Transcriptional plasticity of microglia during intraocular inflammation

Student thesis: Doctoral ThesisDoctor of Philosophy (PhD)

Abstract

Microglia are a tissue-resident immune cell of the central nervous system known to possess functions involved in immune surveillance and tissue homeostasis. Transcriptomics has characterised microglia and enabled discovery of microglial heterogeneity in addition to core microglial transcriptional programmes. However, investigation of microglia during severe inflammatory contexts has been challenging because no markers reliably discriminate them from the monocyte populations that ingress during inflammation. Nonetheless, candidate markers have been identified; these show promise in specific microglial identification yet remain to be widely validated.
Within the literature, there are conflicting reports on how microglia regulate or promote inflammation depending on the tissue insult. However, it is well-recognised that the homeostatic state of microglia is altered yet it remains unknown if this state is restored post-resolution. Furthermore, understanding the plasticity of microglial responses to both acute and persistent inflammation within the eye will help to determine the extent to which different pathways are perturbed.
The purpose of this thesis was to investigate the transcriptional changes that occur in retinal microglia in response to inflammation and whether the homeostatic threshold remains perturbed after acute and/or chronic inflammation.
The data presented herein demonstrates how an ultra-low input mRNA-Seq approach was optimised and validated to permit transcriptomic assessment of low numbers of cells isolated from individual retinas. The Cx3cr1CreER:R26-tdTomato mouse line was then validated as microglial-specific during inflammation. mRNA-Seq was utilised to profile the temporal kinetics of the microglial transcriptome in the acute endotoxin-induced uveitis (EIU) model. Restoration of the microglial homeostatic state was confirmed, and key marker changes were orthogonally validated. Furthermore, C5AR1 was validated as a marker for differentiating microglial subsets during inflammation. The next steps have begun to examine microglial behaviour in experimental autoimmune uveitis (EAU), a model of chronic inflammation, and new approaches are being optimised to better understand tissue heterogeneity.
Date of Award21 Jan 2021
Original languageEnglish
Awarding Institution
  • The University of Bristol
SupervisorDavid A Copland (Supervisor), Colin Chu (Supervisor) & Andrew David Dick (Supervisor)

Keywords

  • microglia
  • transcriptome
  • uveitis
  • retina
  • resolution
  • mRNA-Seq
  • lipopolysaccharide (LPS)
  • heterogeneity

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