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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 are also involved in LPS/TNF induced NO generation in macrophages.(A) WT and SphK-1++ macrophages were stimulated with LPS with and without S1P/DHS and NO was quantified at indicated time intervals. (B, C) Sphk-1 knockdown inhibits LPS and TNF-α induced generation of NO in macrophages. Both control and Sphk-1 knockdown macrophages were stimulated with LPS (B) and TNF-α (C) and NO was quantified at indicated time intervals. (D) The effect of Sphk-1 knockdown was validated in LPS or TNF-α induced generation of NO and expression of iNOs proteins at different time intervals. Shown here is the representative blot from two independent experiments. Data in all figures are represented as mean of µM ± SEM from three independent experiments. The dotted line in all figures represents the constitutive NO titre in macrophages. The values above this line represent the actual titre of NO induced by various treatments. **Indicates p≤0.01; * indicates p≤0.05).
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pone-0010657-g005: Sphk-1 are also involved in LPS/TNF induced NO generation in macrophages.(A) WT and SphK-1++ macrophages were stimulated with LPS with and without S1P/DHS and NO was quantified at indicated time intervals. (B, C) Sphk-1 knockdown inhibits LPS and TNF-α induced generation of NO in macrophages. Both control and Sphk-1 knockdown macrophages were stimulated with LPS (B) and TNF-α (C) and NO was quantified at indicated time intervals. (D) The effect of Sphk-1 knockdown was validated in LPS or TNF-α induced generation of NO and expression of iNOs proteins at different time intervals. Shown here is the representative blot from two independent experiments. Data in all figures are represented as mean of µM ± SEM from three independent experiments. The dotted line in all figures represents the constitutive NO titre in macrophages. The values above this line represent the actual titre of NO induced by various treatments. **Indicates p≤0.01; * indicates p≤0.05).

Mentions: To further demonstrate the involvement of SphK-1 in NO generation via innate signaling pathways, we investigated the effect of Sphk-1 inhibition on LPS induced NO generation. To show this, WT and SphK-1++ macrophages were stimulated with LPS in the presence of DHS and/or S1P and the NO titre was quantified. Treatment of LPS stimulated macrophages with DHS inhibited LPS induced NO generation significantly (Fig. 5A). The NO titers remained consistently higher in SphK-1++ stimulated macrophages in comparison to WT macrophages (Fig. 5A). Although exogenous supplementation of macrophages with S1P did not affect LPS induced NO generation in either of these macrophages (Fig. 5A), it inhibited M. smegmatis induced NO significantly in both (Fig. S4A). SiRNA knockdown of Sphk-1 also inhibited either LPS (Fig. 5B) or TNF-α (Fig. 5C) induced NO titre significantly as well as expression of iNOs proteins in comparison to either mock transfected (scrambled siRNA) or untransfected control macrophages (Fig. 5D). Simultaneous treatment of LPS stimulated/infected and S1P supplemented macrophages with DHS again diminished NO titre in both these hosts (Fig. S4A). In comparison to WT macrophages, SphK-1++ macrophages produced significantly higher NO upon their stimulation with TNF-α and IFN-γ both in the presence or absence of iNOS modulators (Fig. S4B, C). Immunoblot analysis also revealed a downregulation in the expression of iNOs proteins by DHS in LPS/TNFα/IFN-γ stimulated macrophages (Fig. S4D). Control sphingolipids were unable to modulate either LPS (Fig. S5A) or TNF-α (Fig. S5B) induced NO titre in macrophages and excluded the Sphk-1 unspecific effect on NO generation. Next we additionally validated the role of Sphk-1 in NO in mouse primary macrophages. For that purpose we isolated Mac-1+ mouse peritoneal macrophages from C57BL6/j mice as per the method described. Treatment of LPS stimulated (Fig. 6A) or infected (Fig. 6B) CD11b+ mouse peritoneal macrophage with DHS reduced NO titre in these macrophages. To further confirm the novel role of SphK-1 on NO generation, we compared the NO titre among stimulated WT and SphK-1 KO Mac-1+ peritoneal macrophages. As expected, Sphk-1 KO peritoneal macrophages produced significantly less NO in comparison to WT macrophages upon their stimulation with either LPS or TNF-α (Fig. 6C). These results verified and confirmed the novel and specific regulation of SphK-1 in the generation of NO in macrophages.


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 are also involved in LPS/TNF induced NO generation in macrophages.(A) WT and SphK-1++ macrophages were stimulated with LPS with and without S1P/DHS and NO was quantified at indicated time intervals. (B, C) Sphk-1 knockdown inhibits LPS and TNF-α induced generation of NO in macrophages. Both control and Sphk-1 knockdown macrophages were stimulated with LPS (B) and TNF-α (C) and NO was quantified at indicated time intervals. (D) The effect of Sphk-1 knockdown was validated in LPS or TNF-α induced generation of NO and expression of iNOs proteins at different time intervals. Shown here is the representative blot from two independent experiments. Data in all figures are represented as mean of µM ± SEM from three independent experiments. The dotted line in all figures represents the constitutive NO titre in macrophages. The values above this line represent the actual titre of NO induced by various treatments. **Indicates p≤0.01; * indicates p≤0.05).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2871783&req=5

pone-0010657-g005: Sphk-1 are also involved in LPS/TNF induced NO generation in macrophages.(A) WT and SphK-1++ macrophages were stimulated with LPS with and without S1P/DHS and NO was quantified at indicated time intervals. (B, C) Sphk-1 knockdown inhibits LPS and TNF-α induced generation of NO in macrophages. Both control and Sphk-1 knockdown macrophages were stimulated with LPS (B) and TNF-α (C) and NO was quantified at indicated time intervals. (D) The effect of Sphk-1 knockdown was validated in LPS or TNF-α induced generation of NO and expression of iNOs proteins at different time intervals. Shown here is the representative blot from two independent experiments. Data in all figures are represented as mean of µM ± SEM from three independent experiments. The dotted line in all figures represents the constitutive NO titre in macrophages. The values above this line represent the actual titre of NO induced by various treatments. **Indicates p≤0.01; * indicates p≤0.05).
Mentions: To further demonstrate the involvement of SphK-1 in NO generation via innate signaling pathways, we investigated the effect of Sphk-1 inhibition on LPS induced NO generation. To show this, WT and SphK-1++ macrophages were stimulated with LPS in the presence of DHS and/or S1P and the NO titre was quantified. Treatment of LPS stimulated macrophages with DHS inhibited LPS induced NO generation significantly (Fig. 5A). The NO titers remained consistently higher in SphK-1++ stimulated macrophages in comparison to WT macrophages (Fig. 5A). Although exogenous supplementation of macrophages with S1P did not affect LPS induced NO generation in either of these macrophages (Fig. 5A), it inhibited M. smegmatis induced NO significantly in both (Fig. S4A). SiRNA knockdown of Sphk-1 also inhibited either LPS (Fig. 5B) or TNF-α (Fig. 5C) induced NO titre significantly as well as expression of iNOs proteins in comparison to either mock transfected (scrambled siRNA) or untransfected control macrophages (Fig. 5D). Simultaneous treatment of LPS stimulated/infected and S1P supplemented macrophages with DHS again diminished NO titre in both these hosts (Fig. S4A). In comparison to WT macrophages, SphK-1++ macrophages produced significantly higher NO upon their stimulation with TNF-α and IFN-γ both in the presence or absence of iNOS modulators (Fig. S4B, C). Immunoblot analysis also revealed a downregulation in the expression of iNOs proteins by DHS in LPS/TNFα/IFN-γ stimulated macrophages (Fig. S4D). Control sphingolipids were unable to modulate either LPS (Fig. S5A) or TNF-α (Fig. S5B) induced NO titre in macrophages and excluded the Sphk-1 unspecific effect on NO generation. Next we additionally validated the role of Sphk-1 in NO in mouse primary macrophages. For that purpose we isolated Mac-1+ mouse peritoneal macrophages from C57BL6/j mice as per the method described. Treatment of LPS stimulated (Fig. 6A) or infected (Fig. 6B) CD11b+ mouse peritoneal macrophage with DHS reduced NO titre in these macrophages. To further confirm the novel role of SphK-1 on NO generation, we compared the NO titre among stimulated WT and SphK-1 KO Mac-1+ peritoneal macrophages. As expected, Sphk-1 KO peritoneal macrophages produced significantly less NO in comparison to WT macrophages upon their stimulation with either LPS or TNF-α (Fig. 6C). These results verified and confirmed the novel and specific regulation of SphK-1 in the generation of NO in macrophages.

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