Dissertation
Prevention and treatment of neonatal necrotizing enterocolitis and sepsis using antimicrobial peptides and probiotics
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2022
DOI: 10.25820/etd.006489
Abstract
Preterm birth rates are increasing. Preterm birth is associated with an underdevelopment of the gastrointestinal tract that results in a more permissive barrier. Also, preterm infants have an immune system that functions differently from that of term infants or adults, which results in a hyper- or hypo- inflammatory response to stimuli that cannot be immunomodulated. Necrotizing enterocolitis (NEC) and neonatal sepsis both are diseases of mainly preterm infants due to these complications. Despite decades of research, NEC and neonatal sepsis remain difficult to predict, clinically diagnose, and treat. There is need for better prevention and treatment strategies, which fueled this research. We utilized three different types of murine models to test these effects. In all models, P14-P16 mice are utilized as we have shown that in terms of intestinal gene regulation, these mice mimic a preterm infant at the cusp of viability (22-24 weeks’ gestation). Our standard NEC model involves disruption of Paneth cells with intraperitoneal injection with dithizone in wild type mice or diphtheria toxin in PC-DTR mice. Paneth cells play an important role in intestinal innate immunity and premature infants that develop NEC have decreased abundance of Paneth cells compared to infants that do not develop NEC. Following disruption of Paneth cells mice are given a gavage of Klebsiella pneumoniae (K. pneumoniae). This is meant to mimic the significant increase in Enterobacteriaceae species that occurs in infants shortly before NEC onset. In a complimentary model we developed, gavage of K. pneumoniae is replaced with formula feeding as formula fed infants are at a significantly higher risk to develop NEC than their breast-fed counterparts. Finally, we developed a neonatal sepsis model that involves modification of the standard cecal slurry model. In our model, we replace an adult murine cecal slurry with NEC’teria, a combination of bacteria isolated from an infant who died from NEC.
Our results demonstrate that LL-37 and hBD2 are effective at both preventing and treating NEC in the Paneth cell disruption with bacterial dysbiosis NEC model. The mechanisms of action between the two AMPs have similarities and differences. Both AMPs elicit improved epithelial wound healing, but while hBD2 provided measurable effects on K. pneumoniae in vivo, LL-37 did not. Additionally, we demonstrated that hBD2 influenced the CCL2/CCR2 axis, which is important for recruitment of macrophages and monocytes to the site of infection. While both AMPs had significant impacts on NEC, neither was able to improve mortality in our neonatal sepsis model. This can at least be partially explained by the inability of the AMPs to directly impact NEC’teria in vitro.
In addition, we found that the probiotic Bifidobacterium longum subspecies infantis (B. infantis) EVC001 decreased the incidence and severity of NEC in our Paneth cell disruption with bacterial dysbiosis NEC model. We demonstrated that although B. infantis EVC001 did not decrease injury scores in the Paneth cell disruption with RMS formula NEC model, it decreased the inflammatory milieu. Supplementation with B. infantis EVC001 also decreased sepsis induced mortality in our neonatal sepsis model, which we did not see with our AMP experiments.
Together, our work demonstrates that both AMPs and the probiotic B. infantis EVC001 show promise in the prevention or treatment of neonatal sepsis and NEC. Historically, prevention and treatment strategies for NEC and neonatal sepsis tested in animal models have had mixed effects when translated to clinic. We expect these findings will help inform future studies to understand more about the mechanisms of action for these prevention and treatment strategies and ensure these findings are generalizable and translatable to the clinical setting.
Details
- Title: Subtitle
- Prevention and treatment of neonatal necrotizing enterocolitis and sepsis using antimicrobial peptides and probiotics
- Creators
- Shiloh Rae Lueschow
- Contributors
- Steven J McElroy (Advisor)Patrick M Schlievert (Advisor)Jennifer R Bermick (Committee Member)Craig D Ellermeier (Committee Member)Dominique H Limoli (Committee Member)Kai Wang (Committee Member)Mary E Wilson (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Microbiology
- Date degree season
- Spring 2022
- Publisher
- University of Iowa
- DOI
- 10.25820/etd.006489
- Number of pages
- xxi, 334 pages
- Copyright
- Copyright 2022 Shiloh Rae Lueschow
- Language
- English
- Description illustrations
- illustrations (some color)
- Description bibliographic
- Includes bibliographical references (pages 303-334).
- Public Abstract (ETD)
With premature birth on the rise, an increasing number of infants are at risk each year for neonatal associated diseases such as necrotizing enterocolitis (NEC) and neonatal sepsis. Unfortunately, both diseases are difficult to predict, clinically diagnose, and treat. This means many infants are exposed to risk for development of disabilities or death associated with each disease. Using established and newly developed mouse NEC models as well as a newly developed mouse neonatal sepsis model, we tested the efficacy of applying treatment and prevention strategies using the antimicrobial peptides (AMPs) hBD2 and LL-37 as well as probiotic Bifidobacterium longum subspecies infantis EVC001. While the AMPs were effective at preventing and treating NEC, they were ineffective at impacting our sepsis model. In contrast, B. infantis EVC001 effectively lessened NEC injury and other morbidities, while also decreasing sepsis induced mortality.
Further, we found differences in the mechanism of action for each therapeutic. hBD2 improves epithelial wound healing, decreases levels of the pathogen Klebsiella pneumoniae, and influences the CCL2/CCR2 axis, which is important in both our NEC model and in human infants. LL-37 elicits an improvement in epithelial wound healing but does not appear to significantly influence the microbiome. B. infantis EVC001 altered the microbiome, influenced the inflammation, and produced supernatants that improved wound healing. Future work aims to expound upon these mechanisms to determine whether these therapeutics can translate to clinical approaches to prevent and treat NEC and neonatal sepsis.
- Academic Unit
- Microbiology and Immunology
- Record Identifier
- 9984271255602771
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