Mycobacterium tuberculosis/Mycobacterium bovis triggered different variations in lipid composition of Bovine Alveolar Macrophages

Abstract

The lipid composition performs important functions in interaction between macropha-ge and Mycobacterium tuberculosis (MTB)/Mycobacterium bovis (MB). Current understanding regarding the lipid responses of bovine alveolar macrophage (BAM) to MTB/MB is quite limited. The present study conducted lipidomics and transcriptome to assess alterations in BAM lipid compositions upon MB and MTB infection. We found that both MTB and MB induced glycerophospholipids accumulation in BAM, and MTB induced more alterations in lipid composition. MTB could affect the contents of various lipids, especially ceramide phosphocholines, polystyrene (PS) (17:0/0:0), testolic acid and testosterone acetate. Meanwhile, MB particularly induced accumulation of 1-alkyl,2-acylglycerophosphoinositols. Both MB and MTB suppressed the contents of palmitoleamide, N-ethyl arachidonoyl amine, N-(1,1-dimethyl-2-hydroxy-ethyl) arachidonoyll amine, eicosanoyl-EA, and PS (O-18:0/17:0) in BAM. Additionally, transcriptome analysis revealed that only MTB triggered genes involved in immune signaling and lipid related pathways in BAM. And MTB mainly activated genes CXCL2 and CXCL3 relevant to NOD-like receptor, IL-17 and TNF to further induce lipid accumulation in BAM, which in turn promoted the formation of foam cells. Meanwhile, time course RT-qPCR results showed that MTB was recognized by BAM to triggered dramatic immune responses, whereas MB could effectively escape the recognition system of BAM, leading rearrangement of lipid metabolisms in BAM at early infection stage. Altogether, the results of the present study provided evidence for changes in lipid metabolism of MTB/MB attacked BAM and contributed to the detection and treatment of zoonotic tuberculosis.

Figure 1

Overview of the alteration of lipid metabolisms of BAM following MTB and MB attacks. (A–C) Volcano plots of content alterations of lipids in each pairwise comparison (A: MB. vs.CK; B: MTB. vs. CK; C: MB. vs. MTB). The gray points are lipids without significant alteration. The upregulated and downregulated lipids (|Log1.2(Foldchange)|> 1.0; P < 0.05) are labeled by red and green colors, respectively. (D) Principal component analysis represents the degree of separation of all samples and the stability between reproductions of each sample groups. Each circle represents a sample, the circles labeled by the same color indicates the samples with the same treatment.

Figure 2

Specific pattern of lipids contents in BAM was induced by MTB- and MB-infection. (A) Up-set diagrams representing the overlap of identified differentially expressed lipids in each pairwise comparison. (B) Pattern of content variation of differential lipids. The highly increased lipids were shown in red, and the decreased lipids were labeled by green. (C) VIP scores of lipids among three experiment treatments. (D) Heatmap displaying the level of top 15 lipids (based on VIP score) in each treatment. The scale showed the normalized mean peak area of lipids in each group, and the origin value were shown in cell.

Figure 3

Pathway enrichment analysis on MTB- and MB-induced lipids. (A, B) Relative abundance of lipids associated with the MTB or MB attacks. Green and red colors indicate metabolites with relative low and high abundance, respectively (A: MTB-induced lipids; B: MB-induced lipids). (A) Pathways associated with MB and MTB infection. Blue represents MB-induced pathways, while red represents MTB-induced ones (P < 0.05).

Figure 4

Classification of MTB- and MB-induced lipids. (A, B) The pie chart represents the main lipid super-classes associated with MTB- (B) and MB-infection (A). The area represents the percentage of each class. (C, D) Lipid main class enrichment analysis under MTB (B) and MB (A) attacks, respectively. The size of circle represents the lipid number in the enriched categories (P < 0.05).

Figure 5

Differentially expressed genes and functional enrichment analysis of BAM infected by MTB and MB. (A, C) Volcanic map of gene expression in BAM after MB (A) and MTB (B) infections (Log1.5FC < − 1.0 or > 1.0; q-value < 0.05). (B, D) All differentially expressed genes infected by MB (B) and MTB (D) were divided into three GO categories (i.e., cell composition, molecular function, and biological process). Transcriptomic datasets were collected from published articles, as shown in Table 1.

Figure 6

Co-expression network and pathway enrichment analysis of key genes involved in the BAM lipid metabolism. (A) Up-set diagrams representing the overlap of identified differentially expressed genes in both MTB- and MB-infected groups. (B) Enrichment results of differentially expressed genes associated with MTB-infected BAM. The color shows the significance of pathways. The number of genes involved in the pathway is represented by the dot size. (C) Protein–protein interaction network between genes involved in lipid metabolism and signaling pathways. Each circle represented an individual protein. (D) Pathway enrichment result of genes associated with MB infection. Transcriptomic datasets were collected from published articles, as shown in Table 1.

Figure 7

Estimation of the genes relevant to immunity, pathogen recognition and lipid metabolisms in BAM following MTB or MB challenges at time course. (A) Heatmap displays the expression levels of genes relevant to immunity, pathogen recognition and lipid metabolisms in MTB-infected BAM at time course. (B) The expression levels of representative genes in MB-infected BAM at time course. Scale represents the Z-score of normalized Foldchange value of each genes compared to CK (0 h infection). The up- and down-regulated genes are shown in red and blue, respectively. Two-tailed t tests are used for all statistical analysis, P < 0.05.