Regulation and relevance of circadian leukocyte dynamics for skin immune homeostasis

Project Information

CIRCADYN

Grant agreement ID: 101001233

DOI

10.3030/101001233

EC signature date 8 April 2021

Start date 1 October 2021

End date 30 September 2026

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 999 963,00

EU contribution

€ 1 999 963,00

Coordinated by

UNIVERSITE DE GENEVE
Switzerland

Periodic Reporting for period 2 - CIRCADYN (Regulation and relevance of circadian leukocyte dynamics for skin immune homeostasis)

Reporting period: 2023-04-01 to 2024-09-30

Summary of the context and overall objectives of the project

How circadian dynamics affect leukocyte turnover in the skin, a critical epithelial barrier for pathogens and one of the primary sites of initial activation of the immune system, is unknown. The ground-breaking impact of this project is defining the mechanisms by which circadian leukocyte turnover in the skin contributes to tissue homeostasis and how it governs general immunity in steady state and inflammation. Our research will provide insights into fundamental regulations of the immune system and the key initial events that determine the strength and rhythmicity of immune responses.

Research gap: Leukocytes home rhythmically to peripheral tissues, yet it is unknown how circadian dynamics in immigration interact with leukocyte egress, proliferation and cell death to contribute and fine-tune leukocyte homeostasis and turnover. Furthermore, it is unknown what mechanisms orchestrate these rhythms and how tissues and the immune system are affected if rhythmicity is lost. It is unknown what purpose this oscillation serves and how it might be of clinical use.
Overall hypothesis: Leukocytes in the skin exhibit oscillations in egress, proliferation, cell death and/or homing, which together results in an oscillatory leukocyte cellularity. These local oscillations can be synchronized via the SNS and serve to maintain peripheral barriers by protecting them from damage and infection.

Overall objective: We will identify the rhythmic leukocyte dynamics that contribute to circadian leukocyte turnover in tissues. We will define how this is orchestrated by the SNS and what purpose it serves.
Specific Objectives:

Objective 1: To characterize circadian leukocyte dynamics in the skin

Objective 2: To investigate the role of the sympathetic nervous system in circadian leukocyte dynamics in the skin

Objective 3: To define the relevance of rhythmic leukocyte dynamics in the skin

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

We have thus far performed several analyses to assess the functional role and relevance of circadian immune parameters in the skin. We could demonstrate that the initial immune response (hours) to an insult in the skin results in a rhythmic process that can drive adaptive immunity in the downstream draining lymph node. We moved to a skin tumor (melanoma) model to assess clinical significance. Here, we showed that tumor cells inoculated subcutaneously grow better or worse, depending on the time-of-day of their engraftment (Wang et al., Nature 2023). This response is dependent on the adaptive immune system and the circadian clock in dendritic cells, which populate the skin and migrate to the draining lymph nodes. Ultimately, this knowledge can be exploited therapeutically, by performing vaccinations at the most immune-responsive time of the day, to maximize the anti-tumor effect. We have discussed these circadian vaccination regimes in a recent review, underlining the importance of rhythms in therapy design (Wang et al., Science Immunology 2022).

We have extended on these initial observation to show that skin tumors harvested at different times of the day show time-of-day differences in their immune cell number and composition (Wang et al., Cell 2024). Specifically, as defined in objective 1, we found that leukocyte homing to these skin tumors is highly time-of-day dependent but that apoptosis or proliferation do not appear to greatly contribute to these changes. Whether emigration of leukocytes is rhythmic is currently the focus of our investigations. These differences can be exploited therapeutically by infusing tutor-specific T cells at the optimal homing time of the day.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

In this proposal we present ambitious and innovative concepts and technologies that will have significant scientific impact for the understanding into the modulation of the immune system in health and disease. Circadian timing in the adaptive immune system has recently gained attention as an important gating mechanism for immune responses. Leukocyte trafficking to and from the lymph node is highly time-of-day dependent, and this rhythm can greatly influence the strength of the adaptive immune response. Lymph nodes serve as draining sites for peripheral tissues but whether rhythms in these upstream sites govern downstream lymph node responses are completely unknown. Our hypothesis is that in the skin, which provides an epithelial cell barrier to the environment, circadian dynamics in leukocyte immigration, egress, proliferation and/or cell death contribute to a functional epithelial barrier. The mechanisms of this phenomenon are completely unexplored. Furthermore, the relevance of this rhythmicity for the body is unclear. The impact of this proposal lies in the meticulous analysis of circadian immune cell dynamics of the biggest organ of the body, the skin. We propose to unravel the mechanism and interactions of circadian leukocyte dynamics in this tissue using a strong and inter-disciplinary team of researchers (in proteomics, circadian biology, the microbiome and mathematical modeling), rigorous genetic models (lineage-specific and inducible knock-outs and knock-ins) and technically innovative experimental approaches (lineage-specific circadian phosphoproteomics and circadian, single-cell sequencing of skin leukocytes, combined approaches of genetic, pharmacological and local surgical ablation of sympathetic tone as well as site-specific analyses of the microbiome on skin leukocyte oscillations). The quantification and mathematical modeling of leukocyte dynamics in the skin will provide us with the relevant information on the best time and target of adjuvant delivery and allow for better vaccination design. Thus, our research – intersected between the domains of immunology and chronobiology – will go beyond the current state of the art and will provide an in-depth understanding of circadian rhythmicity in immune cell function in the skin, which we aim to harness for the future development of time-tailored therapies targeting adaptive immune responses.

In clinical practice, circadian oscillations are generally not considered. Our data published in Nature 2023 goes beyond the current state of the art, indicating that time of day should matter in clinical practice, such as when treating tumor patients. We are expecting to provide further molecular insights into the steady-state immune oscillations in the skin until the end of the project and aim to leverage this in better vaccine design. We have now provided further evidence for time-of-day changes in skin anti-tutor immunity by showing that antibodies directed against immune-checkpoint inhibitors provide time-of-day benefits, based on their time of infusion (Cell 2024). This is unexpected, given that these drugs exhibit long half-lives. We are now investigating the underlying mechanisms.

Overview of Dendritic cell circadian trafficking in the skin