Thymoquinone (TQ) inhibits the replication of intracellular Mycobacterium tuberculosis in macrophages and modulates nitric oxide production

Abstract

Background: Human tuberculosis, which is caused by the pathogen Mycobacterium tuberculosis, remains a major public health concern. Increasing drug resistance poses a threat of disease resurgence and continues to cause considerable mortality worldwide, which necessitates the development of new drugs with improved efficacy. Thymoquinone (TQ), an essential compound of Nigella sativa, was previously reported as an active anti-tuberculosis agent.

Methods: In this study, the effects of TQ on intracellular mycobacterial replication are examined in macrophages. In addition, its effect on mycobacteria-induced NO production and pro-inflammatory responses were investigated in Mycobacterium tuberculosis (MTB)-infected Type II human alveolar and human myeloid cell lines.

Results: TQ at concentrations ranging from 12.5 to 25 μg/mL and 6.25 to 12.5 μg/mL reduced intracellular M. tuberculosis H37Rv and extensively drug-resistant tuberculosis (XDR-TB) 72 h post-infection in RAW 264.7 cells. TQ treatment also produced a concentration-dependent reduction in nitric oxide production in both H37Rv and XDR-TB infected RAW 264.7 cells. Furthermore, TQ reduced the expression of inducible nitric oxide synthase (iNOS) and pro-inflammatory molecules such as tumor necrosis factor-alpha (TNF-α) and interlukin-6 (IL-6) in H37Rv-infected cells and eventually reduced pathogen-derived stress in host cells.

Conclusions: TQ inhibits intracellular H37Rv and XDR-TB replication and MTB-induced production of NO and pro-inflammatory molecules. Therefore, along with its anti-inflammatory effects, TQ represents a prospective treatment option to combat Mycobacterium tuberculosis infection.

Keywords: Intracellular killing; Mycobacterium tuberculosis; Nitric oxide; Thymoquinone; Xdr-Tb.

Fig. 1

In vitro efficacy data showing the intracellular killing effect of TQ in M. tuberculosis-infected macrophages. Raw 264.7 macrophages were infected with H37Rv and XDR-TB for 2 h at an MOI of 1:10 at 37 °C with 5% CO₂ followed by drug treatment for 3 days. a H37Rv burden (log₁₀ CFU) in H37Rv-infected macrophages after TQ and INH treatment. b XDR-TB burden (log₁₀ CFU) in H37Rv-infected cells after TQ and INH treatment. Data represent the mean ± SD of 3 independent experiments done in triplicate. *P < 0.05 by Student’s t-test. In acid-fast staining, methylene blue was used to stain macrophages, while red colored carbol fuchsin stains the bacteria. c-I Uninfected RAW 264.7 cells. c-II RAW 264.7 cells infected with H37Rv. d-I Uninfected RAW 264.7 cells. d-II RAW 264.7 cells infected with XDR-TB. Raw 264.7 macrophages were infected with GFP-H37Ra for 3 h at an MOI of 1:1 at 37 °C with 5% CO₂ followed by drug treatment for 5 days. e Comparative TQ efficacy in an in vitro model, along with INH

Fig. 2

Modulation of MTB-induced NO production by TQ in M. Tuberculosis-infected macrophages. Raw 264.7 macrophages were infected with H37Rv and XDR-TB for 2 h at an MOI of 1:10 at 37 °C with 5% CO₂, followed by drug treatment for 3 days. Nitric oxide (μM) was measured using Griess reagent. a Effect of TQ on nitric oxide production in H37Rv-infected Raw 264.7 cells. b Effect of TQ on nitric oxide production in XDR-TB-infected Raw 264.7 cells. Data represent the mean ± SD of 3 independent experiments done in triplicate. *P < 0.05, ** < 0.01, *** < 0.009 by Student’s t-test

Fig. 3

The effect of TQ on MTB-induced NO expression in Type II human alveolar A549 cells. A549 cells were infected with H37Rv for 3 h at an MOI of 1:10 at 37 °C with 5% CO₂, followed by drug treatment for 24 h. a Cytotoxicity of TQ on A549 cells. b Effect of TQ on nitric oxide production in H37Rv-infected A549 cells. Data represent the mean ± SD of 3 independent experiments done in triplicate. * P < 0.05 by Student’s t-test. c Western blot result showing the changes in iNOS protein expression upon TQ treatment; representative result from a triplicate experiment. d qRT-PCR result showing the changes in iNOS mRNA expression. e Changes in IL-6 mRNA expression. f Changes in TNF-α mRNA expression due to TQ treatment. Data represent the mean ± SD of triplicates of an individual experiment and are representative of 3 independent experiments. *P < 0.05 by a two-tailed Student’s t-test

Fig. 4

The effect of TQ on MTB-induced NO expression in human monocyte THP-1 cells. THP-1 monocytes were differentiated into macrophages by 200 nM PMA treatment. The cells were imaged at ×10 magnification with an inverted microscope (ZEISS; Axiovert 25). a-I THP-1 monocytes, undifferentiated. a-II THP-1 macrophages, differentiated. Cells were infected with H37Rv for 3 h at an MOI of 1:10 at 37 °C with 5% CO₂, followed by drug treatment for 24 h. b q-RT-PCR result showing changes in iNOS mRNA expression. c Changes in IL-6 mRNA expression. d Changes in TNF-α mRNA expression due to TQ treatment. Data represent the mean ± SD of triplicates of an individual experiment and are representative of 3 independent experiments. *P < 0.05, ** < 0.005 by two-tailed Student’s t-test