When life begins too early

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Not yet ready for this world: Every year, thousands of babies in Germany are born many weeks too early and often have to struggle for months. Their chances of survival have continuously improved over recent decades. Hardly any infant born before the 28th week of pregnancy survived just a few decades ago, whereas babies are now considered potentially viable from the 23rd week on. However, there is a high risk of infections with life-threatening complications, and sepsis is one of the most common causes of death.

“The threat of infection is particularly high in very small premature babies,” says LMU physician Prof. Markus Sperandio. “For them, a bacterial infection can lead to death within hours. For this reason, they often have to be treated with antibiotics.” Before switching to a career in research, the physiologist and former pediatrician and neonatologist worked at Heidelberg Children’s Hospital for eight years. During that time, he often was confronted with the susceptibility of premature babies to infection and this problem never left his mind ever since. As such, it is no coincidence that he and his team are investigating precisely this subject at LMU’s Biomedical Center.

Fetal immune cells do not adhere

It began with a visit at the renowned Center for Blood Research at Harvard Medical School in Boston, Massachusetts during his postdoctoral time in the US. While there, Sperandio came across a new mouse model that was developed at that time allowing researchers for the first time to microscopically study inflammation processes and the behavior of immune cells in the smallest blood vessels of mouse fetuses. “After returning to Heidelberg, I refined this model and subsequently established and further developed it at LMU.” In this way, he was able to demonstrate that important cells of the innate immune system – a population of white blood cells known as neutrophil granulocytes or neutrophils – do not work properly neither in the fetus nor in newborn mice, when compared with neutrophils from adults. In contrast to neutrophils of adults, neutrophils from fetuses and newborns do not sufficiently attach to the walls of blood vessels and extravasate into the surrounding tissue. This process is necessary to trigger an inflammatory response and thus initiate immune defense.

It turned out to be the same story with humans, as Sperandio’s team discovered when it investigated neutrophils isolated from the umbilical cord blood of premature and full-term infants. Because babies do not keep to birth schedules, obtaining these samples is often difficult to plan. “In the case of premature babies, the circumstances of birth are frequently so dramatic that nobody has time to think about getting samples for us.” This makes Sperandio all the more grateful for the excellent collaboration with the Neonatology Department and the Women’s Clinic at University of Munich Hospital, where the physicians assisted with the collection of samples.

The researchers found that the functional limitations of neutrophils extracted from the umbilical cord were more pronounced the shorter the pregnancy had lasted. To their surprise, they discovered that the functional development of the neutrophils follows a cellular program that very strongly depends on the baby’s postconceptional age – that is to say, the time that has passed since conception. Interestingly, the postnatal age, which begins after birth, was not so significant here. “We had expected that after premature birth, the immune system would be pushed by the new environment outside the maternal uterus such that the neutrophils would quickly switch to the adult mode. But this is precisely what did not happen,” says Sperandio. Even after a full-term birth, it takes some time before immune cells fully reach their normal functionality.

Balance of signaling pathways shifted into one direction

In the present study, the researchers set out to understand which mechanisms are behind the defective ‘docking’ to the vascular wall. To this end, they compared the gene activity of fetal neutrophils from premature babies and full-term babies with that of adult neutrophils. “By means of this so-called transcriptomic analysis, we found some really interesting differences,” says Sperandio. “Compared to adults, many genes that dampen the inflammatory response are upregulated in the neutrophils of both premature and full-term neonates. This means the cells are effectively switched off.”

Particularly affected are signals that are transmitted via the important NF-κB signaling pathway in the cell. This signaling pathway plays a key role in immune and inflammatory responses, but also, for example, in embryonic development. It contains two possible signaling pathways, a canonical one which promotes inflammation and a non-canonical one which counteracts inflammation (as well as performing other functions such as promoting the development of lymph nodes in fetuses). The activity of these two pathways must therefore be finely balanced for the regulation of immune responses. “Our experiments have shown that in fetal neutrophils, this balance is shifted in the direction of the anti-inflammatory pathway,” says Sperandio. “Ultimately, this results in the upregulation of key molecules that strongly counteract inflammation. One of those molecules is A20, for instance, which stabilizes the anti-inflammatory pathway and blocks the pro-inflammatory one.”

Downregulation of the immune system is physiologically useful in the fetus

Under normal circumstances, this blocking of neutrophils in the fetus serves to ensure optimal development. Firstly, fetuses do not need the same aggressive immune defenses in the protected and sterile womb as adults do. Secondly, some of the same mechanisms that are important for brain development before birth, for example, are employed to activate immune cells after birth. If not only neurons but also neutrophils were to strongly react to this during fetal development, “things could get muddled up,” conjectures Sperandio.

Given the high mortality of premature babies from infections, new treatment possibilities would be very welcome. The discovery that factors in the blood such as the molecule A20 are involved in the regulation of neutrophils could potentially contribute to the development of new therapeutic options. A theoretical possibility in the case of an imminent premature birth could be to treat the mother with drugs that reach the fetus through the placenta and stimulate the development of neutrophils to some extent. Cortisone medication is already administered in this way to accelerate lung maturation when a premature birth appears imminent.

A largely unexplored research field

Nevertheless, therapeutic use is still a long way off, emphasizes Sperandio. Part of the reason for this is the lack of experience accumulated in this area. “Our study is the first to demonstrate that these signaling pathways are shifted in fetuses. Indeed, the whole field of perinatal immunology worldwide is still largely unexplored.” A new research alliance by the German Research Foundation (DFG), of which Sperandio is a member, plans to forge ahead into this territory. As part of the work of the new Transregional Collaborative Research Center 359 “Perinatal Development of Immune Cell Topology (PILOT),” scientists want to get to the bottom of mechanisms that are decisive for the development of the immune system before and around the time of birth.

Another question that intrigues Sperandio, for instance, is how the tolerance between fetus and mother works. “You have to realize that the fetus is actually foreign tissue. We’re still lacking a really good explanation of why this tissue is not rejected.” There are parallels with this fetomaternal tolerance in cancer cells, for example, which outsmart the immune system by various stratagems. The physician is also fascinated by scarless repair mechanisms that work up until shortly after birth, but then cease to function. Newborn mice, for example, can still repair minor injuries to the cardiac muscle without scars. And even in human newborns, fingertips injured during a C-section can scarlessly regenerate to a certain extent. “I suspect that we can learn an incredible amount, including for adult medicine, from the fetal period and the postpartum transition period.”

Ina Rohwedder et al.: A20 and the non-canonical NF-κB pathway are key regulators of neutrophil recruitment during fetal ontogeny. JCI Insight 2023