Investigating the impact of TREM2 deficiency on microglial responses and microvascular changes during hypoxia in vivo

Abstract

Chronic hypoxia associated with neurovascular dysfunction is a putative contributor to CNS diseases. Microglial reactivity to hypoxia-related triggers is increasingly recognised in shaping CNS disease trajectory, potentially via effects on microvascular integrity. However, how microglia respond to hypoxia as an isolated stimulus in vivo, and what signalling pathways are employed in relation to the microvasculature remains elusive. Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglial immunoreceptor with key roles in orchestrating microglial reactivity. Our unpublished work in a model of chronic cerebral hypoperfusion showed mice lacking TREM2 have compromised microvascular integrity concomitant with diminished microglial adaptive responses, including blunted transcription of hypoxia signalling genes. Therefore, mice were exposed to 8-9% O₂ in inspired air to investigate the involvement of TREM2 in microglial reactivity and altered microvascular status during brain hypoxia.

Young C57BL/6J mice were used to validate and characterise the experimental model, which revealed that hypoxia induces cerebral microbleeds. Immunofluorescence, flow cytometry and bulk RNA sequencing were then used to assess microglial phenotype, brain immune cell abundance and vascular integrity in Trem2+/+ and Trem2-/- mice following 4 days of hypoxia.

Our results suggest TREM2 signalling does not regulate vascular integrity in hypoxia in adult (6-8 month) mice, but it may play a role in limiting hypoxia-induced microbleed loads in aged (14-17 month) mice. Microglial reactivity following hypoxia was detected at a morphological and transcriptional level, and it included upregulation of interferon, cell cycle, chemotaxis and glycolysis-related genes. Tissue-level examination revealed confinement of microglial responses to the brain region with highest microbleed burden, thus indicating reactivity is primarily driven by microvascular pathology. Surprisingly, TREM2 deficiency did not impact microglial transcriptional alterations in hypoxia (or at least not to the level that exceeds baseline differences in gene expression).

Findings presented in this thesis advance our understanding of microglial responses during brain hypoxia. While TREM2 is not a critical regulator of transcriptional responses during hypoxia in adult microglia, its signalling may attenuate hypoxia-induced microvascular pathology in the aged brain. Future studies are needed to explore this dichotomy. Such studies could assess contributions of TREM2 to niche-restricted microglial subsets emerging in hypoxia across the lifespan, and test if microglia influence brain adaptations to hypoxic stress via other, TREM2-independent mechanisms.