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

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).

The Mechanisms of Action of Hyperbaric Oxygen in Restoring Host Homeostasis during Sepsis.

The perception of sepsis has shifted over time; however, it remains a leading cause of death worldwide. Sepsis is now recognized as an imbalance in host cellular functions triggered by the invading pathogens, both related to immune cells, endothelial function, glucose and oxygen metabolism, tissue repair and restoration. Many of these key mechanisms in sepsis are also targets of hyperbaric oxygen (HBO2) treatment. HBO2 treatment has been shown to improve survival in clinical studies on patients with necrotizing soft tissue infections as well as experimental sepsis models. High tissue oxygen tension during HBO2 treatment may affect oxidative phosphorylation in mitochondria. Oxygen is converted to energy, and, as a natural byproduct, reactive oxygen species are produced. Reactive oxygen species can act as mediators, and both these and the HBO2-mediated increase in oxygen supply have the potential to influence the cellular processes involved in sepsis. The pathophysiology of sepsis can be explained comprehensively through resistance and tolerance to infection. We argue that HBO2 treatment may protect the host from collateral tissue damage during resistance by reducing neutrophil extracellular traps, inhibiting neutrophil adhesion to vascular endothelium, reducing proinflammatory cytokines, and halting the Warburg effect, while also assisting the host in tolerance to infection by reducing iron-mediated injury and upregulating anti-inflammatory measures. Finally, we show how inflammation and oxygen-sensing pathways are connected on the cellular level in a self-reinforcing and detrimental manner in inflammatory conditions, and with support from a substantial body of studies from the literature, we conclude by demonstrating that HBO2 treatment can intervene to maintain homeostasis.

Investigating the Effects of Hyperbaric Oxygen Treatment in Necrotizing Soft Tissue Infection With Transcriptomics and Machine Learning (the HBOmic Study): Protocol for a Prospective Cohort Study With Data Validation.

BACKGROUND: Necrotizing soft tissue infections (NSTIs) are complex multifactorial diseases characterized by rapid bacterial proliferation and progressive tissue death. Treatment is multidisciplinary, including surgery, broad-spectrum antibiotics, and intensive care; adjunctive treatment with hyperbaric oxygen (HBO2) may also be applied. Recent advances in molecular technology and biological computation have given rise to new approaches to infectious diseases based on identifying target groups defined by activated pathophysiological mechanisms. OBJECTIVE: We aim to capture NSTI disease signatures and mechanisms and responses to treatment in patients that receive the highest standard of care; therefore, we set out to investigate genome-wide transcriptional responses to HBO2 treatment during NSTI in the host and bacteria. METHODS: The Effects of Hyperbaric Oxygen Treatment Studied with Omics (HBOmic) study is a prospective cohort study including 95 patients admitted for NSTI at the intensive care unit of Copenhagen University Hospital (Rigshospitalet), Denmark, between January 2013 and June 2017. All participants were treated according to a local protocol for management of NSTI, and biological samples were obtained and stored according to a standard operational procedure. In the proposed study, we will generate genome-wide expression profiles of whole-blood samples and samples of infected tissue taken before and after HBO2 treatment administered during the initial acute phase of infection, and we will analyze the profiles with unsupervised hierarchical clustering and machine learning. Differential gene expression will be compared in samples taken before and after HBO2 treatment (N=85), and integration of profiles from blood and tissue samples will be performed. Furthermore, findings will be compared to NSTI patients who did not receive HBO2 treatment (N=10). Transcriptomic data will be integrated with clinical data to investigate associations and predictors. RESULTS: The first participant was enrolled on July 27, 2021, and data analysis is expected to begin during autumn 2022, with publication of results immediately thereafter. CONCLUSIONS: The HBOmic study will provide new insights into personalized patient management in NSTIs. TRIAL REGISTRATION: ClinicalTrials.gov NCT01790698; https://clinicaltrials.gov/ct2/show/NCT01790698. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/39252.

Hyperbaric oxygen treatment impacts oxidative stress markers in patients with necrotizing soft-tissue infection.

Necrotizing soft-tissue infection (NSTI) is a rare, severe, and fast-progressing bacterial infection associated with a high risk of developing sepsis or septic shock. Increasing evidence indicates that oxidative stress is crucial in the development and progression of sepsis, but its role in NSTI specifically has not been investigated. Some patients with NSTI receive hyperbaric oxygen (HBO2) treatment as the restoration of oxidative stress balance is considered an important mechanism of action, which HBO2 facilitates. However, a gap in knowledge exists regarding the effect of HBO2 treatment on oxidative stress in patients with NSTI. In the present observational study, we aimed to investigate HBO2 treatment effects on known markers of oxidative stress in patients with NSTI. We measured plasma myeloperoxidase (MPO), superoxide dismutase (SOD), heme oxygenase-1 (HO-1) and nitrite+nitrate in 80 patients with NSTI immediately before and after their first HBO2 treatment, and on the following day. We found that HBO2 treatment was associated with a significant increase in MPO and SOD by a median of 3.4 and 8.8 ng/mL, respectively. Moreover, we observed an HBO2 treatment-associated increase in HO-1 in patients presenting with septic shock (n=39) by a median of 301.3 pg/mL. All markers were significantly higher in patients presenting with septic shock compared to patients without shock, and all markers correlated with disease severity. High baseline SOD was associated with 90-day mortality. In conclusion, HBO2 treatment was associated with an increase in MPO and SOD in patients with NSTI, and oxidative stress was more pronounced in patients with septic shock.

Soluble ICAM-1 is modulated by hyperbaric oxygen treatment and correlates with disease severity and mortality in patients with necrotizing soft-tissue infection.

The inflammatory response in patients with necrotizing soft-tissue infection (NSTI) is excessive and often causes collateral damage, thereby worsening disease severity and prognosis. Shedding of endothelial adhesion molecules may be a key regulatory mechanism to modulate the inflammatory response in patients with septic NSTI. Hyperbaric oxygen (HBO2) treatment has demonstrated an effect on adhesion molecules. However, endothelial shedding and its association with NSTI disease severity and prognosis is not fully understood. We hypothesized that shedding of intercellular adhesion molecule-1, and the resulting release of the soluble isoform soluble intercellular adhesion molecule-1 (sICAM-1), is modified by HBO2 treatment, and that sICAM-1 concentrations are associated with severity of disease and mortality in patients with NSTI. We measured sICAM-1 in 80 patients with NSTI immediately before and after first session of HBO2 treatment as well as on the following day. We found an overall sICAM-1 level of 594 ng/mL [interquartile range (IQR) 406-817]. HBO2 significantly (P = 0.01) increased sICAM-1 by a median of 45.1 ng/mL, which remained elevated until the following day; this effect was more pronounced in patients with septic shock. Furthermore, sICAM-1 was significantly correlated with disease severity [simplified acute physiology score II (SAPS II); ρ = 0.24, P = 0.04] and low sICAM-1 was found to be an independent predictor for 90-day mortality in age-sex-SAPS II-adjusted analysis (odds ratio 14.0, 95% CI 1.82-341.4, P = 0.03). These results support the hypothesis that endothelial shedding is an important pathophysiological mechanism in NSTI and suggest that HBO2 treatment may induce immunomodulatory effects that potentially decreases collateral damage and mortality.NEW & NOTEWORTHY HBO2 treatment may be a promising immunomodulatory agent by increasing sICAM-1, thereby lowering risk of collateral damage, especially in the most critically ill patients. sICAM-1 is associated with disease severity in NSTI as emphasized by significant correlations with SAPS II. Low sICAM-1 levels are an independent risk factor of 90-day mortality and appeared to give a good level of diagnostic accuracy, suggesting that sICAM-1 can be used as a prognostic biomarker for NSTI.

Effects of adding adjunctive hyperbaric oxygen therapy to standard wound care for diabetic foot ulcers: a protocol for a systematic review with meta-analysis and trial sequential analysis.

INTRODUCTION: Diabetic foot ulcer represents a major health problem globally. Preliminary studies have indicated that systemic treatment of diabetic foot ulcer patients with hyperbaric oxygen therapy have beneficial effects on wound healing, risk of amputation, glycaemic control, atherosclerosis, inflammatory markers and other clinical and laboratory parameters. This protocol for a systematic review aims at identifying the beneficial and harmful effects of adding hyperbaric oxygen therapy to standard wound care for diabetic foot ulcers. METHODS AND ANALYSIS: This protocol was performed following the recommendations of the Cochrane Collaboration and the eight-step assessment procedure suggested by Jakobsen and colleagues. We plan to include all relevant randomised clinical trials assessing the effects of hyperbaric oxygen therapy in the treatment of diabetic foot ulcer versus any control group with any intervention defined as standard wound care or similar, together with sham interventions. Our primary outcome will be: all-cause mortality, serious adverse events and quality of life. Our secondary outcomes will be: healing of index wound, major amputation and wound infection. Any eligible trial will be assessed and classified as either high risk of bias or low risk of bias, and our conclusions will be based on trials with low risk of bias. The analyses of the extracted data will be performed using Review Manager 5 and Trial Sequential Analysis. For both our primary and secondary outcomes, we will create a 'Summary of Findings' table and use GRADE (Grading of Recommendations Assessment, Development and Evaluation) assessment to assess the quality of the evidence. ETHICS AND DISSEMINATION: We use publicly accessible documents as evidence, there is no participant involvement at an individual level and an institutional ethics approval is not required. The results of the review will be sought published in a peer-reviewed journals, also in the event of insignificant results or null results, and thereby it will be disseminated to clinicians and public available. PROSPERO REGISTRATION NUMBER: CRD42019139256.

The clinical use of hyperbaric oxygen in the treatment of Danish patients with diabetic foot ulcers.

INTRODUCTION: Patients with diabetic foot ulcers (DFU) suffer from diabetes-related complications and comor-bidities. Hyperbaric oxygen therapy (HBOT) is a treatment modality with limited capacity used in the treatment of DFUs. It is important to ensure that HBOT is offered to patients who are suitable for this treatment regarding effect, compliance and life expectancy. The objective of the present study was to describe the population of patients with DFU who were referred to HBOT in Denmark in the 1999-2016 period. METHODS: All patients with DFU who were treated at the HBOT chamber in Copenhagen during the study period were considered. Patients with an invalid social security number or an incorrect diagnosis were excluded. Data on comor-bidities, amputation and death were extracted from the Danish National patient Registry and the Danish Civil Registration System. Continuous data were described as median values and binary data were described as proportions. The probability estimate for survival and amputation was investigated by constructing Kaplan-Meier curves. RESULTS: The cohort included 148 patients. Patients were mainly referred from the Capital Region (92%) and multi-disciplinary wound care centres were the primary referring departments (67%). Comorbidity rates were high with an initial median Charlson Comorbidity Index score of five. The five-year amputation and mortality estimates after referral were 73.5% and 51.8%, respectively. CONCLUSIONS: The study showed that Danish DFU patients who are offered HBOT are in advanced stages of their disease, and the referral hinges on local factors such as geography and the referring source rather than on standardised procedures. FUNDING: none. TRIAL REGISTRATION: not relevant.