NOREPINEPHRINE MODULATES DAILY CLOCK EXPRESSION IN EX VIVO SPLENIC MACROPHAGES.

CARGNELLUTTI E; ANZULOVICH AC

MENDOZA

Congreso; XL REUNION CIENTIFICA DE LA SOCIEDAD DE BIOLOGIA DE CUYO; 2022

The time of day is critical to define the nature of the immune response, since a dysregulation of this mechanism can lead to inflammatorydiseases or immunodeficiencies. In mammals, the central clock in the suprachiasmatic nucleus (SCN) synchronizes cell-autonomous clocksto the sunlight. The splenic macrophages (MΦ) phagocytes and eliminates circulating pathogens, and orchestrate the development of thespecific acquired immune response. However, the central circadian regulation of these splenic cells has not been completely elucidatedyet. 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 order to study the role of NE on theregulation 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 conditions and ad-libitum food/water intake until the experiment. Toanalyze 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 h period (ZT2, ZT6, ZT10, ZT14, ZT18and ZT22) and spleen was aseptically removed for ex vivo cultures. The BMAL1 and ACTIN protein level, were analyzed by Western blotfrom 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 Cosinormethod. Since BMAL-1 modulates some MΦ’s functions through the direct control of Rev-Erb α, which in turns represses Bmal-1expression through the accessory loop of the molecular clock, the relative quantification of this gene was evaluated by q-PCR, using s28as reference gene. In this case, cDNA was obtained from ex vivo splenic adherents cells cultivated from control and sympathectomizedrats, at ZT6, ZT14 and ZT 18. The Student t test was used for comparison of data between both groups. The splenic MΦ from control ratsshowed a daily oscillation of BMAL1 (% rhythm: 71.8), with its 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 clockfactor, compared to control. On the order hand, sympathectomized rats show a significant higher Rev-Erbα expression, at the three analyzedZTs (p > 0.05), compared to control group. Our results would indicate that exists a SCN regulation on the molecular clock in splenicadherent cells, through the NE sympathetic pathway.