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

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. We analysed 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 found. We observed the best correlation of the DNA methylomes when both isolation methods captured cells from the lower parts 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. 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.Funding Agencies|Forskningsradet Sydostra Sverige [FORSS-932096]; Swedish Research CouncilSwedish Research CouncilEuropean Commission [2015-02593, 2018-02961]; Swedish Heart Lung FoundationSwedish Heart-Lung Foundation [20150709, 20180613]; Medical Infection and Inflammation Center (MIIC) at Linkoping University; Forskningsradet i Sydostra Sverige [FORSS-932096]; Hjart-Lungfonden [20150709, 20180613]; VetenskapsradetSwedish Research Council [2015-02593, 2018-02961]</p

The spectrum of tuberculosis described as differential DNA methylation patterns in alveolar macrophages and alveolar T cells

Background: Host innate immune cells have been identified as key players in the early eradication of Mycobacterium tuberculosis and in the maintenance of an anti-mycobacterial immune memory, which we and others have shown are induced through epigenetic reprogramming. Studies on human tuberculosis immunity are dominated by those using peripheral blood as surrogate markers for immunity. We aimed to investigate DNA methylation patterns in immune cells of the lung compartment by obtaining induced sputum from M. tuberculosis- exposed subjects including symptom-free subjects testing positively and negatively for latent tuberculosis as well as patients diagnosed with active tuberculosis. Alveolar macrophages and alveolar T cells were isolated from the collected sputum and DNA methylome analyses performed (Illumina Infinium Human Methylation 450 k).Results: Multidimensional scaling analysis revealed that DNA methylomes of cells from the tuberculosis-exposed subjects and controls appeared as separate clusters. The numerous genes that were differentially methylated between the groups were functionally connected and overlapped with previous findings of trained immunity and tuberculosis. In addition, analysis of the interferon-gamma release assay (IGRA) status of the subjects demonstrated that the IGRA status was reflected in the DNA methylome by a unique signature.Conclusions: This pilot study suggests that M. tuberculosis induces epigenetic reprogramming in immune cells of the lung compartment, reflected as a specific DNA methylation pattern. The DNA methylation signature emerging from the comparison of IGRA-negative and IGRA-positive subjects revealed a spectrum of signature strength with the TB patients grouping together at one end of the spectrum, both in alveolar macrophages and T cells. DNA methylation-based biosignatures could be considered for further development towards a clinically useful tool for determining tuberculosis infection status and the level of tuberculosis exposure.Funding Agencies|Linkoeping University; Forskningsradet Sydoestra Sverige; Swedish Research Council [FORSS-932096]; Swedish Heart Lung Foundation [2015-02593, 2018-02961, 2018-04246, 106-2018-FONDECYT]; CONCYTEC-PROCIENCIA [2018-05973, 20150709]; Board of Research at the Karolinska Institute, Stockholm [20180613]; World Infection Fund</p

A differential DNA methylome signature of pulmonary immune cells from individuals converting to latent tuberculosis infection

Tuberculosis (TB), caused by Mycobacterium tuberculosis, spreads via aerosols and the first encounter with the immune system is with the pulmonary-resident immune cells. The role of epigenetic regulations in the immune cells is emerging and we have previously shown that macrophages capacity to kill M. tuberculosis is reflected in the DNA methylome. The aim of this study was to investigate epigenetic modifications in alveolar macrophages and T cells in a cohort of medical students with an increased risk of TB exposure, longitudinally. DNA methylome analysis revealed that a unique DNA methylation profile was present in healthy subjects who later developed latent TB during the study. The profile was reflected in a different overall DNA methylation distribution as well as a distinct set of differentially methylated genes (DMGs). The DMGs were over-represented in pathways related to metabolic reprogramming of macrophages and T cell migration and IFN-gamma production, pathways previously reported important in TB control. In conclusion, we identified a unique DNA methylation signature in individuals, with no peripheral immune response to M. tuberculosis antigen who later developed latent TB. Together the study suggests that the DNA methylation status of pulmonary immune cells can reveal who will develop latent TB infection.Funding Agencies|Swedish Research CouncilSwedish Research CouncilEuropean Commission [2018-04246, 2018-05973]; Consejo Nacional de Ciencia, Tecnologia e Innovacion Tecnologica CONCYTEC; Medical Infection and Inflammation Center (MIIC) at Linkoping University; Cienciactiva [106-2018-FONDECYT]</p

The effect of BCG vaccination on alveolar macrophages obtained from induced sputum from healthy volunteers

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