Background.The number of hematopoietic stem/progenitor cells (HSPCs) in peripheral blood (PB) undergoes a circadian oscillation, with the peak occurring in the early morning hours and the nadir at night, and, as nicely demonstrated, this peak has been attributed to the enhanced tonus of the vegetative nervous system in the early morning hours (Nature 2008, 452, 442-447). Moreover, our group has demonstrated that release of HSPCs from bone marrow (BM) into PB is regulated during stress- or pharmacology-induced mobilization by activation of three ancient serum proteolytic cascades, the complement cascade (ComC), the coagulation cascade (CoaC), and the fibrynolytic cascade (FibC) (Stem Cell Rev. 2018; 14:677-685). Since it is known that the ComC, CoaC, and FibC show circadian activation at late night/early morning hours due to deep sleep hypoxia, regulation of the circadian oscillation of HSPC numbers in PB becomes more complex. Moreover, as we recently demonstrated, an important role in egress of HSPCs from BM into PB is played by purinergic signaling involving adenosine triphosphate (ATP) released from cells, which, as signaling mediators in the extracellular space, activate the Nlrp3 inflammasome in hematopoietic cells (Leukemia 2019; 33:815-825). Activation of the Nlrp3 inflammasome induces a state of sterile inflammation in the BM microenvironment and activates the ComC, CoaC, and FibC. Hypothesis. Since Nlrp3 inflammasome activation regulates egress of HSPCs from BM into PB by inducing BM sterile inflammation and activation of the ComC, CoaC, and FibC undergoes circadian activation, we became interested in whether Nlrp3 protein complex orchestrates circadian changes in the number of HSPCs circulating in PB.Materials and Methods. To address this important question, we studied the circadian oscillation in the number of circulating HSPCs in mice. Mice were accustomed to alternating periods of 12 hours light and 12 hours darkness. Light was turned on at 6 AM (ZT0), and the numbers of circulating white blood cells (WBCs), Sca-1+kit+Lin- HSCs, Sca-1+Lin-CD45+ HSCs, clonogenic CFU-GM progenitors, and non-hematopoietic Sca-1+Lin-CD45- cells (VSELs) were measured at 7 AM (ZT1), 11 AM (ZT5), 7 PM (ZT13), and 3 AM (ZT21). At the same time points, we evaluated expression of the Nlrp3 inflammasome at the mRNA level; Nlrp3 activation by measuring Nlrp3 inflammasome activation markers, such as interleukin-1beta, interleukin-18, and Hmgb1, at the mRNA and protein levels; ComC activation (by C5a ELISA); CoaC activation (by thrombin/antithrombin ELISA); and FibC activation (by plasmin/antiplasmin complex ELISA). To confirm the role of the Nlrp3 inflammasome in the circadian oscillation of HSPCs released into PB, we inhibited its activity by employing the specific small-molecule inhibitor MCC950. Results. We observed circadian changes in the expression and activation of the Nlrp3 inflammasome, with a peak in the early morning hours at ZT1 that preceded the peak in the number of circulating HSPCs at ZT5. This increase in activation of the Nlrp3 inflammasome and the number of circulating cells in WT animals was preceded by an increase in C5a concentration in PB at ZT1 as well as activation of the CoaC and FibC at ZT21. As expected, inhibition of the Nlrp3 inflammasome by MCC950 inhibited circadian oscillation of circulating HSPCs in PB. Conclusions. Our study confirms circadian activation of the Nlrp3 inflammsome due to the ComC, CoaC, and FibC in mice at late-night/early-morning hours preceding the release of HSPCs from BM into PB. The fact that we observed significant decrease in circadian changes in the number of circulating cells in PB in mice exposed to an Nlrp3 inflammasome inhibitor confirms its pivotal role in executing circadian release of HSPCs from BM into PB. Moreover, the fact that mice exposed to an Nlrp3 inhibitor show defective activation of the ComC and normal activation of the the CoaC and FibC indicates that, of the ancient proteolytic cascades tested, the ComC is the major player regulating Nlrp3 inflammasome-dependent circadian egress of HSPCs.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.