Functional characterisation of a novel immune modulatory molecule from Fasciola hepatica

Field	Value	Language
dc.contributor.author	Alvarado, R
dc.date.accessioned	2015-06-03T05:20:47Z
dc.date.available	2015-06-03T05:20:47Z
dc.date.issued	2014
dc.identifier.uri	http://hdl.handle.net/10453/35990
dc.description	University of Technology, Sydney. Faculty of Science.	en_US
dc.description.abstract	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.	en_US
dc.format	Thesis (PhD)	en_US
dc.language.iso	en	en_US
dc.relation	https://opus.lib.uts.edu.au/bitstream/10453/35990/13/02whole.pdf
dc.rights	info:eu-repo/semantics/openAccess
dc.rights	The author owns the copyright in this thesis including all reproduction and reuse rights for the work. The work may not be altered without the permission of the copyright owner. Attribution is essential when quoting or paraphrasing from this thesis.
dc.rights	au.edu.uts.lib/ppc
dc.rights	© 2014 Raquel Alvarado
dc.title	Functional characterisation of a novel immune modulatory molecule from Fasciola hepatica	en_US
dc.type	Thesis
utslib.copyright.status	open_access

Abstract:

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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