Circadian Rhythms in Anticancer Immunity: Mechanisms and Treatment Opportunities.

Circadian rhythms of approximately 24 h have emerged as important modulators of the immune system. These oscillations are important for mounting short-term, innate immune responses, but surprisingly also long-term, adaptive immune responses. Recent data indicate that they play a central role in antitumor immunity, in both mice and humans. In this review, we discuss the evolving literature on circadian antitumor immune responses and the underlying mechanisms that control them. We further provide an overview of circadian treatment regimens-chrono-immunotherapies-that harness time-of-day differences in immunity for optimal efficacy. Our aim is to provide an overview for researchers and clinicians alike, for a better understanding of the circadian immune system and how to best harness it for chronotherapeutic interventions. This knowledge is important for a better understanding of immune responses per se and could revolutionize the way we approach the treatment of cancer and a range of other diseases, ultimately improving clinical practice.

Circadian tumor infiltration and function of CD8+ T cells dictate immunotherapy efficacy.

The quality and quantity of tumor-infiltrating lymphocytes, particularly CD8+ T cells, are important parameters for the control of tumor growth and response to immunotherapy. Here, we show in murine and human cancers that these parameters exhibit circadian oscillations, driven by both the endogenous circadian clock of leukocytes and rhythmic leukocyte infiltration, which depends on the circadian clock of endothelial cells in the tumor microenvironment. To harness these rhythms therapeutically, we demonstrate that efficacy of chimeric antigen receptor T cell therapy and immune checkpoint blockade can be improved by adjusting the time of treatment during the day. Furthermore, time-of-day-dependent T cell signatures in murine tumor models predict overall survival in patients with melanoma and correlate with response to anti-PD-1 therapy. Our data demonstrate the functional significance of circadian dynamics in the tumor microenvironment and suggest the importance of leveraging these features for improving future clinical trial design and patient care.

Mesenchymal stem cell: keystone of the hematopoietic stem cell niche and a stepping-stone for regenerative medicine.

Mesenchymal stem cells (MSCs) are self-renewing precursor cells that can differentiate into bone, fat, cartilage, and stromal cells of the bone marrow. Recent studies suggest that MSCs themselves are critical for forming a niche that maintains hematopoietic stem cells (HSCs). The ease by which human MSC-like and stromal progenitor cells can be isolated from the bone marrow and other tissues has led to the rapid development of clinical investigations exploring their anti-inflammatory properties, tissue preservation capabilities, and regenerative potential. However, the identity of genuine MSCs and their specific contributions to these various beneficial effects have remained enigmatic. In this article, we examine the definition of MSCs and discuss the importance of rigorously characterizing their stem cell activity. We review their role and that of other putative niche constituents in the regulation of bone marrow HSCs. Additionally, how MSCs and their stromal progeny alter immune function is discussed, as well as potential therapeutic implications.

Circadian clocks guide dendritic cells into skin lymphatics.

Migration of leukocytes from the skin to lymph nodes (LNs) via afferent lymphatic vessels (LVs) is pivotal for adaptive immune responses1,2. Circadian rhythms have emerged as important regulators of leukocyte trafficking to LNs via the blood3,4. Here, we demonstrate that dendritic cells (DCs) have a circadian migration pattern into LVs, which peaks during the rest phase in mice. This migration pattern is determined by rhythmic gradients in the expression of the chemokine CCL21 and of adhesion molecules in both mice and humans. Chronopharmacological targeting of the involved factors abrogates circadian migration of DCs. We identify cell-intrinsic circadian oscillations in skin lymphatic endothelial cells (LECs) and DCs that cogovern these rhythms, as their genetic disruption in either cell type ablates circadian trafficking. These observations indicate that circadian clocks control the infiltration of DCs into skin lymphatics, a process that is essential for many adaptive immune responses and relevant for vaccination and immunotherapies.

Small-molecule CBP/p300 histone acetyltransferase inhibition mobilizes leukocytes from the bone marrow via the endocrine stress response.

Mutations of the CBP/p300 histone acetyltransferase (HAT) domain can be linked to leukemic transformation in humans, suggestive of a checkpoint of leukocyte compartment sizes. Here, we examined the impact of reversible inhibition of this domain by the small-molecule A485. We found that A485 triggered acute and transient mobilization of leukocytes from the bone marrow into the blood. Leukocyte mobilization by A485 was equally potent as, but mechanistically distinct from, granulocyte colony-stimulating factor (G-CSF), which allowed for additive neutrophil mobilization when both compounds were combined. These effects were maintained in models of leukopenia and conferred augmented host defenses. Mechanistically, activation of the hypothalamus-pituitary-adrenal gland (HPA) axis by A485 relayed shifts in leukocyte distribution through corticotropin-releasing hormone receptor 1 (CRHR1) and adrenocorticotropic hormone (ACTH), but independently of glucocorticoids. Our findings identify a strategy for rapid expansion of the blood leukocyte compartment via a neuroendocrine loop, with implications for the treatment of human pathologies.

Plasmacytoid dendritic cells control homeostasis of megakaryopoiesis.

Platelet homeostasis is essential for vascular integrity and immune defence1,2. Although the process of platelet formation by fragmenting megakaryocytes (MKs; thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of MKs by their progenitor cells (megakaryopoiesis) remains unclear3,4. Here we use intravital imaging to track the cellular dynamics of megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as homeostatic sensors that monitor the bone marrow for apoptotic MKs and deliver IFNα to the MK niche triggering local on-demand proliferation and maturation of MK progenitors. This pDC-dependent feedback loop is crucial for MK and platelet homeostasis at steady state and under stress. pDCs are best known for their ability to function as vigilant detectors of viral infection5. We show that virus-induced activation of pDCs interferes with their function as homeostatic sensors of megakaryopoiesis. Consequently, activation of pDCs by SARS-CoV-2 leads to excessive megakaryopoiesis. Together, we identify a pDC-dependent homeostatic circuit that involves innate immune sensing and demand-adapted release of inflammatory mediators to maintain homeostasis of the megakaryocytic lineage.

Dendritic cells direct circadian anti-tumour immune responses.

The process of cancer immunosurveillance is a mechanism of tumour suppression that can protect the host from cancer development throughout its lifetime1,2. However, it is unknown whether the effectiveness of cancer immunosurveillance fluctuates over a single day. Here we demonstrate that the initial time of day of tumour engraftment dictates the ensuing tumour size across mouse cancer models. Using immunodeficient mice as well as mice lacking lineage-specific circadian functions, we show that dendritic cells (DCs) and CD8+ T cells exert circadian anti-tumour functions that control melanoma volume. Specifically, we find that rhythmic trafficking of DCs to the tumour draining lymph node governs a circadian response of tumour-antigen-specific CD8+ T cells that is dependent on the circadian expression of the co-stimulatory molecule CD80. As a consequence, cancer immunotherapy is more effective when synchronized with DC functions, shows circadian outcomes in mice and suggests similar effects in humans. These data demonstrate that the circadian rhythms of anti-tumour immune components are not only critical for controlling tumour size but can also be of therapeutic relevance.

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.

Circadian Effects on Vascular Immunopathologies.

Circadian rhythms exert a profound impact on most aspects of mammalian physiology, including the immune and cardiovascular systems. Leukocytes engage in time-of-day-dependent interactions with the vasculature, facilitating the emigration to and the immune surveillance of tissues. This review provides an overview of circadian control of immune-vascular interactions in both the steady state and cardiovascular diseases such as atherosclerosis and infarction. Circadian rhythms impact both the immune and vascular facets of these interactions, primarily through the regulation of chemoattractant and adhesion molecules on immune and endothelial cells. Misaligned light conditions disrupt this rhythm, generally exacerbating atherosclerosis and infarction. In cardiovascular diseases, distinct circadian clock genes, while functioning as part of an integrated circadian system, can have proinflammatory or anti-inflammatory effects on these immune-vascular interactions. Here, we discuss the mechanisms and relevance of circadian rhythms in vascular immunopathologies.

Time-of-Day-Dependent Trafficking and Function of Leukocyte Subsets.

The number of leukocytes circulating in blood in mammals is under circadian control (i.e., ∼24h). We summarize here latest findings on the mechanisms governing leukocyte migration from the blood into various organs, focusing on the distinct leukocyte subtype- and tissue-specific molecules involved. We highlight the oscillatory expression patterns of adhesion molecules, chemokines, and their receptors that are expressed on endothelial cells and leukocytes, and which are crucial regulators of rhythmic leukocyte recruitment. We also discuss the relevance of clock genes for leukocyte function and migration. Finally, we compare immune cell rhythms under steady-state conditions as well as during inflammation and disease, and we postulate how these findings provide potential new avenues for therapeutic intervention.

Neutrophils Recirculate through Lymph Nodes to Survey Tissues for Pathogens.

The adaptive immune function of lymph nodes is dependent on constant recirculation of lymphocytes. In this article, we identify neutrophils present in the lymph node at steady state, exhibiting the same capacity for recirculation. In germ-free mice, neutrophils still recirculate through lymph nodes, and in mice cohoused with wild microbiome mice, the level of neutrophils in lymph nodes increases significantly. We found that at steady state, neutrophils enter the lymph node entirely via L-selectin and actively exit via efferent lymphatics via an S1P dependent mechanism. The small population of neutrophils in the lymph node can act as reconnaissance cells to recruit additional neutrophils in the event of bacterial dissemination to the lymph node. Without these reconnaissance cells, there is a delay in neutrophil recruitment to the lymph node and a reduction in swarm formation following Staphylococcus aureus infection. This ability to recruit additional neutrophils by lymph node neutrophils is initiated by LTB4. This study establishes the capacity of neutrophils to recirculate, much like lymphocytes via L-selectin and high endothelial venules in lymph nodes and demonstrates how the presence of neutrophils at steady state fortifies the lymph node in case of an infection disseminating through lymphatics.

Acute mental stress drives vascular inflammation and promotes plaque destabilization in mouse atherosclerosis.

AIMS: Mental stress substantially contributes to the initiation and progression of human disease, including cardiovascular conditions. We aim to investigate the underlying mechanisms of these contributions since they remain largely unclear. METHODS AND RESULTS: Here, we show in humans and mice that leucocytes deplete rapidly from the blood after a single episode of acute mental stress. Using cell-tracking experiments in animal models of acute mental stress, we found that stress exposure leads to prompt uptake of inflammatory leucocytes from the blood to distinct tissues including heart, lung, skin, and, if present, atherosclerotic plaques. Mechanistically, we found that acute stress enhances leucocyte influx into mouse atherosclerotic plaques by modulating endothelial cells. Specifically, acute stress increases adhesion molecule expression and chemokine release through locally derived norepinephrine. Either chemical or surgical disruption of norepinephrine signalling diminished stress-induced leucocyte migration into mouse atherosclerotic plaques. CONCLUSION: Our data show that acute mental stress rapidly amplifies inflammatory leucocyte expansion inside mouse atherosclerotic lesions and promotes plaque vulnerability.

Adrenergic nerves govern circadian leukocyte recruitment to tissues.

The multistep sequence leading to leukocyte migration is thought to be locally regulated 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. Constitutive leukocyte adhesion and migration in murine bone marrow (BM) and skeletal-muscle microvasculature fluctuated with circadian peak values at night. Migratory oscillations, altered by experimental jet lag, were implemented by perivascular SNS fibers acting on ß-adrenoreceptors expressed on nonhematopoietic cells and leading to tissue-specific, differential circadian oscillations in the expression of endothelial cell adhesion molecules and chemokines. We showed that these rhythms have physiological consequences through alteration of hematopoietic cell recruitment and overall survival in models of septic shock, sickle cell vaso-occlusion, and BM transplantation. These data provide unique insights in the leukocyte adhesion cascade and the potential for time-based therapeutics for transplantation and inflammatory diseases.

Neutrophil ageing is regulated by the microbiome.

Blood polymorphonuclear neutrophils provide immune protection against pathogens, but may also promote tissue injury in inflammatory diseases. Although neutrophils are generally considered to be a relatively homogeneous population, evidence for heterogeneity is emerging. Under steady-state conditions, neutrophil heterogeneity may arise from ageing and replenishment by newly released neutrophils from the bone marrow. Aged neutrophils upregulate CXCR4, a receptor allowing their clearance in the bone marrow, with feedback inhibition of neutrophil production via the IL-17/G-CSF axis, and rhythmic modulation of the haematopoietic stem-cell niche. The aged subset also expresses low levels of L-selectin. Previous studies have suggested that in vitro-aged neutrophils exhibit impaired migration and reduced pro-inflammatory properties. Here, using in vivo ageing analyses in mice, we show that neutrophil pro-inflammatory activity correlates positively with their ageing whilst in circulation. Aged neutrophils represent an overly active subset exhibiting enhanced αMß2 integrin activation and neutrophil extracellular trap formation under inflammatory conditions. Neutrophil ageing is driven by the microbiota via Toll-like receptor and myeloid differentiation factor 88-mediated signalling pathways. Depletion of the microbiota significantly reduces the number of circulating aged neutrophils and dramatically improves the pathogenesis and inflammation-related organ damage in models of sickle-cell disease or endotoxin-induced septic shock. These results identify a role for the microbiota in regulating a disease-promoting neutrophil subset.

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.

Arteriolar niches maintain haematopoietic stem cell quiescence.

Cell cycle quiescence is a critical feature contributing to haematopoietic stem cell (HSC) maintenance. Although various candidate stromal cells have been identified as potential HSC niches, the spatial localization of quiescent HSCs in the bone marrow remains unclear. Here, using a novel approach that combines whole-mount confocal immunofluorescence imaging techniques and computational modelling to analyse significant three-dimensional associations in the mouse bone marrow among vascular structures, stromal cells and HSCs, we show that quiescent HSCs associate specifically with small arterioles that are preferentially found in endosteal bone marrow. These arterioles are ensheathed exclusively by rare NG2 (also known as CSPG4)(+) pericytes, distinct from sinusoid-associated leptin receptor (LEPR)(+) cells. Pharmacological or genetic activation of the HSC cell cycle alters the distribution of HSCs from NG2(+) periarteriolar niches to LEPR(+) perisinusoidal niches. Conditional depletion of NG2(+) cells induces HSC cycling and reduces functional long-term repopulating HSCs in the bone marrow. These results thus indicate that arteriolar niches are indispensable for maintaining HSC quiescence.

Cathepsin G Controls Arterial But Not Venular Myeloid Cell Recruitment.

BACKGROUND: Therapeutic targeting of arterial leukocyte recruitment in the context of atherosclerosis has been disappointing in clinical studies. Reasons for such failures include the lack of knowledge of arterial-specific recruitment patterns. Here we establish the importance of the cathepsin G (CatG) in the context of arterial myeloid cell recruitment. METHODS: Intravital microscopy of the carotid artery, the jugular vein, and cremasteric arterioles and venules in Apoe-/-and CatG-deficient mice (Apoe-/-Ctsg-/-) was used to study site-specific myeloid cell behavior after high-fat diet feeding or tumor necrosis factor stimulation. Atherosclerosis development was assessed in aortic root sections after 4 weeks of high-fat diet, whereas lung inflammation was assessed after inhalation of lipopolysaccharide. Endothelial deposition of CatG and CCL5 was quantified in whole-mount preparations using 2-photon and confocal microscopy. RESULTS: Our observations elucidated a crucial role for CatG during arterial leukocyte adhesion, an effect not found during venular adhesion. Consequently, CatG deficiency attenuates atherosclerosis but not acute lung inflammation. Mechanistically, CatG is immobilized on arterial endothelium where it activates leukocytes to firmly adhere engaging integrin clustering, a process of crucial importance to achieve effective adherence under high-shear flow. Therapeutic neutralization of CatG specifically abrogated arterial leukocyte adhesion without affecting myeloid cell adhesion in the microcirculation. Repetitive application of CatG-neutralizing antibodies permitted inhibition of atherogenesis in mice. CONCLUSIONS: Taken together, these findings present evidence of an arterial-specific recruitment pattern centered on CatG-instructed adhesion strengthening. The inhibition of this process could provide a novel strategy for treatment of arterial inflammation with limited side effects.

Hydroxyurea and a cGMP-amplifying agent have immediate benefits on acute vaso-occlusive events in sickle cell disease mice.

Inhibition of leukocyte adhesion to the vascular endothelium represents a novel and important approach for decreasing sickle cell disease (SCD) vaso-occlusion. Using a humanized SCD-mouse-model of tumor necrosis factor-α-induced acute vaso-occlusion, we herein present data demonstrating that short-term administration of either hydroxyurea or the phosphodiesterase 9 (PDE9) inhibitor, BAY73-6691, significantly altered leukocyte recruitment to the microvasculature. Notably, the administration of both agents led to marked improvements in leukocyte rolling and adhesion and decreased heterotypic red blood cell-leukocyte interactions, coupled with prolonged animal survival. Mechanistically, these rheologic benefits were associated with decreased endothelial adhesion molecule expression, as well as diminished leukocyte Mac-1-integrin activation and cyclic guanosine monophosphate (cGMP)-signaling, leading to reduced leukocyte recruitment. Our findings indicate that hydroxyurea has immediate beneficial effects on the microvasculature in acute sickle-cell crises that are independent of the drug's fetal hemoglobin-elevating properties and probably involve the formation of intravascular nitric oxide. In addition, inhibition of PDE9, an enzyme highly expressed in hematopoietic cells, amplified the cGMP-elevating effects of hydroxyurea and may represent a promising and more tissue-specific adjuvant therapy for this disease.