Circadian rhythms in ex-vivo spleen macrophages

RAMIREZ M; CAMPOS LE; ALFONSO JO; ANZULOVICH AC; CARGNELUTTI E

Mendoza

Congreso; XXXVI Reunión Científica Anual de la Sociedad de Biología de Cuyo; 2018

Sociedad de Biología de Cuyo

Accumulating evidence links the immune system and circadian regulation. Spleen macrophages (MΦ) play acritical role in innateimmunity, acting as sentinels to fight against circulating pathogens, and orchestrating thedevelopment of the specific acquiredimmune response. However, the temporal organization of those processesand the significance of circadian regulation in thespleen, have not been completely elucidated yet.Communication between the central clock in the suprachiasmatic nucleus andperipheral tissues occurs throughthe autonomic nervous system. Other authors reported daily oscillation of norepinephrine inspleen. Based on this,our general objective is to study the circadian rhythms of clock and clock-controlled genes in the spleenMΦ. 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) andspleens were aseptically removed for ex vivo cultures of MΦ. In order tooptimize these cellular cultures in our lab, we testeddifferent incubation times (2 h and 24 h) and the number ofinitial total spleen cells (2 x 106; 4 x 106 and 8 x 106 splenocytes).Once we defined optimal experimentalconditions of our model, BMAL1 protein levels were analyzed from adhered cells bywestern blot, and normalizedagainstβ-actin. We observed BMAL1 displays a circadian rhythm (p