Comparative transcriptomic analysis of mouse macrophages infected with live attenuated vaccine strains of Mycobacterium tuberculosis

Abstract

The BCG vaccine has been used against tuberculosis (TB) for over a hundred years; however, it does not protect adults from pulmonary TB. To develop alternative vaccines against TB, we generated Mycobacterium tuberculosis H37Rv (Mtb)-derived vaccine strains by rationally deleting key virulent genes, resulting in single (SKO; ΔfbpA), double (DKO; ΔfbpA-ΔsapM), triple (TKO-D; ΔfbpA-ΔsapM-ΔdosR and TKO-Z; ΔfbpA-ΔsapM-Δzmp1), and quadruple (QKO; ΔfbpA-ΔsapM-Δzmp1-dosR) strains. To understand how macrophages, the host cells that defend against infection and process antigens for presentation to immune cells, respond to these vaccine strains, we performed transcriptomic analyses of mouse bone marrow-derived macrophages (BMDMs) infected with these strains. The transcriptomic data were compared with similar data obtained from macrophages infected with Mtb H37Rv and BCG. Our analyses revealed that genes associated with various immune and cell signaling pathways, such as NF-kappa B signaling, TNF signaling, cytokine-cytokine receptor interaction, chemokine signaling, hematopoietic cell lineage, Toll-like receptor signaling, IL-17 signaling, Th1 and Th2 cell differentiation, Th17 cell differentiation, and T cell receptor signaling were differentially expressed in BMDMs infected with our vaccine strains. Enhanced expression of cytokines and chemokines, including proinflammatory cytokines such as TNF-α, IL-6, GM-CSF, and IL-1, which are essential for the immune response against Mtb infection, was also observed in BMDMs infected with these strains. In particular, BMDMs infected with all vaccine strains exhibited a significant upregulation of genes associated with the IL-17 pathway. These results may indicate that our vaccine strains could induce a protective immune response against TB.

Keywords: BCG; IL-17; Mtb-vaccines; RNA-sequencing; immune signaling; macrophages; mouse; transcripts.

Figure 1

Schematics showing RNA-Seq workflow and data analysis. Fresh BMDMs were isolated from the female wild-type C57BL/6J mice and infected with respective vaccine strains or left uninfected. Following a 4-hour phagocytosis period, the BMDMs were washed with D-PBS and cultured in fresh DMEM supplemented with 10% fetal bovine serum for an additional 24 and 72 hours. RNA was isolated and subjected to eukaryotic mRNA enrichment at each time point. Subsequently, cDNA libraries were prepared, followed by adapter ligation and amplification for Illumina sequencing. The RNA-Seq data were aligned to the mouse transcriptome, and differential gene expression (DEG) analysis was performed. The figure was generated using BioRender.

Figure 2

Transcriptome profiling of mouse BMDMs infected with vaccine strains in comparison to control. (A, D), Heatmap of the differentially expressed genes at 24 h and 72 h; (B, E), Venn diagram of the differentially expressed genes showing the number of overlapping and unique genes among groups at 24 h and 72 h; (C, F), Volcano plot showing distribution of p values and log₂ fold change of differentially expressed genes as green (downregulated), red (upregulated) and black (Not significant) at 24 h and 72 h with respective bar graphs in percentage.

Figure 3

Transcriptome profiling of mouse BMDMs infected with vaccine strains in comparison to Mtb H37Rv. (A, D), Heatmap of the differentially expressed genes at 24 h and 72 h; (B, E), Venn diagram of the differentially expressed genes showing the number of overlapping and unique genes among groups at 24 h and 72 h; (C, F), Volcano plot showing differentially expressed genes as green (downregulated), red (upregulated) and black (Not significant) at 24 h and 72 h with respective bar graphs in percentage.

Figure 4

KEGG pathway analysis in upregulated differentially expressed transcripts of mouse BMDMs infected with vaccine strains versus H37Rv at 24 h post-infection. Dot plots illustrate the top 30 enriched pathways in upregulated DEGs in BCG, SKO, DKO, TKOD, TKOZ, and QKO compared to H37Rv control. Dot plots measure fold enrichment, where the dot size reflects the total number of genes in each pathway, and the gradient color indicates statistical significance expressed as −log₁₀ (P).

Figure 5

KEGG pathway analysis in upregulated differentially expressed transcripts of mouse BMDMs infected with vaccine strains versus H37Rv at 72 h post-infection. Dot plots illustrate the top 30 enriched pathways in upregulated DEGs in BCG, SKO, DKO, TKOD, TKOZ, and QKO compared to H37Rv control. Dot plots measure fold enrichment, where the dot size reflects the total number of genes in each pathway enriched, and the gradient color indicates statistical significance expressed as −log₁₀ (P).

Figure 6

KEGG pathway analysis in downregulated differentially expressed transcripts of mouse BMDMs infected with vaccine strains versus H37Rv at 24 h post-infection. Dot plots illustrate the top 30 enriched pathways in downregulated DEGs in BCG, SKO, DKO, TKOD, TKOZ, and QKO compared to H37Rv control. Dot plots measure fold enrichment, where the dot size reflects the total number of genes in each category, and the gradient color indicates statistical significance expressed as −log₁₀ (P).

Figure 7

KEGG pathway analysis in downregulated differentially expressed transcripts of mouse BMDMs infected with vaccine strains versus H37Rv at 72 h post-infection. Dot plots illustrate the top 30 enriched pathways in downregulated DEGs in BCG, SKO, DKO, TKOD, TKOZ, and QKO compared to H37Rv control. Dot plots measure fold enrichment, where the dot size reflects the total number of genes in each category, and the gradient color indicates statistical significance expressed as −log₁₀ (P).

Figure 8

Gene ontology analysis for biological processes in the differentially upregulated transcripts of mouse BMDMs infected with vaccine strains versus H37Rv at 24 h post-infection. Dot plots illustrate the top 30 enriched biological processes in upregulated DEGs in BCG, SKO, DKO, TKOD, TKOZ, and QKO compared to H37Rv control. Dot plots measure fold enrichment, where dot size reflects the total number of genes in each biological process, and the gradient color indicates statistical significance expressed as −log₁₀ (P).

Figure 9

Gene ontology analysis for biological processes in the differentially upregulated transcripts of mouse BMDMs infected with vaccine strains versus H37Rv at 72 h post-infection. Dot plots illustrate the top 30 enriched biological processes in upregulated DEGs in BCG, SKO, DKO, TKOD, TKOZ, and QKO compared to H37Rv control. Dot plots measure fold enrichment, where the dot size reflects the total number of genes in each biological process, and the gradient color indicates statistical significance expressed as −log₁₀ (P).

Figure 10

Gene ontology analysis for biological processes in the differentially downregulated transcripts of mouse BMDMs infected with vaccine strains versus H37Rv at 24 h post-infection. Dot plots illustrate the top 30 enriched biological processes of downregulated DEGs in BCG, SKO, DKO, TKOD, TKOZ, and QKO compared to H37Rv control. Dot plots measure fold enrichment, where the dot size reflects the total number of genes in each biological process, and the gradient color indicates statistical significance expressed as −log₁₀ (P).

Figure 11

Gene ontology analysis for biological processes in the differentially downregulated transcripts of mouse BMDMs infected with vaccine strains versus H37Rv at 72 h post-infection. Dot plots illustrate the top 30 enriched biological processes in downregulated DEGs in BCG, SKO, DKO, TKOD, TKOZ, and QKO compared to H37Rv control. Dot plots measure fold enrichment, where the dot size reflects the total number of genes in each biological process, and the gradient color indicates statistical significance expressed as −log₁₀ (P).

Figure 12

IL17 signaling pathway is differentially regulated in vaccine groups in comparison with H37Rv. (A, C) Heat map depicts the green–red gradient that reflects relative gene expression among vaccine groups at 24 h and 72 h. (B, D) Gene expression levels of Csf2, Csf3, ilß, ptgs2, and lcn2 in BMDMs infected with vaccine strains at 24 and 72 hours as determined by qRT-PCR. Data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test.

Figure 13

qPCR Validation of differentially regulated pathways in vaccine groups comparison with H37Rv. (A, B) Gene expression levels of Csf1, tnf, slc7a2, lta, ddit4, and dapk2 in vaccine strains infected BMDMs at 24 h and 72 h as determined by qRT-PCR. Data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test.