H3K27ME3 AS SUPPRESSOR MARK OF ASTROCYTIC PRO-INFLAMMATORY GENES

VIDOS C; RAMOS AJ; VILLARREAL ALEJANDRO

Mar del Plata

Congreso; REUNIÓN CONJUNTA SAIC SAB AAFE AACYTAL 2023; 2023

SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA (SAIC)

Astrocytes respond to brain injury through a phenomenon called reactiveastrogliosis, in which a pro-inflammatory astrocytic phenotypewas described. Astrocyte pathological conversion with a pro-inflammatorygain of function involves stable transcriptomic changes followingactivation of transcription factor NF-kB. We have shown thatpro-inflammatory gain of function correlates with increased histoneacetylation and that these molecular mechanisms are dependent onmicroglia abundance and microglia soluble factors.We aimed here to establish a two-step treatment protocol that allowsus to address molecular changes in astrocytes exposed to microglialsoluble signals, in microglia-depleted cultures. We are currentlyfocused on studying mechanisms involving histone 3 methylation atlysine 27 which is a repressive mark for gene expression.We exposed primary cultures of mixed glial cells (containing astrocytesand microglia) to lipopolysaccharide (LPS) during 24hs toobtain a pro-inflammatory conditioned medium (PCM). We then exposedastrocyte enriched cultures (microglia-depleted) to the PCMfrom 1-6hs with a 1h interval to address NFkB activation, and during24, 48 and 72 hs to address morphological changes and pro-inflammatorymarker expression.Using immunofluorescence, we observed after PCM treatment atime-dependent increase in NFkB (p65 subunit) nuclear translocation,an increase in complement 3 protein (C3) immunoreactivity andincreased astrocyte reactive phenotype. We did not observe majorchanges in global H3K27me3 after PCM treatment but inhibition ofthe JMJD3 histone demethylase for H3K27me3 showed less C3 immunoreactivityat 48 hs.These preliminary results validate the two-step treatment protocolas a paradigm to address pro-inflammatory phenotype of astrocytesin microglia-depleted cultures. We will further address changes inH3K27me3 enrichment at the promoters of pro-inflammatory gene(e.g. C3) using ChIP-qPCR using this two-step protocol