HHuman health outcomes often vary from day to day. For example, in the morning vaccination It may elicit a more effective immune response than the afternoon jab.1 Similarly, people are more susceptible to: infectious disease at certain times of the day.2
Such fluctuations are orchestrated by the circadian clock, which regulates oscillations in gene expression. Experiments conducted in cells and laboratory animals have shown that such viruses are present in large numbers in the liver, where they are involved in vaccine responses and infections. circadian dependent genes.3 However, some aspects of drug metabolism and immune responses are unique to humans and difficult to investigate due to a lack of experimental systems.
“We knew that the liver had its own circadian rhythm that was independent of the brain’s central clock,” he says. Liliana Mancio Silvaa parasitologist at the Pasteur Institute. “We wanted to see if we could mimic the liver’s circadian oscillations in vitro.”
Mancio Silva teams up with a biomedical engineer Sangeeta Bhatia at Massachusetts Institute of Technology in vitro human liver modelthey explained it Science progresses.4 In characterizing liver cells within the system, researchers identified genes involved in drug metabolism and susceptibility to infection that are under circadian control. This model mimics the organ’s circadian rhythm and provides researchers with a platform to study the influence of circadian genes on human liver function and improve drug development.
Experiments from engineered human livers (parasite and host cell nuclei in blue) show that circadian oscillations control 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. By fine-tuning the culture conditions, the circadian clock gene basic helix-loop-helix ARNT-like protein 1 (BMAL1), which helps coordinate the expression of several other genes, the research team generated liver cells that developed synchronized circadian oscillations and persisted for 10 weeks.
Equipped with a system to study the periodic fluctuations of liver cells, researchers wondered how circadian rhythms affect gene expression. They analyzed the transcriptomes of these cells and found that more than 380 genes are cyclically expressed, and that the majority of these genes are related to drug metabolism, inflammatory responses, and immune responses. I discovered it.
One of these periodically expressed genes, cytochrome P450 3A4 (CYP3A4) encodes drug-metabolizing enzymes. Cytochrome P450 This family is responsible for approximately three-quarters of all drug metabolic reactions in humans.5 They found that CYP3A4 enzyme activity occurred in waves, suggesting that the pharmacokinetics of the drug may vary depending on the time of the day.
To test this, the research team treated liver cells with either the lipid-lowering drug atorvastatin or the nonsteroidal painkiller acetaminophen. These drugs are toxic to the liver at high doses because they are metabolized by CYP3A4 to toxic byproducts. In the treated cells, increased cell death was observed with higher levels of CYP3A4, suggesting that optimizing the time of drug administration may minimize drug side effects.
“[These results] “This is the culmination of 20 years of predictions.” satchidananda pandais a chronobiologist at the Salk Institute for Biological Studies but was not involved in the study. He noted that researchers have previously shown periodic expression of drug-metabolizing genes in mice, adding, “However, actual experiments showing whether cytochrome P450 gene activity cycles in the human liver are difficult. There was no such thing.”
He added: “The technical advance in this paper is to keep human liver cells alive for 10 weeks,” which allows Mancio-Silva and her team to demonstrate that the activity of drug-metabolizing enzymes in humans is cyclical. For the first time, we were able to prove that it was true.
But Panda pointed out that one of the limitations is that the system is continuously exposed to glucose, which does not mimic the fasting and feeding cycles that occur physiologically. Nevertheless, this is the closest the researchers have come to recreating the human liver’s circadian system, he said.
He noted that it will be important for future research to explore circadian regulation beyond drug metabolism genes. CYP3A4.
Mancio-Silva and his team turned to using an in vitro system to investigate how circadian genes influence liver immunity and infection. They discovered that interferon, a protein that fights viruses in the body, stimulates a subset of genes that exhibit oscillating expression patterns. When the research team exposed liver cells to the parasite that causes malaria. Plasmodium falciparum They observed that cells were more susceptible to infection when genes that control the immune response were downregulated.
This observation did not surprise Mancio Silva. “We know that malaria has a circadian component,” she says. She explained that the mosquitoes that spread malaria bite humans at night, when the human immune response is downregulated, delivering the parasite.
These findings not only inform researchers how to better tailor antimalarial drug treatments, but also highlight how experimental biologists need to consider time as a factor when studying the liver. I am. “These results also give us confidence in the liver model we used,” Mancio-Silva said. “We can really faithfully recreate the human liver.”
