the ES of L4 males (Hp males) and 218 in the ES of L4 females (Hp females) that developed in control conditions. Most of the proteins (123) were detected in all groups. Unique proteins identified in the ES of HpC females included annexin, lysozyme-2, apyrase, and galectin. Venom allergen/Ancylostoma-secreted protein-like, transthyretin-like family proteins, and galectins were found in the secretome of HpC males but not in the secretome of control males. These molecules may be responsible for the therapeutic effects of nematodes in DSS-induced colitis.

Nematode-Induced Growth Factors Related to Angiogenesis in Autoimmune Disease Attenuation.

Accumulating data suggest an important role of growth factors in autoimmune diseases and parasitic nematode infections. Nematodes are used in clinical studies of autoimmune diseases and parasite-derived molecules are widely studied for their therapeutic potential in various types of disorders. However, the effect of nematode infection on growth factors in autoimmune disorders has not been studied. The objective of this study was to evaluate the influence of infection with the intestinal nematode Heligmosomoides polygyrus in murine autoimmune models on the production of growth factors. Here, the level of a variety of growth factors related mainly to angiogenesis was evaluated by protein array in the intestinal mucosa of C57BL/6 dextran sodium sulfate-induced colitic mice and in cerebral spinal fluid of experimental autoimmune encephalomyelitis (EAE) mice infected with nematodes. In addition, vessel formation was evaluated in the brains of EAE mice infected with H. polygyrus. A significant influence of nematode infection on the level of angiogenic factors was observed. Parasitic infection of colitic mice resulted in upregulation of mucosal AREG, EGF, FGF-2, and IGFBP-3 in the intestine of the host and better adaptation (infectivity). In EAE mice, infection increased the level of FGF-2 and FGF-7 in CSF. In addition, remodeling of brain vessels was observed, with a higher density of long vessels. Nematode-derived factors are promising tools to fight autoimmune diseases and to study angiogenesis.

Heterogeneity of adherent and non­adherent JAWS II dendritic cells.

Dendritic cells (DCs) are crucial in the development of immune responses. DC JAWS II is a murine cell line frequently used in DC studies. These cells are grown in two cell fractions: Adherent and non-adherent. The present study aimed to compare these two fractions in both immature and lipopolysaccharide (LPS)-activated JAWS II cells. The present study analysed the condition, phenotype, antigen uptake capability, signalling properties and the influence on the activity of T cells using flow cytometry, mixed cell reaction and ELISA methods. Adherent immature JAWS II cells exhibited increased endocytosis and decreased activation of the Pi3K signalling pathway. After LPS activation, adherent JAWS II cells exhibited increased expression levels of CD80 and CD86 costimulatory molecules, increased endocytosis and an elevated ability to induce T cell proliferation, compared with non-adherent cells. These results demonstrated that the two fractions of JAWS II adherent and non-adherent cells exhibited different properties and this should be taken into account in the planning of research.

Importance of TGFß in Cancer and Nematode Infection and Their Interaction-Opinion.

Historically, there has been little interaction between parasitologists and oncologists, although some helminth infections predispose to the development of tumours. In addition, both parasites and tumours need to survive immune attack. Recent research suggests that both tumours and parasites suppress the immune response to increase their chances of survival. They both co-opt the transforming growth factor beta (TGFß) signalling pathway to modulate the immune response to their benefit. In particular, there is concern that suppression of the immune response by nematodes and their products could enhance susceptibility to tumours in both natural and artificial infections.

Nematode galectin binds IgE and modulates mast cell activity.

Mast cell degranulation is the major mechanism influencing establishment and survival of the abomasal nematode Teladorsagia circumcincta and probably many other gastrointestinal nematodes. Host galectins-3 and -9 have been shown to bind IgE and positively and negatively influence mast cell degranulation. As incoming nematodes produce large amounts of galectin, we hypothesised that nematode galectin competes with host galectin and inhibits mast cell degranulation. ELISA was used to show that nematode galectin reduced total IgE activity. Galectin also reduced the binding of sheep IgE to the surface of a mast cell line and decreased the release of LCT-4 and Beta hexosaminidase but not MMP-9. These results indicate that nematode galectin influences mast cell degranulation and identify a potential immunomodulatory mechanism used by nematodes to enhance their establishment and survival.

Reduced Expression of PD-1 in Circulating CD4+ and CD8+ Tregs Is an Early Feature of RRMS.

Altered regulatory T cell (Treg) function could contribute to MS. The expression of activating and inhibitory receptors influences the activity of Tregs. Our aim was to investigate T cell phenotypes in relapsing-remitting MS (RRMS) patients at an early phase of the disease. We examined the influence of demographic parameters on the distribution of CD4+ and CD8+ T cell subclasses by generalized linear modeling. We also studied the expression of the following markers-CTLA-4, GITR, PD-1, FoxP3, Helios, CD28, CD62L, CD103-on T cell subsets from peripheral blood with a 14-color flow cytometry panel. We used an antibody array to define the profiles of 34 Th1/Th2/Th17 cytokines in the serum. Expression of PD-1 and GITR on CD4+ and CD8+ Tregs was decreased in RRMS patients. The proinflammatory factors IFN-γ, IL-17, IL-17F, TGFß-1, TGFß-3, IL-1SRII, IL-12 p40, sgp130, IL-6sR were significantly increased in RRMS patients. Therefore, a deficiency of PD-1 and GITR immune checkpoints on CD4+ and CD8+ Tregs is a feature of RRMS and might underlie impaired T cell control.

Cyclosporine A, in Contrast to Rapamycin, Affects the Ability of Dendritic Cells to Induce Immune Tolerance Mechanisms.

Following organ transplantation, it is essential that immune tolerance is induced in the graft recipient to reduce the risk of rejection and avoid complications associated with the long-term use of immunosuppressive drugs. Immature dendritic cells (DCs) are considered to promote transplant tolerance and may minimize the risk of graft rejection. The aim of the study was to evaluate the effects of immunosuppressive agents: rapamycin (Rapa) and cyclosporine A (CsA) on generation of human tolerogenic DCs (tolDCs) and also to evaluate the ability of these cells to induce mechanisms of immune tolerance. tolDCs were generated in the environment of Rapa or CsA. Next, we evaluated the effects of these agents on surface phenotypes (CD11c, MHC II, CD40, CD80, CD83, CD86, CCR7, TLR2, TLR4), cytokine production (IL-4, IL-6, IL-10, IL-12p70, TGF-ß), phagocytic capacity and resistant to lipopolysaccharide activation of these DCs. Moreover, we assessed ability of such tolDCs to induce T cell activation and apoptosis, Treg differentiation and production of Th1- and Th2-characteristic cytokine profile. Data obtained in this study demonstrate that rapamycin is effective at generating maturation-resistant tolDCs, however, does not change the ability of these cells to induce mechanisms of immune tolerance. In contrast, CsA affects the ability of these cells to induce mechanisms of immune tolerance, but is not efficient at generating maturation-resistant tolDCs.

Galectins - Important players of the immune response to CNS parasitic infection.

Galectins are a family of proteins that bind ß-galactosides and play key roles in a variety of cellular processes including host defense and entry of parasites into the host cells. They have been well studied in hosts but less so in parasites. As both host and parasite galectins are highly upregulated proteins following infection, galectins are an area of increasing interest and their role in immune modulation has only recently become clear. Correlation of CNS parasitic diseases with mental disorders as a result of direct or indirect interaction has been observed. Therefore, galectins produced by the parasite should be taken into consideration as potential therapeutic agents.

The production of excretory-secretory molecules from Heligmosomoides polygyrus bakeri fourth stage larvae varies between mixed and single sex cultures.

BACKGROUND: Excretory-secretory (ES) products are crucial in maintaining helminths in the host. Consequently, the proteins of ES are potential vaccine molecules and potential therapeutic agents for autoimmune diseases. Heligmosomoides polygyrus bakeri, a gastrointestinal parasite of mice, is a model of hookworm infection in humans. ES produced by both sexes of H. polygyrus bakeri L4 stage cultured separately shows different immunomodulatory properties than ES obtained when both sexes are cultured together. Accordingly, the objective of this study was to identify and compare the excretory-secretory molecules from single-sex and mixed cultures. METHODS: The composition of ES of male and female L4 stage nematodes in the presence (cultured together) or absence (cultured alone) of the opposite sex was examined. Proteins were identified using mass spectrometry. The functions of identified proteins were explored with Blast2GO. RESULTS: A total of 258 proteins derived from mixed larval culture in the presence of sex pheromones were identified, 160 proteins from pure female cultures and 172 from pure male cultures. Exposure of nematodes to the sex pheromones results in abundant production of proteins with immunomodulatory properties such as Val proteins, acetylcholinesterases, TGF-ß mimic 9 and HpARI. Proteins found only in ES from mixed larval cultures were TGF-ß mimics 6 and 7 as well as galectin. CONCLUSIONS: The presence of the opposite sex strongly influences the composition of ES products, probably by chemical (pheromone) communication between individuals. However, examination of the composition of ES from various conditions gives an opportunity for searching for new potentially therapeutic compounds and anthelminthics as well as components of vaccines. Manipulation of the nematode environment might be important for the studies on the immunomodulatory potential of nematodes.

The interaction of host and nematode galectins influences the outcome of gastrointestinal nematode infections.

Galectins are a family of proteins that bind ß-galactosides and play key roles in a variety of cellular processes including host defence. They have been well studied in hosts but less so in gastrointestinal nematodes. Both host and parasite galectins are present in the gastrointestinal tract following infection. Parasite galectins can both bind antibody, especially highly glycosylated IgE and be bound by antibody. Parasite galectins may act as molecular sponges that soak up antibody. Host galectins promote mast cell degranulation while parasite galectins inhibit degranulation. Host and parasite galectins can also bind mucins and influence mucus viscosity. As the protective response against gastrointestinal nematode infection is partly dependent on IgE mediated mast cell degranulation and mucus, the interactions between host and parasite galectins play key roles in determining the outcome of infection.

Immunomodulatory potential of nematodes against dendritic cells is dependent on intestinal inflamation.

The mouse intestinal parasite Heligmosomoides polygyrus demonstrates adaptation to the inflammatory milieu as a result of colitis induced by dextran sulphate sodium (DSS). Nematodes from mice with colitis had different effects on dendritic cells than nematodes from mice without colitis. Immature JAWSII cells pre-exposed to L4 stage H. polygyrus from DSS-treated mice were adoptively transferred to mice with induced colitis. After two days, a higher disease activity index, macroscopic damage score and colon histology score were observed. MLN T cells isolated nine days after transfer demonstrated proinflammatory IFN-γ and IL-17 production. Transfer of JAWSII stimulated with male or female L4 larvae from a control invasion resulted in a slight improvement of colitis; in addition, dendritic cells exposed to H. polygyrus female L4 larvae, provoked migration of CD8+CD25+ T cells from MLN to the colon. Nematodes from an inflammatory environment changed cytokine production by dendritic cells. Inflammatory milieu changing nematode immunomodulatory activity affects dendritic cell functions, which offers new insight into the helminth-host relationship.

The intestinal milieu influences the immunoproteome of male and female Heligmosomoides polygyrus bakeri L4 stage.

The gastrointestinal nematode Heligmosomoides polygyrus bakeri shows enhanced survival in mice with colitis. As the antibody response plays an important role in antiparasitic immunity, antibodies against male and female L4 H. polygyrus were examined in mice with and without colitis. Levels of specific antibodies in the mucosa and serum were determined by enzyme-linked immunosorbent assay and immunogenic proteins of male and female parasites were identified using 2D electrophoresis and mass spectrometry. The function of identified proteins was explored with Blast2Go. Nematodes in mice with colitis induced higher levels of specific immunoglobulin G (IgG1) and IgA, a lower level of IgE in the small intestine and a higher level of IgE in serum against female L4. Infected mice with colitis recognized 12 proteins in male L4 and 10 in female L4. Most of the recognized proteins from male L4 were intermediate filament proteins, whereas the proteins from female L4 were primarily actins and galectins. Nematodes from mice with colitis were immunogenically different from nematodes from control mice. This phenomenon gives new insights into helminth therapy as well as host-parasite interactions.

L4 stage Heligmosomoides polygyrus prevents the maturation of dendritic JAWS II cells.

Helminths and their products are strong candidates for the treatment of autoimmunological disorders and allergies. Being a key population of antigen-presenting cells, dendritic cells play a crucial role in the therapeutic potential of worms. The study compares the effects of live pre-male and pre-female L4 stage Heligmosomoides polygyrus administration on the maturation and activation of the JAWS II line of immature dendritic cells. On stimulation with L4 stage H. polygyrus, JAWS II cells acquire semi-mature status and induce Th2 and regulatory responses in vitro. The strongest immunosuppressive effect on JAWS II cells was observed following stimulation with both sexes of nematodes together; this was manifested as immature dendritic cell morphology, proliferation inhibition, cell cycle change, decreased translocation of NF-κB into the nucleus, and lower expression of surface cellular costimulatory molecules CD80, CD86 and MHC I. However, greater production of proinflammatory (IL-12p70, TNF-α, IL-6) and Th2 response-promoting cytokines (IL-4) was observed by JAWS II following exposure to both sexes compared to male or female larvae alone. Sex had no influence on the viability, apoptosis process or endocytosis abilities of the JAWS II cell line. The findings indicate that the presence of only a single sex of the parasite influences a developed response, resulting in reduced proinflammatory and an antiparasitic reaction.