The interactions between inflammasome activation and induction of autophagy following Pseudomonas aeruginosa infection

Jabir, Majid Sakhi (2014) The interactions between inflammasome activation and induction of autophagy following Pseudomonas aeruginosa infection. PhD thesis, University of Glasgow.

Abstract

Introduction
Autophagy is a cellular process whereby elements within cytoplasm become engulfed within membrane vesicles and trafficked to fuse with lysosomes. This is a common cellular response to starvation, allowing non-essential cytoplasmic contents to be recycled in times of energy deprivation. However, autophagy also plays an important role in immunity and inflammation, where it promotes host defence and down-regulates inflammation. A specialised bacterial virulence mechanism, the type III secretion system (T3SS) in Pseudomonas aeruginosa (PA), an extracellular bacterium, is responsible for the activation of the inflammasome and IL-1β production, a key cytokine in host defence. The relationship between inflammasome activation and induction of autophagy is not clear.
Hypothesis and aims
The central hypothesis is that induction of autophagy occurs following PA infection and that this process will influence inflammasome activation in macrophages. Our aims were to determine the role of the T3SS in the induction of autophagy in macrophages following infection with PA, and to investigate the effects of autophagy on inflammasome activation and other pro-inflammatory pathways following infection with these bacteria.
Materials and methods
Primary mouse bone marrow macrophages BMDMs were infected with PA, in vitro. Induction of autophagy was determined using five different methods: - electron microscopy, immunostaining of the autophagocytic marker LC3, FACS, RT-PCR assays for autophagy genes, and post-translational conjugation of phosphatidylethanoloamine (PE) to LC3 using Western blot. Inflammasome activation was measured by secretion of active IL-1β and caspase-1 using ELISA and Western blot. Functional requirements of proteins were determined using knockout animals
or SiRNA mediated knockdown.
Result and Conclusions
PA induced autophagy that was not dependent on a functional T3SS but was dependent on TLR4 and the signaling molecule TRIF. PA infection also strongly induced activation of the inflammasome which was absolutely dependent on a functional T3SS. We found that inhibition of inflammasome activation increased autophagy, suggesting that the inflammasome normally inhibits this process. Further experiments showed that this inhibitory effect was due to the proteolytic action of caspase-1 on the signaling molecule TRIF. Using a construct of TRIF with a mutation in the proteolytic cleavage site, prevented caspase-1 cleavage and increased autophagy. TRIF is also involved in the production of interferon-β following infection. We also found that caspase-1 cleavage of TRIF down-regulated this pathway as well. Caspase-1 mediated inhibition of TRIF-mediated signaling is a novel pathway in the inflammatory response to infection. It is potentially amenable to therapeutic intervention. Recognition of a pathogen infection is a key function of the innate immune system that allows an appropriate defensive response to be initiated. One of the most important innate immune defences is provided by a multi-subunit cytoplasmic platform termed the inflammasome that results in production of the cytokine IL-1β.
The human pathogen Pseudomonas aeruginosa activates the inflammasome following infection in a process that is dependent on a specialized bacterial virulence apparatus, the type III secretory system (T3SS). Here, we report the novel finding that this infection results in mitochondrial damage and release of mitochondrial DNA into the cytoplasm. This initiates activation of an inflammasome based on the protein NLRC4. Autophagy induced during infection removes damaged mitochondria and acts to down-regulate NLRC4 activation following infection. Our results highlight a new pathway in innate immune activation following infection with a pathogenic bacterium that could be exploited to improve outcomes following infection.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Inflammasome activations, Autophagy, T3SS, Pseudomonas aeruginosa
• Subjects: Q Science > QR Microbiology; Q Science > QR Microbiology > QR180 Immunology; R Medicine > R Medicine (General)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
• Supervisor's Name: Evans, Prof. Tom
• Date of Award: 2014
• Depositing User: Dr. Majid Sakhi Jabir Jabir
• Unique ID: glathesis:2014-5331
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 06 Aug 2014 10:01
• Last Modified: 06 Aug 2014 10:06
• URI: https://theses.gla.ac.uk/id/eprint/5331