Mycobacterium tuberculosis Infection Induces HDAC1-Mediated Suppression of IL-12B Gene Expression in Macrophages

Abstract

Downregulation of host gene expression is one of the many strategies employed by intracellular pathogens such as Mycobacterium tuberculosis (MTB) to survive inside the macrophages and cause disease. The underlying molecular mechanism behind the downregulation of host defense gene expression is largely unknown. In this study we explored the role of histone deacetylation in macrophages in response to infection by virulent MTB H37Rv in manipulating host gene expression. We show a significant increase in the levels of HDAC1 with a concomitant and marked reduction in the levels of histone H3-acetylation in macrophages containing live, but not killed, virulent MTB. Additionally, we show that HDAC1 is recruited to the promoter of IL-12B in macrophages infected with live, virulent MTB, and the subsequent hypoacetylation of histone H3 suppresses the expression of this gene which plays a key role in initiating Th1 responses. By inhibiting immunologically relevant kinases, and by knockdown of crucial transcriptional regulators, we demonstrate that protein kinase-A (PKA), CREB, and c-Jun play an important role in regulating HDAC1 level in live MTB-infected macrophages. By chromatin immunoprecipitation (ChIP) analysis, we prove that HDAC1 expression is positively regulated by the recruitment of c-Jun to its promoter. Knockdown of HDAC1 in macrophages significantly reduced the survival of intracellular MTB. These observations indicate a novel HDAC1-mediated epigenetic modification induced by live, virulent MTB to subvert the immune system to survive and replicate in the host.

Keywords: CREB; THP-1 macrophages; c-jun; epigenetic modifications; host-pathogen; interleukin-12.

Figure 1

Status of expression of HDAC1 and HDAC2 in macrophages upon MTB infection. (A) HDAC1 and (B) HDAC2 expression at 12 h; (C) HDAC1 and (D) HDAC2 expression at 24 h. Each result represents the mean ± SD of data from three experiments. ^**p < 0.05.

Figure 2

Differential expression of HDAC1 upon MTB infection. (A) Immuno-cytochemical imaging shows expression of HDAC1 in variously infected macrophages at 12 h; (B) 24 h post infection (PI); (C) at 12 h PI, levels of HDAC1 are low in MTB-infected macrophage cells; (D) at 24 h PI, levels of HDAC1 are high in MTB-infected macrophages; (E) levels of HDAC1 expression depend on the bacterial load inside the macrophages at 24 h PI; (F–H) densitometric analysis of HDAC1 bands normalized with that of histone H3. Quantitative data of HDAC1 protein is expressed as the mean of ratio of densitometric values of HDAC1 to histone H3 bands ± SD for three independent experiments. **p < 0.05.

Figure 3

Kinetics of HDAC1 in macrophages upon MTB infection. (A) No significant change in the levels of HDAC1 in uninfected macrophages at 0, 6, 12, 18, 24, and 48 h PI, (B) HDAC1 level increases and peaks at 24 h in the case of macrophages infected with live, virulent MTB, (C) HDAC1 level drops gradually in macrophages containing heat-killed MTB. (D–F) Densitometric analysis of HDAC1 bands normalized with histone H3. Quantitative data of HDAC1 protein is expressed as the mean of ratio of densitometries of HDAC1 to histone H3 bands ± SD for three independent experiments. Levels of HDAC1 protein increased significantly (^**P < 0.05 vs. 0 h) only in the case of macrophages infected with live, virulent MTB.

Figure 4

Histone H3 acetylation status in macrophages upon MTB infection. (A) Increase in histone H3-acetylation status in MTB-infected macrophages at 12 h PI is visualized by confocal microscopy; (B) decrease in histone H3-acetylation in MTB-infected macrophages at 24 h PI; (C,D) different levels of acetylated histone H3 at 12 h and 24 h PI by western blot. (E,F) Densitometric analysis of H3Ac bands normalized with histone H3. Quantitative data of HDAC1 protein is expressed as the mean of ratio of densitometries of HDAC1 to histone H3 bands ± SD for three independent experiments. ^**p < 0.05.

Figure 5

HDAC1 downregulates the IL-12B expression in macrophages 24 h post-MTB infection. (A) Status of HDAC1 and H3-Ac on the IL12B promoter by ChIP; (B) qPCR of IL-12B; (C) ELISA of IL-12p40. (D) IL-12 expression is upregulated in MTB-infected macrophages upon HDAC inhibition; (E) IL-12 expression is upregulated in MTB-infected macrophages when HDAC1 is knocked down. One of three independent experiments is shown for (A); for (B–E) data are the mean ± SD of three independent experiments. ^**p < 0.05.

Figure 6

HDAC1 and IL-12p40 status in PBMC-derived macrophages upon MTB infection. (A) HDAC1 level increases in macrophages upon infection with live virulent MTB (B) Levels of IL-12p40 are significantly low in macrophages infected with live virulent MTB, compared to those cells treated with killed bacteria. One of three independent experiments is shown in case of (A); for (B) data are the mean ± SD of three independent experiments. The numbers indicated below the blots are the normalized densitometry values of the bands calculated by imageJ software. ^**p < 0.05.

Figure 7

HDAC1 levels in macrophages upon MTB infection in the presence of kinase inhibitors, and after knock-down of transcriptional regulators. (A) HDAC1 levels are significantly low when PKA is inhibited, (B) HDAC1 levels are significantly low when CREB and C-jun are knocked down and a marginal difference is observed in the case of macrophages in which SOX5 is knocked down. (C,D) Densitometric analysis of HDAC1 bands normalized with that of histone H3. Quantitative data of HDAC1 protein is expressed as the mean of ratio of densitometric values of HDAC1 to histone H3 bands ± SD for three independent experiments. ^**p < 0.05.

Figure 8

Recruitment of c-Jun on HDAC1 promoter in macrophages during MTB infection. (A) Sequence analysis of HDAC1 promoter region (−269 to +101 retrieved from Eukaryotic Promoter Database) revealed the presence of two putative consensus binding sequence (dissimilarity < 15%) for transcription factor c-Jun. c-Jun binding sites, PCR- amplified region, and TSS are indicated; (B) ChIP analysis for the recruitment of c-JUN on HDAC1 promoter. Data from one of three independent experiments are shown.

Figure 9

Survival of MTB decreases when HDAC1 is knocked down in macrophages. THP-1 monocytes (1 × 10⁶ cells) seeded in a 6-well plate were treated with scrambled siRNA and siHDAC1, and infected with MTB. Macrophages not treated with siRNA but infected with MTB were also kept as control. The number of bacteria recovered from the macrophages 4 h after infection was considered as the number of bacteria that gained entry into the macrophages (and the number at 0 h for studying the viability inside macrophages); and subsequent isolation of bacilli from the macrophages was carried out at 24, 48, and 72 h. The number of viable bacilli in each of the wells was assayed by plating on a 7H10 agar plates and incubating the plates at 37°C, and counting the colony forming unit (cfu). Data shown here are the mean ± SD of three independent experiments.

Figure 10

Proposed model for the upregulation of HDAC1 in macrophages infected with MTB. PKA phosphorylates CREB leading to the transcriptional activation of c-Jun gene. c-Jun is recruited onto the HDAC1 promoter of macrophages infected with live, virulent MTB. HDAC1 is recruited to the promoters of proinflamatory genes resulting in transcriptional repression.