Mots-Clés

single cell RNAsequencing, sepsis

Description

Integrating single-cell RNA sequencing analysis to define respiratory epithelial cells functional plasticity during experimental sepsis

• Description

Sepsis-induced immunosuppression confers increased susceptibility to secondary pulmonary bacterial infections. As frontline defense, it is essential to understand the role of airway epithelial cells during sepsis-induced immunosuppression. 

We raise the hypothesis that sepsis-induced alterationsin airway epithelium contribute to post-septic susceptibility towards secondary pulmonary bacterial infections. The main goal of our project is to address the role of airway epithelium in post-septic lung defense.

Our team established an experimental double-hitmodel in which mice subjected to non-pulmonary sepsis by polymicrobial peritonitis by Cecal ligation and puncture (CLP)exhibit major susceptibility to secondary P. aeruginosapulmonary infection. We generated consistent preliminary data that demonstrate that bronchial and alveolar epithelial cells are deeply impaired in their barrier and immune functions in response to remote abdominal sepsis.

The traditional view of airway epithelium has been revised these past years thanks to single-cell RNA sequencing studies that revealed cellular heterogeneity and identified novel cell subpopulations and different cell states according to health and disease.

A critical question raised is the cellular heterogeneity of airway epithelial cells following post-sepsis injury. Epithelial immune functions and regeneration properties after tissue injury would be of special interest. Indeed, restoring epithelial function after sepsis-induced damage requires a better understanding of how these newly identified epithelial cell subtypes and cell states orchestrate the immune response and tissue repair following injury induced by sepsis.

We will use the single-cell RNA-seq expression data from GEO with accession number GSE207651 (cf doi: 10.3389/fimmu.2022.981784).The mice underwent the Cecal ligation and puncture (CLP) or sham surgery. The mice in Sham group and CLP group were sacrificed 24 hours after operation, while the CLP-48h group were sacrificed at 48 hours after operation. Three sham group specimens, three CLP specimens, three CLP-48h specimens were mixed for single-cell RNA sequencing. The prepared single cell suspensions were subjected to Magnetic-activated cell sorting (MACS) using Miltenyi mouse CD45 magnetic beads according to the manufacturer's recommendations. The CD45- and CD45+ cell suspensions were mixed 1:1 for single-cell sequencing.

This work will likely generate new mechanistic insights in the pathophysiology of post-injury pneumonia, to ultimately identify new therapeutics based on functional restoration of the airway epithelium.

• Objectives

Cellular composition of epithelial cells will be investigated in sham and septic lung tissue. Our main aim is to analysethe heterogeneity of airway epithelial cells to unravel the functional plasticity of these cells following experimental sepsis. This will be achieved through four specific objectives:

1. Epithelial cells clustering based on differences in marker genes:

Which sub-clusters are enriched/reduced following CLP. Visualization of epithelial cells subclusters associated with the sepsis phenotype.

2. Lineage dedifferentiation trajectory (pseudotime analysis):

Prediction of epithelial cells differentiation trajectories with Monocle.

3. Functional enrichment analysis:

DEG and enriched GO functions of upregulated genes in epithelial sub-clusters.

Pathway activity : AUCell

4.Cell-cell communication analysis:

We will investigate the network of epithelial-epithelial or -immune cells interactions using ICELLNET (Vassili Soumelis lab, https://github.com/soumelis-lab/ICELLNET) and/or Cell Phone DB2 (Roser Vento-Tormo et al., Nature 2018) tools.

Dot plots showing ligand-receptor pairs of cytokines (interaction weghts/strenght) between epithelial cells and other immune cell groups.

The continuation of this work is foreseen in an application for funding from the ANR that has just been submitted.