NOREPINEPHRINE MODULATES DAILY RHYTHMS IN EX VIVO SPLENIC MACROPHAGES

RAMIRES M; DELGADO SM; ANZULOVICH AC; CARGNELLUTTI E

San Luis

Congreso; XXXVII REUNION CIENTIFICA DE LA SOCIEDAD DE BIOLOGIA DE CUYO; 2019

The splenic macrophages (MΦ) phagocytes and eliminates circulating pathogens and orchestrate the development of the specificacquired immune response. Even though circadian effects on the immune system have been documented, the circadian regulation inthe spleen has not been completely elucidated yet. In mammals, the central clock in the suprachiasmatic nucleus (SCN) of theanterior hypothalamus synchronizes cell-autonomous clocks. Communication between SCN and spleen occurs by the sympatheticnervous system (SNS), through nerves that release norepinephrine (NE) in areas of T and MΦ cells. Previously, other authorsreported the daily oscillation of NE in the spleen. Our focus was to determine whether rhythmic expression of the molecular clock inthe splenic MΦ is regulated by SNS innervation. For this purpose, we disrupted the autonomic innervation to the spleen of 4-monthold male Holtzman rats, by splenic injections of guanethidine. Guanethidine has several effects on peripheral sympathetic neuronsincluding the blockade of neural transmission, depletion of neuronal NE stores, and blockade of the reuptake of NE into the neurons.Animals were maintained under 12 h-light: 12 h-dark conditions and ad libitum food/water intake until the experiment. First, weevaluated the effects of local guanethidine injection on spleen macroscopic appearance, size, weight, and lymphocyte numbers.Control animals received intrasplenic saline solution injection. The Student‟s t-test was used for comparison of data between bothgroups. Second, to study the NE temporal impact on the molecular clock of splenic MΦ, ten days after injection of saline solution orguanethidine, control (N = 4/ZT) and sympathectomized rats (N = 3/ZT) were euthanized at different times during a 24 h period(ZT2, ZT6, ZT10, ZT14, ZT18, and ZT22) and spleen was aseptically removed for ex vivo cultures. The clock transcription factor,BMAL1, and ACTIN were analyzed by Western blot from splenic adherent cells. Time-point data were computed by one-wayanalysis of variance (ANOVA) and followed by Tukey‟s post hoc test. Further, chronobiologic statistics were used for validatingtemporal changes as rhythms. Thus, each series of data were analyzed by the Cosinor method. We found no significant differences inthe spleen macroscopic appearance, size, weight, and lymphocyte number between both treated groups, suggesting localguanethidine injections did not have any toxicity on spleen cells. The splenic MΦ from control rats showed a diary oscillation ofBMAL1 (% rhythm: 71.8), with its acrophase occurring in the middle of the light period. In contrast, the ex vivo splenic MΦ from guanethidine-treated animals lost the diary oscillation of BMAL1. Our results would indicate regulation of the molecular clock insplenic adherent cells by the SCN, through the NE sympathetic pathway.