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Sphingosine kinase-1 (SphK-1) regulates Mycobacterium smegmatis infection in macrophages.

Prakash H, Lüth A, Grinkina N, Holzer D, Wadgaonkar R, Gonzalez AP, Anes E, Kleuser B - PLoS ONE (2010)

Bottom Line: This led to a reduction in the generation of NO and secretion of TNF-alpha in infected macrophages.Enhanced NO generation and expression of iNOs in SphK-1++ infected macrophages demonstrated their M-1(bright) phenotype of these macrophages.These findings thus suggested a novel antimycobacterial role of SphK-1 in macrophages.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany. h.prakash@dkfz-heidelberg.de

ABSTRACT
Sphingosine kinase-1 is known to mediate Mycobacterium smegmatis induced inflammatory responses in macrophages, but its role in controlling infection has not been reported to date. We aimed to unravel the significance of SphK-1 in controlling M. smegmatis infection in RAW 264.7 macrophages. Our results demonstrated for the first time that selective inhibition of SphK-1 by either D, L threo dihydrosphingosine (DHS; a competitive inhibitor of Sphk-1) or Sphk-1 siRNA rendered RAW macrophages sensitive to M. smegmatis infection. This was due to the reduction in the expression of iNOs, p38, pp-38, late phagosomal marker, LAMP-2 and stabilization of the RelA (pp-65) subunit of NF-kappaB. This led to a reduction in the generation of NO and secretion of TNF-alpha in infected macrophages. Congruently, overexpression of SphK-1 conferred resistance in macrophages to infection which was due to enhancement in the generation of NO and expression of iNOs, pp38 and LAMP-2. In addition, our results also unraveled a novel regulation of p38MAPK by SphK-1 during M. smegmatis infection and generation of NO in macrophages. Enhanced NO generation and expression of iNOs in SphK-1++ infected macrophages demonstrated their M-1(bright) phenotype of these macrophages. These findings thus suggested a novel antimycobacterial role of SphK-1 in macrophages.

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SphK-1 regulates M. smegmatis infection induced NO generation in macrophages.(A) WT macrophages were infected with M. smegmatis with and without DHS for indicated time intervals and their culture supernatants were collected to measure inducible NO as NO2 as described. (B) Sphk-1 knockdown inhibits M. smegmatis induced NO generation. Both control and Sphk-1 siRNA knockdown macrophages were infected with M. smegmatis and NO was quantified in their culture supernatant at indicated time intervals. (C) Complementation of NO by SphK-1 overexpression in macrophages. Both WT and SphK-1++ macrophages were infected with M. smegmatis with and without DHS and NO was quantified in the culture supernatants at indicated time points. (D) SphK-1 overexpression restores the functional integrity of NO response in infected macrophages. Both WT and SphK-1++ macrophages were infected with increasing doses of M. smegmatis and co-stimulated with LPS (positive control). The culture supernatants were collected at indicated time intervals and NO was quantified. The dotted line in all figures represents the constitutive NO titre in macrophages. The values above this line represent the actual titre of NO being induced by various treatments. ** Indicates p≤0.01; * indicates p≤0.05.
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pone-0010657-g004: SphK-1 regulates M. smegmatis infection induced NO generation in macrophages.(A) WT macrophages were infected with M. smegmatis with and without DHS for indicated time intervals and their culture supernatants were collected to measure inducible NO as NO2 as described. (B) Sphk-1 knockdown inhibits M. smegmatis induced NO generation. Both control and Sphk-1 siRNA knockdown macrophages were infected with M. smegmatis and NO was quantified in their culture supernatant at indicated time intervals. (C) Complementation of NO by SphK-1 overexpression in macrophages. Both WT and SphK-1++ macrophages were infected with M. smegmatis with and without DHS and NO was quantified in the culture supernatants at indicated time points. (D) SphK-1 overexpression restores the functional integrity of NO response in infected macrophages. Both WT and SphK-1++ macrophages were infected with increasing doses of M. smegmatis and co-stimulated with LPS (positive control). The culture supernatants were collected at indicated time intervals and NO was quantified. The dotted line in all figures represents the constitutive NO titre in macrophages. The values above this line represent the actual titre of NO being induced by various treatments. ** Indicates p≤0.01; * indicates p≤0.05.

Mentions: In order to clarify the potential reason for sensitization of Sphk-1 inhibited macrophages to infection, we investigated the effect of SphK-1 inhibition on M. smegmatis infection induced NO generation in macrophages due to the fact that NO is a well known marker for classically activated M-1 professional macrophages and is involved in antibacterial defenses [40]–[45]. In macrophages, NO is produced by the iNOs enzyme and capable of neutralizing a wide variety of mycobacterial membrane lipids and other components and indeed is involved in maturation of phagosomes during mycobacterial infection [46]–[48]. During the first 4 h time period of infection, no significant increase in NO was observed in the infected macrophages. The NO titre increased slightly at the 9th h post infection and significantly at the 24th h post infection in comparison to the uninfected control (Fig. 4A). Inhibition of SphK-1 significantly reduced M. smegmatis induced NO titre during the course of infection (Fig. 4A). siRNA knockdown of Sphk-1 also reduced infection induced NO titre significantly in comparison to mock (scrambled) transfected or untransfected control (Fig. 4B). Interestingly, Sphk-1++ macrophages, in comparison to WT macrophages, produced significantly higher NO in response to infection (Fig. 4C). Moreover, SphK-1 overexpression replenished DHS mediated loss of NO in macrophages (Fig. 4C). As expected, control sphingolipids could not modulate infection induced NO titre in macrophages (Fig. S3C).


Sphingosine kinase-1 (SphK-1) regulates Mycobacterium smegmatis infection in macrophages.

Prakash H, Lüth A, Grinkina N, Holzer D, Wadgaonkar R, Gonzalez AP, Anes E, Kleuser B - PLoS ONE (2010)

SphK-1 regulates M. smegmatis infection induced NO generation in macrophages.(A) WT macrophages were infected with M. smegmatis with and without DHS for indicated time intervals and their culture supernatants were collected to measure inducible NO as NO2 as described. (B) Sphk-1 knockdown inhibits M. smegmatis induced NO generation. Both control and Sphk-1 siRNA knockdown macrophages were infected with M. smegmatis and NO was quantified in their culture supernatant at indicated time intervals. (C) Complementation of NO by SphK-1 overexpression in macrophages. Both WT and SphK-1++ macrophages were infected with M. smegmatis with and without DHS and NO was quantified in the culture supernatants at indicated time points. (D) SphK-1 overexpression restores the functional integrity of NO response in infected macrophages. Both WT and SphK-1++ macrophages were infected with increasing doses of M. smegmatis and co-stimulated with LPS (positive control). The culture supernatants were collected at indicated time intervals and NO was quantified. The dotted line in all figures represents the constitutive NO titre in macrophages. The values above this line represent the actual titre of NO being induced by various treatments. ** Indicates p≤0.01; * indicates p≤0.05.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2871783&req=5

pone-0010657-g004: SphK-1 regulates M. smegmatis infection induced NO generation in macrophages.(A) WT macrophages were infected with M. smegmatis with and without DHS for indicated time intervals and their culture supernatants were collected to measure inducible NO as NO2 as described. (B) Sphk-1 knockdown inhibits M. smegmatis induced NO generation. Both control and Sphk-1 siRNA knockdown macrophages were infected with M. smegmatis and NO was quantified in their culture supernatant at indicated time intervals. (C) Complementation of NO by SphK-1 overexpression in macrophages. Both WT and SphK-1++ macrophages were infected with M. smegmatis with and without DHS and NO was quantified in the culture supernatants at indicated time points. (D) SphK-1 overexpression restores the functional integrity of NO response in infected macrophages. Both WT and SphK-1++ macrophages were infected with increasing doses of M. smegmatis and co-stimulated with LPS (positive control). The culture supernatants were collected at indicated time intervals and NO was quantified. The dotted line in all figures represents the constitutive NO titre in macrophages. The values above this line represent the actual titre of NO being induced by various treatments. ** Indicates p≤0.01; * indicates p≤0.05.
Mentions: In order to clarify the potential reason for sensitization of Sphk-1 inhibited macrophages to infection, we investigated the effect of SphK-1 inhibition on M. smegmatis infection induced NO generation in macrophages due to the fact that NO is a well known marker for classically activated M-1 professional macrophages and is involved in antibacterial defenses [40]–[45]. In macrophages, NO is produced by the iNOs enzyme and capable of neutralizing a wide variety of mycobacterial membrane lipids and other components and indeed is involved in maturation of phagosomes during mycobacterial infection [46]–[48]. During the first 4 h time period of infection, no significant increase in NO was observed in the infected macrophages. The NO titre increased slightly at the 9th h post infection and significantly at the 24th h post infection in comparison to the uninfected control (Fig. 4A). Inhibition of SphK-1 significantly reduced M. smegmatis induced NO titre during the course of infection (Fig. 4A). siRNA knockdown of Sphk-1 also reduced infection induced NO titre significantly in comparison to mock (scrambled) transfected or untransfected control (Fig. 4B). Interestingly, Sphk-1++ macrophages, in comparison to WT macrophages, produced significantly higher NO in response to infection (Fig. 4C). Moreover, SphK-1 overexpression replenished DHS mediated loss of NO in macrophages (Fig. 4C). As expected, control sphingolipids could not modulate infection induced NO titre in macrophages (Fig. S3C).

Bottom Line: This led to a reduction in the generation of NO and secretion of TNF-alpha in infected macrophages.Enhanced NO generation and expression of iNOs in SphK-1++ infected macrophages demonstrated their M-1(bright) phenotype of these macrophages.These findings thus suggested a novel antimycobacterial role of SphK-1 in macrophages.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany. h.prakash@dkfz-heidelberg.de

ABSTRACT
Sphingosine kinase-1 is known to mediate Mycobacterium smegmatis induced inflammatory responses in macrophages, but its role in controlling infection has not been reported to date. We aimed to unravel the significance of SphK-1 in controlling M. smegmatis infection in RAW 264.7 macrophages. Our results demonstrated for the first time that selective inhibition of SphK-1 by either D, L threo dihydrosphingosine (DHS; a competitive inhibitor of Sphk-1) or Sphk-1 siRNA rendered RAW macrophages sensitive to M. smegmatis infection. This was due to the reduction in the expression of iNOs, p38, pp-38, late phagosomal marker, LAMP-2 and stabilization of the RelA (pp-65) subunit of NF-kappaB. This led to a reduction in the generation of NO and secretion of TNF-alpha in infected macrophages. Congruently, overexpression of SphK-1 conferred resistance in macrophages to infection which was due to enhancement in the generation of NO and expression of iNOs, pp38 and LAMP-2. In addition, our results also unraveled a novel regulation of p38MAPK by SphK-1 during M. smegmatis infection and generation of NO in macrophages. Enhanced NO generation and expression of iNOs in SphK-1++ infected macrophages demonstrated their M-1(bright) phenotype of these macrophages. These findings thus suggested a novel antimycobacterial role of SphK-1 in macrophages.

Show MeSH
Related in: MedlinePlus