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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 TNF-α secretion in macrophages.(A) Both WT and SphK-1++ macrophages were infected with M. smegmatis with and without DHS. The TNF-α titre was quantified in their culture supernatants until 4 h post infection. (B,C) Delay in the secretion of TNF-α by S1P in macrophages. The macrophages were infected with M. smegmatis with and without DHS (20 µM) and S1P (5 µM), and their culture supernatants were collected at 9 h (B) and 24 h (C) post infection and TNF-α titre was quantified. (D) The macrophages were stimulated by LPS with and without S1P and DHS, TNF titre was quantified at 9 h (D) and 24 h (E) post treatment. (F) Sphk-1 overexpression delays in the secretion of TNF-α by infected macrophage upon LPS co-stimulation. Both WT and Sphk-1++ macrophages were infected with M. smegmatis and co-stimulated with LPS for indicated time intervals. The TNF titre was quantified. Data are represented as a mean ρg/ml ± SEM from three independent experiments. ** Indicates p≤0.01; * indicates p≤0.05.
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pone-0010657-g008: SphK-1 regulates TNF-α secretion in macrophages.(A) Both WT and SphK-1++ macrophages were infected with M. smegmatis with and without DHS. The TNF-α titre was quantified in their culture supernatants until 4 h post infection. (B,C) Delay in the secretion of TNF-α by S1P in macrophages. The macrophages were infected with M. smegmatis with and without DHS (20 µM) and S1P (5 µM), and their culture supernatants were collected at 9 h (B) and 24 h (C) post infection and TNF-α titre was quantified. (D) The macrophages were stimulated by LPS with and without S1P and DHS, TNF titre was quantified at 9 h (D) and 24 h (E) post treatment. (F) Sphk-1 overexpression delays in the secretion of TNF-α by infected macrophage upon LPS co-stimulation. Both WT and Sphk-1++ macrophages were infected with M. smegmatis and co-stimulated with LPS for indicated time intervals. The TNF titre was quantified. Data are represented as a mean ρg/ml ± SEM from three independent experiments. ** Indicates p≤0.01; * indicates p≤0.05.

Mentions: TNF-α is a well known trigger of acute inflammatory response during mycobacterial infection and largely secreted by activated macrophages [57], [58]. Therefore, we investigated the effect of SphK-1 modulation on TNF-α secretion also. Treatment of infected macrophages with DHS significantly reduced M. smegmatis induced TNF-α secretion in WT infected macrophages in comparison to infected controls (Fig. 8A). This observation explained to some extent the most probable reason for the sensitization of Sphk-1inhibited macrophages to infection. On the basis of increased resistance to infection, we predicted enhanced TNF-α secretion in Sphk-1++ infected macrophages over WT infected macrophages. While we were able to detect higher TNF-α secretion in SphK-1++ infected macrophages over WT infected macrophages at 1 h post infection, this remained insignificant (data not shown). Interestingly, and contrary to our expectations, the TNF-α secretion in Sphk-1++ infected macrophages was significantly reduced in comparison to WT infected macrophages during later time points of infection (Fig. 8A). This observation revealed negative regulation of Sphk-1 over-expression on TNF-α secretion. In order to complement Sphk-1 inhibition mediated loss in TNF-α secretion in macrophages, we supplemented infected macrophages exogenously with S1P (SK reaction product). Surprisingly, this supplementation delayed TNF-α secretion in the infected macrophages at 9 h (Fig. 8B), which was restored at 24 h post infection (Fig. 8C).


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 TNF-α secretion in macrophages.(A) Both WT and SphK-1++ macrophages were infected with M. smegmatis with and without DHS. The TNF-α titre was quantified in their culture supernatants until 4 h post infection. (B,C) Delay in the secretion of TNF-α by S1P in macrophages. The macrophages were infected with M. smegmatis with and without DHS (20 µM) and S1P (5 µM), and their culture supernatants were collected at 9 h (B) and 24 h (C) post infection and TNF-α titre was quantified. (D) The macrophages were stimulated by LPS with and without S1P and DHS, TNF titre was quantified at 9 h (D) and 24 h (E) post treatment. (F) Sphk-1 overexpression delays in the secretion of TNF-α by infected macrophage upon LPS co-stimulation. Both WT and Sphk-1++ macrophages were infected with M. smegmatis and co-stimulated with LPS for indicated time intervals. The TNF titre was quantified. Data are represented as a mean ρg/ml ± SEM from three independent experiments. ** Indicates p≤0.01; * indicates p≤0.05.
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Related In: Results  -  Collection

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pone-0010657-g008: SphK-1 regulates TNF-α secretion in macrophages.(A) Both WT and SphK-1++ macrophages were infected with M. smegmatis with and without DHS. The TNF-α titre was quantified in their culture supernatants until 4 h post infection. (B,C) Delay in the secretion of TNF-α by S1P in macrophages. The macrophages were infected with M. smegmatis with and without DHS (20 µM) and S1P (5 µM), and their culture supernatants were collected at 9 h (B) and 24 h (C) post infection and TNF-α titre was quantified. (D) The macrophages were stimulated by LPS with and without S1P and DHS, TNF titre was quantified at 9 h (D) and 24 h (E) post treatment. (F) Sphk-1 overexpression delays in the secretion of TNF-α by infected macrophage upon LPS co-stimulation. Both WT and Sphk-1++ macrophages were infected with M. smegmatis and co-stimulated with LPS for indicated time intervals. The TNF titre was quantified. Data are represented as a mean ρg/ml ± SEM from three independent experiments. ** Indicates p≤0.01; * indicates p≤0.05.
Mentions: TNF-α is a well known trigger of acute inflammatory response during mycobacterial infection and largely secreted by activated macrophages [57], [58]. Therefore, we investigated the effect of SphK-1 modulation on TNF-α secretion also. Treatment of infected macrophages with DHS significantly reduced M. smegmatis induced TNF-α secretion in WT infected macrophages in comparison to infected controls (Fig. 8A). This observation explained to some extent the most probable reason for the sensitization of Sphk-1inhibited macrophages to infection. On the basis of increased resistance to infection, we predicted enhanced TNF-α secretion in Sphk-1++ infected macrophages over WT infected macrophages. While we were able to detect higher TNF-α secretion in SphK-1++ infected macrophages over WT infected macrophages at 1 h post infection, this remained insignificant (data not shown). Interestingly, and contrary to our expectations, the TNF-α secretion in Sphk-1++ infected macrophages was significantly reduced in comparison to WT infected macrophages during later time points of infection (Fig. 8A). This observation revealed negative regulation of Sphk-1 over-expression on TNF-α secretion. In order to complement Sphk-1 inhibition mediated loss in TNF-α secretion in macrophages, we supplemented infected macrophages exogenously with S1P (SK reaction product). Surprisingly, this supplementation delayed TNF-α secretion in the infected macrophages at 9 h (Fig. 8B), which was restored at 24 h post infection (Fig. 8C).

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