Figures and data
TGR5 is expressed in BM hematopoietic stem and progenitor cells (HSPCs) and differentiated myeloid populations
A, representative flow cytometry gating strategy used to identify HSPCs and GFP positivity in TGR5:GFP mice and their wild-type controls. B, frequencies of GFP+ cells in the lineage−cKit+Sca1+ (LKS) population in the BM of 8-12-week-old male TGR5:GFP mice and their controls (n=9 for wild-type control mice, n=11 for TGR5:GFP mice). C, frequencies of GFP+ cells in myeloid progenitors, common myeloid progenitors (CMP), granulocyte-monocyte progenitors (GMP) and megakaryocyte-erythrocyte progenitors (MEP) in the BM of 8-12-week-old male TGR5:GFP mice and their wild-type controls (n=9 for wild-type control mice, n=11 for TGR5:GFP mice). D, immunodetection of GFP+ cells by DAB immunohistochemistry in BM of 8-12-week-old male TGR5:GFP mice (n=2 for wild-type control mice, n=3 for TGR5:GFP mice). Scale bar: 50 µm in low magnification images, 20 µm in the corresponding digitally zoomed images. Results represent the mean ± s.e.m., n represents biologically independent replicates. Two-tailed Student’s t-test (B and C) was used for statistical analysis. P values (exact value) are indicated.
Loss of TGR5 does not impair steady-state hematopoiesis
A, frequencies of hematopoietic stem and progenitor (HSPCs) populations in the BM of 8-12-week-old Tgr5+/+ and Tgr5−/− male mice expressed as percentage of cells over total live cells acquired (n=16 for Tgr5+/+ and n= 15 for Tgr5−/− mice). B, total number of cells per one leg (hip, femur and tibia) for HSPC populations in the BM of 8-12-week-old Tgr5+/+ and Tgr5−/− male mice (n=16 for Tgr5+/+ and n= 15 for Tgr5−/− mice). C, total number of CD45+ cells per one leg (hip, femur and tibia) in the BM of 8-12-week-old Tgr5+/+ and Tgr5−/− male mice (n=16 for Tgr5+/+ and n= 15 for Tgr5−/− mice). D-H, complete blood counts in peripheral blood of 8-12-week-old Tgr5+/+ and Tgr5−/− male mice; myeloid cells (D), lymphocytes (E), red blood cells (F), hemoglobin (G), and platelets (H). I, workflow depicting the primary competitive transplant setting. J, flow cytometry analysis of peripheral blood chimerism of serial transplantation experiments (n=8, 8-12-week-old Tgr5+/+ and Tgr5−/− male donors transplanted into 3 CD45.1 recipient mice) at 3 and 16 weeks post-transplant. Axis represents % CD45.2 donor cells over the sum of CD45.2 donor and CD45.1/.2 competitor events (results for secondary and tertiary transplants in supplementary figures). Results represent the mean ± s.e.m., n represents biologically independent replicates Two-tailed Student’s t-test was used for statistical analysis. P values (exact value) are indicated, ns indicates non-significance.
Loss of TGR5 alters the BM microenvironment
A, representative native µCT-derived 3D reconstructions of the trabecular structure in the distal femoral metaphysis of 8-12-week-old Tgr5+/+ and Tgr5−/− male mice. B, µCT-derived bone morphometry measurements of the trabecular structure in the distal femoral metaphysis of 8-12-week-old Tgr5+/+ and Tgr5−/− male mice (n=17 for both Tgr5+/+ and Tgr5−/− mice); shown are bone volume/total volume (BV/TV), trabecular thickness (Tb. Th), trabecular separation (Tb. Sp) and trabecular number (Tb. N) C, representative contrast-enhanced µCT-derived 3D reconstructions of osmium tetroxide (OsO4)-stained BMAT in the tibias of 8-12-week-old Tgr5+/+ and Tgr5−/−male mice fed chow diet. D, quantification of the OsO4-stained bone marrow adipose tissue (BMAT) content in chow diet-fed, 8-12-week-old Tgr5+/+ and Tgr5−/− male mice (n=8 for both Tgr5+/+ and Tgr5−/−mice). OsO4-stained BMAT proximal of tibiofibular junction was classified as “proximal BMAT”; OsO4-stained BMAT distal of tibiofibular junction was classified as “distal BMAT”. E, representative contrast-enhanced µCT-derived 3D reconstructions of OsO4-stained BMAT in the tibias of 1-year-old Tgr5+/+ and Tgr5−/− male mice. F, quantification of the OsO4-stained BMAT content in 1-year-old Tgr5+/+ and Tgr5−/− male mice (n=12 for Tgr5+/+ and n=10 for Tgr5−/− mice). Proximal and distal BMAT were defined as in D. G, representative contrast-enhanced µCT-derived 3D reconstructions of OsO4-stained BMAT in the tibias of 20-week-old Tgr5+/+ and Tgr5−/−male mice fed high-fat diet. H, quantification of the OsO4-stained BMAT content in 20-week-old Tgr5+/+ and Tgr5−/−male mice fed for 12 weeks with high-fat diet (n=8 for both Tgr5+/+ and Tgr5−/− mice). Proximal and distal BMAT were defined as in D. Results represent the mean ± s.e.m., n represents biologically independent replicates. Two-tailed Student’s t-test (B, D, F, H) was used for statistical analysis. P values (exact value) are indicated, ns indicates no statistical significance.
Loss of TGR5 leads to accumulation of adipocyte progenitors and hastens hematopoietic recovery upon irradiation and BM transplantation
A, spectrophotometric quantification of Oil red O (ORO)-stained BMSCs after 7 days of adipocytic differentiation (n=3 for both Tgr5+/+ and Tgr5−/−). B, percentage of adipocyte progenitor cells (APCs) in the CD45−Ter119−CD31− BM stromal gate (n=5 for bothTgr5+/+ and Tgr5−/−). C, number of fibroblast colony-forming units (CFU-Fs) obtained from 1 million total BM cells (n=3 both for Tgr5+/+ and Tgr5−/−). D, Schematic depiction of the inverse chimera transplantation setup. E, survival of BM transplant recipients (n=20 for both Tgr5+/+ and Tgr5−/− at D0, 8-12-week-old male mice in 2 independent experiments). F-K peripheral blood recovery evaluated longitudinally by complete blood counts for platelets (F), white blood cells (WBCs) (G), neutrophils (H), monocytes (I), lymphocytes (J) and red blood cells (RBCs) (K) (n=7 for Tgr5+/+ and n=12 Tgr5−/− 8-12-week-old male recipient mice). Results represent the mean ± s.e.m. All in vitro experiments were conducted on cells obtained from 8-12-week-old Tgr5+/+ and Tgr5−/− male mice. One-way ANOVA with Bonferroni multiple test correction (A), two-tailed Student’s t-test (B, C), Mantel-Cox test (E) and two-way ANOVA with Holm-Šídák multiple comparison correction (F-K) were used for statistical analysis. For E, baseline values (Day 0) were not considered for the analysis of the recovery. P values (exact value) are indicated, ns indicates non-significance
TGR5 is expressed in spleen, thymus and differentiated hematopoietic populations
A, B, immunodetection of GFP+ cells by DAB immunohistochemistry in spleen (A) and thymus (B) of 8-12-week-old male TGR5:GFP mice (n=2 for wild-type control mice, n=3 for TGR5:GFP mice). Scale bar: 50 µm in low magnification images, 20 µm in the corresponding digitally zoomed images. C, representative flow cytometry gating strategy used to identify differentiated population in peripheral blood and GFP positivity in TGR5:GFP mice. D, frequencies of GFP+ cells in peripheral blood cell populations of 8-12-week-old male TGR5:GFP mice and their wild-type controls (n=2 for wild-type control mice, n=3 for TGR5:GFP mice, axis represents % GFP+ cells within the viable CD45+ cell gate) Results represent the mean ± s.e.m., n represents biologically independent replicates.
Loss of TGR5 does not impair steady-state hematopoiesis
A, number of colonies per 10.000 total BM CD45+ cells (A) and per leg (B) (n=16 for Tgr5+/+ and n= 15 for Tgr5−/− mice). C, flow cytometry analysis of peripheral blood chimerism of serial BM transplantation experiments (n=8, 8-12-week-old Tgr5+/+ and Tgr5−/− male donors transplanted into 3 CD45.1 recipient mice each) at 3 and 16 weeks post-transplant. Results represent the mean ± s.e.m., n represents biologically independent replicates Two-tailed Student’s t-test was used for statistical analysis. ns indicates non-significance.
Loss of TGR5 alters the BM microenvironment
A, representative native µCT-derived 3D reconstructions of the cortical bone in the mid-femoral diaphysis of 8-12-week-old Tgr5+/+ and Tgr5−/− male mice. B, µCT-derived bone morphometry measurements of the cortical bone in the mid-femoral diaphysis of 8-12-week-old Tgr5+/+ and Tgr5−/− male mice (n=17 for both Tgr5+/+ and Tgr5−/− mice); shown are the total cross-sectional area inside the periosteal envelope (Tt.Ar), cortical bone area (Ct.Ar), cortical area fraction (Ct.Ar/Tt.Ar) and average cortical thickness (Ct. Th). C, representative native µCT-derived 3D reconstructions of the trabecular structures in the fourth lumbar vertebrae (L4) of 8-12-week-old Tgr5+/+ and Tgr5−/− male mice. D, µCT-derived bone morphometry measurements of the trabecular structures in L4 of 8-12-week-old Tgr5+/+ and Tgr5−/− male mice (n=17 for both Tgr5+/+ and Tgr5−/− mice); shown are bone volume/total volume (BV/TV), trabecular thickness (Tb. Th), trabecular separation (Tb. Sp) and trabecular number (Tb. N). E, representative images obtained from contrast-enhanced µCT scanning of decalcified OsO4-stained spines depicting lipid content in the red-to-yellow marrow transition in the caudal spine (CA) of 12-week-old male Tgr5+/+ and Tgr5−/− mice. F, sagittal view of a representative µCT-derived 3D reconstructions of OsO4-stained BMAT in the second caudal vertebrae (CA2) of 12-week-old Tgr5+/+ and Tgr5−/−male mice fed chow diet. G, quantification of the OsO4-stained BMAT for the first to third caudal vertebra (CA1-CA3) in the caudal red-to-yellow transition in chow diet-fed, 12-week-old Tgr5+/+ and Tgr5−/− male mice (n=3 for both Tgr5+/+ and Tgr5−/− mice). Results represent the mean ± s.e.m., n represents biologically independent replicates. H, representative hematoxylin & eosin (H&E) histological sections of CA1-CA3 from a cohort independent from the animals shown in E (n=4 for both Tgr5+/+ and Tgr5−/− mice). Scale bar in E and H = 500 µm. Results represent the mean ± s.e.m., n represents biologically independent replicates. Two-tailed Student’s t-test (B, D, G) was used for statistical analysis. P values (exact value) are indicated, ns indicates no statistical significance.
Loss of TGR5 leads to accumulation of adipocyte progenitors
A, representative images of Oil red O (ORO)-stained BMSCs after 7 days of adipocytic differentiation. B-C, digital holographic microscopy imaging of BMSCs after 7 days of adipocytic differentiation. Optical path difference (OPD) quantification, indicative of the degree of lipidation (B) and representative images; lipid inclusions appear as bright areas in this technique (C). D-E, alkaline phosphatase (ALP) (D) and alizarin red (AR) (E) on top of ALP stains of BMSCs after 7 days of osteoblastic differentiation. F, representative flow cytometry gating strategy for the CD45-Ter119-CD31-BM stroma population and adipocyte progenitor cells (APCs) in the BM stroma gate. G, representative images for fibroblast colony-forming units (CFU-F) assay. All cells were obtained from 8-12-week-old Tgr5+/+ and Tgr5−/− male mice. One-way ANOVA with Holm-Šídák multiple test correction (B) was used for statistical analysis. P values (exact value) are indicated.
