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.

Development of Acanthocheilonema viteae in Meriones shawi: Absence of microfilariae and production of active ES-62.

AIMS: ES-62 is a well-studied anti-inflammatory molecule secreted by L4-adult stage Acanthocheilonema viteae. We maintain the life cycle of A viteae using Meriones libycus as the definitive host. Here, we investigated whether the full life cycle could be maintained, and functional ES-62 produced, in a related jird species-Meriones shawi. METHODS AND RESULTS: Adult worms were produced in comparable numbers in the two species, but very few microfilariae (MF) were observed in the M shawi bloodstream. M shawi ES-62 produced ex vivo was functional and protective in a mouse model of arthritis. Myeloid-derived cells from naïve and infected jirds of both species were compared with respect to ROS production and osteoclast generation, and some differences between the two species in both the absence and presence of infection were observed. CONCLUSIONS: The life cycle of A viteae cannot be successfully completed in M shawi jirds but L3 stage worms develop to adulthood and produce functional ES-62. Preliminary investigation into jird immune responses suggests that infection can differentially modulate myeloid responses in the two species. However, species-specific reagents are required to understand the complex interplay between A viteae and its host and to explain the lack of circulating MF in infected M shawi jirds.

Dendritic cells provide a therapeutic target for synthetic small molecule analogues of the parasitic worm product, ES-62.

ES-62, a glycoprotein secreted by the parasitic filarial nematode Acanthocheilonema viteae, subverts host immune responses towards anti-inflammatory phenotypes by virtue of covalently attached phosphorylcholine (PC). The PC dictates that ES-62 exhibits protection in murine models of inflammatory disease and hence a library of drug-like PC-based small molecule analogues (SMAs) was synthesised. Four sulfone-containing SMAs termed 11a, 11e, 11i and 12b were found to reduce mouse bone marrow-derived dendritic cell (DC) pathogen-associated molecular pattern (PAMP)-induced pro-inflammatory cytokine production, inhibit NF-κB p65 activation, and suppress LPS-induced up-regulation of CD40 and CD86. Active SMAs also resulted in a DC phenotype that exhibited reduced capacity to prime antigen (Ag)-specific IFN-γ production during co-culture with naïve transgenic TCR DO.11.10 T cells in vitro and reduced their ability, following adoptive transfer, to prime the expansion of Ag-specific T lymphocytes, specifically TH17 cells, in vivo. Consistent with this, mice receiving DCs treated with SMAs exhibited significantly reduced severity of collagen-induced arthritis and this was accompanied by a significant reduction in IL-17+ cells in the draining lymph nodes. Collectively, these studies indicate that drug-like compounds that target DCs can be designed from parasitic worm products and demonstrate the potential for ES-62 SMA-based DC therapy in inflammatory disease.

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.