To further understand the mechanism through which the molecular clock influences the innate immune cell function

Abstract

The antimicrobial immune response demonstrates circadian rhythmicity, with the host being able to effect enhanced bacterial clearance during the active phase, times of high physical activity. This adaptation likely evolved to limit enhanced antimicrobial responses to times when the threat of encountering infection is greatest. These circadian rhythms are regulated at the molecular level through a complex network composed of a highly conserved set of clock proteins that function as part of a transcription-translation feedback loop. Understanding the molecular mechanism of the circadian-regulated immune response may reveal new treatment strategies for infections/inflammatory diseases. While a lot is known about the importance of these clock genes for the immune response, whether they influence neutrophil bactericidal activity, the most abundant immune cell that provides the first line of defence against invading pathogens, remains unknown. These questions were addressed by taking advantage of the genetic tractability, live imaging potential and the evolutionarily conserved biology of the zebrafish model. Recent research has shown that the innate immune response to bacterial infection (Salmonella Typhimurium) in larval zebrafish is under circadian control and is regulated by the light-responsive core clock gene period2 (per2), with Per2 playing a protective role in response to infection. In this study, we show that Per2 operates in a cellautonomous fashion within neutrophils and is essential for their antibacterial activity. Neutrophils deficient in per2 (isolated from per2-/- mutant zebrafish line) were incapable of sufficiently killing phagocytosed bacteria, had decreased phagocytic potential and reactive oxygen species (ROS) (a fundamental antibacterial weapon), when transplanted into WT recipients. Analysis of the transcriptome of per2-/- neutrophils suggests that these phenotypes may be the result of decreased expression of high mobility group box 1a (hmgb1a), that encodes a well-known immune regulatory protein. While Per2 provides a protective effect within neutrophils during infection, we show that its binding partner Cry1a displays an opposing role whereby it acts to repress the antimicrobial function of neutrophils. Through generating a cry1a-/- mutant, we show that cry1a-/- larvae exhibited enhanced survival to bacterial challenge and possess enhanced neutrophil phagocytic and bactericidal capacity through elevated ROS production. These results revealed a new role for Cry1a in regulating neutrophil function during infection. By performing various rescue experiments involving supplementing per2-/- larvae with per2 mRNA that lacks the Cry protein-interacting domain, we also shown that Per2 interaction with Cry proteins is necessary to rescue the diminished survival observed in per2-/- larvae following infection. In light of this, we propose that the molecular mechanism by which Per2 promotes an increased bactericidal role within neutrophils may be through inhibiting Cry-mediated repression of antimicrobial genes, possibly hmgb1a. To the best of our knowledge, this is the first time two core clock genes have been shown to control neutrophil antibacterial activity. It also supports the value of the zebrafish model system in revealing new insights into cell-specific functions of circadian clock components.