Journal: JCI insight

Article Title: The homing of exogenous hair follicle mesenchymal stem cells into hair follicle niches.

doi: 10.1172/jci.insight.173549

Figure Lengend Snippet: Figure 8. Chemokine mediates the homing of exogenous hfMSCs into follicle niches. (A) RT-qPCR experiments showed the upregulated chemokines after depilation of NOD/SCID dorsal skin (n = 3). (B) Transwell chemotaxis assays were performed to detect the migratory response of DPCs and DSCs to chemokines at different concentrations (namely, control: 0 ng/mL; low: 5 ng/mL; medium: 50 ng/mL; and high: 500 ng/mL) (n = 5). (C) Wound healing assays were performed to detect the chemotaxis effects of CXCL2, CXCL13, and CXCL16 on DPCs and DSCs (concentrations are the same as in B) (n = 3). One-way ANOVA followed by Bonferroni’s post hoc test. Data reported as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: For the neutralizing CXCL13 study, 50 μg/mL CXCL13 neutralizing Ab (R&D Systems, Bio-Techne; catalog AF470) was mixed with 2 × 106 DPCs/ DSCs, and the mixture was intradermally injected into depilated back skin of adult NOD/SCID mice.

Techniques: Quantitative RT-PCR, Chemotaxis Assay, Control

Journal: JCI insight

Article Title: The homing of exogenous hair follicle mesenchymal stem cells into hair follicle niches.

doi: 10.1172/jci.insight.173549

Figure Lengend Snippet: Figure 9. Chemokine receptor mediates the homing of exogenous hfMSCs into follicle niches. (A) Dorsal skin sections were harvested 1, 2, 4, and 6 days after depilation. (The control group comprised a section of shaved dorsal skin.) Immunostaining showed the expression of CXCL13, CXCL16, and CXCL2 (representative of 3 experiments). (B and C) RT-qPCR experiments showed the expression of receptors related to upregulated chemokines in low-passage and high-passage DPCs/DSCs (n = 3). Two-tailed Student’s t test. Data reported as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. (D) Venn diagram shows the intersection of the downregulated chemokine receptors in high-passage DPCs/DSCs. (E) Immunostaining shows the expression of CXCR5 in low-passage and high-passage DPCs/DSCs (representative of 3 experiments). Scale bars: 100 μm in A and E.

Article Snippet: For the neutralizing CXCL13 study, 50 μg/mL CXCL13 neutralizing Ab (R&D Systems, Bio-Techne; catalog AF470) was mixed with 2 × 106 DPCs/ DSCs, and the mixture was intradermally injected into depilated back skin of adult NOD/SCID mice.

Techniques: Control, Immunostaining, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: JCI insight

Article Title: The homing of exogenous hair follicle mesenchymal stem cells into hair follicle niches.

doi: 10.1172/jci.insight.173549

Figure Lengend Snippet: Figure 10. The CXCL13/CXCR5 axis mediates the homing of exogenous hfMSCs into follicle niches. (A) RT-qPCR experiments show the mRNA expression of CXCR5 in DPCs/DSCs after transfection with lentiviral vector expressing CXCR5 shRNA (n = 3). (B) Immunostaining shows the protein expression of CXCR5 in DPCs/DSCs after transfection with lentiviral vector expressing CXCR5 shRNA4 (representative of 3 experiments). (C) DPCs/DSCs transfected with lentiviral vector expressing CXCR5-shRNA4 were injected into the dorsal skin of depilated NOD/SCID mice. (D) DPCs/DSCs mixed with CXCL13 neutralizing Ab were injected into the dorsal skin of depilated NOD/SCID mice. HFs containing exogenous DPCs (green; arrow) or DSCs (green; arrow) were immunos- tained for alkaline phosphatase (ALP) (red) (C and D). (E and F) Number of HFs containing EGFP+ transplanted cells and number of homing EGFP+ cells per ×100 original magnification field of view, as shown in C and D (n = 7 skin sections from 4 mice per group). Statistical comparisons were performed using 1-way ANOVA followed by Bonferroni’s post hoc test (A) and 2-tailed Student’s t test (E and F). Scale bars: 100 μm. Data are reported as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: For the neutralizing CXCL13 study, 50 μg/mL CXCL13 neutralizing Ab (R&D Systems, Bio-Techne; catalog AF470) was mixed with 2 × 106 DPCs/ DSCs, and the mixture was intradermally injected into depilated back skin of adult NOD/SCID mice.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation, shRNA, Immunostaining, Injection

Journal: Frontiers in Immunology

Article Title: CXCL13 promotes thermogenesis in mice via recruitment of M2 macrophage and inhibition of inflammation in brown adipose tissue

doi: 10.3389/fimmu.2023.1253766

Figure Lengend Snippet: Identification of CXCL13 as an elevated chemokine in brown adipose tissue in response to cold stimulation. (A) Venn chart of RNA-seq database to screen candidate upregulated molecules preferentially expressed in the BAT in response to thermogenic activation (right) and the list of genes upregulated in all five databases (left). (B) Eight-week-old C57BL6/J male mice were exposed to the cold (6°C) for 1 day or 7 days while the control mice were set at the 22 °C room temperature (RT). mRNA levels of the Cxcl13 gene in the BAT and SWAT (n=4-6/group) were analyzed. Western blot analysis of CXCL13 protein in the BAT (C) and the SWAT (D) of the mice exposed to cold or RT (n=3-4/group). (E) Quantification of CXCL13 protein in (C, D) . Expression of Cxcl13 mRNA in SVF and mature adipocytes of the BAT (F) and SWAT (G) (n=4-6/group). Western blot analysis of CXCL13 protein in the SVF of BAT (H) and the SWAT (I) of the mice exposed to cold or RT (n=3-4/group). (J) Violin plots displaying the log2 expression levels for Cxcl13 in Lin+ cells. MAC, macrophage; MONO, monocyte; DEND, dendritic cell; NKT, natural killer T-cell; Tlym, T lymphocyte; Blym, B lymphocyte; RET, reticulocyte; NEUT, neutrophil; VEC, vascular endothelial cell; Lin-, lineage-negative cells; RBC, red blood cell. (K) Violin plots displaying the log2 expression levels for Cxcl13 in Lin- cells. ASC, adipose tissue stromal cell; VEC, vascular endothelial cell; VSMC, vascular smooth muscle cells; Prolif/Diff, proliferating/differentiating cells. (L) The mRNA expression of Cxcl13 in primary brown adipocytes was treated with different concentrations of Forskolin for 6 hours (n=3-5/group). Data were presented as mean ± S.E.M. * p < 0.05, ** p <0.01, *** p <0.001.

Article Snippet: Mouse anti-CXCL13/BLC/BCA-1 antibody (AF470) and CXCL13 ELISA Kits (MCX130) were purchased from RDSystems Inc. (USA).

Techniques: RNA Sequencing Assay, Activation Assay, Western Blot, Expressing

Journal: Frontiers in Immunology

Article Title: CXCL13 promotes thermogenesis in mice via recruitment of M2 macrophage and inhibition of inflammation in brown adipose tissue

doi: 10.3389/fimmu.2023.1253766

Figure Lengend Snippet: CXCL13 induced BAT activation and promoted thermogenesis in lean mice. Normal-diet-fed C57BL6/J male mice were treated with CXCL13 or PBS through local BAT injection for 3 days. (A) Representative thermal images (left) and surface temperature ratio of the interscapular region (right) in PBS and CXCL13 injected mice (n=4-5/group). (B) Heat production of PBS and CXCL13 injected mice. Values represent measurements of 24 hours (n=3-4/group, left). Quantitative measurements of heat production in the mice (right). (C) Respiratory exchange ratio (RER) of PBS and CXCL13 injected mice. Values represent measurements of 24 hours (n=3-4/group, left). Quantitative measurements of RER in the mice (right). (D) mRNA levels of thermogenic genes and Cxcr5 in the BAT (n=4-5/group). (E) Western blot analysis of UCP1 and PRDM16 protein in the BAT (n=3-4/group, left). Quantitative relative measurements of proteins to ACTIN (right). (F) Representative images of UCP1 immunohistochemical staining of the BAT from PBS and CXCL13 injected groups (left) and the statistical analysis (right, n=3/group). Data were presented as mean ± S.E.M. * p < 0.05, ** p <0.01.

Article Snippet: Mouse anti-CXCL13/BLC/BCA-1 antibody (AF470) and CXCL13 ELISA Kits (MCX130) were purchased from RDSystems Inc. (USA).

Techniques: Activation Assay, Injection, Western Blot, Immunohistochemistry, Staining

Journal: Frontiers in Immunology

Article Title: CXCL13 promotes thermogenesis in mice via recruitment of M2 macrophage and inhibition of inflammation in brown adipose tissue

doi: 10.3389/fimmu.2023.1253766

Figure Lengend Snippet: CXCL13 promoted thermogenesis and enhanced cold tolerance in diet-induced obese (DIO) mice. High-fat diet (HFD)-induced obese C57BL6/J male mice were locally injected with CXCL13 or PBS in the BAT for 3 days. (A) Changes of rectal temperature during 3 days of PBS or CXCL13 injection (n=6-8/group). (B) Representative thermal images (left) and BAT region temperature in PBS and CXCL13 injected mice (right) (n=6-8/group). (C) Heat production of PBS and CXCL13 injected obese mice (n=3-4/group, left) and quantification of the light and night period (right). (D) RER of PBS and CXCL13 injected obese mice (n=3-4/group, left) and quantification of the light and night period (right). (E) Changes in rectal temperature during a 6-hour cold tolerance test (n=6-8/group). (F) Representative thermal images (left) and BAT region temperature in PBS and CXCL13-injected mice (right) after 6 hours of cold exposure (n=4/group). (G) mRNA levels of thermogenic genes in the BAT (n=6-8/group). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse anti-CXCL13/BLC/BCA-1 antibody (AF470) and CXCL13 ELISA Kits (MCX130) were purchased from RDSystems Inc. (USA).

Techniques: Injection

Journal: Frontiers in Immunology

Article Title: CXCL13 promotes thermogenesis in mice via recruitment of M2 macrophage and inhibition of inflammation in brown adipose tissue

doi: 10.3389/fimmu.2023.1253766

Figure Lengend Snippet: CXCL13 induced M2 macrophage migration in brown adipose tissue. Normal-diet-fed C57BL6/J male mice were treated with CXCL13 or PBS for 3 days using BAT injection. (A) Relative mRNA levels of genes indicative of M2 and M1 macrophage genes in the BAT (n=3-5/group). (B) Serum TNFα level after BAT CXCL13 injection (n=5/group). (C) Representative images of TNFα immunohistochemical staining of the BAT (left) and the statistical analysis (right, n=3/group). (D) Western blot analysis of TNFα protein in the BAT (n=3/group, left). Quantitative measurements of TNFα proteins to ACTIN (right). (E) Flow cytometry gating schemes for subset distribution of macrophages in SVF of the BAT. (F) The percentage of F4/80+CD11b+ (total macrophage) within the CD45+ cells (left), and CD11c+ cells (M1 macrophage) or CD206+ cells (M2 macrophage) within the CD45+F4/80+CD11b+ population from the BAT (right) (n=4/group) (G) Representative images of CD206 immunohistochemical staining of the BAT and statistical analysis (n=3/group). * p < 0.05, ** p < 0.01.

Article Snippet: Mouse anti-CXCL13/BLC/BCA-1 antibody (AF470) and CXCL13 ELISA Kits (MCX130) were purchased from RDSystems Inc. (USA).

Techniques: Migration, Injection, Immunohistochemistry, Staining, Western Blot, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: CXCL13 promotes thermogenesis in mice via recruitment of M2 macrophage and inhibition of inflammation in brown adipose tissue

doi: 10.3389/fimmu.2023.1253766

Figure Lengend Snippet: CXCL13 down-regulated the expression of the M1 macrophage pro-inflammatory gene and up-regulated the expression of M2 macrophage marker genes in vitro . Bone Marrow-derived Macrophages (BMDM) were cultured and treated with different dosages of CXCL13 for 12 h. (A) Activated M1 macrophages were treated with different concentrations of CXCL13 (n=3-4/group). (B) Activated M2 macrophages were treated with different concentrations of CXCL13 (n=4-6/group). (C) The TNFα content in the supernatant of M1 macrophages culture medium treated with 50 ng/mL CXCL13 (n=3-4/group). (D) M1 (D) and M2 (E) macrophages migration assay induced by 50 ng/mL CXCL13 or control medium. Cells were fixed and stained by hematoxylin. (F) Quantification analysis of cells in figures D and E (n=3/group). (G) M1 and M2 macrophages migration. M1 or M2 macrophages were cultured with complete medium (CM), pre-adipocyte CM, pre-adipocyte CM + IgG or pre-adipocyte CM + anti-CXCL13 for 20 hrs. Cells were fixed and stained by hematoxylin (n=3/group). (H) Diagram of the co-culture system of primary brown adipocytes and M2 macrophages. (I) mRNA levels of thermogenic genes in the brown adipocytes treated with different concentrations of CXCL13 (n=4-6/group). (J) mRNA levels of thermogenic genes in the primary brown adipocytes co-cultured with M2 macrophages and treated with different concentrations of CXCL13 (n=4-6/group). (K) mRNA levels of lipogenesis genes in the primary brown adipocytes co-cultured with M2 macrophages and treated with different concentrations of CXCL13 (n=4-6/group). (L) mRNA levels of lipolysis genes in the primary brown adipocytes co-cultured with M2 macrophages and treated with different concentrations of CXCL13 (n=4-6/group). (M) mRNA levels of fatty acid oxidation genes in the primary brown adipocytes co-cultured with M2 macrophages and treated with different concentrations of CXCL13 (n=4-6/group). (N) Western blot analysis of Lipogenic (ACC), lipolysis (HSL and ATGL), and thermogenic (UCP1) protein levels in the primary brown adipocytes co-cultured with M2 macrophages and treated with 50 ng/mL CXCL13 (n=4/group, left) Quantitative measurements of proteins to ACTIN (right). * p <0.05, ** p <0.01,*** p <0.001.

Article Snippet: Mouse anti-CXCL13/BLC/BCA-1 antibody (AF470) and CXCL13 ELISA Kits (MCX130) were purchased from RDSystems Inc. (USA).

Techniques: Expressing, Marker, In Vitro, Derivative Assay, Cell Culture, Migration, Staining, Co-Culture Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: CXCL13 promotes thermogenesis in mice via recruitment of M2 macrophage and inhibition of inflammation in brown adipose tissue

doi: 10.3389/fimmu.2023.1253766

Figure Lengend Snippet: Proposed model of cold elevated CXCL13 expression in thermogenesis of brown adipose tissue in mice. Cold exposure induces elevation of CXCL13 in the adipose tissue stromal cell (ASC) of SVF. CXCL13 can promote brown adipocyte thermogenesis by inducing M2 macrophage activation and marker gene ( Arg1 , Clec10a , Mrc1 ) expression, and promoting cellular migration. CXCL13 can also suppress inflammation by down-regulating TNFα expression in M1 macrophages. The upward arrows represent up-regulation and the downward arrows represent down-regulation.

Article Snippet: Mouse anti-CXCL13/BLC/BCA-1 antibody (AF470) and CXCL13 ELISA Kits (MCX130) were purchased from RDSystems Inc. (USA).

Techniques: Expressing, Activation Assay, Marker, Migration