Poster CC5 - Multiomic single nuclei sequencing of the uterus in an inflammation induced model of preterm birth

Friday, October 25, 2024

3:30 PM – 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

DM

Davell M Carter

Graduate Student
Johns Hopkins Medical Institutions
Baltimore, Maryland, United States

Co-Author(s)

JO

Jairo Ortiz, MD

Senior Research Specialist
Johns Hopkins Center for Nanomedicine, United States
YZ

Yvonne Zhaii

Graduate Student
Johns Hopkins University, United States
JQ

Jiang Qian, PhD

Professor
Johns Hopkins University, United States

Primary Investigator(s)

Laura Ensign, B.S., PhD (she/her/hers)

Marcella E. Woll Professor
Johns Hopkins University
BALTIMORE, Maryland, United States

Introduction: Preterm birth (PTB) is defined as birth before 37 weeks gestation and affects 15 million babies annually. Medical care for preterm neonates also is associated with a significant cost burden, totaling about $26 billion in the United States alone. Unfortunately, therapeutic options for preventing PTB are limited. We previously described methods to enhance delivery to the reproductive tract for new preventative treatment approach in a mouse model of inflammation- induced PTB that led to the delivery of live pups. While vaginal administration nanosuspensions of histone deacetylase inhibitors (HDACi) in the form of a mucoinert nanosuspension was effective, the mechanism was not yet clear. Advancements in high throughput sequencing have allowed for characterization of gene expression and histone accessibility at the resolution of single cells. Given that gestational tissues are extremely heterogeneous, we sought to characterize gene expression and histone accessibility at the single nuclei resolution. Preliminary analysis highlighted differences at the multiomic level between healthy pregnant mice and mice exposed to intrauterine inflammation with and without treatment to prevent PTB. Mechanistic understanding may elucidate novel treatment option of prevention of PTB.

Materials and

Methods: Timed pregnant CD1 mice underwent laparotomy on gestational day 15 (E15). Mice received an intrauterine injection of lipopolysaccharide (LPS) between the two distal pups in both uterine horns. Control groups received intrauterine injections of saline on E15. Following surgery mice were vaginally dosed with a mucoinert Trichostatin A (TSA) nanosuspension of the HDACi in combination with a mucoinert nanosuspension of progesterone (P4), or vehicle. Eight hours post dosing, mice were sacrificed, to collect uterus snap frozen. The uterus was processed into a single nuclei suspension for single nuclei analysis. Single nuclei were pooled together by condition and submitted for 10x genomics multiome sequencing by core facilities. All subsequent analysis was performed in R studio version 2023.09.0+463 and Seurat package version 5.

Results, Conclusions, and Discussions: In mouse gestational tissues our analysis identified distinct clusters of varying cell populations. Differentially expressed gene (DEG) analysis unveiled top expressed genes between pregnant mice and mice exposed to intrauterine inflammation on E15, and changes with drug treatment gave further insight into mechanistic underpinnings in PTB. Notably, the NF-κB pathway was enriched in the uterus. Additionally, the binding motif of the transcriptional factor NF-κB is enriched in the up-regulated peaks as revealed by snATAC-Seq. Drug treatment with TSA/P4 downregulated Tumor necrosis factor alpha (TNF-α) associated proteins receptor, activator of nuclear factor κ B (RANK), tumor necrosis factor, alpha induced protein 3 (TNFAIP3), as well as various chemokines. On E15, there were 21 identifiable cell types in the CD1 mouse uterus across saline treated, LPS treated, and drug treated conditions. At 8 hours post intrauterine injection of LPS, elements of the NF-κB pathway were highly enriched. Vaginal co-treatment with TSA/P4, which is effective in preventing PTB in the mouse model, resulted in decreased expression of the proteins RANK, TNFAI3, and CXCL2. Single nuclei sequencing of the CD1 mouse uterus after intrauterine LPS exposure revealed activation of the NF-κB pathway on a gene expression level. Vaginal co- treatment with TSA/P4 revealed downregulation of 3 genes in the NF-κB pathway as well as TNF-α. Further studies will need to be performed to further validate changes in gene expression and downstream effects. Future experiments work will further interrogate changes in gene expression in addition to chromatin accessibility across the time course of gestation.

Acknowledgements (Optional):