hourHuman health outcomes often show daily fluctuations. For example, morning vaccination It may trigger a more effective immune response than a jab in the afternoon.1 Likewise, people are more susceptible to diseases such as: infection At certain times of the day.2
This variability is coordinated by the biological clock, which regulates oscillations of gene expression. Experiments on cells and laboratory animals have shown that these substances are abundant in the liver, where they play a role in vaccine response and infection. Circadian dependent genes.3 However, some aspects of drug metabolism and immune responses are unique to humans and difficult to investigate due to lack of experimental systems.
“We knew that the liver has its own circadian rhythm that is independent of our brain’s central clock,” he said. Liliana Mancio-SilvaParasitologist at the Pasteur Institute. “We wanted to know if we could mimic the circadian oscillations of the liver in vitro.”
Mancio-Silva collaborates with biomedical engineers Sangeeta Bhatia Developed at the Massachusetts Institute of Technology In vitro human liver modelThey explained in . Science advances.4 Characterizing the system’s hepatocytes, the researchers identified genes involved in drug metabolism and susceptibility to infections under circadian regulation. This model, which mimics the organ’s circadian rhythm, provides researchers with a platform to study the impact of circadian genes on human liver function and improve drug development.
Experiments with engineered human livers (blue are parasite nuclei and host cells) show that circadian oscillations regulate hepatocyte infection. Plasmodium falciparum (green)
Liliana Mancio and Eliana Real
To develop the new system, the researchers obtained liver cells from individual donors and cultured them with fibroblasts, which provide structural support. Circadian clock gene basic helix-loop-helix ARNT-like protein 1 (BMAL1), which helps coordinate the expression of several different genes, the team created liver cells that developed synchronized circadian oscillations that lasted for 10 weeks.
Researchers with systems to study cyclical changes in liver cells have wondered how circadian rhythms affect gene expression. They analyzed the transcriptomes of these cells and found that more than 380 genes were expressed periodically and that most of these genes were involved in drug metabolism, inflammation, and immune responses.
One of these periodically expressed genes, cytochrome P450 3A4 (CYP3A4) encodes a drug-metabolizing enzyme. Cytochrome P450 This family is responsible for approximately three quarters of all drug metabolism reactions in humans.5 They found that CYP3A4 enzyme activity occurs in waves, suggesting that the drug’s pharmacokinetics may vary depending on the time of day.
To test this, the team treated liver cells with the lipid-lowering drug atorvastatin or the nonsteroidal painkiller acetaminophen. This drug is harmful to the liver at high doses because it is metabolized by CYP3A4 to toxic byproducts. In treated cells, they observed that higher levels of CYP3A4 correlated with greater cell death, suggesting that optimizing drug administration time may minimize drug side effects.
“[These results] “It is the culmination of 20 years of predictions,” he said. Sachidananda PandaA chronobiologist at the Salk Institute for Biological Studies, he was not involved in the study. He noted that researchers have previously shown cyclical expression of drug metabolism genes in mice. “But there have been no actual experiments showing whether cytochrome P450 gene activity is circulating in the human liver.”
He added: “The technical innovation of this paper is keeping human liver cells alive for 10 weeks.” This allowed Mancio-Silva and her team to demonstrate for the first time that drug-metabolizing enzyme activity in humans is cyclical.
One limitation, however, is that the system is constantly bathed in glucose, which does not mimic the fasting-feeding cycle that occurs physiologically, Panda noted. Still, this is the closest researchers have come to summarizing the circadian system among humans, he said.
He noted that it will be important in future studies to explore circadian regulation beyond drug metabolism genes. CYP3A4.
Mancio-Silva and the team turned to using in vitro systems to investigate how circadian genes affect liver immunity and infection. They found that interferon, the body’s virus-fighting protein, stimulates a subset of genes that show oscillating expression patterns. When the research team exposed liver cells to the parasite that causes malaria Plasmodium falciparum They observed that when genes that control the immune response were downregulated, cells became more susceptible to infection.
This observation did not surprise Mancio-Silva. “We know that malaria has a circadian component,” she said. Mosquitoes that spread malaria bite humans and transmit the parasite at night, when the human immune response is downregulated, she explained.
These findings not only tell researchers how to better manage antimalarial drug treatment, but also highlight how experimental biologists must consider time a factor when studying the liver. “These results increase our confidence in the liver model used,” Mancio-Silva said. “We can truly and faithfully recreate the human liver.”
