NOREPINEPHRINE MODULATES CLOCK IN EX VIVO SPLENIC MACROPHAGES

ANZULOVICH, A. C.; CARGNELUTTI, E.

Congreso; XXXIX REUNIÓN CIENTÍFICA ANUAL DE LA SOCIEDAD DE BIOLOGÍA DE CUYO; 2021

ABSTRACTThe time of day is critical to define the nature of the immune response,since a dysregulation of this mechanism can lead to inflammatory diseases orimmunodeficiencies. In mammals, the central clock in the suprachiasmaticnucleus (SCN) synchronizes cell-autonomous clocks to the sunlight. The splenicmacrophages (MΦ) phagocytes and eliminates circulatingpathogens, and orchestrate the development of the specific acquired immuneresponse. However, the central circadian regulation of these splenic cells hasnot been completely elucidated yet. Communication between SCN and spleen occursby the sympathetic nervous system (SNS), through nerves that releasenorepinephrine (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 guanethidineadministration. Animals were maintaining under 12h-light: 12h-dark conditionsand ad-libitum food/water intake until the experiment. To analyze the NEtemporal impact on molecular clock of splenic MΦ, tendays after injection of saline solution or guanethidine, control (N=4/ZT) andsympathectomized rats (N=3/ZT) were euthanized at different times during a 24 hperiod (ZT2, ZT6, ZT10, ZT14, ZT18 and ZT22) and spleen was aseptically removedfor ex vivo cultures. The BMAL1 and ACTIN protein level, were analyzed byWestern blot from splenic adherent cells. Time point data were compute by 1-wayanalysis 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 MΦ?s functions through the direct control of Rev-Erb α, whichin turns represses Bmal-1 expression through the accessory loop of themolecular clock, the relative quantification of this gene was evaluated byq-PCR, using s28 as reference gene. In this case, cDNA was obtained fromex vivo splenic adherents cells cultivated from control andsympathectomized rats, at ZT6, ZT14 and ZT 18. The Student t test wasused for comparison of data between both groups. The splenic MΦfrom 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 the24h-oscillation of BMAL1 and showed significant lower levels of this clockfactor, compared to control. On the order hand, sympathectomized rats show a significanthigher Rev-Erbα expression, at the three analyzed ZTs (p >0.05), compared to control group. Our results would indicate that exists a SCNregulation on the molecular clock in splenic adherent cells, through the NEsympathetic pathway.