[Role of the ES-62 protein derived from Acanthocheilonema viteae in regulation of immune dysregulation diseases: a review].

ES-62 is a phosphorylcholine-containing, 62 kDa glycoprotein derived from the excretory-secretory product of Acanthocheilonema viteae, which is effective for the prevention and treatment of immune dysregulation diseases through triggering activation of immune cells, such as dendritic cells, mononuclear macrophages and regulatory B cells and mediating immune responses. Recently, the role of the ES-62 protein in the management of allergic, autoimmune and metabolic diseases has been paid much attention. This review summarizes the regulatory role of the ES-62 protein in immune dysregulation diseases and the underlying mechanisms, so as to provide insights into future experimental studies.

Immunomodulatory properties of ES-62, a phosphorylcholine-containing glycoprotein secreted by Acanthocheilonema viteae.

Filarial nematodes are parasites that have the ability to persist in their hosts for extended periods of time due to the employment of various mechanisms to divert or down-regulate the host's immune responses. One of these mechanisms is the production of immunomodulatory excretory-secretory (ES) products. This review will discuss the properties of one such product, ES-62, which over the years, has been shown to interact with and modulate the activities of a variety of cells of the immune system including B and T lymphocytes, dendritic cells, macrophages and mast cells. Overall, ES-62 diverts the immune system towards an anti-inflammatory phenotype and consistent with this it has been shown to have therapeutic potential in models of inflammatory disease associated with autoimmunity and allergy.

Receptor usage by the Acanthocheilonema viteae-derived immunomodulator, ES-62.

ES-62 is an immunomodulatory phosphorylcholine (PC)-containing glycoprotein secreted by the rodent filarial nematode Acanthocheilonema viteae. Previously, the use of knockout mice has revealed the effects of ES-62 on macrophages and dendritic cells to be dependent on TLR4. However, it is possible that ES-62 may interact with additional proteins on the surfaces of target cells and hence that cells may vary with respect to receptor usage. In this study, we identified by molecular weight, proteins that interact with ES-62 and found differences amongst the immune system cells studied. Thus, whereas lymphocytes appear to have two major interacting proteins of ∼135 and ∼82 kDa, U937 monocytes only contain an ES-62-binding protein of the latter molecular weight. Binding to the proteins on B cells and U937 cells was blocked by PC, suggesting a critical role for this ES-62 moiety in facilitating interaction. Finally, ES-62 binding is followed by internalization in both macrophages and B cells but only in the former was absence of TLR4 found to block internalization. These findings are consistent with differences in receptor usage by ES-62 amongst different cell-types.

Stage-specific and species-specific differences in the production of the mRNA and protein for the filarial nematode secreted product, ES-62.

Previous studies have shown that the secreted phosphorylcholine-containing glycoprotein of filarial nematodes, ES-62, is only present in the post-infective life-cycle stages, but that the mRNA is transcribed throughout the worm's life-cycle. The aim of this current study was to investigate whether the presence or absence of protein expression simply reflects differences in mRNA abundance. To this end, we investigated the relative abundance of ES-62 using TaqMan real time RT-PCR, in different life-cycle stages of 2 model filarial nematode parasites, Acanthocheilonema viteae and Brugia pahangi. For B. pahangi, microfilariae, infective larvae and adult worms were each found to have approximately similar levels of ES-62 mRNA. However, the corresponding stages of A. viteae differed greatly from each other with a pattern of increased mRNA production with maturation. As a rule A. viteae had higher levels of ES-62 mRNA than B. pahangi, and this was particularly noticeable in the adult stage where the difference was approximately 3500-fold higher. However, this significant difference in mRNA abundance was not reflected in the quantity of ES-62 protein secreted by the adult worms of each species, as A. viteae only secreted approximately 3 times as much ES-62 as B. pahangi. Thus, overall, the results obtained from this study indicate that ES-62 protein production does not solely reflect mRNA levels, and also suggest that the 2 nematodes may employ different mechanisms for regulating protein production.

Expression of the filarial nematode phosphorylcholine-containing glycoprotein, ES62, is stage specific.

ES62, an immunomodulatory phosphorylcholine-containing glycoprotein secreted by the rodent filarial nematode Acanthocheilonema viteae, has previously been shown to be produced by L4 larvae and adult worms only. However, homologous sequences to ES62 have recently been found in L1 and L3 cDNA libraries of certain human filarial nematodes. Therefore, the various stages of A. viteae were re-examined and it was again found that only the post-L3 stages secreted ES62. Synthesis but not secretion by earlier stages was ruled out by examination of the protein content of whole worm extracts and by immunoelectron microscopy. However, examination by PCR of the mRNA for ES62 revealed that it was found in the L1 and L3 larvae. This may explain why homologous sequences to ES62 have been found in Brugia malayi and Onchocerca volvulus larval cDNA libraries. It also suggests that filarial nematodes, in general, may secrete ES62. To obtain evidence for this, we investigated production by Brugia pahangi, a close relation of B. malayi. We found that ES62 was indeed secreted but, as with A. viteae, only by the post-L3 stages, although again the mRNA for ES62 could be detected in the earlier stages. Overall our results suggest that production of ES62 is not species specific, that it is indeed stage specific, and that this may be due to post-transcriptional control of expression.

Investigation of the nature of potential phosphorylcholine donors for filarial nematode glycoconjugates.

Filarial nematodes produce proteins containing phosphorylcholine (PC) covalently attached to N-linked glycans. Our previous work has suggested that transfer of PC might be dependent on a metabolite of the Kennedy pathway of phospholipid biosynthesis. In this study we have investigated whether the end product of this pathway, phosphatidylcholine, and in addition, sphingomyelin, could act as PC donors. Pulse-chase experiments employing [3H]choline as radiolabel, ruled out sphingomyelin, as the Acanthocheilonema viteae PC-containing protein, ES-62, was radiolabelled 20-30 min prior to the lipid. Phosphatidylcholine however was labelled immediately before ES-62 increasing the possibility that it could act as donor. This was further investigated by radiolabelling phosphatidylcholine synthesised via an alternative pathway such that other metabolites in the Kennedy pathway were not labelled. Specifically, we labelled the choline component of phosphatidylcholine using both [3H]serine and [3H]S-adenosyl methionine (SAM). Incubation of worms with [3H]serine failed to result in labelling of the PC component of ES-62 whereas the presence of [3H]SAM in the medium led to labelling of ES-62 but only 24 h after labelling of phosphatidylcholine. As ES-62 is labelled within minutes of phosphatidylcholine when employing [3H]choline as radiolabel, this suggests that labelling of ES-62 when using SAM is not due to direct transfer of PC from phosphatidylcholine. It is therefore concluded that neither sphingomyelin nor phosphatidylcholine act as PC donors for filarial nematode glycoproteins. The analysis of PC-containing metabolites and products from A. viteae additionally revealed the presence of PC-substituted glycolipids that were also radiolabelled by the use of [14C]choline. The kinetics of radiolabelling however differed from that observed in the case of ES-62, sphingomyelin and phosphatidylcholine in so far as labelled glycolipids were first detectable hours rather than minutes after addition of [14C]choline.

Cystatins of filarial nematodes up-regulate the nitric oxide production of interferon-gamma-activated murine macrophages.

Cystatins of two filarial nematodes were studied with regard to their capacity to up-regulate the production of nitric oxide (NO) in vitro, and the effects were analysed. Recombinant cystatin of the human pathogenic filaria Onchocerca volvulus and of the rodent filaria Acanthocheilonema viteae significantly enhanced the NO production of interferon (IFN)-gamma-activated macrophages of BALB/c and C3H/HeJ mice. Truncated cystatins lacking the N-terminal protease inhibitory active site, and showing marginal protease inhibitory activity, up-regulated the NO production to the same extent as the full-length proteins, indicating that the effect on the NO production is independent of cysteine protease inhibition. NO did not contribute to the suppression of proliferative T cell responses exerted by filarial cystatins, as shown in other studies, since NO synthase inhibitors did not restore proliferative responses. The up-regulation of NO production induced by filarial cystatins was partly dependent on the production of interleukin-10 and tumour necrosis factor-alpha, since depletion of both cytokines by antibodies led to a diminution of the enhanced NO production by 22-48%. Our data suggest that filarial cystatins are potent triggers of the production of NO, a mediator which was shown to have a role as an effector molecule against filarial worms in vitro and in vivo.

Effects of 2-deoxy-D-glucose on Acanthocheilonema viteae: rodent filariids as studied by multinuclear NMR spectroscopyt.

A well known glucose antimetabolite, 2-deoxy glucose (2DG) widely used in chemotherapy of cancer along with radiation, was evaluated as an antifilarial agent by nuclear magnetic resonance. The uptake and metabolism of 2DG in the experimental filarial infection Acanthocheilonema viteae was studied by in vivo multinuclear NMR. An unusually long retention time of 2DG6P within these parasites was observed on continuous 31P NMR monitoring, along with a decrease in ATP levels. These results led to therapeutic investigation in A. viteae infected host Mastomys coucha. 2DG showed a remarkable adulticidal activity (73.6%) with 50% sterilization of surviving female worms at a dose of 250 mg/kg x 5, p.o. NMR observations and activity profile substantiate the findings of one another, directed towards the hitting of bioenergetic machinery of A. viteae by macrofilaricidal agent (2DG).

Mechanisms underlying the transfer of phosphorylcholine to filarial nematode glycoproteins--a possible role for choline kinase.

Phosphorylcholine (PC) is a common constituent of proteins secreted by filarial nematodes. As this substance has been shown to interfere with immune responses, we are interested in designing strategies for blocking its attachment. Towards this end, we are investigating the mechanism of incorporation of PC into filarial molecules and in the present manuscript we describe experiments relating to elucidating the source of PC for attachment. Synthesis of phosphatidylcholine in eukaryotic organisms can occur by a mechanism involving the transfer of PC from CDP-choline to diacylglycerol (the Kennedy pathway). By (i) measuring transfer of radio-isotope labelled PC from CDP-choline to parasite molecules and (ii) employing inhibitors of CDP-choline synthesis, we have investigated whether CDP-choline can act as a source of PC for transfer to ES-62, a major secreted glycoprotein of the rodent filarial nematode Acanthocheilonema viteae. Although we can find no evidence of this, we show that attachment of PC is blocked by hemicholinium-3, an inhibitor of choline kinase, the first enzyme in the Kennedy pathway. Thus, at least the first step in this pathway--phosphorylation of choline, would appear to be necessary for attachment of PC to ES-62.

Acanthocheilonema viteae: stage-specific expression of G-protein alpha-subunits.

We have previously demonstrated by Western blot analysis that the adult stage of the filarial nematode Acanthocheilonema viteae expresses the alpha-subunits of heterotrimeric G-proteins corresponding to GS and Gq. We now show, using the same technique, that these two alpha-subunits are not detectable in the microfilaria stage of the parasite. Conversely, microfilariae contain Go, an alpha-subunit not expressed by the adult worm. No other G-protein alpha-subunits were found in microfilariae by Western blotting. However, reverse transcriptase-polymerase chain reaction (RT-PCR) with degenerate G-protein oligonucleotide primers, followed by hybridisation analysis, using oligonucleotides specific for individual G-protein alpha-subunits, not only confirmed expression of Go, but also detected Gi1 and G11 alpha-subunits. G-protein expression in infective larvae was also investigated by RT-PCR analysis: this stage of the organism was found to resemble the adult more than the microfilaria but differed from the adult in that GS was absent and Gi3 was present. The significance of these stage-specific differences in G-protein expression is discussed with respect to their possible role in parasite development and survival.

Studies on the site and mechanism of attachment of phosphorylcholine to a filarial nematode secreted glycoprotein.

We have recently shown that the immunomodulatory substance phosphorylcholine (PC) is covalently attached to ES-62, a major secreted protein of the filarial nematode parasite Acanthocheilonema viteae, via an N-linked glycan. Linkage of PC to N-glycans is previously unreported, and hence we have investigated the biochemical events underlying it. PC addition was found by pulse-chase experiments to be a fairly early event during intracellular transport, occurring within 40-60 min of protein synthesis. Biosynthetic labeling/immunoprecipitation experiments revealed that addition of PC to ES-62 was blocked by (i) brefeldin A, an inhibitor of trafficking of newly synthesized proteins from the endoplasmic reticulum (ER) to the Golgi, (ii) 1-deoxynorijirimycin, an inhibitor of glucosidase activity in the ER, and (iii) 1-deoxymannojirimycin, an inhibitor of mannosidase I in the cis Golgi. Swainsonine, an inhibitor of mannosidase II in the medial Golgi, did not affect PC addition. Taken together these data indicate that PC attachment is a post-ER event which is dependent on generation of an appropriate substrate during oligosaccharide processing. Furthermore, they strongly suggest that PC addition takes place in the medial Golgi and that the substrate for addition is the 3-linked branch of Man5GlcNAc3 or Man3GLcNAc3.

Characterization of heterotrimeric G-proteins in adult Acanthocheilonema viteae.

Heterotrimeric G-proteins have been found in eukaryotic cells, from yeast to humans, but have received little attention, to date, with respect to parasitic organisms. We now present the first report of the characterization of heterotrimeric G-proteins expressed in a filarial nematode, Acanthocheilonema viteae. Using a combination of (i) affinity labelling with [alpha-32P]GTP; (ii) ADP-ribosylation with cholera toxin and pertussis toxin; (iii) Western blotting with a panel of anti-G-protein antibodies; and (iv) reverse transcriptase-PCR with degenerate G-protein oligonucleotide primers followed by hybridization analysis using oligonucleotides specific for individual G-protein subunits, we demonstrate that adult A. viteae expresses homologues of the beta 1- and/or beta 2-like subunits and alpha-subunits of the Gs, G1, Gq and G12 subfamilies found in mammals. The role which these G-proteins may play in the biology of the organism is discussed.

Prevention of attachment of phosphorylcholine to a major excretory-secretory product of Acanthocheilonema viteae using tunicamycin.

ES-62, a major excretory-secretory (ES) product of Acanthocheilonema viteae, consists of a protein backbone with N-linked carbohydrate and the immunomodulatory group phosphorylcholine (PC); it can, therefore, be biosynthetically labeled with radioactive leucine, glucosamine, or choline. Incubation of worms with tunicamycin results in an ES product whose secretion is partially blocked, which demonstrates reduced molecular weight when employing leucine as radiolabel, and which lacks radioactivity when employing glucosamine or choline as label. Furthermore, the retained ES product can be detected in somatic extracts of parasites exposed to tunicamycin, by its reactivity for antibodies against the whole parasite product but not by antibodies against PC alone. These results support the idea that PC is attached to ES-62 via an N-linked glycan and hence are consistent with the recent observation that PC can be removed from ES-62 by the sugar-cleaving enzyme, N-glycosidase F. The implications of this structural information with respect to designing inhibitors of PC attachment for use as chemotherapeutic agents, and also the advantage of such material in raising antibodies to filarial ES, are discussed.

Polyamine uptake by a rodent filariid, Acanthocheilonema viteae (Nematoda: Filarioidea).

Helminth parasites lack the capacity to synthesize polyamines de novo. With the view to characterise alternative pools for the replenishment of polyamines, uptake of spermidine and spermine, were studied in Acanthocheilonema viteae, a parasite known to infect rodents. Motile worms recovered from the subcutaneous tissues of experimentally infected Mastomys natalensis were incubated in vitro with radiolabelled polyamines for the uptake assays. A time-dependent, temperature-sensitive, energy-requiring and saturable-uptake of the polyamines was observed. Male worms exhibited better uptake than the females and spermidine influx occurred at a higher rate than for spermine. A marginal competition of spermine with spermidine uptake and vice-versa was noticed while putrescine did not compete with uptake of either polyamines. Methyl-glyoxal-bis-guanyl hydrazone and Berenil caused significant inhibition of spermidine as well as spermine uptake. Subcutaneous tissues of A. viteae infected animals exhibited markedly higher levels of polyamines compared with the tissues obtained from healthy animals. Filarial worms are thus equipped with multiple polyamine transport systems which may aid their growth and survival within the host.

Effect of antibiotics on protein synthesis in filarial parasites.

Acanthocheilonema viteae, Litomosoides carinii and Setaria cervi were found to actively synthesize proteins in vitro. Different centrifugation fractions and their TCA-precipitable fractions were assessed for the distribution of newly synthesized proteins. Penicillin and streptomycin inhibited the process in A. viteae. The synthesis in S. cervi was susceptible to puromycin, chloramphenicol, cycloheximide, neomycin and polymyxin B. The process in L. carinii was strongly blocked by puromycin while chloramphenicol had no significant effect.

Changes in the surface composition after transmission of Acanthocheilonema viteae third stage larvae into the jird.

This study describes the dynamics and the biochemical nature of changes in the surface of the filarial nematode Acanthocheilonema viteae after its transmission into the vertebrate host. Vector-derived third-stage larvae (mL3) were inoculated into naive Meriones unguiculatus and recovered from the tissues at different times post-infection until their moult to fourth-stage larvae (L4). Surface-specific labelling with fluoresceinated lectins revealed that the larvae are covered by a carbohydrate envelope. Although the mL3 envelope was strongly reduced one day after transmission, new surface carbohydrates appeared until the onset of moulting, some of which could also be identified on the surface of L4. In general, surface carbohydrates were partially shed by moving larvae, suggesting a loose association of these components in the epicuticle. The fate of cuticular lipids and proteins of L3 and L4 was monitored by external 125I-labelling and differential extraction of the components. Thin-layer chromatography of surface-labelled lipids revealed only minor changes 1 day after parasite transmission. Afterwards the number of lipids accessible to label decreased further until moulting was complete. Two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis of surface-labelled proteins showed a consistent surface exposure of mL3 specific proteins until 1 day post-infection. Thereafter, the composition of surface-labelled proteins changed rapidly, resembling that of the L4 as early as several days before moulting. During this period individual differences in the composition of surface proteins were evident.

Polyamine metabolism in some helminth parasites.

Polyamine levels of some helminth parasites were analyzed by reverse phase HPLC of benzoyl derivatives. Setaria cervi, Acanthocheilonema viteae, Hymenolepis nana, H. diminuta, and Ascaridia galli contained higher levels of spermine than spermidine while in Ancylostoma ceylanicum and Nippostrongylus brasiliensis the spermidine levels were higher than spermine; putrescine was either absent or present in minor quantities. The enzymes of polyamine biosynthesis viz., ornithine decarboxylase, S-adenosyl methionine (SAM)-decarboxylase, and arginine decarboxylase were present in very low to negligible amounts in all the parasites examined. A. ceylanicum exhibited high activity of ornithine amino transferase (OAT) and catalyzed appreciable decarboxylation of ornithine. The ornithine decarboxylating activity of A. ceylanicum was localized in the particulate fraction containing mitochondria, not inhibited by alpha-difluoromethyl ornithine, the specific inhibitor of ornithine decarboxylase (ODC), but inhibited in the presence of glutamate, suggesting the involvement of mitochondrial OAT rather than a true ODC in ornithine decarboxylation in this parasite. Significant activity of polyamine oxidase was also detected in helminth parasites. The absence of polyamine biosynthesizing enzymes in helminth parasites suggests their dependence on hosts for uptake and interconversion of polyamines, providing a potential target for chemotherapy.

An investigation of fructose utilization in Acanthocheilonema viteae.

The capacity of Acanthocheilonema viteae to metabolize fructose was investigated in vitro. In common with other filarial species A. viteae oxidized fructose to lactate but its rate of consumption was only 40% of the glucose-containing control value. Fructose was not incorporated into glycogen. Release of 14CO2 from [U-14C]fructose was not detected in the presence of glucose and was about 40% of the glucose-containing value under conditions where fructose was the sole hexose substrate. Fructose consumption and lactate excretion increased in proportion to the external concentration of fructose. However, worm viability was not maintained in fructose over a 120 h in vitro incubation. In the presence of fructose, protein synthesis (measured incorporation of [35S]methionine into acid-insoluble material) was reduced compared to the glucose-containing control group; but was significantly greater than the value obtained under glucose-free conditions.

Glucose transport in Acanthocheilonema viteae.

The uptake of glucose by Acanthocheilonema viteae was studied in vitro. The process was selective for the D-isomer and saturatable with a Km of 2 mM. The rate of glucose transport/utilization was inhibited by 2-deoxyglucose, mannose, 5-thioglucose and dipyridamole but, unlike mammalian systems, was not impaired by cytochalasin B, phloretin, phloridzin, 3-O-methylglucose and 4,6-ethylideneglucose. A potential chemotherapeutic advantage of selectively inhibiting filarial glucose transport exists for the following reasons. (1) The glucose transporter present in A. viteae was shown to be different from the one present in some mammalian systems. (2) Incubation under glucose-free conditions led to glycogen depletion, loss of motility and worm death. (3) Worms maintained in vitro for more than 18 h without glucose did not survive when implanted into gerbils.