Intercellular Communication Between Extracellular Vesicles and Murine Marrow Cells Is Influenced By Circadian Rhythm

Introduction: Extracellular vesicles (EVs) are membrane-bound, subcellular fragments that contain DNA, RNA, protein and lipids, and play an important role in intercellular communication. They have been implicated in myriad physiologic and pathologic processes within the hematopoietic system including thrombosis and leukemia, and as such, have tremendous therapeutic potential if manipulated appropriately. In order to fully harness their clinical potential, it will be important to more fully characterize the factors that modulate their biogenesis and function. Numerous factors are thought to influence EVs and their ability to communicate with target marrow cells, but little is known about how circadian oscillations alter EV function. Using a well-established model of lung-derived EV modulation of marrow phenotype, we report here our studies exploring the effects of circadian rhythm on the ability of lung-derived EVs to alter the transcriptome of bone marrow target cells.

Methods: C57BL/6 mice were housed on a 12-hour light/dark schedule. For the first set of experiments, lungs, blood and bone marrow were harvested (6-16 mice/time) at three different circadian time-points (8,12 and 24 hours after light onset (HALOs). EVs were isolated from each tissue at each time-point and quantified by nanoparticle tracking analysis. Second, to determine if circadian rhythm influences the ability of lung-derived EVs to modulate the transcriptome of target whole bone marrow cells (WBM), lungs were harvested (3 mice/time-point x 2 experiments) at 4,12,16, and 24 hours after light onset and co-cultured, across a cell-impermeable membrane, with murine WBM. The WBM was harvested after 24hrs in co-culture and analyzed for the induction of pulmonary specific mRNA expression by RT-PCR. Finally, to determine if altering the circadian timepoint of the target whole bone marrow influenced the ability of EVs to modulate marrow phenotype, lungs harvested from mice sacrificed at HALO 2 were cultured for 48hrs. EVs were isolated from the lung-conditioned media by ultracentrifugation. WBM was then harvested at different circadian time-points (HALO 4, 8, 12, 16, 20, and 24; 3 mice/time-point x 2 experiments), cultured with or without the lung-derived EVs for 24hrs, and then analyzed for pulmonary-specific mRNAs by RT-PCR.

Results: There were changes in EV quantity related to circadian timepoint. There were significantly increased numbers of EVs generated from lungs harvested at HALO 24 and HALO 8 when compared to HALO 16. Similarly, marrow harvested at HALO 8 had significantly increased number of EVs as compared to WBM harvested at HALO 16 and 24. In addition to circadian-related differences in EV quantity, there were clear oscillations in the expression patterns of pulmonary specific mRNAs when both the circadian timepoint of the lung donor and the circadian timepoint of the recipient marrow cells were altered. If lung was harvested at HALO 12 or HALO 16, the co-cultured WBM showed significantly increased expression of pulmonary-specific mRNAs when compared to WBM co-cultured with lung harvested at HALO 4 or HALO 24. Likewise, when WBM harvested at different circadian time-points was incubated with lung-derived EVs, there was significantly increased expression of surfactant A, surfactant B, and aquaporin 5 when WBM was harvested at HALO 16 when compared to HALO 12 or HALO 24.

Conclusions: Based on these data, we conclude that circadian rhythm is likely an important modulator of EV-mediated intercellular communication with whole bone marrow cells and warrants more detailed study.