Application of single cell multiomics points to changes in chromatin accessibility near calcitonin receptor like receptor and a possible role for adrenomedullin in the post-shock lung

Abstract

Introduction: Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a commonly occurring sequelae of traumatic injury resulting from indirect insults like hypovolemic shock and/or extrapulmonary sepsis. The high lethality rate associated with these pathologies outlines the importance of clarifying the "priming" effects seen in the post-shock lung microenvironment, which are understood to bring about a dysregulated or overt immune response when triggered by a secondary systemic infectious/septic challenge culminating in ALI. In this pilot project, we test the hypothesis that application of a single cell multiomics approach can elucidate novel phenotype specific pathways potentially contributing to shock-induced ALI/ARDS.

Methods: Hypovolemic shock was induced in C57BL/6 (wild-type), PD-1, PD-L1, or VISTA gene deficient male mice, 8-12 weeks old. Wild-type sham surgeries function as negative controls. A total of 24-h post-shock rodents were sacrificed, their lungs harvested and sectioned, with pools prepared from 2 mice per background, and flash frozen on liquid nitrogen. N = 2 biological replicates (representing 4 mice total) were achieved for all treatment groups across genetic backgrounds. Samples were received by the Boas Center for Genomics and Human Genetics, where single cell multiomics libraries were prepared for RNA/ATAC sequencing. The analysis pipeline Cell Ranger ARC was implemented to attain feature linkage assessments across genes of interest.

Results: Sham (pre-shock) results suggest high chromatin accessibility around calcitonin receptor like receptor (CALCRL) across cellular phenotypes with 17 and 18 feature links, exhibiting positive correlation with gene expression between biological replicates. Similarity between both sample chromatin profiles/linkage arcs is evident. Post-shock wild-type accessibility is starkly reduced across replicates where the number of feature links drops to 1 and 3, again presenting similar replicate profiles. Samples from shocked gene deficient backgrounds displayed high accessibility and similar profiles to the pre-shock lung microenvironment.

Conclusion: High pre-shock availability of DNA segments and their positive correlation with CALCRL gene expression suggests an apparent regulatory capacity on transcription. Post-shock gene deficient chromatin profiles presented similar results to that of pre-shock wild-type samples, suggesting an influence on CALCRL accessibility. Key changes illustrated in the pre-ALI context of shock may allow for additional resolution of "priming" and "cellular pre-activation/pre-disposition" processes within the lung microenvironment.

Keywords: ALI; ARDS; multiomics; sepsis; shock; single cell; trauma.

FIGURE 1

Illustrates workflow involved in data acquisition. Surgical techniques, sample harvesting, sequencing library preparation, and data analysis make up this process (N = 2/group).

FIGURE 2

All samples in this multiomics approach are presented here in T-SNE plot format from Loupe Browser data visualization software. Wild-type sham/hemorrhaged as well as gene deficient hemorrhaged profiles are depicted. Samples are bracketed by individual biological replicates. Colored datapoints are clustered according to similarities in transcriptional profiles (N = 2/group).

FIGURE 3

Depicts graph based projections (A) for all clusters in a representative sample [wild-type hem (1)], and its log2 fold change expression profile (B) for Calcrl.

FIGURE 4

(A) Depicts a heat map of top significant genes upregulated per cluster (log2 fold change) in a representative sample [wild-type hem (1)]. Violin plots show relative log2 fold change expression profiles for markers used to identify endothelial cells in this sample: Calcrl (B), Tek (C), and Tmem100 (D).

FIGURE 5

All feature links are depicted (within 1 Mb of Calcrl) (A) for representative sample wild-type hem (1). A single feature link is highlighted (B), then shown at higher resolution at this specific locus chr2:84184437-84185292 (C). The genomic location of Calcrl is denoted by the bracketed dashed line below the linkage arcs. (D) Displays average number of Calcrl unique molecular identifiers (UMIs), or cut sites per cell at this locus (E) for cells within each identified phenotype, presented in bar graph format.

FIGURE 6

Feature linkage assessment of wild-type pre (A) and post-shock (B) lung microenvironments of Calcrl. Blue linkage arcs are displayed at the top of each sample plot; relative strength of correlation between accessible loci and Calcrl enrichment is conveyed via arc height. Colored peak information below arcs displays accessibility based on cells in individual clusters. The abundance of cells with accessible chromatin is represented by peak heights (N = 2/group).

FIGURE 7

Feature linkage assessment of hemorrhaged gene deficient backgrounds. PD-1^–/– (A), PD-L1^–/– (B) and VISTA^–/– (C). Data is displayed as represented in Figure 6.