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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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NFκB is required for Mcl-1 expression induced by S. aureus. (a, b) hMDMs were pretreated for 30 min with Bay 11-7095 (40 μM) followed by S. aureus infection at an MOI of 50. After the indicated times, RNA and protein were extracted and Mcl-1 expression levels were determined by qRT-PCR and immunoblot (a and b, resp.). Data represent the means ± SD from three separate experiments. *P < 0.05. (c) The effect of NFκB inhibition on IL-6 expression was measured by qRT-PCR. Shown are the mean values calculated from the results of five independent real-time reactions using hMDMs derived from different donors. Bars represent mean relative expression ± SD; *P < 0.05.
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fig6: NFκB is required for Mcl-1 expression induced by S. aureus. (a, b) hMDMs were pretreated for 30 min with Bay 11-7095 (40 μM) followed by S. aureus infection at an MOI of 50. After the indicated times, RNA and protein were extracted and Mcl-1 expression levels were determined by qRT-PCR and immunoblot (a and b, resp.). Data represent the means ± SD from three separate experiments. *P < 0.05. (c) The effect of NFκB inhibition on IL-6 expression was measured by qRT-PCR. Shown are the mean values calculated from the results of five independent real-time reactions using hMDMs derived from different donors. Bars represent mean relative expression ± SD; *P < 0.05.

Mentions: A variety of intracellular signalling pathways are activated by pathogens. Among them, activation of NFκB has been shown to be critical for cytoprotection of infected cells [19]. Moreover, S. aureus is a potent inducer of NFκB activity as was confirmed in infected macrophages by EMSA (Supplement Figure 4). To determine the effect of inhibition of the NFκB pathway on Mcl-1 expression in hMDMs, macrophages were infected with S. aureus followed by incubation for 6 h with a specific NFκB-inhibitor, and then the levels of MCL1 were assessed in comparison to untreated infected cells. As seen in Figure 6(a), inhibition of the NFκB pathway abrogated the S. aureus-induced increase in MCL1 gene transcription. This effect was confirmed at the protein level as well (Figure 6(b)). These results strongly suggested that Mcl-1 expression in S. aureus-infected cells is dependent on NFκB. Since IL-6 transcription is upregulated in an NFκB-dependent manner [20], we investigated the possibility that NFκB stimulated MCL1 expression indirectly, via IL-6. S. aureus-induced production of IL-6 in infected macrophages was abrogated by treatment with Bay 11-7095, the NFκB-specific cell-permeable inhibitor, which indicated that IL-6 production was absolutely dependent on NFκB (Figure 6(c)). Taken together, these findings indicate that IL-6-mediated Mcl-1 expression in infected macrophages is regulated through the NFκB pathway.


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)

NFκB is required for Mcl-1 expression induced by S. aureus. (a, b) hMDMs were pretreated for 30 min with Bay 11-7095 (40 μM) followed by S. aureus infection at an MOI of 50. After the indicated times, RNA and protein were extracted and Mcl-1 expression levels were determined by qRT-PCR and immunoblot (a and b, resp.). Data represent the means ± SD from three separate experiments. *P < 0.05. (c) The effect of NFκB inhibition on IL-6 expression was measured by qRT-PCR. Shown are the mean values calculated from the results of five independent real-time reactions using hMDMs derived from different donors. Bars represent mean relative expression ± SD; *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569898&req=5

fig6: NFκB is required for Mcl-1 expression induced by S. aureus. (a, b) hMDMs were pretreated for 30 min with Bay 11-7095 (40 μM) followed by S. aureus infection at an MOI of 50. After the indicated times, RNA and protein were extracted and Mcl-1 expression levels were determined by qRT-PCR and immunoblot (a and b, resp.). Data represent the means ± SD from three separate experiments. *P < 0.05. (c) The effect of NFκB inhibition on IL-6 expression was measured by qRT-PCR. Shown are the mean values calculated from the results of five independent real-time reactions using hMDMs derived from different donors. Bars represent mean relative expression ± SD; *P < 0.05.
Mentions: A variety of intracellular signalling pathways are activated by pathogens. Among them, activation of NFκB has been shown to be critical for cytoprotection of infected cells [19]. Moreover, S. aureus is a potent inducer of NFκB activity as was confirmed in infected macrophages by EMSA (Supplement Figure 4). To determine the effect of inhibition of the NFκB pathway on Mcl-1 expression in hMDMs, macrophages were infected with S. aureus followed by incubation for 6 h with a specific NFκB-inhibitor, and then the levels of MCL1 were assessed in comparison to untreated infected cells. As seen in Figure 6(a), inhibition of the NFκB pathway abrogated the S. aureus-induced increase in MCL1 gene transcription. This effect was confirmed at the protein level as well (Figure 6(b)). These results strongly suggested that Mcl-1 expression in S. aureus-infected cells is dependent on NFκB. Since IL-6 transcription is upregulated in an NFκB-dependent manner [20], we investigated the possibility that NFκB stimulated MCL1 expression indirectly, via IL-6. S. aureus-induced production of IL-6 in infected macrophages was abrogated by treatment with Bay 11-7095, the NFκB-specific cell-permeable inhibitor, which indicated that IL-6 production was absolutely dependent on NFκB (Figure 6(c)). Taken together, these findings indicate that IL-6-mediated Mcl-1 expression in infected macrophages is regulated through the NFκB pathway.

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