Secondary bacterial superinfections have been a significant source of debilitating morbidity and mortality outcomes during both seasonal influenza outbreaks and historical pandemics. As many as 40% of those infected with influenza that develop a secondary bacterial infection such as S. aureus or S. pneumoniae will succumb to the infection. The complex relationships between the immune system, the pathogens, and host response makes this facet of biomedical research a topic of continual discoveries. During superinfection, there is a novel hypothesis that the failure of resolution causes a cascade of uncontrolled inflammatory responses leading to excessive morbidity or death. Here, we investigate a specific metabolite receptor, PPARα, and how its induction during superinfection affects the host immune response and the ability to resolve the infection. During superinfections, previous studies from our lab has found that the CYP450 metabolites are produced in abundance compared to their respective levels during singular viral or bacterial infections. These metabolites furthermore induced PPARα, which has been found to enhance necroptosis during superinfection conditions. Understanding programming of cell death provides insights on the resulting inflammation during an active infection. We further examined PPARα properties by inducing it without the presence of pathogens and found PPAR’s mechanisms to be context dependent. When PPARα is stimulated solely with a chemical agonist WY14643, the induction drives macrophages to apoptosis. When we started examining PPARα induction while inducing programmed cell death profiles, we found that the effect of PPARα to be uninfluential to apoptosis, but highly influential in necroptotic cell death. Latest studies associate very long chain fatty acid accumulation in cells undergoing necroptosis, and fatty acid isolation and analysis was completed. Fatty acid isolation showed an accumulation of ceramides and a significant decrease of both vital eicosanoid precursors and cell membrane associated phospholipids.
In these studies, we next examined how induction of PPARα affects macrophage immune response and ultimately hinders resolution of infection. In vivo animal studies showed that macrophages were polarizing toward anti-inflammatory M2 phenotypes during the superinfection. When flow cytometry studies were performed to examine if metabolites stimulating PPARα were responsible for this change, we found that macrophages were polarizing to the M2b phenotype. This finding has been highly intriguing to our studies, as M2b macrophages are most abundantly present when resolution occurs in influenza infection and mice have successfully produced anti-influenza antibodies. Further examination was done to see how macrophage immune response was affected. Using Nanostring and examining PPARα activated macrophages via microscopy, several cytokines and chemokines for immune response were dampened. Microscopy specifically showed the nuclear localization of NFκB is effectively diminished during PPAR activation, demonstrating that the immune response is impaired. Finally, macrophage function was considered and analyzed by CFU assays and live cell microscopy. Both indicated that phagocytosis was impaired in macrophages, but microscopy elucidated that there was a lack of bacterial killing due to PPAR activation.
Understanding the mechanisms of superinfection and how to effectively ameliorate them has potential to not only reduce the amount of morbidity and mortality around influenza each year, but advance the understandings of how these different systems come together in other immune contexts. The understanding of programmed cell-death, inflammatory gene networks, immune response and function all have changed by lipid profiling and how these metabolites can influence traditionally well studied systems. Having a greater appreciation for how metabolites induce immune, transcriptional, or cellular changes, as well as how metabolites and lipid profiling can be altered would be groundbreaking for inflammation research of several diseases and conditions that are not fully understood. / Biomedical Sciences
| Identifer | oai:union.ndltd.org:TEMPLE/oai:scholarshare.temple.edu:20.500.12613/7222 |
| Date | January 2021 |
| Creators | Lucarelli, Ronald, 0000-0002-5628-5338 |
| Contributors | Tam, Vincent, Gallucci, Stefania, Kilpatrick, Laurie, Tükel, Çagla, Ma, Amy T. |
| Publisher | Temple University. Libraries |
| Source Sets | Temple University |
| Language | English |
| Detected Language | English |
| Type | Thesis/Dissertation, Text |
| Format | 112 pages |
| Rights | IN COPYRIGHT- This Rights Statement can be used for an Item that is in copyright. Using this statement implies that the organization making this Item available has determined that the Item is in copyright and either is the rights-holder, has obtained permission from the rights-holder(s) to make their Work(s) available, or makes the Item available under an exception or limitation to copyright (including Fair Use) that entitles it to make the Item available., http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | http://dx.doi.org/10.34944/dspace/7201, Theses and Dissertations |
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