Transcriptional regulators SP110 and SP140 modulate inflammatory response genes in Mycobacterium tuberculosis-infected human macrophages

Abstract

Understanding the functions of human transcriptional regulatory genes SP110 and SP140 during Mycobacterium tuberculosis infection is crucial; in a mouse model, homologous genes Sp110 and Sp140 have been shown to negatively regulate inflammatory response genes, including the type I interferon (IFN) response. The reduction of these genes in mice is associated with susceptibility to M. tuberculosis infection and the development of necrotizing granulomatous lesions. To investigate the involvement of SP110 and SP140 in human inflammatory response, we analyzed their regulatory manner in THP-1 macrophages infected with M. tuberculosis. Genome-wide transcriptional profiling revealed that the depletion of SP110 and/or SP140 impaired the induction of gene expression associated with inflammatory responses, including IFN response genes, although it had little effect on the intracellular proliferation of M. tuberculosis. By contrast, genes related to phosphorylation were upregulated in infected macrophages with SP110 and/or SP140 knockdown, but downregulated in infected control macrophages without their knockdown. Reverse transcription-quantitative PCR and ELISA further confirmed the impairment of the induction of IFN response genes by the depletion of SP110 and/or SP140 in M. tuberculosis-infected macrophages. These findings suggest that human SP110 and SP140 act as positive regulators for genes associated with inflammatory responses in M. tuberculosis-infected macrophages.

Importance: Tuberculosis (TB) is one of the most serious infectious diseases, with high morbidity and mortality worldwide. C3HeB/FeJ mice are widely utilized for evaluating anti-TB drugs because their drug sensitivity and pathology during M. tuberculosis infection resemble those of human TB, including the development of necrotizing granulomas. Downregulation of the transcriptional regulatory genes Sp110 and Sp140 in C3HeB/FeJ mice has been demonstrated to activate gene expression associated with inflammatory responses during M. tuberculosis infection, resulting in susceptibility to the infection. Here, we examined the regulatory manner of SP110 and SP140 using transcriptomic analysis in M. tuberculosis-infected human macrophages. Depletion of SP110 and/or SP140 in M. tuberculosis-infected THP-1 macrophages impaired the induction of gene expression associated with inflammatory responses, including interferon response genes, compared with that in control macrophages. These results suggest that human SP110 and SP140 act as positive regulators for genes associated with inflammatory responses upon M. tuberculosis infection.

Keywords: Mycobacterium tuberculosis; RNA sequencing; SP110; SP140; interferon response; macrophage; oxidative phosphorylation; transcriptional factor.

Fig 1

Knockdown of SP110 and/or SP140 by siRNA treatment in THP-1 macrophages. (A) Design of siRNA targeting SP110 and SP140. Exon sequences targeted by siRNA molecules are highlighted. (B) Knockdown efficacy of SP110 and/or SP140 genes. PMA-stimulated THP-1 cells were treated with siRNA targeting SP110 and/or SP140. The expression levels of SP110 and SP140 in transfected cells were investigated by RT-qPCR (n = 4). Two molecules were used for siRNA targeting SP110 (SP110-1 and SP110-2). Four molecules were mixed and used for siRNA targeting SP140 (SP140). SP110-1 and SP140 were mixed and used for knockdown targeting SP110 and SP140 (SP110−1 + SP140). The values of –dCT are shown as gene expression (gene exp.). ***P < 0.001, as determined by ANOVA with Dunnett’s test.

Fig 2

Intracellular proliferation of M. tuberculosis and viability of infected macrophages treated with siRNA targeting SP110 and/or SP140. PMA-stimulated THP-1 cells were treated with siRNA targeting SP110 and/or SP140, followed by infection with M. tuberculosis. (A) Intracellular proliferation of M. tuberculosis within macrophages. M. tuberculosis-infected macrophages treated with siRNA targeting the indicated genes were harvested at 24 and 48 h post-infection (p.i.). The values of bacterial numbers were determined by CFU assays. Mean and SD values of bacterial numbers are also presented from four independent experiments (n = 4). (B) Viability of macrophages. The viability of M. tuberculosis-infected macrophages treated with siRNA targeting the indicated genes was measured using a LIVE/DEAD fixable stain. Mean and SD values of the proportion of live cells are presented (n = 3). *: P < 0.05, as determined by ANOVA with Tukey–Kramer multiple comparison test. (C) Growth activity of macrophages. The growth activity of uninfected and M. tuberculosis-infected macrophages treated with siRNA targeting the indicated genes was measured by MTT assay. Mean and SD values of the optical absorbance are presented (n = 6).

Fig 3

Genome-wide analysis of the gene expression profiles of M. tuberculosis-infected macrophages derived from C3HeB/FeJ and CH/HeN mice. (A) Gene expression of Sp110 and Sp140 in BMMs. Count per million (CPM) of Sp110 and Sp140 genes in BMMs from C3HeB/FeJ (FeJ) and C3H/HeN (HeN) mice are indicated. (B) Comparison of gene expression in M. tuberculosis-infected BMMs between C3HeB/FeJ and C3H/HeN mice. BMMs from FeJ and HeN mice were infected with M. tuberculosis for 24 h (n = 2). RNA was extracted and subjected to messenger RNA sequencing (mRNA-seq). Gene set enrichment analysis (GSEA) was conducted on M. tuberculosis-infected BMMs from C3HeB/FeJ (FeJ) and C3H/HeN (HeN) mice. Significantly enriched hallmarks are depicted, representing both activated and suppressed hallmarks in M. tuberculosis-infected BMMs from C3HeB/FeJ mice compared with those from C3H/HeN mice. The bar size indicates the normalized enrichment score (NES) for activated and repressed hallmarks in FeJ mice. The color scale indicates the adjusted P value. NES, normalized enrichment score. P. adjust, adjusted P values. (C) Heatmap visualization of the expression of IFN response genes. The major genes were obtained from the enriched GSEA hallmark gene sets of IFN-alpha and IFN-gamma responses. The top 20 genes were selected using adjusted P values, followed by hierarchical clustering analysis. Annotation bars indicate infection with M. tuberculosis and BMMs from FeJ or HeN mice.

Fig 4

Genome-wide analysis of the gene expression profiles of M. tuberculosis-infected macrophages treated with siRNA targeting SP110 and/or SP140. (A) GOBP enrichment analysis for macrophages. THP-1 macrophages treated with siRNA against SP110 and/or SP140 were infected with M. tuberculosis for 24 h (n = 5). RNA was extracted and subjected to mRNA-seq. The list includes upregulated (G1, G3, G5, and G7) and downregulated (G2, G4, G6, and G8) DEGs of M. tuberculosis-infected macrophages (Mtb) compared with those in uninfected macrophages (UN) treated with siRNA targeting control (G1 and G2), SP110 (G3 and G4), SP140 (G5 and G6), or SP110 and SP140 (G7 and G8). G1; 654 genes, G2; 113 genes, G3; 761 genes, G4; 544 genes, G5; 213 genes, G6; 16 genes, G7; 2342 genes, and G8; 2092 genes. The significant GOBP terms for upregulated and downregulated DEGs in treated macrophages are shown. GeneRatio, the gene ratio of the indicated GO term in DEG. P. adjust, adjusted P values. (B) GSEA of M. tuberculosis-infected macrophages. Significantly enriched hallmarks are shown, representing both activated and suppressed hallmarks in M. tuberculosis-infected macrophages compared with those in uninfected macrophages. The bar size indicates the normalized enrichment score (NES) for activated and suppressed hallmarks in infected macrophages. Target genes for siRNA treatment are also indicated. The color scale indicates the adjusted P value. NES, normalized enrichment score. p. adjust, adjusted P values.

Fig 5

Comparison of gene expression among M. tuberculosis-infected macrophages treated with siRNA targeting SP110 and/or SP140. (A) GSEA was conducted on M. tuberculosis-infected macrophages treated with siRNA targeting control and indicated genes. Significantly enriched hallmarks are depicted, representing both activated and suppressed hallmarks in M. tuberculosis-infected macrophages treated with siRNA targeting SP110 and/or SP140 compared with those in uninfected macrophages treated with siRNA. (B) GSEA of M. tuberculosis-infected macrophages treated with siRNA for SP140 compared with those treated with siRNA for SP110. Significantly enriched hallmarks are depicted, representing both activated and suppressed hallmarks in M. tuberculosis-infected macrophages treated with siRNA for SP140 compared with those in M. tuberculosis-infected macrophages treated with siRNA for SP110. The bar size indicates the NES for activated and repressed hallmarks in SP110- and/or SP140-knockdown macrophages. The color scale indicates the adjusted P value. NES, normalized enrichment score. P. adjust, adjusted P values.

Fig 6

Heatmap visualization and validation of expression in selected IFN response genes. (A) Heatmap visualization of the expression of IFN response genes. The major genes were obtained from the enriched GSEA hallmark gene sets of IFN-alpha and IFN-gamma responses. The top 20 genes were selected using adjusted P values, followed by hierarchical clustering analysis. Annotation bars indicate infection with M. tuberculosis and siRNA treatment conditions. (B, C) Gene expression related to IFN response pathways was investigated by RT-qPCR. THP-1 macrophages treated with siRNA against SP110 and/or SP140 were infected with M. tuberculosis for 24 h (n = 6). RNA was extracted and subjected to RT-qPCR. (B) SP110 and SP140 expression, (C) IFN response genes. The values of −dCT are depicted as gene expression (gene exp.). *P < 0.05, **P < 0.01, ***P < 0.001, as determined by ANOVA with Dunnett’s test.

Fig 7

Reduction of CXCL10 secretion by knockdown of SP110 and/or SP140. ELISA for secreted CXCL10 in M. tuberculosis-infected THP-1 macrophages with SP110 and/or SP140 knockdown. THP-1 macrophages treated with siRNA against SP110 and/or SP140 were infected with M. tuberculosis for 24 h (n = 6). Culture media were collected and subjected to ELISA for CXCL10. **P < 0.01, ***P < 0.001, as determined by ANOVA with Dunnett’s test.