biostudies-arrayexpressAdam EwingHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16151Tuberculosis (TB) remains a leading cause of illness and death globally. Type 2 diabetes (T2D) patients are more likely to become infected with Mycobacterium tuberculosis (M.tb). When they are infected, they are more likely to develop active TB disease and to present with more severe TB and those who receive TB treatment are more likely to experience adverse treatment outcomes. Genomic DNA (gDNA) was isolated from unstimulated whole blood and bronchoalveolar lavage (BAL) cells. Analysis of methylation arrays was carried out following the Bioconductor ‘methylationArrayAnalysis’ workflow, using the IlluminaHumanMethylationEPICanno.ilm10b4.hg19 annotation set for Illumina MethylationEPIC arrays. Probes were excluded where detection p-value was <0.01, where they were located on the Y chromosome, or were annotated as containing SNPs or known to be cross-reacting. Probes on the X chromosome were retained as only females were included in this analysis. Background correction was carried out via the normal-exponential out-of-band (Noob) method and probe intensities were quantile normalized. To identify differentially methylated positions (DMPs), the data was filtered using the criteria shown in extended data figure 8. To permit comparison between DMPs a secondary cutoff of uncorrected p<0.01 and beta values >0.8 and <0.2 was retained. Gene set enrichment testing was carried out via the “gsameth” function in the missMethyl package using hallmark and GO gene sets from the MSigDB Molecular Signatures Database. The Hallmark (H) gene sets represent well-defined biological states or processes, whereas the GO (C5) set consists of genes annotated by the same ontology term.biostudies-arrayexpressHybridization - Bisulfite converted DNA was amplified, enzymatically fragmented, precipitated and hybridized onto Illumina Infinium HumanMethylationEPIC BeadChips following the Infinium protocol.Sample Collection - Heparinized whole blood was centrifuged at 800xg for 12min to remove the plasma and to collect the buffy coats. Buffy coats were resuspended in PBS (Lonza) containing 1mM EDTA (Sigma14 Aldrich) and 1% human serum albumin (Sigma-Aldrich), layered onto Ficoll-Paque-Plus (GE Healthcare) and centrifuged at 600xg for 30min at RT. PBMCs were collected, and washed twice with RPMI-HEPES, first by centrifuging at 600xg for 4min at 4°C and then at 150xg for 8min at 4°C to remove excess platelets. BAL was performed using a fiberoptic bronchoscopy. Two hundred ml of saline solution was instilled in aliquots of 50ml each and aspirated to collect BAL from the lower airways and alveoli. Upon collection, 2ml of BAL was sent to the National Health Laboratory Service to perform cytospins and May-Grunwald Giemsa staining to determine differential cell counts. Approximately 90% [95% CI=80-95 (no-T2D) and 70-95 (T2D)] of cells were macrophages (supplementary figure 1a). Cytology was performed by microscopy using a Zeiss Axioskop 2 Plus Ergonomic Trinocular Microscope with Zeiss ZEN lite v2.0 software. The remaining BAL was centrifuged at 200xg for 10min at 4°C to pellet and wash cells with RPMI-HEPES (Sigma-Aldrich).Nucleic Acid Extraction - Genomic DNA (gDNA) was isolated, using the DNeasy Blood & Tissue kit (Qiagen), according to the manufacturer’s instructions. Red blood cells (RBCs) were lysed using the eBioscience RBC Lysis Buffer (ThermoFisher), and the leukocytes used as input material. The eluted gDNA was quantified using a Nanodrop 2000c spectrophotometer (ThermoFisher). 250-1000ng gDNA was bisulfite converted using the Zymo EZ DNA Methylation Kit (Zymo Research).Labeling - Bisulfite converted DNA was amplified, enzymatically fragmented, precipitated and hybridized onto Illumina Infinium HumanMethylationEPIC BeadChips following the Infinium protocol.Scaning - Illumina Infinium MethylationEPIC BeadChips were scanned using the Illumina iScan system and raw methylation data from 865,860 CpG sites exported as IDAT files.MIAME ScoreRaw DataOrganizationAssays and DataProcessed DataMAGE-TAB FilesArray DesignsData Transformation - Analysis of methylation arrays was carried out following the Bioconductor ‘methylationArrayAnalysis’ workflow, using the IlluminaHumanMethylationEPICanno.ilm10b4.hg19 annotation set for Illumina MethylationEPIC arrays. Background correction was carried out via the Noob method and probe intensities were quantile normalized.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsType 2 diabetes (T2D) increases susceptibility to tuberculosis (TB) with the underlying mechanisms remaining unknown. To determine whether immune dysfunction in the lung contributes to TB susceptibility, we obtained paired human alveolar macrophages (HAMs) and monocyte-derived macrophages (MDMs) from TB-exposed individuals with/without T2D. Upon infection with <i>Mycobacterium tuberculosis</i> (<i>M.tb),</i> T2D-HAMs had more <i>M.tb</i> growth and produced more TNF. There were fewer neutrophils in the bronchoalveolar lavage of T2D patients which was inversely correlated with <i>M.tb</i> growth. Both T2D-HAMs and MDMs expressed less CD32, with T2D patients having fewer M1-like MDMs. T2D-MDMs produced less IL-1RA and CSF2. Overall <i>M.tb</i>-induced gene expression was delayed in T2D-HAMs, but genes involved in negative regulation of neutrophil migration were upregulated. T2D-HAM DNA was hypermethylated compared to control HAMs, however genes linked to TNF signalling were hypomethylated. We show here the first in-depth analysis of T2D-HAMs providing an explanation for more severe TB in T2D patients.methylation profiling by arrayHomo sapiensHUMAN ALVEOLAR MACROPHAGE FUNCTION IS IMPAIRED IN TUBERCULOSIS CONTACTS WITH DIABETESAdam EwingLéanie Kleynhans, Carine Kunsevi-Kilola, Happy Tshivhula, Tariq Webber, Alana Keyser, Nicole Prins, Candice I Snyders, Ayanda Shabangu, Virginie Rozot, Martin Kidd, Hao Zhang, Hong Cai, Yufeng Wang, Adam D Ewing, Stephanus T Malherbe, Abul K Azad, Eusondia Arnett, Blanca I Restrepo, Larry S Schlesinger, Katharina RonacherfalseDNA Methylation of gDNA from blood and bronchoalveolar lavage of tuburbulosis patients with and without type 2 diabetes via Illumina Infinium MethylationEPIC arrayTuberculosis (TB) remains a leading cause of illness and death globally. Type 2 diabetes (T2D) patients are more likely to become infected with Mycobacterium tuberculosis (M.tb). When they are infected, they are more likely to develop active TB disease and to present with more severe TB and those who receive TB treatment are more likely to experience adverse treatment outcomes. Genomic DNA (gDNA) was isolated from unstimulated whole blood and bronchoalveolar lavage (BAL) cells. Analysis of methylation arrays was carried out following the Bioconductor ‘methylationArrayAnalysis’ workflow, using the IlluminaHumanMethylationEPICanno.ilm10b4.hg19 annotation set for Illumina MethylationEPIC arrays. Probes were excluded where detection p-value was <0.01, where they were located on the Y chromosome, or were annotated as containing SNPs or known to be cross-reacting. Probes on the X chromosome were retained as only females were included in this analysis. Background correction was carried out via the normal-exponential out-of-band (Noob) method and probe intensities were quantile normalized. To identify differentially methylated positions (DMPs), the data was filtered using the criteria shown in extended data figure 8. To permit comparison between DMPs a secondary cutoff of uncorrected p<0.01 and beta values >0.8 and <0.2 was retained. Gene set enrichment testing was carried out via the “gsameth” function in the missMethyl package using hallmark and GO gene sets from the MSigDB Molecular Signatures Database. The Hallmark (H) gene sets represent well-defined biological states or processes, whereas the GO (C5) set consists of genes annotated by the same ontology term.2025-12-14T00:00:00Z2026-05-27T13:24:41.003Z2025-11-17T11:24:41.844ZE-MTAB-1615139649174EFO_0002944EFO_0003814EFO_0003813EFO_0002759EFO_0005518EFO_0003816EFO_000381510.21203/rs.3.rs-5489046/v1