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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 overexpression confers resistance to M. smegmatis infection in macrophages.(A) Sphk-1 was overexpressed in macrophages and validated by western blot, immunofluorescence and competitive S1P titers in WT and SphK-1++ macrophages. (B) Sphk-1 overexpression confers resistance to infection. Both WT and SphK-1 ++macrophages were infected with M. smegmatis and mycobacterial killing was observed up to 24 h post infection. (C) The cells under section (B) were treated with DHS and the effect on M. smegmatis killing was again evaluated up to 24 h post infection. (D) S1P regulates mycobacterial growth in macrophages. The cells under section (B) were supplemented with S1P (5 µM) and the effect of S1P on mycobacterial infection was monitored during the first 4 h time period. Data are represented as mean of CFU ± SEM from three independent experiments. ** Indicates p≤0.01; * indicates p≤0.05.
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pone-0010657-g002: SphK-1 overexpression confers resistance to M. smegmatis infection in macrophages.(A) Sphk-1 was overexpressed in macrophages and validated by western blot, immunofluorescence and competitive S1P titers in WT and SphK-1++ macrophages. (B) Sphk-1 overexpression confers resistance to infection. Both WT and SphK-1 ++macrophages were infected with M. smegmatis and mycobacterial killing was observed up to 24 h post infection. (C) The cells under section (B) were treated with DHS and the effect on M. smegmatis killing was again evaluated up to 24 h post infection. (D) S1P regulates mycobacterial growth in macrophages. The cells under section (B) were supplemented with S1P (5 µM) and the effect of S1P on mycobacterial infection was monitored during the first 4 h time period. Data are represented as mean of CFU ± SEM from three independent experiments. ** Indicates p≤0.01; * indicates p≤0.05.

Mentions: To further substantiate whether an upregulation of SphK-1 would counteract bacterial killing, SphK-1 was overexpressed in macrophages (Fig. 2A) and these macrophages (Sphk-1++) were infected with M. smegmatis. Interestingly and expectedly, SphK-1 overexpression conferred resistance to infection in Sphk-1++ macrophages in comparison to WT infected macrophages (Fig. 2B). The increased resistance of SphK-1++ infected macrophages was abolished by DHS treatment (Fig. 2C). Treatment of both WT and Sphk-1++ infected macrophages with S1P (SK reaction product) enhanced bacterial killing in both WT and SphK-1++ infected macrophages. This effect was found more pronounced in SphK-1++ infected macrophages than in WT infected macrophages (Fig. 2D). These observations confirmed the involvement of SphK-1 in controlling M. smegmatis infection 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 overexpression confers resistance to M. smegmatis infection in macrophages.(A) Sphk-1 was overexpressed in macrophages and validated by western blot, immunofluorescence and competitive S1P titers in WT and SphK-1++ macrophages. (B) Sphk-1 overexpression confers resistance to infection. Both WT and SphK-1 ++macrophages were infected with M. smegmatis and mycobacterial killing was observed up to 24 h post infection. (C) The cells under section (B) were treated with DHS and the effect on M. smegmatis killing was again evaluated up to 24 h post infection. (D) S1P regulates mycobacterial growth in macrophages. The cells under section (B) were supplemented with S1P (5 µM) and the effect of S1P on mycobacterial infection was monitored during the first 4 h time period. Data are represented as mean of CFU ± SEM from three independent experiments. ** Indicates p≤0.01; * indicates p≤0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0010657-g002: SphK-1 overexpression confers resistance to M. smegmatis infection in macrophages.(A) Sphk-1 was overexpressed in macrophages and validated by western blot, immunofluorescence and competitive S1P titers in WT and SphK-1++ macrophages. (B) Sphk-1 overexpression confers resistance to infection. Both WT and SphK-1 ++macrophages were infected with M. smegmatis and mycobacterial killing was observed up to 24 h post infection. (C) The cells under section (B) were treated with DHS and the effect on M. smegmatis killing was again evaluated up to 24 h post infection. (D) S1P regulates mycobacterial growth in macrophages. The cells under section (B) were supplemented with S1P (5 µM) and the effect of S1P on mycobacterial infection was monitored during the first 4 h time period. Data are represented as mean of CFU ± SEM from three independent experiments. ** Indicates p≤0.01; * indicates p≤0.05.
Mentions: To further substantiate whether an upregulation of SphK-1 would counteract bacterial killing, SphK-1 was overexpressed in macrophages (Fig. 2A) and these macrophages (Sphk-1++) were infected with M. smegmatis. Interestingly and expectedly, SphK-1 overexpression conferred resistance to infection in Sphk-1++ macrophages in comparison to WT infected macrophages (Fig. 2B). The increased resistance of SphK-1++ infected macrophages was abolished by DHS treatment (Fig. 2C). Treatment of both WT and Sphk-1++ infected macrophages with S1P (SK reaction product) enhanced bacterial killing in both WT and SphK-1++ infected macrophages. This effect was found more pronounced in SphK-1++ infected macrophages than in WT infected macrophages (Fig. 2D). These observations confirmed the involvement of SphK-1 in controlling M. smegmatis infection 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