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The role of Mcl-1 in S. aureus-induced cytoprotection of infected macrophages.

Koziel J, Kmiecik K, Chmiest D, Maresz K, Mizgalska D, Maciag-Gudowska A, Mydel P, Potempa J - Mediators Inflamm. (2013)

Bottom Line: Consistent with this, we proved a crucial role of Mcl-1 in S. aureus-induced cytoprotection, since silencing of MCL1 by siRNA profoundly reversed the cytoprotection of infected cells leading to apoptosis.Increased MCL1 expression in infected cells was associated with enhanced NFκB activation and subsequent IL-6 secretion, since the inhibition of both NFκB and IL-6 signalling pathways abrogated Mcl-1 induction and cytoprotection.Finally, we confirmed our observation in vivo in murine model of septic arthritis showing the association between the severity of arthritis and Mcl-1 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Ul. Gronostajowa 7, 30-387 Kraków, Poland. joanna.koziel@uj.edu.pl

ABSTRACT
As a facultative intracellular pathogen, Staphylococcus aureus invades macrophages and then promotes the cytoprotection of infected cells thus stabilizing safe niche for silent persistence. This process occurs through the upregulation of crucial antiapoptotic genes, in particular, myeloid cell leukemia-1 (MCL-1). Here, we investigated the underlying mechanism and signal transduction pathways leading to increased MCL-1 expression in infected macrophages. Live S. aureus not only stimulated de novo synthesis of Mcl-1, but also prolonged the stability of this antiapoptotic protein. Consistent with this, we proved a crucial role of Mcl-1 in S. aureus-induced cytoprotection, since silencing of MCL1 by siRNA profoundly reversed the cytoprotection of infected cells leading to apoptosis. Increased MCL1 expression in infected cells was associated with enhanced NFκB activation and subsequent IL-6 secretion, since the inhibition of both NFκB and IL-6 signalling pathways abrogated Mcl-1 induction and cytoprotection. Finally, we confirmed our observation in vivo in murine model of septic arthritis showing the association between the severity of arthritis and Mcl-1 expression. Therefore, we propose that S. aureus is hijacking the Mcl-1-dependent inhibition of apoptosis to prevent the elimination of infected host cells, thus allowing the intracellular persistence of the pathogen, its dissemination by infected macrophages, and the progression of staphylococci diseases.

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Effect of Mcl-1 expression on cytoprotection induced by S. aureus in hMDMs. (a) Human macrophages were treated with an MCL1 siRNA (siMCL1) or a nonspecific siRNA (siNS). At 24 h following transfection macrophages were infected with S. aureus at an MOI 1 : 50. After additional 24 h cells were collected and Mcl-1 expression was detected by immunoblot. Data are representative of three separate experiments using hMDMs derived from different donors. (b) The increase in caspase-3 activity (RFU/min) induced by STS in MCL1 knockdown macrophages infected with S. aureus. Twenty-four hours after treatment with siRNA, hMDMs were infected with S. aureus (24 h), followed by treatment with STS at a concentration of 1 μM for 18 h. The measurement of caspase-3 activity (RFU/min) in cell lysates was performed using DEVD-AFC as a substrate. The figure is representative of three experiments, using hMDMs cultures obtained from different donors. Light bars—STS, dark bars—Sa + STS. Data represent means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. (c) Increased susceptibility to the cytotoxic effects of staurosporine (STS) in MCL1 knockdown macrophages infected with S. aureus. Twenty-four hours after treatment with siRNA, hMDMs were infected with S. aureus (24 h), followed by treatment with STS at a concentration of 1 μM for 24 h. Plasma membrane permeabilisation or cell lysis induced in the hMDMs was assessed by measuring LDH activity in the culture medium. LDH activity in the media of cells treated only with STS was arbitrarily set as 100%. Results were calculated based on data (± SD) from three separate experiments. *P < 0.05.
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fig4: Effect of Mcl-1 expression on cytoprotection induced by S. aureus in hMDMs. (a) Human macrophages were treated with an MCL1 siRNA (siMCL1) or a nonspecific siRNA (siNS). At 24 h following transfection macrophages were infected with S. aureus at an MOI 1 : 50. After additional 24 h cells were collected and Mcl-1 expression was detected by immunoblot. Data are representative of three separate experiments using hMDMs derived from different donors. (b) The increase in caspase-3 activity (RFU/min) induced by STS in MCL1 knockdown macrophages infected with S. aureus. Twenty-four hours after treatment with siRNA, hMDMs were infected with S. aureus (24 h), followed by treatment with STS at a concentration of 1 μM for 18 h. The measurement of caspase-3 activity (RFU/min) in cell lysates was performed using DEVD-AFC as a substrate. The figure is representative of three experiments, using hMDMs cultures obtained from different donors. Light bars—STS, dark bars—Sa + STS. Data represent means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. (c) Increased susceptibility to the cytotoxic effects of staurosporine (STS) in MCL1 knockdown macrophages infected with S. aureus. Twenty-four hours after treatment with siRNA, hMDMs were infected with S. aureus (24 h), followed by treatment with STS at a concentration of 1 μM for 24 h. Plasma membrane permeabilisation or cell lysis induced in the hMDMs was assessed by measuring LDH activity in the culture medium. LDH activity in the media of cells treated only with STS was arbitrarily set as 100%. Results were calculated based on data (± SD) from three separate experiments. *P < 0.05.

Mentions: As we described previously S. aureus protects infected macrophages, both human and murine, against induced cell death [7]. Thus, to further determine whether the survival of infected macrophages in response to proapoptotic stimulants was dependent on Mcl-1 synthesis, RNA interference with siRNA was used to selectively silence MCL1 gene expression. Treatment of cells with an MCL1-specific siRNA, but not a nonspecific control siRNA, resulted in specific and efficient suppression of Mcl-1 protein levels at 24–72 h after-transfection (data not shown) without effect on macrophages viability. The infection of macrophages 24 h after-transfection has not influenced the low level of already silenced protein within 24–48 h after infection (Figure 4(a)). The increasing caspase-3 activity (Figure 4(b)) and a lactate dehydrogenase leaking from macrophages (Figure 4(c)) revealed that the knockdown of MCL1 expression significantly attenuated the S. aureus-exerted cytoprotection of cells in a staurosporine-induced cell death model. Our data also indicates (Supplementary Figure 2) that silencing of Mcl-1 in S. aureus-infected macrophages partly ablates the cytoprotection against spontaneous cell death. This observation confirmed that Mcl-1 plays an important role in preventing apoptosis in S. aureus-infected human macrophages.


The role of Mcl-1 in S. aureus-induced cytoprotection of infected macrophages.

Koziel J, Kmiecik K, Chmiest D, Maresz K, Mizgalska D, Maciag-Gudowska A, Mydel P, Potempa J - Mediators Inflamm. (2013)

Effect of Mcl-1 expression on cytoprotection induced by S. aureus in hMDMs. (a) Human macrophages were treated with an MCL1 siRNA (siMCL1) or a nonspecific siRNA (siNS). At 24 h following transfection macrophages were infected with S. aureus at an MOI 1 : 50. After additional 24 h cells were collected and Mcl-1 expression was detected by immunoblot. Data are representative of three separate experiments using hMDMs derived from different donors. (b) The increase in caspase-3 activity (RFU/min) induced by STS in MCL1 knockdown macrophages infected with S. aureus. Twenty-four hours after treatment with siRNA, hMDMs were infected with S. aureus (24 h), followed by treatment with STS at a concentration of 1 μM for 18 h. The measurement of caspase-3 activity (RFU/min) in cell lysates was performed using DEVD-AFC as a substrate. The figure is representative of three experiments, using hMDMs cultures obtained from different donors. Light bars—STS, dark bars—Sa + STS. Data represent means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. (c) Increased susceptibility to the cytotoxic effects of staurosporine (STS) in MCL1 knockdown macrophages infected with S. aureus. Twenty-four hours after treatment with siRNA, hMDMs were infected with S. aureus (24 h), followed by treatment with STS at a concentration of 1 μM for 24 h. Plasma membrane permeabilisation or cell lysis induced in the hMDMs was assessed by measuring LDH activity in the culture medium. LDH activity in the media of cells treated only with STS was arbitrarily set as 100%. Results were calculated based on data (± SD) from three separate experiments. *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: Effect of Mcl-1 expression on cytoprotection induced by S. aureus in hMDMs. (a) Human macrophages were treated with an MCL1 siRNA (siMCL1) or a nonspecific siRNA (siNS). At 24 h following transfection macrophages were infected with S. aureus at an MOI 1 : 50. After additional 24 h cells were collected and Mcl-1 expression was detected by immunoblot. Data are representative of three separate experiments using hMDMs derived from different donors. (b) The increase in caspase-3 activity (RFU/min) induced by STS in MCL1 knockdown macrophages infected with S. aureus. Twenty-four hours after treatment with siRNA, hMDMs were infected with S. aureus (24 h), followed by treatment with STS at a concentration of 1 μM for 18 h. The measurement of caspase-3 activity (RFU/min) in cell lysates was performed using DEVD-AFC as a substrate. The figure is representative of three experiments, using hMDMs cultures obtained from different donors. Light bars—STS, dark bars—Sa + STS. Data represent means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. (c) Increased susceptibility to the cytotoxic effects of staurosporine (STS) in MCL1 knockdown macrophages infected with S. aureus. Twenty-four hours after treatment with siRNA, hMDMs were infected with S. aureus (24 h), followed by treatment with STS at a concentration of 1 μM for 24 h. Plasma membrane permeabilisation or cell lysis induced in the hMDMs was assessed by measuring LDH activity in the culture medium. LDH activity in the media of cells treated only with STS was arbitrarily set as 100%. Results were calculated based on data (± SD) from three separate experiments. *P < 0.05.
Mentions: As we described previously S. aureus protects infected macrophages, both human and murine, against induced cell death [7]. Thus, to further determine whether the survival of infected macrophages in response to proapoptotic stimulants was dependent on Mcl-1 synthesis, RNA interference with siRNA was used to selectively silence MCL1 gene expression. Treatment of cells with an MCL1-specific siRNA, but not a nonspecific control siRNA, resulted in specific and efficient suppression of Mcl-1 protein levels at 24–72 h after-transfection (data not shown) without effect on macrophages viability. The infection of macrophages 24 h after-transfection has not influenced the low level of already silenced protein within 24–48 h after infection (Figure 4(a)). The increasing caspase-3 activity (Figure 4(b)) and a lactate dehydrogenase leaking from macrophages (Figure 4(c)) revealed that the knockdown of MCL1 expression significantly attenuated the S. aureus-exerted cytoprotection of cells in a staurosporine-induced cell death model. Our data also indicates (Supplementary Figure 2) that silencing of Mcl-1 in S. aureus-infected macrophages partly ablates the cytoprotection against spontaneous cell death. This observation confirmed that Mcl-1 plays an important role in preventing apoptosis in S. aureus-infected human macrophages.

Bottom Line: Consistent with this, we proved a crucial role of Mcl-1 in S. aureus-induced cytoprotection, since silencing of MCL1 by siRNA profoundly reversed the cytoprotection of infected cells leading to apoptosis.Increased MCL1 expression in infected cells was associated with enhanced NFκB activation and subsequent IL-6 secretion, since the inhibition of both NFκB and IL-6 signalling pathways abrogated Mcl-1 induction and cytoprotection.Finally, we confirmed our observation in vivo in murine model of septic arthritis showing the association between the severity of arthritis and Mcl-1 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Ul. Gronostajowa 7, 30-387 Kraków, Poland. joanna.koziel@uj.edu.pl

ABSTRACT
As a facultative intracellular pathogen, Staphylococcus aureus invades macrophages and then promotes the cytoprotection of infected cells thus stabilizing safe niche for silent persistence. This process occurs through the upregulation of crucial antiapoptotic genes, in particular, myeloid cell leukemia-1 (MCL-1). Here, we investigated the underlying mechanism and signal transduction pathways leading to increased MCL-1 expression in infected macrophages. Live S. aureus not only stimulated de novo synthesis of Mcl-1, but also prolonged the stability of this antiapoptotic protein. Consistent with this, we proved a crucial role of Mcl-1 in S. aureus-induced cytoprotection, since silencing of MCL1 by siRNA profoundly reversed the cytoprotection of infected cells leading to apoptosis. Increased MCL1 expression in infected cells was associated with enhanced NFκB activation and subsequent IL-6 secretion, since the inhibition of both NFκB and IL-6 signalling pathways abrogated Mcl-1 induction and cytoprotection. Finally, we confirmed our observation in vivo in murine model of septic arthritis showing the association between the severity of arthritis and Mcl-1 expression. Therefore, we propose that S. aureus is hijacking the Mcl-1-dependent inhibition of apoptosis to prevent the elimination of infected host cells, thus allowing the intracellular persistence of the pathogen, its dissemination by infected macrophages, and the progression of staphylococci diseases.

Show MeSH
Related in: MedlinePlus