Comparative transcriptomic analysis of THP-1-derived macrophages infected with Mycobacterium tuberculosis H37Rv, H37Ra and BCG

Abstract

Tuberculosis (TB) remains a worldwide healthcare concern, and the exploration of the host-pathogen interaction is essential to develop therapeutic modalities and strategies to control Mycobacterium tuberculosis (M.tb). In this study, RNA sequencing (transcriptome sequencing) was employed to investigate the global transcriptome changes in the macrophages during the different strains of M.tb infection. THP-1 cells derived from macrophages were exposed to the virulent M.tb strain H37Rv (Rv) or the avirulent M.tb strain H37Ra (Ra), and the M.tb BCG vaccine strain was used as a control. The cDNA libraries were prepared from M.tb-infected macrophages and then sequenced. To assess the transcriptional differences between the expressed genes, the bioinformatics analysis was performed using a standard pipeline of quality control, reference mapping, differential expression analysis, protein-protein interaction (PPI) networks, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Q-PCR and Western blot assays were also performed to validate the data. Our findings indicated that, when compared to BCG or M.tb H37Ra infection, the transcriptome analysis identified 66 differentially expressed genes in the M.tb H37Rv-infected macrophages, out of which 36 genes were up-regulated, and 30 genes were down-regulated. The up-regulated genes were associated with immune response regulation, chemokine secretion, and leucocyte chemotaxis. In contrast, the down-regulated genes were associated with amino acid biosynthetic and energy metabolism, connective tissue development and extracellular matrix organization. The Q-PCR and Western blot assays confirmed increased expression of pro-inflammatory factors, altered energy metabolic processes, enhanced activation of pro-inflammatory signalling pathways and increased pyroptosis in H37Rv-infected macrophage. Overall, our RNA sequencing-based transcriptome study successfully identified a comprehensive, in-depth gene expression/regulation profile in M.tb-infected macrophages. The results demonstrated that virulent M.tb strain H37Rv infection triggers a more severe inflammatory immune response associated with increased tissue damage, which helps in understanding the host-pathogen interaction dynamics and pathogenesis features in different strains of M.tb infection.

Keywords: Mycobacterium tuberculosis; infection; macrophage; regulation; transcriptome.

FIGURE 1

Enrichment analysis of THP‐1‐infected H37Rv strain DEGs in high‐throughput RNA sequencing. A, The heat map showing the relative transcript levels of the differential genes in THP‐1 cells uninfected and infected with H37Rv M.tb strains shows the expression pattern changes after infection. B, Volcano plot showing considerably up‐regulated (red dots) and down‐regulated genes (blue dots). C, NC vs H37Rv GO biological process enrichment analysis of significant DEGs. D, NC vs H37Rv KEGG pathway enrichment analysis of significant DEGs

FIGURE 2

DEGs PPI networks. A, PPI network showing the up‐regulated DEGs from infected H37Rv strain group compared with the control group. B, PPI network showing the down‐regulated DEGs from infected H37Rv strains group compared with the control group. The colour representation Kmeans clustering (network is clustered into a specified number of clusters)

FIGURE 3

Enrichment analysis of THP‐1‐infected H37Ra strain DEGs in high‐throughput RNA sequencing. A, The heat map showing the relative transcript levels of the differential genes in THP‐1 cells uninfected and infected with H37Ra M.tb strains shows that the expression pattern changes after infection. B, Volcano plot showing considerably up‐regulated (red dots) and down‐regulated genes (blue dots). C, NC vs H37Ra GO biological process enrichment analysis of significant DEGs. D, NC vs H37Ra KEGG pathway enrichment analysis of significant DEGs

FIGURE 4

DEGs PPI networks. A, PPI network showing the up‐regulated DEGs from infected H37Ra strain group compared with the control group. B, PPI network showing the down‐regulated DEGs from the infected H37Ra strain group compared with the control group. The colour representation Kmeans clustering (network is clustered into a specified number of clusters)

FIGURE 5

Enrichment analysis of THP‐1‐infected BCG strain DEGs in high‐throughput RNA sequencing. A, The heat map showing the relative transcript levels of the differential genes in THP‐1 cells uninfected and infected with BCG Mycobacterium tuberculosis strains displays the expression pattern changes after infection. B, Volcano plot showing considerably up‐regulated (red dots) and down‐regulated genes (blue dots). C, NC vs BCG GO biological process enrichment analysis of significant DEGs. D, NC vs BCG KEGG pathway enrichment analysis of significant DEGs

FIGURE 6

DEGs PPI networks. A, PPI network showing the up‐regulated DEGs from infected BCG strain group compared with the control group. B, PPI network showing the down‐regulated DEGs from infected BCG strain group compared with the control group. The colour representation K means clustering (network is clustered into a specified number of clusters)

FIGURE 7

The heat map, Venn diagram, GO and KEGG classification of the DEGs screening. A, Shows the number of DEGs in three different datasets and the crossing area indicates the cross‐DEGs in different datasets. Including NC vs H37Rv (orange), NC vs H37Ra (green) and NC vs BCG (red). B, The heat map shows the relative transcript levels of the 133 differential genes in THP‐1 cells infected with Mycobacterium tuberculosis H37Rv, HA36Ra and BCG strains. C, Common 133 differential genes GO biological process enrichment analysis of significant DEGs. D, Common 133 differential genes KEGG pathway enrichment analysis of significant DEGs

FIGURE 8

The heat map GO and KEGG of the DEGs screening. A, The heat map showing the relative transcript levels of the 66 differential genes in THP‐1 cells infected with Mycobacterium tuberculosis H37RV strains. B, NC vs H37Rv GO biological process enrichment analysis of 66 significant DEGs. C, NC vs H37Rv KEGG pathway enrichment analysis of 66 significant DEGs

FIGURE 9

Comparative analysis of pro‐inflammatory factors and immune regulatory gene expression in different strains of MTB‐infected THP‐1 cells by Q‐PCR. H37RV, H37Ra and BCG‐infected THP‐1cell mRNA was extracted, and the expression abundance of IL‐1β, TNF‐α, IL‐10, CCL2, CCL3, CCL4, CD14, CD36, TLR4, CXCL8, CSF2 and IRF9 was determined by RT‐qPCR. The comparative analysis of transcriptional expression levels of these cytokines, chemokines and immune regulatory factors was assessed and shown in the graph. Data are expressed as the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

FIGURE 10

Detection of the signalling transduction pathway activation in different strains of MTB‐infected THP‐1 cells by Western blot. A, The protein expression of p‐P65, p‐STAT1, p‐ERK, p‐JNK, Caspase‐1, Cleaved‐Caspase‐1, Caspase‐3, Cleaved‐Caspase‐3 and GAPDH in different strains of MTB‐infected THP‐1 cells was assessed by Western blot. B, The graph shows the comparison of the relative intensity of the protein expression in the Western blot; the relative intensity of each band was calculated after normalization with the intensity of GAPDH in a blot, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001