Sepsis Derived Exosomes Transfer Functional DNA Methyltransfereases with Resultant Immunosuppression and Gene Silencing

Author(s):
Jon Wisler; Kanhaiya SIngh; Helen Hou; Chandan Sen

Background:

Each year sepsis affects over 750,000 individuals with a high mortality of 30-40%.  Following a septic insult patients exhibit profound immunosuppresion, which is associated with increased short and long-term mortality.  Patients that survive the initial insult have high rates of secondary infections with worsened mortality.  We  have previously demonstrated that multiple components of epigenetic modification are involved in the generation of this immunosuppressed phenotype.  Specifically, DNA methylation via DNA Methyltransferases (DNMTs) acts to silence various genes during sepsis.  Circulating exosomes are important in the transfer of cellular information and signal amplification.  They are known to alter recipient cell function in diseases like sepsis.

Hypothesis:

We hypothesize that circulating exosomes contain DNMT protein that is transferred to recipient cells and results in promoter methylation and gene silencing.

Methods:

Circulating exosomes were isolated from patients with sepsis.  DNMT 1, 3A, and 3B levels were determined using an ELISA based assay.  Colormetric assays were used to assess activity levels.  In order to assess functional transfer, naive monocytes were treated with sepsis derived exosomes.  Methylation was assessed using bisulfate sequencing.

Results:

Significant elevations in DNMT 1, 3A, and 3B expression were seen on days 1, 3, and 5 of sepsis.  These increases correlated with increased activity.  When transferred to naive monocytes, DNMT activity is retained and methylation events occur with significant increases in promoter methylation seen at TNF-alpha.  Cultured monocyte derived exosomes were found to contain high levels of DNMT 1, 3A, and 3B when stimulated with endotoxin suggesting these as the source for the high level of activity within exosomes of patients with sepsis.  These exosomes are taken up by naive cells with resultant methylation events and gene silencing.

Conclusions:

These data demonstrate that sepsis derived circulating exosomes contain high levels of DNMTs and that these DNMTs are transferred to recipient cells with resultant methylation at promoters of genes involved in the inflammatory response.  This results in gene silencing and subsequent immunosuppression.  This is important in that it identifies circulating exosomes as a key process in the development of sepsis and sepsis related immunosuppression.