Health outcomes in humans often show daily variations. For instance, morning vaccinations may trigger a more effective immune response than a jab in the afternoon.1 Similarly, people are more susceptible to infections at certain times of the day.2
Such variability is orchestrated by circadian clocks that regulate oscillations in gene expression. Experiments conducted in cells and lab animals have shown that the liver, which plays a role in vaccine responses and infections, displays a large number of such circadian-dependent genes.3 However, some aspects of drug metabolism and immune reactions are unique to humans and difficult to investigate due to a lack of experimental systems.
“We knew that the liver has its own circadian rhythm that is independent of the central clock in our brain,” said Liliana Mancio-Silva, a parasitologist at the Pasteur Institute. “We wanted to know whether we can mimic the liver circadian oscillations in vitro.”
Mancio-Silva teamed up with biomedical engineer Sangeeta Bhatia at Massachusetts Institute of Technology to develop an in vitro human liver model, which they described in Science Advances.4 In characterizing the hepatocytes in their system, the researchers identified the genes involved in drug metabolism and susceptibility to infections that are under circadian control. The model, which mimics the circadian rhythm of the organ, provides researchers with a platform to study the influence of circadian genes on human liver functions and improve drug development.
To develop their new system, the researchers obtained liver cells from individual donors and cultured them alongside fibroblasts, which provided structural support. By tweaking the culture conditions to express the circadian clock gene basic helix-loop-helix ARNT-like protein 1 (BMAL1), which helps orchestrate the expression of several other genes, the team generated liver cells that developed synchronized circadian oscillations that were sustained for 10 weeks.
Equipped with a system to study cyclic variations in liver cells, the researchers wondered how circadian rhythms affected gene expression. They analyzed the transcriptomes of these cells and found that more than 380 genes were expressed cyclically and that a majority of these genes were related to drug metabolism and inflammatory and immune responses.
One of these cyclically expressed genes, cytochrome P450 3A4 (CYP3A4) encodes a drug-metabolizing enzyme in the cytochrome P450 family, which is responsible for about three-quarters of all drug metabolism reactions in humans.5 They found that CYP3A4 enzymatic activity occurred in waves, suggesting that drug pharmacokinetics may differ depending on the time of the day.
To test this, the team treated hepatic cells with either the lipid-lowering drug atorvastatin or the non-steroidal analgesic acetaminophen. These drugs are harmful to the liver at high doses due to their metabolism by CYP3A4 into toxic byproducts. In the treated cells, they observed that higher levels of CYP3A4 correlated with greater cell death, suggesting that adverse drug effects may be minimized by optimizing the time at which the drug is administered.
“[These results] are the culmination of predictions that were going on for 20 years,” said Satchidananda Panda, a chronobiologist at the Salk Institute for Biological Studies, who was not involved with the study. He noted that researchers had previously shown the cyclic expression of drug-metabolizing genes in mice, “But there was no real experiment showing whether cytochrome P450 gene activity cycles in the human liver.”
He added, “The technological breakthrough in this paper is keeping human liver cells alive for 10 weeks,” which enabled Mancio-Silva and her team to prove, for the first time, that drug-metabolizing enzyme activities in humans are cyclical.
However, one limitation, Panda noted, was that the system is continuously bathed in glucose, which doesn’t mimic the fasting-feeding cycle that happens physiologically. Nevertheless, this is the closest researchers have gotten to recapitulating the human liver’s circadian system, he said.
He noted that it will be important for future studies to explore the circadian control of drug-metabolizing genes other than CYP3A4.
Mancio-Silva and the team switched gears to use the in vitro system to investigate how circadian genes influence immunity and infection in the liver. They found that interferons, the body’s virus-fighting proteins, stimulated a subset of genes that exhibited oscillating expression patterns. When the research team exposed liver cells to the malaria-causing parasite Plasmodium falciparum they observed that cells were more susceptible to infection when the genes regulating the immune response were downregulated.
This observation did not surprise Mancio-Silva. “We know that malaria has a circadian component,” she said. Malaria-spreading mosquitoes bite humans and deliver the parasites at night when the human immune response is downregulated, she explained.
These findings not only inform researchers how to better administer anti-malarial drug treatment, but they also highlight how experimental biologists must consider time as a factor when studying the liver. “These results also give us confidence in the liver model used,” Mancio-Silva said. “We can truly and faithfully recapitulate the human liver.”
- Long JE, et al. Morning vaccination enhances antibody response over afternoon vaccination: A cluster-randomised trial. Vaccine. 2016;34(24):2679-2685.
- Tognini P, et al. Circadian coordination of antimicrobial responses. Cell Host Microbe. 2017;22(2):185-192.
- Tahara Y, Shibata S. Circadian rhythms of liver physiology and disease: Experimental and clinical evidence. Nat Rev Gastroenterol Hepatol. 2016;13(4):217-226.
- March S, et al. Autonomous circadian rhythms in the human hepatocyte regulate hepatic drug metabolism and inflammatory responses. Sci Adv. 2024;10(17):eadm9281.
- Waring RH. Cytochrome P450: Genotype to phenotype. Xenobiotica. 2020;50(1):9-18.
