A novel anti-mycobacterial function of mitogen-activated protein kinase phosphatase-1

Abstract

Background: Mycobacterium tuberculosis (MTB) is a major cause of morbidity and mortality in the world. To combat against this pathogen, immune cells release cytokines including tumor necrosis factor-alpha (TNF-alpha), which is pivotal in the development of protective granulomas. Our previous results showed that Bacillus Calmette Guerin (BCG), a mycobacterium used as a model to investigate the immune response against MTB, stimulates the induction of TNF-alpha via mitogen-activated protein kinase (MAPK) in human blood monocytes. Since MAPK phosphatase-1 (MKP-1) is known to regulate MAPK activities, we examined whether MKP-1 plays a role in BCG-induced MAPK activation and cytokine expression.

Results: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1. Additionally, the induction of MKP-1 was regulated by p38 MAPK and extracellular signal-regulated kinase 1 and 2 (ERK1/2). Surprisingly, when MKP-1 expression was blocked by its specific siRNA, there was a significant decrease in the levels of phospho-MAPK (p38 MAPK and ERK1/2) and TNF-alpha inducible by BCG.

Conclusions: Since TNF-alpha is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity.

Figure 1

Different inducers show differential induction of MKP-1. (A) Primary human blood monocytes were stimulated with LPS (50 ng/ml), Pam3Cys (50 ng/ml), or Poly IC (100 μg/ml) for 1 (open bars) and 3 (black bars) hours. Phosphate buffered saline (PBS) was used as a reagent control. RNA was harvested and MKP-1 mRNA was measured by QPCR method. (B) Primary human monocytes were treated with LPS (50 ng/ml) for the indicated time points and RNA samples were harvested. MKP-1 levels were studied by using QPCR. Independent experiments were done on blood cells from 4 different donors, and the results are shown as mean ± SD. * = p < 0.05.

Figure 2

BCG induces MKP-1 expression. (A) Human monocytes were treated with BCG (MOI = 1 CFU/cell) for the indicated time points and RNA was harvested. MKP-1 level was assayed by using QPCR. Results are shown as mean ± SD from 3 different donors. * = p < 0.05. (B) Cells were treated as in (A) for the indicated time points and proteins were collected. MKP-1 protein levels were measured by Western blotting. Independent experiments were done on monocytes from 3 different donors and one representative set of results is shown. The intensities of the protein bands were determined by using Bio-Rad Quantity One imaging software. The intensities of MKP-1 were normalized to the corresponding actin. The values in parenthesis are the relative normalized intensities compared to those of untreated cells.

Figure 3

BCG-induced MKP-1 expression is dependent on p38 MAPK and ERK1/2. Human monocytes were pretreated with several inhibitors including PD98059 (20 μM), SB203580 (100 nM), SP600125 (100 nM), and CAPE (5 μg/ml) for 1 hour. BCG (MOI = 1 CFU/cell) was then added for 1 hour and RNA was harvested. Levels of MKP-1 were measured by QPCR. Percentage change was defined as the percentage of the fold induction of (BCG + inhibitor) over the fold induction of BCG without the inhibitor. Sample of BCG was set as 100% for comparisons. Independent experiments were performed on blood cells from 4 different donors and the results are shown as mean ± SD. * = p < 0.05.

Figure 4

Transfection of MKP-1 siRNA into primary human blood monocytes can increase LPS-induced TNF-α level while decrease BCG-induced TNF-α expression. Cells were first transfected with control (ctrl) or MKP-1 siRNA (MKP-1 si, 200 nM) for 24 hours and then treated with different inducers as indicated. (A) After transfection, cells were treated with Mock (M) or BCG (B, MOI = 1 CFU/cell) for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR. (B) Monocytes were treated as in (A) for the indicated time points and MKP-1 proteins were measured by Western blotting. (C) Cells were treated with LPS (50 ng/ml) for the indicated time points. RNA was harvested, and levels of MKP-1 and TNF-α were measured by RT-PCR. For both (B) and (C), the intensities of the PCR/protein bands were determined by using Bio-Rad Quantity One imaging software. The intensities of bands were normalized to the corresponding control. The values in parenthesis are the relative normalized intensities compared to those of the control siRNA-transfected cells without other treatment. (D) BCG (MOI = 1 CFU/cell) was added for 3 hours. RNA was harvested and levels of TNF-α, IL-6 and IL-10 were measured by QPCR. Percentage change was defined as the percentage of the fold induction of (MKP-1 si + BCG) over the fold induction of (ctrl + BCG). Results of (ctrl + BCG) were set as 100%. For (A)-(C), independent experiments were done on monocytes from 3 different donors and one representative set of results is shown. For (D), experiments were performed on 4 different donors and the results are shown as mean ± SD. * = p < 0.05.

Figure 5

BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA. Transfection was done as in Figure 4, and BCG (MOI = 1 CFU/cell) was added for the indicated time points. Protein samples were harvested and levels of phospho-p38 MAPK (p-p38), phospho-ERK1/2 (p-ERK1/2) and actin were measured by Western blotting. Independent experiments were done on monocytes from 4 different donors and one representative set of results is shown. The intensities of the protein bands were determined by using Bio-Rad Quantity One imaging software. The intensities of phospho-proteins were normalized to the corresponding actin. The values in parenthesis are the relative normalized intensities compared to those of control siRNA-transfected cells without other treatment.

Figure 6

MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. LPS model was provided according to literature findings (Left). In this scenario, LPS activates MKP-1, which in turn dephosphorylates and deactivates phospho-p38 MAPK, resulting in less TNF-α induction. However, the situation in DHP-HSA activation of DUSP2 is more complicated (Middle), since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the de-repression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression. The unexpected antimycobacterial role of MKP-1 (Right) may be explained by events similar to the DUSP2 effects. In this case (Right), there was an inhibition of unknown pathways or kinases downstream of MKP-1, and the unknown factor in turn inhibits MAPKs activation leading to more TNF-α induction. The details and kinase targets are yet to be identified.