Abstract
Human alveolar macrophages (HAM) are primary bacterial niche and immune response cells during Mycobacterium tuberculosis (M.tb) infection, and human blood monocyte-derived macrophages (MDM) are a model for investigating M.tb-macrophage interactions. Here, we use a targeted RNA-Seq method to measure transcriptome-wide changes in RNA expression patterns of freshly obtained HAM (used within 6 h) and 6 day cultured MDM upon M.tb infection over time (2, 24 and 72 h), in both uninfected and infected cells from three donors each. The Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (AmpliSeq) uses primers targeting 18,574 mRNAs and 2,228 non-coding RNAs (ncRNAs) for a total of 20,802 transcripts. AmpliSeqTM yields highly precise and reproducible gene expression profiles (R2 >0.99). Taking advantage of AmpliSeq’s reproducibility, we establish well-defined quantitative RNA expression patterns of HAM versus MDM, including significant M.tb-inducible genes, in networks and pathways that differ in part between MDM and HAM. A similar number of expressed genes are detected at all time-points between uninfected MDM and HAM, in common pathways including inflammatory and immune functions, but canonical pathway differences also exist. In particular, at 2 h, multiple genes relevant to the immune response are preferentially expressed in either uninfected HAM or MDM, while the HAM RNA profiles approximate MDM profiles over time in culture, highlighting the unique RNA expression profile of freshly obtained HAM. MDM demonstrate a greater transcriptional response than HAM upon M.tb infection, with 2 to >10 times more genes up- or down-regulated. The results identify key genes involved in cellular responses to M.tb in two different human macrophage types. Follow-up bioinformatics analysis indicates that approximately 30% of response genes have expression quantitative trait loci (eQTLs in GTEx), common DNA variants that can influence host gene expression susceptibility or resistance to M.tb, illustrated with the TREM1 gene cluster and IL-10.
Highlights
Susceptibility to tuberculosis (TB) varies substantially between individuals
We further compared the AmpliSeq data obtained here from monocytederived macrophages (MDM) and Human alveolar macrophages (HAM) with RNA-Seq results from an equal number of blood samples downloaded from Genotype-Tissue Expression (GTEx) [44] (Fig 1, panel B)
While only a small portion of significant differentially expressed (DE) genes overlaps between MDM and HAM, we find a larger portion of highly expressed genes (>2,000 reads per million (RPM), !1.5-fold change) overlap between MDM and HAM (Table 1), while substantial differences remain
Summary
Susceptibility to tuberculosis (TB) varies substantially between individuals While both genetic and environmental factors play essential roles in an individual’s susceptibility to M.tb infection or active TB [1,2,3,4,5,6,7,8,9,10], candidate gene and genome-wide association studies (GWAS) indicate the presence of single nucleotide polymorphisms (SNPs) in host genes that affect susceptibility to TB [1,2,3,4,5,6,7,8, 11]. We use next-generation RNA sequencing targeting RNA regions with specific primers (AmpliSeq Whole Transcriptome Human Gene Expression, 20,802 transcripts) This approach is reported to yield highly reproducible transcriptomes [30]. We apply AmpliSeq for the first time to measure transcriptomes in M.tbinfected MDM and HAM
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