Circadian rhythms in ex vivo spleen macrophages

RAMIRES, M; CAMPOS, L.E.; ALFONSO, J.O; ANZULOVICH, A. C.; CARGNELUTTI, E.

Congreso; XXXVI REUNIÓN CIENTÍFICA ANUAL DE LA SOCIEDAD DE BIOLOGÍA DE CUYO; 2018

CIRCADIAN RHYTHMS IN EX VIVO SPLEEN MACROPHAGESAccumulating evidence links the immune system and circadian regulation. Spleen macrophages (MΦ) play a critical role in innate immunity, acting as sentinels to fight against circulating pathogens, and orchestrating the development of the specific acquired immune response. However, the temporal organization of those processes and the significance of circadian regulation in the spleen, have not been completely elucidated yet.Communication between the central clock in the suprachiasmatic nucleus and peripheral tissues occurs through the autonomic nervous system. Other authors reported daily oscillation of norepinephrine in spleen. Based on this, our general objective is to study the circadian rhythms of clock and clock-controlled genes in the spleen MΦ.Male Holtzman rats were maintained under 12h-light: 12h-dark conditions, and ad-libitum food/water intake, from weaning. Sixteen-week-old animals were euthanized at different times during a 24 h period (ZT2, ZT6, ZT10, ZT14, ZT18 and ZT22) and spleens were aseptically removed for ex vivo cultures of MΦ. In order to optimize these cellular cultures in our lab, we tested different incubation times (2 h and 24 h) and the number of initial total spleen cells (2 x 106; 4 x 106 and 8 x 106 splenocytes). Once we defined optimal experimental conditions of our model, BMAL1 protein levels were analyzed from adhered cells by western blot, and normalized against β-actin. We observed BMAL1 displays a circadian rhythm (p <0.001) in the spleen MΦ of rat. The rhythm´s parameters were: mesor 0.92 ± 0.05; acrophase 08:52 ± 01.58; amplitude 0.27 ± 0.05. BMAL1 circadianrhythm in the ex vivo MΦ, would impact in the temporal organization of the immune activity of these cells. Probably, BMAL1 peak occurring at middle of the day triggers the expression of clock-controlled genes involved in the defense of the organism against pathogens, during the diurnal (activity) period.