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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 Alters the Biophysical Properties and Composition of Cellular Membranes(A–D) Membrane fluidity measurements. (A) Confocal images pseudocolored based on GP values (see color bar). Left: shCTRL; right: shSMPDL3B. Scale bar, 20 μm. (B) GP value distribution from representative experiment. Mean ± SEM, n = 7. (C) Fraction of ordered membrane of RAW264.7 relative to shCTRL. Mean ± SD of at least three biological replicates is shown. (D) Fraction of ordered membrane of BMDMs relative to WT. Mean ± SD, n = 5. Figure is representative of two biological replicates.(E) Lipids were extracted from control (shCTLR) or SMPDL3B-depleted (shSMPDL3B) RAW264.7 macrophages and analyzed by MS for glycerophospho- and sphingolipids. Bubble plots represent mean log2-transformed fold-change differences between cell lines. SM, sphingomyelin; GluCer, glucosylceramide; Cer, ceramide; PC, (lyso-) phosphatidylcholine; PE, (lyso-) phosphatidylethanolamine; PA, phosphatidic acid; PI, (lyso-) phosphatidylinositol; PS, (lyso-) phosphatidylserine; PG, phosphatidylglycerol. Data are representative of two biological replicates each consisting of five technical replicates.(F) Relative lipid levels for ceramide in control (shCTRL) and SMPDL3B-depleted RAW264.7 macrophages (shSMPDL3B). Cer d18:1/16:0 is abbreviated as C16:0, Cer d18:1/22:0 as C22:0, Cer d18:1/24:0 as C24:0, and Cer d18:1/24:1 as C24:1. Data represent mean ± SEM of two biological replicates each consisting of five technical replicates. ∗p ≤ 0.05.(G) Control (shCTRL) and SMPDL3B-depleted RAW264.7 macrophages (shSMPDL3B) were pretreated for 30 min with vehicle (VEH) or the indicated synthetic ceramide species (15 μM) and then stimulated with 100 ng/ml LPS for 8 hr. IL-6 release was measured by ELISA.Data show mean ± SD of technical triplicates and is representative of at least two independent experiments. See Figure S4.
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fig4: SMPDL3B Alters the Biophysical Properties and Composition of Cellular Membranes(A–D) Membrane fluidity measurements. (A) Confocal images pseudocolored based on GP values (see color bar). Left: shCTRL; right: shSMPDL3B. Scale bar, 20 μm. (B) GP value distribution from representative experiment. Mean ± SEM, n = 7. (C) Fraction of ordered membrane of RAW264.7 relative to shCTRL. Mean ± SD of at least three biological replicates is shown. (D) Fraction of ordered membrane of BMDMs relative to WT. Mean ± SD, n = 5. Figure is representative of two biological replicates.(E) Lipids were extracted from control (shCTLR) or SMPDL3B-depleted (shSMPDL3B) RAW264.7 macrophages and analyzed by MS for glycerophospho- and sphingolipids. Bubble plots represent mean log2-transformed fold-change differences between cell lines. SM, sphingomyelin; GluCer, glucosylceramide; Cer, ceramide; PC, (lyso-) phosphatidylcholine; PE, (lyso-) phosphatidylethanolamine; PA, phosphatidic acid; PI, (lyso-) phosphatidylinositol; PS, (lyso-) phosphatidylserine; PG, phosphatidylglycerol. Data are representative of two biological replicates each consisting of five technical replicates.(F) Relative lipid levels for ceramide in control (shCTRL) and SMPDL3B-depleted RAW264.7 macrophages (shSMPDL3B). Cer d18:1/16:0 is abbreviated as C16:0, Cer d18:1/22:0 as C22:0, Cer d18:1/24:0 as C24:0, and Cer d18:1/24:1 as C24:1. Data represent mean ± SEM of two biological replicates each consisting of five technical replicates. ∗p ≤ 0.05.(G) Control (shCTRL) and SMPDL3B-depleted RAW264.7 macrophages (shSMPDL3B) were pretreated for 30 min with vehicle (VEH) or the indicated synthetic ceramide species (15 μM) and then stimulated with 100 ng/ml LPS for 8 hr. IL-6 release was measured by ELISA.Data show mean ± SD of technical triplicates and is representative of at least two independent experiments. See Figure S4.

Mentions: To evaluate the impact of SMPDL3B, a lipid-associated enzyme, on the cellular membrane environment, we measured membrane fluidity of macrophages. The lipid phase of membranes in intact cells can be studied using fluorescent probes that alter their spectral emission properties dependent on lipid packing and can thus reflect membrane order (Owen et al., 2012). The generalized polarization (GP) function can be used to calculate a normalized ratio between the measured intensities of ordered and unordered fractions in fluorescence microscopy images and therefore allows the quantification of changes in membrane order. In line with a role for SMPDL3B in membrane biology, microscopy-based measurements of membrane fluidity using the fluorescent probe di-4-ANEPPDHQ revealed a strong decrease of membrane order (i.e., increase of membrane fluidity) in SMPDL3B-depleted RAW264.7 macrophages (Figures 4A–4C). Treatment with the cholesterol-extracting agent methyl-β-cyclodextrin (MβCD) also led to reduced measureable membrane order and served as a positive control for this assay system. As an increase in membrane fluidity associated with SMPDL3B knockdown might affect the integrity of lipid rafts found on the cell surface, we measured the concentration of the raft marker GM1 ganglioside by flow cytometry. Interestingly, the concentration of GM1 was not affected by knockdown of Smpdl3b, suggesting no general modulatory effect on lipid raft abundance (Figure S4A). Highly consistent with the data obtained in RAW264.7 cells, also Smpdl3b-deficient BMDMs showed a decrease in membrane order, indeed assigning a role to SMPDL3B in membrane biology (Figure 4D).


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 Alters the Biophysical Properties and Composition of Cellular Membranes(A–D) Membrane fluidity measurements. (A) Confocal images pseudocolored based on GP values (see color bar). Left: shCTRL; right: shSMPDL3B. Scale bar, 20 μm. (B) GP value distribution from representative experiment. Mean ± SEM, n = 7. (C) Fraction of ordered membrane of RAW264.7 relative to shCTRL. Mean ± SD of at least three biological replicates is shown. (D) Fraction of ordered membrane of BMDMs relative to WT. Mean ± SD, n = 5. Figure is representative of two biological replicates.(E) Lipids were extracted from control (shCTLR) or SMPDL3B-depleted (shSMPDL3B) RAW264.7 macrophages and analyzed by MS for glycerophospho- and sphingolipids. Bubble plots represent mean log2-transformed fold-change differences between cell lines. SM, sphingomyelin; GluCer, glucosylceramide; Cer, ceramide; PC, (lyso-) phosphatidylcholine; PE, (lyso-) phosphatidylethanolamine; PA, phosphatidic acid; PI, (lyso-) phosphatidylinositol; PS, (lyso-) phosphatidylserine; PG, phosphatidylglycerol. Data are representative of two biological replicates each consisting of five technical replicates.(F) Relative lipid levels for ceramide in control (shCTRL) and SMPDL3B-depleted RAW264.7 macrophages (shSMPDL3B). Cer d18:1/16:0 is abbreviated as C16:0, Cer d18:1/22:0 as C22:0, Cer d18:1/24:0 as C24:0, and Cer d18:1/24:1 as C24:1. Data represent mean ± SEM of two biological replicates each consisting of five technical replicates. ∗p ≤ 0.05.(G) Control (shCTRL) and SMPDL3B-depleted RAW264.7 macrophages (shSMPDL3B) were pretreated for 30 min with vehicle (VEH) or the indicated synthetic ceramide species (15 μM) and then stimulated with 100 ng/ml LPS for 8 hr. IL-6 release was measured by ELISA.Data show mean ± SD of technical triplicates and is representative of at least two independent experiments. See Figure S4.
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fig4: SMPDL3B Alters the Biophysical Properties and Composition of Cellular Membranes(A–D) Membrane fluidity measurements. (A) Confocal images pseudocolored based on GP values (see color bar). Left: shCTRL; right: shSMPDL3B. Scale bar, 20 μm. (B) GP value distribution from representative experiment. Mean ± SEM, n = 7. (C) Fraction of ordered membrane of RAW264.7 relative to shCTRL. Mean ± SD of at least three biological replicates is shown. (D) Fraction of ordered membrane of BMDMs relative to WT. Mean ± SD, n = 5. Figure is representative of two biological replicates.(E) Lipids were extracted from control (shCTLR) or SMPDL3B-depleted (shSMPDL3B) RAW264.7 macrophages and analyzed by MS for glycerophospho- and sphingolipids. Bubble plots represent mean log2-transformed fold-change differences between cell lines. SM, sphingomyelin; GluCer, glucosylceramide; Cer, ceramide; PC, (lyso-) phosphatidylcholine; PE, (lyso-) phosphatidylethanolamine; PA, phosphatidic acid; PI, (lyso-) phosphatidylinositol; PS, (lyso-) phosphatidylserine; PG, phosphatidylglycerol. Data are representative of two biological replicates each consisting of five technical replicates.(F) Relative lipid levels for ceramide in control (shCTRL) and SMPDL3B-depleted RAW264.7 macrophages (shSMPDL3B). Cer d18:1/16:0 is abbreviated as C16:0, Cer d18:1/22:0 as C22:0, Cer d18:1/24:0 as C24:0, and Cer d18:1/24:1 as C24:1. Data represent mean ± SEM of two biological replicates each consisting of five technical replicates. ∗p ≤ 0.05.(G) Control (shCTRL) and SMPDL3B-depleted RAW264.7 macrophages (shSMPDL3B) were pretreated for 30 min with vehicle (VEH) or the indicated synthetic ceramide species (15 μM) and then stimulated with 100 ng/ml LPS for 8 hr. IL-6 release was measured by ELISA.Data show mean ± SD of technical triplicates and is representative of at least two independent experiments. See Figure S4.
Mentions: To evaluate the impact of SMPDL3B, a lipid-associated enzyme, on the cellular membrane environment, we measured membrane fluidity of macrophages. The lipid phase of membranes in intact cells can be studied using fluorescent probes that alter their spectral emission properties dependent on lipid packing and can thus reflect membrane order (Owen et al., 2012). The generalized polarization (GP) function can be used to calculate a normalized ratio between the measured intensities of ordered and unordered fractions in fluorescence microscopy images and therefore allows the quantification of changes in membrane order. In line with a role for SMPDL3B in membrane biology, microscopy-based measurements of membrane fluidity using the fluorescent probe di-4-ANEPPDHQ revealed a strong decrease of membrane order (i.e., increase of membrane fluidity) in SMPDL3B-depleted RAW264.7 macrophages (Figures 4A–4C). Treatment with the cholesterol-extracting agent methyl-β-cyclodextrin (MβCD) also led to reduced measureable membrane order and served as a positive control for this assay system. As an increase in membrane fluidity associated with SMPDL3B knockdown might affect the integrity of lipid rafts found on the cell surface, we measured the concentration of the raft marker GM1 ganglioside by flow cytometry. Interestingly, the concentration of GM1 was not affected by knockdown of Smpdl3b, suggesting no general modulatory effect on lipid raft abundance (Figure S4A). Highly consistent with the data obtained in RAW264.7 cells, also Smpdl3b-deficient BMDMs showed a decrease in membrane order, indeed assigning a role to SMPDL3B in membrane biology (Figure 4D).

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