Researchers identify missing link between intestinal disease and brain injury in premature babies

Physicians have long known that necrotizing enterocolitis (NEC), a potentially fatal infectious condition that destroys the lining of premature babies, is often linked to the development of severe brain injury in these infants. surviving. However, it is not yet known in what ways the diseased intestine communicates “its destruction of the newborn’s brain.

Now, working with mice, researchers at Johns Hopkins Medicine and the University of Lausanne in Switzerland have identified that missing link – an immune system cell they say will travel from the slice to the brain and attack cells rather than the defense as usual. .

The team’s findings are published Jan. 6, 2021, in the journal Science Translational Medicine.

Seen in as many as 12% of babies weighing less than 3.5 pounds at birth, NEC is a rapidly progressing gastrointestinal crisis in which bacteria invade the colon wall and causing inflammation that can destroy healthy tension at the site. If enough cells become necrotic (die) until a hole is formed in the intestinal wall, bacteria can enter the bloodstream and cause life-threatening sepsis.

In a 2018 mouse study, researchers at Johns Hopkins Medicine and the Fred Hutchinson Cancer Research Center found that animals with NEC produce a protein called receptor-like receptor 4 (TLR4) that binds bacteria in the digestion and elimination of the intestinal destruction. They also concluded that TLR4 simultaneously activates immune cells in the brain called microglia, leading to white matter loss, brain injury and reduced mental function. It was not clear how the two are connected.

For this latest study, the researchers speculated that CD4 + T lymphocytes – immune system cells also known as helper T cells – may be the link. CD4 + T cells get the nickname “helper” because they help another type of immune cell called B lymphocyte (or B cell) to respond to superficial proteins – antigens – on cells that under attack by foreign invaders such as bacteria or viruses. Activated by the CD4 + T cells, B cells immediately become plasma cells that make antibodies to identify the infectious cells to get rid of the body or memory cells that “remember” the biochemistry of the antigen for faster response to future attacks.

CD4 + T cells also send out chemical messages that bring another type of T cell – called a killer T cell – to the area until the targeted infectious cells are removed. However, if this activity takes place in the wrong place or at the wrong time, the signals can inadvertently direct the killer T cells to attack healthy cells in their place.

We knew from comparing the brains of infants with NEC with those of infants who died from other causes that the former had accumulations of CD4 + T cells and showed increased microglial activity. We suspected that these T cells came from NEC-inflamed regions of the spleen and tried to confirm it by using newborn mice as a model of what happens in human babies. “

David Hackam, MD, Ph.D., Lead Author Study, Chief Surgeon, Johns Hopkins Children’s Center and Professor of Surgery, Johns Hopkins University School of Medicine

In the first of a series of experiments, the researchers stimulated NEC in infant mice and then examined their brains. As expected, the figs showed a significant increase in CD4 + T cells as well as higher levels of protein associated with increased microglial activity. In a follow-up test, the researchers showed that mice with NEC had a weak blood-brain barrier – the biological wall that normally blocks bacteria, viruses and other dangerous substances that infect circulation in the bloodstream from reaching the central nervous system. This, the researchers said, could explain how CD4 + T cells from the cleft could travel to the brain.

Next, the researchers concluded that the accumulation of CD4 + T cells was responsible for the brain damage observed by NEC. They did this first by blocking the movement of the helper T cells into the brain and then in a separate test, neutralizing the T cells by binding them to a specially designed antibody. In all cases, microglial activity was suppressed and white matter in the brain was retained.

To further explain the role of CD4 + T cells in brain injury, the researchers extracted T cells from the brains of mice with NEC and inserted them into the brains of implanted mice. bred so that both T and B lymphocytes did not. Compared to control mice that did not receive any T cells, the mice that received the lymphocytes had higher levels of the chemical markers that attracted killed T cells. The researchers also noticed the activity of the microglia, inflammation in the brain and loss of white matter – all signs of brain injury.

The researchers then tried to better explain how the accumulating CD4 + T cells destroy white matter – actually a fat called myelin covering and protects neurons in the brain, and enables communication between them. To do this, they used organoids, laboratory-grown mouse brain cells to mimic the entire brain. Brain-derived CD4 + T cells from mice with NEC were added to these laboratory “mini-brains” and then examined for several weeks.

Hackam and his colleagues found that a specific chemical signal from the T cells – cytokine (an inflammatory protein) called interferon-gamma (IFN-gamma) – increased in the organs as the level of myelin decreased. This activity was not observed in the organs that received CD4 + T cells from mice without NEC.

After adding IFN-gamma to the organs, the researchers observed the same increased levels of inflammation and myelin reduction seen in mice with NEC. When they applied a neutralizing antibody IFN-gamma, cytokine production was significantly reduced, inflammation was reduced and white matter was partially restored.

The researchers concluded that IFN-gamma directs the process leading to NEC-related brain injuries. Their finding was confirmed when a study of brain bones from mice with NEC showed higher levels of IFN-gamma than in interferons from mice without the disease.

Next, the researchers investigated whether CD4 + T cells could migrate from the cleft to the brain of mice with NEC. To do this, they obtained CD4 + T cells from the womb of infant mice with and without NEC. Both cell types were introduced into the brains of infantile mice in two groups – one set that could produce the Rag1 protein and one that could not. Rag1-deficient mice do not have mature T or B lymphocytes.

The Rag1-deficient mice that received gut-based T helper cells from mice with NEC showed the same features of brain injury seen in the previous trials. T cells from both mice with and without NEC did not cause brain injury in mice with Rag1, and T cells from mice without NEC did not cause in mice with Rag1 deficiency. This showed that the helper T cells derived from gut from mice with NEC were the only ones that could cause brain injury.

In a second experiment, gut-derived T cells from mice with and without NEC were introduced into the peritoneum – the membrane lining the abdominal cavity – of Rag1-deficient mice. Only brain intestinal T cells from mice with NEC caused brain injury.

This finding was confirmed by genetically following the same extracts from both brain and gut T-derived lymphocytes from mice with and without NEC. The sequence of T helper cells from mice with NEC was, on average, 25% genetically similar while those from mice without NEC were only 2% combined.

In a final test, the researchers blocked IFN-gamma by itself. By doing this it provided great protection against the development of brain injury in mice with true NEC. This suggests, the researchers say, a therapeutic approach that may be beneficial for premature babies with the condition.

“Our research strongly suggests that NEC-induced congenital T helper cells can migrate to the brain and cause damage,” Hackam said. “It has been previously shown that the mouse model in the Our study closely matches what happens in humans, so we believe this is the most likely way in which NEC-related brain injuries will develop in premature babies. “

Based on these findings, Hackam argues that measures could prevent this type of brain injury, including medications to prevent INF-gamma action.