cd73 ko mice  (MedChemExpress)

Journal: Molecular Metabolism

Article Title: CD73 promotes the immunoregulatory functions of hepatic Tregs through enzymatic and nonenzymatic pathways in MASLD development

doi: 10.1016/j.molmet.2025.102131

Figure Lengend Snippet: CD73 expression on Tregs is increased during MASLD progression . (A–B). The mRNA levels of Nt5e in the livers of the MCD- or CDHFD-fed mice were determined by real-time PCR. (C–D). The Nt5e mRNA levels in hepatocytes or liver MNCs of the MCD- or CDHFD-fed mice were measured by real-time PCR. (E). CD73 expressions on liver CD4 + T, CD8 + T, NK cells, neutrophils, monocytes, and Kupffer cells were determined in NCD- and CDHFD-fed mice. (F). CD73 expressions on blood CD4 + T, CD8 + T, NK, and NKT cells were measured in NCD- and CDHFD-fed mice. (G). CD73 expressions on liver CD4 + T, CD8 + T, NK cells, neutrophils, monocytes, and Kupffer cells were determined in the NCD- and MCD-fed mice. (H). CD73 expressions on blood CD4 + T, CD8 + T, NK, and NKT cells were measured in the MCD-fed mice. (I). Representative flow cytometry plots of CD73 expression on Tregs (CD4 + CD25 + CD127 - ) in the livers or blood of the CDHFD-fed mice. (J–K). Statistical analysis of the percentages of CD73 + Tregs in the liver or blood of the CDHFD- and MCD-fed mice. (L). CD73 concentration in plasma was determined. (M). The ratio of CD73 + Tregs in PBMCs (left) and the concentration of soluble CD73 in plasma (right) from the healthy controls or MASLD patients. n = 5–21 per group. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: FFAs with or without 100 ng/ml recombinant mouse TRAIL protein (1121-TL; R&D) were added to Tregs for 48 h. Tregs were also incubated with 500 ng/ml of recombinant mouse CD73 protein (4488-EN; R&D) or control IgG for 6 h, followed by treatment with 200 μM FFAs and 100 ng/ml of recombinant mouse TRAIL protein for another 48 h. ATP (A6419; Sigma–Aldrich), AMP (A9396; Sigma–Aldrich), or adenosine (ADO) (A4036; Sigma–Aldrich) was separately added to Tregs from WT or Cd73 KO mice for 24 h. For the experiments with inhibitors, Tregs were separately incubated with 10 μM of the p38 inhibitor SB203580 (HY-10256; MCE), 2.5 μM of the AKT inhibitor MK2206 (HY-10358; MCE), 10 μM of the ERK1/2 inhibitor PD98059 (HY-12028; MCE), or 15 μM of the GATA2 inhibitor K-7174 (HY-12743; MCE) for 1 h and then stimulated with 200 μM FFAs for another 48 h.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Concentration Assay

Journal: Molecular Metabolism

Article Title: CD73 promotes the immunoregulatory functions of hepatic Tregs through enzymatic and nonenzymatic pathways in MASLD development

doi: 10.1016/j.molmet.2025.102131

Figure Lengend Snippet: Cd73 knockout decreases intrahepatic Treg survival and immunomodulatory activity in CDHFD-induced MASLD model. (A). Body weight of the NCD- or CDHFD-fed WT or Cd73 knockout mice. (B). Plasma ALT, AST, and TBIL levels were determined. (C). Fasting plasma glucose levels were determined in the NCD- or CDHFD-fed mice. (D). Representative H&E, Oil Red O, and α-SMA staining and quantification of liver histology staining. (E). The relative mRNA expressions of proinflammatory cytokines ( Ifng , Il17 , and Tnfa ) in the liver were measured. (F). Fibrosis-related genes ( Col3a1 , Col1a1 , and Acta2 ) were assessed by real-time PCR. (G). Hydroxyproline levels in liver tissues from the NCD- or CDHFD-fed mice. (H–I). Ratio of Tregs among liver CD4 + T cells and CD45 + cells. (J). Apoptosis (Annexin V, Caspase-3 activity) and proliferation (Ki67, BCL-2) of liver Tregs were detected by flow cytometry. (K). Representative flow cytometry plots and statistical analysis of Granzyme Bpositive Tregs in the liver. (L–N). Proportion of CTLA4 + , IL-10 + , and CD69 + Tregs in each group of mice. n = 5 per group. ∗ P < 0.05, ∗∗ P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: FFAs with or without 100 ng/ml recombinant mouse TRAIL protein (1121-TL; R&D) were added to Tregs for 48 h. Tregs were also incubated with 500 ng/ml of recombinant mouse CD73 protein (4488-EN; R&D) or control IgG for 6 h, followed by treatment with 200 μM FFAs and 100 ng/ml of recombinant mouse TRAIL protein for another 48 h. ATP (A6419; Sigma–Aldrich), AMP (A9396; Sigma–Aldrich), or adenosine (ADO) (A4036; Sigma–Aldrich) was separately added to Tregs from WT or Cd73 KO mice for 24 h. For the experiments with inhibitors, Tregs were separately incubated with 10 μM of the p38 inhibitor SB203580 (HY-10256; MCE), 2.5 μM of the AKT inhibitor MK2206 (HY-10358; MCE), 10 μM of the ERK1/2 inhibitor PD98059 (HY-12028; MCE), or 15 μM of the GATA2 inhibitor K-7174 (HY-12743; MCE) for 1 h and then stimulated with 200 μM FFAs for another 48 h.

Techniques: Knock-Out, Activity Assay, Staining, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: Molecular Metabolism

Article Title: CD73 promotes the immunoregulatory functions of hepatic Tregs through enzymatic and nonenzymatic pathways in MASLD development

doi: 10.1016/j.molmet.2025.102131

Figure Lengend Snippet: CD73 maintains Treg survival and immunomodulatory activity, protecting against MASLD progression. (A). The Rag1 −/− mice were adoptively transferred with WT mouse-derived CD3 + T cells (W/WT Treg group) or CD3 + T cells without Tregs (W/O Treg group). In the W/ Cd73 KO Treg group, Rag1 −/− mice received CD3 + T cells without Tregs (from WT mice) together with Cd73 KO Tregs (from Cd73 KO mice). The above recipient mice and control Rag1 −/− mice not receiving cell transfer were subsequently fed MCD for 4 weeks. Created in https://BioRender.com . (B–C). Plasma ALT and AST levels were determined. (D). H&E and Oil Red O staining of representative paraffin-embedded liver sections. (E). Quantification of liver H&E and Oil Red O staining. (F). The body weight of mice at a different time. (G). The cytokine levels in plasma were measured by ELISA. (H) Ratio of CD4 + FOXP3 + Tregs in liver CD4 + T cells. (I, J) Ratio of Annexin V + , Tim-3 + , CTLA4 + Tregs in the livers of transplanted Rag1 −/− mice. (K). Ratio of monocytes (CD11b high F4/80 low ) in liver CD45 + Ly6G − cells. (L). The level of TNF-α secreted by monocytes was detected. (M). Ratio of Th1 (IFN-γ + ) or Th17 (IL-17 + ) in CD4 + T cells. n = 3–5 per group. ∗ P < 0.05, ∗∗ P < 0.01. $: A comparison between group Rag1 KO and W/O Treg with P < 0.05; $$: A comparison between group Rag1 KO and W/O Treg with P < 0.01; #: A comparison between group W/O Treg and W/WT Treg with P < 0.05; ##: A comparison between group W/O Treg and W/WT Treg with P < 0.01; &: A comparison between group W/WT Treg and W/ Cd73 KO Treg with P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: FFAs with or without 100 ng/ml recombinant mouse TRAIL protein (1121-TL; R&D) were added to Tregs for 48 h. Tregs were also incubated with 500 ng/ml of recombinant mouse CD73 protein (4488-EN; R&D) or control IgG for 6 h, followed by treatment with 200 μM FFAs and 100 ng/ml of recombinant mouse TRAIL protein for another 48 h. ATP (A6419; Sigma–Aldrich), AMP (A9396; Sigma–Aldrich), or adenosine (ADO) (A4036; Sigma–Aldrich) was separately added to Tregs from WT or Cd73 KO mice for 24 h. For the experiments with inhibitors, Tregs were separately incubated with 10 μM of the p38 inhibitor SB203580 (HY-10256; MCE), 2.5 μM of the AKT inhibitor MK2206 (HY-10358; MCE), 10 μM of the ERK1/2 inhibitor PD98059 (HY-12028; MCE), or 15 μM of the GATA2 inhibitor K-7174 (HY-12743; MCE) for 1 h and then stimulated with 200 μM FFAs for another 48 h.

Techniques: Activity Assay, Derivative Assay, Control, Staining, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Molecular Metabolism

Article Title: CD73 promotes the immunoregulatory functions of hepatic Tregs through enzymatic and nonenzymatic pathways in MASLD development

doi: 10.1016/j.molmet.2025.102131

Figure Lengend Snippet: FFAs increase CD73 expression on Tregs via p38/Gata2 signaling pathway. (A). GSEA of Tregs from MCD-versus NCD-fed mice was performed. (B) After the addition of FFAs for 48 h, the expression of CD73 on Tregs was measured by flow cytometry. (C) Phosphorylated AKT activity in the Tregs after FFAs treatment. (D). MAPK signaling was detected in FFAs-stimulated Tregs by flow cytometry. (E). Tregs isolated from WT mice were preincubated with p38 inhibitor (SB203580), AKT inhibitor (MK2206), or ERK1/2 inhibitor (PD98059) for 1 h followed by FFAs treatment for 48 h, and the CD73 expression was detected by flow cytometry. (F). Transcription factor-binding sites of the Nt5e promoter region were predicted through the JASPAR CORE database and intersected with DEGs of Tregs from MCD- versus NCD-fed mice. (G). Relative mRNA levels of transcription factors ( Runx2, Gata2, Sox9 , and Tcf4 ) were determined by real-time PCR. (H–I). Runx2 and Gata2 expression was detected in Tregs incubated with p38 inhibitor (SB203580) followed by FFAs stimulation. (J). CUT&Tag assays of GATA2 binding sites showed a peak distribution around the Nt5e promoter region in the FFAs-treated Tregs. (K). The enrichment of GATA2 binding sites in the Nt5e promoter region was confirmed by real-time PCR, and the data were normalized to those of IgG. (L). CD73 expression on Tregs with pre-treatment of Gata2 inhibitor (K7174) and subsequent FFA stimulation. n = 4–5 per group. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: FFAs with or without 100 ng/ml recombinant mouse TRAIL protein (1121-TL; R&D) were added to Tregs for 48 h. Tregs were also incubated with 500 ng/ml of recombinant mouse CD73 protein (4488-EN; R&D) or control IgG for 6 h, followed by treatment with 200 μM FFAs and 100 ng/ml of recombinant mouse TRAIL protein for another 48 h. ATP (A6419; Sigma–Aldrich), AMP (A9396; Sigma–Aldrich), or adenosine (ADO) (A4036; Sigma–Aldrich) was separately added to Tregs from WT or Cd73 KO mice for 24 h. For the experiments with inhibitors, Tregs were separately incubated with 10 μM of the p38 inhibitor SB203580 (HY-10256; MCE), 2.5 μM of the AKT inhibitor MK2206 (HY-10358; MCE), 10 μM of the ERK1/2 inhibitor PD98059 (HY-12028; MCE), or 15 μM of the GATA2 inhibitor K-7174 (HY-12743; MCE) for 1 h and then stimulated with 200 μM FFAs for another 48 h.

Techniques: Expressing, Flow Cytometry, Activity Assay, Isolation, Binding Assay, Real-time Polymerase Chain Reaction, Incubation

Journal: Molecular Metabolism

Article Title: CD73 promotes the immunoregulatory functions of hepatic Tregs through enzymatic and nonenzymatic pathways in MASLD development

doi: 10.1016/j.molmet.2025.102131

Figure Lengend Snippet: CD73 protects Tregs from AMP-induced toxicity, while the degradation product adenosine promotes Treg survival and function in the presence of FFAs. (A–C). The plasma concentrations of ATP, AMP, and adenosine (ADO) were measured in the CDHFD- or MCD-fed WT and Cd73 KO mice, as well as the MCD-fed Treg-transplanted Rag1 −/− mice. (D) Relative AMP and ADO levels in the supernatant after the addition of ATP to cultures of WT or Cd73 KO Tregs. (E). Apoptosis of CD4 + T cells without Tregs was detected after ADO treatment with or without FFAs treatment. (F). Apoptosis and TNF-α secretion were measured in ADO-stimulated monocytes with or without FFAs treatment. (G). Apoptosis of ADO-stimulated Tregs was detected by flow cytometry. (H). The expression of immunomodulatory molecules (CTLA4, Perforin, IL-10, Granzyme B, and TGF-β) on WT Tregs were detected after ADO stimulation with or without FFAs treatment. (I–K). Treg apoptosis (Annexin V and 7AAD) and IL-10 expression were compared between WT and Cd73 KO mice after AMP supplement with or without FFAs treatment. (L) The relative mRNA expression of Bcl2, Ctla4, Gzmb, Prf1, Il10 , and Tgfb in Tregs from WT and Cd73 KO mice was determined after AMP supplement with or without FFAs treatment. n = 4–5 per group. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: FFAs with or without 100 ng/ml recombinant mouse TRAIL protein (1121-TL; R&D) were added to Tregs for 48 h. Tregs were also incubated with 500 ng/ml of recombinant mouse CD73 protein (4488-EN; R&D) or control IgG for 6 h, followed by treatment with 200 μM FFAs and 100 ng/ml of recombinant mouse TRAIL protein for another 48 h. ATP (A6419; Sigma–Aldrich), AMP (A9396; Sigma–Aldrich), or adenosine (ADO) (A4036; Sigma–Aldrich) was separately added to Tregs from WT or Cd73 KO mice for 24 h. For the experiments with inhibitors, Tregs were separately incubated with 10 μM of the p38 inhibitor SB203580 (HY-10256; MCE), 2.5 μM of the AKT inhibitor MK2206 (HY-10358; MCE), 10 μM of the ERK1/2 inhibitor PD98059 (HY-12028; MCE), or 15 μM of the GATA2 inhibitor K-7174 (HY-12743; MCE) for 1 h and then stimulated with 200 μM FFAs for another 48 h.

Techniques: Flow Cytometry, Expressing

Journal: Molecular Metabolism

Article Title: CD73 promotes the immunoregulatory functions of hepatic Tregs through enzymatic and nonenzymatic pathways in MASLD development

doi: 10.1016/j.molmet.2025.102131

Figure Lengend Snippet: CD73 inhibits Treg apoptosis by suppressing the TRAIL-DR5 interaction. (A). The relative mRNA expression of Tnfrsf10b (DR5) and Tnfsf10 (TRAIL) in the liver was measured by real-time PCR. (B). Plasma content of TRAIL in the CDHFD- or NCD-fed mice. (C). DR5 expression on liver Tregs. (D). Relative apoptotic levels were compared between CD4 + T cells without Tregs and Tregs followed by FFAs treatment with or without TRAIL. (E). Relative apoptotic levels were compared among CD73 high , CD73 int and CD73 neg Tregs after FFAs treatment with or without TRAIL. (F–G). Relative Annexin V and Caspase-3 activity were measured in the WT and Cd73 KO Tregs treated with FFAs with or without TRAIL. (H). Tregs were blocked with CD73 protein or IgG for 6 h, followed by the addition of FFAs and TRAIL for 48 h. The proportions of Annexin V + Tregs after TRAIL supplement. (I). CD73 on Tregs limits inflammation and maintains homeostasis during MASLD progression (Created in https://BioRender.com ). n = 3–6 per group. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: FFAs with or without 100 ng/ml recombinant mouse TRAIL protein (1121-TL; R&D) were added to Tregs for 48 h. Tregs were also incubated with 500 ng/ml of recombinant mouse CD73 protein (4488-EN; R&D) or control IgG for 6 h, followed by treatment with 200 μM FFAs and 100 ng/ml of recombinant mouse TRAIL protein for another 48 h. ATP (A6419; Sigma–Aldrich), AMP (A9396; Sigma–Aldrich), or adenosine (ADO) (A4036; Sigma–Aldrich) was separately added to Tregs from WT or Cd73 KO mice for 24 h. For the experiments with inhibitors, Tregs were separately incubated with 10 μM of the p38 inhibitor SB203580 (HY-10256; MCE), 2.5 μM of the AKT inhibitor MK2206 (HY-10358; MCE), 10 μM of the ERK1/2 inhibitor PD98059 (HY-12028; MCE), or 15 μM of the GATA2 inhibitor K-7174 (HY-12743; MCE) for 1 h and then stimulated with 200 μM FFAs for another 48 h.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Activity Assay

Journal: Molecular Metabolism

Article Title: CD73 promotes the immunoregulatory functions of hepatic Tregs through enzymatic and nonenzymatic pathways in MASLD development

doi: 10.1016/j.molmet.2025.102131

Figure Lengend Snippet: CD73 expression on Tregs is increased during MASLD progression . (A–B). The mRNA levels of Nt5e in the livers of the MCD- or CDHFD-fed mice were determined by real-time PCR. (C–D). The Nt5e mRNA levels in hepatocytes or liver MNCs of the MCD- or CDHFD-fed mice were measured by real-time PCR. (E). CD73 expressions on liver CD4 + T, CD8 + T, NK cells, neutrophils, monocytes, and Kupffer cells were determined in NCD- and CDHFD-fed mice. (F). CD73 expressions on blood CD4 + T, CD8 + T, NK, and NKT cells were measured in NCD- and CDHFD-fed mice. (G). CD73 expressions on liver CD4 + T, CD8 + T, NK cells, neutrophils, monocytes, and Kupffer cells were determined in the NCD- and MCD-fed mice. (H). CD73 expressions on blood CD4 + T, CD8 + T, NK, and NKT cells were measured in the MCD-fed mice. (I). Representative flow cytometry plots of CD73 expression on Tregs (CD4 + CD25 + CD127 - ) in the livers or blood of the CDHFD-fed mice. (J–K). Statistical analysis of the percentages of CD73 + Tregs in the liver or blood of the CDHFD- and MCD-fed mice. (L). CD73 concentration in plasma was determined. (M). The ratio of CD73 + Tregs in PBMCs (left) and the concentration of soluble CD73 in plasma (right) from the healthy controls or MASLD patients. n = 5–21 per group. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Male C57BL/6 Cd73 knockout (KO) (stock no. 018986), Foxp3 GFP + transgenic (stock no. 023800), C57BL/6-GFP (stock no. 004353), and Rag1 KO (stock no. 002216) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Concentration Assay, Clinical Proteomics

Journal: Molecular Metabolism

Article Title: CD73 promotes the immunoregulatory functions of hepatic Tregs through enzymatic and nonenzymatic pathways in MASLD development

doi: 10.1016/j.molmet.2025.102131

Figure Lengend Snippet: Cd73 knockout decreases intrahepatic Treg survival and immunomodulatory activity in CDHFD-induced MASLD model. (A). Body weight of the NCD- or CDHFD-fed WT or Cd73 knockout mice. (B). Plasma ALT, AST, and TBIL levels were determined. (C). Fasting plasma glucose levels were determined in the NCD- or CDHFD-fed mice. (D). Representative H&E, Oil Red O, and α-SMA staining and quantification of liver histology staining. (E). The relative mRNA expressions of proinflammatory cytokines ( Ifng , Il17 , and Tnfa ) in the liver were measured. (F). Fibrosis-related genes ( Col3a1 , Col1a1 , and Acta2 ) were assessed by real-time PCR. (G). Hydroxyproline levels in liver tissues from the NCD- or CDHFD-fed mice. (H–I). Ratio of Tregs among liver CD4 + T cells and CD45 + cells. (J). Apoptosis (Annexin V, Caspase-3 activity) and proliferation (Ki67, BCL-2) of liver Tregs were detected by flow cytometry. (K). Representative flow cytometry plots and statistical analysis of Granzyme Bpositive Tregs in the liver. (L–N). Proportion of CTLA4 + , IL-10 + , and CD69 + Tregs in each group of mice. n = 5 per group. ∗ P < 0.05, ∗∗ P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Male C57BL/6 Cd73 knockout (KO) (stock no. 018986), Foxp3 GFP + transgenic (stock no. 023800), C57BL/6-GFP (stock no. 004353), and Rag1 KO (stock no. 002216) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Knock-Out, Activity Assay, Clinical Proteomics, Staining, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: Molecular Metabolism

Article Title: CD73 promotes the immunoregulatory functions of hepatic Tregs through enzymatic and nonenzymatic pathways in MASLD development

doi: 10.1016/j.molmet.2025.102131

Figure Lengend Snippet: CD73 maintains Treg survival and immunomodulatory activity, protecting against MASLD progression. (A). The Rag1 −/− mice were adoptively transferred with WT mouse-derived CD3 + T cells (W/WT Treg group) or CD3 + T cells without Tregs (W/O Treg group). In the W/ Cd73 KO Treg group, Rag1 −/− mice received CD3 + T cells without Tregs (from WT mice) together with Cd73 KO Tregs (from Cd73 KO mice). The above recipient mice and control Rag1 −/− mice not receiving cell transfer were subsequently fed MCD for 4 weeks. Created in https://BioRender.com . (B–C). Plasma ALT and AST levels were determined. (D). H&E and Oil Red O staining of representative paraffin-embedded liver sections. (E). Quantification of liver H&E and Oil Red O staining. (F). The body weight of mice at a different time. (G). The cytokine levels in plasma were measured by ELISA. (H) Ratio of CD4 + FOXP3 + Tregs in liver CD4 + T cells. (I, J) Ratio of Annexin V + , Tim-3 + , CTLA4 + Tregs in the livers of transplanted Rag1 −/− mice. (K). Ratio of monocytes (CD11b high F4/80 low ) in liver CD45 + Ly6G − cells. (L). The level of TNF-α secreted by monocytes was detected. (M). Ratio of Th1 (IFN-γ + ) or Th17 (IL-17 + ) in CD4 + T cells. n = 3–5 per group. ∗ P < 0.05, ∗∗ P < 0.01. $: A comparison between group Rag1 KO and W/O Treg with P < 0.05; $$: A comparison between group Rag1 KO and W/O Treg with P < 0.01; #: A comparison between group W/O Treg and W/WT Treg with P < 0.05; ##: A comparison between group W/O Treg and W/WT Treg with P < 0.01; &: A comparison between group W/WT Treg and W/ Cd73 KO Treg with P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Male C57BL/6 Cd73 knockout (KO) (stock no. 018986), Foxp3 GFP + transgenic (stock no. 023800), C57BL/6-GFP (stock no. 004353), and Rag1 KO (stock no. 002216) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Activity Assay, Derivative Assay, Control, Clinical Proteomics, Staining, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Molecular Metabolism

Article Title: CD73 promotes the immunoregulatory functions of hepatic Tregs through enzymatic and nonenzymatic pathways in MASLD development

doi: 10.1016/j.molmet.2025.102131

Figure Lengend Snippet: FFAs increase CD73 expression on Tregs via p38/Gata2 signaling pathway. (A). GSEA of Tregs from MCD-versus NCD-fed mice was performed. (B) After the addition of FFAs for 48 h, the expression of CD73 on Tregs was measured by flow cytometry. (C) Phosphorylated AKT activity in the Tregs after FFAs treatment. (D). MAPK signaling was detected in FFAs-stimulated Tregs by flow cytometry. (E). Tregs isolated from WT mice were preincubated with p38 inhibitor (SB203580), AKT inhibitor (MK2206), or ERK1/2 inhibitor (PD98059) for 1 h followed by FFAs treatment for 48 h, and the CD73 expression was detected by flow cytometry. (F). Transcription factor-binding sites of the Nt5e promoter region were predicted through the JASPAR CORE database and intersected with DEGs of Tregs from MCD- versus NCD-fed mice. (G). Relative mRNA levels of transcription factors ( Runx2, Gata2, Sox9 , and Tcf4 ) were determined by real-time PCR. (H–I). Runx2 and Gata2 expression was detected in Tregs incubated with p38 inhibitor (SB203580) followed by FFAs stimulation. (J). CUT&Tag assays of GATA2 binding sites showed a peak distribution around the Nt5e promoter region in the FFAs-treated Tregs. (K). The enrichment of GATA2 binding sites in the Nt5e promoter region was confirmed by real-time PCR, and the data were normalized to those of IgG. (L). CD73 expression on Tregs with pre-treatment of Gata2 inhibitor (K7174) and subsequent FFA stimulation. n = 4–5 per group. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Male C57BL/6 Cd73 knockout (KO) (stock no. 018986), Foxp3 GFP + transgenic (stock no. 023800), C57BL/6-GFP (stock no. 004353), and Rag1 KO (stock no. 002216) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Expressing, Flow Cytometry, Activity Assay, Isolation, Binding Assay, Real-time Polymerase Chain Reaction, Incubation

Journal: Molecular Metabolism

Article Title: CD73 promotes the immunoregulatory functions of hepatic Tregs through enzymatic and nonenzymatic pathways in MASLD development

doi: 10.1016/j.molmet.2025.102131

Figure Lengend Snippet: CD73 protects Tregs from AMP-induced toxicity, while the degradation product adenosine promotes Treg survival and function in the presence of FFAs. (A–C). The plasma concentrations of ATP, AMP, and adenosine (ADO) were measured in the CDHFD- or MCD-fed WT and Cd73 KO mice, as well as the MCD-fed Treg-transplanted Rag1 −/− mice. (D) Relative AMP and ADO levels in the supernatant after the addition of ATP to cultures of WT or Cd73 KO Tregs. (E). Apoptosis of CD4 + T cells without Tregs was detected after ADO treatment with or without FFAs treatment. (F). Apoptosis and TNF-α secretion were measured in ADO-stimulated monocytes with or without FFAs treatment. (G). Apoptosis of ADO-stimulated Tregs was detected by flow cytometry. (H). The expression of immunomodulatory molecules (CTLA4, Perforin, IL-10, Granzyme B, and TGF-β) on WT Tregs were detected after ADO stimulation with or without FFAs treatment. (I–K). Treg apoptosis (Annexin V and 7AAD) and IL-10 expression were compared between WT and Cd73 KO mice after AMP supplement with or without FFAs treatment. (L) The relative mRNA expression of Bcl2, Ctla4, Gzmb, Prf1, Il10 , and Tgfb in Tregs from WT and Cd73 KO mice was determined after AMP supplement with or without FFAs treatment. n = 4–5 per group. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Male C57BL/6 Cd73 knockout (KO) (stock no. 018986), Foxp3 GFP + transgenic (stock no. 023800), C57BL/6-GFP (stock no. 004353), and Rag1 KO (stock no. 002216) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Clinical Proteomics, Flow Cytometry, Expressing

Journal: Molecular Metabolism

Article Title: CD73 promotes the immunoregulatory functions of hepatic Tregs through enzymatic and nonenzymatic pathways in MASLD development

doi: 10.1016/j.molmet.2025.102131

Figure Lengend Snippet: CD73 inhibits Treg apoptosis by suppressing the TRAIL-DR5 interaction. (A). The relative mRNA expression of Tnfrsf10b (DR5) and Tnfsf10 (TRAIL) in the liver was measured by real-time PCR. (B). Plasma content of TRAIL in the CDHFD- or NCD-fed mice. (C). DR5 expression on liver Tregs. (D). Relative apoptotic levels were compared between CD4 + T cells without Tregs and Tregs followed by FFAs treatment with or without TRAIL. (E). Relative apoptotic levels were compared among CD73 high , CD73 int and CD73 neg Tregs after FFAs treatment with or without TRAIL. (F–G). Relative Annexin V and Caspase-3 activity were measured in the WT and Cd73 KO Tregs treated with FFAs with or without TRAIL. (H). Tregs were blocked with CD73 protein or IgG for 6 h, followed by the addition of FFAs and TRAIL for 48 h. The proportions of Annexin V + Tregs after TRAIL supplement. (I). CD73 on Tregs limits inflammation and maintains homeostasis during MASLD progression (Created in https://BioRender.com ). n = 3–6 per group. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Male C57BL/6 Cd73 knockout (KO) (stock no. 018986), Foxp3 GFP + transgenic (stock no. 023800), C57BL/6-GFP (stock no. 004353), and Rag1 KO (stock no. 002216) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Clinical Proteomics, Activity Assay

Journal: Cell Research

Article Title: 40 Hz light flickering promotes sleep through cortical adenosine signaling

doi: 10.1038/s41422-023-00920-1

Figure Lengend Snippet: a Schematic drawing depicting the production of Adenosine (Ado) from ATP via CD73 enzymes. b Extracellular adenosine increase (as detected by GRAB Ado ) caused by 40 Hz light flickering in V1 of WT and CD73 -KO mice. c Quantified fluorescence of GRAB Ado1.0 Δ F/F0 in response to 40 Hz light flickering in CD73 -KO mice ( n = 6/group). d Schematic drawing depicting the release of Ado via ENTs and the pharmacological inhibition of ENTs blocking activity. e Pretreatment with dipyridamole (30 min before light flickering) abolished 40 Hz flickering-induced extracellular adenosine generation during light flickering and after stimulation cessation. f Group summary of GRAB Ado1.0 Δ F/F0 in response to 40 Hz light flickering application of dipyridamole ( n = 5–6/group). g Description of metabolomic screening for dipyridamole administration h Total tissue (intracellular and extracellular) adenosine levels in V1, as assessed by HPLC analysis, after 40 Hz light flickering with or without pretreatment with dipyridamole ( n = 14/group). * P < 0.05, comparing the dipyridamole-treated group with the vehicle-treated group. i Schematic drawing depicting the release of Ado via ENTs and blocking ENT1 activity in ENT1 -KO mice. j 40 Hz flickering induced a robust and sustained increase of the extracellular adenosine generation in both WT and ENT1 -KO mice, with slightly lesser induction in ENT1 -KO than WT mice at later time points. k Group summary of GRAB Ado1.0 ΔF/F0 in response to 40 Hz light flickering from ENT1 -KO mice ( n = 5–6/group). l Schematic drawing depicting the release of Ado via ENTs and blocking ENT2 activity by ENT2 -KO mice. m 40 Hz flickering-induced extracellular adenosine generation was robust and sustained in WT but largely abolished in ENT2 -KO mice. n Group summary of Ado1.0 Δ F / F 0 in response to 40 Hz light flickering from ENT2 -KO mice ( n = 6–7/group). The data are presented as mean ± SEM, ** P < 0.01, * P < 0.05, Student’s t -test; WT vs KO; dipyridamole-treated group vs vehicle group.

Article Snippet: C57BL/6 WT mice were purchased from institute-approved vendors (Beijing Vital River Laboratory Animal Technology Co., Ltd.). vGluT2-IRES-CRE mice JAX Strain (016963) and Nt5e ( CD73 ) KO ( CD73 -KO) mice (JAX Strain 018986) were obtained from Jackson Laboratory.

Techniques: Fluorescence, Inhibition, Blocking Assay, Activity Assay