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Cell-type specific mechanisms regulating rhythms in leukocyte homing

Periodic Reporting for period 4 - CIRCODE (Cell-type specific mechanisms regulating rhythms in leukocyte homing)

Reporting period: 2020-03-01 to 2020-08-31

Leukocytes are the key components of the immune system that fight infections and provide tissue repair, yet their migration patterns throughout the body over the course of a day are completely unknown. Circadian, ~24 hour rhythms are emerging as important novel regulators of immune cell migration and function, which impacts inflammatory diseases such as myocardial infarction and sepsis. Altering leukocyte tissue infiltration and activation at the proper times provides an option for therapy that would maximize the clinical impact of drugs and vaccinations and minimize side effects. The overall objectives of this project were thus to provide detailed insights into the recruitment patterns of various leukocyte subsets over the course of the day and discover the molecular mechanisms responsible for these effects.

In this project, we have created a four-dimensional map of leukocyte migration to organs in time and space and investigated the molecular mechanisms that regulate cell-type specific rhythms in leukocyte trafficking. We have defined the daily oscillating molecular signatures of adhesion molecules on leukocytes and endothelial cells and have thus identified a circadian traffic code that dictates the rhythmic migration of leukocyte subsets to specific organs under steady-state and inflammatory conditions. We have assessed the impact of lineage-specific arrhythmia on immune homeostasis and leukocyte trafficking using an innovative combination of novel genetic tools. Together, we have created a resource for reference with respect to the principal components governing leukocyte trafficking across the day. This will be valuable to the field to assess leukocyte migration in vivo and provide the fundamental layout to target specific molecules in order to alter the trafficking of leukocyte subsets to specific organs at any given time of the day.
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 the gene 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.
We have identified a broad and rhythmic program that governs the migration patterns of leukocyte subsets throughout the body over the course of the day. We have further defined that an organ- and leukocyte-subset-specific functional rhythmic signature exists, consisting of pro-migratory factors on endothelial cells and leukocytes. Our data demonstrate that rhythmicity in both blood vessels and leukocytes contributes to this process given that a genetically induced lack of a functional clock in either ablates time-of-day differences. We have thus identified an extensive, time-of-day-dependent trafficking zip code that guides migration of leukocytes to organs. Our observations are of particular relevance since we demonstrate rhythms in leukocyte homing to extend to humans.

We have identified the time-of-day dependent traffic routes of leukocytes in the organism. We have characterized the cell-type-specific circadian expression profiles of pro-migratory factors in leukocytes and endothelial cells. We have identified daily rhythms in the expression of pro-migratory factors on leukocyte subsets and endothelial cells of different organs. We have additionally identified a rhythmic and cell-type specific recruitment profile for leukocyte subsets to different organs and identified the oscillatory patterns of genes of the circadian clock in different cell lineages. We have published these results in two Immunity papers in 2017 and 2018, in a Circulation paper in 2019, as well as in several overview articles. We have engaged in many scientific conferences and seminars, allowing the dissemination of these results to a wide audience, including members of the general public and industry. We are now in the process of harnessing these insights to develop chronopharmacological, i.e. time-of-day-dependent, immuno-therapies in the aim to treat or prevent multiple inflammatory diseases.
It had been incompletely understood how leukocytes – the critical effector cells of the immune system – circulate within the body over the course of a day. In this project, we have defined a novel circadian homing code comprised of molecules involved in the migration of leukocytes across the whole body in time. The project combined the disciplines of immunology and chronobiology to obtain unprecedented information in time and space of circadian leukocyte trafficking and investigating how immune-cell specific oscillations are generated at the molecular level, which is of broad impact for both fields.

We have focused the work schedule of this proposal on innovative ideas and technologies that will have significant clinical and scientific impact on immune homeostasis and disease. The idea that circadian rhythms regulate the inflammatory response through the rhythmic expression of pro-migratory molecules on endothelial cells and leukocytes is unprecedented and of broad impact for immunology and chronobiology: We have performed innovative proteomics approaches to identify a trafficking code that determines leukocyte distribution in the body in time and space. We also identified the molecular basis underlying these oscillations using a combination of novel genetic models that allow the induction of cell-type specific arrhythmias. By adding time as a newly identified and critical 4th dimension that impacts leukocyte trafficking we were able to identify novel regulators of leukocyte homing at specific times. We dissected the mechanisms using rigorous genetic models (general vs. cell-inducible knockouts), as well as technically innovative experimental approaches (circadian proteomics of cell surface proteins and quantitative mapping of endothelial cell adhesion molecules in organs). The identification of a circadian code for leukocyte trafficking in health and disease represents major advances that will lead to new ways to modulate immune cell migration and function. Thus, our work program went beyond the current state-of-the-art to form the molecular basis to develop new time-based interventions that have the potential to enhance the general efficacies of drugs and inflammatory therapies. For example, administration of antibodies that block the infiltration of inflammatory leukocyte subtypes to a specific organ at the time when it predominantly occurs would maximize its therapeutic impact while minimizing side effects.