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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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SFN showed a decrease in mycobacterial burden in infected macrophages. THP-1-derived macrophages were pretreated with DMSO or 10 μM SFN 3 h before infection with M. abscessus for 3 h. The unincorporated mycobacteria were incubated in culture medium containing 250 μg/ml amikacin for 1 h before thorough washes. The cells were then incubated in medium with 50 μg/ml amikacin for the indicated time period (0, 1, 3, and 7 days). Intracellular mycobacteria were released by cell lysing in ice-cold water, serially diluted, and seeded on agar plates. Colony-forming units were counted 5 days after incubation at 37 °C. The graph represents the means±S.E.M. of two independent experiments done in triplicates. *P<0.03, **P<0.01.
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fig2: SFN showed a decrease in mycobacterial burden in infected macrophages. THP-1-derived macrophages were pretreated with DMSO or 10 μM SFN 3 h before infection with M. abscessus for 3 h. The unincorporated mycobacteria were incubated in culture medium containing 250 μg/ml amikacin for 1 h before thorough washes. The cells were then incubated in medium with 50 μg/ml amikacin for the indicated time period (0, 1, 3, and 7 days). Intracellular mycobacteria were released by cell lysing in ice-cold water, serially diluted, and seeded on agar plates. Colony-forming units were counted 5 days after incubation at 37 °C. The graph represents the means±S.E.M. of two independent experiments done in triplicates. *P<0.03, **P<0.01.

Mentions: Since M. abscessus has been previously shown to proliferate more favorably in oxidative stress conditions,5 we sought to determine the effect of Nrf2 activation on the bactericidal activity of macrophages against M. abscessus. THP-1-derived macrophages were infected with Mabs for 3 h at a multiplicity of infection of 10 bacilli for 1 cell, thoroughly washed and incubated in amikacin and SFN supplemented medium. After the indicated post infection times, macrophages were lysed and live internalized mycobacteria were quantified using the colony counting method (colony-forming unit, CFU). The results showed an increase in Mabs viability and proliferation over the 7- day observation period in DMSO-pretreated cells (Figure 2). Interestingly, SFN-pretreated macrophages infected with Mabs yielded a 2-fold decrease in CFU at day 3 post infection and a 3-fold decrease at day 7 post infection compared with DMSO-treated cells. These data suggest that SFN may have an inhibitory effect on mycobacterial proliferation in macrophages.


Caspase-independent apoptosis in infected macrophages triggered by sulforaphane via Nrf2/p38 signaling pathways
SFN showed a decrease in mycobacterial burden in infected macrophages. THP-1-derived macrophages were pretreated with DMSO or 10 μM SFN 3 h before infection with M. abscessus for 3 h. The unincorporated mycobacteria were incubated in culture medium containing 250 μg/ml amikacin for 1 h before thorough washes. The cells were then incubated in medium with 50 μg/ml amikacin for the indicated time period (0, 1, 3, and 7 days). Intracellular mycobacteria were released by cell lysing in ice-cold water, serially diluted, and seeded on agar plates. Colony-forming units were counted 5 days after incubation at 37 °C. The graph represents the means±S.E.M. of two independent experiments done in triplicates. *P<0.03, **P<0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4979433&req=5

fig2: SFN showed a decrease in mycobacterial burden in infected macrophages. THP-1-derived macrophages were pretreated with DMSO or 10 μM SFN 3 h before infection with M. abscessus for 3 h. The unincorporated mycobacteria were incubated in culture medium containing 250 μg/ml amikacin for 1 h before thorough washes. The cells were then incubated in medium with 50 μg/ml amikacin for the indicated time period (0, 1, 3, and 7 days). Intracellular mycobacteria were released by cell lysing in ice-cold water, serially diluted, and seeded on agar plates. Colony-forming units were counted 5 days after incubation at 37 °C. The graph represents the means±S.E.M. of two independent experiments done in triplicates. *P<0.03, **P<0.01.
Mentions: Since M. abscessus has been previously shown to proliferate more favorably in oxidative stress conditions,5 we sought to determine the effect of Nrf2 activation on the bactericidal activity of macrophages against M. abscessus. THP-1-derived macrophages were infected with Mabs for 3 h at a multiplicity of infection of 10 bacilli for 1 cell, thoroughly washed and incubated in amikacin and SFN supplemented medium. After the indicated post infection times, macrophages were lysed and live internalized mycobacteria were quantified using the colony counting method (colony-forming unit, CFU). The results showed an increase in Mabs viability and proliferation over the 7- day observation period in DMSO-pretreated cells (Figure 2). Interestingly, SFN-pretreated macrophages infected with Mabs yielded a 2-fold decrease in CFU at day 3 post infection and a 3-fold decrease at day 7 post infection compared with DMSO-treated cells. These data suggest that SFN may have an inhibitory effect on mycobacterial proliferation in macrophages.

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