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The Lipid-Modifying Enzyme SMPDL3B Negatively Regulates Innate Immunity.

Heinz LX, Baumann CL, Köberlin MS, Snijder B, Gawish R, Shui G, Sharif O, Aspalter IM, Müller AC, Kandasamy RK, Breitwieser FP, Pichlmair A, Bruckner M, Rebsamen M, Blüml S, Karonitsch T, Fauster A, Colinge J, Bennett KL, Knapp S, Wenk MR, Superti-Furga G - Cell Rep (2015)

Bottom Line: Lipid metabolism and receptor-mediated signaling are highly intertwined processes that cooperate to fulfill cellular functions and safeguard cellular homeostasis.Increased cellular responses could be reverted by re-introducing affected ceramides, functionally linking membrane lipid composition and innate immune signaling.Taken together, our results identify the membrane-modulating enzyme SMPDL3B as a negative regulator of TLR signaling that functions at the interface of membrane biology and innate immunity.

View Article: PubMed Central - PubMed

Affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria.

No MeSH data available.


Related in: MedlinePlus

SMPDL3B Is a Negative Regulator of TLR Signaling(A) BMDMs or BMDCs were stimulated with 100 ng/ml LPS or 1 μM CpG-DNA for the indicated time and relative expression of Smpdl3b was measured by RT-PCR.(B) Expression of SMPDL3B and tubulin in wild-type and Smpdl3b-deficient BMDMs was analyzed by western blot.(C and E) BMDMs or BMDCs from wild-type (WT) or Smpdl3b-deficient (KO) mice were stimulated with 100 ng/ml LPS for the indicated time, and relative expression of KC was measured by RT-PCR.(D and F) BMDMs or BMDCs from wild-type (WT) or Smpdl3b-deficient (KO) mice were stimulated with LPS for 8 hr, and supernatants were analyzed for KC by ELISA.(G) The phosphorylation status of p38, JNK, and ERK and protein levels of IκBα in the lysates of control (shCTRL) and SMPDL3B-depleted RAW264.7 cells (shS3B) upon stimulation with LPS were analyzed by western blot.(A and C–F) Data show mean ± SD of technical triplicates and are representative of at least two independent experiments. (G) Data are representative of two independent experiments. See also Figure S2.
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fig2: SMPDL3B Is a Negative Regulator of TLR Signaling(A) BMDMs or BMDCs were stimulated with 100 ng/ml LPS or 1 μM CpG-DNA for the indicated time and relative expression of Smpdl3b was measured by RT-PCR.(B) Expression of SMPDL3B and tubulin in wild-type and Smpdl3b-deficient BMDMs was analyzed by western blot.(C and E) BMDMs or BMDCs from wild-type (WT) or Smpdl3b-deficient (KO) mice were stimulated with 100 ng/ml LPS for the indicated time, and relative expression of KC was measured by RT-PCR.(D and F) BMDMs or BMDCs from wild-type (WT) or Smpdl3b-deficient (KO) mice were stimulated with LPS for 8 hr, and supernatants were analyzed for KC by ELISA.(G) The phosphorylation status of p38, JNK, and ERK and protein levels of IκBα in the lysates of control (shCTRL) and SMPDL3B-depleted RAW264.7 cells (shS3B) upon stimulation with LPS were analyzed by western blot.(A and C–F) Data show mean ± SD of technical triplicates and are representative of at least two independent experiments. (G) Data are representative of two independent experiments. See also Figure S2.

Mentions: SMPDL3B expression was prominently observed in macrophages and DCs (Figure S1G). Consistent with a possible role for this enzyme in the course of inflammatory processes, Smpdl3b transcription in bone marrow-derived macrophages (BMDMs) and DCs (BMDCs) was robustly induced upon TLR stimulation (Figure 2A). To further study the role of this protein in primary cells, we decided to generate Smpdl3b-deficient mice. The knockout mice were viable and did not have any overt developmental phenotype. Interestingly, BMDMs and BMDCs from Smpdl3b-deficient mice showed higher expression and release of the chemokine KC/CXCL1 upon stimulation with TLR agonists in comparison to wild-type cells (Figures 2B–2F and S2A). In line with this, knockdown of Smpdl3b in RAW264.7 macrophages increased the release of interleukin 6 (IL-6) upon treatment with lipopolysaccharide (LPS), CpG-DNA (CpG), and imiquimod (IMQ) (Figures S2B and S2C). To evaluate whether other important membrane-dependent events that occur early upon TLR activation were affected by SMPDL3B depletion, we measured the internalization rates of TLR4 and the phagocytic uptake of CpG (Figures S2D–S2F). Endocytosis of TLR4 upon LPS stimulation was unchanged in RAW264.7 macrophages or primary BMDMs depleted or deficient in Smpdl3b, suggesting that the enhanced release of cytokines in SMPDL3B-depleted cells was not caused by retaining TLR4 at the plasma membrane or malfunctioning of receptor endocytosis (Figures S2D and S2E). Also, uptake of fluorescently labeled Cy3-CpG was unaltered in knockdown cells further indicating that endocytic functions were intact (Figure S2F). Taken together, these data show that Smpdl3b expression is induced upon TLR stimulation and reveal that SMPDL3B negatively affects TLR-dependent responses.


The Lipid-Modifying Enzyme SMPDL3B Negatively Regulates Innate Immunity.

Heinz LX, Baumann CL, Köberlin MS, Snijder B, Gawish R, Shui G, Sharif O, Aspalter IM, Müller AC, Kandasamy RK, Breitwieser FP, Pichlmair A, Bruckner M, Rebsamen M, Blüml S, Karonitsch T, Fauster A, Colinge J, Bennett KL, Knapp S, Wenk MR, Superti-Furga G - Cell Rep (2015)

SMPDL3B Is a Negative Regulator of TLR Signaling(A) BMDMs or BMDCs were stimulated with 100 ng/ml LPS or 1 μM CpG-DNA for the indicated time and relative expression of Smpdl3b was measured by RT-PCR.(B) Expression of SMPDL3B and tubulin in wild-type and Smpdl3b-deficient BMDMs was analyzed by western blot.(C and E) BMDMs or BMDCs from wild-type (WT) or Smpdl3b-deficient (KO) mice were stimulated with 100 ng/ml LPS for the indicated time, and relative expression of KC was measured by RT-PCR.(D and F) BMDMs or BMDCs from wild-type (WT) or Smpdl3b-deficient (KO) mice were stimulated with LPS for 8 hr, and supernatants were analyzed for KC by ELISA.(G) The phosphorylation status of p38, JNK, and ERK and protein levels of IκBα in the lysates of control (shCTRL) and SMPDL3B-depleted RAW264.7 cells (shS3B) upon stimulation with LPS were analyzed by western blot.(A and C–F) Data show mean ± SD of technical triplicates and are representative of at least two independent experiments. (G) Data are representative of two independent experiments. See also Figure S2.
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fig2: SMPDL3B Is a Negative Regulator of TLR Signaling(A) BMDMs or BMDCs were stimulated with 100 ng/ml LPS or 1 μM CpG-DNA for the indicated time and relative expression of Smpdl3b was measured by RT-PCR.(B) Expression of SMPDL3B and tubulin in wild-type and Smpdl3b-deficient BMDMs was analyzed by western blot.(C and E) BMDMs or BMDCs from wild-type (WT) or Smpdl3b-deficient (KO) mice were stimulated with 100 ng/ml LPS for the indicated time, and relative expression of KC was measured by RT-PCR.(D and F) BMDMs or BMDCs from wild-type (WT) or Smpdl3b-deficient (KO) mice were stimulated with LPS for 8 hr, and supernatants were analyzed for KC by ELISA.(G) The phosphorylation status of p38, JNK, and ERK and protein levels of IκBα in the lysates of control (shCTRL) and SMPDL3B-depleted RAW264.7 cells (shS3B) upon stimulation with LPS were analyzed by western blot.(A and C–F) Data show mean ± SD of technical triplicates and are representative of at least two independent experiments. (G) Data are representative of two independent experiments. See also Figure S2.
Mentions: SMPDL3B expression was prominently observed in macrophages and DCs (Figure S1G). Consistent with a possible role for this enzyme in the course of inflammatory processes, Smpdl3b transcription in bone marrow-derived macrophages (BMDMs) and DCs (BMDCs) was robustly induced upon TLR stimulation (Figure 2A). To further study the role of this protein in primary cells, we decided to generate Smpdl3b-deficient mice. The knockout mice were viable and did not have any overt developmental phenotype. Interestingly, BMDMs and BMDCs from Smpdl3b-deficient mice showed higher expression and release of the chemokine KC/CXCL1 upon stimulation with TLR agonists in comparison to wild-type cells (Figures 2B–2F and S2A). In line with this, knockdown of Smpdl3b in RAW264.7 macrophages increased the release of interleukin 6 (IL-6) upon treatment with lipopolysaccharide (LPS), CpG-DNA (CpG), and imiquimod (IMQ) (Figures S2B and S2C). To evaluate whether other important membrane-dependent events that occur early upon TLR activation were affected by SMPDL3B depletion, we measured the internalization rates of TLR4 and the phagocytic uptake of CpG (Figures S2D–S2F). Endocytosis of TLR4 upon LPS stimulation was unchanged in RAW264.7 macrophages or primary BMDMs depleted or deficient in Smpdl3b, suggesting that the enhanced release of cytokines in SMPDL3B-depleted cells was not caused by retaining TLR4 at the plasma membrane or malfunctioning of receptor endocytosis (Figures S2D and S2E). Also, uptake of fluorescently labeled Cy3-CpG was unaltered in knockdown cells further indicating that endocytic functions were intact (Figure S2F). Taken together, these data show that Smpdl3b expression is induced upon TLR stimulation and reveal that SMPDL3B negatively affects TLR-dependent responses.

Bottom Line: Lipid metabolism and receptor-mediated signaling are highly intertwined processes that cooperate to fulfill cellular functions and safeguard cellular homeostasis.Increased cellular responses could be reverted by re-introducing affected ceramides, functionally linking membrane lipid composition and innate immune signaling.Taken together, our results identify the membrane-modulating enzyme SMPDL3B as a negative regulator of TLR signaling that functions at the interface of membrane biology and innate immunity.

View Article: PubMed Central - PubMed

Affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria.

No MeSH data available.


Related in: MedlinePlus