Glucocorticoid signaling regulates expression of the EBI3 subunit of IL-27 in neonatal macrophages: Implications for antenatal corticosteroid therapy

This study demonstrates that glucocorticoid signaling directly upregulates the expression of the IL-27 subunit EBI3 in neonatal macrophages via nuclear translocation of the glucocorticoid receptor, suggesting that antenatal corticosteroid therapy may inadvertently increase immunosuppressive IL-27 levels and compromise survival during neonatal sepsis.

The Gist

The Big Picture: Why Are Newborns So Vulnerable?

Imagine a newborn baby's immune system is like a newly built security team in a skyscraper. It's eager to learn, but it's also very cautious. In fact, it's too cautious.

When a baby is born, their immune system is flooded with a "calm down" signal called IL-27. Think of IL-27 as a super-powerful noise-canceling headphone that the security team wears. While this helps the baby not to overreact to harmless things (like dust or food), it also makes it hard for them to hear the alarm bells when a real bad guy (like a bacteria) tries to break in. This is why newborns are so susceptible to serious infections.

This study asked a simple question: Why does this "noise-canceling headphone" (IL-27) stay turned up so loud in babies, and can we turn it down?

The researchers investigated two main suspects:

1. The "Hard Drive" (DNA Methylation): Did the baby's genetic code have the volume knob stuck on high because of how the DNA was written?
2. The "Remote Control" (Hormones): Is there a hormone signal telling the baby's cells to keep the volume up?

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Investigation #1: The Hard Drive (DNA Methylation)

The Theory:
Imagine DNA as a library of instruction manuals. Sometimes, the library puts a "Do Not Read" sticker (methylation) on certain pages to silence them. The researchers thought maybe, in adults, the "IL-27" manual had a "Do Not Read" sticker, but in babies, the sticker was missing, so the volume stayed high.

The Findings:
The scientists looked at the "sticker status" of the genes in baby macrophages (the immune cells) versus adult macrophages.

• Result: They found that the "IL-27" manual actually had more stickers (methylation) on it in the babies than in the adults!
• The Twist: This was the opposite of what they expected. If the stickers were there to silence the gene, why was the gene so loud in babies?
• Conclusion: The "Do Not Read" stickers weren't the reason the volume was high. The "Hard Drive" wasn't the culprit.

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Investigation #2: The Remote Control (Hormones)

The Theory:
If the hard drive wasn't the problem, maybe someone was holding the remote control. The researchers looked at two hormones that are very high during pregnancy and birth: Progesterone (the pregnancy hormone) and Glucocorticoids (stress hormones that help baby lungs mature).

They wondered: Is one of these hormones pressing the "Volume Up" button on the IL-27 gene?

The Findings:

• The Players: The baby's immune cells had a receptor (a socket) for Glucocorticoids, but they didn't have a socket for Progesterone.
• The Experiment: They exposed baby immune cells to synthetic Glucocorticoids (like Dexamethasone, a drug often given to moms before preterm birth to help baby lungs grow).
• The Result: As soon as the Glucocorticoids arrived, the cells immediately started cranking up the production of EBI3.
  • Note: IL-27 is made of two parts, like a sandwich: p28 and EBI3. The hormone didn't change the p28 part, but it turned the EBI3 part up to maximum volume.
• The Proof: They watched the cells under a microscope. When the hormone arrived, the "Glucocorticoid Receptor" (the remote control receiver) jumped from the outside of the cell into the nucleus (the control room) and physically grabbed the switch for the EBI3 gene.

The Analogy:
Think of the Glucocorticoid as a Key. The baby's immune cell has a lock (the receptor). When the Key turns, it opens the door to the control room and flips a switch that says, "Make more EBI3!"

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The "Wait a Minute" Moment

The researchers then asked: "Okay, so the hormone CAN turn the volume up. But is the baby's body naturally doing this all the time?"

They tested this by looking at mice that were genetically engineered to lack the Glucocorticoid receptor in their immune cells.

• Surprise: Even without the receptor, the baby mice still had high levels of IL-27.
• Meaning: While Glucocorticoids can turn the volume up, the natural, steady-state high volume in babies isn't caused by the baby's own hormones. There must be another reason why the volume is high naturally.

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The Real-World Implication: The "Double-Edged Sword"

Here is the most important part for parents and doctors:

The Scenario:
When a mom is at risk of having a baby too early (preterm labor), doctors often give her corticosteroids (like Dexamethasone). This is a life-saving treatment that helps the baby's lungs mature so they can breathe outside the womb.

The Risk:
This study shows that while these steroids help the lungs, they also act like a master volume knob for the baby's immune system.

1. The steroid enters the baby's system.
2. It tells the immune cells to make more EBI3.
3. More EBI3 means more IL-27.
4. More IL-27 means the baby's immune system is even more suppressed.

The Metaphor:
Giving steroids to a preterm baby is like giving them a life vest (for their lungs) that is also made of heavy lead (for their immune system). The life vest keeps them from drowning, but the lead makes it harder for them to swim if a shark (infection) shows up.

Summary for Everyone

1. Newborns have a "quiet" immune system because of high levels of IL-27.
2. DNA stickers (methylation) aren't the reason for this quietness.
3. Glucocorticoid hormones (like the ones used in medical treatments) act as a remote control that can make the immune system even quieter by boosting IL-27.
4. The Takeaway: While corticosteroid therapy is vital for saving preterm babies' lungs, doctors need to be aware that it might accidentally make these babies even more vulnerable to infections. Future treatments might need to balance helping the lungs while protecting the immune system.

Technical Summary

1. Problem Statement

Neonates, particularly preterm infants, suffer from disproportionately high mortality rates due to invasive bacterial infections. A key factor in this susceptibility is an immature immune profile characterized by a Th2 bias and elevated levels of the immunosuppressive cytokine IL-27. IL-27 is a heterodimer composed of two subunits, p28 and EBI3. Previous studies established that IL-27 levels are elevated in neonatal macrophages compared to adults and that high IL-27 compromises survival during neonatal sepsis.

The central scientific question addressed in this study is: What regulatory mechanisms drive the elevated baseline expression of IL-27 in neonatal macrophages? The authors investigated two primary hypotheses:

1. Epigenetic Regulation: Whether decreased DNA methylation in neonates (relative to adults) leads to increased IL-27 gene expression.
2. Hormonal Regulation: Whether steroid hormones prevalent during pregnancy and the neonatal period (specifically glucocorticoids and progesterone) regulate IL-27 expression via hormone response elements (HREs).

2. Methodology

The study employed a multi-faceted approach combining human clinical samples, murine genetic models, and molecular biology techniques:

• Sample Collection:
  • Human: Cord blood mononuclear cells (CBMCs) from healthy term newborns and peripheral blood mononuclear cells (PBMCs) from adult donors. Cells were differentiated into macrophages in vitro.
  • Murine: Neonatal (≤8 days) and adult C57BL/6J mice, including wild-type (WT) and myeloid-specific Glucocorticoid Receptor Knockout (GRKO; LysM-Cre x Nr3c1fl/fl) mice.
• Epigenetic Analysis (Methyl-Seq):
  • Whole-genome enzymatic methyl-seq (EM-seq) was performed on neonatal and adult macrophages to identify Differentially Methylated Regions (DMRs).
  • Functional Validation: Macrophages were treated with the DNA methyltransferase inhibitor 5-azacitidine to assess if demethylation alters IL-27 expression.
• Hormonal Stimulation:
  • Macrophages were treated with Dexamethasone (synthetic glucocorticoid) or Progesterone at varying concentrations and time points.
  • Antagonist Studies: Cells were pre-treated with RU486 (mifepristone), a competitive antagonist for both Glucocorticoid Receptor (GR) and Progesterone Receptor (PR), to determine receptor specificity.
• Molecular Characterization:
  • qPCR: Measured mRNA expression of IL27p28, EBI3, NR3C1 (GR), and PGR.
  • Immunofluorescence & Confocal Microscopy: Visualized GR translocation from cytoplasm to nucleus in response to dexamethasone.
  • Chromatin Immunoprecipitation (ChIP): Assessed direct binding of GR to the EBI3 promoter region.
  • Genetic Models: Compared IL-27 expression in WT vs. GRKO neonatal mice to determine the role of endogenous GR signaling.

3. Key Results

A. Epigenetic Regulation (Methylation)

• Global Methylation: Adult macrophages exhibited higher global methylation levels compared to neonates, consistent with developmental maturation.
• IL-27p28 Locus: The promoter region of IL27p28 contained DMRs that were more highly methylated in neonates than in adults.
• EBI3 Locus: No significant DMRs were found in the EBI3 promoter between neonates and adults.
• Functional Impact: Treatment with 5-azacitidine (demethylation) increased IL27p28 expression in adult macrophages but had no effect on neonatal macrophages.
• Conclusion: While methylation differences exist, they do not explain the elevated baseline IL-27 in neonates; in fact, the neonatal IL27p28 promoter is less methylated, yet expression is high. This suggests methylation is not the primary driver of the neonatal phenotype.

B. Hormonal Regulation (Glucocorticoids vs. Progesterone)

• Receptor Expression: Neonatal macrophages express the Glucocorticoid Receptor (GR) but do not express the Progesterone Receptor (PR).
• Stimulation Effects:
  • Dexamethasone: Significantly induced EBI3 gene expression in both human and murine neonatal macrophages in a dose-dependent manner. It did not significantly affect IL27p28.
  • Progesterone: Did not alter EBI3 or IL27p28 expression in neonatal macrophages (likely due to lack of PR).
• Mechanism of Action:
  • Dexamethasone induced rapid nuclear translocation of GR.
  • ChIP-qPCR confirmed that GR directly binds to the glucocorticoid response element (GRE) in the EBI3 promoter region upon stimulation.
  • RU486 pretreatment blocked the dexamethasone-induced upregulation of EBI3, confirming GR-dependency.

C. Endogenous Regulation in Neonates

• Genetic Knockout: In neonatal mice lacking GR specifically in myeloid cells (GRKO), baseline levels of IL27p28 and EBI3 in the spleen and bone marrow-derived macrophages (BMDMs) were not significantly different from Wild-Type littermates.
• Conclusion: While exogenous glucocorticoids can upregulate EBI3, endogenous glucocorticoid levels in the neonatal period are insufficient to drive the high baseline levels of IL-27 observed in vivo. Other factors must contribute to the steady-state elevation.

4. Key Contributions

1. Subunit-Specific Regulation: The study demonstrates that the two subunits of IL-27 are regulated by distinct mechanisms. IL27p28 expression appears linked to developmental methylation changes, while EBI3 is directly regulated by glucocorticoid signaling.
2. Direct Transcriptional Control: The authors provide direct evidence (via ChIP and nuclear translocation assays) that the Glucocorticoid Receptor binds to the EBI3 promoter to drive transcription.
3. Clarification of Progesterone Role: The study corrects previous assumptions about progesterone driving IL-27 in neonates by showing that neonatal macrophages lack PR and do not respond to progesterone, whereas they are highly responsive to glucocorticoids.
4. Distinction between Endogenous and Exogenous: The research clarifies that while the capacity for GC-induced IL-27 exists, natural neonatal GC levels do not fully account for baseline IL-27 elevation, suggesting a complex interplay of factors.

5. Significance and Clinical Implications

The findings have profound implications for Antenatal Corticosteroid (ACS) therapy, a standard clinical intervention given to pregnant women at risk of preterm labor to accelerate fetal lung maturity.

• The Risk: Since ACS (e.g., dexamethasone) directly upregulates the EBI3 subunit of IL-27 in neonatal macrophages, and high IL-27 is known to suppress protective immune responses against bacterial sepsis, there is a potential unintended consequence of this therapy.
• The Mechanism: Administering corticosteroids may inadvertently increase IL-27 levels in the newborn, potentially exacerbating susceptibility to invasive bacterial infections in an already vulnerable population.
• Future Directions: The study suggests that clinical protocols for corticosteroid administration in pregnancy and neonates should consider the immunosuppressive risks associated with elevated IL-27. It highlights the need for further preclinical models to balance lung development benefits against infection risks and suggests IL-27 as a potential therapeutic target for neonatal sepsis.

Summary: This paper elucidates that while DNA methylation patterns differ between neonates and adults, they do not solely explain high neonatal IL-27. Instead, the study identifies glucocorticoid signaling as a potent, direct regulator of the EBI3 subunit. This finding raises critical safety considerations regarding the use of corticosteroids in pregnancy, as they may inadvertently compromise neonatal immunity by elevating IL-27 levels.