Role of innate immune complement and toll-like receptor signalling in neurodegeneration

Lee, Jia Yu (2014). Role of innate immune complement and toll-like receptor signalling in neurodegeneration PhD Thesis, School of Biomedical Sciences, The University of Queensland. doi:10.14264/uql.2014.244

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Author Lee, Jia Yu
Thesis Title Role of innate immune complement and toll-like receptor signalling in neurodegeneration
School, Centre or Institute School of Biomedical Sciences
Institution The University of Queensland
DOI 10.14264/uql.2014.244
Publication date 2014
Thesis type PhD Thesis
Supervisor Trent Woodruff
Peter Noakes
Simon Phipps
Stephen Taylor
Total pages 131
Language eng
Subjects 0606 Physiology
0601 Biochemistry and Cell Biology
Formatted abstract
Neuroinflammation is one of the hallmarks that contribute to the pathogenesis of neurodegenerative diseases. The innate immune system is the first line of response against pathogens and tissue injuries in the central nervous system (CNS). Chronic activation of the immune system leads to dysregulation of inflammatory responses, which could contribute to the progressive loss of neurons seen in neurodegenerative disease. Recent studies have shown the two major components of the innate immune system, the complement system, and the toll-like receptor (TLR) system, have a role in many neurodegenerative diseases such as Alzheimer’s disease and amyotrophic lateral sclerosis (ALS). Receptors from these two systems are present on microglial cells and can modulate the inflammatory state of the CNS. Complement factor 5a (C5a) receptor (C5aR) and TLR4 are known to synergise to enhance inflammation outside of the CNS, however, to date, no studies have investigated whether these two systems also interact in neuroinflammatory disease. Therefore, this project initially investigated the interplay between C5aR and TLR4, in inducing pro-inflammatory cytokine release from microglia cells in vitro, and how this interplay may contribute to brain inflammatory responses in an acute in vivo mouse model of sepsis. Previous studies from our laboratories have shown that inhibition of C5aR reduces microglial inflammation, and increases survival in the mouse SOD1G93A transgenic model of ALS. However, a role for TLR4 in this model has not been investigated, and was also characterized in this project. C5a induces its major pro-inflammatory effects through binding to the C5aR. The current study extended previous findings of an interplay between C5aR and TLR4 in peripheral immune cells, to the major inflammatory cell of the CNS, microglia. Our data suggests that the magnitude of C5a-induced cytokine generation may differ depending on the activation state of microglia cells. BV-2 cells, an activated microglia cell line, showed markedly increased pro-inflammatory cytokine generation upon co-administration of C5a and the TLR4 ligand lipopolysaccharide (LPS). However, a more modest pro-inflammatory synergy was observed in primary microglia cells isolated from early post-natal mouse brains. This suggests, that the full enhancement of TLR4-induced cytokine generation by C5a, may require a relevant priming/differentiation stimulus of microglia to take on a major pro-inflammatory phenotype. Such priming stimuli may occur in neurodegenerative disease. To further investigate the role of C5aR-TLR4 in inflammatory responses in vivo, a model of LPS-induced neuroinflammation was next established. Systemic administration of LPS in wild-type mice caused a significant increase in C5a receptor (C5aR and C5L2) expression, along with several pro-inflammatory cytokines in the brain after 24 hours. Using mice deficient in C5aR, these pro-inflammatory cytokines were significantly decreased in the brain compared to wild-type mice after LPS exposure. This suggests that C5a-C5aR interaction in the brain, enhances TLR-4 induced neuroinflammation in vivo. Finally, a role for TLR4 in the hSOD1G93A mouse model of ALS was investigated. TLR4 can initiate an inflammatory response upon exposure to host-derived ligands; damage-associated molecular patterns (DAMPs) such as HMGB1, which are released during tissue injury. In the current study, TLR4 expression was significantly increased at the end-stage of disease in hSOD1G93A mice, and was found to be expressed on glia cells (microglia and astrocytes) and motor neurons in the lumbar spinal cord. This was correlated with increases in C5aR expression (located on microglia), in these same ages. HMGB1 was also significantly increased in the lumbar spinal cord of diseased hSOD1G93A mice, suggesting a potential for TLR4 activation in disease progression. To explore this, hSOD1G93A mice deficient in TLR4 (hSOD1G93A x TLR4-/-) were generated, and these mice were examined for behavior and survival alongside TLR4-sufficient hSOD1G93A mice (hSOD1G93A x TLR4+/+). There was no significant change in motor function between these two groups throughout disease progression, however, TLR4-deficient hSOD1G93A mice had significantly increased survival compared to TLR4-sufficient hSOD1G93A mice. This demonstrates that TLR4 plays a pathogenic role in the hSOD1G93A model of ALS. In summary, this work has demonstrated that microglial C5a receptor activation modulates TLR4 inflammatory responses, with enhancement of LPS-induced cytokine release in vitro. In vivo, C5aR also enhanced inflammatory responses in the brain following systemic administration of LPS. Previous studies have shown that C5aR also plays a pathogenic role in the chronic hSOD1G93A model of ALS. The present study now shows that TLR4 also contributes to disease pathology in this model. Collectively, these results suggest that TLR4-C5aR interplay can enhance neuroinflammatory responses in vivo, and thereby may contribute synergistically to enhance neurodegeneration. As such, the TLR4-C5aR signalling axis is a potential future therapeutic target in neuroinflammatory diseases such as ALS. Future studies could investigate the combined inhibition of TLR4 and C5aR in the hSOD1G93A mice, which may lead to a greater survival outcome, than inhibition of each alone.  
Keyword Neurodegeneration

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Created: Mon, 04 Aug 2014, 02:45:19 EST by Jia Yu Lee on behalf of University of Queensland Graduate School