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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) Mabs induces Nrf2 protein expression level in THP-1-derived macrophages. Protein expression levels were normalized to β-actin in the total protein extracts, and to Lamin A/C in the nuclear extracts. (b) RT-qPCR: Nrf2 signaling pathway is activated by M. abscessus and SFN. The Nrf2 targets HO-1 and NQO1 mRNA expression levels were normalized to the housekeeping gene ubiquitin. Data shown are the means±S.E.M. of three independent experiments done in triplicates. (c) Protein expression levels of HO-1 and NQO1 were detected by immunoblotting. Densitometric quantification of protein signals was normalized to β-actin.
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fig1: (a) Mabs induces Nrf2 protein expression level in THP-1-derived macrophages. Protein expression levels were normalized to β-actin in the total protein extracts, and to Lamin A/C in the nuclear extracts. (b) RT-qPCR: Nrf2 signaling pathway is activated by M. abscessus and SFN. The Nrf2 targets HO-1 and NQO1 mRNA expression levels were normalized to the housekeeping gene ubiquitin. Data shown are the means±S.E.M. of three independent experiments done in triplicates. (c) Protein expression levels of HO-1 and NQO1 were detected by immunoblotting. Densitometric quantification of protein signals was normalized to β-actin.

Mentions: To determine the effect of Mabs infection on the antioxidant pathway, we sought to define the expression level of Nrf2 in macrophages derived from phorbol 12-myristate 13-acetate (PMA)-differentiated human THP-1 cells. Macrophages were pretreated 3 h with SFN before infection with Mabs. Mabs infection induced Nrf2 protein expression level 24 h post infection 2.8-fold higher as compared with that of DMSO (Figure 1a). Nrf2 expression level in macrophages treated with SFN was increased more than 3-fold compared with DMSO-treated cells. Interestingly, Mabs infection in SFN-treated macrophages strongly increased Nrf2 protein level to 10.5-fold. Nrf2 activation is confirmed by analyzing nuclear proteins extracted from SFN-pretreated and/or Mabs-infected macrophages. Immunoblots revealed that Nrf2 is translocated to the nucleus in SFN-pretreated macrophages (4.7-fold increase) and confirmed the strong increase in Nrf2 previously seen in total protein extracts in SFN-pretreated/Mabs-infected cells (6.2-fold increase). Interestingly, infection with Mabs alone augmented Nrf2 in the total protein extracts but is not reflected in the nuclear Nrf2.


Caspase-independent apoptosis in infected macrophages triggered by sulforaphane via Nrf2/p38 signaling pathways
(a) Mabs induces Nrf2 protein expression level in THP-1-derived macrophages. Protein expression levels were normalized to β-actin in the total protein extracts, and to Lamin A/C in the nuclear extracts. (b) RT-qPCR: Nrf2 signaling pathway is activated by M. abscessus and SFN. The Nrf2 targets HO-1 and NQO1 mRNA expression levels were normalized to the housekeeping gene ubiquitin. Data shown are the means±S.E.M. of three independent experiments done in triplicates. (c) Protein expression levels of HO-1 and NQO1 were detected by immunoblotting. Densitometric quantification of protein signals was normalized to β-actin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4979433&req=5

fig1: (a) Mabs induces Nrf2 protein expression level in THP-1-derived macrophages. Protein expression levels were normalized to β-actin in the total protein extracts, and to Lamin A/C in the nuclear extracts. (b) RT-qPCR: Nrf2 signaling pathway is activated by M. abscessus and SFN. The Nrf2 targets HO-1 and NQO1 mRNA expression levels were normalized to the housekeeping gene ubiquitin. Data shown are the means±S.E.M. of three independent experiments done in triplicates. (c) Protein expression levels of HO-1 and NQO1 were detected by immunoblotting. Densitometric quantification of protein signals was normalized to β-actin.
Mentions: To determine the effect of Mabs infection on the antioxidant pathway, we sought to define the expression level of Nrf2 in macrophages derived from phorbol 12-myristate 13-acetate (PMA)-differentiated human THP-1 cells. Macrophages were pretreated 3 h with SFN before infection with Mabs. Mabs infection induced Nrf2 protein expression level 24 h post infection 2.8-fold higher as compared with that of DMSO (Figure 1a). Nrf2 expression level in macrophages treated with SFN was increased more than 3-fold compared with DMSO-treated cells. Interestingly, Mabs infection in SFN-treated macrophages strongly increased Nrf2 protein level to 10.5-fold. Nrf2 activation is confirmed by analyzing nuclear proteins extracted from SFN-pretreated and/or Mabs-infected macrophages. Immunoblots revealed that Nrf2 is translocated to the nucleus in SFN-pretreated macrophages (4.7-fold increase) and confirmed the strong increase in Nrf2 previously seen in total protein extracts in SFN-pretreated/Mabs-infected cells (6.2-fold increase). Interestingly, infection with Mabs alone augmented Nrf2 in the total protein extracts but is not reflected in the nuclear Nrf2.

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.