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An optical nanoreporter of endolysosomal lipid accumulation reveals enduring effects of diet on hepatic macrophages in vivo

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Science Translational Medicine  03 Oct 2018:
Vol. 10, Issue 461, eaar2680
DOI: 10.1126/scitranslmed.aar2680
  • Fig. 1 Characterization of the ssCTTC3TTC-(9,4) optical reporter.

    (A) Photoluminescence excitation emission plot of ssCTTC3TTC-(9,4). a.u., arbitrary units. (B) Nanotube emission center wavelength ± SD in cell culture medium with 1% FBS at near-saturating concentrations of BSA (20 mg/ml), salmon testes double-stranded DNA (1 mg/ml), CMC (5 mg/ml), mPEG-phosphoethanolamine 18:0 (mPEG-PE 18:0; 5.93 mM), PEG-cholesterol (5.93 mM), or mPEG-ceramide (5.93 mM). Error bars represent SD from n = 3 technical replicates. (C) Frames from molecular dynamics simulations showing equilibrated structures of the ssCTTC3TTC-(9,4) nanotube complex in the presence of cholesterol and sphingomyelin. (D) Water density as a function of distance from the surface of ssCTTC3TTC-(9,4) nanotube complexes, in the presence of sphingomyelin or cholesterol, or with no lipids present. (E) Overlays of transmitted light and NIR hyperspectral images of the reporter in RAW 264.7 macrophages cultured in complete cell culture medium with or without U18666A (3 μg/ml), Lalistat 3a2 (10 μM), or imipramine hydrochloride (10 μM). (F) Histogram of emission center wavelengths from all pixels with NIR emission from the NIR hyperspectral images of cells under the conditions described in (E).

  • Fig. 2 Reporter biodistribution.

    (A) Quantification of fluorescence over time in different regions of mice after intravenous injection of ssCTTC3TTC-SWCNT. Results are averaged for five mice, and error bars represent SD. Insets represent the average fluorescence intensity (Int.) in the specified region immediately after injection. Error bars have been removed for clarity. (B) Fluorescence image of the reporter in vivo in a mouse, 24 hours after intravenous injection (200 ng). (C) Fluorescence image of the reporter in mouse organs ex vivo, 24 hours after injection as in (B). (D) Quantification of ssCTTC3TTC-SWCNT emission spectra ex vivo obtained with a NIR spectroscope. ****P < 0.0001 versus other groups, one-way analysis of variance (ANOVA) with Dunnett’s post-test. Error bars represent SD. n = 3 mice per group. (E) Quantification of ssCTTC3TTC-SWCNT emission in isolated murine KCs, hepatocytes (HPs), and hepatic stellate cells (HSCs), 24 hours after intravenous injection. ****P < 0.001, one-way ANOVA with Tukey’s post-test. Error bars represent SEM. n = 49 to 62 cells per group.

  • Fig. 3 Noninvasive detection of lysosomal storage disorders in vivo.

    (A) In vivo emission spectra of the reporter from live wild-type (WT) and ASMKO mice. (B) Box plot of reporter center wavelength from spectra shown in (A). Individual data points are overlaid with diamonds of the same color, indicating repeated spectra taken from a single mouse. (C) Box plot of reporter center wavelength from the livers of WT and ASMKO mice ex vivo. Individual data points are overlaid, with each point representing a different mouse. (D) Overlay of transmitted light and NIR hyperspectral image of nanotube complexes in resected sections of frozen liver tissue from WT and ASMKO mice. Scale bar, 20 μm. (E) Histogram of emission center wavelengths of all pixels from NIR hyperspectral images of frozen sections of resected liver tissue from WT and ASMKO mice. (F) Mean center wavelength of reporter emission from frozen sections of resected liver tissue imaged with NIR hyperspectral microscopy. Error bars represent SD. (G) In vivo emission spectra of the reporter from live WT and Npctm(I1061T)Dso mice. (H) Box plot of reporter center wavelength from spectra shown in (G). Individual data points are overlaid with diamonds of the same color, indicating repeated spectra taken from a single mouse. (I) Box plot of reporter center wavelength from the livers of WT and Npctm(I1061T)Dso mice ex vivo. Individual data points are overlaid, with each point representing a different mouse. (J) Overlay of transmitted light and NIR hyperspectral image of nanotube complexes in resected sections of frozen liver tissue from WT and Npctm(I1061T)Dso mice. Scale bar, 20 μm. (K) Histogram of emission center wavelengths of all pixels from NIR hyperspectral images of frozen sections of resected liver tissue from WT and Npctm(I1061T)Dso mice. (L) Mean center wavelength of reporter emission taken from frozen sections of resected liver tissue with NIR hyperspectral microscopy. Error bars represent SD. *P < 0.05, **P < 0.01, ***P < 0.001, t test with Welch’s correction. n = 3 mice per group.

  • Fig. 4 Dynamic detection of oxLDL accumulation.

    (A) Schematic of experimental procedure used in (B) to (D), shown with a photograph of the NIR in vivo spectroscope (right) used for data acquisition. (B) Mean center wavelength of reporter emission from C57BL/6 mice injected with oxLDL. Error bars represent SD. (C) Overlay of transmitted light and NIR hyperspectral image of reporter emission in vivo before and after oxLDL injection in mice. (D) Trajectories of endolysosomal lipid accumulation from mice injected with oxLDL (200 μg) or phosphate-buffered saline (PBS). Error bars represent SD. ***P < 0.001, ****P < 0.0001 compared to control, one-way ANOVA with Dunnett’s post-test. n = 3 mice per experimental group and n = 8 mice for control mice injected with PBS (0 μg of oxLDL).

  • Fig. 5 Measurement of KC endolysosomal lipid flux in a NAFLD model.

    (A) H&E-stained liver tissue from C57BL/6 mice fed either the WD or SC for 1 or 3 months. (B) Quantification of steatotic area observed in H&E sections from SC or WD mice. Ten to 20 sections were observed per mouse for a total of 30 to 100 sections per group. (C) Box plots of reporter center wavelengths of in vivo emission spectra of the reporter from WD- or SC-fed mice. Individual data points are overlaid with diamonds of the same color, indicating repeated spectra taken from a single mouse. (D) Mean center wavelength of reporter emission from the mouse livers in (C) ex vivo. Error bars represent SD. (E) Mean center wavelength of reporter emission from live WD- or SC-fed mice at various time points. Error bars represent SD. n.s., not significant. (F) Reporter center wavelength in live C57BL/6 mice over time with SC or WD feeding. Separate injections of reporter were used to obtain signal on days 0 and 15. Solid lines indicate averages, and error bars represent SD. *P < 0.05, ***P < 0.001, one-way ANOVA with Sidak’s post-test (A to E) or a t test with Welch’s correction (F). n = 3 to 5 mice per group.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/461/eaar2680/DC1

    Materials and Methods

    Fig. S1. Two-dimensional photoluminescence plots of isolated SWCNT chiralities.

    Fig. S2. Identification of ssCTTC3TTC-(9,4) as a potential probe for endolysosomal lipid accumulation.

    Fig. S3. Representative emission spectra of ssCTTC3TTC-(9,4) at a neutral and acidic pH.

    Fig. S4. ssCTTC3TTC-(9,4) photoluminescent response in solution.

    Fig. S5. Atomic force microscopy of ssCTTC3TTC-(9,4) complexes.

    Fig. S6. Initial REMD configurations.

    Fig. S7. Analysis of REMD simulations.

    Fig. S8. Mean emission wavelength of ssCTTC3TTC-(9,4) in live RAW 264.7 macrophages treated with inhibitors.

    Fig. S9. Effect of inhibitors on ssCTTC3TTC-(9,4) emission spectra in solution.

    Fig. S10. Reporter response to HPβCD treatment in MEFs.

    Fig. S11. Assessing the effect of ssCTTC3TTC-(9,4) on lipoprotein hydrolysis.

    Fig. S12. NIR in vivo spectroscope.

    Fig. S13. Representative images of liver tissue from wild-type and ASMKO mice.

    Fig. S14. Representative images of hepatic tissues from wild-type and ASMKO mice stained with the hepatocyte marker CK8/18.

    Fig. S15. Representative images of hepatic tissue from wild-type and ASMKO mice stained with the KC marker F4/80.

    Fig. S16. Representative images from H&E-stained tissues from the livers of wild-type and Npctm(I1061T)Dso mice.

    Fig. S17. Representative images of hepatic tissue from wild-type and Npctm(I1061T)Dso mice stained with the hepatocyte marker CK8/18.

    Fig. S18. Representative images of hepatic tissues from wild-type and Npctm(I1061T)Dso mice stained with the KC marker F4/80.

    Fig. S19. Exponential fits of the decrease in ssCTTC3TTC-(9,4) emission wavelength after intravenous injection of 200 μg of oxLDL in mice.

    Fig. S20. Testing the effects of elevated lipid concentrations on reporter emission in whole blood.

    Fig. S21. Weights of male C57BL/6 mice fed SC or WD with HFCS.

    Fig. S22. Representative images of hepatic tissues stained with CK8/18 from mice fed SC or WD with HFCS for 1 month.

    Fig. S23. Representative images of hepatic tissues stained with F4/80 from mice fed SC or WD with HFCS for 1 month.

    Fig. S24. Representative images of hepatic tissues stained with CK8/18 from mice fed SC or WD with HFCS for 3 months.

    Fig. S25. Representative images of hepatic tissues stained with F4/80 from mice fed SC or WD with HFCS for 3 months.

    Fig. S26. Representative images of H&E-stained liver tissues from C57BL/6 mice fed SC or WD with HFCS for 1 month.

    Fig. S27. Representative H&E-stained liver tissues from mice fed SC or WD with HFCS for 3 months.

    Fig. S28. In vivo emission spectra of the reporter from the liver of mice fed SC or WD with HFCS for 1 to 3 months.

    Fig. S29. Analysis of intercellular heterogeneity of KC lipid content in vivo.

    Fig. S30. Reporter presence in relation to lipogranulomas.

    Fig. S31. Weight and serum chemistry measurements of mice fed SC or WD with HFCS for 3 months.

    Fig. S32. Serum chemistry measurements and weights of reporter- and vehicle-injected mice in a model of hepatic injury.

    Fig. S33. Relative hepatic expression of genes involved in the innate immune response after injection with ssCTTC3TTC-(9,4) or PBS.

    Fig. S34. Representative images of H&E-stained liver tissue from C57BL/6 mice fed SC or WD with HFCS for 2 weeks.

    Fig. S35. Representative images of liver tissue stained with Oil Red O neutral lipid stain.

    Fig. S36. Representative images of hepatic tissue from mice fed SC or WD with HFCS for 2 weeks stained with the hepatocyte marker CK8/18.

    Fig. S37. Representative images of hepatic tissue from mice fed SC or WD with HFCS for 2 weeks stained with the KC marker F4/80.

    Fig. S38. Representative H&E sections of livers from mice shown in Fig. 5E.

    Table S1. Average reporter emission center wavelengths measured from wild-type and ASMKO mice in vivo, ex vivo¸ and in frozen sections of resected liver tissue.

    Table S2. Average reporter emission center wavelengths measured from wild-type and Npctm(I1061T)Dso mice in vivo, ex vivo, and in frozen sections of resected liver tissue.

    Table S3. Individual mouse data.

    References (4756)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Two-dimensional photoluminescence plots of isolated SWCNT chiralities.
    • Fig. S2. Identification of ssCTTC3TTC-(9,4) as a potential probe for endolysosomal lipid accumulation.
    • Fig. S3. Representative emission spectra of ssCTTC3TTC-(9,4) at a neutral and acidic pH.
    • Fig. S4. ssCTTC3TTC-(9,4) photoluminescent response in solution.
    • Fig. S5. Atomic force microscopy of ssCTTC3TTC-(9,4) complexes.
    • Fig. S6. Initial REMD configurations.
    • Fig. S7. Analysis of REMD simulations.
    • Fig. S8. Mean emission wavelength of ssCTTC3TTC-(9,4) in live RAW 264.7 macrophages treated with inhibitors.
    • Fig. S9. Effect of inhibitors on ssCTTC3TTC-(9,4) emission spectra in solution.
    • Fig. S10. Reporter response to HPβCD treatment in MEFs.
    • Fig. S11. Assessing the effect of ssCTTC3TTC-(9,4) on lipoprotein hydrolysis.
    • Fig. S12. NIR in vivo spectroscope.
    • Fig. S13. Representative images of liver tissue from wild-type and ASMKO mice.
    • Fig. S14. Representative images of hepatic tissues from wild-type and ASMKO mice stained with the hepatocyte marker CK8/18.
    • Fig. S15. Representative images of hepatic tissue from wild-type and ASMKO mice stained with the KC marker F4/80.
    • Fig. S16. Representative images from H&E-stained tissues from the livers of wild-type and Npctm(I1061T)Dso mice.
    • Fig. S17. Representative images of hepatic tissue from wild-type and Npctm(I1061T)Dso mice stained with the hepatocyte marker CK8/18.
    • Fig. S18. Representative images of hepatic tissues from wild-type and Npctm(I1061T)Dso mice stained with the KC marker F4/80.
    • Fig. S19. Exponential fits of the decrease in ssCTTC3TTC-(9,4) emission wavelength after intravenous injection of 200 μg of oxLDL in mice.
    • Fig. S20. Testing the effects of elevated lipid concentrations on reporter emission in whole blood.
    • Fig. S21. Weights of male C57BL/6 mice fed SC or WD with HFCS.
    • Fig. S22. Representative images of hepatic tissues stained with CK8/18 from mice fed SC or WD with HFCS for 1 month.
    • Fig. S23. Representative images of hepatic tissues stained with F4/80 from mice fed SC or WD with HFCS for 1 month.
    • Fig. S24. Representative images of hepatic tissues stained with CK8/18 from mice fed SC or WD with HFCS for 3 months.
    • Fig. S25. Representative images of hepatic tissues stained with F4/80 from mice fed SC or WD with HFCS for 3 months.
    • Fig. S26. Representative images of H&E-stained liver tissues from C57BL/6 mice fed SC or WD with HFCS for 1 month.
    • Fig. S27. Representative H&E-stained liver tissues from mice fed SC or WD with HFCS for 3 months.
    • Fig. S28. In vivo emission spectra of the reporter from the liver of mice fed SC or WD with HFCS for 1 to 3 months.
    • Fig. S29. Analysis of intercellular heterogeneity of KC lipid content in vivo.
    • Fig. S30. Reporter presence in relation to lipogranulomas.
    • Fig. S31. Weight and serum chemistry measurements of mice fed SC or WD with HFCS for 3 months.
    • Fig. S32. Serum chemistry measurements and weights of reporter- and vehicle-injected mice in a model of hepatic injury.
    • Fig. S33. Relative hepatic expression of genes involved in the innate immune response after injection with ssCTTC3TTC-(9,4) or PBS.
    • Fig. S34. Representative images of H&E-stained liver tissue from C57BL/6 mice fed SC or WD with HFCS for 2 weeks.
    • Fig. S35. Representative images of liver tissue stained with Oil Red O neutral lipid stain.
    • Fig. S36. Representative images of hepatic tissue from mice fed SC or WD with HFCS for 2 weeks stained with the hepatocyte marker CK8/18.
    • Fig. S37. Representative images of hepatic tissue from mice fed SC or WD with HFCS for 2 weeks stained with the KC marker F4/80.
    • Fig. S38. Representative H&E sections of livers from mice shown in Fig. 5E.
    • Table S1. Average reporter emission center wavelengths measured from wild-type and ASMKO mice in vivo, ex vivo¸ and in frozen sections of resected liver tissue.
    • Table S2. Average reporter emission center wavelengths measured from wild-type and Npctm(I1061T)Dso mice in vivo, ex vivo, and in frozen sections of resected liver tissue.
    • References (4756)

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    Other Supplementary Material for this manuscript includes the following:

    • Table S3 (Microsoft Excel format). Individual mouse data.

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