Calendula officinalis Triterpenoid Saponins Impact the Immune Recognition of Proteins in Parasitic Nematodes.

The influence of triterpenoid saponins on subcellular morphological changes in the cells of parasitic nematodes remains poorly understood. Our study examines the effect of oleanolic acid glucuronides from marigold (Calendula officinalis) on the possible modification of immunogenic proteins from infective Heligmosomoides polygyrus bakeri larvae (L3). Our findings indicate that the triterpenoid saponins alter the subcellular morphology of the larvae and prevent recognition of nematode-specific proteins by rabbit immune-IgG. TEM ultrastructure and HPLC analysis showed that microtubule and cytoskeleton fibres were fragmented by saponin treatment. MASCOT bioinformatic analysis revealed that in larvae exposed to saponins, the immune epitopes of their proteins altered. Several mitochondrial and cytoskeleton proteins involved in signalling and cellular processes were downregulated or degraded. As possible candidates, the following set of recognised proteins may play a key role in the immunogenicity of larvae: beta-tubulin isotype, alpha-tubulin, myosin, paramyosin isoform-1, actin, disorganized muscle protein-1, ATP-synthase, beta subunit, carboxyl transferase domain protein, glutamate dehydrogenase, enolase (phosphopyruvate hydratase), fructose-bisphosphate aldolase 2, tropomyosin, arginine kinase or putative chaperone protein DnaK, and galactoside-binding lectin. Data are available via ProteomeXchange with identifier PXD024205.

The antiparasitic activity of avenacosides against intestinal nematodes.

Avena sativa L., 1753 (Poaceae) is used as feed for livestock and as a crop rotation agent. The purpose of the study was to examine the molecular mechanisms behind the antihelminth activity of the oat saponins avenacoside B (AveB) and 26-desglucoavenacoside B (26DGAveB) by evaluating their effect on Heligmosomoides bakeri, a parasitic nematode of mice. The avenacosides AveB and 26DGAveB were separated and purified from A. sativa green leaves, and their mycotoxic activity was confirmed against the fungus Trichoderma harzianum. The anti-nematode activity of the avenacosides was measured by egg hatching assay. In the surviving L3 larvae exposed to avenacosides, the expression of CED-9, a protein of the apoptosis pathway, was identified by Western blotting. The protein profile of L3 larvae was monitored by High Performance Liquid Chromatography (HPLC). The action of saponins on glycoprotein pump (Pgp) activity in L3 larvae was compared to that of the pump blocker Verapamil (VPL). A mouse model was used to measure the infectivity of L3 larvae exposed to AveB and 26DGAveB, and the outcome of the immune response. Both compounds induced morphological changes in larvae and blocked Pgp activity; however, only 26DGAveB provoked expression of CED-9. The infected mice displayed changes in the molecular pattern of larval proteins and enhanced IL-4 production, indicating that avenacosides reduced the infectivity of H. bakeri larvae. In avenacosides, the residue without glucose at the C26 position demonstrated greater anti-nematode activity. Our findings indicate that A. sativa compounds are natural products with anti-parasitic activity.

Changes in Heligmosomoides polygyrus glycoprotein pattern by saponins impact the BALB/c mice immune response.

Saponins of marigold (Calendula officinalis), in particular derivatives of 3-O-monoglucuronide of oleanolic acid, are able to reduce infectivity of Heligmosomoides polygyrus in mice. The purpose of this study was to understand the immune activation provoked by third-stage larvae exposed to marigold glucuronides. We also examined the pattern of glycosylation of larval antigens which appeared to be crucial for induction of cytokine production in BALB/c mice; higher concentrations of IL-6, IFN-γ, IL-10 and TNF-α were observed in serum or intestine one week post infection. Three weeks later, in the chronic phase of infection, cells in culture were able to produce IL-6, IFN-γ, TNF-α and IL-17. Restimulation of cells with H. polygyrus antigen resulted in reduced production of IL-6, and TNF-α. The pattern of cytokine production co-existed with reduced expression of terminal glucose, α-linked mannose, N-acetyl-galactosamine, ß-galactose, N-acetyl-glucosamine and α-fucose in several protein bands. Galactose, as a new terminal carbohydrate residue appeared in 20-24kDa protein bands. The number of immunogenic epitopes in parasitic antigens was reduced; only three protein bands of 56, 26 and 12kDa were recognized by IgG1. These studies provide a model system to find the glycosylated molecules expressed on nematodes that improve establishment and survival and characterize cytokine production in mice infected with larvae exposed to saponin. Identification of these molecules is the first step in the recognition of key antigenic epitopes able to induce protective or tolerogenic immune responses.

[Nematode infections in mice--an experimental model of immunoregulation]. / Zarazenia nicieniami u myszy--eksperymentalny model immunoregulacji.

There has been a substantial increase in the incidence of autoimmune and allergic diseases in Western countries in the past few decades. However, in the geographic area endemic for parasitic helminth infections, such diseases remain relatively rare. It has been hypothesized that helminths may protect against immune disorders that have been observed in urbanized area. Studies on rodents infected with nematodes Trichinella spiralis, Heligmosomoides polygyrus, Nippostrongylus brasiliensis and Trichuris muris have provided considerable information about immune mechanisms in aspects of host-parasite interaction and immunoregulation. Helminths inducing a long-lasting asymptomatic infection are regarded as major modifiers of the host immune system. Parasitic worms can establish and reproduce in mammalian hosts switching off inflammation and inducing a tolerant response to parasitic antigens. In this review we summarized recent information on the immunoregulation during nematode infection and mechanisms used by nematodes, including the induction of regulatory T cells and apoptosis in the host. The innate immune response seems to determine the different sensitivity of mice to nematode infection. In this review we also discuss results of our own studies on H. polygyrus, demonstrating that it induces different mechanisms in different strains of mice which might play important role in the modulation of immune response. In the slow responder mice apoptosis would play a key role in the outcome of immune response. Contrary to that, in fast responder mice a defensive inflammatory response is mostly down-regulated via endogenous opioids pathway. Understanding the molecular mechanisms that mediate the effects that helminths have on the immune system will provide information that can be exploited to prevent inflammatory diseases.