Functional characterisation of a novel immune modulatory molecule from Fasciola hepatica

Publication Type:
Thesis
Issue Date:
2014
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The ability of tissue dwelling helminth parasites to induce chronic long term infections, is enabled by the establishment of T helper 2/ regulatory T cell (Th2/Treg) immune responses within their mammalian hosts. Such responses prevent the expulsion of the parasites, whilst simultaneously avoiding excessive inflammation/fibrosis arising within the host, as a consequence of tissue damage induced by helminth migration. Importantly, helminths excrete and secrete a series of molecules (collectively known as ES products), which not only play major roles in parasite biology, but also exert direct immune modulatory functions, promoting the establishment of Th2/Treg immunity. The trematode, Fasciola hepatica, is an excellent model of helminth-mediated immune modulation, because it induces a very rapid switch towards Th2 responses in its mammalian hosts and inhibits Th1 immunity. Fractionation of the ES products of F. hepatica has identified three major immune modulatory components: the protease cathepsin L1, the antioxidant peroxiredoxin, and a previously uncharacterised peptide, FhHDM-1. Structural analysis of FhHDM-1 revealed a close resemblance to the cathelicidin, LL-37, a well characterised mammalian immune-modulating peptide. Therefore, a putative immune modulatory role for FhHDM-1 was explored in this project. Immunofluorescent confocal microscopy demonstrated that FhHDM-1 interacted with macrophage lipid rafts, prior to being actively internalised by cholesterol- and cytoskeletal network-dependent endocytosis, with progressive compartmentalisation of the peptide into early endosomes and endolysosomal vesicles. Flow cytometry studies indicated that, once internalised, FhHDM-1 enhanced the rate of endocytosis of dextran by macrophages. Despite this, FhHDM-1 was found to impair the acidification of macrophage endolysosomes and as a consequence, the efficient processing and subsequent presentation of ovalbumin to T cells was prevented, as assessed by decreased detection of digested fluorescent ovalbumin and reduced IL-2 secretion by transgenic CD4⁺ T cells. Additionally, FhHDM-1 impaired NLRP3 inflammasome activation by lysosomal disruptive agents in macrophages. This was found to be a consequence of reduced cathepsin B activity (due to FhHDM-1 induced suboptimal lysosomal acidification), which was incapable of stimulating inflammasome complex formation, thus avoiding IL-1β and caspase-1 cleavage. These findings suggest that by targeting endolysosomal activity, FhHDM-1 limits macrophage function. Therefore, the current study is the first to demonstrate that FhHDM-1 possesses immune modulatory properties, which are directed by a mechanism not previously described for a helminth-secreted cathelicidin-like peptide.
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