Multi-Organ Transcriptome Dynamics in a Mouse Model of Cecal Ligation and Puncture-Induced Polymicrobial Sepsis

Abstract

Purpose During sepsis, an excessive inflammatory immune reaction contributes to multi-organ dysfunction syndrome (MODS), a critical condition associated with high morbidity and mortality; however, the molecular mechanisms driving MODS remain elusive. Methods We used RNA sequencing to characterize transcriptional changes in the early phase of sepsis, at 6, 12, 24 hour time points in lung, kidney, liver, and heart tissues, in a cecal ligation and puncture (CLP)-induced polymicrobial sepsis murine model. Results The CLP surgery induced significant changes (adj. p -value<0.05) in expression of hundreds of transcripts in the four organs tested, with the highest number exceeding 2,000 differentially expressed genes (DEGs) in all organs at 12 hours post-CLP. Over-representation analysis by functional annotations of DEGs to the Reactome database revealed the immune system, hemostasis, lipid metabolism, signal transduction, and extracellular matrix remodeling biological processes as significantly altered in at least two organs, while metabolism of proteins and RNA were revelaed as being liver tissue specific in the early phase of sepsis. Conclusion RNA sequencing across organs and time-points in the CLP murine model allowed us to study the trajectories of transcriptome changes demonstrating alterations common across multiple organs as well as biological pathways altered in an organ-specific manner. These findings could pave new directions in the research of sepsis-induced MODS and indicate new sepsis treatment strategies.

Selective Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibition by the SCH772984 Compound Attenuates In Vitro and In Vivo Inflammatory Responses and Prolongs Survival in Murine Sepsis Models

“…We chose the heart, kidney, lung, and liver tissues for the RNA-Seq survey upon SCH772984 treatment as those organs are frequently affected in the course of sepsis [ 37 ]. Our recent RNA-seq transcriptomic survey of the same tissues 6 h, 12 h, and 24 h post-CLP procedure in mice indicated that the alteration of the immune system pathways is common across multiple organs, and the highest number of significant DEGs in those organs, when compared to sham-operated mice, is observed at 12 h [ 38 ]. Based on this observation and the fact that CLP mice start manifesting clinical signs of sepsis at around 12 h post-CLP [ 39 ], we selected the 6 h and 12 h time points to capture early molecular events of SCH772984 action.…”

CryoGrid-PIXUL-RNA: High throughput RNA isolation platform for tissue transcript analysis

“…Next, we tested the above protocol using either proteinase K or TRIzol extraction from CryoCore samples taken from CryoTray-stored frozen livers of either LPS (endotoxin) or PBS (control) treated mice ( Fig.3A ). Isolated RNA was used in Matrix RT-qPCR using exon-spanning primers to the housekeeping Actb , and multiorgan sepsis-inducible Ngal ( Lcn2 ) genes (48,51). The CTs for Actb were much higher (lower mRNA levels) with the proteinase K method compared to TRIzol ( Fig.3B ).…”

“…Matrix-RT-qPCR was used first to compare the PK buffer vs TRIzol protocol ( Fig.6A ) to assess the well-described organ-specific, sepsis-induced, and housekeeping mRNA expression patterns (1,48,51). Data are shown as both CTs and as a ratio to the Rpl32 housekeeping mRNA ( Fig.6B ).…”

“…Ngal (Lcn2 ) expression was induced by LPS treatment in all four organs as illustrated by decreased CT and increased ratio to Rpl32 . Elevated LPS endotoxin-induced Ngal/Lcn2 expression in liver as well as inducible expression across multiple organs has also been shown in the mouse cecal ligation and puncture (CLP) sepsis model (48). The results obtained with the PIXUL-PK buffer were indistinguishable from those with the PIXUL-TRIzol method, suggesting that the two methods are equivalent when measuring expression of these genes by RT-qPCR ( Fig.6 ).…”

“…3A). Isolated RNA was used in Matrix RT-qPCR using exon-spanning primers to the housekeeping Actb, and multiorgan sepsis-inducible Ngal (Lcn2) genes (48,51). The CTs for Actb were much higher (lower mRNA levels) with the proteinase K method compared to TRIzol (Fig.…”