Limits...
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.

No MeSH data available.


(a) SFN does not interfere with phagosomal maturation. The pH acidification was determined by quantifying FITC signal intensities, which is pH dependent in THP-1 infected with FITC-labeled Mabs-mCherry. The pH-independent mCherry fluorochrome was used as an internal control to estimate the amount of labeled bacteria per well. Graph shows one representative experiment out of ten. Data represent the means±S.E.M. of 10 independent experiments done in triplicates. (b) ROS response in M. abscessus-infected macrophages. THP-1-derived cells were infected and ROS was detected using H2DCFDA fluorescence labeling. Data acquisition (n=5000 events) and analysis were performed using the MARKII imaging flow cytometer. The graph represents the means±S.E.M. of three independent experiments. *P<0.05 compared with DMSO-treated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4979433&req=5

fig3: (a) SFN does not interfere with phagosomal maturation. The pH acidification was determined by quantifying FITC signal intensities, which is pH dependent in THP-1 infected with FITC-labeled Mabs-mCherry. The pH-independent mCherry fluorochrome was used as an internal control to estimate the amount of labeled bacteria per well. Graph shows one representative experiment out of ten. Data represent the means±S.E.M. of 10 independent experiments done in triplicates. (b) ROS response in M. abscessus-infected macrophages. THP-1-derived cells were infected and ROS was detected using H2DCFDA fluorescence labeling. Data acquisition (n=5000 events) and analysis were performed using the MARKII imaging flow cytometer. The graph represents the means±S.E.M. of three independent experiments. *P<0.05 compared with DMSO-treated cells.

Mentions: Numerous pathogens including M. tuberculosis have developed complex mechanisms to survive and proliferate in host phagocytes by interfering with the phagosomal maturation process thus blocking the fusion of phagosomes with lysosomes and the generation of phagolysosomes. This impediment prevents exposition of the ingested bacteria to reactive oxygen metabolites, lysosomal hydrolases, and general acidification of the phagolysosome to pH below 5.0, allowing the use of macrophages as proliferation reservoir.13 To determine whether Mabs or/and SFN has an effect on phagosomal maturation, phagosome acidification assay was used. Mabs expressing mCherry fluorochrome was surface labeled with the pH-sensitive fluorescein-5-isothiocyanate (FITC) and were used to infect THP-1-derived macrophages. FITC, which emission intensities are pH dependent, was used to determine phagosomal acidification, while mCherry, a pH-independent fluorochrome, was used as an internal indicator of the number of bacteria (Figure 3a). Our results showed that Mabs efficiently inhibited phagosomal acidification in DMSO treated macrophages (pH=6.27±0.07). Interestingly, reduction in mycobacterial burden in SFN-treated macrophages (Figure 2) was not due to a decrease in phagosomal pH since the phagosomes of SFN-treated macrophages remained stable at pH 6.17±0.14 (Figure 3a).


Caspase-independent apoptosis in infected macrophages triggered by sulforaphane via Nrf2/p38 signaling pathways
(a) SFN does not interfere with phagosomal maturation. The pH acidification was determined by quantifying FITC signal intensities, which is pH dependent in THP-1 infected with FITC-labeled Mabs-mCherry. The pH-independent mCherry fluorochrome was used as an internal control to estimate the amount of labeled bacteria per well. Graph shows one representative experiment out of ten. Data represent the means±S.E.M. of 10 independent experiments done in triplicates. (b) ROS response in M. abscessus-infected macrophages. THP-1-derived cells were infected and ROS was detected using H2DCFDA fluorescence labeling. Data acquisition (n=5000 events) and analysis were performed using the MARKII imaging flow cytometer. The graph represents the means±S.E.M. of three independent experiments. *P<0.05 compared with DMSO-treated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: (a) SFN does not interfere with phagosomal maturation. The pH acidification was determined by quantifying FITC signal intensities, which is pH dependent in THP-1 infected with FITC-labeled Mabs-mCherry. The pH-independent mCherry fluorochrome was used as an internal control to estimate the amount of labeled bacteria per well. Graph shows one representative experiment out of ten. Data represent the means±S.E.M. of 10 independent experiments done in triplicates. (b) ROS response in M. abscessus-infected macrophages. THP-1-derived cells were infected and ROS was detected using H2DCFDA fluorescence labeling. Data acquisition (n=5000 events) and analysis were performed using the MARKII imaging flow cytometer. The graph represents the means±S.E.M. of three independent experiments. *P<0.05 compared with DMSO-treated cells.
Mentions: Numerous pathogens including M. tuberculosis have developed complex mechanisms to survive and proliferate in host phagocytes by interfering with the phagosomal maturation process thus blocking the fusion of phagosomes with lysosomes and the generation of phagolysosomes. This impediment prevents exposition of the ingested bacteria to reactive oxygen metabolites, lysosomal hydrolases, and general acidification of the phagolysosome to pH below 5.0, allowing the use of macrophages as proliferation reservoir.13 To determine whether Mabs or/and SFN has an effect on phagosomal maturation, phagosome acidification assay was used. Mabs expressing mCherry fluorochrome was surface labeled with the pH-sensitive fluorescein-5-isothiocyanate (FITC) and were used to infect THP-1-derived macrophages. FITC, which emission intensities are pH dependent, was used to determine phagosomal acidification, while mCherry, a pH-independent fluorochrome, was used as an internal indicator of the number of bacteria (Figure 3a). Our results showed that Mabs efficiently inhibited phagosomal acidification in DMSO treated macrophages (pH=6.27±0.07). Interestingly, reduction in mycobacterial burden in SFN-treated macrophages (Figure 2) was not due to a decrease in phagosomal pH since the phagosomes of SFN-treated macrophages remained stable at pH 6.17±0.14 (Figure 3a).

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.