Author ORCID Identifier

https://orcid.org/0000-0002-6167-4848

Semester

Fall

Date of Graduation

2025

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Cory Robinson

Committee Member

Jennifer Franko

Committee Member

Joshua Mattila

Committee Member

Michael Hu

Committee Member

Timothy Eubank

Abstract

The first month of life is a critical and vulnerable developmental stage, characterized by a heightened risk of life-threatening infections. Each year, 1.3 million newborns succumb to neonatal bacterial sepsis. The immune system during the neonatal period has unique features that lead to inefficiencies at clearing infection. Early life immunity is characterized by a Th2-biased profile with decreased IL‑12, IFN-γ, and TNF-α, along with increased IL‑6 and IL‑23. Additionally, IL‑27, an immunosuppressive cytokine, is also elevated during this period and further contributes to susceptibility to infection. Early diagnosis and intervention are critical in administering effective care in newborns and there is an emergent need for novel therapeutics for treating cases of neonatal sepsis. IL‑27 is an ideal target as previous reports have shown that mice lacking IL‑27 signaling fare better in an Escherichia coli-induced model of neonatal bacterial sepsis.

Pursuing IL‑27 as a therapeutic target requires a comprehensive understanding of the biology. Epigenetic and hormonal influences were explored as potential mechanisms for baseline increased IL‑27. Global methylation of CpG islands in the promoter region of the IL‑27 genes in adult and cord blood-derived macrophages were compared to assess epigenetic factors as a mechanism of IL‑27 production. Adult macrophages displayed more methylation overall, but IL‑27p28 was more methylated in neonatal macrophages. Hormonal influences were also evaluated due to previous publications showing progesterone leading to increased IL‑27 in adult macrophages. Both progesterone and glucocorticoids (GCs) increase in the mother throughout pregnancy and remain elevated following birth in the newborn. Neonatal macrophages expressed glucocorticoid receptor (GR), but not progesterone receptor (PR) and produced EBI3 in a dose-dependent manner when stimulated with dexamethasone, a synthetic GC. However, there were no significant differences in IL‑27p28 or EBI3 expression in neonatal macrophages or spleens from GRKO and WTfl animals. This suggested endogenous levels of GC were insufficient to influence gene expression.

The detailed IL‑27 producer profile during neonatal bacterial sepsis was generated using a reporter mouse strain that expressed eGFP under control of the IL‑27p28 promoter. Neonatal IL‑27p28eGFP mice were infected with a luciferase-expressing strain of O1:K1:H7 E. coli. Whole animal imaging identified the spleen, liver, and lungs as tissues with high levels of bacteria, thus, these tissues were pulled for analysis by flow cytometry, which showed the liver with both the highest number of IL‑27 producers and increase in these cells during infection. Phenotypic analysis and single-cell RNA sequencing identified Kupffer cells, monocytes, monocyte-derived cells, and emerging populations of neutrophils and dendritic cells as the leading IL‑27 producers. The transcriptome highlighted changes in inflammatory and bactericidal processes as well as metabolic shifts within the cells. Comparison of the bacterial burden per cell from IL‑27 producer and non-producer cells displayed reduced bacterial clearance by IL‑27 producers. Ultimately, these data supported the hypothesis of IL‑27-producing cells at infection sites representing a complex population of myeloid cells that impair control of infection.

The intent behind these studies was to understand signals and mechanisms that regulate IL-27 expression levels in neonates and further characterize the role of IL‑27 in early life immunity. These findings provide valuable insight into neonatal immune biology and sets the stage for additional studies on mechanisms of IL‑27 production and effects of IL‑27 producers in severe infections like sepsis. This work is of particular significance to medical providers by providing additional information on the influence of GCs on IL-27 and cells responsible for making IL-27. Together, these data support progress toward IL-27 antagonization as a therapeutic approach for neonatal infection and immunity.

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