No abstract

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 DNMTmRNAs 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 TNFa 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 TNFa 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.

Chronic pancreatitis (CP) is a fibro-inflammatory syndrome in individuals who develop persistent pathological responses to parenchymal injury or stress. Novel therapeutic or dietary interventions that could lessen inflammation in this disease could significantly improve quality of life in patients with CP. Complex dietary foods like soy and tomatoes are composed of active metabolites with anti-inflammatory effects. Data from our group reports that bioactive agents in soy and tomatoes can reduce pro-inflammatory cytokines and suppressive immune populations. Additionally, our team has developed a novel soy-tomato juice currently being studied in healthy individuals with no toxicities, and good compliance and bioavailability. Thus, we hypothesize that administration of a soy-tomato enriched diet can reduce inflammation and severity of CP. C57BL/6 mice were injected intraperitoneally with 50 μg/kg caeurlein (7 hourly injections, twice weekly) for 6 weeks to induce CP. After 4 weeks of caerulein injections, mice were administered a control or a soy-tomato enriched diet for 2 weeks. Disease severity was measured via immunohistochemical analysis of pancreata measuring loss of acini, fibrosis, inflammation, and necrosis. Serum lipase and amylase levels were analyzed at the end of the study. Inflammatory factors in the serum and pancreas, and immune populations in the spleen of mice were analyzed by cytokine multiplex detection, qRT-PCR, and flow cytometry respectively. Infra-red (IR) sensing of mice was used to monitor spontaneous activity and distress of mice. Mice fed a soy-tomato enriched diet had a significantly reduced level of inflammation and severity of CP (p = 0.032) compared to mice administered a control diet with restored serum lipase and amylase levels (p < 0.05). Mice with CP fed a soy-tomato diet had a reduction in inflammatory factors (TNF-α, IL-1β, IL-5) and suppressive immune populations (myeloid-derived suppressor cells; MDSC) compared to control diet fed mice (p < 0.05). Infra-red sensing to monitor spontaneous activity of mice showed that soy-tomato enriched diet improved total activity and overall health of mice with CP (p = 0.055) and CP mice on a control diet were determined to spend more time at rest (p = 0.053). These pre-clinical results indicate that a soy-tomato enriched diet may be a novel treatment approach to reduce inflammation and pain in patients with CP.