Dissertation
Effects of Helicobacter pylori induced neutrophil subtype differentiation on chemotaxis and phagosome maturation
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Autumn 2022
DOI: 10.25820/etd.006753
Abstract
Helicobacter pylori is gastric pathogen that colonizes 50% of the global population and evokes a histological gastritis that often remains asymptomatic. This infection is characterized by a neutrophil dominant inflammatory response with increased cell density resulting in increased disease severity. Often persisting for the life of the individual, this pathogenic infection can induce peptic ulcer disease, gastric adenocarcinoma, and even mucosa-associated lymphoid tissue (MALT) lymphoma. Remarkably, the neutrophil dominant immune response fails to resolve infection instead resulting in accumulation of heavily infected cells. Our lab discovered that H. pylori infection prevents neutrophil antimicrobial killing mechanisms by regulating cell respiratory burst, however the mechanisms by which this is achieved, and resultant accumulation of cells is unknown.
We demonstrate within this thesis that H. pylori infection causes defective PMN migration, possibly explaining enhanced accumulation of these cells at sites of infection. Our data reveal failed uropod retraction in a 2D model, which is characterized by aberrant actin structures and mis-localized myosin machinery. This uropod retraction phenotype resembled the effects of myosin machinery inhibitors, however, analysis of myosin phosphorylation by immunoblotting showed that his aspect of the myosin activation cycle significantly increased during infection. Interestingly, adhesion of infected PMNs and subcellular localization of the integrin CD11b/CD18 were unchanged. Furthermore, H. pylori the gastric epithelial virulence effectors tested were dispensable to defective chemotaxis even though we determined that this is an active process that is not recapitulated by formalin-killed bacteria. Thus, as migration is an essential neutrophil function, our phenotype indicates that infected neutrophils exhibit altered cell function.
The next aim of my thesis was to determine the effects of H. pylori on another essential function, phagosome maturation. PMN phagosome maturation during infection results in bacterial killing and degradation through delivery of cytotoxic peptides, enzymes, and reactive oxygen species (ROS) to these compartments. As H. pylori displays intracellular persistence, we examined phagocytosis and phagosome maturation during this infection. This revealed failed phagosome maturation through inhibited granule-phagosome fusion, yet, interestingly, granule exocytosis was globally enhanced after 24 hours of infection. Infection was further characterized by CEACAM recruitment to the forming phagosome, in addition to phagosome accumulation of the autophagy protein LC3B. Taken together, the data in this thesis support a model of altered PMN function that results in failed bacterial clearance and infection persistence despite a robust cellular response.
Details
- Title: Subtitle
- Effects of Helicobacter pylori induced neutrophil subtype differentiation on chemotaxis and phagosome maturation
- Creators
- Allan Scot Prichard
- Contributors
- Lee-Ann H Allen (Advisor)Jason Barker (Committee Member)Craig Ellermeier (Committee Member)Prajwal Gurung (Committee Member)Mary Weber (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Microbiology
- Date degree season
- Autumn 2022
- Publisher
- University of Iowa
- DOI
- 10.25820/etd.006753
- Number of pages
- xix, 157 pages
- Copyright
- Copyright 2022 Allan Scot Prichard
- Language
- English
- Description illustrations
- Illustrations, charts, graphs, tables
- Description bibliographic
- Includes bibliographical references (pages 137-157).
- Public Abstract (ETD)
The human immune system is designed to quickly identify foreign invading pathogens, such as bacteria, and mount a swift response to clear them while minimizing damage to healthy tissue. Neutrophils are one of the most abundant immune cells in the body and are considered the first line of defense against invading bacteria. During an infection, neutrophils migrate toward bacteria and then use a variety of mechanisms to kill and degrade them. This can be achieved by ingestion of bacteria followed by their intracellular destruction, or release of net-like structures that capture and kill bacteria extracellularly. After neutrophils kill bacteria, they proceed to die and are efficiently cleared by other immune cells. This clearance process prevents the build-up of harmful compounds that would otherwise cause tissue damage.
Over millennia, invading bacteria have discovered ways to survive neutrophil killing mechanisms to continue infection in the body. Some bacteria even choose harsh environments in the body to infect where it is more difficult for neutrophils to function. One such bacterium, Helicobacter pylori, lives in the acidic stomach environment where it replicates extensively and kills stomach cells. While neutrophils move toward this infection and eat the invading bacteria, the cell killing mechanisms are insufficient during Helicobacter pylori infection and there is a massive accumulation of heavily infected neutrophils.
We hypothesize that Helicobacter pylori infection of neutrophils is preventing essential cell functions in the body for effective bacterial killing. The goal of this research was to determine how Helicobacter pylori infection regulates neutrophil migration and killing mechanisms to persist in the host. The research presented here establishes that migration is significantly delayed during infection and that the eating mechanism fails to kill and degrade bacteria due to failed delivery of harmful compounds. Together, this work enhances our understanding of how Helicobacter pylori survives the neutrophil response and hijacks these cells to further its own ends.
- Academic Unit
- Microbiology and Immunology
- Record Identifier
- 9984362959102771
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