Thesis
The composition and function of extracellular vesicles in leishmaniasis
University of Iowa
Master of Science (MS), University of Iowa
Autumn 2020
DOI: 10.17077/etd.005737
Abstract
Visceral leishmaniasis (VL) is a potentially fatal disease caused by Leishmania donovani or L. infantum. Parasites reside quiescently in macrophages. Despite their intracellular nature, even localized forms of the disease caused by these parasites are characterized by profound systemic effects. Leishmania-infected macrophages, like all eukaryotic cells studied, release small, protein- and RNA-laden extracellular vesicles, many of which are sometimes called exosomes, which facilitate intercellular communication. It is unknown how the systemic effects of Leishmaniasis are caused. This thesis is based on the hypothesis that exosomes released from infected macrophages mediate at least some of the systemic immune changes in VL. The data presented support this hypothesis and raise new questions as to the pathways by which this is achieved.
Chapter 2 provides evidence that infection by L. infantum alters the protein composition of exosomes released by human macrophages. In order for the overall hypothesis to be correct, exoproteomes of infected macrophages must differ from uninfected macrophages. In experiments begun by previous student in the Wilson lab, Patrick Kelly, monocyte derived macrophages (MDMs) from 4 healthy human donors were infected with L. infantum and extracellular parasites removed. Exosomes were collected and exoproteomes were isotope labeled and identified by LC-MS/MS. Data were exported in MaxQuant 1.6.3.3, and analyzed with Perseus 1.6.2.3.
After filtering for quality and for proteins found in all 5 technical replicates of at least one donor at least one infection status, 244 were quantified. Pairwise comparison revealed significant differences between donors (FDR 0.05, S0=1), and between infected and uninfected states. 2-way ANOVA (p<0.05) revealed 100 proteins differed significantly by infection status. According to DAVID analysis, proteins found to be present at significantly different levels based on infection status included proteins associated with pathways that have been termed, inflammation, lipid metabolism, and the regulation of chromatin. This led to us to hypothesize that the pathology seen in leishmaniasis may be, in-part, due to mis-regulation of chromatin, and that MDMs exposed to exosomes derived from infected cells would show the same M2-like quiescent state that MDMs exposed to L. infantum parasites exhibit.
In chapter 3, we subsequently queried the potential functions of these exosomes if released systemically in host, which might lead to some of the systemic effects of leishmaniasis. As a means of modeling the effects of exosomes released from macrophages containing intracellular parasites, for instance macrophages of the liver and spleen in visceral leishmaniasis, we set out to evaluate this was through the application of exosomes isolated from infected or uninfected human monocytes on MDMs isolated from the same donor. We hypothesized that MDMs exposed to exosomes derived from infected cells would show the same M2-like quiescent state that MDMs exposed to L. infantum parasites exhibit. We do not have data for these experiments at this time. Plans for the continuation of these experiments, as well as other likely pathways of investigation revealed by the data presented in chapter 2, are discussed in chapter 4.
Our data supported our overall hypothesis that exosomes released from infected macrophages mediate some systemic immune changes in VL and raised new questions as to the pathways by which this is achieved.
Details
- Title: Subtitle
- The composition and function of extracellular vesicles in leishmaniasis
- Creators
- M. Murphy Keller
- Contributors
- Mary Wilson (Advisor)Christine Petersen (Committee Member)Robert Piper (Committee Member)
- Resource Type
- Thesis
- Degree Awarded
- Master of Science (MS), University of Iowa
- Degree in
- Interdisciplinary Studies
- Date degree season
- Autumn 2020
- DOI
- 10.17077/etd.005737
- Publisher
- University of Iowa
- Number of pages
- xv, 87 pages
- Copyright
- Copyright 2020 M. Murphy Keller
- Language
- English
- Description illustrations
- illustrations (some color)
- Description bibliographic
- Includes bibliographical references (page 58-61).
- Public Abstract (ETD)
Leishmania spp. parasites cause the disease leishmaniasis, including Visceral leishmaniasis (VL), which is usually fatal in humans without treatment. There are an estimated 0.5 million new reported cases and 50,000 deaths caused by VL annually. Current treatments are associated with toxicity, and many are administered intravenously and thus cumbersome in endemic regions. There is emerging drug resistance, and the drugs frequently have costs higher than the average annual household income of afflicted individuals. Novel approaches to disease management, therefore, are needed.
Like most cells, human cells infected by leishmaniasis release small packets of cellular information called exosomes. Exosomes play a role in cellular communication both under normal circumstances and during disease. We hypothesized that exosomes released from infected human cells play a role in the progression of disease. The data presented support this hypothesis and raise new questions as to how the exosomes do so.
Chapter 2 provides evidence that infection by L. infantum alters the protein composition of exosomes released by human macrophages. Proteins that were found to be present at significantly different levels based on infection status included proteins associated with inflammation, viral uptake, lipid metabolism, and the regulation of chromatin. Chapter 3 describes preliminary experiments that have been begun to test the function of exosomes during disease.
- Academic Unit
- Interdisciplinary Studies Program
- Record Identifier
- 9984036791702771
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