Mast cell subsets and their functional modulation by the Acanthocheilonema viteae product ES-62

ES-62, an immunomodulator secreted by filarial nematodes, exhibits therapeutic potential in mouse models of allergic inflammation, at least in part by inducing the desensitisation of Fc휀RI-mediated mast cell responses. However, in addition to their pathogenic roles in allergic and autoimmune diseases, mast cells are important in fighting infection, wound healing, and resolving inflammation, reflecting that mast cells exhibit a phenotypic and functional plasticity. We have therefore characterised the differential functional responses to antigen (via Fc휀RI) and LPS and their modulation by ES-62 of the mature peritoneal-derived mast cells (PDMC; serosal) and those of the connective tissue-like mast cells (CTMC) and themucosal-likemast cells derived from bone marrow progenitors (BMMC) as a first step to produce disease tissue-targeted therapeutics based on ES-62 action. All three mast cell populations were rendered hyporesponsive by ES-62 and whilst the mechanisms underlying such desensitisation have not been fully delineated, they reflect a downregulation of calcium and PKC훼 signalling. ES-62 also downregulatedMyD88 and PKC훿 in mucosal-type BMMC but not PDMC, the additional signals targeted in mucosal-type BMMC likely reflecting that these cells respond to antigen and LPS by degranulation and cytokine secretion whereas PDMC predominantly respond in a degranulationbased manner

Protective effect of small molecule analogues of the Acanthocheilonema viteae secreted product ES-62 on oxazolone-induced ear inflammation

ES-62 is the major secreted protein of the rodent filarial nematode Acanthocheilonema viteae. The molecule contains covalently attached phosphorylcholine (PC) residues, which confer anti-inflammatory properties on ES-62, underpinning the idea that drugs based on this active moiety may have therapeutic potential in human diseases associated with aberrant inflammation. Here we demonstrate that two synthetic small molecule analogues (SMAs) of ES-62 termed SMA 11a and SMA 12b are protective in the oxazolone-induced acute allergic contact dermatitis mouse model of skin inflammation, as measured by a significant reduction in ear inflammation following their administration before oxazolone sensitisation and before oxazolone challenge. Furthermore, it was found that when tested, 12b was effective at reducing ear swelling even when first administered before challenge. Histological analysis of the ears showed elevated cellular infiltration and collagen deposition in oxazolone-treated mice both of which were reduced by treatment with the two SMAs. Likewise, the oxazolone-induced increase in IFNγ mRNA in the ears was reduced but no effect on other cytokines investigated was observed. Finally, no influence on the mast cell populations in the ear was observed

The carbohydrate-linked phosphorylcholine of the parasitic nematode product ES-62 modulates complement activation

Parasitic nematodes manufacture various carbohydratelinked phosphorylcholine (PCh)-containing molecules, including ES-62, a protein with an N-linked glycan terminally substituted with PCh. The PCh component is biologically important because it is required for immunomodulatory effects. We showed that most ES-62 was bound to a single protein, C-reactive protein (CRP), in normal human serum, displaying a calcium-dependent, high-avidity interaction and ability to form large complexes. Unexpectedly, CRP binding to ES-62 failed to efficiently activate complement as far as the C3 convertase stage in comparison with PCh-BSA and PCh-containing Streptococcus pneumoniae cell wall polysaccharide. C1q capture assays demonstrated an ES-62-CRP-C1q interaction in serum. The three ligands all activated C1 and generated C4b to similar extents. However, a C2a active site was not generated following ES-62 binding to CRP, demonstrating that C2 cleavage was far less efficient for ES-62-containing complexes. We proposed that failure of C2 cleavage was due to the flexible nature of carbohydrate-bound PCh and that reduced proximity of the C1 complex was the reason that C2 was poorly cleaved. This was confirmed using synthetic analogues that were similar to ES-62 only in respect of having a flexible PCh. Furthermore, ES-62 was shown to deplete early complement components, such as the rate-limiting C4, following CRP interaction and thereby inhibit classical pathway activation. Thus, flexible PCh-glycan represents a novel mechanism for subversion of complement activation. These data illustrate the importance of the rate-limiting C4/C2 stage of complement activation and reveal a new addition to the repertoire of ES-62 immunomodulatory mechanisms with possible therapeutic applications

Protective effect of small molecule analogues of the Acanthocheilonema viteae secreted product ES-62 on oxazolone-induced ear inflammation

ES-62 is the major secreted protein of the rodent filarial nematode Acanthocheilonema viteae. The molecule contains covalently attached phosphorylcholine (PC) residues, which confer anti-inflammatory properties on ES-62, underpinning the idea that drugs based on this active moiety may have therapeutic potential in human diseases associated with aberrant inflammation. Here we demonstrate that two synthetic small molecule analogues (SMAs) of ES-62 termed SMA 11a and SMA 12b are protective in the oxazolone-induced acute allergic contact dermatitis mouse model of skin inflammation, as measured by a significant reduction in ear inflammation following their administration before oxazolone sensitisation and before oxazolone challenge. Furthermore, it was found that when tested, 12b was effective at reducing ear swelling even when first administered before challenge. Histological analysis of the ears showed elevated cellular infiltration and collagen deposition in oxazolone-treated mice both of which were reduced by treatment with the two SMAs. Likewise, the oxazolone-induced increase in IFNγ mRNA in the ears was reduced but no effect on other cytokines investigated was observed. Finally, no influence on the mast cell populations in the ear was observed

The immunomodulatory parasitic worm product ES-62 reduces lupus-associated accelerated atherosclerosis in a mouse model.

ES-62 is an anti-inflammatory phosphorylcholine-containing glycoprotein secreted by the filarial nematode Acanthocheilonema viteae. Accelerated atherosclerosis frequently occurs in systemic lupus erythematosus, resulting in substantial cardiovascular morbidity and mortality. We examined the effects of ES-62 in the gld.apoE(-/-) mouse model of this condition. Treatment with ES-62 did not substantially modulate renal pathology but caused decreased anti-nuclear autoantibody levels. Moreover, a striking 60% reduction in aortic atherosclerotic lesions was observed, with an associated decrease in macrophages and fibrosis. We believe that these latter findings constitute the first example of a defined parasitic worm product with therapeutic potential in atherosclerosis: ES-62-based drugs may represent a novel approach to control accelerated atherosclerosis in systemic lupus erythematosus

The role of individual protein kinase C isoforms in mouse mast cell function and their targeting by the immunomodulatory parasitic worm product, ES-62

ES-62, a glycoprotein secreted by the filarial nematode Acanthocheilonema viteae, has been shown to modulate the immune system through subversion of signal transduction pathways operating in various immune system cells. With respect to human bone marrow-derived mast cells (BMMCs), ES-62 was previously shown to inhibit FcϵRI-mediated mast cell functional responses such as degranulation and pro-inflammatory cytokine release through a mechanism involving the degradation of PKC-α. At the same time, it was noted that the worm product was able to degrade certain other PKC isoforms but the significance of this was uncertain. In this study, we have employed PKC isoform KO mice to investigate the role of PKC-α, -β -ϵ, and -θ in mouse BMMCs in order to establish their involvement in mast cell-mediated responses and also, if their absence impacts on ES-62’s activity. The data obtained support that in response to antigen cross-linking of IgE bound to FcϵRI, pro-inflammatory cytokine release is controlled in part by a partnership between one conventional and one novel isoform with PKC-α and -θ acting as positive regulators of IL-6 and TNF-α production, while PKC-β and ϵ act as negative regulators of such cytokines. Furthermore, ES-62 appears to target certain other PKC isoforms in addition to PKC-α to inhibit cytokine release and this may enable it to more efficiently inhibit mast cell responses

The helminth product, ES-62, protects against airway inflammation by resetting the Th cell phenotype

We previously demonstrated inhibition of ovalbumin (OVA)-induced allergic airway hyper-responsiveness in the mouse using ES-62, a phosphorylcholine-containing glycoprotein secreted by the filarial nematode, Acanthocheilonema viteae. This inhibition correlated with ES-62-induced mast cell desensitisation, although the degree to which this reflected direct targeting of mast cells remained unclear as suppression of the Th2 phenotype of the inflammatory response, as measured by eosinophilia and IL-4 levels in the lungs, was also observed. We now show that inhibition of the lung Th2 phenotype is reflected in ex vivo analyses of draining lymph node recall cultures and accompanied by a decrease in the serum levels of total and OVA-specific IgE. Moreover, ES-62 also suppresses the lung infiltration by neutrophils that is associated with severe asthma and is generally refractory to conventional anti-inflammatory therapies, including steroids. Protection against Th2-associated airway inflammation does not reflect induction of regulatory T cell (Treg) responses (there is no increased IL-10 or Foxp3 expression) but rather a switch in polarisation towards increased T-bet expression and IFNγ production. This ES-62-driven switch in the Th1/Th2 balance is accompanied by decreased IL-17 responses, a finding in line with reports that IFNγ and IL-17 are counter-regulatory. Consistent with ES-62 mediating its effects via IFNγ-mediated suppression of pathogenic Th2/Th17 responses, we found that neutralising anti-IFNγ antibodies blocked protection against airway inflammation in terms of pro-inflammatory cell infiltration, particularly by neutrophils and lung pathology. Collectively, these studies indicate that ES-62, or more likely small molecule analogues, could have therapeutic potential in asthma, in particular for those subtypes of patients (e.g. smokers, steroid-resistant) who are refractory to current treatments

An investigation of the ability of the secreted filarial nematode product ES-62 to inhibit TH1 responses to heterologous molecules

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