Helen S. Goodridge

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.

Differential regulation of interleukin-12 p40 and p35 induction via Erk mitogen-activated protein kinase-dependent and -independent mechanisms and the implications for bioactive IL-12 and IL-23 responses

Bioactive interleukin (IL)‐12 is a 70 000‐molecular weight (MW) heterodimeric cytokine comprising p40 and p35 chains. However, p40 can also form homodimers that antagonize bioactive IL‐12 or heterodimerize with p19 to form IL‐23, which exhibits overlapping yet distinct functions to that of IL‐12. We now define distinct signalling mechanisms that regulate lipopolysaccharide (LPS)‐mediated induction of IL‐12 p40 and p35 in macrophages and which may therefore provide therapeutic targets for precise and specific fine‐tuning of cytokine responses. Thus, whilst LPS‐induced p38 mitogen‐activated protein kinase (MAPkinase) activation is required for the induction of both p40 and p35 subunits, Erk MAPkinase signalling mediates negative feedback regulation of p40, but not p35, production. Such Erk MAPkinase activation is downstream of calcium influx and targets LPS‐induced IL‐12 p40 transcription by suppressing the synthesis of the transcription factor, interferon regulatory factor‐1 (IRF‐1). In contrast, negative regulation of the p35 subunit of IL‐12 occurs via a calcium‐dependent, but Erk‐independent, mechanism, which is likely to involve nuclear factor (NF)‐κB signalling. Finally, the importance of both Erk and p38 MAPkinases in differentially regulating IL‐12 p40 and p35 production is underscored by each being targeted by ES‐62, a product secreted by parasitic filarial nematodes to polarize the immune system towards an anti‐inflammatory phenotype conducive to their survival.

Modulation of Macrophage Cytokine Production by ES-62, a Secreted Product of the Filarial Nematode Acanthocheilonema viteae

Parasite survival and host health may depend on the ability of the parasite to modulate the host immune response by the release of immunomodulatory molecules. Excretory-secretory (ES)-62, one such well-defined molecule, is a major secreted protein of the rodent filarial nematode Acanthocheilonema viteae, and has homologues in human filarial nematodes. Previously we have shown that ES-62 is exclusively associated with a Th2 Ab response in mice. Here we provide a rationale for this polarized immune response by showing that the parasite molecule suppresses the IFN-γ/LPS-induced production, by macrophages, of bioactive IL-12 (p70), a key cytokine in the development of Th1 responses. This suppression of the induction of a component of the host immune response extends to the production of the proinflammatory cytokines IL-6 and TNF-α, but not NO. The molecular mechanism underlying these findings awaits elucidation but, intriguingly, the initial response of macrophages to ES-62 is to demonstrate a low and transient release of these cytokines before becoming refractory to further release induced by IFN-γ/LPS. The relevance of our observations is underscored by the finding that macrophages recovered from mice exposed to “physiological” levels of ES-62 by the novel approach of continuous release from implanted osmotic pumps in vivo were similarly refractory to release of IL-12, TNF-α, IL-6, but not NO, ex vivo. Therefore, our results suggest that exposure to ES-62 renders macrophages subsequently unable to produce Th1/proinflammatory cytokines. This likely contributes to the generation of immune responses with an anti-inflammatory Th2 phenotype, a well-documented feature of filarial nematode infection.

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.

Immunomodulatory properties of Ascaris suum glycosphingolipids – phosphorylcholine and non‐phosphorylcholine‐dependent effects

Immunomodulatory properties of phosphorylcholine (PC)‐containing glycosphingolipids from Ascaris suum were investigated utilizing immune cells from BALB/c mice. Proliferation of splenic B cells induced either via F(ab′)2 fragments of anti‐murine Ig (anti‐Ig) or LPS was significantly reduced when the glycosphingolipids were present in the culture medium. However whereas the LPS‐mediated effect was dependent on the PC moiety of the glycosphingolipids, the result generated when using anti‐Ig was not. Analysis of cell cycle status and mitochondrial potential indicated that the combination of the glycosphingolipids and anti‐Ig reduced B cell proliferation, at least in part, by inducing apoptosis. Consistent with the observed suppression of B cell activation/cell cycle progression, investigation of the effect of glycosphingolipid pre‐exposure on mitogenic B cell signal transduction pathways activated by anti‐Ig, revealed a PC‐independent inhibitory effect on dual (thr/tyr) phosphorylation and activation of ErkMAPKinase. The glycosphingolipids were also investigated for their inhibitory effect on LPS/IFN‐γ induced Th1/pro‐inflammatory cytokine production by peritoneal macrophages. It was found that IL‐12 p40 production was inhibited and in an apparently PC‐dependent manner. Overall these data indicate that PC‐containing glycosphingolipids of A. suum appear to have at least two immunomodulatory constituents – PC and an as yet unknown component.

Signalling mechanisms underlying subversion of the immune response by the filarial nematode secreted product ES-62

Secretion of immunomodulatory molecules is a key strategy employed by pathogens to enable their survival in host organisms. For example, arthropod‐transmitted filarial nematodes, which achieve longevity within the infected host by suppressing and modulating the host immune response, produce excretory–secretory (ES) products that have been demonstrated to possess immunomodulatory properties. In this review we discuss the immunomodulatory effects of the phosphorylcholine‐containing filarial nematode‐secreted glycoprotein ES‐62 and describe the intracellular signal transduction pathways it targets to achieve these effects.

In vivo activation of murine peritoneal B1 cells by the filarial nematode phosphorylcholine-containing glycoprotein ES-62

Mice were subcutaneously implanted with osmotic pumps loaded with ES‐62, an immunomodulatory phosphorylcholine (PC)‐containing glycoprotein secreted by filarial nematodes. The concentration of ES‐62 was set to give a serum level within the range found for PC‐containing molecules during natural filarial nematode infection of humans. Peritoneal B1 cells were recovered from the mice and the effect of exposure to ES‐62 on a number of parameters determined ex vivo. B1 cells exposed to ES‐62 showed an increase in spontaneous proliferation that was enhanced by ex vivo exposure to F(ab′)2fragments of anti‐IgM antibodies (anti‐IgM), to activate via the antigen receptor, or LPS. Consistent with this, cell‐cycle analysis indicated that cells pre‐exposed to ES‐62 showed increased cell‐cycle progression following stimulation with anti‐IgM. Pre‐exposed cells also showed an increase in both spontaneous and anti‐IgM induced IL‐10 secretion. Taken together, these data indicate that ES‐62 activates murine B1 cells in vivo. Conversely, we have previously shown conventional (B2) B cells to be rendered hypo‐responsive by in vivo exposure to ES‐62 and the different effect on the two cell types is discussed in relation to the nature of the antibody response arising during filarial nematode infection.

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 worms 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.

Subversion of immune cell signal transduction pathways by the secreted filarial nematode product, ES-62.

Filarial nematodes achieve longevity within the infected host by suppressing and modulating the host immune response. To do this, the worms actively secrete products that have been demonstrated to possess immunomodulatory properties. In this article we discuss the immunomodulatory effects of the phosphorylcholine-containing filarial nematode secreted glycoprotein ES-62. In particular we describe how it modulates intracellular signal transduction pathways in a number of different cells of the immune system, in particular B-lymphocytes, T-lymphocytes, macrophages and dendritic cells.