INVESTIGADORES
CARGNELUTTI ethelina
congresos y reuniones científicas
Título:
NOREPINEPHRINE MODULATES CLOCK IN EX VIVO SPLENIC MACROPHAGES
Autor/es:
CARGNELUTTI, E.; ANZULOVICH, A. C.
Reunión:
Congreso; XL REUNIÓN CIENTÍFICA ANUAL DE LA SOCIEDAD DE BIOLOGÍA DE CUYO; 2022
Resumen:
The time of day is critical to define the nature of the immune response,since a dysregulation of this mechanism can lead to inflammatory diseases or immunodeficiencies. In mammals, the central clock in the suprachiasmaticnucleus (SCN) synchronizes cell-autonomous clocks to the sunlight. The splenic macrophages (MΦ) phagocytes and eliminates circulating pathogens, and orchestrate the development of the specific acquired immuneresponse. However, the central circadian regulation of these splenic cells has not been completely elucidated yet. Communication between SCN and spleen occurs by the sympathetic nervous system (SNS), through nerves that release norepinephrine (NE) in areas of MΦ cells. Previously, other authors reported daily oscillation of NE in spleen. In orderto study the role of NE on the regulation of the molecular clock of spleen MΦ, we have developed a rat model of local sympathetic denervation by guanethidine administration. Animals were maintaining under 12h-light: 12h-dark conditions and ad-libitum food/water intake until the experiment. To analyze the NE temporal impact on molecular clock of splenic MΦ, ten days after injection of saline solution or guanethidine, control (N=4/ZT) and sympathectomized rats (N=3/ZT) were euthanized at different times during a 24 hperiod (ZT2, ZT6, ZT10, ZT14, ZT18 and ZT22) and spleen was aseptically removed for ex vivo cultures. The BMAL1 and ACTIN protein level, were analyzed by Western blot from splenic adherent cells. Time point data were compute by 1-way analysis of variance (ANOVA) and followed by Tukey post hoc test. Further, chronobiologic statistics were used for validating temporal changes as rhythms.Thus, each series of data were analyzed by Cosinor method. Since BMAL-1modulates some MΦs functions through the direct control of Rev-Erb α, which in turns represses Bmal-1 expression through the accessory loop of the molecular clock, the relative quantification of this gene was evaluated by q-PCR, using s28 as reference gene. In this case, cDNA was obtained from ex vivo splenic adherents cells cultivated from control and sympathectomized rats, at ZT6, ZT14 and ZT 18. The Student t test was used for comparison of data between both groups. The splenic MΦ from control rats showed a daily oscillation of BMAL1 (% rhythm: 71.8), withits acrophase occurring at the middle of the light period. Noteworthy, the exvivo splenic MΦ from guanethidine-treated animals lost the 24h-oscillation of BMAL1 and showed significant lower levels of this clock factor, compared to control. On the order hand, sympathectomized rats show a significant higher Rev-Erbα expression, at the three analyzed ZTs (p >0.05), compared to control group. Our results would indicate that exists a SCN regulation on the molecular clock in splenic adherent cells, through the NE sympathetic pathway.