Differential DNA methylation of potassium channel KCa3.1 and immune signalling pathways is associated with infant immune responses following BCG vaccination

www.nature.com/scientificreports OPEN Received: 14 February 2018 Accepted: 21 August 2018 Published: xx xx xxxx Differential DNA methylation of potassium channel KCa3.1 and immune signalling pathways is associated with infant immune responses following BCG vaccination Mateusz Hasso-Agopsowicz Steven G. Smith1 1 , Thomas J. Scriba2, Willem A. Hanekom2, Hazel M. Dockrell1 & Bacillus Calmette–Guérin (BCG) is the only licensed vaccine for tuberculosis (TB) and induces highly variable protection against pulmonary disease in different countries. We hypothesised that DNA methylation is one of the molecular mechanisms driving variability in BCG-induced immune responses. DNA methylation in peripheral blood mononuclear cells (PBMC) from BCG vaccinated infants was measured and comparisons made between low and high BCG-specific cytokine responders. We found 318 genes and 67 pathways with distinct patterns of DNA methylation, including immune pathways, e.g. for T cell activation, that are known to directly affect immune responses. We also highlight signalling pathways that could indirectly affect the BCG-induced immune response: potassium and calcium channel, muscarinic acetylcholine receptor, G Protein coupled receptor (GPCR), glutamate signalling and WNT pathways. This study suggests that in addition to immune pathways, cellular processes drive vaccine-induced immune responses. Our results highlight mechanisms that require consideration when designing new TB vaccines. Mycobacterium bovis Bacillus Calmette–Guérin (BCG) is the only licensed vaccine against tuberculosis (TB). BCG protects against severe childhood cases of miliary TB and tuberculous meningitis1. However, the protection afforded by the vaccine against pulmonary TB varies across geographical regions with studies reporting vaccine efficacy ranging from 0% in India2 to 80% in the United Kingdom (UK)3. Reasons for these variations remain unknown. Possible explanations include comorbidities4, pre-exposure to environmental mycobacteria5, and genetics6. Previous studies in Malawi7, The Gambia8, Indonesia9 and the UK8,10 have shown disparate immune responses across these populations, with a dominant production of IFNγ in the UK and greater production of T helper (Th)2 cytokines in Malawi and The Gambia10. Multiple efforts are being undertaken to identify correlates of protection following BCG vaccination11,12. The importance of IFNγ has been highlighted in numerous studies8,13–16, however, the Th1 boosting candidate TB vaccine MVA85A (Modified Vaccinia virus Ankara expressing Ag85A from M. tuberculosis) failed to improve on protection afforded by BCG in vaccinated infants17; a large South African study did not find that BCG specific Th1 response correlated with risk of TB disease18; however further analysis of the MVA85A clinical trial showed that the frequencies of cells producing BCG-specific IFNγ was associated with a reduced risk of developing disease16. 1 Department of Immunology and Infection, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, United Kingdom. 2South African Tuberculosis Vaccine Initiative and Institute of Infectious Disease and Molecular Medicine, Division of Immunology, Department of Pathology, University of Cape Town, Institute of Infectious Diseases and Molecular Medicine, Rondebosch, 7701, Cape Town, South Africa. Correspondence and requests for materials should be addressed to M.H.-A. (email: ) or S.G.S. (email: ) ScIenTIFIc Reports | (2018) 8:13086 | DOI:10.1038/s41598-018-31537-9 1 www.nature.com/scientificreports/ Another unknown factor in the immune response to the BCG vaccine is the molecular mechanism that drives these immune responses. Recent studies highlight the role of transcriptomics19–21 as means of identifying the key mechanisms and the importance of measuring RNA profiles in vaccine trials. Epigenetics, a mechanism known to play a role in regulation of gene expression, is another mechanism of growing importance. Specifically, DNA methylation of CpG dinucleotides in mammals is known to regulate gene expression and subsequent protein production22,23. In-vitro methylation of herpes thymidine kinase (tk) genes resulted in in-vivo downregulation of gene expression24 and methylation of the O6-methylguanine-DNA-methyltransferase (MGMT) gene was negatively correlated with its protein concentration in humans25. In the context of immune responses, two recent studies have examined the role of epigenetic regulation, gene expression and protein production in responses to Hepatitis B26 and Influenza27 vaccines. Several differentially methylated genes were found between low and high immune responders to Hep B vaccine and DNA methylation was correlated with gene expression and protein levels for multiple genes following influenza vaccination. Lastly, recent evidence shows that DNA methylation of macrophages is correlated with anti-mycobacterial activity in BCG vaccinated participants28. We have examined whether the DNA methylation profile of peripheral blood mononuclear cells (PBMC) in BCG vaccinated infants is associated with the magnitude of BCG specific immune responses. We found novel pathways and genes that were differentially methylated between high and low BCG responders amongst South African infants who received BCG vaccination at birth. This knowledge will allow us to understand molecular mechanisms that drive vaccine-induced immune responses, paving the way to design better and more effective TB vaccines. Results Sample collection and processing. We used 60 archived frozen PBMC samples from a previous study18. 36.7% were female, 83.3% Cape Mixed Ancestry, 13.3% Black African and 3.3% Asian. All participants received the Japanese BCG vaccine at birth and their blood was collected at 10 weeks post vaccination. An increase in cytokine production after BCG stimulation is not correlated with cell phenotype. To investigate the magnitude of immune responses to the BCG vaccine, we measured the cytokine production after stimulation with BCG (SSI strain, 1.2 × 106 organisms/ml) or Staphylococcus Enterotoxin B (SEB) using an intracellular cytokine staining (ICS) assay. We looked at cytokine responses in the PBMC population (see gating strategy in Fig. 1A). There was a consistent increase in the production of all cytokines after BCG stimulation and IFNγ, TNFα, IL2 but not IL8 and IL4/5/13 after SEB stimulation (Fig. 1B). We examined whether the observed magnitude of immune responses was due to changes in T cell frequency and conducted a Spearman correlation analysis of frequencies of IFNγ-expressing cells following BCG stimulation with other immune parameters. The frequencies of BCG-specific IFNγ cells (hereafter called IFNγ BCG) were correlated with IL4/5/13, IL2 and IL8 secreted following stimulation with BCG but not with any T cell population measured (Table S1). This indicates that in this assay, the BCG ind (...truncated)