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

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.

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(a) Annexin V-FITC assay in THP-1-derived macrophages pretreated with MAPK inhibitors SB203580, SP600125, and PD98059. Infected cells were collected 24 h post infection. Data were from two independent experiments of n=10 000 events that were imaged and analyzed using MARKII imaging cytometer. *P<0.03 compared with SFN-treated cells. (b) THP-1-derived macrophages were pretreated with MAPK inhibitors before SFN pretreatment and Mabs infection. Mycobacteria were collected 48 h after infection by lysing cells in ice-cold water, serially diluted, and seeded on agar plates. CFUs were obtained 5 days after incubation at 37 °C. *P<0.03 compared with scramble transfected cells. (c) Caspase-independent cell death pathway activated by SFN and M. abscessus infection in THP-1-derived macrophages.
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fig8: (a) Annexin V-FITC assay in THP-1-derived macrophages pretreated with MAPK inhibitors SB203580, SP600125, and PD98059. Infected cells were collected 24 h post infection. Data were from two independent experiments of n=10 000 events that were imaged and analyzed using MARKII imaging cytometer. *P<0.03 compared with SFN-treated cells. (b) THP-1-derived macrophages were pretreated with MAPK inhibitors before SFN pretreatment and Mabs infection. Mycobacteria were collected 48 h after infection by lysing cells in ice-cold water, serially diluted, and seeded on agar plates. CFUs were obtained 5 days after incubation at 37 °C. *P<0.03 compared with scramble transfected cells. (c) Caspase-independent cell death pathway activated by SFN and M. abscessus infection in THP-1-derived macrophages.

Mentions: To determine whether p38 signaling pathway had a central role in the increase in cell apoptosis induced by SFN and Mabs infection, THP-1-derived macrophages were pretreated with MAPK inhibitors of p38, JNK, and ERK, before pretreatment with SFN and Mabs infection, and annexin V-FITC labeling was performed. A significant decrease was observed in apoptosis in SB203580- and SP600125-pretreated macrophages compared with SFN-pretreated cells, while no significant difference was observed in PD98059-pretreated cells (Figure 8a).


Caspase-independent apoptosis in infected macrophages triggered by sulforaphane via Nrf2/p38 signaling pathways
(a) Annexin V-FITC assay in THP-1-derived macrophages pretreated with MAPK inhibitors SB203580, SP600125, and PD98059. Infected cells were collected 24 h post infection. Data were from two independent experiments of n=10 000 events that were imaged and analyzed using MARKII imaging cytometer. *P<0.03 compared with SFN-treated cells. (b) THP-1-derived macrophages were pretreated with MAPK inhibitors before SFN pretreatment and Mabs infection. Mycobacteria were collected 48 h after infection by lysing cells in ice-cold water, serially diluted, and seeded on agar plates. CFUs were obtained 5 days after incubation at 37 °C. *P<0.03 compared with scramble transfected cells. (c) Caspase-independent cell death pathway activated by SFN and M. abscessus infection in THP-1-derived macrophages.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4979433&req=5

fig8: (a) Annexin V-FITC assay in THP-1-derived macrophages pretreated with MAPK inhibitors SB203580, SP600125, and PD98059. Infected cells were collected 24 h post infection. Data were from two independent experiments of n=10 000 events that were imaged and analyzed using MARKII imaging cytometer. *P<0.03 compared with SFN-treated cells. (b) THP-1-derived macrophages were pretreated with MAPK inhibitors before SFN pretreatment and Mabs infection. Mycobacteria were collected 48 h after infection by lysing cells in ice-cold water, serially diluted, and seeded on agar plates. CFUs were obtained 5 days after incubation at 37 °C. *P<0.03 compared with scramble transfected cells. (c) Caspase-independent cell death pathway activated by SFN and M. abscessus infection in THP-1-derived macrophages.
Mentions: To determine whether p38 signaling pathway had a central role in the increase in cell apoptosis induced by SFN and Mabs infection, THP-1-derived macrophages were pretreated with MAPK inhibitors of p38, JNK, and ERK, before pretreatment with SFN and Mabs infection, and annexin V-FITC labeling was performed. A significant decrease was observed in apoptosis in SB203580- and SP600125-pretreated macrophages compared with SFN-pretreated cells, while no significant difference was observed in PD98059-pretreated cells (Figure 8a).

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