Altered microRNA profiles in bronchoalveolar lavage fluid exosomes in asthmatic patients

Abstract

Background: Asthma is characterized by increased airway narrowing in response to nonspecific stimuli. The disorder is influenced by both environmental and genetic factors. Exosomes are nanosized vesicles of endosomal origin released from inflammatory and epithelial cells that have been implicated in asthma. In this study we characterized the microRNA (miRNA) content of exosomes in healthy control subjects and patients with mild intermittent asthma both at unprovoked baseline and in response to environmental challenge.

Objective: To investigate alterations in bronchoalveolar lavage fluid (BALF) exosomal miRNA profiles due to asthma, and following subway air exposure.

Methods: Exosomes were isolated from BALF from healthy control subjects (n = 10) and patients with mild intermittent asthma (n = 10) after subway and control exposures. Exosomal RNA was analyzed by using microarrays containing probes for 894 human miRNAs, and selected findings were validated with quantitative RT-PCR. Results were analyzed by using multivariate modeling.

Results: The presence of miRNAs was confirmed in exosomes from BALF of both asthmatic patients and healthy control subjects. Significant differences in BALF exosomal miRNA was detected for 24 miRNAs with a subset of 16 miRNAs, including members of the let-7 and miRNA-200 families, providing robust classification of patients with mild nonsymptomatic asthma from healthy subjects with 72% cross-validated predictive power (Q(2) = 0.72). In contrast, subway exposure did not cause any significant alterations in miRNA profiles.

Conclusion: These studies demonstrate substantial differences in exosomal miRNA profiles between healthy subjects and patients with unprovoked, mild, stable asthma. These changes might be important in the inflammatory response leading to bronchial hyperresponsiveness and asthma.

FIG 1

Flow cytometric analysis of BALF exosomes displayed expression of MHCII and CD63 surface markers, whereas MHC class I, CD54, and CD86 were not detected. Results are shown as the MFI for the detected molecule divided by the MFI for the isotype control. Data represent means + SEMs (n = 10 in each group). AC, Asthmatic patients after control exposure; AS, asthmatic patients after subway exposure; HC, healthy control subjects after control exposure; HS, healthy control subjects after subway exposure.

FIG 2

OPLS analysis of the asthmatic versus healthy groups at baseline resulted in a robust separation between groups (scores plot, left panel). The optimized model of 16 miRNAs generated a highly significant classification model (P = 1.4 × 10⁻⁵, CV-ANOVA), with a predictive power of 73% (R² = 0.77, Q² = 0.73). The loadings for the predictive component of the 16 miRNAs are shown in the middle panel. Correlation analysis with PLS showed a strong correlation between the expression pattern of the 24 miRNAs significantly altered between the 2 groups (q < 0.05) and FEV₁ in asthmatic subjects (PLS inner relation, R² = 0.74; right panel). AC, Asthmatic patients after control exposure; HC, healthy control subjects after control exposure.

FIG 3

OPLS analysis of the asthmatic versus healthy groups after subway exposure yielded a significant (P = .049, CV-ANOVA) separation, although with some overlap between the 2 groups (scores plot, left panel). The optimal model consisting of 11 miRNA variables (loadings, right panel) resulted in 35% predictive power (R² = 0.40, Q² = 0.35). AS, Asthmatic patients after subway exposure; HS, healthy control subjects after subway exposure.

FIG 4

Comparison of the 2 OPLS models for classification of the asthmatic and healthy groups after control exposure (x-axis) and subway exposure (y-axis) through an SUS plot revealed high correlation between the models (R² = 0.98). Clustering of the variables along the diagonal indicates that the miRNA alterations driving the separation between the 2 groups were the same at baseline and after subway exposure.

FIG 5

OPLS analysis based on maximal allergy response using the 16 biomarker miRNAs identified in Fig 2 showed a strong separation according to the expected groups, with 2 outliers from the asthmatic group clustering with the healthy control subjects. These 2 patients had no (subject 4; allergy response = 0) or very mild (subject 34; allergy response = 2) atopic responses, in contrast to the other asthmatic subjects, who all had an allergy response of greater than 4 to at least 1 of the allergens tested. The apparent relation between the alterations in miRNA profile detected at baseline levels to allergic asthma were verified by the high correlation between the allergen response model and the asthma diagnosis model in the SUS plot (R² = 0.98, lower panel). Notably, these subjects had stable, asymptomatic, mild intermittent asthma, with no allergen challenge for at least 3 weeks.