recombinant mouse gdf15 protein  (R&D Systems)

Journal: bioRxiv

Article Title: Long-Term Intestinal Epithelial Remodeling Induced by Acute Protein-Energy Malnutrition

doi: 10.1101/2025.10.20.683425

Figure Lengend Snippet: A-B) Comparison of transcriptional PEM effects in intestinal epithelial cells (IEC, blue) and organoids (yellow) isolated of mice from two separate dietary PEM-recovery intervention experiments during (A) acute PEM and (B) after recovery. The dot plot depicts the Log 2 [fold-changes PEM_4w/10w vs Ctrl_4w/10w] whereas the Venn diagram depicts the overlap in up- and downregulated DEGs. C) Number of differentially (hypo- and hyper-) methylated positions (DMPs) detected by BeadCHiP array in small intestinal epithelial cells from mice during acute PEM (4 weeks) and after recovery (10 weeks) and respective controls. D) Shared DMPs between acute PEM (4 weeks) and recovery (10 weeks). E) Predicted transcription factor binding sites enriched in observed DMPs. Dot size is proportional to the odds ratio and color corresponds to the p-value of enrichment. Top selected transcription factors are visualized. F) Schematic depiction of gene expression - DNA methylation integration analysis. Spearman’s rank correlation coefficient between the methylation intensity and gene expression values was calculated between the DMPs located within a range of 5kb before or after the transcription start site of the respective DEG. G) Venn Diagram depicting the overlaps between shared DEGs between PEM_4w and Rec_10w and DEGs correlated with their nearby DMPs, identifying 31 candidate DEGs linked with DMPs including GDF15 that has been linked to body mass regulation.

Article Snippet: Organoids were divided into experimental groups and stimulated with 1 μg E. coli derived recombinant mouse GDF15 (R&D Systems, 8944-GD) per 500 μl organoid medium for up to 120h, 20 μM of the pan-PPAR agonist Lanifibranor (MedChemExpress, HY-104049) for 24h to 96h or 1 μM 9(S)-HODE (Sigma Aldrich, SML0503) for 24h to 96h.

Techniques: Comparison, Isolation, Methylation, Binding Assay, Gene Expression, DNA Methylation Assay

Journal: bioRxiv

Article Title: Long-Term Intestinal Epithelial Remodeling Induced by Acute Protein-Energy Malnutrition

doi: 10.1101/2025.10.20.683425

Figure Lengend Snippet: A) Expression of Gdf15 , Pparg and Pparbd were assessed in small intestinal tissue of both CONVR and GF mice subjected to PEM and after recovery. *p < 0.05, ***p < 0.001 and ****p < 0.0001, ns = non-significant. Significance testing was performed using Wilcoxon-Mann-Whitney-Test. B) Schematic drawing of organoid intervention experiment. Murine intestinal organoids were first cultured in ENR-CV stem cell organoid medium and then Paneth cell differentiation was induced using ENR-CD or PEM-CD medium. Stimulants (recombinant GDF15 [1 µg/ml], pan-PPAR-agonist Lanifibranor [20 µM] or the lipid 9-HODE [1 µM] were added during initial ENR-CD and PEM-CD culture and kept throughout the experimental duration. All experiments were performed independently at least twice in triplicates. C-E) Relative Lyz1 , Defa5 and Gdf15 mRNA expression in organoids during Paneth cell differentiation and stimulated with (C) recombinant GDF15, (D) the Pan-PPAR-agonist Lanifibranor or the lipid (E) 9-HODE. Note that PPAR-activation boosts GDF15 levels thereby blocking Paneth cell differentiation and the bacterial PEM metabolite 9-HODE also suppresses Paneth cell differentiation. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001 and ns = not significant using Mann-Whitney U test.

Article Snippet: Organoids were divided into experimental groups and stimulated with 1 μg E. coli derived recombinant mouse GDF15 (R&D Systems, 8944-GD) per 500 μl organoid medium for up to 120h, 20 μM of the pan-PPAR agonist Lanifibranor (MedChemExpress, HY-104049) for 24h to 96h or 1 μM 9(S)-HODE (Sigma Aldrich, SML0503) for 24h to 96h.

Techniques: Expressing, MANN-WHITNEY, Cell Culture, Cell Differentiation, Recombinant, Activation Assay, Blocking Assay

Journal: bioRxiv

Article Title: Long-Term Intestinal Epithelial Remodeling Induced by Acute Protein-Energy Malnutrition

doi: 10.1101/2025.10.20.683425

Figure Lengend Snippet: Expression of Lyz1 and Gdf15 were assessed in intestinal organoids stimulated with the (A) pan-PPAR-agonist Lanifibranor [20 µM] or the (B) lipid 9-HODE [1 µM] during Paneth cell differentiation. Note that stimulation with Lanifibranor or 9-HODE did not alter Lyz1 or Gdf15 expression in contrast to stimulation during PEM as shown in .

Article Snippet: Organoids were divided into experimental groups and stimulated with 1 μg E. coli derived recombinant mouse GDF15 (R&D Systems, 8944-GD) per 500 μl organoid medium for up to 120h, 20 μM of the pan-PPAR agonist Lanifibranor (MedChemExpress, HY-104049) for 24h to 96h or 1 μM 9(S)-HODE (Sigma Aldrich, SML0503) for 24h to 96h.

Techniques: Expressing, Cell Differentiation

Journal: bioRxiv

Article Title: De novo and scaffold-based design of GDF15 binders for cancer cachexia diagnostics and therapeutics

doi: 10.1101/2025.09.03.673894

Figure Lengend Snippet: a. Cryo-EM structure of the extracellular GDF15–GFRAL–RET complex (PDB: 6Q2J) showing a 2:2:2 stoichiometry, wherein the dimeric GDF15 bridges two GFRAL co-receptors and two RET receptors. b. Binding interfaces of GDF15 with GFRAL (top) and RET (bottom). Key interacting residues are shown as sticks. Hydrophobic hotspot residues used for binder design are highlighted in pink. c. Target sites for GDF15 binder design. The convex surface engaging GFRAL (site A, red) and the concave surface contacting RET (site B, light blue) are highlighted. Insets show electrostatic surface potentials of each site (white, hydrophobic; blue, positive charge; red, negative charge). d. Workflow of binder design using three scaffold generation strategies: Scaffold Grafting (SG), Diffusion-based de novo Design, and Scaffold-Search and Grafting (SSG).

Article Snippet: Recombinant human or mouse GDF15 (#9279-GD or #8944-GD, R&D Systems) was prepared at a stock concentration of 250 μg/mL and subsequently diluted in pooled human serum (Sigma #H6914) or mouse serum (Abbkine #BMS0070), resulting in a final concentration of up to 5 μg/mL (≤500 nM).

Techniques: Cryo-EM Sample Prep, Binding Assay, Diffusion-based Assay

Journal: bioRxiv

Article Title: De novo and scaffold-based design of GDF15 binders for cancer cachexia diagnostics and therapeutics

doi: 10.1101/2025.09.03.673894

Figure Lengend Snippet: a. Workflow of binder design via scaffold grafting. The GFRAL D2 domain was extracted as the initial scaffold to generate SG A 1 and SG A 2. SG A 2 was further optimized by scaffold-guided partial diffusion, resulting in five variants (SG A 2-1 to SG A 2-5). b. Extracted GFRAL D2 scaffold (residues 129∼211) from the GFRAL extracellular domain. The N- and C-termini, helices, and disulfide bonds are indicated. c. Backbone RMSD distribution of 100 ProteinMPNN-designed variants relative to the parental GFRAL D2 scaffold (mean RMSD = 0.85 Å). d. Structural alignment of the initial scaffold GFRAL D2 with SG A 1 (left) and SG A 2 (right). RMSD with AF3-predicted structure and AF3 scores (pAE_interaction and pLDDT) for the binder/GDF15 complex are indicated, with the N- and C-termini labeled. e. Structural comparison of GFRAL-D2, SG A 1, and SG A 2 at the binding interface. Residues that enhance binding, commonly observed in both SG A 1 and SG A 2, are indicated. The rightmost panel shows the superposition of three scaffolds, highlighting α5 displacement. f. Superposition of SG A 1 and SG A 2, showing a unique electrostatic interaction in SG A 2. g. Structural alignment of SG A 2 with its partial diffusion-derived variants. RMSD with AF2-predicted structure and AF2 scores (pAE_interaction and pLDDT) for the binder/GDF15 complex are indicated, with the N- and C-termini labeled. h. Backbone RMSD distribution of partial diffusion–derived variants relative to the SG A 2 scaffold (mean RMSD = 4.6 Å). i. Structural model of SG A 2-4 at the binding interface. Conserved binding residues in α5 (D69, Q72, L73, Q76), retained in SG A 2-4 variants compared to SG A 2, are highlighted. j. Binding interface comparison of SG A 2 and SG A 2-4, highlighting the shifted α1 position and distinct interacting residues on this helix.

Article Snippet: Recombinant human or mouse GDF15 (#9279-GD or #8944-GD, R&D Systems) was prepared at a stock concentration of 250 μg/mL and subsequently diluted in pooled human serum (Sigma #H6914) or mouse serum (Abbkine #BMS0070), resulting in a final concentration of up to 5 μg/mL (≤500 nM).

Techniques: Diffusion-based Assay, Labeling, Comparison, Binding Assay, Derivative Assay

Journal: bioRxiv

Article Title: De novo and scaffold-based design of GDF15 binders for cancer cachexia diagnostics and therapeutics

doi: 10.1101/2025.09.03.673894

Figure Lengend Snippet:

Article Snippet: Recombinant human or mouse GDF15 (#9279-GD or #8944-GD, R&D Systems) was prepared at a stock concentration of 250 μg/mL and subsequently diluted in pooled human serum (Sigma #H6914) or mouse serum (Abbkine #BMS0070), resulting in a final concentration of up to 5 μg/mL (≤500 nM).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: De novo and scaffold-based design of GDF15 binders for cancer cachexia diagnostics and therapeutics

doi: 10.1101/2025.09.03.673894

Figure Lengend Snippet: a. Workflow of de novo binder design using RF diffusion. A total of 1,728 initial scaffolds (50∼90 a.a) were generated, sequence-designed (three sequences per each backbone), and computationally filtered using in silico evaluation metrics. The top five binders (DE A 1–DE A 5) were structurally analyzed and experimentally validated by expression, purification, and binding analysis. The best-performing DE A 3 was further optimized by scaffold-guided partial diffusion, resulting in seven variants (DE A 3-1 to DE A 3-7). b. Distribution of helix counts in RFdiffusion-generated scaffolds to analyze structural diversity. c. AF2-predicted structural models of the five selected de novo binder candidates in complex with the GDF15 dimer. Binder lengths, pAE_interaction, and pLDDT values are indicated, with the N- and C-termini labeled. d. SDS-PAGE analysis of binders (DE A 1–5, left; DE A 3-1 to DE A 3-7, right) after E. coli expression and affinity purification. e. Binding interface comparison of DE A 3 (left) and DE A 3-5 (right) with GDF15. Key interacting residues are shown as sticks and labeled.

Article Snippet: Recombinant human or mouse GDF15 (#9279-GD or #8944-GD, R&D Systems) was prepared at a stock concentration of 250 μg/mL and subsequently diluted in pooled human serum (Sigma #H6914) or mouse serum (Abbkine #BMS0070), resulting in a final concentration of up to 5 μg/mL (≤500 nM).

Techniques: Diffusion-based Assay, Generated, Sequencing, In Silico, Expressing, Purification, Binding Assay, Labeling, SDS Page, Affinity Purification, Comparison

Journal: bioRxiv

Article Title: De novo and scaffold-based design of GDF15 binders for cancer cachexia diagnostics and therapeutics

doi: 10.1101/2025.09.03.673894

Figure Lengend Snippet: a. The RET domain segment (residues 586–622) was tested as an initial scaffold for site B binders design. b. AF2-predicted structures of 100 RET-derived variants by sequence design. Only disulfide-constrained β-strands remained folded. c. In silico filtering of 500 de novo backbones generated by RFdiffusion with site B hotspot constraints (W225, W228, M253, and Y297). No candidates satisfied filtering thresholds (pLDDT > 85 and pAE_interaction < 10; red box). d. Workflow of the scaffold search and grafting (SSG) strategy. The GDF15 structure was used as a query in the DALI server to search the Protein Data Bank (PDB) for natural scaffolds with similar topology and surface geometry. Candidate scaffolds were then subjected to scaffold-guided partial diffusion and sequence design. e. Representative scaffold candidates identified from the DALI server search: Follistatin/Activin A (PDB 2B0U), BMP9 pro-complex (mature domain + prodomain) (PDB 4YCI), BMP2/RGMA (PDB 4UHY), TGF-β3/GC-1008 antibody (PDB 3EO1), and BMP2/BMP2 receptor A (PDB 1ES7). RMSD relative to GDF15 is indicated. f. Structural comparison of GDF15/RET (green/pink) and BMP2/RGMA (olive/purple) complexes. GDF15 and BMP2 show overall similarity (RMSD = 2.5 Å), but interacting partners differ topologically. g. Structural alignment of RGMA with RGMA-derived binder variants (SSG B 1 to SSG B 5). RMSD with AF2-predicted structures and AF2 scores (pAE_interaction and pLDDT) for the binder/GDF15 complex are indicated. h. SDS-PAGE analysis of binders (SSG B 1-SSG B 5) after E. coli expression and affinity purification.

Article Snippet: Recombinant human or mouse GDF15 (#9279-GD or #8944-GD, R&D Systems) was prepared at a stock concentration of 250 μg/mL and subsequently diluted in pooled human serum (Sigma #H6914) or mouse serum (Abbkine #BMS0070), resulting in a final concentration of up to 5 μg/mL (≤500 nM).

Techniques: Derivative Assay, Sequencing, In Silico, Generated, Diffusion-based Assay, Comparison, SDS Page, Expressing, Affinity Purification

Journal: bioRxiv

Article Title: De novo and scaffold-based design of GDF15 binders for cancer cachexia diagnostics and therapeutics

doi: 10.1101/2025.09.03.673894

Figure Lengend Snippet: a, b. Schematic illustration (a) and structural model (b) of the BAT biosensor (SmBiT-GDF15 binder-LgBiT) for GDF15 detection in the “OFF” and “ON” states. The BAT biosensor consists of a designed GDF15 binder (red) flanked by SmBiT at the N-terminus (cyan, 1, SmBiT) and LgBiT at the C-terminus (blue, 2, LgBiT). In the absence of GDF15, two split luciferase fragments remain apart (“OFF” state with only background activity). Upon GDF15 binding, steric constraints bring two split luciferase fragments into proximity, enabling fragment complementation and restoring NanoLuc activity (“ON” state). The lengths of linker1 and linker2 (b, left) are key determinants for background signals in the absence of GDF15. c. SDS-PAGE analysis of SmBiT-DE A 3-LgBiT with various linker combinations after E.coli expression and affinity purification. d. Screening of linker combinations for SmBiT-DE A 3-LgBiT using a luminescence assay. Signal-to-noise ratios (luminescence intensity of each construct divided by that of the control without GDF15) are shown, with optimal linker combinations highlighted in red. e, g. Luminescent signals of SmBiT-DE A 3-LgBiT with 0-10 linkers (e) and of SmBiT-DE A 3-5-LgBiT with 5-5 linkers (g). Luminescence (arbitrary units, AU) is plotted against various concentrations of human or mouse GDF15 (n = 3). The linear detection range is indicated with a red box (0–10 nM). f. Sequence alignment of human and mouse GDF15. Conserved residues at site A are marked with black circles; species-specific substitutions at the interface are highlighted with green circles. h. AF3-predicted structures of DE A 3 or DE A 3-5 bound to human or mouse GDF15. Per-residue pLDDT values are color-coded according to the scale bar.

Article Snippet: Recombinant human or mouse GDF15 (#9279-GD or #8944-GD, R&D Systems) was prepared at a stock concentration of 250 μg/mL and subsequently diluted in pooled human serum (Sigma #H6914) or mouse serum (Abbkine #BMS0070), resulting in a final concentration of up to 5 μg/mL (≤500 nM).

Techniques: Luciferase, Activity Assay, Binding Assay, SDS Page, Expressing, Affinity Purification, Luminescence Assay, Construct, Control, Sequencing, Residue

Journal: bioRxiv

Article Title: De novo and scaffold-based design of GDF15 binders for cancer cachexia diagnostics and therapeutics

doi: 10.1101/2025.09.03.673894

Figure Lengend Snippet: a. Schematic diagram of Fc-fused SG A 2-4 binder (SG A 2-4-Fc). b. SEC profile of SG A 2-4-Fc on a Superdex® 200 Increase 10/300 GL column (left) and SDS-PAGE analysis of elution fractions (right). c. SPR analysis of SG A 2-4-Fc and ponsegromab binding to immobilized GDF15. Sensorgrams are shown for analytes ranging from 5 to 50 nM (SG A 2-4-Fc or ponsegromab). d. Inhibition of GDF15-induced RET, AKT, and ERK phosphorylation in HEK293T cells stably expressing GFRAL and RET. Cells were co-treated with GDF15 (10 nM, 246 ng/ml) and either SG A 2-4-Fc or ponsegromab (100 nM; 7.8 μg/ml for SG A 2-4-Fc or 14.6 μg/ml for ponsegromab) for 30 mins. Phosphorylation was quantified relative to total protein (RET, AKT, and ERK each), normalized to the GDF15-only condition (n = 3). Statistical significance was determined using unpaired t-test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant). e. Dose-dependent inhibition of GDF15-induced SRE-luciferase activity by SG A 2-4-Fc or ponsegromab in HEK293T cells co-expressing GFRAL, RET, and an SRE-Luc2 reporter. Data were normalized to GDF15-induced luminescence (100%), and IC 50 values were determined by non-linear regression fitting in GraphPad Prism.

Article Snippet: Recombinant human or mouse GDF15 (#9279-GD or #8944-GD, R&D Systems) was prepared at a stock concentration of 250 μg/mL and subsequently diluted in pooled human serum (Sigma #H6914) or mouse serum (Abbkine #BMS0070), resulting in a final concentration of up to 5 μg/mL (≤500 nM).

Techniques: SDS Page, Binding Assay, Inhibition, Phospho-proteomics, Stable Transfection, Expressing, Luciferase, Activity Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Transcription factor EGR2 alleviates autoimmune uveitis via activation of GDF15 to modulate the retinal microglial phenotype.

doi: 10.1073/pnas.2316161121

Figure Lengend Snippet: Fig. 5. GDF15 is a target of EGR2. (A) Volcano plot of DEGs in vehicle-LPS+IFN-γ and oeEGR2-LPS+IFN-γ cells. (B) GO analysis of Vehicle-LPS+IFN-γ and oeEGR2- LPS+IFN-γ groups. (C) KEGG pathway enrichment analysis in the Vehicle-LPS+IFN-γ and oeEGR2-LPS+IFN-γ groups. (D) Heatmap of significant DEGs following EGR2 overexpression. (E) The RNA-seq results were verified by RT-qPCR (n = 3). **P < 0.01; ***P < 0.001; unpaired Student’s t test. (F) Western blotting to confirm the protein expression level of GDF15 in the Vehicle-LPS+IFN-γ and oeEGR2-LPS+IFN-γ groups (n = 3). **P < 0.01; unpaired Student’s t test. (G) Genomic tracks for ChIP-seq around GDF15. (H) Potential EGR2 binding sites of GDF15 promoter. (I) ChIP-PCR to validate primers spanning predicted GDF15 promoter sequences (n = 3). ***P < 0.001; unpaired Student’s t test.

Article Snippet: Recombinant GDF15 protein (R&D Systems, USA, Catalog #8944- GD- 025) was injected into the mouse vitreous with a Hamilton syringe at 10 μM with a total volume of 1 μL on day 8 after EAU modeling, and the control group was injected with the same volume of vehicle solution by the same method.

Techniques: Over Expression, RNA Sequencing, Quantitative RT-PCR, Western Blot, Expressing, ChIP-sequencing, Binding Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Transcription factor EGR2 alleviates autoimmune uveitis via activation of GDF15 to modulate the retinal microglial phenotype.

doi: 10.1073/pnas.2316161121

Figure Lengend Snippet: Fig. 6. GDF15 knockdown reversed the effects of EGR2 in microglia. (A) EGFP expression in HMC3 cells transfected with lentivirus. (Scale bar, 100 μm.) (B and C) Knockdown efficiency of GDF15 was assessed by RT-qPCR and western blotting (n = 3). ns, P > 0.05; **P < 0.01; ***P < 0.001; unpaired Student’s t test. (D and E) RT–qPCR and western blotting assays for the detection of iNOS, IL-1β, and TNF-α expression in the Vehicle1, oeEGR2, oeEGR2+shGDF15, and oeEGR2+Vehicle2 groups (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; one-way ANOVA. (F and G) Transwell images and wound healing images of the abovementioned groups (n = 3). (Scale bar, 100 μm.) *P < 0.05; **P < 0.01; ***P < 0.001; one-way ANOVA. (H) EdU assay of the Vehicle1, oeEGR2, oeEGR2+shGDF15, and oeEGR2+Vehicle2 groups (n = 3). (Scale bar, 100 μm.) **P < 0.01; ***P < 0.001; one-way ANOVA.

Article Snippet: Recombinant GDF15 protein (R&D Systems, USA, Catalog #8944- GD- 025) was injected into the mouse vitreous with a Hamilton syringe at 10 μM with a total volume of 1 μL on day 8 after EAU modeling, and the control group was injected with the same volume of vehicle solution by the same method.

Techniques: Knockdown, Expressing, Transfection, Quantitative RT-PCR, Western Blot, EdU Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Transcription factor EGR2 alleviates autoimmune uveitis via activation of GDF15 to modulate the retinal microglial phenotype.

doi: 10.1073/pnas.2316161121

Figure Lengend Snippet: Fig. 7. Recombinant GDF15 alleviated EAU progression in vivo. (A) Schematic of animal experimental design. (B and C) Anterior chamber inflammation and retinal histopathological staining of WT+EAU, CKO+EAU, and CKO+EAU+rbGDF15 groups. Clinical scores and pathology scores were also shown (n = 9). (Scale bar, 100 μm.) *P < 0.05; **P < 0.01; Kruskal–Wallias test. (D) Retinal microglia fluorescence staining in the WT+EAU, CKO+EAU, and CKO+EAU+rbGDF15 groups (n = 3). (Scale bar, 50 μm.) (E) Protein expression of iNOS, IL-1β, and TNF-α in retina detected using western blotting (n = 6). *P < 0.05; **P < 0.01; ***P < 0.001; one-way ANOVA.

Article Snippet: Recombinant GDF15 protein (R&D Systems, USA, Catalog #8944- GD- 025) was injected into the mouse vitreous with a Hamilton syringe at 10 μM with a total volume of 1 μL on day 8 after EAU modeling, and the control group was injected with the same volume of vehicle solution by the same method.

Techniques: Recombinant, In Vivo, Staining, Fluorescence, Expressing, Western Blot

Journal: Respiratory research

Article Title: GDF15 ameliorates sepsis-induced lung injury via AMPK-mediated inhibition of glycolysis in alveolar macrophage.

doi: 10.1186/s12931-024-02824-z

Figure Lengend Snippet: Fig. 1 GDF15 ameliorated sepsis-induced lung injury and inflammation. The C57BL/6 mice were intraperitoneally injected with LPS (5 mg/kg) 1 h after rmGDF15 administration (50 ng/kg, i.p.) (n = 4). (A) Immunofluorescent staining of the co-localization of F4/80 and GDF15 in lung tissues. Scale bar = 10 μm. (B) The total protein concentration in BALF was measured. (C) H&E staining of lung tissues. (D) IHC staining showed the infiltration of Ly6G + neu trophils in lung tissues. (E-H) The levels of IL-6 and TNFα in BALF and plasma were tested by ELISA (n = 4). (I) The levels of CXCL1 in plasma were measured by ELISA (n = 4). Data were expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001

Article Snippet: Mouse recombinant GDF15 (#10596- GD-025) was purchased from R&D Systems (Minnesota, USA).

Techniques: Injection, Staining, Protein Concentration, Immunohistochemistry, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Respiratory research

Article Title: GDF15 ameliorates sepsis-induced lung injury via AMPK-mediated inhibition of glycolysis in alveolar macrophage.

doi: 10.1186/s12931-024-02824-z

Figure Lengend Snippet: Fig. 2 GDF15 inhibited glycolysis via activating AMPK in AMs. (A, B) MH-S were pretreated with 2-DG (5 mM) before stimulated with LPS (1 µg/mL) for 6 h. The mRNA expressions of IL-6 and TNFα were determined by RT-qPCR (n = 3). (C, D) The concentrations of IL-6 and TNFα in the supernatants were measured by ELISA (n = 3). (E, F) MH-S were pretreated with rmGDF15 (200 ng/mL) prior to LPS (1 µg/mL) stimuli. The protein levels of p-AMPK and AMPK were detected using western blot. (G, H) MH-S were pretreated with compound C before rmGDF15 treatment and LPS stimulation. The concentration of IL-6 and TNFα in the supernatants were measured by ELISA (n = 3). (I) MH-S were pretreated with rmGDF15 prior to LPS stimuli. Lactate concentration in plasma samples was measured (n = 3). (J, K) The protein levels of glycolytic enzymes were detected using western blot. (L) MH-S were pretreated with rmGDF15 before LPS stimuli, followed by the measurement of glycolysis rate for 80 min. Rot&AA: Rotenone & antimycin A; Oligo: Oligomycin. (M) The protein levels of phosphorylated AMPK as well as glycolytic enzymes in primary AMs isolated from BALF of septic mice with or without rmGDF15 administration were determined by western blot. (N-O) MH-S were pretreated with AMPK inhibitor (compound C, 5 µM) before rmGDF15 treatment and LPS stimulation. The protein levels of phosphorylated AMPK as well as glycolytic enzymes were detected using western blot. comp C: compound C. Data were expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001

Article Snippet: Mouse recombinant GDF15 (#10596- GD-025) was purchased from R&D Systems (Minnesota, USA).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Concentration Assay, Clinical Proteomics, Isolation

Journal: Respiratory research

Article Title: GDF15 ameliorates sepsis-induced lung injury via AMPK-mediated inhibition of glycolysis in alveolar macrophage.

doi: 10.1186/s12931-024-02824-z

Figure Lengend Snippet: Fig. 3 GDF15 inhibited MAPK/NF-κB signaling via AMPK activation. (A, B) MH-S were pretreated with rmGDF15 prior to LPS stimuli. The protein levels of p-p65, p65, p-p38, and p38 were detected using western blot. (C, D) MH-S were pretreated with compound C before rmGDF15 treatment and LPS stimu lation. The protein levels of p-p65, p65, p-p38, and p38 were detected using western blot. Data were expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001

Article Snippet: Mouse recombinant GDF15 (#10596- GD-025) was purchased from R&D Systems (Minnesota, USA).

Techniques: Activation Assay, Western Blot

Journal: Respiratory research

Article Title: GDF15 ameliorates sepsis-induced lung injury via AMPK-mediated inhibition of glycolysis in alveolar macrophage.

doi: 10.1186/s12931-024-02824-z

Figure Lengend Snippet: Fig. 4 Glycolysis inhibition exhibited anti-inflammatory effect on AMs during sepsis via inducing GDF15 expression. The transcriptome analysis was performed on MH-S stimulated with LPS (1 µg/mL) after 2-DG (5 mM) pretreatment (n = 3). (A) Volcano plot of differently expressed genes. (B) MH-S were stimulated with LPS (1 µg/mL) after 2-DG (5 mM) treatment. The mRNA expressions of GDF15 were detected by RT-qPCR (n = 3). (C) The protein levels of GDF15 were detected using western blot. (D) The secreted levels of GDF15 in the supernatants were measured by ELISA (n = 3). (E, F) The concentration of IL-6 and TNFα in the supernatants were measured by ELISA (n = 3). (G) MH-S were transfected with GDF15-specific siRNA or scrambled siRNA before 2-DG (5 mM) pretreatment and LPS (1 µg/mL) stimuli. The mRNA levels of GDF15 were analyzed by RT-qPCR (n = 3). (H, I) The concentrations of IL-6 and TNFα in the culture medium were measured by ELISA (n = 3). (K, L) The C57BL/6 mice were i.p. injected with LPS (5 mg/kg) with or without 2-DG (500 mg/ kg, i.p.) pretreatment for 1 h. (J) Immunofluorescent co-staining of F4/80 and GDF15 in lung tissues. Scale bar = 10 μm. The levels of GDF15 in plasma and BALF were measured by ELISA (n = 4). (M-P) The levels of IL-6 and TNFα in plasma and BALF were measured by ELISA (n = 4). (Q) H&E staining of lung tis sues. (R) IHC staining showed the infiltration of Ly6G + neutrophils in lung tissues. Data were expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001

Article Snippet: Mouse recombinant GDF15 (#10596- GD-025) was purchased from R&D Systems (Minnesota, USA).

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, Transfection, Injection, Staining, Clinical Proteomics, Immunohistochemistry

Journal: Respiratory research

Article Title: GDF15 ameliorates sepsis-induced lung injury via AMPK-mediated inhibition of glycolysis in alveolar macrophage.

doi: 10.1186/s12931-024-02824-z

Figure Lengend Snippet: Fig. 5 eIF2⍺-ATF4 signaling mediated 2-DG-related GDF15 expression in AMs. (A) KEGG analysis of genes regulated by both LPS stimulation and 2-DG treatment. (B) Heatmap of differently expressed genes in the pathway related to protein processing in endoplasmic reticulum (ER). (C) GSEA analysis showing the primary enrichment of ER stress signaling in 2-DG-regulated genes. (D) The protein levels of phosphorylated eIF2α and ATF4 in LPS-stimu lated MH-S with or without 2-DG pretreatment were analyzed by western blot. (E) The protein levels of phosphorylated eIF2α and ATF4 in AMs isolated from BALF of septic mice with or without 2-DG pretreatment were analyzed by western blot. (F, G) MH-S were transfected with ATF4-specific siRNA or scrambled siRNA before 2-DG (5 mM) pretreatment and LPS (1 µg/mL) stimuli. The mRNA level of ATF4 was detected by RT-qPCR (n = 3). The concentra tions of IL-6 in the culture medium were measured by ELISA (n = 3). (H-J) The mRNA levels of ATF4 and GDF15 were detected by RT-qPCR (n = 3). The protein levels of GDF15 in MH-S were detected by western blot. (K, L) MH-S were treated with LPS (1 µg/ml) after Salubrinal (50 µM) pretreatment. The mRNA level of GDF15 were detected by RT-qPCR (n = 3). The secreted levels of GDF15 in the supernatants were measured by ELISA (n = 3). Sal = Salubrinal. Data were expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001

Article Snippet: Mouse recombinant GDF15 (#10596- GD-025) was purchased from R&D Systems (Minnesota, USA).

Techniques: Expressing, Western Blot, Isolation, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Respiratory research

Article Title: GDF15 ameliorates sepsis-induced lung injury via AMPK-mediated inhibition of glycolysis in alveolar macrophage.

doi: 10.1186/s12931-024-02824-z

Figure Lengend Snippet: Fig. 6 The increased plasma levels of GDF15 in patients with sepsis were positively correlated with clinical prognostic indicators. (A) The plasma levels of GDF15 in septic patients and healthy controls were measured by ELISA. (B-F) Correlation analysis of plasma GDF15 and CRP, PCT, LDHA, lactate, and SOFA scores in patients with sepsis

Article Snippet: Mouse recombinant GDF15 (#10596- GD-025) was purchased from R&D Systems (Minnesota, USA).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Respiratory research

Article Title: GDF15 ameliorates sepsis-induced lung injury via AMPK-mediated inhibition of glycolysis in alveolar macrophage.

doi: 10.1186/s12931-024-02824-z

Figure Lengend Snippet: Fig. 7 Schematic illustration of GDF15 regulation in sepsis-induced inflammatory response of AMs. Glycolysis inhibition promoted GDF15 expression via activating eIF2⍺-ATF4 axis. In turn, GDF15 inhibited glycolysis and MAPKs/NF-κB signaling through AMPK activation, thereby alleviating the inflammatory response and lung injury in sepsis

Article Snippet: Mouse recombinant GDF15 (#10596- GD-025) was purchased from R&D Systems (Minnesota, USA).

Techniques: Inhibition, Expressing, Activation Assay

Journal: Frontiers in Immunology

Article Title: Identification of a distinct cluster of GDF15 high macrophages induced by in vitro differentiation exhibiting anti-inflammatory activities

doi: 10.3389/fimmu.2024.1309739

Figure Lengend Snippet: Immunofluorescence double labeling showing the existence of GDF15 high macrophages in lung tissues. (A) Results obtained in healthy human lung tissues from 3 independent subjects. Two representative microscopic fields were shown. Arrowheads indicated the CD68 + GDF15 high macrophages. (B) Results obtained in human lung tissues with COPD from 4 independent subjects. Arrowheads indicated the CD68 + GDF15 high macrophages. (C) Results obtained in rat lung tissues without and with experimental PAH. The nuclei were counterstained with DAPI (blue). White arrowheads indicated CD68 + GDF15 high macrophages. The red box highlighted the presence of CD68 + GDF15 high macrophages (white arrowheads); the green box highlighted the presence of CD68 + GDF15 low macrophages (red arrowheads). The bar graphs showed the absolute and relative abundances of CD68 + GDF15 high macrophages in normal and PAH lungs. Data were expressed as mean ± SEM. * P < 0.05, unpaired t -test.

Article Snippet: Recombinant GDF15 (#10596-GD) and recombinant interferon (IFN)-γ (#585-IF) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Immunofluorescence, Labeling

Journal: Frontiers in Immunology

Article Title: Identification of a distinct cluster of GDF15 high macrophages induced by in vitro differentiation exhibiting anti-inflammatory activities

doi: 10.3389/fimmu.2024.1309739

Figure Lengend Snippet: GDF15 high macrophages could be derived by in vitro differentiation of mononuclear cells. (A, B) Immunofluorescence staining and flow cytometry results confirming that in vitro differentiation of human peripheral blood mononuclear cells (PBMNCs) with GM-CSF for 7 days yielded CD68 + macrophages. (C, D) Flow cytometry and immunofluorescence double labeling results showing that the PBMNC-derived macrophages contained a minor population of GDF15 high cells (arrowheads in D ) (example from 3 independent experiments). (E, F) Fluorescence microscopy and flow cytometry data showing that GM-CSF differentiation of rat bone marrow mononuclear cells (BMMNCs) in vitro yielded macrophages (GFP expressing) of a high purity (~90%). CD68pro-GFP rats had a GFP transgene under the control of CD68 promoter. Cells from normal rats showing no GFP fluorescence served as a negative control (left panel in E ). (G, H) Flow cytometry and immunofluorescence double labeling data (from 3 independent experiments) showing that the BMMNC-derived macrophages (from CD68pro-GFP rats) contained a minor population of GDF15 high cells (arrowheads in H ). The flow cytometry data in panels (C, G) were from cells gated for GFP + . The nuclei were counterstained with DAPI (blue).

Article Snippet: Recombinant GDF15 (#10596-GD) and recombinant interferon (IFN)-γ (#585-IF) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Derivative Assay, In Vitro, Immunofluorescence, Staining, Flow Cytometry, Labeling, Fluorescence, Microscopy, Expressing, Control, Negative Control

Journal: Frontiers in Immunology

Article Title: Identification of a distinct cluster of GDF15 high macrophages induced by in vitro differentiation exhibiting anti-inflammatory activities

doi: 10.3389/fimmu.2024.1309739

Figure Lengend Snippet: Flow cytometry results showing that GDF15 high macrophages did not exhibit a typical M1 or M2 phenotype. Experiments were performed in human PBMNC-derived macrophages, using CD86, CD80 and IL-1β as the M1 markers, and CD206, CD163 and IL-4 as the M2 markers. Data were from a single test using pooled samples from 4 healthy volunteers.

Article Snippet: Recombinant GDF15 (#10596-GD) and recombinant interferon (IFN)-γ (#585-IF) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Flow Cytometry, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Identification of a distinct cluster of GDF15 high macrophages induced by in vitro differentiation exhibiting anti-inflammatory activities

doi: 10.3389/fimmu.2024.1309739

Figure Lengend Snippet: Molecular characterization of human PBMNC-derived GDF15 high macrophages with scRNA-seq. (A) Graphical outline of the experimental procedure. (B) UMAP plots showing the identified cell sub-populations (C1 to C7) based on the scRNA-seq data from total 73,768 cells pooled from samples of 3 healthy volunteers, 3 PAH patients harboring mutations in BMPR2 gene, and 3 PAH patients without BMPR2 mutations. The putative nomenclatures for C1 to C7 were given below the graph. The numbers 0 to 13 demarcated the initial cell clusters obtained with the default clustering process of Seurat. (C) Violin plots showing expression patterns of the identified marker genes for C1 to C7. The horizontal bars represented median values. (D) UMAP plots showing expression patterns of the top 10 genes that were overexpressed in GDF15 high macrophages (C5) as compared to GDF15 low cells. (E) UMAP plots showing expression patterns of the top 9 genes encoding secreted proteins which were overexpressed in GDF15 high macrophages as compared to GDF15 low cells. (F) Cell-cell communication network map created using CellChat showing the possible effector cells of the GDF15 high macrophage. (G, H) Predicted ligand-receptor pairs potentially involved in the signaling of reciprocal communications between GDF15 high macrophage and other cell types as listed in (F) .

Article Snippet: Recombinant GDF15 (#10596-GD) and recombinant interferon (IFN)-γ (#585-IF) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Derivative Assay, Expressing, Marker

Journal: Frontiers in Immunology

Article Title: Identification of a distinct cluster of GDF15 high macrophages induced by in vitro differentiation exhibiting anti-inflammatory activities

doi: 10.3389/fimmu.2024.1309739

Figure Lengend Snippet: GDF15 high macrophages exhibited reduced inflammatory activation in vitro . (A) Expression patterns of potential substitute cell surface markers for GDF15 based on the scRNA-seq data. (B) Flow cytometry results showing that rat BMMNC-derived macrophages contained a minor fraction of TNFSF9 high cells, whose expression level was correlated with that of GDF15 (from 3 independent experiments). (C) Flow cytometry verification of the correlation between TNFSF9 and GDF15 expressions in human PBMNC-derived macrophages (from 2 independent experiments). (D) Real-time PCR results showing that GDF15 high macrophages (H) exhibited reduced expressions of TNF-α, IL-1β and IL-6 in response to LPS stimulation (1 μg/mL for 6 hr), as compared to GDF15 low cells (L). Rat BMMNC-derived macrophages were FACS purified using TNFSF9 as a substitute marker for GDF15, and primed with IFN-γ (10 ng/mL for 12 hr). (E) Boyden chamber cell migration assay showing that GDF15 high macrophages (H) exhibited reduced migratory activity as compared to GDF15 low cells (L) in the absence and presence of LPS stimulation. (F) Representative fluorescent microscopic images and quantitative data showing that GDF15 high macrophages exhibited reduced phagocytic activity in the presence of LPS stimulation as compared to GDF15 low cells. Phagocytosis was assessed by internalization of fluorochrome-labeled latex beads (orange color). The macrophages were from CD68pro-GFP rats. Data were mean ± SEM. * P < 0.05, one-way ANOVA. NS, no significance.

Article Snippet: Recombinant GDF15 (#10596-GD) and recombinant interferon (IFN)-γ (#585-IF) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, In Vitro, Expressing, Flow Cytometry, Derivative Assay, Real-time Polymerase Chain Reaction, Purification, Marker, Cell Migration Assay, Activity Assay, Labeling

Journal: Frontiers in Immunology

Article Title: Identification of a distinct cluster of GDF15 high macrophages induced by in vitro differentiation exhibiting anti-inflammatory activities

doi: 10.3389/fimmu.2024.1309739

Figure Lengend Snippet: GDF15 high macrophages exerted anti-inflammatory effects via paracrine mechanisms. (A-C) RAW264.7 cells co-cultured with rat BMMNC-derived GDF15 high (H) or GDF15 low (L) macrophages were left untreated or stimulated with LPS for 4 hr. Results for the expression of pro-inflammatory cytokines ( A , real-time PCR), cell migratory activity (B) , and phagocytic activity (C) were shown. (D-F) Unsorted rat BMMNC-derived macrophages co-cultured with rat GDF15 high (H) or GDF15 low (L) macrophages were left untreated or stimulated with LPS for 4 hr. Results for the expression of pro-inflammatory cytokines ( D , real-time PCR), cell migratory activity (E) , and phagocytic activity (F) were shown. Data were mean ± SEM. * P < 0.05, one-way ANOVA. NS, no significance.

Article Snippet: Recombinant GDF15 (#10596-GD) and recombinant interferon (IFN)-γ (#585-IF) were purchased from R&D Systems (Minneapolis, MN, USA).

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

Journal: Frontiers in Immunology

Article Title: Identification of a distinct cluster of GDF15 high macrophages induced by in vitro differentiation exhibiting anti-inflammatory activities

doi: 10.3389/fimmu.2024.1309739

Figure Lengend Snippet: GDF15 might be a macrophage-derived anti-inflammatory factor. (A) Real-time PCR results showing that treatment with exogenous GDF15 (20 ng/mL) inhibited LPS-induced expression of pro-inflammatory cytokines in RAW264.7 cells. (B) Flow cytometry results showing that GDF15 treatment had no effects on phagocytosis in RAW264.7 cells without or with LPS stimulation. (C) Representative images and quantitative data of Boyden chamber assay showing that exogenous GDF15 inhibited migration of LPS-challenged RAW264.7 cells. Cells on the membrane were stained with Giemsa. (D) Effects of conditioned medium from GDF15 high macrophages (H), as compared to the medium from GDF15 low cells (L), on the expression of pro-inflammatory cytokines in RAW264.7 cells. All experiments were performed in the presence of LPS stimulation. α-GDF15, GDF15neutralizing antibody; IgG, non-specific immunoglobulin control. (E) The same experiments as those in D carried out in rat BMMNC-derived macrophages. Data were mean ± SEM. * P < 0.05, one-way ANOVA; † P < 0.05, unpaired t -test. NS, no significance.

Article Snippet: Recombinant GDF15 (#10596-GD) and recombinant interferon (IFN)-γ (#585-IF) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Derivative Assay, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Boyden Chamber Assay, Migration, Membrane, Staining, Control

Journal: Frontiers in Immunology

Article Title: Identification of a distinct cluster of GDF15 high macrophages induced by in vitro differentiation exhibiting anti-inflammatory activities

doi: 10.3389/fimmu.2024.1309739

Figure Lengend Snippet: Detection of GDF15 high macrophages in various human tissues. GDF15 high macrophages (arrowheads) were identified using immunofluorescence double labeling with anti-CD68 (green color) and anti-GDF15 (red color) antibodies in (A) colon tissues from both healthy subjects and patients with ulcerative colitis, (B) kidneys (the normal peri-tumor tissue) (tested in one sample only) and (C) atherosclerotic plaques in the carotid artery (representative data from 6 independent samples showing similar results). The nuclei were counterstained with DAPI (blue). Data were mean ± SEM. NS, no significance (unpaired t -test).

Article Snippet: Recombinant GDF15 (#10596-GD) and recombinant interferon (IFN)-γ (#585-IF) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Immunofluorescence, Labeling

Journal: Frontiers in Immunology

Article Title: Identification of a distinct cluster of GDF15 high macrophages induced by in vitro differentiation exhibiting anti-inflammatory activities

doi: 10.3389/fimmu.2024.1309739

Figure Lengend Snippet: Results of scRNA-seq analysis showing the existence of GDF15 high clusters in rat lung macrophages. (A) Top, UMAP plots showing scRNA-seq clustering of freshly isolated lung alveolar macrophages and interstitial macrophages from rats (3 independent samples from Control group and 3 from experimental PAH group). Total 18977 alveolar macrophage cells (11842 from Control and 7135 from PAH) and 15357 interstitial macrophage cells (6768 from Control and 8589 from PAH) were analyzed. Bottom, patterns of GDF15 expression showing that clusters 2/4 in alveolar and clusters 1/3 in interstitial macrophages were GDF15 high . (B) Comparison of the percentage prevalence of GDF15 high alveolar and interstitial macrophages between control and PAH animals. (C) Venn diagrams showing the numbers of differentially expressed genes between GDF15 high versus GDF15 low macrophages from rat lungs (Control group only) and from human PBMNCs.

Article Snippet: Recombinant GDF15 (#10596-GD) and recombinant interferon (IFN)-γ (#585-IF) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Isolation, Control, Expressing, Comparison