Investigating the effects of inflammatory mediators on the neuroinflammation response

Sawkulycz, Xenia (2023) Investigating the effects of inflammatory mediators on the neuroinflammation response. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Ischemic stroke is a devastating event which further stimulates a cascade of inflammatory events in the brain, known as neuroinflammation to occur which is important for repair. However, this neuroinflammation is a double-edged sword that can also have a detrimental impact within the brain. Numerous inflammatory markers are known to be stimulated following an ischemic event and one of interest for this study is C reactive protein (CRP) and its isoforms monomeric c reactive protein (mCRP) and native c reactive protein (nCRP). Several different cell types were used throughout this work which included U937 monocytes which were also differentiated into M0 macrophages. Finally, human microglia cell line HMC-3 cell were also used. Lipopolysaccharide (LPS) was used as a positive inflammatory mediator through this work. Pro-inflammatory effects were observed when different cell types were treated with mCRP or nCRP to test for any inflammatory effects. The liver x receptor agonist (GW3965) was selected for its anti-inflammatory properties and current promising research as a therapeutic in ischemic stroke. Micro-fragment adipose tissue (MFAT) conditioned media was used to test its potential anti-inflammatory effect. Levels of inflammatory activity were measured by several different methods including enzyme-linked immunosorbent assay (ELISA), immunofluorescent staining, aggregation analysis and qPCR. Results from this study showed that mCRP can alter the morphology and physiological function of both U937 monocytes and HMC-3 microglia cells. Increased expression of important pro-inflammatory cytokines was observed, including interleukin 1 beta (IL-1b), interleukin 6 (IL-6), tumour necrosis factor alpha (TNFa), highlighting mCRP pro-inflammatory activity. The LXR agonist (GW3965) significantly reduced key proinflammatory proteins after HMC-3 microglia cells were stimulated with either LPS or mCRP inflammatory mediators. However, no up regulation or down regulation was observed in gene expression at any time point after treatment. This study demonstrated that mCRP acts as a proinflammatory mediator on HMC-3 and U937 monocytes. MFAT, was also seen to have key antiinflammatory properties with secreting key cytokines and chemokines. An in vitro inflammatory cell model was used to determine MFAT condition medium can attenuate inflammatory cytokine up regulation. MFAT attenuated the up regulation of protein and gene expression levels of key cytokines (TNFa, IL-1b, IL-6, and IL-10) which are involved in neuroinflammation after an ischemic event such as stroke. In conclusion, this study demonstrated that mCRP could cause morphological and behaviour changes in either U937 monocytes or HMC-3 microglia cells. Further to this mCRP demonstrated pro-inflammatory activity, whilst the use of GW3965 inhibited its pro- inflammatory activity, therefore reducing TNF-α, IL-6, IL-1b, MCP-1, RANTES. On another note, MFAT secreted key cytokines and chemokines into conditioned media (IL-1ra, Il-1b, IL-4)