Mitochondrial Function in Peripheral Blood Mononuclear Cells (PBMC) Is Enhanced, Together with Increased Reactive Oxygen Species, in Severe Asthmatic Patients in Exacerbation

Asthma is a chronic inflammatory lung syndrome with an increasing prevalence and a rare but significant risk of death. Its pathophysiology is complex, and therefore we investigated at the systemic level a potential implication of oxidative stress and of peripheral blood mononuclear cells’ (PBMC) mitochondrial function. Twenty severe asthmatic patients with severe exacerbation (GINA 4–5) and 20 healthy volunteers participated at the study. Mitochondrial respiratory chain complexes activities using different substrates and reactive oxygen species (ROS) production were determined in both groups by high-resolution respirometry and electronic paramagnetic resonance, respectively. Healthy PBMC were also incubated with a pool of plasma of severe asthmatics or healthy controls. Mitochondrial respiratory chain complexes activity (+52.45%, p = 0.015 for VADP) and ROS production (+34.3%, p = 0.02) were increased in asthmatic patients. Increased ROS did not originate mainly from mitochondria. Plasma of severe asthmatics significantly increased healthy PBMC mitochondrial dioxygen consumption (+56.8%, p = 0.031). In conclusion, such asthma endotype, characterized by increased PMBCs mitochondrial oxidative capacity and ROS production likely related to a plasma constituent, may reflect activation of the immune system. Further studies are needed to determine whether increased PBMC mitochondrial respiration might have protective effects, opening thus new therapeutic approaches

Circulating eosinophils in asthma differ in their gene expression profile when compared to healthy subjects.

<p>Heat map of hierarchical clustering of the top expressed genes of circulating eosinophils from subjects with asthma (<i>n</i> = 4) <i>vs</i> healthy controls (<i>n</i> = 3). The horizontal dendrogram represents the relationship between asthmatic and healthy subjects. The vertical dendrogram represents the relationship between the expression levels of each gene across all the samples. Over-expressed genes are shown in red and under-expressed genes are depicted in green.</p

Gene ontology categories with an enrichment score p<0.01 and > 10% of gene concordance identified using the DAVID database.

<p>Gene ontology categories with an enrichment score p<0.01 and > 10% of gene concordance identified using the DAVID database.</p

RT-qPCR of selected genes.

<p>Gene expression was determined by RT-qPCR in eosinophils isolated from subjects with asthma and healthy controls. Results were normalized to <i>β-actin</i> and expressed as fold change compared with samples from healthy controls. Results are presented as means and SEM. *p<0.05; **p<0.01 (2-tailed non parametric Mann-Whitney).</p

Eosinophils from asthmatic subjects display a similar transcriptional profile as eosinophils from other hypereosinophilic conditions.

<p>Scatterplots are based on fold changes of selected genes that are differentially expressed at least in one hypereosinophilic disease (p< 0.01). Diagonal: estimation of the density of log fold changes (lfc) for each variable. Upper elements: estimation of the bivariate density of each couple of variables. In red: genes with a lfc changed to the same direction in all the conditions. In black: genes with a lfc changed to the opposite direction in at least one couple of variables. With a cut-off for the lfc of 0.15, more than 95% of the probe sets have identical changes. Ast, asthma; Asp, pulmonary aspergillosis; Der, dermatological disease; Par, parasitosis.</p

Subject characteristics.

<p>Subject characteristics.</p

Eosinophil isolation by FACS generates high quality RNA.

<p><b>(A)</b> Flow cytometry gating strategy for the identification of eosinophils. Eosinophils were identified among a granulocyte suspension (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141740#sec002" target="_blank">Methods</a>) as a CD16 fluorescence negative cell population <b>(B)</b> Purity of sorted eosinophils as assessed by cytospin preparation was close to 100%. <b>(C)</b> Bioanalyzer RNA profile with RNA integrity number (RIN) of an eosinophil sample. All RNA samples included in the expression analysis had a RIN > 8.</p