The effects of ES-62 on DC maturation and effector function

Parasitic filarial nematodes are often tolerated in their human hosts for decades, with little evidence of pathology, due to parasite-induced immune modulation. The consensus of opinion, to date, is that such parasites achieve prolonged survival as a result of their active modulation of the host’s immune system by secreted immunomodulatory molecules. Consistent with this, the excretory-secretory protein of Acanthocheilonema vitae, ES-62, has been shown to exhibit immunomodulatory activities that are broadly Th2/anti-inflammatory in nature. Dendritic cells are specialised antigen (Ag)-presenting cells critical for initiating and regulating immune responses. They stimulate T cells and produce cytokines that have important immuneregulatory functions. To date, more than 6 different DC subtypes have been identified that arise from at least two different cell lineages. These subtypes are not only phenotypically distinct but they are also thought to perform different functions and be restricted to certain lymphoid organs. Thus, it was proposed that ES-62 might differentially affect individual DC subtypes and thereby polarise the immune response towards a Th2/anti-inflammatory phenotype. Consistent with this hypothesis, when splenic cDCs were exposed to ES-62, slightly different cytokine profiles were produced by the individual DC subtypes. However, when such cDC subtypes were used to prime Ag-specific T cells, although minor differences could be detected, the overall T cell response displayed a Th2/Treg phenotype. The ability of prior exposure to ES-62 to prevent LPS-matured DC subtypes from inducing Th1 polarisation was also investigated. Although such DCs showed reduced development of a Th1/pro-inflammatory phenotype, only weak Th2 effector responses could be seen suggesting that whilst ES-62 induced a Th2/anti-inflammatory-promoting phenotype with all cDC subsets tested, this maturation could be at least partially reversed/overcome by a subsequent strong pro-inflammatory signal. In addition, the effect of ES-62 on maturation of the pDC phenotype, and consequent ability to induce Th2/anti-inflammatory responses, was investigated. Similarly, the effects of CpG, which is known to activate pDCs, and LPS, a TLR4 ligand which is reported to have no effect on such pDCs, and the ability of such differentially activated pDC phenotypes to prime Ag-specific T cells were studied. Whilst only low levels of cytokines were produced by pDC upon stimulation with any of these molecules, ES-62-exposed pDCs were able to induce Th2-like responses, whereas both CpG- and LPS-matured pDCs induced pro-inflammatory phenotypes. Therefore to investigate the ability of ES-62 to inhibit such responses, pDCs were pre-exposed to ES-62 and subsequently matured with either CpG or LPS. Interestingly, while ES-62+LPS-matured pDCs could promote a Th2 phenotype, pre-exposure to ES-62 could only partially inhibit the pro-inflammatory phenotype induced by GpG. DCs are the main Ag-presenting cells in the body. They express a variety of receptors, including pattern recognition receptors, such as Toll–like receptors (TLRs), C-type lectin receptors (CLRs) and also receptors belonging to the Mannose Receptor group. Furthermore, DCs also express the platelet activating factor receptor (PAF receptor). ES-62 has previously been shown to depend on the presence of TLR4 for exhibiting its effects on bmDC and macrophages, although it has been determined that the receptor does not need to be functional. Thus, it was believed that ES-62 might signal through a different co-receptor or might even be able to utilize a different receptor in cells, such as pDCs, which naturally lack expression of TLR4. Thus, in addition to further investigating the role of TLR4-signalling in the transduction of ES-62-mediated immunomodulation, signalling through two other receptors, namely the mannose receptor (MR) and the PAF receptor, were also investigated. In these studies, it was shown that, in cells expressing TLR4, ES-62 depends on the presence of this receptor, and its downstream adaptor molecule MyD88, to activate DCs and to subsequently induce T cell priming. Whilst no loss of function, in terms of cytokine secretion, could be detected in DCs deficient for the MR or the PAF receptor, ES-62-matured DCs from PAF receptor KO mice appeared to prime slightly stronger Th2/anti-inflammatory responses. Furthermore, whilst LPS treatment of such PAF receptor KO-DCs induced cytokine production similar to that seen with WT DCs, they were unable to prime T cells. Thus, these results indicated that LPS-driven Th1-polarisation is at least partially dependent on signalling through the PAF receptor and that this receptor might be a possible target for ES-62 mediated inhibition of pro-inflammatory signalling. Finally the potential therapeutic role of ES-62 in two murine models of disease, namely collagen-induced arthritis (CIA) and asthma was investigated. In the CIA model, both prophylactic and therapeutic application of the parasite product in vivo has been shown to be beneficial, resulting in delayed onset and reduced severity of disease or a reduction in the severity of existing inflammation, respectively. Thus, it was decided to further investigate the mechanisms underlying such immunomodulation by studying the effects of ES-62 on individual DC subtypes from mice in this disease model. One of the most interesting findings during these experiments was the apparent lack of double negative (DN) DCs recovered from spleens from CIA model mice. However, investigations of the effects of ES-62 on the maturation of the individual subtypes did not reveal any significant differences between individual DC subtypes. Furthermore, when these cells were cultured with T cells from CIA model mice, no significant Th1 or Th2 cytokine production could be found, and collectively the cytokine profile looked very similar to that of disease-associated DCs cultured on their own. By contrast, investigation of the effect of ES-62-treatment in asthma model mice showed that ES-62 was able to drive two different responses. In the lung, draining lymph nodes and the spleen, ES-62 inhibited inflammation and Th2 development, by reducing Th2 cytokines, whereas it promoted Th2 development in peripheral lymph nodes. Interestingly although clinical improvement has been associated with an increase in lung levels of IL10, a Treg-associated cytokine, and increased levels were found in spleen cell cultures following ES-62 treatment of “asthma” model mice, no increase in natural Tregs could be found. Thus to further dissect the mechanisms by which ES-62 mediates its anti-inflammatory action in this disease model, bone-marrow derived DCs from “asthma” model mice treated with ES-62 or PBS in vivo were analysed for cytokine production and for their ability to prime Th cells, either in an immature or LPS-matured state. The results of this study showed that whilst “asthma” group bmDCs did not induce production of pro-inflammatory cytokines, they were able to prime T cells towards a Th2 phenotype. Interestingly, this Th2-like response was reduced if bmDCs had been treated with ES-62 in vivo. By contrast LPS maturation of such “asthma” group bmDCs, and, to a much lesser extent, ES-62 treated “asthma” bmDCs, resulted in increased production of pro-inflammatory cytokines. Furthermore while these “asthma” group DCs induced a slightly reduced Th2 response, the reverse was true for bmDCs from “asthma” mice exposed to ES-62 in vivo. Collectively, the data obtained from the asthma model showed that ES-62 is capable of driving differential immune responses at distinct sites within an animal. In summary, the results presented in this thesis provide novel information about the mechanisms underlying the modulation of T cell responses by ES-62-exposed DC subtypes, and may have identified a putative pathway by which ES-62 might inhibit pro-inflammatory signalling. Furthermore, these studies provide a better understanding of the therapeutic potential of this parasite molecule in inflammatory diseases such as CIA and asthma and collectively, they have demonstrated the potent immunomodulatory effects of ES-62.