Exosomes from patients with septic shock convey miRNAs related to inflammation and cell cycle regulation: new signaling pathways in sepsis?

Background Exosomes isolated from plasma of patients with sepsis may induce vascular apoptosis and myocardial dysfunction by mechanisms related to inflammation and oxidative stress. Despite previous studies demonstrating that these vesicles contain genetic material related to cellular communication, their molecular cargo during sepsis is relatively unknown. In this study, we evaluated the presence of microRNAs (miRNAs) and messenger RNAs (mRNAs) related to inflammatory response and redox metabolism in exosomes of patients with septic shock. Methods Blood samples were collected from 24 patients with septic shock at ICU admission and after 7 days of treatment. Twelve healthy volunteers were used as control subjects. Exosomes were isolated by ultracentrifugation, and their miRNA and mRNA content was evaluated by qRT-PCR array. Results As compared with healthy volunteers, exosomes from patients with sepsis had significant changes in 65 exosomal miRNAs. Twenty-eight miRNAs were differentially expressed, both at enrollment and after 7 days, with similar kinetics (18 miRNAs upregulated and 10 downregulated). At enrollment, 35 differentially expressed miRNAs clustered patients with sepsis according to survival. The pathways enriched by the miRNAs of patients with sepsis compared with control subjects were related mostly to inflammatory response. The comparison of miRNAs from patients with sepsis according to hospital survival demonstrated pathways related mostly to cell cycle regulation. At enrollment, sepsis was associated with significant increases in the expression of mRNAs related to redox metabolism (myeloperoxidase, 64-fold; PRDX3, 2.6-fold; SOD2, 2.2-fold) and redox-responsive genes (FOXM1, 21-fold; SELS, 16-fold; GLRX2, 3.4-fold). The expression of myeloperoxidase mRNA remained elevated after 7 days (65-fold). Conclusions Exosomes from patients with septic shock convey miRNAs and mRNAs related to pathogenic pathways, including inflammatory response, oxidative stress, and cell cycle regulation. Exosomes may represent a novel mechanism for intercellular communication during sepsis. Electronic supplementary material The online version of this article (10.1186/s13054-018-2003-3) contains supplementary material, which is available to authorized users.


Blood Sample Collection, Processing and Plasmatic Cytokines Measurement
Blood sample (30 mL) was collected on EDTA tubes and maintained at 4°C until being processed (maximum of 2 hours). Protease inhibitors (3 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin and 1 µg/ml pepstatin) were added and plasma was stored frozen at -80°C. Plasma was filtered through a 0.22 µm filter and microvesicles were isolated by ultracentrifugation at 120,000g for 2h30 at 4º C (SW 28 Ti Rotor, Optima L-90K Ultracentrifugue, Beckman Coulter, Fullerton, USA) (4). Supernatants were discarded and the pellets were washed twice with PBS.
MicroRNA and total RNA were extracted using the miRNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's protocol. RNA quantity and quality were assessed by spectrophotometry (NanoVue, GE Healthcare, Munich, Germany) and microchip electrophoresis (Bioanalyzer 2100, Agilent Technologies, Palo Alto, United States) respectively.

Western Blotting
Protein was extracted from 1 ml of ultracentrifugated plasma (120,000g for 2h30) using DE buffer (20 mM Tris-HCL, 1 mM EDTA, 1 mM EGTA, 12 mM 2mercaptoethanol, 10% glycerol, 1% Triton-X 100) (5) with protease inhibitor mix (GE Healthcare, Uppsala, Sweden). Total protein was quantified by Bradford and 5 µg of protein per sample were separated by 12% SDS-PAGE. Proteins were transferred to nitrocellulose and blotted with the polyclonal antibody against Flotillin-1 (ABcam ab41927, Cambridge, UK). Coomassie blue staining of the membrane was used as a loading control for the protein extracts (6). The quantification of Flotillin-1 was performed by densitometry analysis using the ImageJ software. Each band was reduced from the gel background.

Exosomes' size distribution profiles and concentration measurements
Circulating exosomes from six patients with samples collected at enrollment and seven days later were quantified and sized. Exosomes were isolated from 100µL of plasma as described above. Three videos of 60 seconds were recorded and analyzed using NanoSight LM10 and NTA software (NanoSight Ltd, Amesbury, UK).

Flow Cytometry Analysis
The pellet concentrated with exosomes from eight septic patients at each time point and eight healthy controls isolated as described was thawed in room temperature and 10 µL of each sample was used in the staining protocol. We used 1-

Analysis of microRNA and inflammatory gene expression
Thirty nanograms of total RNA was used to reverse transcribed the miRNAs using specific stem-loop primers. The preamplification reaction products were analyzed by qPCR using the TaqMan

Analysis of gene expression associated with Oxidative Stress
The PCR array platform used in these experiments comprises 84 genes associated with Oxidative Stress and Antioxidant Defense (PAHS065-A, SABiosciences/Qiagen, Valencia, USA). Twenty-one patients and ten healthy donors were evaluated. Following the manufacturer's instructions, 100ng of RNA were used for the reverse transcription followed by pre-amplification of cDNA using specific primers. In order to avoid variation, PCR plates were prepared in high precision robotic pipetting (QI Agility, Qiagen). Quantitative real time PCR was done in the 7300HT thermocycler (Life Technologies). The data were processed considering the same baseline (3-15) and threshold (0.2) for each gene using the SDS 1.3 software.

qPCR Data Analysis
All Cycle Threshold (Ct) values were analyzed in Statminer v5 (Integromics, Granada, Spain). According to the manufacturer's instructions, Ct values greater than or equal to 32 were excluded for miRNAs and immune studies. For oxidative stress studies, the cutoff value was established at 35 according to the manufacturer's instructions. For the miRNA analysis, the geNorm method was used to identify the best reference controls and the median of miR17, miR20a and miR106a was used for the data normalization. The expression levels of immune and stress oxidative genes were normalized to 18S rRNA (7) and beta-actin, respectively. We calculated the relative gene expression data by the 2^DDCt method (8).