Research ArticleMETABOLIC DISEASE

Neuropilin-1 expression in adipose tissue macrophages protects against obesity and metabolic syndrome

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Science Immunology  16 Mar 2018:
Vol. 3, Issue 21, eaan4626
DOI: 10.1126/sciimmunol.aan4626
  • Fig. 1 NRP1-expressing macrophages accumulate in adipose tissue during DIO and regulate weight gain and glucose tolerance.

    (A) Nrp1 expression levels in populations of eosinophils, neutrophils, monocytes, and macrophages (n = 1 to 4). MHCII, major histocompatibility complex class II; LN, lymph node; MF, macrophage. (B) Flow cytometry analysis of NRP1+ ATMs isolated from VAT of WT mice fed either an NCD or a HFD for 10 weeks, represented as percent (left) or total number of cells per gram of tissue (right) (n = 9). (C) Flow cytometry analysis of NRP1+ monocytes isolated from WT mice fed either an NCD or a HFD for 4 weeks, represented as percent (left) or MFI (right) (n = 5). (D) Representative VAT immunohistochemistry of LysM-Cre/ROSA26EYFPfl/fl mice fed NCD (left), HFD for 10 weeks (middle), and HFD for 22 weeks (right) labeled for NRP1, lectin, and perilipin (magnification, ×30; scale bars, 50 μm; top), and magnification with additional DAPI stain (magnification, ×60; scale bars, 20 μm; bottom). (E) Flow cytometry analysis of NRP1+ ATMs isolated from VAT of LysM-Cre-Nrp1+/+ (control) and LysM-Cre-Nrp1fl/fl mice, represented as percent (left) or total number of cells per gram of tissue (right) (n = 6). (F) Weight gain of control, LysM-Cre-Nrp1fl/fl, and WT on NCD for 30 weeks (n = 4 to 9). EchoMRI of control and LysM-Cre-Nrp1fl/fl mice 18 weeks on NCD (age-matched controls to HFD mice) (n = 4). (G) GTT (n = 12, two experiments) and insulin tolerance test (ITT) (n = 6 to 7, two experiments) of control and LysM-Cre-Nrp1fl/fl mice 18 weeks on NCD. (H) Weight gain of control, LysM-Cre-Nrp1fl/fl, Nrp1fl/fl, and WT on HFD for 18 weeks (n = 4 to 14). EchoMRI and DEXA scan of control and LysM-Cre-Nrp1fl/fl mice 10 weeks on HFD (n = 4). (I) GTT (n = 9 to 10, two experiments) and ITT (n = 6, two experiments) of control and LysM-Cre-Nrp1fl/fl mice 16 weeks on HFD (n = 3). White arrows denote NRP1+ ATMs, and white arrowheads denote NRP1+ CLSs. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s unpaired t test (B, C, and E) or two-way ANOVA with Bonferroni post hoc test (F to I).

  • Fig. 2 NRP1-expressing myeloid cells influence adipocyte hypertrophy, development of fatty liver, and CLSs.

    LysM-Cre-Nrp1+/+ (control) and LysM-Cre-Nrp1fl/fl mice were fed a HFD for 10 weeks. (A) Representative immunohistochemistry of perilipin-labeled VAT (left) (magnification, ×10; scale bar, 100 μm) and quantification of adipocyte density and size (right) (n = 3 to 4). (B) Liver photomicrographs (left) and liver weights (right, n = 8). (C) Oil Red O stain of liver sections (magnification, ×10; scale bar, 100 μm). (D) Immunohistochemistry of F4/80+-labeled VAT and quantification of CLSs (n = 13) (magnification, ×10; scale bars, 100 μm). *P < 0.05, **P < 0.01, ***P < 0.001, Student’s unpaired t test.

  • Fig. 3 NRP1-expressing myeloid cells contribute to adipose tissue vascularization.

    (A) Angiogenesis GSEA of WT or LysM-Cre-Nrp1fl/fl peritoneal macrophages and (B) heat map (n = 2 to 3). NES, normalized enrichment score; FDR, false discovery rate. (C) Representative lectin stain of VAT in control and LysM-Cre-Nrp1fl/fl mice fed NCD for 18 weeks (age-matched controls to HFD mice) (magnification, ×30; scale bar, 50 μm) (left) and quantification of vessel length, area, and lacunarity (right) (n = 6 to 7 per group). A.U., arbitrary units. (D) Lectin stain of VAT in control or LysM-Cre-Nrp1fl/fl 18-week-old mice fed HFD for 10 weeks (magnification, ×30; scale bar, 50 μm) (left) with quantification for vessel length, area, and lacunarity (n = 6 to 11 per group). (E) Pimonidazole adduct staining of control or LysM-Cre-Nrp1fl/fl mice after 10 weeks on HFD (magnification, ×10; scale bar, 100 μm) (left) and quantification of hypoxyprobe stain (right) (n = 7 to 8). *P < 0.05, **P < 0.01, ***P < 0.001, Student’s unpaired t test.

  • Fig. 4 Macrophage-resident NRP1 mitigates cytokine release and proinflammatory polarization.

    (A) Schematic representation of ATM isolation. SVC, stromal vascular cell. (B) qPCRs of Il-6, Tnf-α, and Il-1α in ATMs isolated from 10-week HFD-fed control and LysM-Cre-Nrp1fl/fl mice (n = 4 to 7, four experiments). Heat map (C) and GSEA (D) of inflammatory response genes in WT and LysM-Cre-Nrp1fl/fl peritoneal macrophages (n = 2 to 3 per group). (E to H) Flow cytometry analysis from VAT of LysM-Cre-Nrp1+/+ (control) or LysM-Cre-Nrp1fl/fl mice fed a HFD for 10 weeks representing (E) ATMs in percent (left) or total number of cells per gram of tissue (right) (n = 13 to 17, two experiments). (F) Neutrophils in percent (left) or total number of cells per gram of tissue (right) (n = 6, two experiments). n.s., not significant. (G) CD206 ATMs in percent or total number of cells per gram of tissue (left), CD206+ ATMs in percent or total number of cells per gram of tissue (middle), or C206 MFI (right) (n = 6, two experiments). (H) FACS dot plot of CD206+ and CD206 ATMs. (I) Plasma TNF-α, IL-1β, and IFN-γ expression of 10-week HFD control and LysM-Cre-Nrp1fl/fl mice (n = 5 to 7 per group). *P < 0.05, ***P < 0.001, Student’s unpaired t test.

  • Fig. 5 Deficiency in myeloid-resident NRP1 affects systemic metabolism.

    Daily food intake (A) and total beam breaks (B) of control and LysM-Cre-Nrp1fl/fl mice after 18 weeks on a regular diet. Daily food intake (C) and total beam breaks (D) of control and LysM-Cre-Nrp1fl/fl of 18-week-old mice after 10 weeks on HFD. VO2 (E), heat production (F), and RER (G) of control and LysM-Cre-Nrp1fl/fl mice 18 weeks on a regular diet. VO2 (H), heat production (I), and RER (J) of control and LysM-Cre-Nrp1fl/fl of 18-week-old mice after 10 weeks on HFD. Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s unpaired t test (A and C) and two-way ANOVA with Bonferroni post hoc test (B and D to J) (n = 24, four mice per group). All graph points represent an average calculated over 7 days of measurements.

  • Fig. 6 Macrophage-resident NRP1 promotes FA uptake.

    (A) FA uptake of ATMs isolated from control and LysM-Cre-Nrp1fl/fl mice (n = 4 to 5, two experiments). (B) BODIPY FA analog uptake of peritoneal macrophages isolated from control and LysM-Cre-Nrp1fl/fl mice (n = 7 to 8, two experiments). (C) BODIPY uptake within VAT, liver, plasma, and heart of HFD-fed control and LysM-Cre-Nrp1fl/fl mice (n = 6 per group). (D) Oil Red O stain and quantification of control and LysM-Cre-Nrp1fl/fl macrophages incubated in adipocyte-conditioned medium (ACM) (magnification, ×63; scale bars, 10 μm) (n = 32 to 35 per group). (E to H) Volcano plots of changes in RNA-seq expression of peritoneal macrophages from LysM-Cre-Nrp1fl/fl compared with WT with (E) MSigDB hallmark gene sets of FA metabolism (M5935), (F) adipogenesis (M5905), (G) mitochondrial FA β-oxidation (M14690), and (H) lipid digestion, mobilization, and transport (M1023). *P < 0.05, **P < 0.01, ***P < 0.001, Student’s unpaired t test.

  • Fig. 7 Deficiency in macrophage-resident NRP1 shifts metabolism to glycolysis.

    (A) ECAR of control and LysM-Cre-Nrp1fl/fl macrophages at basal levels, followed by sequential treatment (arrows) of glucose (Glc), oligomycin (O), and 2-DG (n = 3, two experiments). (B) Nonglycolytic acidification, glycolysis, glycolytic capacity, glycolytic reserve (n = 3 to 12) and (C) basal OCR/ECAR ratio of control and LysM-Cre-Nrp1fl/fl macrophages (n = 8 to 9). (D) OCR of control and LysM-Cre-Nrp1fl/fl macrophages cultured in BSA or palmitate, followed by sequential treatment (arrows) with oligomycin (O), FCCP, and rotenone plus antimycin (R + A) (n = 2 to 3, two experiments). Baseline OCR (n = 6 to 9) (E) and maximal respiration OCR (F) of control and LysM-Cre-Nrp1fl/fl macrophages treated with BSA or palmitate (n = 4 to 6). (G) ECAR of BSA- and palmitate-treated control or LysM-Cre-Nrp1fl/fl macrophages (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA with Bonferroni post hoc test (A), Student’s unpaired t test (B, C, E, and G), and one-way ANOVA (F).

  • Fig. 8 Transfer of NRP1-expressing bone marrow improves the metabolic phenotype of LysM-Cre-Nrp1fl/fl mice.

    (A) Schematic of bone marrow transfer. (B) GTT of bone marrow chimeras before HFD feeding: LysM-Cre-Nrp1+/+ or LysM-Cre-Nrp1fl/fl plus CD45.1 bone marrow (left; n = 4 to 7 per group, two experiments), and CD45.1 plus LysM-Cre-Nrp1+/+ or LysM-Cre-Nrp1fl/fl bone marrow (right; n = 6 to 7, two experiments). (C) Flow cytometry analysis showing reconstitution of CD45.1+ and CD45.2+ ATMs and B and T lymphocytes isolated from chimera VAT (n = 2 to 4). (D) GTT of LysM-Cre-Nrp1+/+ or LysM-Cre-Nrp1fl/fl mice plus CD45.1 bone marrow (left; n = 6 to 7 per group, two experiments) and GTT of CD45.1 mice plus LysM-Cre-Nrp1+/+ or LysM-Cre-Nrp1fl/fl bone marrow (right; n = 5 to 7 per group) on HFD. (E) Percent weight gain when on HFD of LysM-Cre-Nrp1+/+ or LysM-Cre-Nrp1fl/fl mice plus CD45.1 bone marrow (left; n = 8 per group) and percent weight gain of CD45.1 mice plus LysM-Cre-Nrp1+/+ or LysM-Cre-Nrp1fl/fl bone marrow (right; n = 5 to 7 per group). (F) Recapitulative schematic of NRP1+ and NRP1 ATMs in adipose tissue homeostasis. *P < 0.05, ***P < 0.001, two-way ANOVA with Bonferroni post hoc test (C and D).

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/21/eaan4626/DC1

    Experimental Procedures

    Fig. S1. NRP1-expressing ATMs accumulate in DIO.

    Fig. S2. Deficiency in myeloid-resident NRP1 influences systemic metabolism.

    Fig. S3. Macrophage-resident NRP1 promotes FA uptake.

    Fig. S4. Gating scheme for ATMs.

    Fig. S5. Transfer of NRP1-expressing bone marrow.

    Table S1. Fluorophore-conjugated antibodies used for flow cytometry.

    Table S2. Primer sets used for reverse transcription PCR.

    Table S3. Raw data.

  • Supplementary Materials

    Supplementary Material for:

    Neuropilin-1 expression in adipose tissue macrophages protects against obesity and metabolic syndrome

    Ariel Molly Wilson, Zhuo Shao, Vanessa Grenier, Gaëlle Mawambo, Jean-François Daudelin, Agnieszka Dejda, Frédérique Pilon, Natalija Popovic, Salix Boulet, Célia Parinot, Malika Oubaha, Nathalie Labrecque, Vincent de Guire, Mathieu Laplante, Guillaume Lettre, Florian Sennlaub, Jean-Sebastien Joyal, Michel Meunier, Przemyslaw Sapieha*

    *Corresponding author. Email: mike.sapieha{at}umontreal.ca

    Published 16 March 2018, Sci. Immunol. 3, eaan4626 (2018)
    DOI: 10.1126/sciimmunol.aan4626

    This PDF file includes:

    • Experimental Procedures
    • Fig. S1. NRP1-expressing ATMs accumulate in DIO.
    • Fig. S2. Deficiency in myeloid-resident NRP1 influences systemic metabolism.
    • Fig. S3. Macrophage-resident NRP1 promotes FA uptake.
    • Fig. S4. Gating scheme for ATMs.
    • Fig. S5. Transfer of NRP1-expressing bone marrow.
    • Table S1. Fluorophore-conjugated antibodies used for flow cytometry.
    • Table S2. Primer sets used for reverse transcription PCR.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S3 (Microsoft Excel format). Raw data.

    Files in this Data Supplement:

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