TGR5 is expressed in BM hematopoietic stem, in progenitor cells (HSPCs) and in 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) Frequency of GFP+ cells in the Lineage−cKit+Sca1+ (LKS) population in the BM of 8- to 12-week-old TGR5:GFP male mice and their controls (n=9 for wild-type mice, n=11 for TGR5:GFP mice). (C) Frequency of GFP+ cells in hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), granulocyte-monocyte progenitors (GMPs), common myeloid progenitors (CMPs) and megakaryocyte-erythrocyte progenitors (MEPs) in the BM of the mice described in B. (D) Frequency of GFP+ cells in megakaryocyte progenitor (MkProgs) in the BM of the mice described in B. (E) Frequency of GFP+ cells in the different stages of erythropoiesis in the BM of 8- to 12-week-old male TGR5:GFP mice and their wild-type controls (n=3 for wild-type mice, n=4 for TGR5:GFP mice); axis represents % GFP+ cells within the parental population. (F) Frequency of GFP+ cells in peripheral blood cell populations of 8- to 12-week-old male TGR5:GFP mice and their wild-type controls (n=5 for wild-type mice, n=7 for TGR5:GFP mice). (G) Immunodetection of GFP+ cells by DAB IHC in BM of 8- to 12-week-old TGR5:GFP male mice (n=2 for wild-type 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. Unpaired, two-tailed Student’s t-test (B, C, D), multiple two-tailed Student’s t-test with Holm-Sidak’s multiple comparison correction (E, F) was used for statistical analysis. P values are indicated.

Loss of TGR5 does not impair steady state hematopoiesis but compromises hematopoietic progenitor function in stress hematopoiesis.

(A) Frequency of hematopoietic stem and progenitor (HSPC) populations in the BM of 8-to 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 the mice described in A. (C) Total number of CD45+ cells per one leg (hip, femur and tibia) of the mice described in A. (D-H) Complete blood counts in peripheral blood of 8- to 12-week-old Tgr5+/+ (n=10) and Tgr5−/− (n=9) 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 in primary transplantation recipients (n=8, 8- to 12-week-old Tgr5+/+ and Tgr5−/− male donors transplanted into 2-3 CD45.1 recipient mice, treated as technical replicates per donor) at 3 (short-term progenitor readout) and 16 weeks (stem cell contribution readout) post-transplant. Axis represents % CD45.2 donor cells over the sum of CD45.2 donor and CD45.1/.2 competitor events. Dashed line refers to the expected contribution. Results represent the mean ± s.e.m., n represents biologically independent replicates unpaired, two-tailed Student’s t-test (C, D, F, J) was used for statistical analysis. P values (exact value) are indicated, ns indicates non-significance.

Loss of TGR5 alters the BM microenvironment in male but not in female mice.

(A) Representative native µCT-derived 3D reconstructions of the trabecular structure in the distal femoral metaphysis of 8- to 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- to 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 number (Tb.N.), trabecular thickness (Tb.Th.) and trabecular separation (Tb.Sp.). (C) Representative contrast-enhanced µCT-derived 3D reconstructions of osmium tetroxide (OsO4)-stained BMAT in the tibias of 8- to 12-week-old Tgr5+/+ and Tgr5−/− male mice. (D) Quantification of the OsO4-stained bone marrow adipose tissue (BMAT) content in 8- to 12-week-old Tgr5+/+ and Tgr5−/− male mice (n=8 for both Tgr5+/+ and Tgr5−/− mice). OsO4-stained regulated BMAT proximal of tibiofibular junction was classified as “proximal rBMAT”; OsO4-stained constitutive BMAT distal of tibiofibular junction was classified as “distal cBMAT”. (E) Representative native µCT-derived 3D reconstructions of the trabecular structure in the distal femoral metaphysis of 8-week-old Tgr5+/+ and Tgr5−/− female mice. (F) µCT-derived bone morphometry measurements of the trabecular structure in the distal femoral metaphysis of 8-week-old Tgr5+/+ and Tgr5−/− female mice (n=8 for both Tgr5+/+ and Tgr5−/− mice); parameters shown analogously to B. (G) Representative contrast-enhanced µCT-derived 3D reconstructions of OsO4-stained BMAT in the tibias of 8-week-old Tgr5+/+ and Tgr5−/− female mice. (H) Quantification of the OsO4-stained BMAT content in 8-week-old Tgr5+/+ and Tgr5−/− female mice (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. Unpaired, 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 alters the BM microenvironment and blunts BM HSPC myeloid bias in 1-year-old male mice.

(A) Representative native µCT-derived 3D reconstructions of the trabecular structure in the distal femoral metaphysis of 1-year-old Tgr5+/+ and Tgr5−/− male mice. (B) µCT-derived bone morphometry measurements of the trabecular structure in the distal femoral metaphysis of 1-year-old Tgr5+/+ and Tgr5−/− male mice (n=12 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 bone marrow adipose tissue (BMAT) in the tibias of 1-year-old Tgr5+/+ and Tgr5−/− male mice. (D) Quantification of the OsO4-stained BMAT content 1-year-old Tgr5+/+ and Tgr5−/− male mice (n=12 for Tgr5+/+ and n=11 for Tgr5−/− mice). OsO4-stained regulated BMAT proximal of tibiofibular junction was classified as “proximal rBMAT”; OsO4-stained constitutive BMAT distal of tibiofibular junction was classified as “distal cBMAT”. (E) Representative gating strategy for common myeloid progenitors (CMP) and common lymphoid progenitors (CLP). (F) Frequency of common lymphoid progenitors (CLP), common myeloid progenitors (CMP) and ratio of CMP-to-CLP cells in the BM of 8- to 12-week-old (calculated from the data presented in Figure 2A-B) and 1-year-old Tgr5+/+ and Tgr5−/− male mice (n=12 for both Tgr5+/+ and Tgr5−/− mice). Results represent the mean ± s.e.m., n represents biologically independent replicates. Unpaired, two-tailed Student’s t-test (B, D, F) and 2-way ANOVA test with Holm-Sidak’s multiple comparison correction (CMP/CLP ratio in F) were used for statistical analysis. Exact p-values are indicated; ns indicates non-significance.

Loss of TGR5 alters the BM microenvironment in HFD-fed male mice.

(A) Representative native µCT-derived 3D reconstructions of the trabecular structure in the distal femoral metaphysis of Tgr5+/+ and Tgr5−/− male mice fed with a high-fat diet (HFD) for 12 weeks. (B) µCT-derived bone morphometry measurements of the trabecular structure in the distal femoral metaphysis of HFD-fed Tgr5+/+ and Tgr5−/− male mice (20-week-old at the end of the intervention, n=24 for Tgr5+/+ and n=19 for Tgr5−/−); 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 bone marrow adipose tissue (BMAT) in the tibias of Tgr5+/+ and Tgr5−/− male mice fed with a HFD for 12 weeks. (D) Quantification of the OsO4-stained BMAT content of mice Tgr5+/+ and Tgr5−/− male mice fed with a HFD for 12 weeks (n=9 for Tgr5+/+ and n=10 for Tgr5−/− mice). OsO4-stained regulated BMAT proximal of tibiofibular junction was classified as “proximal rBMAT”; OsO4-stained constitutive BMAT distal of tibiofibular junction was classified as “distal cBMAT”. Results represent the mean ± s.e.m., n represents biologically independent replicates. Unpaired, two-tailed Student’s t-test (B, D) was used for statistical analysis. Exact p-values are indicated; ns indicates non-significance.

TGR5 is expressed in BM stroma, and its loss do not cause an intrinsic BMSC differentiation defect in vitro.

(A) Representative flow cytometry gating strategy used to identify the stroma-enriched CD45−Ter119−CD31− gate and the GFP of the cells contained in BM of TGR5:GFP mice. (B) Frequency of GFP+ cells in the stroma-enriched population of 8- to 12-week-old male TGR5:GFP mice and their wild-type controls (n=15 for wild-type mice, n=17 for TGR5:GFP mice, axis represents % GFP+ cells within the parental population). (C,D) Representative images (C) and spectrophotometric quantification (D) of Oil red O (ORO)-stained BMSCs after 7 days of adipocytic differentiation (n=3 for both Tgr5+/+ and Tgr5−/− mice). (E,F) Digital holographic microscopy imaging of BMSCs after 7 days of adipocytic differentiation showing representative images; lipid inclusions appear as bright white areas in this technique (E), and optical path difference (OPD) quantification, indicative of the degree of lipidation (F). (G,H) Alkaline phosphatase (ALP) (G) and alizarin red (AR) (H) performed after ALP stains of BMSCs after 7 days of osteoblastic differentiation. All cells were obtained from 8- to 12-week-old Tgr5+/+ and Tgr5−/− male mice. Results represent the mean ± s.e.m., n represents biologically independent replicates. Unpaired, two-tailed Students t-test (B) and one-way ANOVA with Tukey’s multiple test correction (D) or Holm-Šídák’s multiple test correction (F) were used for statistical analysis. P values (exact value) are indicated.

Loss of TGR5 leads to accumulation of adipocyte progenitors and hastens hematopoietic recovery upon irradiation and BM transplantation.

(A) Left: representative flow cytometry gating strategy for adipocyte progenitor cells (APC) in the BM stroma gate. Right: percentage of adipocyte progenitor cells (APC) in the CD45−Ter119−CD31− BM stromal gate (n=5 for both Tgr5+/+ and Tgr5−/− mice). (B) Left: Representative images for fibroblast colony-forming unit (CFU-F) assay. Right: number of CFU-F obtained from 1 million total BM cells (n=3 both for Tgr5+/+ and Tgr5−/− mice). Cells were obtained from 8- to 12-week-old male mice. (C) Schematic depiction of the inverse chimera transplantation setup. (D) Survival of BM transplant recipients (n=20 for both Tgr5+/+ and Tgr5−/− at D0, 8- to 12-week-old male mice in 2 independent experiments). (E-J) Peripheral blood recovery evaluated longitudinally by complete blood counts for platelets (E), white blood cells (WBC) (F), neutrophils (G), monocytes (H), lymphocytes (I) and red blood cells (RBCs) (J) (n=7 for Tgr5+/+ and n=12 Tgr5−/− 8- to 12-week-old male recipient mice). Results represent the mean ± s.e.m., n represents biologically independent replicates. Unpaired, two-tailed Student’s t-test (A, B), Mantel-Cox’s test (D) and two-way ANOVA with Holm-Šídák’s multiple comparison correction (E-J) were used for statistical analysis. For D, baseline values (Day 0) were not considered for the analysis of the recovery. P values (exact value) are indicated.

TGR5 is expressed in hematopoietic progenitors in the bone marrow.

(A) Representative flow cytometry gating strategy used to identify megakaryocyte progenitors (MkProgs) in BM and their GFP positivity in TGR5:GFP mice. (B) Representative flow cytometry gating strategy used to identify cells in the different stages of erythropoiesis in BM and their GFP positivity in TGR5:GFP mice.

TGR5 is expressed in spleen, thymus and differentiated hematopoietic populations.

(A) Representative flow cytometry gating strategy used to identify differentiated populations in peripheral blood and GFP positivity in TGR5:GFP mice. (B, C) Immunodetection of GFP+ cells by DAB IHC in spleen (B) and thymus (C) of 8- to 12-week-old male TGR5:GFP mice (n=2 for wild-type mice, n=3 for TGR5:GFP mice). Scale bar: 50 µm in low magnification images, 20 µm in the corresponding digitally zoomed images.

Loss of TGR5 does not impair steady-state hematopoiesis but compromises hematopoietic progenitor function in stress hematopoiesis.

(A, B) Number of colonies per 10.000 BM CD45+ cells (A) and per leg (B) (n=16 for both Tgr5+/+ and Tgr5−/− mice). (C) Frequency of hematopoietic stem and progenitor (HSPC) populations in the BM of 8-week-old Tgr5+/+ and Tgr5−/− female mice expressed as percentage of cells over total live cells acquired (n=8 for both Tgr5+/+ and Tgr5−/− mice). (D-H) Complete blood counts in peripheral blood of 8-week-old Tgr5+/+ and Tgr5−/− female mice; myeloid cells (D), lymphocytes (E), red blood cells (F), hemoglobin (G), and platelets (H). (I-K) Flow cytometry analysis of peripheral blood chimerism expressed as the percentage of CD45.2 (I), CD45.2 lymphoid (J) and CD45.2 myeloid (K) cells 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, 8 and 16 weeks post-transplant. Results represent the mean ± s.e.m., n represents biologically independent replicates. Unpaired, two-tailed Student’s t-test was used for statistical analysis. *, p<0.05, ns indicates non-significance.

Loss of TGR5 did not alter the femoral cortical parameters or the spine microarchitecture.

(A) Representative native µCT-derived 3D reconstructions of the cortical bone in the mid-femoral diaphysis of 8- to 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- to 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- to 12-week-old Tgr5+/+ and Tgr5−/− male mice. (D) µCT-derived bone morphometry measurements of the trabecular structures in L4 of 8- to 12-week-old Tgr5+/+ and Tgr5−/− male mice (n=10 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 native µCT-derived 3D reconstructions of the cortical bone in the mid-femoral diaphysis of 8-week-old Tgr5+/+ and Tgr5−/− female mice. (F) µCT-derived bone morphometry measurements of the cortical bone in the mid-femoral diaphysis of 8-week-old Tgr5+/+ and Tgr5−/− female mice (n=8 for both Tgr5+/+ and Tgr5−/− mice); parameters shown analogously to B. (G) Representative native µCT-derived 3D reconstructions of the trabecular structures in the fourth lumbar vertebrae (L4) of 8-week-old Tgr5+/+ and Tgr5−/− female mice. (H) µCT-derived bone morphometry measurements of the trabecular structures in L4 of 8-week-old Tgr5+/+ and Tgr5−/− female mice (n=8 for both Tgr5+/+ and Tgr5−/− mice); parameters shown analogously to D. Results represent the mean ± s.e.m., n represents biologically independent replicates. Unpaired, 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 alters the BM microenvironment in the caudal spine.

(A) 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 14-week-old male Tgr5+/+ and Tgr5−/− mice. (B) Sagittal view of a representative contrast-enhanced µCT-derived 3D reconstruction of OsO4-stained BMAT in the second caudal vertebrae (CA2) of 14-week-old Tgr5+/+ and Tgr5−/− male mice. (C) Quantification of the OsO4-stained BMAT for the first to third caudal vertebra (CA1-CA3) in the caudal red-to-yellow transition in 14-week-old Tgr5+/+ and Tgr5−/− male mice (n=3 for both Tgr5+/+ and Tgr5−/− mice). (D) Representative hematoxylin & eosin (H&E) histological sections of CA1-CA3 from a cohort independent from the animals shown in A-C (n=4 for both Tgr5+/+ and Tgr5−/− mice, 11- to 13-week-old). Scale bar in A and D = 500 µm. Results represent the mean ± s.e.m., n represents biologically independent replicates. Unpaired, two-tailed Student’s t-test (C) was used for statistical analysis. P values (exact value) are indicated, ns indicates no statistical significance.

Loss of TGR5 slightly decreases vertebral and femoral cortical mineralized tissue in 1-year-old male mice.

(A) Representative native µCT-derived 3D reconstructions of the cortical bone in the mid-femoral diaphysis of 1-year-old Tgr5+/+ and Tgr5−/− male mice. (B) µCT-derived bone morphometry measurements of the cortical bone in the mid-femoral diaphysis of 1-year-old Tgr5+/+ and Tgr5−/− male mice (n=12 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 1-year-old Tgr5+/+ and Tgr5−/− male mice. (D) µCT-derived bone morphometry measurements of the trabecular structures in L4 of 1-year-old Tgr5+/+ and Tgr5−/− male mice (n=12 for both Tgr5+/+ and Tgr5−/− mice); shown are bone volume/total volume (BV/TV), trabecular thickness (Tb.Th.), trabecular number (Tb.N.) and trabecular separation (Tb.Sp.). Results represent the mean ± s.e.m., n represents biologically independent replicates. Unpaired, two-tailed Student’s t-test (B, D) was used for statistical analysis. P values (exact value) are indicated, ns indicates no statistical significance.

Loss of TGR5 does not affect femoral cortical parameters and blunts BM HSPC myeloid bias in HFD-fed male mice.

(A) Representative native µCT-derived 3D reconstructions of the cortical bone in the mid-femoral diaphysis of HFD-fed (12 weeks) Tgr5+/+ and Tgr5−/− male mice. (B) µCT-derived bone morphometry measurements of the cortical bone in the mid-femoral diaphysis of HFD-fed (12 weeks) Tgr5+/+ and Tgr5−/− male mice (n=24 for Tgr5+/+ and n=18 for 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) Frequency of common lymphoid progenitors (CLP), common myeloid progenitors (CMP) and ratio of CMP-to-CLP cells in the BM of CD-fed (calculated from the data presented in Figure 2A, same data as the “8- to 12-week-old” group in Figure 4F) and HFD-fed Tgr5+/+ and Tgr5−/− mice (n=24 for Tgr5+/+ and n=20 for Tgr5−/− mice). Results represent the mean ± s.e.m., n represents biologically independent replicates. Unpaired, two-tailed Student’s t-test was used for statistical analysis except for last panel in C, where a 2-way ANOVA test with Holm-Sidak’s multiple comparison correction was used. P values (exact value) are indicated, ns indicates no statistical significance.

Loss of TGR5 increases in vitro BMSC differentiation into adipocytes.

(A) Representative images of Oil red O (ORO)-stained BMSCs after 7 days of adipocytic differentiation (n=3 for both Tgr5+/+ and Tgr5−/− mice). Scale bar: 50 µm.