Researchers at the University of Illinois Urbana-Champaign, Duke University, and the Chan Zuckerberg Biohub Chicago found that this cellular limbo appears to be driven by inflammation that disrupts RNA splicing, an essential step cells use to turn genetic instructions into working proteins.

The findings, published in Nature Communications, could point toward new ways to diagnose and potentially treat severe alcohol associated liver disease.

Why the Liver Stops Repairing Itself

The liver is unusual among major human organs because it can regenerate after significant damage or even partial removal. Under normal circumstances, surviving liver cells can temporarily change their identity, multiply, and then mature again to restore lost tissue.

That ability can break down in alcohol associated liver disease, which is the leading cause of liver-related mortality worldwide and is linked to roughly 3 million deaths each year.

“We knew that the liver stops functioning and stops regenerating in patients with alcohol-related hepatitis and cirrhosis, even when a patient has discontinued consuming alcohol, but we didn’t know why,” said U. of I. biochemistry professor Auinash Kalsotra, who co-led the study with Duke University School of Medicine professor Anna Mae Diehl. “The only real life-saving treatment option once a patient reaches the liver failure stage in those diseases is transplantation. But if we understood why these livers were failing, maybe we could intervene.”

Kalsotra and Diehl have spent years studying the molecular processes that allow the liver to rebuild itself. Their previous work showed that regenerating liver cells temporarily reprogram which genes they use.

To begin the repair process, mature liver cells revert toward a fetal-like progenitor state. Progenitor cells are less specialized cells that can divide and produce new tissue. After multiplying, the cells normally reverse that process and become mature, fully functioning liver cells again.

That earlier discovery led the researchers to ask what goes wrong with this regenerative cycle in alcohol associated liver disease.

Liver Cells Become Trapped in Limbo

The team compared healthy liver samples with liver tissue from people with alcohol associated hepatitis or cirrhosis. The diseased samples were obtained from Johns Hopkins University Hospital through an initiative supported by the National Institute on Alcohol Abuse and Alcoholism, part of the National Institutes of Health.

A striking pattern quickly emerged.

Cells in the diseased livers had started moving away from their mature state and toward the regenerative state, but they were unable to finish the transition. Instead, they remained trapped between the two.

“They are neither functional adult cells nor proliferative progenitor cells. Since they are not functioning, more pressure builds on the remaining cells. So they try to regenerate, and they’re all ending up in this unproductive quasi-progenitor state, and that’s what is causing liver failure,” said U. of I. graduate students Ullas Chembazhi and Sushant Bangru, the co-first authors of the study.

The result is a damaging cycle. As more cells enter this unproductive state, fewer remain available to carry out the liver’s normal work. The remaining healthy cells then face greater demands and attempt to regenerate, only to risk becoming trapped as well.

RNA Splicing Emerges as a Key Problem

To understand what was preventing the cells from completing regeneration, the researchers examined the proteins being produced inside liver cells as well as the RNA molecules carrying genetic instructions from DNA to the cellular machinery that builds those proteins.

RNA acts as an intermediary between the genetic code stored in DNA and the proteins that perform most of a cell’s work. Before many RNA molecules can be used, pieces of them must be cut and joined together in a process known as RNA splicing.

This editing step matters because different combinations of RNA segments can produce proteins with different functions or direct them to different locations inside a cell.

Instead of simply measuring the total amounts of RNA and protein, as many studies do, Kalsotra’s team used deep RNA sequencing and computational analysis to examine how RNA fragments were being spliced.

“In comparing the samples, we saw RNA was getting misspliced broadly in alcohol-related liver disease, across thousands of genes, and it was affecting major functions of proteins,” said Kalsotra, who also is affiliated with the Carl R. Woese Institute for Genomic Biology at Illinois.

The scale of the problem was substantial. Mis-splicing appeared across thousands of genes, potentially altering how important proteins function throughout damaged liver cells.

A Missing Protein May Help Explain the Damage

The researchers identified one possible driver of these widespread errors: low levels of a protein called ESRP2.

ESRP2 binds to RNA and helps ensure that it is spliced correctly. In alcohol damaged liver cells, the team found that ESRP2 was deficient.

The consequences were not limited to whether a protein was produced. In many cases, the RNA errors altered molecular instructions that tell proteins where inside the cell they need to go.

“Proteins function at a very specific place in the cell, and that is directed by sequences within the protein that take the protein to that particular spot. We found that, in many cases, the sequence that dictates where the protein localizes within a cell was misspliced. That’s why it was important that we did the multiple analyses we did,” said Kalsotra, also a member of the Chan Zuckerberg Biohub Chicago. “There was the same amount of RNA and protein, but the protein was not at the right place to function. Due to missplicing, key proteins that are required for productive liver regeneration were getting stuck in the cytoplasm, when they needed to be in the nucleus.”

The nucleus contains a cell’s DNA and plays a central role in regulating gene activity. The cytoplasm is the surrounding area where many other cellular processes occur. If proteins needed for regeneration remain in the cytoplasm rather than reaching the nucleus, they may be present in normal amounts but unable to perform their intended jobs.

Mouse Experiments Strengthen the ESRP2 Link

To test whether the loss of ESRP2 could actually contribute to the regeneration failure, the researchers studied mice lacking the gene that produces the protein.

Those animals developed patterns of liver injury and failed regeneration that resembled what the scientists observed in people with advanced alcohol related hepatitis.

That raised another important question: Why was ESRP2 reduced in the first place?

The researchers traced the problem back to inflammation.

When alcohol is processed by the liver, it can damage tissue and attract immune cells and liver support cells to the affected areas. According to the study, those cells released high levels of inflammatory factors and growth factors.

The researchers found that these signals suppress both the production and activity of ESRP2.

Blocking Inflammation Restored Normal Splicing

The team then tested whether interrupting one of those inflammatory signals could reverse the problem.

In laboratory cultures of liver cells, the researchers used a molecule that blocks the receptor for one inflammation-promoting factor. After treatment, ESRP2 levels recovered, and RNA splicing became more normal.

That result suggests the pathway could become a potential treatment target. Rather than attempting to replace damaged liver tissue directly, future therapies might try to interrupt the inflammatory signals that prevent cells from completing regeneration.

The researchers also see potential diagnostic uses. Abnormally spliced RNA molecules could potentially serve as biological markers that help identify or monitor alcohol associated liver disease.

“I’m hopeful these findings will become a launching pad for future clinical studies. We can use these mis-spliced RNAs as diagnostic markers or develop treatments that can curb the inflammation. And if we can correct the splicing defects, then maybe we can improve recovery and restore damaged livers,” Kalsotra said.

Research Team and Support

The research team also included U. of I. biochemistry graduate students Diptatanu Das and Subhashis Natua; U. of I. undergraduate students Katelyn Toohill, Ishita Purwar, and Anuprova Bhowmik; Brandon Peiffer and Zhaoli Sun from Johns Hopkins University School of Medicine; Aurelia Leona and Yogesh Goyal from Northwestern University and Rajesh Dutta from Duke University School of Medicine.

The National Institutes of Health, the Chan-Zuckerberg Biohub Chicago, the Duke Endowment and the Muscular Dystrophy Association supported this work. The National Institutes of Health supported this work through grants R01-AA010154, R01-HL126845, R21-HD104039, R01-AA010154, 5R01-DK077794, 1R56-DK1343340 and R24 AA025017.