Katrina M. Houston

A Filarial Nematode-Secreted Product Signals Dendritic Cells to Acquire a Phenotype That Drives Development of Th2 Cells

Although exogeneous “danger” signals such as LPS can activate APC to produce a Th1 response, the nature of events initiating a Th2 response is controversial. We now show that pathogen-derived products have the capacity to induce bone marrow-derived dendritic cell cultures to acquire a phenotype that promotes the differentiation of naive CD4+ T cells toward either a Th1 or Th2 phenotype. Thus, LPS-matured dendritic cells (DC1) promote a Th1 response (increased generation of IFN-γ and reduced production of IL-4) by Ag-stimulated CD4+ T cells from the DO.11.10 transgenic mouse expressing a TCR specific for an OVA peptide (OVA323–339). In contrast, a phosphorylcholine-containing glycoprotein, ES-62, secreted by the filarial nematode, Acanthocheilonema viteae, which generates a Th2 Ab response in vivo, is found to induce the maturation of dendritic cells (DC2) with the capacity to induce Th2 responses (increased IL-4 and decreased IFN-γ). In addition, we show that the switch to either Th1 or Th2 responses is not effected by differential regulation through CD80 or CD86 and that a Th2 response is achieved in the presence of IL-12.

Immunomodulation via Novel Use of TLR4 by the Filarial Nematode Phosphorylcholine-Containing Secreted Product, ES-62

Filarial nematodes, parasites of vertebrates, including humans, secrete immunomodulatory molecules into the host environment. We have previously demonstrated that one such molecule, the phosphorylcholine-containing glycoprotein ES-62, acts to bias the immune response toward an anti-inflammatory/Th2 phenotype that is conducive to both worm survival and host health. For example, although ES-62 initially induces macrophages to produce low levels of IL-12 and TNF-α, exposure to the parasite product ultimately renders the cells unable to produce these cytokines in response to classic stimulators such as LPS/IFN-γ. We have investigated the possibility that a TLR is involved in the recognition of ES-62 by target cells, because phosphorylcholine, a common pathogen-associated molecular pattern, appears to be responsible for many of the immunomodulatory properties of ES-62. We now demonstrate that ES-62-mediated, low level IL-12 and TNF-α production by macrophages and dendritic cells is abrogated in MyD88 and TLR4, but not TLR2, knockout, mice implicating TLR4 in the recognition of ES-62 by these cells and MyD88 in the transduction of the resulting intracellular signals. We also show that ES-62 inhibits IL-12 induction by TLR ligands other than LPS, bacterial lipopeptide (TLR2) and CpG (TLR9), via this TLR4-dependent pathway. Surprisingly, macrophages and dendritic cells from LPS-unresponsive, TLR4-mutant C3H/HeJ mice respond normally to ES-62. This is the first report to demonstrate that modulation of cytokine responses by a pathogen product can be abrogated in cells derived from TLR4 knockout, but not C3H/HeJ mice, suggesting the existence of a novel mechanism of TLR4-mediated immunomodulation.

Characterisation of the phosphorylcholine-containing N-linked oligosaccharides in the excretory-secretory 62 kDa glycoprotein of Acanthocheilonema viteae

The major excretory-secretory product of the rodent filarial nematode Acanthocheilonema viteae is a 62 kDa glycoprotein (ES-62), which has phosphorylcholine, attached to the N-linked carbohydrates. In this paper, we describe structural studies of N-glycans released from ES-62 by peptide N-glycosidase F. Three major classes of N-glycan structures were observed: high mannose type structures; those which had been fully trimmed to the trimannosyl core and were sub-stoichiometrically fucosylated; and those with a trimannosyl core, with and without core fucosylation, carrying between one and four additional N-acetylglucosamine resides. Of the three classes of glycans, only the last was found to be substituted with detectable levels of phosphorylcholine. The implications of these results with respect to the probable glycosylation pathways operating in A. viteae are discussed.

Structural Studies of N-Glycans of Filarial Parasites CONSERVATION OF PHOSPHORYLCHOLINE-SUBSTITUTED GLYCANS AMONG SPECIES AND DISCOVERY OF NOVEL CHITO-OLIGOMERS

N-Type glycans containing phosphorylcholine (PC-glycans), unusual structures found in the important human pathogens filarial nematodes, represent a novel target for chemotherapy. Previous work in our laboratories produced compositional information on the PC-glycan of ES-62, a secreted protein of the rodent parasite Acanthocheilonema viteae. In particular, we established using fast atom bombardment mass spectrometry (MS) analysis that PC was attached to a glycan with a trimannosyl core, with and without core fucosylation, carrying between one and four additional N-acetylglucosamine residues. In the present study, we demonstrate that this structure is conserved among filarial nematodes, including the parasite of humans,Onchocerca volvulus, for which new drugs are most urgently sought. Furthermore, by employing a variety of procedures, including collision-activated dissociation MS-MS analysis and matrix-assisted laser desorption MS analysis, we reveal that surprisingly, filarial nematodes also contain N-linked glycans, the antennae of which are composed of chito-oligomers. To our knowledge, this is the first report of such structures in a eukaryotic glycoprotein.

The phosphorycholine moiety of the filarial nematode immunomodulator ES-62 is responsible for its anti-inflammatory action in arthritis

Objective: In countries where parasitic infections are endemic, autoimmune disease is relatively rare, leading to the hypothesis that parasite-derived immunomodulators may protect against its development. Consistent with this, we have previously demonstrated that ES-62, a 62 kDa phosphorylcholine (PC)-containing glycoprotein that is secreted by filarial nematodes, can exert anti-inflammatory action in the murine collagen-induced arthritis (CIA) model and human rheumatoid arthritis-derived synovial tissue cultures. As a first step to developing ES-62-based drugs, the aim of this study was to determine whether the PC-moiety of ES-62 was responsible for its anti-inflammatory actions. Methods: We compared the anti-inflammatory activity of a PC-free form of recombinant ES-62 (rES-62) and a synthetic PC-ovalbumin conjugate (OVA-PC) with that of native ES-62 in the CIA model and synovial tissues from patients with rheumatoid arthritis. Results: The anti-inflammatory actions of ES-62 in CIA appear to be dependent on the PC moiety as indicated by the reduction in severity of disease and also suppression of collagen-specific T helper 1 cytokine production observed when testing OVA-PC, but not rES-62. Interestingly, the anti-inflammatory activity of PC did not correlate with a reduction in anti-collagen IgG2a levels. Also, the ES-62-mediated suppression of interferon-γ from human patient tissues could be mimicked by OVA-PC but not rES-62 or ovalbumin. Conclusions: In countries where filariasis is endemic the reduced detection of inflammatory diseases, such as rheumatoid arthritis may be because of the anti-inflammatory action of the PC moieties of ES-62. PC may thus provide the starting point for the development of novel, safe immunomodulatory therapies.

Immunomodulatory properties of a phosphorylcholine‐containing secreted filarial glycoprotein

ES‐62 is a phosphorylcholine (PC)‐containing glycoprotein which is secreted by the rodent filarial nematode Acanthocheilonema viteae. A homologue exists in the human filarial nematode Brugia malayi and indeed PC is found attached to glycoproteins of many, if not all, filarial species. At concentrations equivalent to those found for PC‐containing molecules in the bloodstream of parasitized humans, ES‐62 is able to polyclonally activate certain protein tyrosine kinase and mitogen‐activating protein kinase signal‐transduction elements in B and T lymphocytes following in‐vitro exposure. Although this interaction is insufficient to cause lymphocyte proliferation per se, it serves to desensitize the cells to subsequent activation of the phosphoinositide‐3‐kinase, protein kinase C and Ras mitogen‐activating protein kinase pathways and hence also to proliferation via the antigen receptors. The active component of ES‐62 appears to be PC, as the results obtained with ES‐62 are broadly mimicked by PC conjugated to BSA or PC alone. Although PC can also be shown to desensitize B cells following in‐vivo administration, not all cells are affected, as it is still possible to generate an antibody response. Dissection of this response indicates that it is of the Th2 type.

Phosphorylcholine mimics the effects of ES-62 on macrophages and dendritic cells.

Modulation of macrophage/dendritic cell (DC) cytokine production by the filarial nematode phosphorylcholine (PC)‐containing product, ES‐62, is mediated by Toll‐like receptor (TLR) 4 and signal transduction depends on the TLR adaptor MyD88. Intriguingly, comparison of TLR4 knock‐out (ko) mice with TLR4 mutant C3H/HeJ mice indicates that ES‐62 cytokine responses are not dependent on the Pro712 residue of TLR4, which is crucial for the response to bacterial lipopolysaccharide (LPS). Because other immunomodulatory effects of ES‐62 have been attributed to PC we have now investigated, using PC conjugated to ovalbumin (PC‐Ova), whether PC is responsible for the interaction of ES‐62 with TLR4. PC‐Ova mimicked the modulation of interleukin (IL)‐12 production by ES‐62 in a TLR4‐ and MyD88‐dependent manner and as with native ES‐62, PC‐Ova effects were not dependent on Pro712. Furthermore, both native ES‐62 and PC‐Ova suppressed Akt phosphorylation, whereas neither altered the activation of p38 or Erk MAP kinases. To rule out any role for the ES‐62 protein component, we tested a PC‐free recombinant ES‐62 (rES‐62) generated in the yeast Pichia pastoris. Surprisingly, rES‐62 also modulated IL‐12 production, but in a TLR4/MyD88‐independent manner. Furthermore, rES‐62 strongly activated both the p38 and Erk MAP kinases and Akt. However, recent biophysical analysis suggests there are differences in folding/shape between native and rES‐62 and hence data obtained with the latter should be treated with caution. Nevertheless, although our study indicates that PC is likely to be primarily responsible for the modulation of cytokine production observed with native ES‐62, an immunomodulatory role for the protein component cannot be ruled out.

In vivo exposure of murine dendritic cell and macrophage bone marrow progenitors to the phosphorylcholine-containing filarial nematode glycoprotein ES-62 polarizes their differentiation to an anti-inflammatory phenotype

We have previously shown in an in vitro study that the filarial nematode phosphorylcholine (PC)‐containing glycoprotein ES‐62 promotes a murine dendritic cell (DC) phenotype that induces T helper type 2 (Th2) responses. We now show that, in addition to directly priming Th2 responses, ES‐62 can act to dampen down the pro‐inflammatory DC responses elicited by lipopolysaccharide. Furthermore, we also demonstrate that murine DCs and macrophages derived ex vivo from bone marrow cells exposed in vivo to ES‐62 by release from osmotic pumps are hyporesponsive to subsequent stimulation with lipopolysaccharide. These effects can be largely mimicked by exposure to the PC moiety of ES‐62 conjugated to an irrelevant protein. The data we provide are, as far as we aware, the first to show that a defined pathogen product can modulate the developmental pathway of bone marrow cells of the immune system in vivo. Such a finding could have important implications for the use of pathogen products or their derivatives for immunotherapy.

Hyporesponsiveness of murine B lymphocytes exposed to the filarial nematode secreted product ES-62 in vivo

ES‐62 is a phosphorylcholine (PC)‐containing glycoprotein secreted by filarial nematodes, parasites of vertebrates including humans. We have previously demonstrated that pre‐exposure to this molecule in vitro interferes with subsequent B‐cell receptor (BCR)‐dependent activation of murine splenic B lymphocytes. To investigate the significance of this during filarial nematode infection, we now employ mice exposed to ES‐62, at concentrations equivalent to those found for PC‐containing molecules in the bloodstream of parasitized humans, via release from implanted osmotic pumps. Using this approach, we reveal that splenic and lymph node mononuclear cells, and also purified splenic B cells recovered from these mice have reduced ability ex vivo to proliferate in response to BCR ligation. The effect on BCR‐induced proliferation was further investigated with respect to elucidating the mechanism of action of the parasite product and was shown to be associated with impaired signal transduction affecting the ErkMAPkinase pathway. Also, it was found that ES‐62 did not act by promoting apoptosis or by priming for apoptosis following subsequent stimulation, but rather, appeared to render cells hyporesponsive to stimulation. ES‐62 is thus shown for the first time to be a potent modulator of B lymphocyte function in vivo at a concentration relevant to natural filarial nematode infection. This finding considerably strengthens the idea that ES‐62 plays a role in evasion of the immune response during parasitism.

Presence of Phosphorylcholine on a Filarial Nematode Protein Influences Immunoglobulin G Subclass Response to the Molecule by an Interleukin-10-Dependent Mechanism

ABSTRACT The filarial nematode product ES-62 contains phosphorylcholine (PC) covalently attached to N-linked glycans. ES-62 induced high levels of immunoglobulin G1 (IgG1) antibodies, but no IgG2a, to non-PC epitopes of the molecule following subcutaneous injection into BALB/c mice. Conversely, mice given ES-62 lacking PC demonstrated significant production of both IgG subclasses. Thus, PC appears to block production of IgG2a antibodies to other epitopes on the parasite molecule. A role for interleukin-10 (IL-10) in this effect was shown by the ability of IL-10−/− mice to make an IgG2a antibody response to non-PC epitopes of ES-62.