biostudies-arrayexpressLeon MeiHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15473The treatment of Mycobacterium avium (Mav) infection, responsible for over 80% of non-tuberculous mycobacterial pulmonary disease, remains challenging due to rising antibiotic resistance and unsatisfactory success rates. Hence, there is a need for a deeper understanding of host–pathogen interactions to inform the development of alternative therapeutic approaches, like host-directed therapy (HDT), aimed at improving host antimycobacterial defenses.. However, compared to Mycobacterium tuberculosis (Mtb) infections, knowledge of host-pathogen interactions for Mav infection is still limited. To address this knowledge gap, we performed a genome-wide host transcriptomic analysis of Mav-infected primary human macrophages —the primary host cell—alongside Mtb-infected macrophages to leverage insights from Mtb research. Our findings show substantial overlap in the gene expression patterns between Mav-infected and Mtb-infected macrophages, including induction of cytokine responses and modulation of various G-protein coupled receptors (GPCRs) involved in (lipid-mediated) macrophage immune functions. Notable differences were observed in nerve growth factor (NGF) signaling and genes of the GTPase of immunity-associated protein (GIMAP) family. This study laid a foundation for identifying both shared and Mav‑specific host response pathways, providing direction for future investigations into host-pathogen interactions during Mav infection and the identification of novel targets for HDT.biostudies-arrayexpressLibrary Construction - Library preparation was performed by GenomeScan (Leiden, the Netherlands) using the NEBNext Ultra Directional RNA Library prep kit (New England Biolabs, Ipswich, MA, USA) for Illumina according to the manufacturers protocolSequencing - Gene expressions were profiled using the NovaSeq 6000 platform (Illumina, San Diego, CA, USA) by GenomeScan (Leiden, the Netherlands).Nucleic Acid Extraction - Total RNA was extracted with TRIzol reagent and the Direct-zol RNA miniPrep kit according to the manufacturer's protocol (Zymo Research, Leiden, the Netherlands).Sample Treatment - hMDM were infected with M. tuberculosis strain H37Rv or M. avium strain MAC101 at MOI 10, or left uninfected. After 1 h, the cells were treated with 30 μg/ml gentamicin-containing medium for 10 min to inactivate and remove residual extracellular bacteria, after which the medium was refreshed with medium containing 5 μg/mL gentamicin sulfate before cells were incubated at 37°C/5% CO2 until indicated timepoints.Growth Protocol - Isolated CD14+ monocytes from healthy human donors were cultured for 6 days in RPMI + 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 50 ng/ml macrophage colony-stimulating factor at 37°C, 5% CO2. After 6 days, the differentiated human monocyte-derived macrophages (hMDM) with M2 phenotype were harvested using trypsin-EDTA and reseeded in cell culture medium without antibiotics. The cells were cultured for 24 h at 37°C, 5% CO2.Sample Collection - Buffy coats were collected from healthy anonymous Dutch adult donors after written informed consent (Sanquin Blood Bank, Amsterdam, The the Netherlands). CD14+ monocytes were isolated from peripheral blood mononuclear cells using density gradient centrifugation with Ficoll (Pharmacy, LUMC, the Netherlands) and subsequently magnetic-activated cell sorting (MACS) with anti-CD14-coated microbeads (Miltenyi Biotec, Auburn, CA, USA). Total RNA was extracted with TRIzol reagent and the Direct-zol RNA miniPrep kit according to the manufacturer's protocol (Zymo Research, Leiden, the Netherlands).OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - RNA-Seq files were processed using the opensource BIOWDL RNAseq pipeline v5.0.0 (https://zenodo.org/record/5109461#.Ya2yLFPMJhE) developed at the Leiden University Medical Center (Leiden, the Netherlands). This pipeline performs FASTQ preprocessing (including quality control, quality trimming, and adapter clipping), RNA-Seq alignment, read quantification, and optionally transcript assembly. FastQC was used for checking raw read QC. Adapter clipping was performed using Cutadapt (v2.10) with default settings and standard illumina universal adapter “AGATCGGAAGAG”. RNA-Seq reads’ alignment was performed using STAR (v2.7.5a) on GRCh38 human reference genome. umi_tools (v1.1.1) was used to remove PCR duplicates detected with UMIs. The gene read quantification was performed using HTSeq-count (v0.12.4) with setting “–stranded=reverse”. The gene annotation used for quantification was Ensembl version 111. Using the gene read count matrix, CPM was calculated per sample on all annotated genes. Genes with a higher log2CPM than 1 in at least 25% of all samples are kept for downstream analysis.Sequence Alignment - RNA-Seq reads’ alignment was performed using STAR (v2.7.5a) on GRCh38 human reference genome. umi_tools (v1.1.1) was used to remove PCR duplicates detected with UMIs.UnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of total RNAHomo sapiensLeon MeifalseComparative transcriptomic analysis of human macrophages during Mycobacterium avium versus Mycobacterium tuberculosis infectionThe treatment of Mycobacterium avium (Mav) infection, responsible for over 80% of non-tuberculous mycobacterial pulmonary disease, remains challenging due to rising antibiotic resistance and unsatisfactory success rates. Hence, there is a need for a deeper understanding of host–pathogen interactions to inform the development of alternative therapeutic approaches, like host-directed therapy (HDT), aimed at improving host antimycobacterial defenses.. However, compared to Mycobacterium tuberculosis (Mtb) infections, knowledge of host-pathogen interactions for Mav infection is still limited. To address this knowledge gap, we performed a genome-wide host transcriptomic analysis of Mav-infected primary human macrophages —the primary host cell—alongside Mtb-infected macrophages to leverage insights from Mtb research. Our findings show substantial overlap in the gene expression patterns between Mav-infected and Mtb-infected macrophages, including induction of cytokine responses and modulation of various G-protein coupled receptors (GPCRs) involved in (lipid-mediated) macrophage immune functions. Notable differences were observed in nerve growth factor (NGF) signaling and genes of the GTPase of immunity-associated protein (GIMAP) family. This study laid a foundation for identifying both shared and Mav‑specific host response pathways, providing direction for future investigations into host-pathogen interactions during Mav infection and the identification of novel targets for HDT.2026-01-16T00:00:00Z2026-01-16T20:08:20.864Z2025-08-08T16:19:56.248ZE-MTAB-15473ERP178576EFO_0002944EFO_0004170EFO_0009653EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0004184EFO_0003969