Suppression of inflammatory arthritis by the parasitic worm product ES-62 is associated with epigenetic changes in synovial fibroblasts.

ES-62 is the major secreted protein of the parasitic filarial nematode, Acanthocheilonema viteae. The molecule exists as a large tetramer (MW, ~240kD), which possesses immunomodulatory properties by virtue of multiple phosphorylcholine (PC) moieties attached to N-type glycans. By suppressing inflammatory immune responses, ES-62 can prevent disease development in certain mouse models of allergic and autoimmune conditions, including joint pathology in collagen-induced arthritis (CIA), a model of rheumatoid arthritis (RA). Such protection is associated with functional suppression of "pathogenic" hyper-responsive synovial fibroblasts (SFs), which exhibit an aggressive inflammatory and bone-damaging phenotype induced by their epigenetic rewiring in response to the inflammatory microenvironment of the arthritic joint. Critically, exposure to ES-62 in vivo induces a stably-imprinted CIA-SF phenotype that exhibits functional responses more typical of healthy, Naïve-SFs. Consistent with this, ES-62 "rewiring" of SFs away from the hyper-responsive phenotype is associated with suppression of ERK activation, STAT3 activation and miR-155 upregulation, signals widely associated with SF pathogenesis. Surprisingly however, DNA methylome analysis of Naïve-, CIA- and ES-62-CIA-SF cohorts reveals that rather than simply preventing pathogenic rewiring of SFs, ES-62 induces further changes in DNA methylation under the inflammatory conditions pertaining in the inflamed joint, including targeting genes associated with ciliogenesis, to programme a novel "resolving" CIA-SF phenotype. In addition to introducing a previously unsuspected aspect of ES-62's mechanism of action, such unique behaviour signposts the potential for developing DNA methylation signatures predictive of pathogenesis and its resolution and hence, candidate mechanisms by which novel therapeutic interventions could prevent SFs from perpetuating joint inflammation and destruction in RA. Pertinent to these translational aspects of ES-62-behavior, small molecule analogues (SMAs) based on ES-62's active PC-moieties mimic the rewiring of SFs as well as the protection against joint disease in CIA afforded by the parasitic worm product.

[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.

Site-specific glycoproteomic characterization of ES-62: The major secreted product of the parasitic worm Acanthocheilonema viteae.

ES-62 is the major secreted product of the parasitic filarial nematode Acanthocheilonema viteae and has potent anti-inflammatory activities as a consequence of posttranslational decoration by phosphorylcholine (PC). Previously, we showed that ES-62's PC was attached to N-linked glycans, and using fast atom bombardment mass spectrometry, we characterized the structure of the glycans. However, it was unknown at this time which of ES-62's four potential N-glycosylation sites carries the PC-modified glycans. In the present study, we now employ more advanced analytical tools-nano-flow liquid chromatography with high-definition electrospray mass spectrometry-to show that PC-modified glycans are found at all four potential N-glycosylation sites. Also, our earlier studies showed that up to two PC groups were detected per glycan, and we are now able to characterize N-glycans with up to five PC groups. The number per glycan varies in three of the four glycosylation sites, and in addition, for the first time, we have detected PC on the N-glycan chitobiose core in addition to terminal GlcNAc. Nevertheless, the majority of PC is detected on terminal GlcNAc, enabling it to interact with the cells and molecules of the immune system. Such expression may explain the potent immunomodulatory effects of a molecule that is considered to have significant therapeutic potential in the treatment of certain human allergic and autoimmune conditions.

Small Molecule Analogues of the parasitic worm product ES-62 interact with the TIR domain of MyD88 to inhibit pro-inflammatory signalling.

ES-62 is a protein secreted by the parasitic worm Acanthocheilonema viteae that is anti-inflammatory by virtue of covalently attached phosphorylcholine. Previously we have reported that drug-like Small Molecule Analogues (SMAs) of its phosphorylcholine moiety can mimic ES-62 in protecting against disease development in certain mouse models of autoimmune and allergic conditions, due to them causing partial degradation of the TLR/IL-1R adaptor MyD88. We have now taken a molecular modelling approach to investigating the mechanism underlying this effect and this predicts that the SMAs interact directly with the MyD88 TIR domain. Further support for this is provided by assay of LPS-induced MyD88/NF-κB-driven secreted alkaline phosphatase (SEAP) reporter activity in commercially-available stably transfected (TLR4-MD2-NF-κB-SEAP) HEK293 cells, as SMA12b-mediated inhibition of such SEAP activity is blocked by its pre-incubation with recombinant MyD88-TIR domain. Direct binding of SMA12b to the TIR domain is also shown to inhibit homo-dimerization of the adaptor, an event that can explain the observed degradation of the adaptor and inhibition of subsequent downstream signalling. Thus, these new data identify initial events by which drug-like ES-62 SMAs, which we also demonstrate are able to inhibit cytokine production by human cells, homeostatically maintain "safe" levels of MyD88 signalling.

Prophylactic and therapeutic treatment with a synthetic analogue of a parasitic worm product prevents experimental arthritis and inhibits IL-1ß production via NRF2-mediated counter-regulation of the inflammasome.

Rheumatoid arthritis (RA) remains a debilitating autoimmune condition as many patients are refractory to existing conventional and biologic therapies, and hence successful development of novel treatments remains a critical requirement. Towards this, we now describe a synthetic drug-like small molecule analogue, SMA-12b, of an immunomodulatory parasitic worm product, ES-62, which acts both prophylactically and therapeutically against collagen-induced arthritis (CIA) in mice. Mechanistic analysis revealed that SMA-12b modifies the expression of a number of inflammatory response genes, particularly those associated with the inflammasome in mouse bone marrow-derived macrophages and indeed IL-1ß was the most down-regulated gene. Consistent with this, IL-1ß was significantly reduced in the joints of mice with CIA treated with SMA-12b. SMA-12b also increased the expression of a number of genes associated with anti-oxidant responses that are controlled by the transcription factor NRF2 and critically, was unable to inhibit expression of IL-1ß by macrophages derived from the bone marrow of NRF2(-/-) mice. Collectively, these data suggest that SMA-12b could provide the basis of an entirely novel approach to fulfilling the urgent need for new treatments for RA.

Small molecule analogues of the immunomodulatory parasitic helminth product ES-62 have anti-allergy properties.

ES-62, a glycoprotein secreted by the filarial nematode Acanthocheilonema viteae, exhibits anti-inflammatory properties by virtue of covalently attached phosphorylcholine moieties. Screening of a library of ES-62 phosphorylcholine-based small molecule analogues (SMAs) revealed that two compounds, termed 11a and 12b, mirrored the helminth product both in inhibiting mast cell degranulation and cytokine responses in vitro and in preventing ovalbumin-induced Th2-associated airway inflammation and eosinophil infiltration of the lungs in mice. Furthermore, the two SMAs inhibited neutrophil infiltration of the lungs when administered therapeutically. ES-62-SMAs 11a and 12b thus represent starting points for novel drug development for allergies such as asthma.