Key pathways and genes that are altered during treatment with hyperbaric oxygen in patients with sepsis due to necrotizing soft tissue infection (HBOmic study)

Abstract

Background: For decades, the basic treatment strategies of necrotizing soft tissue infections (NSTI) have remained unchanged, primarily relying on aggressive surgical removal of infected tissue, broad-spectrum antibiotics, and supportive intensive care. One treatment strategy that has been proposed as an adjunctive measure to improve patient outcomes is hyperbaric oxygen (HBO₂) treatment. HBO₂ treatment has been linked to several immune modulatory effects; however, investigating these effects is complicated due to the disease's acute life-threatening nature, metabolic and cell homeostasis dependent variability in treatment effects, and heterogeneity with respect to both patient characteristics and involved pathogens. To embrace this complexity, we aimed to explore the underlying biological mechanisms of HBO₂ treatment in patients with NSTI on the gene expression level.

Methods: We conducted an observational cohort study on prospective collected data, including 85 patients admitted to the intensive care unit (ICU) for NSTI. All patients were treated with one or two HBO₂ treatments and had one blood sample taken before and after the intervention. Total RNAs from blood samples were extracted and mRNA purified with rRNA depletion, followed by whole-transcriptome RNA sequencing with a targeted sequencing depth of 20 million reads. A model for differentially expressed genes (DEGs) was fitted, and the functional aspects of the obtained set of genes was predicted with GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of genes and Genomes) enrichment analyses. All analyses were corrected for multiple testing with FDR.

Results: After sequential steps of quality control, a final of 160 biological replicates were included in the present study. We found 394 protein coding genes that were significantly DEGs between the two conditions with FDR < 0.01, of which 205 were upregulated and 189 were downregulated. The enrichment analysis of these DEGs revealed 20 GO terms in biological processes and 12 KEGG pathways that were significantly overrepresented in the upregulated DEGs, of which the term; "adaptive immune response" (GO:0002250) (FDR = 9.88E-13) and "T cell receptor signaling pathway" (hsa04660) (FDR = 1.20E-07) were the most significant. Among the downregulated DEGs two biological processes were significantly enriched, of which the GO term "apoptotic process" (GO:0006915) was the most significant (FDR = 0.001), followed by "Positive regulation of T helper 1 cell cytokine production" (GO:2000556), and "NF-kappa B signaling pathway" (hsa04064) was the only KEGG pathway that was significantly overrepresented (FDR = 0.001).

Conclusions: When one or two sessions of HBO₂ treatment were administered to patients with a dysregulated immune response and systemic inflammation due to NSTI, the important genes that were regulated during the intervention were involved in activation of T helper cells and downregulation of the disease-induced highly inflammatory pathway NF-κB, which was associated with a decrease in the mRNA level of pro-inflammatory factors.

Trial registration: Biological material was collected during the INFECT study, registered at ClinicalTrials.gov (NCT01790698).

Keywords: Differential gene expression; Hyperbaric oxygen treatment; Immune cells; Inflammation; Necrotizing soft tissue infection; Sepsis; Systems medicine; Transcriptomics.

Fig. 1

Study flow diagram. INFECT Systems Medicine to Study NSTI, TIN transcript integrity number, HBO₂ hyperbaric oxygen treatment

Fig. 2

Enriched GO terms and KEGG pathways among differentially expressed genes. A GOchord plot of top 10 GO terms sorted by FDR. Only genes that are assigned to a minimum of 2 terms are displayed. Genes are ranked according to logFC, with red colors signifying upregulated genes and blue colors signifying downregulated genes. Genes symbols are used to describe genes. B Top 10 KEGG pathways sorted by FDR. Color code indicates -10log(FDR); the length of the bar indicates fold enrichment defined as the percentage of differentially expressed genes belonging to the pathway, divided by the corresponding percentage in the background. The size of the bubbles indicates the number of genes in the enriched pathway

Fig. 3

Illustration of the overrepresented genes in the KEGG pathway NF-κB signaling. Simplified version of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (hsa04064); NF-κB signaling pathway. Differentially expressed genes from immediately before to immediately after hyperbaric oxygen treatment that are significantly downregulated are marked in red (FDR < 0.02). Genes are named with their gene symbol