AmpliSeq transcriptome analysis of human alveolar and monocyte-derived macrophages over time in response to Mycobacterium tuberculosis infection

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.

Human Alveolar and Splenic Macrophage Populations Display a Distinct Transcriptomic Response to Infection With Mycobacterium tuberculosis

“…Additionally, the transcriptional signature observed in AMTBs in response to Mtb infection was associated with myeloid cell inflammation and TREM1 signaling as a topscoring canonical pathway across all macrophage populations evaluated in this study (AMCTs, AMTBs, and SMs) ( Figure 6A). Our findings are in line with recently published reports (15), indicating TREM1 as a top inflammatory signaling pathway playing a critical role in controlling inflammatory processes.…”

“…Although the results of previous studies have provided a framework for understanding the consequences of macrophage-Mtb interaction, relatively little data are available regarding human AMs collected from healthy individuals (14,15), while no data have been obtained from AMs from TB patients. So far, most of our knowledge from the interaction of Mtb with MPS cells has been garnered by the use of monocyte-derived macrophages (MDMs) from healthy individuals (14,(16)(17)(18).…”

Alveolar macrophages up-regulate a non-classical innate response toMycobacterium tuberculosisinfectionin vivo

“…Our results suggest that established in vitro models do not adequately replicate the initial host response to Mtb. In addition, it is worth noting that our observations that murine AMs become more pro-inflammatory over time in culture (data not shown) are in concordance with reports that the response of human AMs to Mtb infection evolves from a profile unique to AMs towards one that is more similar to that of monocytes the longer they are cultured in vitro ( 44 ).…”

Alveolar Macrophage Chromatin Is Modified to Orchestrate Host Response to Mycobacterium bovis Infection

“…Considering the dramatic perturbation of the macrophage by intracellular mycobacteria, we and others have demonstrated that bovine and human alveolar macrophage transcriptomes are extensively reprogrammed in response to infection with M. bovis and M. tuberculosis Vegh et al, 2015;Lavalett et al, 2017;Jensen et al, 2018;Malone et al, 2018;Papp et al, 2018). These studies have also revealed that differentially expressed gene sets and dysregulated cellular networks and pathways are functionally associated with many of the macrophage processes described above that can control or eliminate intracellular microbes.…”