Circulating extracellular vesicles as potential biomarkers and mediators of acute respiratory distress syndrome in sepsis

Abstract

The early sequence of respiratory failure events after the onset of sepsis is still unknown. We hypothesize that the lung should signal through circulating extracellular vesicles (EVs) when it is affected by a systemic inflammatory response. Blood samples were obtained from septic patients with (n = 5) and without acute respiratory distress syndrome (ARDS) (n = 13) at 24 h of intensive care unit admission and 3 days later at Sírio-Libanês Hospital. Pulmonary-originated sepsis was not considered. The characteristics of the plasma-isolated EVs were compatible with exosomes. 48 miRNAs were evaluated by real-time PCR. Comparing all samples from patients with sepsis + ARDS to sepsis only, 9 miRNAs are transported in smaller amounts: miR-766 (-35.7, p = 0.002), miR-127 (-23.8, p = 0.001), miR-340 (-13.5, p = 0.006), miR-29b (-12.8, p = 0.001), miR-744 (-7.1, p = 0.05), miR-618 (-4.0, p = 0.02), miR-598 (-3.8, p = 0.035), miR-1260 (-2.5, p = 0.035); and miR-885-5p is expressed at higher levels (9.5; p = 0.028). In paired samples, the set of altered miRNAs is generally different (p < 0.05) between sepsis + ARDS (miR-1183,-1267,-1290,-17,-192,-199a-3p,-25,-485-3p,-518d,-720) or sepsis only (miR-148a,-193a-5p,-199a-3p,-222,-25,-340,744). Bioinformatic analysis showed that when sepsis is associated with ARDS, those differentially expressed miRNAs potentially target messenger RNAs from the Glycoprotein VI/GP6 signaling pathway. Circulating EV-miRNA cargo could be potential biomarkers of lung inflammation during sepsis in patients requiring mechanical ventilation.

Keywords: ARDS; Exosomes; Extracellular microvesicles; MiRNA; Sepsis.

Fig. 1

Concentration of circulating microvesicles in plasma. Patients diagnosed with sepsis or sepsis associated with acute respiratory distress syndrome (ARDS) had blood samples collected in the first 24 h and 72 h after admission to the intensive care unit as controls were considered patients under mechanical ventilation (Control MV) or healthy volunteers. Patients with sepsis of pulmonary origin or with some previous lung diseases were not considered. The microvesicles were isolated by ultracentrifugation and quantified by the NanoSight equipment. The statistical results are indicated as Fig. 1A p = 0.1349, one-way ANOVA; Fig. 1B p > 0.05, two-way ANOVA; Fig. 1C p = 0.6132, Paired T-Test; Fig. 1D p = 0.0175, Paired T-Test.

Fig. 2

Size distribution profile of microvesicles in sepsis or sepsis associated with respiratory insufficiency. The graph represents the concentration of plasma microvesicles according to size. The exosomes were isolated from plasma by ultracentrifugation and evaluated using Nanosight equipment.

Fig. 3

miRNAs altered in sepsis when associated with acute respiratory distress syndrome. Blood samples were collected in the first 24 h and 72 h of ICU admission, and the microvesicles were isolated by ultracentrifugation. The graph shows a volcano plot of the 48 miRNAs evaluated by real-time PCR, showing the expression of each target in patients with sepsis and ARDS (n = 11 samples) compared to patients with only sepsis (n = 25 samples). Nine miRNAs are transported in smaller quantities: miR-766 (−35.7; p = 0.002), miR-127 (−23.8; p = 0.001), miR-340 (−13.5; p = 0.006), miR-29b (−12.8; p = 0.001), miR-744 (−7.1; p = 0.05), miR-618 (−4.0; p = 0.02), miR-598 (−3.8; p = 0.035), miR-1260 (−2.5; p = 0.035). On the other hand, the miR-885-5p is at elevated levels (9.5; p = 0,028). T Test (Cloud software, ThermoFisher). Normalized data with RNU6.

Fig. 4

Molecular profile of 48 miRNAs transported by Exosomes and microvesicles during hospitalization in an intensive care unit. Patients diagnosed with sepsis (chart A, n = 11) or sepsis associated with the discomfort syndrome (graph B, n = 5) were evaluated for the molecular content of their microvesicles circulating by qPCR. The black bars refer to the first 24 h of hospitalization in the ICU, while the grey bars represent 72 h after. The raw Ct data were normalized with snRNU6 (DDCt) expression. Statistical differences are indicated. * P < 0.05, paired Wilcoxon test.

Fig. 5

A. Biological pathways potentially influenced by miRNAs transported by circulating exosomes in patients with sepsis associated with ARDS (n = 11 samples; n = 5 patients) compared to patients with sepsis (n = 25 samples; n = 13 patients). The samples were obtained at the admission to the Intensive care unit (24 h) and 3 days after treatment. B. Possible RNA messengers being regulated by the EVs-miRNAs. miRNAs transported by circulating exosomes in significantly increased amounts (green color) or decreased (red color) and their possible effects on platelets and/or megakaryocytes in patients during hospitalization in a therapy unit when sepsis is associated with respiratory dysfunctions compared to patients with sepsis without lung dysfunction.