Elevated Circadian Period 2: A Missing Beneficial Factor in Sickle Cell Disease By Lowering Pulmonary Inflammation, Iron Overload and Mortality
Authors: Adebiyi Morayo , Zhao Zhaoyang , Manalo Jeanne , Lee Cheng Chi , Xian Wa , Youqiong Ye , McKeon Frank , Culp-Hill Rachel , D'Alessandro Angelo , Kellems Rodney , Yoo Seung-Hee , Han Leng , Xia Yang 
Published: October 2019

Abstract
Sickle-cell disase (SCD) is a life-threatening hemolytic genetic disorder. Chronic hemolysis and elevated inflammation that underlie SCD pathophysiology is difficult to treat in the clinic due to an unclear mechanism. The role of the circadian clock is required for maintaining inflammatory states which is important for proper cellular and organ function. Circadian clocks are regulated by a series of circadian clock genes which have known functions in inflammation, heme and iron metabolism. However, the function of circadian clocks in SCD remains unknown. Here, using an unbiased and robust microarray screen, we found that genes involved in circadian rhythms, inflammatory response, heme and iron metabolism were significantly altered in the lungs of SCD Berkeley transgenic mice compared to C57BL/6 (WT) mice used as controls (N=3, P<0.05, normalized to WT). Period 2 (Per2) gene plays a role in regulating the circadian clock. Since the role of Per2 is not known in SCD, we transplanted bone marrow (BM) of SCD and WT mice to Per2Luciferase (Per2Luc) bioluminescence reporter mice to generate SCD → Per2 Luc and WT→ Per2 Luc mice. Per2 circadian rhythms were determined by amplitude and periodicity in ex-vivo derived lung tissue explants. Although the period of the Per2 rhythms remained persistent throughout three circadian cycles, we demonstrated that the amplitude of circadian rhythm of Per2 was significantly upregulated in SCD mouse lung compared to WT mouse lung (N=3 per group, P<0.05). However, Per2 circadian rhythms in SCD mouse livers and kidneys did not show differences in amplitude or period compared to controls (N= 3 per group). To assess the importance of elevated Per1 and Per2 homologue genes in SCD, we transplanted BM of SCD mice to Per1/Per2 double deficient (Per1/Per2 dKO) and WT mice, respectively. To our surprise, Per1/Per2 dKO transplanted with SCD mouse BM (SCD→ Per1/Per2 dKO) displayed severe irradiation sensitivity and were more susceptible to an early death compared to WT BM transplanted mice. Mechanistically, we revealed that peripheral deletion of Per1/Per2 led to increased systemic inflammation featured with an increase of peripheral white blood cells (WBCs) in SCD→ Per1/Per2 dKO mice compared to SCD→ WT mice (50.47 ± 6.74 and 31.68 ± 1.04, respectfully, N=5 mice per group, P<0.01) without an effect on sickling in both groups of SCD chimeric mice. Moreover, we found that peripheral deletion of Per1/Per2 resulted in severe lung damage characterized by elevated congestion, neutrophil infiltration and secretion of IL-6 in lavage fluid in SCD→ Per1/Per2 dKO mouse lung compared to SCD→ WT mouse lung. Additionally, we identified that peripheral deletion of Per1/Per2 induced iron overload, heme deposits and macrophage infiltration in the lung of SCD→Per1/Per2 dKO mice compared to SCD→ WT mice. In conclusion, we discovered that Per2 is induced in SCD mouse lung but not in other organs such as kidney and liver and that its elevation is beneficial to counteract systemic inflammation, reduce pulmonary inflammatory responses, iron overload, tissue damage and thus increase survival in SCD mice. Overall, our studies reveal new molecular insight applicable to two burgeoning fields, circadian biology and SCD which pave a way for innovative therapeutic avenue for SCD.

Disclosures
D'Alessandro: Omix Technologies inc: Equity Ownership; Hemanext inc: Membership on an entity's Board of Directors or advisory committees.