Exosomal Transfer of DNA Methyl-Transferase mRNA Induces an Immunosuppressive Phenotype in Human Monocytes.

INTRODUCTION: Survivors of sepsis exhibit persistent immunosuppression. Epigenetic events may be responsible for some of these immunosuppressive changes. During sepsis circulating exosomes contain large quantities of DNA methyltransferase (DNMT) mRNAs. We hypothesized that exosomes directly transfer DNMT mRNAs to recipient monocytes with resultant methylation events and immunosuppression. METHODS: Exosomes containing DNMT mRNA were generated by stimulating monocytes with LPS. Confocal microscopy was used to determine uptake kinetics in the presence of pharmacologic inhibition. Expression and packaging of specific DNMT mRNA was controlled using DNMT siRNAs. Whole genome and gene specific methylation was assessed using bisulfite sequencing. Ingenuity pathway analysis was performed to determine the biological function of significance of differentially methylated regions. RESULTS: Exosomes effectively transferred DNMT mRNA to recipient monocytes. Pharmacologic inhibition of exosome uptake prevented this increase in DNMT mRNA expression. Recipient monocytes exhibited hypermethylation changes and gene suppression. siRNAs decreased the packaging of DNMT mRNAs and prevented TNFα gene suppression, restoring immunocompetence. CONCLUSION: These data support a role for exosome-mediated transfer of DNMT mRNA with resultant methylation and gene silencing. Pharmacologic uptake inhibition or targeted siRNA mediated DNMT gene silencing prevented DNMT mRNA transfer and maintained the cell's ability to express TNFα in response to LPS. This highlights the potential therapeutic value of targeting these exosome-mediated epigenetic events to maintain the host immune response during sepsis.

Analyzing Outcomes Among Older Adults With Necrotizing Soft-Tissue Infections in the United States.

BACKGROUND: Necrotizing soft-tissue infections (NSTIs) encompass a group of severe, life-threatening diseases with high morbidity and mortality. Evidence suggests advanced age is associated with worse outcomes. To date, no large data sets exist describing outcomes in older individuals, and risk factor identification is lacking. METHODS: Retrospective data were obtained from the 2015 Medicare 100% sample. Included in the analysis were those aged ≥65 y with a primary diagnosis of an NSTI (gas gangrene, necrotizing fasciitis, cutaneous gangrene, or Fournier's gangrene). Risk factors for in-hospital mortality and discharge disposition were examined. Continuous variables were assessed using central tendency, t-tests, and Wilcoxon rank-sum tests. Categorical variables were assessed using the chi-squared and Fisher's exact tests. Statistical significance was defined as P < 0.05. RESULTS: 1427 patient records were reviewed. 59% of patients were male, and the overall mean age was 75.4±8.6 y. 1385 (97.0%) patients required emergency surgery for their NSTI diagnosis. The overall mortality was 5.3%. Several underlying comorbidities were associated with higher rates of mortality including cancer (OR: 3.50, P = 0.0009), liver disease (OR: 2.97, P = 0.03), and kidney disease (OR: 2.15, P = 0.01). While associated with high in-hospital mortality, these diagnoses were not associated with a difference in the rate of discharge to home compared with skilled nursing or rehab. Overall, patients discharged to skilled nursing facilities or rehab had higher rates of underlying comorbidities than patients who were discharged home (3 or more comorbid illness 84.3% versus 68.6%, P < 0.0001); however, no individual comorbid illness was associated with discharge location. CONCLUSIONS: In our Medicare data set, we identified several medical comorbidities that are associated with increased rates of in-hospital mortality. Patients with underlying cancers had the highest odds of increased mortality. The effect on outcomes of the potentially immunosuppressive cancer treatments in these patients is unknown. These data suggest that patients with underlying illnesses, especially cancer, kidney disease, or liver disease have higher mortalities and are more likely to be discharged to skilled nursing facilities or rehab. It is unclear why these illnesses were associated with these worse outcomes while others including diabetes and heart disease were not. These data suggest that these particular comorbid illnesses may have special prognostic implications, although further analysis is necessary to identify the causative factors.

Proteomic Pathway Analysis of Monocyte-Derived Exosomes during Surgical Sepsis Identifies Immunoregulatory Functions.

Background: Patients with sepsis exhibit significant long-term immunosuppressive sequelae. Monocyte dysfunction is a hallmark of this damage. Circulating exosomes are an important mediator of the systemic signaling events that occur during the septic response; thus, we sought to characterize the contribution of circulating exosomes to the inflammatory process induced during sepsis Methods: Monocyte-derived exosomes were isolated from cultured monocytes from healthy adult donors via stimulation with lipopolysaccharide (LPS) or phosphate-buffered saline (PBS). The proteome was determined by capillary-liquid chromatography-nanospray tandem mass spectrometry (capillary-LC/NT/MS). Using pathway analysis, proteomic networks of exosomes derived from LPS-stimulated monocytes were compared with those isolated from patients with surgical sepsis. Naïve monocytes were then treated with these exosomes and stimulated with LPS to determine the effects on recipient-cell immune function. Results: Proteomic analysis demonstrated 18 differentially expressed proteins (17 down-regulated, one up-regulated) in sepsis-derived exosomes, with 15 differentially expressed proteins (14 down-regulated, one up-regulated) in the LPS-stimulated exosomes. Functional enrichment analysis demonstrated several down-regulated processes, including localization, biogenesis, and metabolic and cellular processes in addition to immune system processes. In LPS-stimulated macrophages, similar down-regulated processes were seen, including metabolic and cellular processes, as well as the response to stimulus. Cells treated with sepsis-derived exosomes or exosomes from LPS-stimulated monocytes demonstrated significant reductions in tumor necrosis factor (TNF)-α generation in response to LPS stimulation. Conclusions: Proteomic analysis of sepsis-derived exosomes and LPS-stimulated, macrophage-derived exosomes exhibited down-regulation of several important protein networks, including the immune response. In addition, human monocytes treated with exosomes from patients with sepsis or LPS-stimulated monocytes demonstrated significant reductions in TNF-α generation in response to LPS stimulation. These data suggest the contribution of circulating exosomes to systemic signaling and immunomodulation during sepsis.

Challenges and opportunities in implementing pharmacogenomics testing in the clinics.

The field of pharmacogenomics aims to incorporate individual patient genomic information into treatment selection. This is a rapidly evolving field with significant clinical promise. Implementation into clinical practice has several challenges that must be overcome. Genomics-based information encompasses large databases and requires expert knowledge for interpretation. Existing research suggests there are already several areas where pharmacogenomics-based decision-making is ripe for adoption into clinical practice. Impediments exist that must be overcome prior to large-scale implementation of existing pharmacogenomics-based therapies. There are several institutions and corporations at the forefront of implementation that are leading the development; however, larger systems-based approaches will be necessary. This article will discuss some of the present successes and future challenges that are necessary to overcome in order to implement a more patient-centered healthcare system.