V-025 | Histone modifications as a molecular response underlying the astroglial pro-inflammatory phenotype

Cellular and Molecular Neurobiology
Author: Camila Vidos | Email: cami.vidos@hotmail.com

Camila Vidos^1°, Alberto Javier Ramos^1°,Alejandro Villarreal^1°2°

^1° Instituto de Biología Celular y Neurociencia “Prof. E. De Robertis” UBA-CONICET, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina
^2° Instituto Tecnológico de Buenos Aires (ITBA).

Under pathological conditions astrocytes become reactive and might acquire a phenotype with a pro-inflammatory gain of function This pathological phenotype is linked to pro-inflammatory NF-kB activation, involving stable morphological and transcriptomic changes. We’ve demonstrated that pro-inflammatory gain of function in astrocytes correlates with increased histone acetylation and depends on microglial activation.
We hypothesize that NF-kB nuclear translocation in astrocytes recruits chromatin-remodeling enzymes like demethylase KDM6B removing the repressive mark H3K27me3 at pro-inflammatory promoters, after engaging microglial soluble factors. To explore these molecular changes in astrocytes influenced by microglial signals, we developed a two-step protocol. Primary mixed glial cultures were exposed to bacterial lipopolysaccharide (LPS) for 24 hours to generate pro-inflammatory conditioned medium (PCM). Subsequently, microglia-depleted astrocyte cultures were exposed to PCM for 1-6 hours (with 1-hour intervals) to monitor NF-kB activation, and for 24, 48, and 72 hours to assess morphological changes and pro-inflammatory markers. Immunofluorescence revealed a time-dependent increase in NF-kB nuclear translocation and complement 3 protein (C3) immunoreactivity. Quantitative PCR showed increased expression of pro-inflammatory genes like IL6 and CCL2. Further investigation will focus on changes in H3K27me3 enrichment at pro-inflammatory gene promoters using ChIP-qPCR.