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IL-17A Promotes Intracellular Growth of Mycobacterium by Inhibiting Apoptosis of Infected Macrophages.

Cruz A, Ludovico P, Torrado E, Gama JB, Sousa J, Gaifem J, Appelberg R, Rodrigues F, Cooper AM, Pedrosa J, Saraiva M, Castro AG - Front Immunol (2015)

Bottom Line: Mechanistically, we show that IL-17 inhibits p53, and impacts on the intrinsic apoptotic pathway, by increasing the Bcl2 and decreasing Bax expression, decreasing cytochrome c release from the mitochondria, and inhibiting caspase-3 activation.The same effect of IL-17 was observed in infected macrophages upon blockade of p53 nuclear translocation.These results reveal a previously unappreciated role for the IL-17/p53 axis in the regulation of mycobacteria-induced apoptosis and can have important implications in a broad spectrum of diseases where apoptosis of the infected cell is an important host defense mechanism.

View Article: PubMed Central - PubMed

Affiliation: Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho , Braga , Portugal ; ICVS/3B's - PT Government Associate Laboratory, University of Minho , Braga , Portugal.

ABSTRACT
The fate of infected macrophages is a critical aspect of immunity to mycobacteria. By depriving the pathogen of its intracellular niche, apoptotic death of the infected macrophage has been shown to be an important mechanism to control bacterial growth. Here, we show that IL-17 inhibits apoptosis of Mycobacterium bovis BCG- or Mycobacterium tuberculosis-infected macrophages thus hampering their ability to control bacterial growth. Mechanistically, we show that IL-17 inhibits p53, and impacts on the intrinsic apoptotic pathway, by increasing the Bcl2 and decreasing Bax expression, decreasing cytochrome c release from the mitochondria, and inhibiting caspase-3 activation. The same effect of IL-17 was observed in infected macrophages upon blockade of p53 nuclear translocation. These results reveal a previously unappreciated role for the IL-17/p53 axis in the regulation of mycobacteria-induced apoptosis and can have important implications in a broad spectrum of diseases where apoptosis of the infected cell is an important host defense mechanism.

No MeSH data available.


Related in: MedlinePlus

IL-17 inhibits macrophage apoptosis induced by mycobacterial infection. BMDM were infected with M. bovis BCG in the presence or absence of IL-17 as indicated. (A) On days 2 and 7 post-infection, the cell viability was assessed by enumerating nuclei. (B) On days 2, 4, and 5 post-M. bovis BCG infection, activation of caspase-3 was assessed by immunofluorescence. (C) At 48 or 72 h post-M. bovis BCG infection, full-length and cleaved forms of caspase-3 protein were determined by western blot. (D,E) At 24 or 48 h post-M. bovis BCG infection, the Bcl2 and Bax mRNA or at 48 or 72 h post-M. bovis BCG infection, the protein were determined by real-time PCR or western blot respectively.(F) Cytochrome c release from the mitochondria was determined 2 days post-infection by flow cytometry. The fold increase of cytochrome c released over NI control was calculated for each independent experiment. Representative images used for (A) and (B) are in Figure S2 in Supplementary Material. Represented are the mean ± SE of four independent experiments. Significance determined by one-way ANOVA (A,B) or Student’s t test (D–F) (*p < 0.05; **p < 0.01; ***p < 0.001).
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Figure 3: IL-17 inhibits macrophage apoptosis induced by mycobacterial infection. BMDM were infected with M. bovis BCG in the presence or absence of IL-17 as indicated. (A) On days 2 and 7 post-infection, the cell viability was assessed by enumerating nuclei. (B) On days 2, 4, and 5 post-M. bovis BCG infection, activation of caspase-3 was assessed by immunofluorescence. (C) At 48 or 72 h post-M. bovis BCG infection, full-length and cleaved forms of caspase-3 protein were determined by western blot. (D,E) At 24 or 48 h post-M. bovis BCG infection, the Bcl2 and Bax mRNA or at 48 or 72 h post-M. bovis BCG infection, the protein were determined by real-time PCR or western blot respectively.(F) Cytochrome c release from the mitochondria was determined 2 days post-infection by flow cytometry. The fold increase of cytochrome c released over NI control was calculated for each independent experiment. Representative images used for (A) and (B) are in Figure S2 in Supplementary Material. Represented are the mean ± SE of four independent experiments. Significance determined by one-way ANOVA (A,B) or Student’s t test (D–F) (*p < 0.05; **p < 0.01; ***p < 0.001).

Mentions: Given that IL-17 modulated p53 protein, this cytokine could impact on the induction of apoptotic programed cell death, an important antimicrobial mechanism operating upon mycobacterial infection (26). Thus, the role of IL-17 on the survival of infected macrophages was investigated. First, the viability of BMDM at different times post-infection with M. bovis BCG was measured. As expected, the survival of infected macrophages decreased over time as compared to non-infected (NI) cells. However, the presence of IL-17 increased the survival of infected macrophages to levels similar to those of NI cells (Figure 3A; Figure S2A in Supplementary Material). Next, BMDM were infected with M. bovis BCG in the absence or presence of IL-17 and the activation of caspase-3, a major apoptotic effector caspase, was determined by immunofluorescence microscopy. We found that the presence of IL-17 resulted in a reduction in the percentage of caspase-3-positive macrophages through day 5 of infection with M. bovis BCG (Figure 3B; Figure S2B in Supplementary Material) or with M. tuberculosis (Figure S2C in Supplementary Material). Additionally, and in line with the immunofluorescence data, we observed a decrease in the protein levels of activated (cleaved) caspase-3 upon addition of IL-17 to BCG-infected macrophages, as determined by western blot (Figure 3C). Since our previous data (Figure 2) showed that IL-17 reduced p53 protein and since p53 is a master regulator of pro- and anti-apoptotic molecules (29), namely Bax and Bcl2, we next explored the impact of IL-17 on these molecules. Upon infection with M. bovis BCG, the transcription of Bcl2 was not much affected when compared to NI cells, whereas that of Bax was significantly increased (Figure 3D). In the presence of IL-17, a marked increase of Bcl2 transcription accompanied by a decrease of Bax mRNA was observed (Figure 3D). The increased Bcl2 transcription observed in the presence of IL-17 was confirmed at the protein level, as measured by western blot (Figure 3E). In contrast, addition of IL-17 did not affect the amount of Bax protein (Figure 3E). In all, in the presence of IL-17, the ratio of Bcl2/Bax proteins was significantly increased, suggesting an anti-apoptotic activity for IL-17. The ratio of Bcl2 to Bax is an important component of the intrinsic apoptotic pathway that regulates the permeabilization of the outer mitochondrial membrane, and thereby the release of cytochrome c. Therefore, the release of cytochrome c in M. bovis BCG-infected macrophages in the presence or absence of IL-17 was determined. We found that cytochrome c release was observed upon infection of macrophages, in line with the activation of the intrinsic apoptotic pathway (Figure 3F). Further, it was clear that cytochrome c release induced by BCG was abrogated in the presence of IL-17 (Figure 3F). These data suggest that IL-17 inhibits the intrinsic apoptotic pathway by altering the Bcl2/Bax ratio and the release of cytochrome c.


IL-17A Promotes Intracellular Growth of Mycobacterium by Inhibiting Apoptosis of Infected Macrophages.

Cruz A, Ludovico P, Torrado E, Gama JB, Sousa J, Gaifem J, Appelberg R, Rodrigues F, Cooper AM, Pedrosa J, Saraiva M, Castro AG - Front Immunol (2015)

IL-17 inhibits macrophage apoptosis induced by mycobacterial infection. BMDM were infected with M. bovis BCG in the presence or absence of IL-17 as indicated. (A) On days 2 and 7 post-infection, the cell viability was assessed by enumerating nuclei. (B) On days 2, 4, and 5 post-M. bovis BCG infection, activation of caspase-3 was assessed by immunofluorescence. (C) At 48 or 72 h post-M. bovis BCG infection, full-length and cleaved forms of caspase-3 protein were determined by western blot. (D,E) At 24 or 48 h post-M. bovis BCG infection, the Bcl2 and Bax mRNA or at 48 or 72 h post-M. bovis BCG infection, the protein were determined by real-time PCR or western blot respectively.(F) Cytochrome c release from the mitochondria was determined 2 days post-infection by flow cytometry. The fold increase of cytochrome c released over NI control was calculated for each independent experiment. Representative images used for (A) and (B) are in Figure S2 in Supplementary Material. Represented are the mean ± SE of four independent experiments. Significance determined by one-way ANOVA (A,B) or Student’s t test (D–F) (*p < 0.05; **p < 0.01; ***p < 0.001).
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Figure 3: IL-17 inhibits macrophage apoptosis induced by mycobacterial infection. BMDM were infected with M. bovis BCG in the presence or absence of IL-17 as indicated. (A) On days 2 and 7 post-infection, the cell viability was assessed by enumerating nuclei. (B) On days 2, 4, and 5 post-M. bovis BCG infection, activation of caspase-3 was assessed by immunofluorescence. (C) At 48 or 72 h post-M. bovis BCG infection, full-length and cleaved forms of caspase-3 protein were determined by western blot. (D,E) At 24 or 48 h post-M. bovis BCG infection, the Bcl2 and Bax mRNA or at 48 or 72 h post-M. bovis BCG infection, the protein were determined by real-time PCR or western blot respectively.(F) Cytochrome c release from the mitochondria was determined 2 days post-infection by flow cytometry. The fold increase of cytochrome c released over NI control was calculated for each independent experiment. Representative images used for (A) and (B) are in Figure S2 in Supplementary Material. Represented are the mean ± SE of four independent experiments. Significance determined by one-way ANOVA (A,B) or Student’s t test (D–F) (*p < 0.05; **p < 0.01; ***p < 0.001).
Mentions: Given that IL-17 modulated p53 protein, this cytokine could impact on the induction of apoptotic programed cell death, an important antimicrobial mechanism operating upon mycobacterial infection (26). Thus, the role of IL-17 on the survival of infected macrophages was investigated. First, the viability of BMDM at different times post-infection with M. bovis BCG was measured. As expected, the survival of infected macrophages decreased over time as compared to non-infected (NI) cells. However, the presence of IL-17 increased the survival of infected macrophages to levels similar to those of NI cells (Figure 3A; Figure S2A in Supplementary Material). Next, BMDM were infected with M. bovis BCG in the absence or presence of IL-17 and the activation of caspase-3, a major apoptotic effector caspase, was determined by immunofluorescence microscopy. We found that the presence of IL-17 resulted in a reduction in the percentage of caspase-3-positive macrophages through day 5 of infection with M. bovis BCG (Figure 3B; Figure S2B in Supplementary Material) or with M. tuberculosis (Figure S2C in Supplementary Material). Additionally, and in line with the immunofluorescence data, we observed a decrease in the protein levels of activated (cleaved) caspase-3 upon addition of IL-17 to BCG-infected macrophages, as determined by western blot (Figure 3C). Since our previous data (Figure 2) showed that IL-17 reduced p53 protein and since p53 is a master regulator of pro- and anti-apoptotic molecules (29), namely Bax and Bcl2, we next explored the impact of IL-17 on these molecules. Upon infection with M. bovis BCG, the transcription of Bcl2 was not much affected when compared to NI cells, whereas that of Bax was significantly increased (Figure 3D). In the presence of IL-17, a marked increase of Bcl2 transcription accompanied by a decrease of Bax mRNA was observed (Figure 3D). The increased Bcl2 transcription observed in the presence of IL-17 was confirmed at the protein level, as measured by western blot (Figure 3E). In contrast, addition of IL-17 did not affect the amount of Bax protein (Figure 3E). In all, in the presence of IL-17, the ratio of Bcl2/Bax proteins was significantly increased, suggesting an anti-apoptotic activity for IL-17. The ratio of Bcl2 to Bax is an important component of the intrinsic apoptotic pathway that regulates the permeabilization of the outer mitochondrial membrane, and thereby the release of cytochrome c. Therefore, the release of cytochrome c in M. bovis BCG-infected macrophages in the presence or absence of IL-17 was determined. We found that cytochrome c release was observed upon infection of macrophages, in line with the activation of the intrinsic apoptotic pathway (Figure 3F). Further, it was clear that cytochrome c release induced by BCG was abrogated in the presence of IL-17 (Figure 3F). These data suggest that IL-17 inhibits the intrinsic apoptotic pathway by altering the Bcl2/Bax ratio and the release of cytochrome c.

Bottom Line: Mechanistically, we show that IL-17 inhibits p53, and impacts on the intrinsic apoptotic pathway, by increasing the Bcl2 and decreasing Bax expression, decreasing cytochrome c release from the mitochondria, and inhibiting caspase-3 activation.The same effect of IL-17 was observed in infected macrophages upon blockade of p53 nuclear translocation.These results reveal a previously unappreciated role for the IL-17/p53 axis in the regulation of mycobacteria-induced apoptosis and can have important implications in a broad spectrum of diseases where apoptosis of the infected cell is an important host defense mechanism.

View Article: PubMed Central - PubMed

Affiliation: Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho , Braga , Portugal ; ICVS/3B's - PT Government Associate Laboratory, University of Minho , Braga , Portugal.

ABSTRACT
The fate of infected macrophages is a critical aspect of immunity to mycobacteria. By depriving the pathogen of its intracellular niche, apoptotic death of the infected macrophage has been shown to be an important mechanism to control bacterial growth. Here, we show that IL-17 inhibits apoptosis of Mycobacterium bovis BCG- or Mycobacterium tuberculosis-infected macrophages thus hampering their ability to control bacterial growth. Mechanistically, we show that IL-17 inhibits p53, and impacts on the intrinsic apoptotic pathway, by increasing the Bcl2 and decreasing Bax expression, decreasing cytochrome c release from the mitochondria, and inhibiting caspase-3 activation. The same effect of IL-17 was observed in infected macrophages upon blockade of p53 nuclear translocation. These results reveal a previously unappreciated role for the IL-17/p53 axis in the regulation of mycobacteria-induced apoptosis and can have important implications in a broad spectrum of diseases where apoptosis of the infected cell is an important host defense mechanism.

No MeSH data available.


Related in: MedlinePlus