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 control of tumor immunosuppression affects efficacy of immune checkpoint blockade.

The circadian clock is a critical regulator of immunity, and this circadian control of immune modulation has an essential function in host defense and tumor immunosurveillance. Here we use a single-cell RNA sequencing approach and a genetic model of colorectal cancer to identify clock-dependent changes to the immune landscape that control the abundance of immunosuppressive cells and consequent suppression of cytotoxic CD8+ T cells. Of these immunosuppressive cell types, PD-L1-expressing myeloid-derived suppressor cells (MDSCs) peak in abundance in a rhythmic manner. Disruption of the epithelial cell clock regulates the secretion of cytokines that promote heightened inflammation, recruitment of neutrophils and the subsequent development of MDSCs. We also show that time-of-day anti-PD-L1 delivery is most effective when synchronized with the abundance of immunosuppressive MDSCs. Collectively, these data indicate that circadian gating of tumor immunosuppression informs the timing and efficacy of immune checkpoint inhibitors.

Programmed 'disarming' of the neutrophil proteome reduces the magnitude of inflammation.

The antimicrobial functions of neutrophils are facilitated by a defensive armamentarium of proteins stored in granules, and by the formation of neutrophil extracellular traps (NETs). However, the toxic nature of these structures poses a threat to highly vascularized tissues, such as the lungs. Here, we identified a cell-intrinsic program that modified the neutrophil proteome in the circulation and caused the progressive loss of granule content and reduction of the NET-forming capacity. This program was driven by the receptor CXCR2 and by regulators of circadian cycles. As a consequence, lungs were protected from inflammatory injury at times of day or in mouse mutants in which granule content was low. Changes in the proteome, granule content and NET formation also occurred in human neutrophils, and correlated with the incidence and severity of respiratory distress in pneumonia patients. Our findings unveil a 'disarming' strategy of neutrophils that depletes protein stores to reduce the magnitude of inflammation.

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.

A Neutrophil Timer Coordinates Immune Defense and Vascular Protection.

Neutrophils eliminate pathogens efficiently but can inflict severe damage to the host if they over-activate within blood vessels. It is unclear how immunity solves the dilemma of mounting an efficient anti-microbial defense while preserving vascular health. Here, we identify a neutrophil-intrinsic program that enabled both. The gene Bmal1 regulated expression of the chemokine CXCL2 to induce chemokine receptor CXCR2-dependent diurnal changes in the transcriptional and migratory properties of circulating neutrophils. These diurnal alterations, referred to as neutrophil aging, were antagonized by CXCR4 (C-X-C chemokine receptor type 4) and regulated the outer topology of neutrophils to favor homeostatic egress from blood vessels at night, resulting in boosted anti-microbial activity in tissues. Mice engineered for constitutive neutrophil aging became resistant to infection, but the persistence of intravascular aged neutrophils predisposed them to thrombo-inflammation and death. Thus, diurnal compartmentalization of neutrophils, driven by an internal timer, coordinates immune defense and vascular protection.

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.

Fugue G Minor: Getting the Lymph Node Ensemble Together with Circadian Rhythm.

A fugue is characterized by the systematic repetition of a principal theme in simultaneous melodic lines. In this issue of Immunity, Druzd et al. (2017) show that a similar phenomenon occurs in lymph nodes (LNs), in which lymphocyte entry and exit is governed by repetitive circadian rhythms.

Light-entrained and brain-tuned circadian circuits regulate ILC3s and gut homeostasis.

Group 3 innate lymphoid cells (ILC3s) are major regulators of inflammation, infection, microbiota composition and metabolism1. ILC3s and neuronal cells have been shown to interact at discrete mucosal locations to steer mucosal defence2,3. Nevertheless, it is unclear whether neuroimmune circuits operate at an organismal level, integrating extrinsic environmental signals to orchestrate ILC3 responses. Here we show that light-entrained and brain-tuned circadian circuits regulate enteric ILC3s, intestinal homeostasis, gut defence and host lipid metabolism in mice. We found that enteric ILC3s display circadian expression of clock genes and ILC3-related transcription factors. ILC3-autonomous ablation of the circadian regulator Arntl led to disrupted gut ILC3 homeostasis, impaired epithelial reactivity, a deregulated microbiome, increased susceptibility to bowel infection and disrupted lipid metabolism. Loss of ILC3-intrinsic Arntl shaped the gut 'postcode receptors' of ILC3s. Strikingly, light-dark cycles, feeding rhythms and microbial cues differentially regulated ILC3 clocks, with light signals being the major entraining cues of ILC3s. Accordingly, surgically or genetically induced deregulation of brain rhythmicity led to disrupted circadian ILC3 oscillations, a deregulated microbiome and altered lipid metabolism. Our work reveals a circadian circuitry that translates environmental light cues into enteric ILC3s, shaping intestinal health, metabolism and organismal homeostasis.

A detective story of intermittent fasting effect on immunity.

Intermittent fasting (IF) refers to periodic fasting routines, that caloric intake is minimized not by meal portion size reduction but by intermittently eliminating ingestion of one or several consecutive meals. IF can instigate comprehensive and multifaceted alterations in energy metabolism, these metabolic channels may aboundingly function as primordial mechanisms that interface with the immune system, instigating intricate immune transformations. This review delivers a comprehensive understanding of IF, paying particular attention to its influence on the immune system, thus seeking to bridge these two research domains. We explore how IF effects lipid metabolism, hormonal levels, circadian rhythm, autophagy, oxidative stress, gut microbiota, and intestinal barrier integrity, and conjecture about the mechanisms orchestrating the intersect between these factors and the immune system. Moreover, the review includes research findings on the implications of IF on the immune system and patients burdened with autoimmune diseases.

Morning administration enhances humoral response to SARS-CoV-2 vaccination in kidney transplant recipients.

Although severe acute respiratory syndrome coronavirus 2 messenger ribonucleic acid (SARS-CoV-2 mRNA) vaccines are effective in kidney transplant recipients (KTRs), their immune response to vaccination is blunted by immunosuppression. Other tools enhancing vaccination response are therefore needed. Interestingly, aligning vaccine administration with circadian rhythms (chronovaccination) has been shown to boost immune response. However, its applicability in KTRs, whose circadian rhythms are likely disrupted by immunosuppressants, remains unclear. To assess the impact of vaccination timing on seroconversion in the KTRs population, we analyzed data from 553 virus-naïve KTRs who received 2 doses of messenger ribonucleic acid (mRNA) vaccine. Bayesian logistic regression was employed, adjusting for previously identified predictors of seroconversion, including allograft function, maintenance immunosuppressants, or time since transplantation. SARS-CoV-2 immunoglobulin G (IgG) levels were measured with a median of 47 days after the second dose. The results did not reveal a reliable effect of timing of the first dose but did indicate that earlier timing for the second dose brings a notable benefit-every 1-hour delay in the application was associated with a 16% reduction in the odds of seroconversion (OR 0.84, 95% CI 0.71, 0.998). Similar results were obtained from quantile regression modeling IgG levels. In conclusion, morning vaccination is emerging as a promising and easily implementable strategy to enhance vaccine response in KTRs.

The influence of circadian rhythms on CD8+ T cell activation upon vaccination: A mathematical modeling perspective.

Circadian rhythms have been implicated in the modulation of many physiological processes, including those associated with the immune system. For example, these rhythms influence CD8+ T cell responses within the adaptive immune system. The mechanism underlying this immune-circadian interaction, however, remains unclear, particularly in the context of vaccination. Here, we devise a molecularly-explicit gene regulatory network model of early signaling in the naïve CD8+ T cell activation pathway, comprised of three axes (or subsystems) labeled ZAP70, LAT and CD28, to elucidate the molecular details of this immune-circadian mechanism and its relation to vaccination. This is done by coupling the model to a periodic forcing function to identify the molecular players targeted by circadian rhythms, and analyzing how these rhythms subsequently affect CD8+ T cell activation under differing levels of T cell receptor (TCR) phosphorylation, which we designate as vaccine load. By performing both bifurcation and parameter sensitivity analyses on the model at the single cell and ensemble levels, we find that applying periodic forcing on molecular targets within the ZAP70 axis is sufficient to create a day-night discrepancy in CD8+ T cell activation in a manner that is dependent on the bistable switch inherent in CD8+ T cell early signaling. We also demonstrate that the resulting CD8+ T cell activation is dependent on the strength of the periodic coupling as well as on the level of TCR phosphorylation. Our results show that this day-night discrepancy is not transmitted to certain downstream molecules within the LAT subsystem, such as mTORC1, suggesting a secondary, independent circadian regulation on that protein complex. We also corroborate experimental results by showing that the circadian regulation of CD8+ T cell primarily acts at a baseline, pre-vaccination state, playing a facilitating role in priming CD8+ T cells to vaccine inputs according to the time of day. By applying an ensemble level analysis using bifurcation theory and by including several hypothesized molecular targets of this circadian rhythm, we further demonstrate an increased variability between CD8+ T cells (due to heterogeneity) induced by its circadian regulation, which may allow an ensemble of CD8+ T cells to activate at a lower vaccine load, improving its sensitivity. This modeling study thus provides insights into the immune targets of the circadian clock, and proposes an interaction between vaccine load and the influence of circadian rhythms on CD8+ T cell activation.

Circadian Influences of Diet on the Microbiome and Immunity.

Host circadian rhythmicity and the timing of feeding are increasingly recognized to cross-regulate and entrain each other, and may play crucial roles in regulating multiple physiological functions including host immunity and metabolic health. Of relevance, these circadian diet-immune interactions may be modulated by the gut microbiota. We review current knowledge linking the circadian clock and dietary timing to host immune-microbiota interactions, exemplifying how this axis may impact on host immunity in health and in a variety of immune-mediated diseases. We also discuss current challenges in reaching mechanistic insights regarding the functions of the diurnally shifting diet-microbiome-host immune axis. We highlight the possible implications of circadian reprogramming by dietary timing patterns as a future intervention to modulate a variety of immune-related diseases.

Circadian clock proteins and immunity.

Immune parameters change with time of day and disruption of circadian rhythms has been linked to inflammatory pathologies. A circadian-clock-controlled immune system might allow an organism to anticipate daily changes in activity and feeding and the associated risk of infection or tissue damage to the host. Responses to bacteria have been shown to vary depending on time of infection, with mice being more at risk of sepsis when challenged ahead of their activity phase. Studies highlight the extent to which the molecular clock, most notably the core clock proteins BMAL1, CLOCK, and REV-ERBα, control fundamental aspects of the immune response. Examples include the BMAL1:CLOCK heterodimer regulating toll-like receptor 9 (TLR9) expression and repressing expression of the inflammatory monocyte chemokine ligand (CCL2) as well as REV-ERBα suppressing the induction of interleukin-6. Understanding the daily rhythm of the immune system could have implications for vaccinations and how we manage infectious and inflammatory diseases.

Conditional Deletions of Hdc Confirm Roles of Histamine in Anaphylaxis and Circadian Activity but Not in Autoimmune Encephalomyelitis.

Histamine is best known for its role in allergies, but it could also be involved in autoimmune diseases such as multiple sclerosis. However, studies using experimental autoimmune encephalomyelitis (EAE), the most widely used animal model for multiple sclerosis, have reported conflicting observations and suggest the implication of a nonclassical source of histamine. In this study, we demonstrate that neutrophils are the main producers of histamine in the spinal cord of EAE mice. To assess the role of histamine by taking into account its different cellular sources, we used CRISPR-Cas9 to generate conditional knockout mice for the histamine-synthesizing enzyme histidine decarboxylase. We found that ubiquitous and cell-specific deletions do not affect the course of EAE. However, neutrophil-specific deletion attenuates hypothermia caused by IgE-mediated anaphylaxis, whereas neuron-specific deletion reduces circadian activity. In summary, this study refutes the role of histamine in EAE, unveils a role for neutrophil-derived histamine in IgE-mediated anaphylaxis, and establishes a new mouse model to re-explore the inflammatory and neurologic roles of histamine.

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.

The circadian clock of CD8 T cells modulates their early response to vaccination and the rhythmicity of related signaling pathways.

Circadian variations of various aspects of the immune system have been described. However, the circadian control of T cells has been relatively unexplored. Here, we investigated the role of circadian clocks in regulating CD8 T cell response to antigen presentation by dendritic cells (DCs). The in vivo CD8 T cell response following vaccination with DCs loaded with the OVA257-264 peptide antigen (DC-OVA) leads to a higher expansion of OVA-specific T cells in response to vaccination done in the middle of the day, compared to other time points. This rhythm was dampened when DCs deficient for the essential clock gene Bmal1 were used and abolished in mice with a CD8 T cell-specific Bmal1 deletion. Thus, we assessed the circadian transcriptome of CD8 T cells and found an enrichment in the daytime of genes and pathways involved in T cell activation. Based on this, we investigated early T cell activation events. Three days postvaccination, we found higher T cell activation markers and related signaling pathways (including IRF4, mTOR, and AKT) after a vaccination done during the middle of the day compared to the middle of the night. Finally, the functional impact of the stronger daytime response was shown by a more efficient response to a bacterial challenge at this time of day. Altogether, these results suggest that the clock of CD8 T cells modulates the response to vaccination by shaping the transcriptional program of these cells and making them more prone to strong and efficient activation and proliferation according to the time of day.

Peripheral immune circadian variation, synchronisation and possible dysrhythmia in established type 1 diabetes.

AIMS/HYPOTHESIS: The circadian clock influences both diabetes and immunity. Our goal in this study was to characterise more thoroughly the circadian patterns of immune cell populations and cytokines that are particularly relevant to the immune pathology of type 1 diabetes and thus fill in a current gap in our understanding of this disease. METHODS: Ten individuals with established type 1 diabetes (mean disease duration 11 years, age 18-40 years, six female) participated in a circadian sampling protocol, each providing six blood samples over a 24 h period. RESULTS: Daily ranges of population frequencies were sometimes large and possibly clinically significant. Several immune populations, such as dendritic cells, CD4 and CD8 T cells and their effector memory subpopulations, CD4 regulatory T cells, B cells and cytokine IL-6, exhibited statistically significant circadian rhythmicity. In a comparison with historical healthy control individuals, but using shipped samples, we observed that participants with type 1 diabetes had statistically significant phase shifts occurring in the time of peak occurrence of B cells (+4.8 h), CD4 and CD8 T cells (~ +5 h) and their naive and effector memory subsets (~ +3.3 to +4.5 h), and regulatory T cells (+4.1 h). An independent streptozotocin murine experiment confirmed the phase shifting of CD8 T cells and suggests that circadian dysrhythmia in type 1 diabetes might be an effect and not a cause of the disease. CONCLUSIONS/INTERPRETATION: Future efforts investigating this newly described aspect of type 1 diabetes in human participants are warranted. Peripheral immune populations should be measured near the same time of day in order to reduce circadian-related variation.

Immune-enhancing effects of glucocorticoids in response to day-night cycles and stress.

Environmental cues such as the day-night cycle or stressors trigger the production of glucocorticoids (GCs) by the adrenal cortex. GCs are well known for their anti-inflammatory effects that suppress the production of inflammatory cytokines and induce the apoptosis of lymphocytes. Recent studies in mice, however, have revealed pro-inflammatory effects. The diurnal oscillation of GCs induces the expression of IL-7 receptor α (IL-7Rα) and C-X-C motif chemokine receptor 4 (CXCR4) at the active phase, which drives the diurnal homing of T cells into lymphoid organs. This accumulation of T cells at the active phase enhances T-cell priming against bacterial infection and antigen immunization, leading to an increase of effector CD8 T cells and antibody production. GCs induced by moderate stress trigger the homing of memory CD8 T cells into the bone marrow and support the maintenance and response of these cells. Thus, endogenous GCs have a self-defense function to enhance adaptive immune responses. By contrast, strong stress induces even higher GC levels and causes chronic inflammation and autoimmunity. Because GCs can enhance the differentiation and function of T-helper 2 (Th2) and Th17 cells, high stress-induced GC levels might enhance inflammation via Th17 cell differentiation. Overall, the positive and negative effects of GCs may regulate the balance between normal immune responses and susceptibility to infections and inflammatory diseases.

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.