Local Adrenergic Nerves Regulate Diurnal Leukocyte Adhesion: Impact In Sickle Cell Disease
Authors: Scheiermann Christoph , Kunisaki Yuya , Jang Jungeun , Zhang Dachuan , Chow Andrew , Lucas Daniel , Frenette Paul S. 
Published: October 2019

Abstract
Abstract 1099
Leukocyte recruitment is a crucial component of immune homeostasis. The sequential multistep process leading to leukocyte migration is thought to be regulated on demand at the inflammatory site. Here, we show that broad systemic programs involving long-range signals from the sympathetic nervous system (SNS) delivered by adrenergic nerves regulate rhythmic recruitment of leukocytes in tissues. While oscillations in the numbers of circulating leukocytes ranged from 4.1 to 7.8 × 106 cells/ml blood (P<0.001) with a peak ∼5h after the onset of light (zeitgeber time, ZT5), numbers of extravascular leukocytes in skeletal muscle exhibited circadian fluctuations whose pattern ran in antiphase with that of blood, peaking at ZT13 (night) and reaching a trough at ZT5 (ZT5 / ZT13: 5.3 ± 0.3 / 7.3 ± 0.3 cells per 100 × 50 μm2 extravascular vessel segment, p<0.001). Investigations of leukocyte-endothelial cell interactions in real-time by multi-channel fluorescence intravital microscopy (MFIM) of the cremasteric muscle microvasculature revealed increased myeloid cell recruitment at night (1.7-fold enhanced adhesion, p<0.05). Migratory oscillations were implemented by local SNS fibers since unilateral neurectomy of the genitofemoral nerve (GFNx), the nerve that innervates this muscle, completely (100%) ablated the night increase in leukocyte recruitment, suggesting an essential role for local innervation of the tissue in this process. Bone marrow chimeras revealed that signals acting on β2 and β3 adrenoreceptors expressed by non-hematopoietic cells were responsible for circadian recruitment leading to fluctuations of intercellular cell adhesion molecule-1 (ICAM-1) expression on endothelial cells (1.7- and 2-fold increase at ZT13 in ICAM-1 mRNA and protein levels, p<0.001 and p<0.05 respectively). ICAM-1 plays a critical role in this activity since Icam1-deficient animals did not exhibit an apparent rhythm in leukocyte infiltration. GFNx also completely abolished the night increase of ICAM-1 expression providing a direct link between SNS input and leukocyte infiltration. Since circadian rhythms are synchronized by light we tested whether alterations in photic input by induction of experimental jetlag would be sufficient to modify rhythms in leukocyte recruitment to the cremaster muscle. Indeed, circadian rhythms in extravascular leukocyte numbers in the tissue and fluctuations of ICAM-1 expression were completely abolished after a 12h jetlag. Oscillations were mediated by the molecular clock since rhythms of leukocyte numbers in both blood and tissue ceased to exist in Bmal1−/− animals kept in constant darkness for three weeks, in contrast to normal rhythms observed in heterozygous and wild-type littermates. Remarkably, circadian rhythms in leukocyte recruitment persisted during inflammation. After administration of TNF-α, circadian rhythms in ICAM-1 expression (1.4-fold increase, p<0.05) and leukocyte recruitment remained apparent, with ZT13 showing significantly higher leukocyte infiltration than ZT5. In contrast, TNF-α-induced leukocyte infiltration (82%) and adhesion molecule expression were strongly dampened when mice were subjected to a jetlag. To test the relevance in a disease model, we evaluated mice with sickle cell disease (SCD), a genetic disorder in which leukocyte adhesion and activation play a critical role in acute vaso-occlusion. Intravital microscopy revealed an increase in the numbers of adherent and extravasated leukocytes when the experiment was performed at night. In addition, we observed a 2-fold increase in the interactions between circulating RBCs and adherent leukocytes mediated by enhanced β2 integrin (Mac-1) activity (1.9-fold, p<0.05) on leukocytes and resulting in a 31% reduction of the mean venular blood flow rates. Strikingly, the overall survival of SCD mice was significantly reduced in mice challenged at night compared to the daytime (mean survival night: 305 min, day: 489 min, p<0.05, Log rank test). In conclusion, these data suggest that circadian rhythms in leukocyte recruitment can influence disease outcome and provide a novel paradigm in the leukocyte adhesion cascade with the potential for time-based therapeutics in inflammatory diseases.

Disclosures:
No relevant conflicts of interest to declare.