Implication of DNA methylation patterns in mouse microglial cell identity.

Microglial cells, the resident immune cells of the brain parenchyma, are responsible for the establishment and maintenance of the brain homeostasis. Belonging to the macrophage lineage, they function first as a part of the innate immune system and thus, have to execute immune related roles. However, microglial cells are also involved in a plethora of central nervous system (CNS) related roles, from synaptic pruning to oligodendrocyte maturation and clearing of myelin. In order to fulfil those roles, microglial cells can adapt in the best way to a specific situation without harming the delicate CNS environment. Microglial cells are constantly monitoring the environment by displaying surface receptors, phagocytosis can be modulated to clear debris or apoptotic cells, but also synapses; cytokine secretion can be cytotoxic or neurotrophic. Microglial cells are highly plastic, capable of reacting to any stimulation that could hamper the proper functioning of the brain. There is emerging evidence that microglial plasticity is related to chromatin modulation, orchestrated by epigenetic mechanisms, such as non coding RNAs, histones modifications and DNA modifications. DNA methylation is one of the most studied epigenetic mechanisms and yet, little is known about its involvement in microglial identity. Regarding the variety of functions realized by microglia, the involvement of microglial misfunction in a various range of neurodegenerative diseases, neurodevelopmental disorders and brain tumors, amongst others, is not surprising. As such, they do represent a promising target for innovative therapeutic approaches, but in order to achieve an efficient exploitation of microglial functions without hampering brain homeostasis, a deep understanding of the microglial core identity and how it can be influenced by the environment, is required. In this thesis, we hypothesized that mouse microglial identity was modulated by DNA methylation pattern reorganization, and, to test that hypothesis, we induced a reprogramming of microglial cell identity and explored genome-wide DNA methylation patterns. We characterized identities generated by treatments with Lipopolysaccharide (LPS), Interferon gamma (IFN-ɣ), Interleukin 4 (IL-4) and a glioma-based conditioned medium within a time frame between 1 and 48 hours. We observed changes in morphology, secretome composition and gene expression, in line with previously characterized inducible features of microglial reprogramming. We further performed Illumina MethylMouse methylation arrays and observed significant variations in DNA methylation patterns by both treatments and time of exposure, especially in the pro-inflammatory conditions. Then, we performed RNA-sequencing (RNA-seq) to explore gene expression modulation by LPS and IFN-ɣ treatments, however no unequivocal link between DNA methylation changes and gene expression could be established. We could nevertheless witness that DNA methylation changes induced by the treatments and associated with microglial reactivity were not only affecting gene promoters, thereby highlighting the meaning of DNA methylation in different parts of the genome such as the distal intergenic regions. In summary, DNA methylation plays an important part in the reprogramming of microglial cells, however further exploration of other epigenetic mechanisms, such as histone modifications and the chromatin accessibility is necessary to unravel the potential innovative therapeutic options targeting microglia cells in neurological diseases.