DNA methylome-based validation of induced sputum as an effective protocol to study lung immunity: construction of a classifier of pulmonary cell types

Abstract

Background: Flow cytometry is a classical approach used to define cell types in peripheral blood. While DNA methylation signatures have been extensively employed in recent years as an alternative to flow cytometry to define cell populations in peripheral blood, this approach has not been tested in lung-derived samples. Here, we compared bronchoalveolar lavage with a more cost-effective and less invasive technique based on sputum induction and developed a DNA methylome-based algorithm that can be used to deconvolute the cell types in such samples. Results: We analyzed the DNA methylome profiles of alveolar macrophages and lymphocytes cells isolated from the pulmonary compartment. The cells were isolated using two different methods, sputum induction and bronchoalveolar lavage. A strong positive correlation between the DNA methylome profiles of cells obtained with the two isolation methods was observed, and in two of the donors, in which the correlation was best, a later analyses demonstrated that those subjects the samples were consistently derived from the lower part of the lungs. We also identified unique patterns of CpG methylation in DNA obtained from the two cell populations, which can be used as a signature to discriminate between the alveolar macrophages and lymphocytes by means of open-source algorithms. We validated our findings with external data and obtained results consistent with the previous findings. Conclusions: Our analysis opens up a new possibility to identify different cell populations from lung samples and promotes sputum induction as a tool to study immune cell populations from the lung.

A DNA methylome biosignature in alveolar macrophages from TB-exposed individuals predicts exposure to mycobacteria

“…Patients with pulmonary TB, participants with occupational- or household-related TB-exposure and healthy controls, with an age ranging from 18 to 53 years, were enrolled at Linköping University Hospital and Linköping University, respectively. Included subjects (please see Table 1 for demographics) donated peripheral blood and induced sputum samples 12 following oral and written informed consent (ethical approval obtained from the regional ethical review board in Linköping, #2016/237-31). The study protocol included questionnaires on respiratory and overall health, the evaluation of IGRA-status and sputum samples for DNA extraction.…”

“…To enable recruitment of control subjects with very low likelihood of previous TB exposure, we performed the study in a low-endemic setting. We established a protocol for the isolation of distinct cell populations from induced sputum 12 , from which DNA could be isolated. Analyses of DNA methylomes of immune cells isolated from lungs and peripheral blood allowed us to identify distinct DNA methylation signatures in TB-exposed individuals.…”

DNA methylomes derived from alveolar macrophages display distinct patterns in latent tuberculosis - implication for interferon gamma release assay status determination

“…Sputum specimen were collected and processed as previously described 39 , with some modifications. The lung immune cells were obtained from each subject (here, salbutamol was avoided) after the nebulization of a hypertonic saline solution (4%) three times, provoking a profound cough via osmosis, producing pulmonal secretions.…”

“…have previously described the method for the isolation of HLA-DR positive alveolar macrophages 39,40 . The alveolar macrophages and T lymphocytes (CD3 positive cells) were isolated according to our protocol 39 using superparamagnetic beads coupled with anti-human CD3 and Pan Mouse IgG antibodies and HLA-DR/human MHC class II antibodies (Invitrogen Dynabeads). An initial positive selection was done with CD3 beads followed by a positive HLA-DR selection.…”

A Differential DNA Methylome Signature of Pulmonary Immune Cells from Individuals Converting to Latent Tuberculosis Infection

“…We hypothesized that this recent exposure to M. tuberculosis in the lung compartment would induce epigenetic alterations in AMs and alveolar T cells. By sputum induction AMs and alveolar T cells were isolated 23 . Using reduced representation of bisulfite sequencing (RRBS) the DNA methylome was investigated and we identified a different global DNA methylation profile in pulmonary immune cells of the study subjects that developed latent TB infection during the study.…”