Despite the importance of signaling lipids, many questions remain about their function because few tools are available for charting lipid gradients in vivo. Here we generated a sphingosine 1-phosphate (S1P) reporter mouse and used this mouse to define the distribution of S1P in the spleen. Unexpectedly, the presence of blood did not serve as a predictor of the concentration of signaling-available S1P. Large areas of the red pulp had low concentrations of S1P, while S1P was sensed by cells inside the white pulp near the marginal sinus. The lipid phosphate phosphatase LPP3 maintained low S1P concentrations in the spleen and enabled efficient shuttling of marginal zone B cells. The exquisitely tight regulation of S1P availability might explain how a single lipid can simultaneously orchestrate the movements of many cells of the immune system.
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We thank members of the Schwab laboratory, J. Cyster, T. Schmidt, J. Green, D. Littman, M. Dustin, S. Koralov, and T. Lu for discussions, and J. Cyster and L. Pitt for critical reading of the manuscript. Supported by the US National Institutes of Health (AI085166 to S.R.S.) and the Pew Charitable Trusts (S.R.S.).
The authors declare no competing financial interests.
Integrated supplementary information
(a) Expression of CD169 on the marginal zone and white pulp macrophages shown in Fig. 1d. Left panel shows CD169 alone, right panel shows CD169, RFP, and GFP.(b) Expression of CD169 on the marginal zone and white pulp macrophages shown in Fig. 1e. Left panel shows CD169 alone, right panel shows CD169, RFP, and GFP.(c) Schematic of spleen architecture. Blue arrows indicate blood flow.
(a) Spleen section from a littermate control not expressing the S1P reporter, with composite and single fluorophore images. Data are representative of sections from 7 mice.(b) Spleen section from a mouse expressing the S1P reporter, with composite and single fluorophore images. A high-resolution file of this image is available at Figshare.com, http://figshare.com/s/19b5989c2fe011e580f506ec4b8d1f61. Data are representative of sections from 7 mice.(c) Single fluorophore and composite images of representative reporter F4/80+ red pulp macrophages and CD169+ marginal zone macrophages. Data are representative of sections from 7 mice.
(a) 3 additional examples of spleen sections from S1P reporter animals, showing the marginal zone/red pulp boundary. High-resolution files of these images are available at Figshare.com, http://figshare.com/s/13df9226309711e5aa0506ec4b8d1f61, http://figshare.com/s/b1617e52309b11e5a77006ec4bbcf141 and http://figshare.com/s/c7a2f6dc309b11e5a77006ec4bbcf141.Data are representative of sections from 12 mice, 7 Mx1-Cre and 5 Lyz2-Cre.(b) Quantification of the ratio of total GFP: total RFP in the marginal zone and red pulp. We expect that some internalized S1PR1-GFP will be degraded, so increased sensing of S1P in the marginal zone relative to the red pulp should result in an overall reduced ratio of GFP:RFP in the marginal zone compared to the red pulp. For each of the areas used in Fig. 2b, the mean intensity of GFP was divided by the mean intensity of RFP to calculate the ratio of GFP:RFP (unlike Fig. 2b, the analysis was not restricted to CD169+ RFP+ or F4/80+ RFP+ pixels). Bars show mean, error bars show SD. Comparisons were done using an unpaired, 2-tailed Student’s t test; ****, p<0.0001. To account for different microscope settings on different days, for each mouse all ratios were normalized such that the average ratio for the marginal zone was 0.5.(c) Calculation of the Pearson’s correlation coefficient between GFP and RFP in the marginal zone and red pulp. We expect that internalization of S1PR1-GFP in the marginal zone will lead to a lower Pearson’s correlation coefficient between GFP and RFP in the marginal zone compared to the red pulp. The graph compiles 3 mice, with 2-4 sections per mouse. Each point represents one section: for each section the average Pearson’s correlation coefficient from at least 5 MZ or RP areas of at least 2x103 or 1x104 square microns respectively was calculated. Bars show mean, error bars show SEM. Comparisons were done using an unpaired, 2-tailed Student’s t test; *, p<0.05.(d) A representative section from a mouse in which the S1P reporter was induced using UBC-CreERT2 (daily doses of tamoxifen for 5 days, with analysis 5 days after the last tamoxifen injection). Data are representative of sections from 2 mice.(e) Schematic showing S1P synthetic and degrading enzymes. Source data
(a) Spleen section from a littermate control not expressing the S1P reporter, with composite and single fluorophore images. Data are representative of sections from 7 mice.(b) Spleen section from a mouse expressing the S1P reporter, with composite and single fluorophore images. A high-resolution file of this image is available at Figshare.com, http://figshare.com/s/90a735dc2fe011e59ed806ec4b8d1f61. Data are representative of sections from 7 mice.
3 additional examples of spleen sections from S1P reporter animals, showing the marginal zone/white pulp boundary. For the top section, boxed images are maximum pixel intensity projections of the indicated CD169+ cells, integrated over a 74-plane z-stack taken at 0.37 μm intervals. Scale bar for individual cells is 5 μm. High-resolution files of these images are available at Figshare.com, http://figshare.com/s/7d0314d22fe111e5bf1f06ec4b8d1f61, http://figshare.com/s/0d8475962fe211e58bc506ec4b8d1f61, http://figshare.com/s/4c6c875e309511e5a35f06ec4b8d1f61 and http://figshare.com/s/7fb05644309611e5a95706ec4bbcf141.Data are representative of sections from 12 mice, 7 Mx1-Cre and 5 Lyz2-Cre.
(a) Validation of in vivo PE labeling. To test whether labeling occurred in vivo or during tissue processing, we processed the spleen of an anti-CD45-PE injected mouse together with the spleen of an uninjected congenically marked mouse; the spleens were disaggregated together in the same dish and stained together in the same tube. No PE labeling was detected on MZB of the uninjected animal. Splenocytes from an unlabeled mouse, processed alone, were included as a negative control for PE staining. Representative of 2 experiments.(b) Experiment design for Fig. 6e.(c) The requirement for LPP3 in B cells is not cell-intrinsic. Mixed bone marrow (BM) chimeras were generated in which lethally-irradiated CD45.1+ hosts received a mixture of either (90% GFP+ wild-type BM + 10% CD45.2+ LPP3-deficient BM) or (90% GFP+ wild-type BM + 10% CD45.2+ littermate control BM). The chimeras were injected intravenously with a PE-conjugated antibody to CD45, sacrificed 5 minutes later, and analyzed by flow cytometry. Graph shows % PEhi MZB from the LPP3 KO or littermate control donor. (There was also no difference in the % PEhi MZB derived from the GFP donor.) Data represent 8 pairs of mice analyzed in 4 experiments from 3 pairs of BM donors. Source data
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Ramos-Perez, W., Fang, V., Escalante-Alcalde, D. et al. A map of the distribution of sphingosine 1-phosphate in the spleen. Nat Immunol 16, 1245–1252 (2015) doi:10.1038/ni.3296
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