Microglia as the triggering sparkle for chromatin remodeling in pro-inflammatory astrocytes

VILLARREAL A; VIDOS C; MONTEVERDE M; CIERI MB; RAMOS AJ

Capital Federal

Congreso; Reunión anual de sociedades de biociencias 2020. Sociedad Argentina de Investigaciones Clínicas; 2020

Sociedad Argentina de Investigación Clínica (SAIC)

Conversion of astrocytes to a pro-inflammatory phenotype leads to exacerbated neuroinflammation and neurotoxicity, therefore understanding this mechanism has become a main interest as a novel pharmacological target. We here aim to understand an epigenetic mechanism which may lead to a sustained astrocyte response expanding inflammation and neuronal death. We exposed primary cultures of cortical astrocytes containing different amounts of microglia (below 1% and up to 20%) to 25 ng/ml Lipopolissacharide (LPS) for different periods of time to promote pro-inflammatory conversion. Astroglial and microglial morphology was analyzed using immunofluorescence. Nuclear localization of p65 subunit was assessed as parameter of NFkB activation using immunofluorescence for p65/GFAP/DAPI or p65/IBA/DAPI. As an indicator of chromatin remodeling, we studied the levels of acetylated histone 3 at lysins 9 and 14 (H3K9K14ac) using immunofluorescence for H3K9K14ac/GFAP/DAPI. This epigenetic mark is known to be promoted by NFkB activation. Pro-inflammatory conversion of astrocytes was confirmed by analyzing expression of pro-inflammatory cytokines. Results show that LPS-induced astroglial conversion towards a pro-inflammatory phenotype evidenced by changes in morphology, activation of NFkB and cytokine expression is microglia-dependent. This astroglial pro-inflammatory phenotype correlates with global changes in nuclear H3K9K14ac only when they are co-cultured with microglia. Our work evidences a mechanism of gene regulation by chromatin remodeling which may underlie long term cellular changes in astrocyte phenotype conversion. Our results suggest a global reconfiguration of the chromatin which could be pharmacologically targeted to reduce neuroinflammation. PICT-2018-00920 (joven), ISN-CAEN_2020 (category B), PICT 2017-2203, PICT 2015-145.