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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 promotes growth of M. bovis BCG and M. tuberculosis in infected BMDM. BMDM were infected with M. bovis BCG (A) or with M. tuberculosis(B) and treated (+) or not (−) with IL-17. (A) Four days post-infection, the bacterial load was assessed. Fifty out of 50 independent experiments are represented in the graph. The mean-fold increase in CFUs induced by IL-17 is 0.4 ± 0.2 log. (B) Three days post-infection, the bacterial load was assessed. Eleven out of 11 independent experiments are represented in the graph. The mean-fold increase in CFUs induced by IL-17 is 0.28 ± 0.18 log. Significance determined by Student’s t test (***p < 0.001). BMDM were left uninfected or infected with M. bovis BCG and treated (+) or not (−) with IL-17. (C) Nitrites production by was measured in the supernatants of the cultures by the Griess method. (D) The mRNA expression of LRG47 was quantified by real-time PCR using the primers (sense 5′-CTCTGGATCAGGGTTTGAGGAGTA-3′; anti-sense 5′-GGAACT GTGATGGTTTCATGATA-3′) and probes (5′-LCred640-AGGTCCACAGACAGCGTCACTCGG-P-3′; 5′-AACCAGAGAGCCTCACCAGG GAGCTGA-FL-3′) and normalized to HPRT. The fold increase of LRG47 mRNA expression over NI control was calculated. Represented are the mean ± SE of three independent experiments. (E,F) At different time points post-infection, supernatants were harvested and the production of TNF (E) and IL-10 (F) assessed by immunoassay. Data point represents a mean of n = 6. Results are from one representative out of three independent experiments.
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Figure 1: IL-17 promotes growth of M. bovis BCG and M. tuberculosis in infected BMDM. BMDM were infected with M. bovis BCG (A) or with M. tuberculosis(B) and treated (+) or not (−) with IL-17. (A) Four days post-infection, the bacterial load was assessed. Fifty out of 50 independent experiments are represented in the graph. The mean-fold increase in CFUs induced by IL-17 is 0.4 ± 0.2 log. (B) Three days post-infection, the bacterial load was assessed. Eleven out of 11 independent experiments are represented in the graph. The mean-fold increase in CFUs induced by IL-17 is 0.28 ± 0.18 log. Significance determined by Student’s t test (***p < 0.001). BMDM were left uninfected or infected with M. bovis BCG and treated (+) or not (−) with IL-17. (C) Nitrites production by was measured in the supernatants of the cultures by the Griess method. (D) The mRNA expression of LRG47 was quantified by real-time PCR using the primers (sense 5′-CTCTGGATCAGGGTTTGAGGAGTA-3′; anti-sense 5′-GGAACT GTGATGGTTTCATGATA-3′) and probes (5′-LCred640-AGGTCCACAGACAGCGTCACTCGG-P-3′; 5′-AACCAGAGAGCCTCACCAGG GAGCTGA-FL-3′) and normalized to HPRT. The fold increase of LRG47 mRNA expression over NI control was calculated. Represented are the mean ± SE of three independent experiments. (E,F) At different time points post-infection, supernatants were harvested and the production of TNF (E) and IL-10 (F) assessed by immunoassay. Data point represents a mean of n = 6. Results are from one representative out of three independent experiments.

Mentions: IL-17 production is induced upon M. bovis BCG and M. tuberculosis infection, with γδ T cells as the major cellular source during the early stage of the infection (19, 25). However, the impact of this cytokine on mycobacterially infected macrophages remains to be investigated. To address this, mouse primary BMDM were infected with M. bovis BCG in the presence or absence of IL-17 and, 4 days after infection, the number of viable bacteria present in each condition was determined. A significantly higher number of viable bacteria were consistently found in macrophages stimulated with IL-17 (Figure 1A). Importantly, this effect was not specific for M. bovis BCG as the ability of macrophages to control M. tuberculosis was also significantly hampered by IL-17 (Figure 1B). These data suggest that IL-17 renders mycobacterially infected macrophages more permissive to bacterial growth. Notably, IL-17 did not interfere with basic macrophage microbicidal mechanisms, such as nitric oxide production (Figure 1C) or LRG47 expression, a marker for phagolysosome formation (Figure 1D). Since TNF and IL-10 play important roles on the regulation of apoptosis in mycobacterially infected macrophages (11, 12), a possible regulation of these molecules by IL-17 was next investigated. However, IL-17 did not impact the production of TNF (Figure 1E) or of IL-10 (Figure 1F) observed in macrophages upon M. bovis BCG infection.


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 promotes growth of M. bovis BCG and M. tuberculosis in infected BMDM. BMDM were infected with M. bovis BCG (A) or with M. tuberculosis(B) and treated (+) or not (−) with IL-17. (A) Four days post-infection, the bacterial load was assessed. Fifty out of 50 independent experiments are represented in the graph. The mean-fold increase in CFUs induced by IL-17 is 0.4 ± 0.2 log. (B) Three days post-infection, the bacterial load was assessed. Eleven out of 11 independent experiments are represented in the graph. The mean-fold increase in CFUs induced by IL-17 is 0.28 ± 0.18 log. Significance determined by Student’s t test (***p < 0.001). BMDM were left uninfected or infected with M. bovis BCG and treated (+) or not (−) with IL-17. (C) Nitrites production by was measured in the supernatants of the cultures by the Griess method. (D) The mRNA expression of LRG47 was quantified by real-time PCR using the primers (sense 5′-CTCTGGATCAGGGTTTGAGGAGTA-3′; anti-sense 5′-GGAACT GTGATGGTTTCATGATA-3′) and probes (5′-LCred640-AGGTCCACAGACAGCGTCACTCGG-P-3′; 5′-AACCAGAGAGCCTCACCAGG GAGCTGA-FL-3′) and normalized to HPRT. The fold increase of LRG47 mRNA expression over NI control was calculated. Represented are the mean ± SE of three independent experiments. (E,F) At different time points post-infection, supernatants were harvested and the production of TNF (E) and IL-10 (F) assessed by immunoassay. Data point represents a mean of n = 6. Results are from one representative out of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588696&req=5

Figure 1: IL-17 promotes growth of M. bovis BCG and M. tuberculosis in infected BMDM. BMDM were infected with M. bovis BCG (A) or with M. tuberculosis(B) and treated (+) or not (−) with IL-17. (A) Four days post-infection, the bacterial load was assessed. Fifty out of 50 independent experiments are represented in the graph. The mean-fold increase in CFUs induced by IL-17 is 0.4 ± 0.2 log. (B) Three days post-infection, the bacterial load was assessed. Eleven out of 11 independent experiments are represented in the graph. The mean-fold increase in CFUs induced by IL-17 is 0.28 ± 0.18 log. Significance determined by Student’s t test (***p < 0.001). BMDM were left uninfected or infected with M. bovis BCG and treated (+) or not (−) with IL-17. (C) Nitrites production by was measured in the supernatants of the cultures by the Griess method. (D) The mRNA expression of LRG47 was quantified by real-time PCR using the primers (sense 5′-CTCTGGATCAGGGTTTGAGGAGTA-3′; anti-sense 5′-GGAACT GTGATGGTTTCATGATA-3′) and probes (5′-LCred640-AGGTCCACAGACAGCGTCACTCGG-P-3′; 5′-AACCAGAGAGCCTCACCAGG GAGCTGA-FL-3′) and normalized to HPRT. The fold increase of LRG47 mRNA expression over NI control was calculated. Represented are the mean ± SE of three independent experiments. (E,F) At different time points post-infection, supernatants were harvested and the production of TNF (E) and IL-10 (F) assessed by immunoassay. Data point represents a mean of n = 6. Results are from one representative out of three independent experiments.
Mentions: IL-17 production is induced upon M. bovis BCG and M. tuberculosis infection, with γδ T cells as the major cellular source during the early stage of the infection (19, 25). However, the impact of this cytokine on mycobacterially infected macrophages remains to be investigated. To address this, mouse primary BMDM were infected with M. bovis BCG in the presence or absence of IL-17 and, 4 days after infection, the number of viable bacteria present in each condition was determined. A significantly higher number of viable bacteria were consistently found in macrophages stimulated with IL-17 (Figure 1A). Importantly, this effect was not specific for M. bovis BCG as the ability of macrophages to control M. tuberculosis was also significantly hampered by IL-17 (Figure 1B). These data suggest that IL-17 renders mycobacterially infected macrophages more permissive to bacterial growth. Notably, IL-17 did not interfere with basic macrophage microbicidal mechanisms, such as nitric oxide production (Figure 1C) or LRG47 expression, a marker for phagolysosome formation (Figure 1D). Since TNF and IL-10 play important roles on the regulation of apoptosis in mycobacterially infected macrophages (11, 12), a possible regulation of these molecules by IL-17 was next investigated. However, IL-17 did not impact the production of TNF (Figure 1E) or of IL-10 (Figure 1F) observed in macrophages upon M. bovis BCG infection.

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