Cellular and Kaposi's sarcoma-associated herpes virus microRNAs in sepsis and surgical trauma

Abstract

Once a patient is in septic shock, survival rates drop by 7.6% for every hour of delay in antibiotic therapy. Biomarkers based on the molecular mechanism of sepsis are important for timely diagnosis and triage. Here, we study the potential roles of a panel of cellular and viral miRNAs as sepsis biomarkers. We performed genome-wide microRNA (miRNA) expression profiling in leukocytes from septic patients and nonseptic controls, combined with quantitative RT-PCR in plasmas from two cohorts of septic patients, two cohorts of nonseptic surgical patients and healthy volunteers. Enzyme-linked immunosorbent assay, miRNA transfection and chromatin immunoprecipitation were used to study the effects of Kaposi sarcoma herpes virus (KSHV) miRNAs on interleukin's secretion. Differences related to sepsis etiology were noted for plasma levels of 10 cellular and 2 KSHV miRNAs (miR-K-10b and miR-K-12-12*) between septic and nonseptic patients. All the sepsis groups had high KSHV miRNAs levels compared with controls; Afro-American patients had higher levels of KSHV-miR-K12-12* than non-Afro-American patients. Both KSHV miRNAs were increased on postoperative day 1, but returned to baseline on day 7; they acted as direct agonists of Toll-like receptor 8 (TLR8), which might explain the increased secretion of the IL-6 and IL-10. Cellular and KSHV miRNAs are differentially expressed in sepsis and early postsurgical patients and may be exploited for diagnostic and therapeutic purposes. Increased miR-K-10b and miR-K12-12* are functionally involved in sepsis as agonists of TLR8, forming a positive feedback that may lead to cytokine dysregulation.

Figure 1

Schematic representation of plasma samples collection from septic, surgical and healthy patients. Work flow representing the plasma sample collection from patients in the three different clinical conditions. For the nonsepsis surgical patients we obtained both plasma and MNC cells

Figure 2

Different cellular miRNAs are significantly deregulated in plasma of sepsis patients compared with normal samples. (a,b) qRT-PCR showing the comparison of miRNAs between sepsis patients and healthy volunteers in FCH cohort (a) and in MDA patients (b) at day 1 after septic shock. (c) miR-486 expression levels in nonsurgical sepsis patients of the FCH subgroup with pulmonary infection (n=9) versus expression levels of patients from MDA with pulmonary infection (n=12). (d) Comparison of miRNAs expression levels between nonsurvivor sepsis patients at FCH and at MDA (***P<0.0001, *P<0.05)

Figure 3

Viral miRNAs are upregulated in plasma of patients after septic shock. (a) qRT-PCR showing the expression levels of KSHV-miR-K12-10b and KSHV-miR-K12-12* in the patients, divided for sepsis type (**P<001, *P<05). (b) qRT-PCR showing the expression levels of KSHV-miR-K12-10b before surgery and on day 1 and day 7 after surgery in the non-paired set of patients. (c) qRT-PCR showing the expression levels of KSHV-miR-K12-10b before surgery and on day 1 and day 7 after surgery in the paired cohort of patients (A=preoperative samples, B=day 1 post surgery samples, C=day 7 post surgery samples)

Figure 4

KSHV miRNAs are direct agonists of TLR8 and they increase cytokine production when co-transfected in a histiocytic lymphoma cell line. (a) Upper panel: Levels of miRNAs expressed as fold change relative to baseline control determined by quantitative RT-PCR to detect the miR-21 (red), KSHV miR-K12-10b (dark green), KSHV miR-K12-12* (blue). miR-155 (purple) and miR-let-7b (light green) in following samples as grouped from left to right: empty vector transfected HEK293 cells treated with scrambled miRNAs (EV SCR), FLAG-TLR8 HEK293 cells treated with the related miRNA. The experiment was done in duplicates. (b) Immunoprecipitation analysis of the experiment shown in a. Immunoblotting for actin serves as control for cell content and specificity of immunoprecipitation. The cell samples are as indicated by the plasmid vectors used for transfection and the miRNAs they were incubated with. (c) Schema representing plasma viral miRNAs acting as direct agonists on TLR8 receptor and leading to increased secretion of interleukins (IL-6, IL-10 and IL-1b). (d) KSHV-miR-K12-10b and KSHV-miR-K12-12* increases the secretion of IL-6 and IL-10. Co-transfection of both viral miRNAs induces secretion of IL-6 and, IL-10 at levels comparable to LPS stimulation