Daily Rhythms Of Neutrophil Activation Programs Host Response To LPS-Induced Infection

Background: Neutrophils are the most abundant leukocytes and act to resolve infection by phagocytosis, degranulation or the formation of neutrophil extracellular traps (NETs). Golan et.al. (Cell Stem Cell, 2018) showed that murine bone marrow hematopoietic stem cells (HSC) primarily differentiate to mature leukocytes (neutrophils and monocytes) during the day. We suspect that this causes a hyper-inflammatory response in the day, therefore making mice more susceptible to bacterial endotoxins in the day (resting phase) compared to night (active phase).

Moreover, the neutrophil response to infection also involves the activation of thrombotic processes. Together, the immune and coagulation system act to locally compartmentalize the bacteria. However, the circadian regulation of coagulation- particularly by innate immune cells is poorly understood.

Materials and Methods: Wild type (WT) C57 BL/6 Mice were housed under 12:12 hours of light-dark cycle, with lights switched on at 6 AM. Mice were challenged at different time points with LPS (10mg/kg) to mimic bacterial sepsis. Reactive Oxygen Species (ROS) were scavenged using NAC. Bone Marrow (BM), Peripheral Blood (PB) and Liver cells were harvested and analyzed by flow cytometry. The Evans Blue Dye Assay was used to assess Bone Marrow vascular endothelial permeability.

Results: We found that exposure to LPS in the day dramatically elevated ROS production by neutrophils and increased mobilization of neutrophils and monocytes into circulation. This corresponds to a marked increase in the levels of pro-inflammatory cytokines in the blood in the afternoon, which may be lethal. But this is in sharp contrast to mice injected at midnight, where the number of activated neutrophils in PB reduce after LPS treatment- due to a significant reduction in BM vascular permeability, probably due to the high levels of the darkness hormone melatonin at night. The reduced inflammatory response at night is accompanied by a very high recruitment of neutrophils to the inflamed liver- possibly due to higher expression of adhesion molecules in the liver at night (He et.al, Immunity, 2018). Inhibiting the differentiation of BM HSC by scavenging available ROS led to a decreased level of neutrophils in the blood after LPS challenge in the afternoon.

Leukocytes also have a pro-coagulant activity by generating thrombin, which can induce further inflammation by activating their G-protein-coupled membrane receptors, protease-activated receptor-1 (PAR-1). We found that PAR-1 deficient mice do not have a higher recruitment of neutrophils to the liver at night. In addition, we established chimeric mice in which hematopoietic cells from the BM of PAR-1 KO mice were transplanted into WT mice and vice versa. Our results show that PAR-1 expression on hematopoietic cells and not in the stromal compartment is responsible for the differences in vascular permeability, as well as neutrophil recruitment.

Conclusion: Here we report our recent efforts to understand daily oscillations in neutrophil phenotype and kinetics, and their role in regulating the coagulation process. Our study could help in designing strategies to better respond and cope with sepsis.