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Miltenyi Biotec anti human siglec 1 pe vio770
Anti Human Siglec 1 Pe Vio770, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 94/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Enhanced TLR7 responses in patients with PC. (A and B) PBMCs from chilblain patients (PC, n = 16) or HD (controls, n = 20) were stimulated with the TLR7-selective agonist IMQ for 24 h. (A) Secreted IFN-α was evaluated in a LEGENDplex assay. (B) IFN-α production in response to IMQ is shown relative to the age of female (F) and male (M) patients or controls. Freshly isolated PBMCs were used in these experiments. (C) PBMCs from chilblain patients (PC, n = 12) or HD (controls, n = 12) were stimulated with the TLR7-selective agonist CL087 and the dual TLR7/8 agonist R848 for 24 h. Secreted IFN-α was evaluated in a LEGENDplex assay. Cryopreserved PBMCs were used in these experiments. (D) ISRE reporter cells (expressing luciferase under the control of an ISRE, THP1-Dual) were treated with supernatants from the CL087-stimulated PBMCs of chilblain patients (PC, n = 12) or HD (controls, n = 12). Luciferase activity was quantified with a luminometer, and RLA is represented as fold induction over untreated reporter cells. (E) THP1-Dual cells were treated with the supernatants of CL087-stimulated PBMCs from chilblain patients (PC, n = 12) or HD (controls, n = 12). The expression of ISG15 and SIGLEC1 was analyzed by flow cytometry with intracellular staining. Graphs show MFI as fold induction over basal levels in THP1 cells. (F) PBMCs from chilblain patients (PC, n = 15) or HD (controls, n = 16) were stimulated with viral nucleic acid surrogates for 24 h. Secreted IFN-α levels were evaluated in a LEGENDplex assay after stimulation with liposome-encapsulated poly(I:C), cGAMP (STING agonist) or poly(dA:dT) (cytosolic DNA sensors), or naked CpG-A (TLR9 agonist). Freshly isolated PBMCs were used in these experiments. (G) PBMCs from chilblain patients (PC) or HD (controls) were stimulated for 24 h with SARS-CoV-2 virus ( n = 8) or IAV ( n = 12). Secreted IFN-α was evaluated in a LEGENDplex assay. Cryopreserved PBMCs were used in these experiments. Adjusted P values in A and C–G were determined in Mann–Whitney tests with the Bonferroni correction for multiple testing. ***P < 0.001; **P < 0.01; *P < 0.05; ns = nonsignificant. IMQ, imiquimod; RLA, relative luciferase activity; MFI, mean fluorescence intensity; IAV, influenza A virus.

Journal: The Journal of Experimental Medicine

Article Title: Enhanced TLR7-dependent production of type I interferon by pDCs underlies pandemic chilblains

doi: 10.1084/jem.20231467

Figure Lengend Snippet: Enhanced TLR7 responses in patients with PC. (A and B) PBMCs from chilblain patients (PC, n = 16) or HD (controls, n = 20) were stimulated with the TLR7-selective agonist IMQ for 24 h. (A) Secreted IFN-α was evaluated in a LEGENDplex assay. (B) IFN-α production in response to IMQ is shown relative to the age of female (F) and male (M) patients or controls. Freshly isolated PBMCs were used in these experiments. (C) PBMCs from chilblain patients (PC, n = 12) or HD (controls, n = 12) were stimulated with the TLR7-selective agonist CL087 and the dual TLR7/8 agonist R848 for 24 h. Secreted IFN-α was evaluated in a LEGENDplex assay. Cryopreserved PBMCs were used in these experiments. (D) ISRE reporter cells (expressing luciferase under the control of an ISRE, THP1-Dual) were treated with supernatants from the CL087-stimulated PBMCs of chilblain patients (PC, n = 12) or HD (controls, n = 12). Luciferase activity was quantified with a luminometer, and RLA is represented as fold induction over untreated reporter cells. (E) THP1-Dual cells were treated with the supernatants of CL087-stimulated PBMCs from chilblain patients (PC, n = 12) or HD (controls, n = 12). The expression of ISG15 and SIGLEC1 was analyzed by flow cytometry with intracellular staining. Graphs show MFI as fold induction over basal levels in THP1 cells. (F) PBMCs from chilblain patients (PC, n = 15) or HD (controls, n = 16) were stimulated with viral nucleic acid surrogates for 24 h. Secreted IFN-α levels were evaluated in a LEGENDplex assay after stimulation with liposome-encapsulated poly(I:C), cGAMP (STING agonist) or poly(dA:dT) (cytosolic DNA sensors), or naked CpG-A (TLR9 agonist). Freshly isolated PBMCs were used in these experiments. (G) PBMCs from chilblain patients (PC) or HD (controls) were stimulated for 24 h with SARS-CoV-2 virus ( n = 8) or IAV ( n = 12). Secreted IFN-α was evaluated in a LEGENDplex assay. Cryopreserved PBMCs were used in these experiments. Adjusted P values in A and C–G were determined in Mann–Whitney tests with the Bonferroni correction for multiple testing. ***P < 0.001; **P < 0.01; *P < 0.05; ns = nonsignificant. IMQ, imiquimod; RLA, relative luciferase activity; MFI, mean fluorescence intensity; IAV, influenza A virus.

Article Snippet: Permeabilized cells were incubated overnight at 4°C with the following antibodies: ISG15 PE (clone 851701, Cat. IC8044P, RRID:AB_3656663, 1:200 dilution; R&D Systems) and SIGLEC1 APC (clone 7-239, Cat. 130-098-645, RRID:AB_2655547, 1:400 dilution; Miltenyi Biotec), in the presence of Human TruStain FcX (AB_2818986; BioLegend).

Techniques: Isolation, Expressing, Luciferase, Control, Activity Assay, Flow Cytometry, Staining, Virus, MANN-WHITNEY, Fluorescence

Related to . (A) PBMCs from chilblain patients (PC, n = 13) or HD (controls, n = 16) were stimulated with the TLR7-selective agonist IMQ for 24 h. Secreted cytokines were evaluated in a LEGENDplex assay. Freshly isolated PBMCs were used in these experiments. (B) THP1-Dual reporter cells were treated with various doses of recombinant IFN-α (rIFN-α2b), ranging from 10 to 1,000 IU/ml. Left panel: luciferase activity was quantified with a luminometer, and RLA is represented as fold induction over untreated reporter cells. Right panels: the expression of ISG15 and SIGLEC1 was analyzed by flow cytometry with intracellular staining. Graphs show MFI as fold induction over basal levels in THP1 cells. The dashed lines represent the mean type I IFN activity levels for supernatants from the TLR7-stimulated leukocytes of HD (black) (controls, n = 12) and chilblain patients (red) (PC, n = 12). (C) PBMCs from chilblain patients (PC, n = 15) or HD (controls, n = 16) were stimulated with viral nucleic acid surrogates for 24 h. Secreted IFN-β levels were evaluated in a LEGENDplex assay after stimulation with liposome-encapsulated poly(I:C), cGAMP (STING agonist) or poly(dA:dT) (cytosolic DNA sensors), or naked CpG-A (TLR9 agonist). Freshly isolated PBMCs were used in these experiments. (D) PBMCs from PC patients or controls were stimulated for 24 h with SARS-CoV-2 ( n = 8) or IAV ( n = 12). Secreted IFN-β levels were evaluated in a LEGENDplex assay. Cryopreserved PBMCs were used in these experiments. (E) PBMCs from PC patients (PC, n = 12) or HD (controls, n = 12) were stimulated for 24 h with HSV-1. Secreted IFN-α and IFN-β levels were evaluated in a LEGENDplex assay. (F) Distribution of lymphoid and myeloid subsets among PBMCs from PC patients (PC, n = 12) and HD (controls, n = 14) was determined by flow cytometry. Data are represented as percentages of total CD45 + cells. CM, central memory T cells; EM, effector memory T cells; EMRA, CD45RA + effector memory T cells; NK, natural killer cells; cDC CD1c + , CD1c + conventional dendritic cells; cDC CD1c − , CD1c − conventional dendritic cells. (G) Frequency of CD123 + BDCA2 + pDCs in PBMCs from PC patients ( n = 12) and controls ( n = 14), as analyzed in F, is shown on a separate graph. Bars represent the mean ± SD. The adjusted P values in A, C–E, and G were determined in Mann–Whitney tests with the Bonferroni correction for multiple testing. *P < 0.05; ns = nonsignificant. IMQ, imiquimod; RLA, relative luciferase activity; MFI, mean fluorescence intensity; IAV, influenza A virus.

Journal: The Journal of Experimental Medicine

Article Title: Enhanced TLR7-dependent production of type I interferon by pDCs underlies pandemic chilblains

doi: 10.1084/jem.20231467

Figure Lengend Snippet: Related to . (A) PBMCs from chilblain patients (PC, n = 13) or HD (controls, n = 16) were stimulated with the TLR7-selective agonist IMQ for 24 h. Secreted cytokines were evaluated in a LEGENDplex assay. Freshly isolated PBMCs were used in these experiments. (B) THP1-Dual reporter cells were treated with various doses of recombinant IFN-α (rIFN-α2b), ranging from 10 to 1,000 IU/ml. Left panel: luciferase activity was quantified with a luminometer, and RLA is represented as fold induction over untreated reporter cells. Right panels: the expression of ISG15 and SIGLEC1 was analyzed by flow cytometry with intracellular staining. Graphs show MFI as fold induction over basal levels in THP1 cells. The dashed lines represent the mean type I IFN activity levels for supernatants from the TLR7-stimulated leukocytes of HD (black) (controls, n = 12) and chilblain patients (red) (PC, n = 12). (C) PBMCs from chilblain patients (PC, n = 15) or HD (controls, n = 16) were stimulated with viral nucleic acid surrogates for 24 h. Secreted IFN-β levels were evaluated in a LEGENDplex assay after stimulation with liposome-encapsulated poly(I:C), cGAMP (STING agonist) or poly(dA:dT) (cytosolic DNA sensors), or naked CpG-A (TLR9 agonist). Freshly isolated PBMCs were used in these experiments. (D) PBMCs from PC patients or controls were stimulated for 24 h with SARS-CoV-2 ( n = 8) or IAV ( n = 12). Secreted IFN-β levels were evaluated in a LEGENDplex assay. Cryopreserved PBMCs were used in these experiments. (E) PBMCs from PC patients (PC, n = 12) or HD (controls, n = 12) were stimulated for 24 h with HSV-1. Secreted IFN-α and IFN-β levels were evaluated in a LEGENDplex assay. (F) Distribution of lymphoid and myeloid subsets among PBMCs from PC patients (PC, n = 12) and HD (controls, n = 14) was determined by flow cytometry. Data are represented as percentages of total CD45 + cells. CM, central memory T cells; EM, effector memory T cells; EMRA, CD45RA + effector memory T cells; NK, natural killer cells; cDC CD1c + , CD1c + conventional dendritic cells; cDC CD1c − , CD1c − conventional dendritic cells. (G) Frequency of CD123 + BDCA2 + pDCs in PBMCs from PC patients ( n = 12) and controls ( n = 14), as analyzed in F, is shown on a separate graph. Bars represent the mean ± SD. The adjusted P values in A, C–E, and G were determined in Mann–Whitney tests with the Bonferroni correction for multiple testing. *P < 0.05; ns = nonsignificant. IMQ, imiquimod; RLA, relative luciferase activity; MFI, mean fluorescence intensity; IAV, influenza A virus.

Techniques: Isolation, Recombinant, Luciferase, Activity Assay, Expressing, Flow Cytometry, Staining, MANN-WHITNEY, Fluorescence, Virus

$(A) Uniform Manifold Approximation and Projection (UMAP) representation of unsupervised clustering of resident F4/80 + CD11b + (CD45 + CD45.2iv − CD3 − CD19 − SiglecF − ) cells sorted from the VAT of young (7,558 cells) and aged (7,139 cells) mice from males and females. (B) Repartition of cells in each cluster as a fraction of total cells (males and females) from young and aged VAT. (C) Violin plots depicting the expression of Siglec1 , Itgax , Cd226 , Cd9 , Cd163 , Folr2 , and Cd38 genes by cluster. (D) Schematic summarizing the cell surface markers used to define seven clusters identified via single-cell RNA-sequencing of resident F4/80 + CD11b + cells (CD45 + CD45.2iv − CD3 − CD19 − SiglecF − ) from the VAT. Abbreviations for each subset and defining cell surface markers are as follows: Nerve-associated-macrophages (NAM, C5): CD169 + CD11c − , Myeloid Precursors-1 (MP-1, C13): CD169 + CD11c + , Vessel-associated Macrophages (VAM, C0): CD38 + FOLR2 + CD11c − CD38 + , Age-associated Macrophages (AAM, C4): CD38 + FOLR2 − CD169 − CD11c − , Lipid-associated Macrophages (LAM, C10): CD9 + CD11c + CD226 − CD163 − CD169 − , Adipose Tissue Macrophages-1 (ATM-1, C3): CD226 + CD11c + CD163 − CD169 − , Dendritic Cells-1 (DC-1, C6): CD163 + CD226 + CD11c + CD163 + CD169 − . (E) Representative flow cytometry plots depicting the proportion of NAM, MP-1, VAM, AAM, LAM, ATM-2, and DC-1 populations in 3-month (n=4) and 21-month (n=5) old females. (F) Quantification of the proportion of NAM, MP-1, VAM, AAM, LAM, ATM-2, and DC-1in the VAT from young and aged females. Data is displayed as the mean +/− SEM and is representative of one independent experiment from 3-month (n=4) and 21-month-old (n=5) females. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and *, p < 0.05; **, p < 0.01, n.s. = not significant.$

Journal: bioRxiv

Article Title: Nerve-associated macrophages control adipose homeostasis across lifespan and restrain age-related inflammation

doi: 10.1101/2024.10.12.618004

Figure Lengend Snippet: (A) Uniform Manifold Approximation and Projection (UMAP) representation of unsupervised clustering of resident F4/80 + CD11b + (CD45 + CD45.2iv − CD3 − CD19 − SiglecF − ) cells sorted from the VAT of young (7,558 cells) and aged (7,139 cells) mice from males and females. (B) Repartition of cells in each cluster as a fraction of total cells (males and females) from young and aged VAT. (C) Violin plots depicting the expression of Siglec1 , Itgax , Cd226 , Cd9 , Cd163 , Folr2 , and Cd38 genes by cluster. (D) Schematic summarizing the cell surface markers used to define seven clusters identified via single-cell RNA-sequencing of resident F4/80 + CD11b + cells (CD45 + CD45.2iv − CD3 − CD19 − SiglecF − ) from the VAT. Abbreviations for each subset and defining cell surface markers are as follows: Nerve-associated-macrophages (NAM, C5): CD169 + CD11c − , Myeloid Precursors-1 (MP-1, C13): CD169 + CD11c + , Vessel-associated Macrophages (VAM, C0): CD38 + FOLR2 + CD11c − CD38 + , Age-associated Macrophages (AAM, C4): CD38 + FOLR2 − CD169 − CD11c − , Lipid-associated Macrophages (LAM, C10): CD9 + CD11c + CD226 − CD163 − CD169 − , Adipose Tissue Macrophages-1 (ATM-1, C3): CD226 + CD11c + CD163 − CD169 − , Dendritic Cells-1 (DC-1, C6): CD163 + CD226 + CD11c + CD163 + CD169 − . (E) Representative flow cytometry plots depicting the proportion of NAM, MP-1, VAM, AAM, LAM, ATM-2, and DC-1 populations in 3-month (n=4) and 21-month (n=5) old females. (F) Quantification of the proportion of NAM, MP-1, VAM, AAM, LAM, ATM-2, and DC-1in the VAT from young and aged females. Data is displayed as the mean +/− SEM and is representative of one independent experiment from 3-month (n=4) and 21-month-old (n=5) females. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and *, p < 0.05; **, p < 0.01, n.s. = not significant.

Article Snippet: Cells were stained with live/dead Aqua viability dye (Invitrogen) or Viakrome 808 viability dye (Beckman Coulter) for 30 minutes, followed by Fc receptor blocking (Invitrogen) for 10 minutes and surface staining for 30 minutes with the following antibodies: CD45-PE-Cy7 (Clone 30-F11, catalog no. 103114), CD11b-APC (Clone M1/70 catalog no. 101212), FOLR2-PE (Clone 10/FR2 catalog no. 153304), CD9-FITC (Clone MZ3 catalog no. 124808), CD226-BV421 (Clone TX42.1 catalog no. 133615), CD11c-BV750 (Clone N418 catalog no. 117357), B220-BV711 (Clone RA3-6B2 catalog no. 103255), Ly6G-FITC (Clone 1A8 catalog no. 127606), Ly6C-AF700 (Clone HK1.4 catalog no. 128024) ( all from BioLegend ); CD45.2-FITC (Clone 104 catalog no. 11-0454-85), F4/80-PE-Texas Red (Clone BM8 catalog no. MF48017), CD163-SB702 (Clone TNKUPJ catalog no. 67-1631-82), CD169-APC-eFluor780 (Clone SER-4 catalog no. 47-5755-82), CD45-APC (Clone 30-F11, catalog no. 17-0451-83), CD169-PE (Clone SER-4 catalog no. 12-5755-80), FOXP3-PE-Cy5.5 (Clone FJK-16s catalog no. 35-5773-82), LYVE-1-Biotin (Clone ALY7 catalog no. 13-0443-82) ( all from Thermo Fisher Scientific ); CD38-BUV737 (Clone 90/CD38 catalog no. 741748), Siglec-F-BV605 (Clone E50-2440 catalog no. 740388), CD11b-BUV395 (Clone M1/70 catalog no. 563553), CD3-BV480 (Clone 17A2 catalog no. 565642), CD4-BUV563 (Clone GK1.5 catalog no. 612923), CD8a-BUV615 (Clone 53-6.7 catalog no. 613004), I-A/I-E-BUV805 (Clone M5/114.15.2 catalog no. 748844), CD25-PE-CF594 (Clone PC61 catalog no. 562694) ( all from BD Biosciences ); and F4/80-StarBrightViolet570 (Clone CI:A3-1 catalog no. MCA497) (from Bio-Rad).

Techniques: Expressing, RNA Sequencing Assay, Flow Cytometry, Two Tailed Test

$(A) Repartition of cells in each cluster as a fraction of total cells from young or aged VAT from males ( top ) and females ( bottom ). (B) Schematic depicting the flow cytometry gating strategy used to define clusters 0, 3, 4, 5, 6, 10, and 13 identified via single cell RNA-sequencing of resident F4/80 + CD11b + (CD45 + CD45.2iv − CD3 − CD19 − ) cells from the visceral white adipose tissue (VAT). Fluorescence minus one (FMO) control(s) were included for the following cell surface markers: CD11b, CD169, CD11c, CD226, CD9, CD163, FOLR2, and CD38. Abbreviations for each population and defining cell surface markers are as follows: Nerve-associated-macrophages (NAM, C5): CD169 + CD11c − , Myeloid Precursors-1 (MP-1, C13): CD169 + CD11c + , Vessel-associated Macrophages (VAM, C0): CD38 + FOLR2 + CD11c − CD38 + , Age-associated Macrophages (AAM, C4): CD38 + FOLR2 − CD169 − CD11c − , Lipid-associated Macrophages (LAM, C10): CD9 + CD11c + CD226 − CD163 − CD169 − , Adipose Tissue Macrophages-1 (ATM-1, C3): CD226 + CD11c + CD163 − CD169 − , Dendritic Cells-1 (DC-1, C6): CD163 + CD226 + CD11c + CD163 + CD169 − . (C) Quantification of cells from clusters 5, 13, 0, 4, 10, 3, and 6 as a fraction of live CD45+ cells per gram of visceral white adipose tissue (VAT). Data is displayed as the mean +/− SEM and is representative of one independent experiment from 3-month (n=4) and 21-month-old (n=5) females. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and *, p < 0.05; **, p < 0.01, n.s. = not significant.$

Journal: bioRxiv

Article Title: Nerve-associated macrophages control adipose homeostasis across lifespan and restrain age-related inflammation

doi: 10.1101/2024.10.12.618004

Figure Lengend Snippet: (A) Repartition of cells in each cluster as a fraction of total cells from young or aged VAT from males ( top ) and females ( bottom ). (B) Schematic depicting the flow cytometry gating strategy used to define clusters 0, 3, 4, 5, 6, 10, and 13 identified via single cell RNA-sequencing of resident F4/80 + CD11b + (CD45 + CD45.2iv − CD3 − CD19 − ) cells from the visceral white adipose tissue (VAT). Fluorescence minus one (FMO) control(s) were included for the following cell surface markers: CD11b, CD169, CD11c, CD226, CD9, CD163, FOLR2, and CD38. Abbreviations for each population and defining cell surface markers are as follows: Nerve-associated-macrophages (NAM, C5): CD169 + CD11c − , Myeloid Precursors-1 (MP-1, C13): CD169 + CD11c + , Vessel-associated Macrophages (VAM, C0): CD38 + FOLR2 + CD11c − CD38 + , Age-associated Macrophages (AAM, C4): CD38 + FOLR2 − CD169 − CD11c − , Lipid-associated Macrophages (LAM, C10): CD9 + CD11c + CD226 − CD163 − CD169 − , Adipose Tissue Macrophages-1 (ATM-1, C3): CD226 + CD11c + CD163 − CD169 − , Dendritic Cells-1 (DC-1, C6): CD163 + CD226 + CD11c + CD163 + CD169 − . (C) Quantification of cells from clusters 5, 13, 0, 4, 10, 3, and 6 as a fraction of live CD45+ cells per gram of visceral white adipose tissue (VAT). Data is displayed as the mean +/− SEM and is representative of one independent experiment from 3-month (n=4) and 21-month-old (n=5) females. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and *, p < 0.05; **, p < 0.01, n.s. = not significant.

Techniques: Flow Cytometry, RNA Sequencing Assay, Fluorescence, Control, Two Tailed Test

(A) Uniform Manifold Approximation and Projection (UMAP) ( left ) and violin plot ( right ) showing enrichment of senescence associated gene transcripts and gene set enrichment score (GSES) per cell. Senescence associated gene transcripts were defined using the SenMayo gene set (SAUL_SEN_MAYO, MM16098). (B) Gene Ontology (GO) analysis showing enrichment of unique biological process (BP) terms by cluster. (C) Dot plot depicting the average expression of genes enriched in NAMs (Cluster 5). Dot size represents the percentage of cells expressing a corresponding gene in a given cluster. (D) Whole mount immunofluorescence confocal microscopy images of visceral adipose tissue (VAT) from adult CD169:mTmG dual reporter mice labeled with the pan-neuronal marker TUBB3. CD169 is labeled with membrane EGFP (mGFP + ) ( green ) while non-recombined structures such as adipocytes, nerves, and blood vessels are labeled with membrane Tomato (mTomato + ) ( red ). Non-recombined structures such as adipocytes, nerves, and blood vessels and can be distinguished by their morphology with adipocytes being round, nerves appearing dense and curved/sinusoidal, and blood vessels appearing straight and hollow. Bottom Left : Single channel and merged image of a CD169 + cell embedded between a mTomato + TUBB3 + nerve. Bottom Right : CD169 + cells on the surface of mTomato + blood vessels and thin TUBB3 + nerve fibers. Data is representative of n = 4 independent experiments using 3- or 4-month-old females.

Journal: bioRxiv

Article Title: Nerve-associated macrophages control adipose homeostasis across lifespan and restrain age-related inflammation

doi: 10.1101/2024.10.12.618004

Figure Lengend Snippet: (A) Uniform Manifold Approximation and Projection (UMAP) ( left ) and violin plot ( right ) showing enrichment of senescence associated gene transcripts and gene set enrichment score (GSES) per cell. Senescence associated gene transcripts were defined using the SenMayo gene set (SAUL_SEN_MAYO, MM16098). (B) Gene Ontology (GO) analysis showing enrichment of unique biological process (BP) terms by cluster. (C) Dot plot depicting the average expression of genes enriched in NAMs (Cluster 5). Dot size represents the percentage of cells expressing a corresponding gene in a given cluster. (D) Whole mount immunofluorescence confocal microscopy images of visceral adipose tissue (VAT) from adult CD169:mTmG dual reporter mice labeled with the pan-neuronal marker TUBB3. CD169 is labeled with membrane EGFP (mGFP + ) ( green ) while non-recombined structures such as adipocytes, nerves, and blood vessels are labeled with membrane Tomato (mTomato + ) ( red ). Non-recombined structures such as adipocytes, nerves, and blood vessels and can be distinguished by their morphology with adipocytes being round, nerves appearing dense and curved/sinusoidal, and blood vessels appearing straight and hollow. Bottom Left : Single channel and merged image of a CD169 + cell embedded between a mTomato + TUBB3 + nerve. Bottom Right : CD169 + cells on the surface of mTomato + blood vessels and thin TUBB3 + nerve fibers. Data is representative of n = 4 independent experiments using 3- or 4-month-old females.

Techniques: Expressing, Immunofluorescence, Confocal Microscopy, Labeling, Marker, Membrane

(A) Whole mount immunofluorescence of VAT from adult CD169:mTmG dual reporter mice labeling the nucleus ( cyan ), the pan-neuronal marker TUBB3 ( blue ), CD169+ macrophages with membrane EGFP (mGFP+) ( green ), and non-recombined structures such as adipocytes, nerves, and blood vessels with membrane Tomato (mTomato+) ( red ). Non-recombined structures such as adipocytes, nerves, and blood vessels can be distinguished by their morphology with adipocytes being round, nerves appearing dense and curved/sinusoidal, and blood vessels appearing straight and hollow. Each panel represents a single plane on the z-axis (z). White arrowheads highlight elongated CD169+ cells. Data is representative of n = 4 independent experiments using 3-4 month-old females. (B) Transmission electron microscopy (TEM) of a nerve bundle isolated from the inguinal white adipose tissue (iWAT). The following structures are labeled: perineurium ( p ), Schwann cells ( sc ), myelinated axons ( * ), unmyelinated axons ( black arrow ), blood vessels ( bv ). Data is representative of n = 2 independent experiments using 3-month-old wildtype males. (C) Transmission electron microscopy (TEM) of a nerve bundle isolated from the inguinal white adipose tissue (iWAT) depicting an elongated nerve-associated macrophage (NAM) ( green ) located between myelinated ( blue ) and unmyelinated ( orange ) axons. Data is representative of n = 2 independent experiments using 3-month-old wildtype males. (D) Transmission electron microscopy (TEM) of a nerve bundle isolated from the inguinal white adipose tissue (iWAT) depicting a nerve-associated macrophage (NAM) ( green ) with pseudopodia and intracellular electron dense material located between myelinated ( blue ) and unmyelinated ( orange ) axons. Data is representative of n = 2 independent experiments using 3-month-old wildtype males. (E) Whole mount immunofluorescence of iWAT labeled with antibodies against the NAM marker ( red , white arrowheads ), the pan-neuronal marker TUBB3 ( blue ), and the myelin marker CNPase (2′,3′-Cyclic-nucleotide 3’-phosphodiesterase) ( green ). Data is representative of n = 1 independent experiment using 3-month-old wildtype males (n = 2). (F) Whole mount immunofluorescence of VAT from adult wildtype males labeled with antibodies against the NAM marker CD169 ( red , white arrowheads ), the pan-neuronal marker TUBB3 ( blue ), and the myelin marker MBP (myelin basic protein) ( green ). Data is representative of n = 1 independent experiment using 3-month-old wildtype males (n = 2). (G) In vivo two-photon imaging of iWAT from adult CD169:mTmG dual reporter mice labeling CD169 + macrophages with membrane EGFP (mGFP + ) ( green ), and non-recombined structures such as adipocytes, nerves, and blood vessels with membrane Tomato (mTomato + ) ( red ). Non-recombined structures such as adipocytes, nerves ( white outline ), and blood vessels ( yellow outline ) and can be distinguished by their morphology with adipocytes being round, nerves appearing dense and curved/sinusoidal, and blood vessels appearing straight and hollow. Yellow arrowheads highlight CD169 + cells interacting with blood vessels and/or nerves, white arrowheads highlight CD169 + cells on large nerve bundles. Data is representative of n = 3 independent experiment using 3-month-old males

Journal: bioRxiv

Article Title: Nerve-associated macrophages control adipose homeostasis across lifespan and restrain age-related inflammation

doi: 10.1101/2024.10.12.618004

Figure Lengend Snippet: (A) Whole mount immunofluorescence of VAT from adult CD169:mTmG dual reporter mice labeling the nucleus ( cyan ), the pan-neuronal marker TUBB3 ( blue ), CD169+ macrophages with membrane EGFP (mGFP+) ( green ), and non-recombined structures such as adipocytes, nerves, and blood vessels with membrane Tomato (mTomato+) ( red ). Non-recombined structures such as adipocytes, nerves, and blood vessels can be distinguished by their morphology with adipocytes being round, nerves appearing dense and curved/sinusoidal, and blood vessels appearing straight and hollow. Each panel represents a single plane on the z-axis (z). White arrowheads highlight elongated CD169+ cells. Data is representative of n = 4 independent experiments using 3-4 month-old females. (B) Transmission electron microscopy (TEM) of a nerve bundle isolated from the inguinal white adipose tissue (iWAT). The following structures are labeled: perineurium ( p ), Schwann cells ( sc ), myelinated axons ( * ), unmyelinated axons ( black arrow ), blood vessels ( bv ). Data is representative of n = 2 independent experiments using 3-month-old wildtype males. (C) Transmission electron microscopy (TEM) of a nerve bundle isolated from the inguinal white adipose tissue (iWAT) depicting an elongated nerve-associated macrophage (NAM) ( green ) located between myelinated ( blue ) and unmyelinated ( orange ) axons. Data is representative of n = 2 independent experiments using 3-month-old wildtype males. (D) Transmission electron microscopy (TEM) of a nerve bundle isolated from the inguinal white adipose tissue (iWAT) depicting a nerve-associated macrophage (NAM) ( green ) with pseudopodia and intracellular electron dense material located between myelinated ( blue ) and unmyelinated ( orange ) axons. Data is representative of n = 2 independent experiments using 3-month-old wildtype males. (E) Whole mount immunofluorescence of iWAT labeled with antibodies against the NAM marker ( red , white arrowheads ), the pan-neuronal marker TUBB3 ( blue ), and the myelin marker CNPase (2′,3′-Cyclic-nucleotide 3’-phosphodiesterase) ( green ). Data is representative of n = 1 independent experiment using 3-month-old wildtype males (n = 2). (F) Whole mount immunofluorescence of VAT from adult wildtype males labeled with antibodies against the NAM marker CD169 ( red , white arrowheads ), the pan-neuronal marker TUBB3 ( blue ), and the myelin marker MBP (myelin basic protein) ( green ). Data is representative of n = 1 independent experiment using 3-month-old wildtype males (n = 2). (G) In vivo two-photon imaging of iWAT from adult CD169:mTmG dual reporter mice labeling CD169 + macrophages with membrane EGFP (mGFP + ) ( green ), and non-recombined structures such as adipocytes, nerves, and blood vessels with membrane Tomato (mTomato + ) ( red ). Non-recombined structures such as adipocytes, nerves ( white outline ), and blood vessels ( yellow outline ) and can be distinguished by their morphology with adipocytes being round, nerves appearing dense and curved/sinusoidal, and blood vessels appearing straight and hollow. Yellow arrowheads highlight CD169 + cells interacting with blood vessels and/or nerves, white arrowheads highlight CD169 + cells on large nerve bundles. Data is representative of n = 3 independent experiment using 3-month-old males

Techniques: Immunofluorescence, Labeling, Marker, Membrane, Transmission Assay, Electron Microscopy, Isolation, In Vivo, Imaging

$(A) Schematic representation of the experimental protocol used to deplete CD169-macrophages using CD169 diphtheria toxin receptor (CD169-DTR) mice. 3-month-old wildtype (WT) (n=6) and CD169-DTR (n=9) female mice were injected intra-peritoneal (i.p) with 40ng/gram diphtheria toxin (DT) on day 0 (D0) followed by 4ng/gram DT on D3, D6, D9, and D12, and sacrificed on D13. (B) Representative flow cytometry plots ( left ) and bar graph ( right ) quantifying nerve-associated macrophages in the stromal vascular fraction (SVF) of VAT from 3-month-old WT and CD169-DTR females treated with DT and sacrificed on D13. Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (C) Tissue wet weight ( top ) and stromal vascular fraction (SVF) cell number ( bottom ) from the VAT of from 3-month-old WT and CD169-DTR females treated with DT and sacrificed on D13. Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (D) Flow cytometry from the VAT of 3-month-old WT and CD169-DTR females treated with DT and sacrificed on D13. Bar plots represent either the proportion of CD45+ and CD45-cells (as a fraction of total live cells) and B-cells, CD8 + T-cells, CD4 + T-cells, Monocytes, Neutrophils, Eosinophils, and MHCII+CD11c+ ATMs or dendritic cells (DCs) (as a fraction of live CD45+). Populations were defined using the cell surface markers and gating strategy outlined in . Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (E) Total expression and phosphorylation levels of lipolysis proteins from the VAT of ad libitum fed or 24-hour fasted 9-month-old WT and CD169-DTR females treated with DT and sacrificed on D8 (n=4 per genotype). (F) Total expression of electron transport chain proteins from the VAT of ad libitum fed or 24-hour fasted 9-month-old WT and CD169-DTR females treated with DT and sacrificed on D8 (n=4 per genotype).$

Journal: bioRxiv

Article Title: Nerve-associated macrophages control adipose homeostasis across lifespan and restrain age-related inflammation

doi: 10.1101/2024.10.12.618004

Techniques: Injection, Flow Cytometry, Two Tailed Test, Expressing

$(A) Change in body weight curves for 3-month-old WT and CD169-DTR females (n=6-9 per genotype) treated with DT and sacrificed on D13. (B) Representative flow cytometry plots quantifying nerve-associated macrophages (NAMs) in the spleen from 3-month-old WT and CD169-DTR females treated with DT and sacrificed on D13 (n=6-9 per genotype). (C) Bar plot quantifying the proportion of nerve-associated macrophages in the spleen from 3-month-old WT and CD169-DTR females treated with DT and sacrificed on D13. Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (D) Spleen tissue wet weight ( top ) and splenocyte cell number ( bottom ) of from 3-month-old WT and CD169-DTR females treated with DT and sacrificed on D13. Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (E) Flow cytometry of the spleen from 3-month-old WT and CD169-DTR females treated with DT and sacrificed on D13. Bar plots represent either the proportion of CD45 + and CD45 − cells (as a fraction of total live cells) and B-cells, CD8 + T-cells, CD4 + T-cells, Monocytes, Neutrophils, Eosinophils, and MHCII + CD11c + ATMs or dendritic cells (DCs) (as a fraction of live CD45 + ). Populations were defined using the cell surface markers and gating strategy outlined in . Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (F) Relative mRNA expression of Adrb3 ( top ) and Pparγ ( bottom ) in the VAT of ad libitum fed 9-month-old WT and CD169-DTR females treated with DT and sacrificed on D8 (n=3-5 per genotype). Data is represented as a mean +/− SEM. Statistical significance was determined via via Student’s two-tailed t-test with α=0.05 and p < 0.05.$

Journal: bioRxiv

Article Title: Nerve-associated macrophages control adipose homeostasis across lifespan and restrain age-related inflammation

doi: 10.1101/2024.10.12.618004

Figure Lengend Snippet: (A) Change in body weight curves for 3-month-old WT and CD169-DTR females (n=6-9 per genotype) treated with DT and sacrificed on D13. (B) Representative flow cytometry plots quantifying nerve-associated macrophages (NAMs) in the spleen from 3-month-old WT and CD169-DTR females treated with DT and sacrificed on D13 (n=6-9 per genotype). (C) Bar plot quantifying the proportion of nerve-associated macrophages in the spleen from 3-month-old WT and CD169-DTR females treated with DT and sacrificed on D13. Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (D) Spleen tissue wet weight ( top ) and splenocyte cell number ( bottom ) of from 3-month-old WT and CD169-DTR females treated with DT and sacrificed on D13. Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (E) Flow cytometry of the spleen from 3-month-old WT and CD169-DTR females treated with DT and sacrificed on D13. Bar plots represent either the proportion of CD45 + and CD45 − cells (as a fraction of total live cells) and B-cells, CD8 + T-cells, CD4 + T-cells, Monocytes, Neutrophils, Eosinophils, and MHCII + CD11c + ATMs or dendritic cells (DCs) (as a fraction of live CD45 + ). Populations were defined using the cell surface markers and gating strategy outlined in . Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (F) Relative mRNA expression of Adrb3 ( top ) and Pparγ ( bottom ) in the VAT of ad libitum fed 9-month-old WT and CD169-DTR females treated with DT and sacrificed on D8 (n=3-5 per genotype). Data is represented as a mean +/− SEM. Statistical significance was determined via via Student’s two-tailed t-test with α=0.05 and p < 0.05.

Techniques: Flow Cytometry, Two Tailed Test, Expressing

(A) Representative schematic depicting the flow cytometry gating strategy used to quantify the depletion efficiency of nerve-associated macrophages (NAMs) and other immune cell subsets in the visceral white adipose tissue (VAT) and spleen of wildtype (WT) and CD169-DTR mice following intraperitoneal diphtheria toxin administration in 3-, 6-, 9-, and 24-month-old females and at the following endpoints: day 8, day 13, day 25, and day 34.

Journal: bioRxiv

Article Title: Nerve-associated macrophages control adipose homeostasis across lifespan and restrain age-related inflammation

doi: 10.1101/2024.10.12.618004

Figure Lengend Snippet: (A) Representative schematic depicting the flow cytometry gating strategy used to quantify the depletion efficiency of nerve-associated macrophages (NAMs) and other immune cell subsets in the visceral white adipose tissue (VAT) and spleen of wildtype (WT) and CD169-DTR mice following intraperitoneal diphtheria toxin administration in 3-, 6-, 9-, and 24-month-old females and at the following endpoints: day 8, day 13, day 25, and day 34.

Techniques: Flow Cytometry

$(A) Schematic representation of the experimental protocol used to deplete CD169-macrophages using CD169 diphtheria toxin receptor (CD169-DTR) mice. 9-month-old Wildtype (WT) and CD169-DTR (n=9-11) female mice were injected intra-peritoneal (i.p) with 40ng/gram diphtheria toxin (DT) on day 0 (D0) followed by 4ng/gram DT on D2 and D4. On day 7 (D7) mice either remained ad libitum fed (n=4-5 per genotype) or were fasted for 24-hours (n=5-6 per genotype) and then scarified on D8. (B) Quantification of nerve-associated macrophages (NAMs) in the stromal vascular fraction (SVF) of VAT from 9-month-old Wildtype (WT) and CD169-DTR females treated with DT that were either ad libitum fed or 24-hour fasted and then sacrificed on D8. Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (C) Relative protein quantification of the following proteins: Hormone-sensitive Lipase (HSL), HSL phosphorylated at Ser563 (p-HSL S563 ), HSL phosphorylated at Ser660 (p-HSL S660 ), Adipose triglyceride lipase (ATGL), and Monoamine-oxidase A (MAOA). Protein lysate is from the VAT of ad libitum fed ( top panel ) or 24-hour fasted ( bottom panel ) 9-month-old WT and CD169-DTR females treated with DT and sacrificed on D8 (n=4 per genotype). Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (D) Relative protein quantification of oxidative phosphorylation proteins: Complex I (CI): NADH:ubiquinone oxidoreductase subunit B8 (NDUFB8), Complex II (CII): succinate dehydrogenase complex, subunit B, iron sulfur (Ip) (SDHB), CIII: ubiquinol cytochrome c reductase core protein 2 (UQCRC2), Complex IV (CIV): mitochondrially encoded cytochrome c oxidase I (MTCO1), Complex V (CV): ATP synthase F1 subunit alpha (ATP5A). Protein lysate is from the VAT of ad libitum fed 9-month-old WT and CD169-DTR females treated with DT and sacrificed on D8 (n=4 per genotype). Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05.$

Journal: bioRxiv

Article Title: Nerve-associated macrophages control adipose homeostasis across lifespan and restrain age-related inflammation

doi: 10.1101/2024.10.12.618004

Figure Lengend Snippet: (A) Schematic representation of the experimental protocol used to deplete CD169-macrophages using CD169 diphtheria toxin receptor (CD169-DTR) mice. 9-month-old Wildtype (WT) and CD169-DTR (n=9-11) female mice were injected intra-peritoneal (i.p) with 40ng/gram diphtheria toxin (DT) on day 0 (D0) followed by 4ng/gram DT on D2 and D4. On day 7 (D7) mice either remained ad libitum fed (n=4-5 per genotype) or were fasted for 24-hours (n=5-6 per genotype) and then scarified on D8. (B) Quantification of nerve-associated macrophages (NAMs) in the stromal vascular fraction (SVF) of VAT from 9-month-old Wildtype (WT) and CD169-DTR females treated with DT that were either ad libitum fed or 24-hour fasted and then sacrificed on D8. Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (C) Relative protein quantification of the following proteins: Hormone-sensitive Lipase (HSL), HSL phosphorylated at Ser563 (p-HSL S563 ), HSL phosphorylated at Ser660 (p-HSL S660 ), Adipose triglyceride lipase (ATGL), and Monoamine-oxidase A (MAOA). Protein lysate is from the VAT of ad libitum fed ( top panel ) or 24-hour fasted ( bottom panel ) 9-month-old WT and CD169-DTR females treated with DT and sacrificed on D8 (n=4 per genotype). Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (D) Relative protein quantification of oxidative phosphorylation proteins: Complex I (CI): NADH:ubiquinone oxidoreductase subunit B8 (NDUFB8), Complex II (CII): succinate dehydrogenase complex, subunit B, iron sulfur (Ip) (SDHB), CIII: ubiquinol cytochrome c reductase core protein 2 (UQCRC2), Complex IV (CIV): mitochondrially encoded cytochrome c oxidase I (MTCO1), Complex V (CV): ATP synthase F1 subunit alpha (ATP5A). Protein lysate is from the VAT of ad libitum fed 9-month-old WT and CD169-DTR females treated with DT and sacrificed on D8 (n=4 per genotype). Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05.

Techniques: Injection, Two Tailed Test

$(A) Schematic representation of the experimental protocol used to deplete CD169-macrophages using CD169 diphtheria toxin receptor (CD169-DTR) mice. 6-month and 24-month-old wildtype (WT) and CD169-DTR female mice were injected intra-peritoneal (i.p) with 40ng/gram diphtheria toxin (DT) on day 0 (D0) followed by 4ng/gram DT on D3, D6, D9, and D12, D15, D18, D21, D24, and sacrificed on D25. (B) Change in body weight curves for 6-month and 24-month-old WT and CD169-DTR females treated with DT and sacrificed on D24. (C) Bar graph of body weight for 6-month and 24-month-old WT and CD169-DTR females (n=6-11 per genotype) treated with DT and sacrificed on D24. Data is represented as a mean +/− SEM. Statistical significance was determined via two-way ANOVA with α=0.05 and p < 0.05. (D) Quantification of free fatty acids (FFA) in the serum of 6-month and 24-month-old WT and CD169-DTR females (n=5-15 per genotype) treated with DT and sacrificed on D24. Data is represented as a mean +/− SEM. Statistical significance was determined via two-way ANOVA with α=0.05 and p < 0.05. (E) Total expression and phosphorylation levels of lipolysis proteins from the VAT of ad libitum 24-month-old WT and CD169-DTR females treated with DT and sacrificed on D25 (n=4 per genotype). (F) Relative mRNA expression of Gdf3 , Adrb3 , and Pparγ genes in the VAT of ad libitum fed 6- and 24-month-old WT and CD169-DTR females treated with DT and sacrificed on D25 (n=5-7 per genotype). Data is represented as a mean +/− SEM. Statistical significance was determined via two-way ANOVA with α=0.05 and p < 0.05. (G) Monocyte chemoattractant protein-1 (MCP-1) and TNFα levels in the serum of ad libitum fed 24-month-old WT and CD169-DTR females treated with DT and sacrificed on D25 (n=5-7 per genotype). Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (H) Flow cytometry from the VAT of 24-month-old WT and CD169-DTR females (n=8-19 per genotype) treated with DT and sacrificed on D25. Bar plots represent either the proportion of CD45 + and CD45 − cells (as a fraction of total live cells) and B-cells, CD8 + T-cells, CD4 + T-cells, Monocytes, Neutrophils, Eosinophils, and MHCII + CD11c + ATMs or dendritic cells (DCs) (as a fraction of live CD45 + ). Populations were defined using the cell surface markers and gating strategy outlined in . Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (I) Relative mRNA expression of inflammasome and pro-inflammatory cytokine genes in the VAT of ad libitum fed 6- and 24-month-old WT and CD169-DTR females treated with DT and sacrificed on D25 (n=5-6 per genotype). Data is represented as a mean +/− SEM. Statistical significance was determined via two-way ANOVA with α=0.05 and p < 0.05.$

Journal: bioRxiv

Article Title: Nerve-associated macrophages control adipose homeostasis across lifespan and restrain age-related inflammation

doi: 10.1101/2024.10.12.618004

Figure Lengend Snippet: (A) Schematic representation of the experimental protocol used to deplete CD169-macrophages using CD169 diphtheria toxin receptor (CD169-DTR) mice. 6-month and 24-month-old wildtype (WT) and CD169-DTR female mice were injected intra-peritoneal (i.p) with 40ng/gram diphtheria toxin (DT) on day 0 (D0) followed by 4ng/gram DT on D3, D6, D9, and D12, D15, D18, D21, D24, and sacrificed on D25. (B) Change in body weight curves for 6-month and 24-month-old WT and CD169-DTR females treated with DT and sacrificed on D24. (C) Bar graph of body weight for 6-month and 24-month-old WT and CD169-DTR females (n=6-11 per genotype) treated with DT and sacrificed on D24. Data is represented as a mean +/− SEM. Statistical significance was determined via two-way ANOVA with α=0.05 and p < 0.05. (D) Quantification of free fatty acids (FFA) in the serum of 6-month and 24-month-old WT and CD169-DTR females (n=5-15 per genotype) treated with DT and sacrificed on D24. Data is represented as a mean +/− SEM. Statistical significance was determined via two-way ANOVA with α=0.05 and p < 0.05. (E) Total expression and phosphorylation levels of lipolysis proteins from the VAT of ad libitum 24-month-old WT and CD169-DTR females treated with DT and sacrificed on D25 (n=4 per genotype). (F) Relative mRNA expression of Gdf3 , Adrb3 , and Pparγ genes in the VAT of ad libitum fed 6- and 24-month-old WT and CD169-DTR females treated with DT and sacrificed on D25 (n=5-7 per genotype). Data is represented as a mean +/− SEM. Statistical significance was determined via two-way ANOVA with α=0.05 and p < 0.05. (G) Monocyte chemoattractant protein-1 (MCP-1) and TNFα levels in the serum of ad libitum fed 24-month-old WT and CD169-DTR females treated with DT and sacrificed on D25 (n=5-7 per genotype). Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (H) Flow cytometry from the VAT of 24-month-old WT and CD169-DTR females (n=8-19 per genotype) treated with DT and sacrificed on D25. Bar plots represent either the proportion of CD45 + and CD45 − cells (as a fraction of total live cells) and B-cells, CD8 + T-cells, CD4 + T-cells, Monocytes, Neutrophils, Eosinophils, and MHCII + CD11c + ATMs or dendritic cells (DCs) (as a fraction of live CD45 + ). Populations were defined using the cell surface markers and gating strategy outlined in . Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (I) Relative mRNA expression of inflammasome and pro-inflammatory cytokine genes in the VAT of ad libitum fed 6- and 24-month-old WT and CD169-DTR females treated with DT and sacrificed on D25 (n=5-6 per genotype). Data is represented as a mean +/− SEM. Statistical significance was determined via two-way ANOVA with α=0.05 and p < 0.05.

Techniques: Injection, Expressing, Two Tailed Test, Flow Cytometry

$(A) Visceral white adipose tissue (VAT) wet weight ( right ) and stromal vascular fraction (SVF) cell number ( left ) from 24-month-old Wildtype (WT) and CD169-DTR females treated with DT and sacrificed on D25 (n=6-13 per genotype). Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (B) Relative protein quantification of the following proteins: Hormone-sensitive Lipase (HSL), HSL phosphorylated at Ser563 (p-HSL S563 ), HSL phosphorylated at Ser660 (p-HSL S660 ), Adipose triglyceride lipase (ATGL), and Monoamine-oxidase A (MAOA). Protein lysate is from the VAT of ad libitum fed 24-month-old WT and CD169-DTR females treated with DT and sacrificed on D25 (n=4 per genotype). Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (C) IL-1β, IL-6, CXCL1, and Eotaxin levels in the serum of ad libitum fed 24-month-old Wildtype (WT) and CD169-DTR females treated with diphtheria toxin (DT) and sacrificed on D25 (n=5-11 per genotype). Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (D) Flow cytometry from the VAT of 24-month-old WT and CD169-DTR females treated with DT and sacrificed on D34. Bar plots represent either the proportion of CD45 + and CD45 − cells (as a fraction of total live cells) and B-cells, CD8 + T-cells, CD4 + T-cells, Monocytes, Neutrophils, Eosinophils, and MHCII + CD11c + ATMs or dendritic cells (DCs) (as a fraction of live CD45 + ). Populations were defined using the cell surface markers and gating strategy outlined in . Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (E) Flow cytometry from the VAT of 24-month-old WT and CD169-DTR females treated with DT and sacrificed on D25 ( top ) or D34 ( bottom ). Bar plots represent either the proportion of regulatory T-cells, gd T-cells, TH1 cells, TH2 cells, and TH17 cells. Populations were defined using the cell markers and gating strategy outlined in . Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05.$

Journal: bioRxiv

Article Title: Nerve-associated macrophages control adipose homeostasis across lifespan and restrain age-related inflammation

doi: 10.1101/2024.10.12.618004

Figure Lengend Snippet: (A) Visceral white adipose tissue (VAT) wet weight ( right ) and stromal vascular fraction (SVF) cell number ( left ) from 24-month-old Wildtype (WT) and CD169-DTR females treated with DT and sacrificed on D25 (n=6-13 per genotype). Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (B) Relative protein quantification of the following proteins: Hormone-sensitive Lipase (HSL), HSL phosphorylated at Ser563 (p-HSL S563 ), HSL phosphorylated at Ser660 (p-HSL S660 ), Adipose triglyceride lipase (ATGL), and Monoamine-oxidase A (MAOA). Protein lysate is from the VAT of ad libitum fed 24-month-old WT and CD169-DTR females treated with DT and sacrificed on D25 (n=4 per genotype). Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (C) IL-1β, IL-6, CXCL1, and Eotaxin levels in the serum of ad libitum fed 24-month-old Wildtype (WT) and CD169-DTR females treated with diphtheria toxin (DT) and sacrificed on D25 (n=5-11 per genotype). Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (D) Flow cytometry from the VAT of 24-month-old WT and CD169-DTR females treated with DT and sacrificed on D34. Bar plots represent either the proportion of CD45 + and CD45 − cells (as a fraction of total live cells) and B-cells, CD8 + T-cells, CD4 + T-cells, Monocytes, Neutrophils, Eosinophils, and MHCII + CD11c + ATMs or dendritic cells (DCs) (as a fraction of live CD45 + ). Populations were defined using the cell surface markers and gating strategy outlined in . Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (E) Flow cytometry from the VAT of 24-month-old WT and CD169-DTR females treated with DT and sacrificed on D25 ( top ) or D34 ( bottom ). Bar plots represent either the proportion of regulatory T-cells, gd T-cells, TH1 cells, TH2 cells, and TH17 cells. Populations were defined using the cell markers and gating strategy outlined in . Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05.

Techniques: Two Tailed Test, Flow Cytometry

$(A) Tissue wet weight of the spleen from 24-month-old Wildtype (WT) and CD169-DTR females treated with DT and sacrificed on D25 (n=8-19 per genotype). Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (B) Bar plot quantifying nerve-associated macrophages in the spleen from 24-month-old WT and CD169-DTR females treated with DT and sacrificed on D13. Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (C) Flow cytometry from the spleen of 24-month-old WT and CD169-DTR females treated with DT and sacrificed on D25. Bar plots represent either the proportion of CD45 + and CD45 − cells (as a fraction of total live cells) and B-cells, CD8 + T-cells, CD4 + T-cells, Monocytes, Neutrophils, Eosinophils, and MHCII + CD11c + ATMs or dendritic cells (DCs) (as a fraction of live CD45 + ). Populations were defined using the cell surface markers and gating strategy outlined in . Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05.$

Journal: bioRxiv

Article Title: Nerve-associated macrophages control adipose homeostasis across lifespan and restrain age-related inflammation

doi: 10.1101/2024.10.12.618004

Figure Lengend Snippet: (A) Tissue wet weight of the spleen from 24-month-old Wildtype (WT) and CD169-DTR females treated with DT and sacrificed on D25 (n=8-19 per genotype). Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (B) Bar plot quantifying nerve-associated macrophages in the spleen from 24-month-old WT and CD169-DTR females treated with DT and sacrificed on D13. Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05. (C) Flow cytometry from the spleen of 24-month-old WT and CD169-DTR females treated with DT and sacrificed on D25. Bar plots represent either the proportion of CD45 + and CD45 − cells (as a fraction of total live cells) and B-cells, CD8 + T-cells, CD4 + T-cells, Monocytes, Neutrophils, Eosinophils, and MHCII + CD11c + ATMs or dendritic cells (DCs) (as a fraction of live CD45 + ). Populations were defined using the cell surface markers and gating strategy outlined in . Data is represented as a mean +/− SEM. Statistical significance was determined via Student’s two-tailed t-test with α=0.05 and p < 0.05.

Techniques: Two Tailed Test, Flow Cytometry

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