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Caspase-independent apoptosis in infected macrophages triggered by sulforaphane via Nrf2/p38 signaling pathways

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ABSTRACT

Mycobacterium abscessus (Mabs), a non-tuberculous mycobacterium, is an emerging and rapidly growing opportunistic pathogen that is frequently found in patients with cystic fibrosis and in immunosuppressed patients. Its high tolerance to antibiotics is of great concern for public health. In this study, our results showed that human THP-1-derived macrophages infected with M. abscessus presented an increase in ROS production and cell necrosis. In addition, M. abscessus infection triggered activation of the Nuclear factor E2-related factor 2 (Nrf2) signaling pathway, and the induction of HO-1 and NQO1 expression levels. Interestingly, pretreatment of macrophages with sulforaphane (SFN), an activator of the antioxidant key regulator Nrf2, followed by M. abscessus infection significantly decreased mycobacterial burden. We demonstrated that this reduction in mycobacterial growth was due to an activation in cell apoptosis in SFN-pretreated and M. abscessus-infected macrophages. Pretreatment with specific MAPK inhibitors, PD98059, SP600125, and SB203580 to ERK, JNK, and p38 respectively, failed to inhibit induction of Nrf2 expression, suggesting that Nrf2 signaling pathway was upstream of MAPK signaling. Activation of cell apoptosis was caspase 3/7 independent but p38 MAPK dependent. Moreover, p38 MAPK induction was abolished in macrophages transfected with Nrf2 siRNA. In addition, p38 inhibitor abolished Nrf2-dependent apoptosis in infected macrophages. Taken together, our results indicate that modulation of the Nrf2 signaling using Nrf2 activators may help potentiate the actual drug therapies used to treat mycobacterial infection.

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Macrophage apoptosis induction by SFN is caspase 3/7 independent. THP-1-derived macrophages pretreated with SFN or DMSO were pretreated for 3 h before mycobacterial infection. Caspase 3/7 activities were determined using FLICA inhibitor probe. (a) FLICA inhibitor probe (green) and Mabs-mCherry (red indicated by white arrows) were excited by a 488-nm laser and a 568-nm laser, respectively. Each individual macrophage was imaged by the 40× objective of the ImageStream MARKII cytometer and data analysis was performed on n=10 000 events. (b) Results were expressed as the percentage of FLICA-positive cells. *P<0.04 compared with DMSO-treated cells, and #P<0.001 compared with SFN-treated cells. (c) Colocalization of Mabs-mCherry in FLICA-positive cells. Data represent the means±S.E.M. of three independent experiments.
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fig6: Macrophage apoptosis induction by SFN is caspase 3/7 independent. THP-1-derived macrophages pretreated with SFN or DMSO were pretreated for 3 h before mycobacterial infection. Caspase 3/7 activities were determined using FLICA inhibitor probe. (a) FLICA inhibitor probe (green) and Mabs-mCherry (red indicated by white arrows) were excited by a 488-nm laser and a 568-nm laser, respectively. Each individual macrophage was imaged by the 40× objective of the ImageStream MARKII cytometer and data analysis was performed on n=10 000 events. (b) Results were expressed as the percentage of FLICA-positive cells. *P<0.04 compared with DMSO-treated cells, and #P<0.001 compared with SFN-treated cells. (c) Colocalization of Mabs-mCherry in FLICA-positive cells. Data represent the means±S.E.M. of three independent experiments.

Mentions: Since activation of the caspase cascade is an essential process in cell apoptosis,15 we sought to determine whether the cell apoptosis observed in SFN-treated and Mabs-infected macrophages was caspase dependent. THP-1 cells treated with SFN were infected with Mabs, and activated caspases 3/7 were assessed using a non-cytotoxic fluorescent inhibitor of caspases (FLICA) probe that binds covalently to active caspases 3 and 7 and analyzed by imaging flow cytometry (Figure 6a). At 24 h post infection, no significant activation of caspases 3/7 was seen in SFN-pretreated, Mabs-infected, and SFN-pretreated–Mabs-infected macrophages compared with DMSO-treated cells (Figure 6b). However, cells incubated 48 h after infection showed a significant increase in caspases 3/7 activation in Mabs-infected cells compared with DMSO-treated cells and in SFN-pretreated–Mabs-infected macrophages compared with SFN-pretreated cells. Treatment with SFN did not modify caspases 3/7 activity in the presence or absence of Mabs. Colocalization of caspases 3/7 positive cells and Mabs-mCherry positive cells confirmed that SFN does not activate caspases 3/7 pathway in infected cells compared with untreated cells (Figure 6c).


Caspase-independent apoptosis in infected macrophages triggered by sulforaphane via Nrf2/p38 signaling pathways
Macrophage apoptosis induction by SFN is caspase 3/7 independent. THP-1-derived macrophages pretreated with SFN or DMSO were pretreated for 3 h before mycobacterial infection. Caspase 3/7 activities were determined using FLICA inhibitor probe. (a) FLICA inhibitor probe (green) and Mabs-mCherry (red indicated by white arrows) were excited by a 488-nm laser and a 568-nm laser, respectively. Each individual macrophage was imaged by the 40× objective of the ImageStream MARKII cytometer and data analysis was performed on n=10 000 events. (b) Results were expressed as the percentage of FLICA-positive cells. *P<0.04 compared with DMSO-treated cells, and #P<0.001 compared with SFN-treated cells. (c) Colocalization of Mabs-mCherry in FLICA-positive cells. Data represent the means±S.E.M. of three independent experiments.
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fig6: Macrophage apoptosis induction by SFN is caspase 3/7 independent. THP-1-derived macrophages pretreated with SFN or DMSO were pretreated for 3 h before mycobacterial infection. Caspase 3/7 activities were determined using FLICA inhibitor probe. (a) FLICA inhibitor probe (green) and Mabs-mCherry (red indicated by white arrows) were excited by a 488-nm laser and a 568-nm laser, respectively. Each individual macrophage was imaged by the 40× objective of the ImageStream MARKII cytometer and data analysis was performed on n=10 000 events. (b) Results were expressed as the percentage of FLICA-positive cells. *P<0.04 compared with DMSO-treated cells, and #P<0.001 compared with SFN-treated cells. (c) Colocalization of Mabs-mCherry in FLICA-positive cells. Data represent the means±S.E.M. of three independent experiments.
Mentions: Since activation of the caspase cascade is an essential process in cell apoptosis,15 we sought to determine whether the cell apoptosis observed in SFN-treated and Mabs-infected macrophages was caspase dependent. THP-1 cells treated with SFN were infected with Mabs, and activated caspases 3/7 were assessed using a non-cytotoxic fluorescent inhibitor of caspases (FLICA) probe that binds covalently to active caspases 3 and 7 and analyzed by imaging flow cytometry (Figure 6a). At 24 h post infection, no significant activation of caspases 3/7 was seen in SFN-pretreated, Mabs-infected, and SFN-pretreated–Mabs-infected macrophages compared with DMSO-treated cells (Figure 6b). However, cells incubated 48 h after infection showed a significant increase in caspases 3/7 activation in Mabs-infected cells compared with DMSO-treated cells and in SFN-pretreated–Mabs-infected macrophages compared with SFN-pretreated cells. Treatment with SFN did not modify caspases 3/7 activity in the presence or absence of Mabs. Colocalization of caspases 3/7 positive cells and Mabs-mCherry positive cells confirmed that SFN does not activate caspases 3/7 pathway in infected cells compared with untreated cells (Figure 6c).

View Article: PubMed Central - PubMed

ABSTRACT

Mycobacterium abscessus (Mabs), a non-tuberculous mycobacterium, is an emerging and rapidly growing opportunistic pathogen that is frequently found in patients with cystic fibrosis and in immunosuppressed patients. Its high tolerance to antibiotics is of great concern for public health. In this study, our results showed that human THP-1-derived macrophages infected with M. abscessus presented an increase in ROS production and cell necrosis. In addition, M. abscessus infection triggered activation of the Nuclear factor E2-related factor 2 (Nrf2) signaling pathway, and the induction of HO-1 and NQO1 expression levels. Interestingly, pretreatment of macrophages with sulforaphane (SFN), an activator of the antioxidant key regulator Nrf2, followed by M. abscessus infection significantly decreased mycobacterial burden. We demonstrated that this reduction in mycobacterial growth was due to an activation in cell apoptosis in SFN-pretreated and M. abscessus-infected macrophages. Pretreatment with specific MAPK inhibitors, PD98059, SP600125, and SB203580 to ERK, JNK, and p38 respectively, failed to inhibit induction of Nrf2 expression, suggesting that Nrf2 signaling pathway was upstream of MAPK signaling. Activation of cell apoptosis was caspase 3/7 independent but p38 MAPK dependent. Moreover, p38 MAPK induction was abolished in macrophages transfected with Nrf2 siRNA. In addition, p38 inhibitor abolished Nrf2-dependent apoptosis in infected macrophages. Taken together, our results indicate that modulation of the Nrf2 signaling using Nrf2 activators may help potentiate the actual drug therapies used to treat mycobacterial infection.

No MeSH data available.


Related in: MedlinePlus