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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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Apoptosis induction by SFN in M. abscessus-infected macrophages. THP-1-derived macrophages pretreated with SFN or DMSO for 3 h were infected with M. abscessus at the MOI of 10:1 for 24 or 48 h. (a) Annexin V-FITC (green) and Mabs-mCherry (red; white arrow) were excited by 488 and 568 nm lasers, respectively, and each individual macrophage was imaged by the 40× objective of the ImageStream MARKII cytometer and data analysis was performed (n=10 000 cells). (b) The percentage of apoptotic cells was determined after the indicated post infection times using Annexin V-FITC labeling. #P<0.03 compared with SFN-treated cells. (c) Colocalization of Mabs-mCherry in annexin V-FITC positive cells. Data represent the means±S.E.M. of three independent experiments. *P<0.04 compared with Mabs-mCherry infected cells.
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fig5: Apoptosis induction by SFN in M. abscessus-infected macrophages. THP-1-derived macrophages pretreated with SFN or DMSO for 3 h were infected with M. abscessus at the MOI of 10:1 for 24 or 48 h. (a) Annexin V-FITC (green) and Mabs-mCherry (red; white arrow) were excited by 488 and 568 nm lasers, respectively, and each individual macrophage was imaged by the 40× objective of the ImageStream MARKII cytometer and data analysis was performed (n=10 000 cells). (b) The percentage of apoptotic cells was determined after the indicated post infection times using Annexin V-FITC labeling. #P<0.03 compared with SFN-treated cells. (c) Colocalization of Mabs-mCherry in annexin V-FITC positive cells. Data represent the means±S.E.M. of three independent experiments. *P<0.04 compared with Mabs-mCherry infected cells.

Mentions: Since SFN did not affect phagosomal acidification nor cell necrosis, we hypothesized that SFN might reduce mycobacterial burden through cell apoptosis. Indeed, one of the mechanisms phagocytes utilize to increase mycobactericidal activity is for the infected macrophages to trigger apoptosis, generating apoptotic bodies that will induce killing by non-infected bystander macrophages.14 To test the apoptotic response of macrophages following mycobacterial infection, THP-1-derived macrophages were infected with Mabs and left incubated for 24 and 48 h. Early cell apoptosis was detected using an Annexin V-FITC probe and quantified by imaging flow cytometry (Figure 5a).


Caspase-independent apoptosis in infected macrophages triggered by sulforaphane via Nrf2/p38 signaling pathways
Apoptosis induction by SFN in M. abscessus-infected macrophages. THP-1-derived macrophages pretreated with SFN or DMSO for 3 h were infected with M. abscessus at the MOI of 10:1 for 24 or 48 h. (a) Annexin V-FITC (green) and Mabs-mCherry (red; white arrow) were excited by 488 and 568 nm lasers, respectively, and each individual macrophage was imaged by the 40× objective of the ImageStream MARKII cytometer and data analysis was performed (n=10 000 cells). (b) The percentage of apoptotic cells was determined after the indicated post infection times using Annexin V-FITC labeling. #P<0.03 compared with SFN-treated cells. (c) Colocalization of Mabs-mCherry in annexin V-FITC positive cells. Data represent the means±S.E.M. of three independent experiments. *P<0.04 compared with Mabs-mCherry infected cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4979433&req=5

fig5: Apoptosis induction by SFN in M. abscessus-infected macrophages. THP-1-derived macrophages pretreated with SFN or DMSO for 3 h were infected with M. abscessus at the MOI of 10:1 for 24 or 48 h. (a) Annexin V-FITC (green) and Mabs-mCherry (red; white arrow) were excited by 488 and 568 nm lasers, respectively, and each individual macrophage was imaged by the 40× objective of the ImageStream MARKII cytometer and data analysis was performed (n=10 000 cells). (b) The percentage of apoptotic cells was determined after the indicated post infection times using Annexin V-FITC labeling. #P<0.03 compared with SFN-treated cells. (c) Colocalization of Mabs-mCherry in annexin V-FITC positive cells. Data represent the means±S.E.M. of three independent experiments. *P<0.04 compared with Mabs-mCherry infected cells.
Mentions: Since SFN did not affect phagosomal acidification nor cell necrosis, we hypothesized that SFN might reduce mycobacterial burden through cell apoptosis. Indeed, one of the mechanisms phagocytes utilize to increase mycobactericidal activity is for the infected macrophages to trigger apoptosis, generating apoptotic bodies that will induce killing by non-infected bystander macrophages.14 To test the apoptotic response of macrophages following mycobacterial infection, THP-1-derived macrophages were infected with Mabs and left incubated for 24 and 48 h. Early cell apoptosis was detected using an Annexin V-FITC probe and quantified by imaging flow cytometry (Figure 5a).

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