Circadian Rhythms in Solid Organ Transplantation

Abstract

Circadian rhythms are daily cycles in physiology that can affect medical interventions. This review considers how these rhythms may relate to solid organ transplantation. It begins by summarizing the mechanism for circadian rhythm generation known as the molecular clock, and basic research connecting the clock to biological activities germane to organ acceptance. Next follows a review of clinical evidence relating time of day to adverse transplantation outcomes. The concluding section discusses knowledge gaps and practical areas where applying circadian biology might improve transplantation success.

Circadian rhythms are daily oscillations in biological function that endow organisms with an intrinsic ability to keep time [1–3]. By enabling organisms to anticipate the day-night cycle regardless of weather conditions, circadian rhythms provide a powerful evolutionary advantage. In any given organism, circadian rhythms occur up and down the biological scale and manifest in clinically important functions like drug metabolism and immunity. There is a growing interest in the concept of “circadian medicine”, where therapies are tailored around patients’ circadian rhythms to improve their effectiveness [2, 4, 5].

This review considers the application of circadian biology to solid organ transplantation, which is little studied to date. It is intended as a primer for practitioners and transplant biologists, beginning with a description of the molecular clock mechanism and concluding with a review of the clinical literature evaluating the time of day as a factor in transplant success.

The molecular clock mechanism.

A biological rhythm is any predictably recurring variation in organism activity over time. Such rhythms are ubiquitous and occur across timescales ranging from milliseconds to decades [6, 7]. By definition, a biological rhythm is “circadian” if it repeats roughly every 24 hours without the need for environmental input to perpetuate the pattern [7, 8]. While not dependent on the environment, circadian rhythms must be responsive to exposures like light onset to align them with the environment, a process called entrainment [8]. The system that generates circadian rhythms is often conceptualized as an “oscillator” or clock that takes cyclical stimuli in the environment as input and imparts 24-hour patterns onto physiology as output (Fig. 1A).

Figure. 1: The circadian system in mammals.

Panel A depicts the “oscillator” paradigm of circadian rhythm generation, in which environmental cues set the phase of an intrinsic and continuously running clock. The clock then imparts a daily structure to biological function, from behavior to biomolecules. Panel B is a schematic of the circadian clock mechanism, composed of transcription factors called “clock genes” that regulate their expression in a feedback loop that takes 24 hours to reinitialize. Panel C depicts the two circadian clock compartments at issue during transplant. The recipient’s circadian system (black) consists of the central CNS pacemaker and peripheral clocks in recipient tissues. Clocks in the donor organ (red), may be set to a different circadian clock time (out of phase) than the recipient clocks at the time of transplantation and lack autonomic innervation that helps to synchronize peripheral clocks.

Recognition of circadian rhythms goes back millennia, but an understanding of how they come about only emerged in the 1990s with genetic experiments in model organisms combined with analyses of patients with heritable “circadian” sleep disorders [9–12]. Using mutant flies whose circadian pattern of activity was either disrupted or altered, it was possible to clone the first “clock” genes (Fig. 1B) [9, 10]. Studies revealed that clock genes are organized into a transcription factor network connected by a series of autoregulatory feedback loops [13]. In mammals, the core element is a heterodimer composed of the proteins BMAL1 and CLOCK that stimulate transcription from promoters and enhancer elements containing “E-box” motifs [14]. Among the hundreds of transactivated genes are the negative regulator genes PER1–3 and CRY1–2, whose protein products form a multimeric complex that moves to the nucleus and inhibits BMAL1/CLOCK. The stability of the PER/CRY complex is modulated by phosphorylation and ubiquitination, thereby speeding up or slowing down its feedback inhibition and allowing the “phase” of the clock to be adjusted [15]. A second group of clock genes called REVERBα/β (or NR1D1/2) are inhibitory transcription factors that form another negative feedback loop by repressing genes containing ROR-responsive elements (RREs), including BMAL1. Positive feedback is supplied by the clock genes RORα–γ (RAR Related Orphan Receptor A–C) that stimulate transcription at RREs and directly compete with REVERBα/β. The cumulative result is that clock genes all oscillate in abundance in a roughly 24-hour cycle and— because they are transcription factors— impart a 24-hour bias to gene expression in general.

Virtually all nucleated cells express clock genes and have a functioning molecular clock. In mammals, roughly 50–85% of protein-coding mRNAs show rhythmic abundance organism-wide, with 5–10% exhibiting rhythms in any one organ [16, 17]. Among genes regulated by the clock are master transcription factors important for metabolic, immune, and redox functions, like nuclear factor IL-3–regulated (NFIL3), the PAR domain basic leucine zipper transcription factors (DBP, TEF, and HLF), the peroxisome proliferator–activated receptors (PPARs) [18–20], and nuclear factor erythroid 2-related factor 2 (NRF2) [21–23]. Beyond gene expression, other biomolecules oscillate as well, including metabolites [24], proteins [25, 26], and covalent modifications like phosphorylation and ubiquitination [27–29]. It follows that cellular physiology has a strong circadian component, especially metabolism and cell division [30, 31].

Moving from the microscopic to the macroscopic, molecular clocks within neighboring cells are kept in register with one another through pulsatile extracellular cues like nutrient availability, corticosteroids, and autonomic tone [32]. The result is a general “internal synchrony” of billions of cell-autonomous circadian clocks within organisms, which allow rhythms in organ physiology to emerge. In mammals, neuro-humoral cues are under CNS control through a circadian “central pacemaker” located in the suprachiasmatic nucleus (SCN) of the ventral hypothalamus. The SCN receives direct light input from retinal ganglion cells and projects to hypothalamic areas, producing daily rhythms of pituitary hormone secretions, wakefulness, appetite, and temperature [32, 33]. Thus, circadian rhythms in organisms are the product of a two-tiered system, consisting of a “local” clock within each cell and rhythmic synchronizing cues broadcast by the CNS, which entrain clocks to the day-night cycle.

Understanding the role of the circadian system in transplantation can be especially challenging, as there are two circadian systems to consider: that of the recipient and that of the donated organ (Fig. 1C). Of the many circadian outputs in physiology, three stand out as particularly germane to the immediate transplantation setting. First, immune system activity is highly rhythmic across the day at virtually every level (Table 1). For example, inflammatory cytokine release from macrophages in response to endotoxin or other microbial products varies markedly based on the time of day. Leukocyte trafficking into organs exhibits a circadian rhythm, hence the WBC burden of transplanted organs could theoretically vary with the time of procurement. On the recipient side, the potential for leukocyte infiltration into a new allograft might vary by the phase in the circadian cycle when transplantation occurs. Finally, T cell activation has a circadian rhythm, and hence the time of transplantation may figure into graft tolerance.

Table 1:

Circadian Clock Regulation of Basic Immune Processes.

Immune Activity	Cell Types	Clock-Dependent Mechanisms	References
Leukocyte Development	Innate Lymphoid Cells	Regulated by clock genes NFIL3, RORa	[89–95]
	TH17 cells	Regulated by clock genes RORa, RORgt, NR1D1	[96–98]
	Treg cells	Accessory clock gene NFIL3 regulates FOXP3	[99]
	Neutrophils	BMAL regulates CXCL2	[52–54]
Leukocyte Trafficking	All	Rhythms in cell adhesion molecule expression on leukocytes and endothelial cells.	[57]
		Rhythms in leukocyte chemotactic factors and receptors.	[56, 57, 100–104]
Innate Immunity	Macrophages	Rhythms in secretion of numerous cytokines and chemokines.	[43–47, 105–107]
		BMAL1 regulates PD-L1promoting immune senescence.	[42, 48]
		Rhythms in phagocytosis associated with oscillations in mitochondrial dynamics.	[108–111]
	NK cells	Rhythms in IFNg, Perforin, and granzyme expression.	[112, 113]
	Neutrophils	Rhythms in NETosis, NADPH oxidase, and phagocytosis.	[53, 55]
	Mast cells/Eosinophils	Rhythms in IgE and fMLP stimulated chemokine secretion.	[114]
Adaptive Immunity	Dendritic cells	Rhythms in activation via TLR ligands.	[69, 70]
		Rhythms in antigen processing which correlate with mitochondrial dynamics.	[115]
		Rhythms in CD80 costimulatory molecule expression.	[116]
	T cells	Rhythms in CD8 cell expansion upon antigen challenge.	[117, 118]
	B cells	Rhythms in antibody titers based on immunization time of day. Correlates with times of higher lymph node cellularity.	[119]
		Rhythm in IgA secretion from intestinal plasma cells.	[120]
		Regulation of BCR signaling by CRY1/2. Deletion of these genes produces spontaneous antibodymediated autoimmune disease in mice.	[121]

Secondly, there are circadian rhythms in the response to tissue ischemia. In animal surgical models, the extent of ischemia-reperfusion injury in the heart and kidney varies with time of day in a circadian manner [34–38]. This may reflect circadian rhythms in antioxidant capacity, including the direct transcriptional regulation of the master regular NRF2 by the circadian clock [21–23]. Additionally, induction of hypoxia-inducible factor 1α (HIF1a) by low oxygen conditions and exercise is enhanced by the clock protein BMAL1 [39, 40]. Neutrophils and macrophages which are key in ischemia reperfusion injury, are under strong circadian regulation. Macrophages exhibit circadian patterns of phagocytosis, chemokine and secretion, as well as cell adhesion molecule expression and trafficking [41–48]. This is important because changes in macrophage activation, abundance, and function play a part in determining the severity of the ischemic injury [49–51]. Similarly, neutrophils exacerbate post-ischemic inflammation and demonstrate circadian regulation in trafficking patterns, cytokine secretion, NETosis, and phagocytosis [52–58].

Finally, there are circadian rhythms in drug metabolism [16, 59]. Multiple P450 enzymes are rhythmically expressed, including CYP3A4 and 2C8 that contribute to the metabolism of immunosuppressive agents like tacrolimus, sirolimus, prednisolone, and mycophenolate motefil [60]. For drugs like prednisone and tacrolimus whose half-lives are shorter than a circadian cycle (about 12 hours in each case), the time of dosing can affect their pharmacodynamics [61–63]. Evidence for clinical relevance exists for asthmatic patients, where the time of inhaled steroid dosing alters the minimum clinically effective dose [64].

Evidence that transplantation time of day matters.

In contrast to basic research studies, the clinical literature assessing circadian rhythms in solid organ transplantation is sparse. There are 16 retrospective studies in PUBMED analyzing associations between transplantation surgery time of day and outcomes (Table 2), and 6 examining the time of organ procurement (Table 3). As a group, the studies differ in terms of the organs considered, sample size, outcomes measured, and the approach to binning times of day. Most studies dichotomously compare “day’ versus “night” transplantation, which has limited sensitivity. Nevertheless, most studies conclude that night-time or early morning transplantation correlates with moderately worse outcomes. These include acute complications like graft dysfunction and long-term graft survival. The evidence is strongest for lung transplantation (4/4 studies), which is biologically plausible given the heavy immune composition of this organ: about 25% of cells in the lung are of hematopoietic origin and the density of neutrophils in the lung is 3 times higher than peripheral blood [65, 66].

Table 2: Association between transplantation time of day and outcomes.

Times of day are expressed in military time.

Organ	Transplantation Time Bins Compared	Sample Size	Duration of Followup	Outcomes	Time Effect (Highest Risk Bin for Adverse Outcomes)	Reference
Lung	7:00–18:59 19:00–6:59	10.545	1 Year	Postoperative Events Airway Dehiscence Patient Survival	− + (19:00–6:59). + 90-day survival (19:00–6:59)	[122]
Lung	0:00–3:59 4:00–7:59 8:00–11:59 12:00–15:59 16:00–19:59 12:00–23:59	563	3 Days	Primary Graft Dysfunction	+ (4:00–7:59)	[80]
Lung	5:00–17:59 18:00–4:59	740	5 Years	Postoperative Events Overall Survival Chronic Rejection-Free Survival	+ (18:00–4:59) + (18:00–4:59) + (18:00–4:59)	[123]
Lung	0:00–5:59 6:00–11:59 12:00–17:59 18:00–23:59	32,841	10 years	Length of Stay Overall Survival Primary Graft Dysfunction	+ (18:00–0:00) + (18:00–0:00) + (18:00–0:00)	[124]
Heart	4:00–1159 12:00–19:59 20:00–3:59	235	3 Years	Delayed Graft Function Postoperative Events Infection Acute Rejection Patient Survival	− − − − − (trend towards worse survival with morning to afternoon)	[125]
Heart	7:00–18:59 19:00–6:59	16,573	1 Year	Post-operative Events Patient Survival	− −	[122]
Liver	8:00–14:00 20:00–2:00	147	Not Specified	Intraoperative Events Delayed Graft Function	+ + (20:00–2:00)	[126]
Liver	7:00–18:59 19:00–6:59	8816	3 Years	Graft Survival	+/− (19:00–6:59 in unadjusted analysis)	[127]
Liver	8:00–17:59 18:00–7:59	350	1 Year	Patient Survival Graft Survival	−	[128]
Kidney	0:00–5:59 6:00–11:59 12:00–17:59 18:00–23:59	726	7 Days	Delayed Graft Function	+ (6:00–11:59) Note: association noted only with organs obtained from brain-dead donors (n=536).	[84]
Kidney	10:00–17:59 18:00–9:59	8962	3 Years	Patient Survival	+ (18:00–9:59)	[129]
Kidney	8:00–19:59 20:00–7:59	215	1 Year	Graft Survival Patient Survival	−	[130]
Kidney	8:00–19:59 20:00–7:59	260	43 Months (Median)	Graft Survival Re-transplantation	+ (20:00–7:59) + (20:00–7:59)	[131]
Kidney	8:00–19:59 20:00–7:59	443	1 Year	Graft Survival Patient Survival	−	[132]
Kidney	8:00–19:59 20:00–7:59	7	1 Year	Delayed Graft Function Serum Creatinine	−	[133]
Kidney	8:00–19:59 20:00–7:59	4519	10 Years	Graft Survival	+(20:00–7:59)	[134]
Kidney	8:00–19:59 20:00–7:59	873	5 Years	Delayed Graft Function Acute Rejection Graft Survival	+(20:00–7:59) − −	[135]

Table 3: Association between organ procurement time of day and transplant outcomes.

Times of day are expressed in military time.

Organ	Procurement Time Bins Compared	Sample Size	Duration of Followup	Outcomes	Time Effect (Highest Risk Bin for Adverse Outcomes)	Reference
Heart	12:00–23:59 0:00–11:59	266 (propensity score matched)	6 Years	Acute Rejection-Free Survival Chronic Rejection-Free Survival Overall Survival	+ (12:00–23:59) − + (12:00–23:59)	[136]
Liver	7:00–18:59 19:00–6:59	8816	3 Years	Graft Survival	+/− (19:00–6:59 in unadjusted analysis)	[127]
Kidney	0:00–5:59 6:00–11:59 12:00–17:59 18:00–23:59	726	7 Days	Delayed Graft Function	+ (6:00–11:59) Note: association noted only with organs obtained from brain-dead donors (n=536).	[84]
Kidney	0:00–5:59 6:00–11:59 12:00–17:59 18:00–23:59	536	7 Days	Delayed Graft Function	−	[77]
Kidney	10:00–17:59 18:00–9:59	8962	3 Years	Patient Survival	+ (18:00–9:59)	[129]
Kidney	12:00–23:59 0:00–11:59	5026	10 Years	Delayed Graft Function eGFR at 1 Year Patient and Graft Survival	+ (12:00–23:59) − + (12:00–23:59)	[137]

Immunologically focused transplant studies rarely consider time-of-day as a biological variable. However, studies repeatedly demonstrate that there is circadian regulation of leukocyte trafficking, cytokine release, and immune cell surface marker upregulation [67–71]. These fluctuations may result in differential immune measurements at different times of day, similar to how transplant patients tend to develop arrhythmia at night [72–75]. Therefore, studies which consider differences in these immune markers may generate spurious results. For example, if a study is performed in mice, and the controls are measured in the morning and the transplant mice are measured in the afternoon, differences in the respective immune landscapes will be significant regardless of treatment. Conversely, replicate experiments performed at different times of day may yield variable results that can be dismissed as falsely negative. Beyond the direct time-of-day effect on the immune landscape, many metabolic systems are dependent on circadian regulated protein and expression levels which could affect drug metabolism and response [4, 5, 62].

Intriguingly, some studies indicate that organ procurement time of day independently correlates with transplant success, implying that circadian rhythms within the donor or donor organ may affect graft performance (Table 3). At first glance, this seems counterintuitive as most solid organ donors are brain-dead and presumably lack a functioning central circadian pacemaker. However, no literature assessing biological rhythms or circadian clock function in the organ donor population is currently available. The nearest correlate would be intensive care unit (ICU) patients, who routinely exhibit intact rhythms in core body temperature and even clock gene expression [76, 77]. A major distinction between ICU patients and healthy individuals is that circadian outputs like temperature rhythms appear unmoored from the day-night cycle and peak at different points of the day from patient to patient [76]. Whether this extends to organ donors is unknown. Of note, organ donors routinely receive supra-physiologic doses of corticosteroids as part of pre-procurement management [78]. Given that corticosteroids are a strong circadian clock synchronizer [79], it’s tempting to speculate this could enable donors to recapitulate physiologic circadian rhythms in the absence of a central pacemaker.

Altogether, clinical observations suggest a time-of-day effect in solid organ transplantation, both respect to the donor organs and the recipient. As with all real-world biological rhythms, the effects observed are likely an amalgam of environmental variations (like differences in hospital staffing or performance across the day) and periodicity in the underlying biology (circadian clocks). Regardless, there is an opportunity to improve transplantation success by better understanding time as a biological variable in organ donors and recipients.

Future Directions.

Clinicians already consider donor-recipient matching based on organ size, infectious history, blood group, and, in the case of hematopoietic stem cells, HLA typing. Circadian biology raises the possibility that temporally matching the circadian systems of donated organs and recipients may improve transplant acceptance. Getting to this point will require a much better mechanistic understanding of how circadian clocks relate to organ transplant biology. It will also require translational research advances to practically apply circadian biology to transplantation process of care. Several topics need parallel development:

Mechanistic research.

While clinical observations are suggestive, there are currently no direct mechanistic studies evaluating how the circadian clock affects transplant pathophysiology like primary graft dysfunction, acute rejection, or chronic rejection. However, multiple tools and model organisms can be applied to this question including clock gene mutant mice, many of which are commercially available. A useful quality of circadian clocks is that they can be experimentally manipulated by adjusting light or feeding cycles, thereby shifting organisms into different “time zones” before employing them as organ donors or recipients. The internal circadian synchrony of model organisms can be disrupted by providing a variable light schedule to create a state of “chronic jet lag”, which can serve to validate observations with clock mutant mice. The existing clinical literature and the prominent immune composition of the lung illustrates the need to explore circadian rhythm contributions to thoracic transplants and subsequent recovery.

Donor circadian biology.

Given the suggestion that the time of organ procurement may be clinically relevant, there may be an opportunity to employ donor circadian biology to affect outcomes. The extent to which biological rhythms persist in organ donors (brain dead or otherwise) is unknown. However, donors routinely receive corticosteroids, which have the potential to synchronize or reboot circadian rhythms in the donor. The pre-procurement period is one of the most practical points in the transplantation process to manipulate circadian rhythms in immune cell trafficking or ischemia tolerance given the degree of clinical control. Circadian clocks have a stereotypical reaction to synchronizing agents like steroids called a “phase response curve”, in which the clocks will either advance, fall back, or do nothing based on the internal clock time when stimulus occurs. To be able to predict how corticosteroid dosing affects circadian clocks in donors, it will be necessary to develop a means of estimating the internal clock time of individual donors. Assays already exist for predicting clock time using peripheral blood or skin samples from healthy individuals, and similar methodologies could be adapted to the donor population.

Organ preservation.

Evidence suggests that hypothermia as mild as 18°C synchronizes circadian clocks, making organ preservation conditions a factor in circadian biology within the allograft [80, 81]. With the increasing popularity of continuous perfusion systems, there is the potential to expose grafts to temperature or hormonal cycles during the period between organ procurement and transplantation. In so doing, it may be possible to shift circadian clocks so that their phases are aligned with the intended recipient. Research is needed to determine whether such an approach can optimize graft resilience against ischemia/reperfusion injury.

Circadian pharmacology.

Despite studies suggesting that time of day affects the pharmacodynamics of immunosuppressants, tailoring the dosing of these drugs around patient circadian rhythms to maximize their therapeutic index is not a part of routine practice. Likely, part of the reason is that the standard practice of tacrolimus drug level monitoring allows clinicians to empirically determine dosing, and there is the potential for increasing non-compliance by making dosing schedules too arduous or complicated for patients. One opportunity may be the dosing of prednisone, a once-a-day drug where night dosing may enable lower amounts to be given to patients based on asthma literature. Additionally, pulse steroid dosing for clinical deterioration may provide differential benefits based on morning versus evening dosing [64]. More research is warranted to investigate this.

Rhythms as a marker of transplant recipient health.

Finally, for transplant recipients to thrive socially they must regain circadian rhythms in activity, sleep, and food intake. Literature suggests that circadian rhythms connected to donor organ physiology, for example, heart rate variation and blood pressure, are lost initially after solid organ transplantation [82–85]. Re-establishment of these rhythms could potentially represent a positive long-term prognostic sign [86]. Conversely, a loss of circadian rhythms in activity or sleep could presage clinical decompensation in transplant recipients, as this is already understood to be a general predictor of future hospitalization [87, 88]. Over-the-counter devices including cell phones can collect actigraphy data and represent a possible way to improve clinical follow-up, especially for recipients living far away from specialized transplant centers.

In summary, circadian rhythms and their underlying clock are likely to be relevant to solid organ transplantation on multiple biological and clinical levels. The topic is wide open for study, and future research will determine how circadian biology can be best leveraged to improve transplant success.