Circadian Expression of Migratory Factors Establishes Lineage-Specific Signatures that Guide the Homing of Leukocyte Subsets to Tissues.

The number of leukocytes present in circulation varies throughout the day, reflecting bone marrow output and emigration from blood into tissues. Using an organism-wide circadian screening approach, we detected oscillations in pro-migratory factors that were distinct for specific vascular beds and individual leukocyte subsets. This rhythmic molecular signature governed time-of-day-dependent homing behavior of leukocyte subsets to specific organs. Ablation of BMAL1, a transcription factor central to circadian clock function, in endothelial cells or leukocyte subsets demonstrated that rhythmic recruitment is dependent on both microenvironmental and cell-autonomous oscillations. These oscillatory patterns defined leukocyte trafficking in both homeostasis and inflammation and determined detectable tumor burden in blood cancer models. Rhythms in the expression of pro-migratory factors and migration capacities were preserved in human primary leukocytes. The definition of spatial and temporal expression profiles of pro-migratory factors guiding leukocyte migration patterns to organs provides a resource for the further study of the impact of circadian rhythms in immunity.

Lymphocyte Circadian Clocks Control Lymph Node Trafficking and Adaptive Immune Responses.

Lymphocytes circulate through lymph nodes (LN) in search for antigen in what is believed to be a continuous process. Here, we show that lymphocyte migration through lymph nodes and lymph occurred in a non-continuous, circadian manner. Lymphocyte homing to lymph nodes peaked at night onset, with cells leaving the tissue during the day. This resulted in strong oscillations in lymphocyte cellularity in lymph nodes and efferent lymphatic fluid. Using lineage-specific genetic ablation of circadian clock function, we demonstrated this to be dependent on rhythmic expression of promigratory factors on lymphocytes. Dendritic cell numbers peaked in phase with lymphocytes, with diurnal oscillations being present in disease severity after immunization to induce experimental autoimmune encephalomyelitis (EAE). These rhythms were abolished by genetic disruption of T cell clocks, demonstrating a circadian regulation of lymphocyte migration through lymph nodes with time-of-day of immunization being critical for adaptive immune responses weeks later.

Artery-Associated Sympathetic Innervation Drives Rhythmic Vascular Inflammation of Arteries and Veins.

BACKGROUND: The incidence of acute cardiovascular complications is highly time-of-day dependent. However, the mechanisms driving rhythmicity of ischemic vascular events are unknown. Although enhanced numbers of leukocytes have been linked to an increased risk of cardiovascular complications, the role that rhythmic leukocyte adhesion plays in different vascular beds has not been studied. METHODS: We evaluated leukocyte recruitment in vivo by using real-time multichannel fluorescence intravital microscopy of a tumor necrosis factor-α-induced acute inflammation model in both murine arterial and venous macrovasculature and microvasculature. These approaches were complemented with genetic, surgical, and pharmacological ablation of sympathetic nerves or adrenergic receptors to assess their relevance for rhythmic leukocyte adhesion. In addition, we genetically targeted the key circadian clock gene Bmal1 (also known as Arntl) in a lineage-specific manner to dissect the importance of oscillations in leukocytes and components of the vessel wall in this process. RESULTS: In vivo quantitative imaging analyses of acute inflammation revealed a 24-hour rhythm in leukocyte recruitment to arteries and veins of the mouse macrovasculature and microvasculature. Unexpectedly, although in arteries leukocyte adhesion was highest in the morning, it peaked at night in veins. This phase shift was governed by a rhythmic microenvironment and a vessel type-specific oscillatory pattern in the expression of promigratory molecules. Differences in cell adhesion molecules and leukocyte adhesion were ablated when disrupting sympathetic nerves, demonstrating their critical role in this process and the importance of ß2-adrenergic receptor signaling. Loss of the core clock gene Bmal1 in leukocytes, endothelial cells, or arterial mural cells affected the oscillations in a vessel type-specific manner. Rhythmicity in the intravascular reactivity of adherent leukocytes resulted in increased interactions with platelets in the morning in arteries and in veins at night with a higher predisposition to acute thrombosis at different times as a consequence. CONCLUSIONS: Together, our findings point to an important and previously unrecognized role of artery-associated sympathetic innervation in governing rhythmicity in vascular inflammation in both arteries and veins and its potential implications in the occurrence of time-of-day-dependent vessel type-specific thrombotic events.

Influence of circadian clocks on adaptive immunity and vaccination responses.

The adaptive immune response is under circadian control, yet, why adaptive immune reactions continue to exhibit circadian changes over long periods of time is unknown. Using a combination of experimental and mathematical modeling approaches, we show here that dendritic cells migrate from the skin to the draining lymph node in a time-of-day-dependent manner, which provides an enhanced likelihood for functional interactions with T cells. Rhythmic expression of TNF in the draining lymph node enhances BMAL1-controlled ICAM-1 expression in high endothelial venules, resulting in lymphocyte infiltration and lymph node expansion. Lymph node cellularity continues to be different for weeks after the initial time-of-day-dependent challenge, which governs the immune response to vaccinations directed against Hepatitis A virus as well as SARS-CoV-2. In this work, we present a mechanistic understanding of the time-of-day dependent development and maintenance of an adaptive immune response, providing a strategy for using time-of-day to optimize vaccination regimes.

Loss of direct adrenergic innervation after peripheral nerve injury causes lymph node expansion through IFN-γ.

Peripheral nerve injury can cause debilitating disease and immune cell-mediated destruction of the affected nerve. While the focus has been on the nerve-regenerative response, the effect of loss of innervation on lymph node function is unclear. Here, we show that the popliteal lymph node (popLN) receives direct neural input from the sciatic nerve and that sciatic denervation causes lymph node expansion. Loss of sympathetic, adrenergic tone induces the expression of IFN-γ in LN CD8 T cells, which is responsible for LN expansion. Surgery-induced IFN-γ expression and expansion can be rescued by ß2 adrenergic receptor agonists but not sensory nerve agonists. These data demonstrate the mechanisms governing the pro-inflammatory effect of loss of direct adrenergic input on lymph node function.