The developed world is experiencing a steep increase in diseases based on aberrant autoimmune inflammatory responses and allergic conditions. By contrast, such diseases are uncommon in countries endemic for parasitic helminths, suggesting that parasite-derived immunomodulators may protect against their development. One such molecule, ES-62, which is a phosphorylcholine (PC)-containing glycoprotein originally identified from the filarial nematode Acanthocheilonema viteae, has shown therapeutic potential in a range of inflammatory diseases. Although its mechanism of action is not fully understood, ES-62 is known to target dendritic cells (DCs) to subvert T helper cell (TH) differentiation and hence modulate the subsequent host immune response to an anti-inflammatory phenotype. ES-62 requires Toll-like receptor 4 (TLR4) to mediate its effects, and here, we have investigated the mechanisms underpinning ES-62-mediated subversion of TLR4-pro-inflammatory signalling as exemplified by its canonical ligand, bacterial endotoxin (lipopolysaccharide, LPS) that results in the activation of the transcription factor, nuclear factor-κB (NF-κB) and consequent secretion of proinflammatory cytokines. This study investigated the mechanism of ES-62-mediated uncoupling of NF-κB activation by its modulation of TLR4 expression and its associated LPS recognising complex TLR4/Myeloid differentiation factor-2 (MD2), as the internalisation, trafficking and degradation of this receptor complex has previously been shown to regulate its responses to different stimuli. ES-62 and LPS were found to differentially modulate the dynamics and expression of the total cellular pool of TLR4, relative to that of the ‘active’ LPS recognising TLR4/MD2 complex. Specifically, ES-62 acted to antagonise the dynamics of the LPS response, providing a mechanism for its induction of TLR4 hyporesponsiveness. In addition, as preliminary studies demonstrated an interaction between ES-62 and the Platelet activating factor receptor (PAFR), the possibility that ES-62 might signal via TLR4 in concert with one or more co-receptors was investigated but the data did not conclusively support a role for the PAFR as a target of ES-62 action. The dissection of the effects of ES-62 on TLR4 signalling was further pursued by defining its modulation and/or sequestration of transduction elements associated with the TLR4 mediated activation of NF-κB. Of particular interest were PKCα, PKCε and PKCδ members of the protein kinase C family that directly interact with TLR4 and its immediate signalling adaptors to mediate proinflammatory cytokine (TNF-α, IL-6 and IL-12p70) 2 production. Several isoforms including PKCα have previously been shown to be targeted in B cells and mast cells for degradation by ES-62. Here, we utilised pharmacological inhibitors of proteosomal and lysosomal protein degradation pathways to demonstrate that whilst LPS induces the turnover of PKCs through the proteosome, ES-62 directs their degradation through the lysosomes. In conjunction with this, ES-62 and LPS were also found to differentially regulate the expression of tumour necrosis factor receptor associated factor-6 (TRAF6) and c-Casitas B lineage lymphoma (c-Cbl), E3 ubiquitn ligases, which act as scaffolds (via Lysine 63 linked polyubiquitination chains) in the activation and stabilisation of NF-κB. However, it was found that ES-62 did not prevent recruitment of these various TLR4 signal transducers upon subsequent recognition of LPS, suggesting that it acts to attenuate hyper-inflammatory responses without leaving an individual immunocompromised to infection. In addition, by mimicking an inflammatory environment with exogenous granulocyte macrophage-colony stimulating factor (GM-CSF), it was found that the actions of ES-62 had more profound effects on the modulation of the aforementioned signal transducers and its range of targets were expanded to include, myeloid differentiation primary responses gene 88 (MyD88), which transmits the early response signals from all the TLR family receptors, except TLR3.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:578575 |
| Date | January 2012 |
| Creators | Eason, Russell John |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/4538/ |
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