DOI: 10.5061/dryad.d2547d86d
title: Data from: Down-regulated GAS6 impairs synovial macrophage efferocytosis and promotes obesity-associated osteoarthritis

description: Obesity has always been considered a significant risk factor in OA
 progression, but the underlying mechanism of obesity-related inflammation
 in OA synovitis remains unclear. The present study found that synovial
 macrophages infiltrated and polarized in the obesity microenvironment and
 identified the essential role of M1 macrophages in impaired macrophage
 efferocytosis using pathology analysis of obesity-associated OA. The
 present study revealed that obese OA patients and Apoe-/- mice showed a
 more pronounced synovitis and enhanced macrophage infiltration in synovial
 tissue, accompanied by dominant M1 macrophage polarization. Obese OA mice
 had more severe cartilage destruction and increased levels of synovial
 apoptotic cells than OA mice in the control group. Enhanced M1-polarized
 macrophages in obese synovium decreased growth arrest-specific 6 (GAS6)
 secretion, resulting in impaired macrophage efferocytosis in synovial
 apoptotic cells. Intracellular contents released by accumulated apoptotic
 cells further triggered an immune response and lead to a release of
 inflammatory factors, such as TNF-α, IL-1β, and IL-6, which induce
 chondrocyte homeostasis dysfunction in obese OA patients. Intra-articular
 injection of GAS6 restored the phagocytic capacity of macrophages, reduced
 the accumulation of local apoptotic cells, and decreased the levels of
 TUNEL- and caspase-3-positive cells, preserving cartilage thickness and
 preventing the progression of obesity-associated OA. Therefore, targeting
 macrophage-associated efferocytosis or intra-articular injection of GAS6
 is a potential therapeutic strategy for obesity-associated OA.
description: # Data from: Down-regulated GAS6 impairs synovial macrophage efferocytosis
 and promotes obesity-associated osteoarthritis ## General information
 **Dataset overview** Data name: eLife_Primary_data.zip A detailed
 description of the experimental design and specific methodology can be
 found in the relevant publication (see below). This dataset contains the
 underlying quantitative data and original images used to generate the
 figures and statistical analyses presented in the study. The study
 investigates the mechanism of obesity-related inflammation in
 osteoarthritis (OA) synovitis, specifically focusing on the role of GAS6
 in macrophage efferocytosis. Sequencing data have been deposited in GEO
 under accession code GSE53986. The data were derived from three main
 experimental approaches: 1. **Human Clinical Samples:** Synovial tissue,
 fluid, and cartilage obtained from patients with/without obesity
 undergoing arthroscopic treatment or total knee arthroplasty. 2. **Animal
 Models:** Destabilization of the medial meniscus (DMM) induced OA models
 in C57BL/6 (standard diet) and Apoe-deficient (Apoe-/-) mice (high-fat
 diet), including intra-articular injection treatments with recombinant
 mouse GAS6 or R428. 3. **In Vitro Experiments:** Efferocytosis assays,
 apoptosis induction, and molecular biology assays (RT-qPCR, ELISA, IF)
 using RAW264.7 cells, BMDM cells, and primary chondrocytes. Data
 collection and processing involved: * **Histological Analysis:** OARSI
 grading and synovitis scoring were performed on stained tissue sections. *
 **Image Analysis:** Immunofluorescence (IF) and TUNEL assay images were
 captured using an Olympus BX43 microscope and analyzed using **ImageJ**
 software. * **Flow Cytometry:** Macrophage phagocytic capacity was
 quantified via flow cytometry. * **Statistical Analysis:** All raw data
 were processed and analyzed using **GraphPad Prism** software. ## Notes
 for files #### Directory structure and file naming The dataset is
 organized into 5 directories, each corresponding to a main figure in the
 publication (e.g., `Figure_1`, `Figure_2`). Within each directory, files
 are categorized into: 1. **Statistical Data:** An Excel file named `Figure
 X-Statistical Data.xlsx` containing the source data for graphs and
 statistical analyses. 2. **Original Images:** Raw microscopy images
 (histology, immunofluorescence) or blots used to generate the figure
 panels. Image files are named to reflect the experimental condition (e.g.,
 `HE_Normal individuals.png`). #### Data formats **1. Statistical Data
 (`.xlsx`)** Each Excel file contains multiple sheets (tabs), where each
 sheet corresponds to a specific sub-panel in the figure. * **Sheet
 Names:** Follow the pattern `Figure [X][Panel] [Variable Name]`. *
 *Example:* `Figure 1C Synovitis Score`, `Figure 1D F4-80 positive cells`.
 * **Data Organization:** * Rows represent individual samples/animals. *
 Columns represent experimental groups (e.g., "Normal",
 "Obese", "Control", "DMM"). * Cells contain
 quantitative values (e.g., OARSI scores, cell counts per field,
 fluorescence intensity, relative mRNA expression). * **Missing Data:**
 Missing values are recorded as empty cells or `N/A`. **2. Image Files
 (`.tif`,** **`.jpg`, etc.)** * **Microscopy images:** Provide raw or
 minimally processed images for histology (Safranin O-fast green, H&E)
 and Immunofluorescence (IF). * **Scale bars:** Scale information is either
 embedded in the image metadata or detailed in the figure legends of the
 related publication. #### Abbreviations used * **DMM:** Destabilization of
 the medial meniscus * **GAS6:** Growth arrest-specific 6 * **rmGAS6:**
 Recombinant mouse GAS6 * **OA:** Osteoarthritis * **IF:**
 Immunofluorescence * **IHC:** Immunohistochemistry * **ACs:** Apoptotic
 cells * **M1:** Pro-inflammatory macrophages * **HE:** Hematoxylin and
 Eosin staining * **SFO:** Safranin O-fast green staining * **Col2:**
 Collagen Type II * **MMP13:** Matrix Metallopeptidase 13 * **ACN:**
 Aggrecan * **C57:** C57BL/6 ``` Dataset_Root ├── Figure_1 ├── Figure_2 ├──
 Figure_3 ├── Figure_4 └── Figure_5 ``` ##### Sub-directories **Figure_1**
 ``` Figure_1/ ├── A ├── B\ ├── Figure 1-figure supplement 1.csv\ └──
 Figure 1-Statistical data.xlsx ``` Contains underlying data for **Figure
 1: Synovial hyperplasia and macrophage polarization in obese OA
 patients**. **Sub-directories: A and B** Contains the original microscopy
 images corresponding to **Figure 1A** and **Figure 1B**. #### Figure 1A
 Images (Histology) * **Content:** "SFO" panels show human
 articular cartilage stained with **Safranin O and Fast Green**;
 "HE" panels show synovial tissue stained with **Hematoxylin and
 Eosin (H&E)**. * **Experimental Groups:** Normal individuals, OA
 patients without obesity, Obese individuals, and OA patients with obesity.
 * **File Naming:** Files are named to reflect the staining and
 experimental group, e.g., `HE_Normal individuals`, `SFO_Normal OA
 patients`. * **Scale bars:** 200 µm (Cartilage), 50 µm (Synovium). ####
 Figure 1B Images (Immunofluorescence) * **Content:** Immunofluorescence
 staining of synovial tissues to identify macrophage polarization. *
 **Markers & Channels:** * **F4/80 (Green):** Total macrophages *
 **iNOS (Red):** M1 macrophage marker * **CD206:** M2 macrophage marker *
 **File Naming:** Files are named by marker and experimental group, e.g.,
 `F480_Normal OA patients`, `INOS_Normal OA patients`, `CD206_Normal
 individuals`. * **Scale bar:** 50 µm. #### Figure 1-figure supplement 1
 Contains data for **Figure 1—figure supplement 1: Blood lipids in
 obesity-related patients and general patients**. * **Data Structure:** *
 **Columns:** Experimental groups (Normal individuals, OA patients without
 obesity, Obese individuals, OA patients with obesity). * **Rows:**
 Clinical parameters including Total cholesterol (TC), Triglyceride (TG),
 and BMI index. #### Figure 1-Statistical Data.xlsx Contains the raw
 quantitative data used for the statistical graphs in **Figure 1C** and
 **Figure 1D**. * **Sheet:** **`Figure 1C Synovitis Score`** *
 **Description:** Quantification of synovitis severity based on
 histological scoring (Krenn score).\ Corresponds to **Panel C**. *
 **Sheet:** **`Figure 1D F4-80 positive cells`** * **Description:**
 Quantification of F4/80 positive macrophages as a proportion of the total
 lining cell population.\ Corresponds to **Panel D**. * **Sheet:**
 **`Figure 1D INOS positive cells`** * **Description:** Quantification of
 iNOS positive (M1) macrophages.\ Corresponds to **Panel D**. * **Sheet:**
 **`Figure 1D CD206 positive cells`** * **Description:** Quantification of
 CD206 positive (M2) macrophages.\ Corresponds to **Panel D**. **Figure_2**
 ``` Figure_2/ ├── A ├── D\ ├── F\ ├── Figure 2-figure supplement 1 ├──
 Figure 2-figure supplement 2 ├── Table │ ├── Table2.csv │ ├── Table3.csv │
 └── Table4.csv └── Figure 2-Statistical data.xlsx ``` Contains underlying
 data for **Figure 2: Cartilage loss, synovial hyperplasia, and macrophage
 polarization in Apoe−/− OA**. * **Sub-directories:** A, D, F, Figure2
 figure supplement1, Figure2 figure supplement2 * Contains the original
 microscopy images corresponding to the panels in **Figure 2** and its
 figure supplements. * **Naming Convention:**\ File names denote the tissue
 type (**C = Cartilage; S = Synovium**), staining method, and experimental
 group. * **File Naming:**\ Files are named by marker and experimental
 group, or named to reflect the staining and experimental group, e.g.,\
 `CD206_Ctrl_Apoe`, `INOS_Ctrl_Apoe`, `C_HE_OA_Apoe`. #### Figure 2A, 2D,
 2F Images * **Histology (Panel A):**\ **Safranin O and Fast Green**
 staining (Cartilage, Scale bar: 200 µm) and **H&E** staining
 (Synovium, Scale bar: 50 µm) of controls and DMM mice. * **IHC (Panel
 D):**\ Immunohistochemical staining for **Aggrecan** (Cartilage) and
 **MMP-13** (Cartilage and Synovium).\ Scale bar: 50 µm. * **IF (Panel
 F):**\ Immunofluorescence for **iNOS** (M1 marker) and **CD206** (M2
 marker) in synovial tissues.\ Scale bar: 50 µm. #### Figure 2–figure
 supplement 1 Images (4 weeks post-surgery) * **Content:**\ H&E
 staining of cartilage and synovial tissue in controls and DMM mice at 4
 weeks post-surgery. * **Location:**\ Directory `Figure2 figure
 supplement1`. #### Figure 2–figure supplement 2 Images (4 weeks
 post-surgery) * **Content:**\ Immunofluorescence of **iNOS** and **CD206**
 in synovial tissues at 4 weeks post-surgery. * **Location:**\ Directory
 `Figure2 figure supplement2`. #### Sub-directory: Table Contains source
 tables describing the animal model specifications. * **Table 2:**\
 Comparison of specifications and energy of ordinary feed and high-fat
 feed. * **Table 3:**\ Lipid status of **APOE−/−** obese mice and
 **C57BL/6** mice. * **Table 4:**\ Weight gain after feeding for 8 weeks.
 #### Figure 2 Statistical Data.xlsx Contains the raw quantitative data
 used for the statistical graphs in **Figure 2** and its supplements. *
 **Main Figure 2 Sheets:** * **`Figure 2B OARSI score`:**\ Quantitative
 analysis of cartilage destruction based on the OARSI scale. * **`Figure 2C
 Krenn synovitis score`:**\ Synovitis scores for joints corresponding to
 Panel A. * **`Figure2E Cartilage_posivitve`** **&**
 **`Figure2E-Synovium_posotive`:**\ Quantification of **MMP-13** positive
 cells in cartilage and synovium. * **`Figure 2G inos-positive`** **&**
 **`Figure2G CD206-positive`:**\ Quantification of M1 and M2 macrophage
 proportions in the synovial lining cell population. * **Supplementary
 Figure Sheets (4 weeks post-surgery):** * **`Figure2 FigureS1B-OARSI`:**\
 OARSI scores at 4 weeks. * **`Figure2 FigureS1B-Krenns score`:**\
 Synovitis scores at 4 weeks. * **`Figure2 FigureS2-INOS positive`:**\ iNOS
 positive cell quantification at 4 weeks. * **`Figure2 FigureS2-CD206
 positive`:**\ CD206 positive cell quantification at 4 weeks. **Figure_3**
 ``` Figure_3/ ├── A ├── C\ ├── F\ ├── Figure 3- figure supplement 2
 primary blots ├── Figure 3- figure supplement 3 ├── Figure 3- figure
 supplement 1.tif ├── Figure 3- figure supplement 1-souce data 1.csv └──
 Figure 3-Statistical data.xlsx ``` Contains underlying data for **Figure
 3: Loss of GAS6 expression in synovium of obese OA patients and Apoe−/− OA
 mice**, along with supplementary data regarding macrophage polarization
 and **AXL receptor expression**. * **Sub-directories:** A, C, F, Figure 3-
 figure supplement 3 * Contains the original immunofluorescence (IF) images
 corresponding to the panels in **Figure 3** and **Figure 3—figure
 supplement 3**. * **File Naming:**\ Files are named by marker and
 experimental group, or named to reflect the staining and experimental
 group, e.g.,\ `GAS6_Normal individuals`, `GAS6_OA_Apoe`, `LPS-24h`. ####
 Figure 3A, 3C, 3F Images (GAS6 Expression) * **Content:**\
 Immunofluorescence staining to visualize **GAS6 expression in
 macrophages**. * **Samples:** * **Human (Panel A):** Normal, Non-obese OA,
 Obese, Obese OA * **Mouse (Panel C):** C57BL/6 and Apoe−/− mice (Control
 vs. DMM) * **Cells (Panel F):** RAW264.7 cells treated with LPS (24/48 hr)
 * **Scale bar:**\ 50 µm #### Figure 3—figure supplement 3 Images (AXL
 Expression) * **Content:**\ Immunofluorescence staining for the GAS6
 receptor **AXL**. * **Samples:** * **Human (Panel A):** Normal, Non-obese
 OA, Obese, Obese OA * **Mouse (Panel C):** C57BL/6 and Apoe−/− mice
 (Control vs. DMM) #### Sub-directory: Figure 3- figure supplement 2
 primary blots Contains raw, uncropped Western Blot images corresponding to
 **Figure 3—figure supplement 2B**. * **Target Proteins:** * GAS6 * iNOS
 (file labeled as NOS2): M1 macrophage marker * CD86: M1 macrophage marker
 * GAPDH: Loading control * **Experimental Conditions:**\ RAW264.7 cells
 after LPS or IL-4 stimulation. #### Figure 3 Statistical Data.xlsx
 Contains the raw quantitative data used for the statistical graphs. *
 **Main Figure 3 Sheets:** * **`Figure3B HomoF480-GAS6-positive`:**\
 Quantification of F4/80-GAS6+ macrophages in human synovium (n = 6). *
 **`Figure3D miceF480-GAS6-positive`:**\ Quantification of F4/80-GAS6+
 macrophages in mouse synovium (n = 6). * **`Figure3E GAS6
 consentration`:**\ ELISA results for GAS6 in synovial fluid of non-obese
 and obese OA patients (n = 13 per group). * **`Figure3G F480-GAS6
 positive`:**\ Quantification of GAS6+ RAW264.7 cells after LPS treatment
 (n = 6). * **Supplementary Figure Sheets:** * **`Figure3 FigureS2A gas6
 mRNA`:**\ Relative mRNA expression of GAS6 in LPS-treated cells (n = 3). *
 **`Figure3 FigureS2C mRNA level`:**\ Relative mRNA expression of
 inflammatory cytokines (IL-1β, IL-6, TNF-α) under LPS/rmGAS6/R428
 treatments (n = 6). * **`Figure3 FigureS3-homo f480 axl`:**\
 Quantification of f480-AXL+ macrophages in human synovium (n = 6). *
 **`Figure3 FigureS2-Mus f480-axl`:**\ Quantification of f480-AXL+
 macrophages in mouse synovium (n = 6). * **Note:**\ Data related to
 **Figure 3—figure supplement 1 (GSE53986 analysis)** is derived from the
 public dataset **GSE53986**. **Figure_4** ``` Figure_4/ ├── A ├── C\ ├──
 E\ ├── H\ ├── Figure 4- figure supplement 1 ├── Figure 4- figure
 supplement 2 ├── Figure 4-figure supplement 3 primary blots └── Figure
 4-Statistical data.xlsx ``` Contains underlying data for **Figure 4:
 Accumulation of apoptotic cells in OA and impaired phagocytic ability of
 M1-polarized macrophages**, along with mechanistic studies on **MerTK
 expression** and rescue experiments. * **Sub-directories:** A, C, E, H,
 Figure 4-figure supplement 1, Figure 4-figure supplement 2 * Contains the
 original immunofluorescence (IF) images and flow cytometry plots. * **File
 Naming:**\ Files are named by marker and experimental group, or named to
 reflect the staining and experimental group, e.g.,\ `Caspase3_Normal
 individuals`, `Caspase3_Ctrl_C57`, `LPS+rmGAS6+R428`. #### In Vivo
 Apoptosis & Receptors (Panels A, C & Figure 4- figure supplement
 1) * **Apoptosis Markers:**\ **Caspase-3** and **TUNEL** staining in
 synovial tissues. * **Receptor Staining:**\ Double staining for **F4/80
 (Red)** and **MER (Green)** to evaluate **MerTK expression** on
 macrophages. * **Samples:** * **Human synovial tissue:** Normal
 individuals, Obese individuals, Normal OA patients, Obese OA patients *
 **Mouse synovial tissue:** Ctrl_C57, Ctrl_Apoe, OA_C57, OA_Apoe, * **File
 Examples:**\ `Caspase3_Normal individuals`, `TUNEL_OA_Apoe`,
 `MER_Ctrl_Apoe` * **Scale bar:**\ 50 µm #### In Vitro Efferocytosis Assays
 (Panels E, H & Figure 4- figure supplement 2) * **Methodology:**\
 Macrophage phagocytosis assay. * **Staining:**\ **F4/80 (Red):**
 Macrophages\ **CFSE (Green):** Apoptotic Thymocytes. Co-localization
 indicates successful engulfment. * **Cells:** * **BMDMs** (Bone
 Marrow-Derived Macrophages) and **RAW264.7 cells** * **Treatments:**\
 Files labeled with conditions like `LPS+rmGAS6`, `LPS+rmGAS6+R428`. *
 **Flow Cytometry:**\ Includes fluorescence-intensity distribution plots.
 #### Sub-directory: Figure 4-figure supplement 3 primary blots Contains
 raw, uncropped Western Blot images corresponding to **Figure 4—figure
 supplement 3**. * **Target Proteins:** * **CD86 & iNOS:** M1
 macrophage markers * **GAPDH:** Loading control * **Condition:**\ RAW264.7
 cells after **R428** (Axl inhibitor) stimulation. #### Figure
 4-Statistical Data.xlsx Contains the raw quantitative data used for the
 statistical graphs. * **In Vivo Quantification (Apoptosis):** *
 **`Figure4B-Caspase3 positive`** **/** **`Figure4B-TUNEL positive`:**\
 Quantification in human synovium (n = 6). * **`Figure4D-Caspase3
 positive`** **/** **`Figure4D-TUNEL positive`:**\ Quantification in mouse
 synovium (n = 6). * **In Vitro Quantification (Efferocytosis &
 mRNA):** * **`Figure 4F effero index`:**\ Phagocytic ability of BMDMs from
 **Apoe−/−** vs **C57BL/6** mice (n = 5). * **`Figure 4G mRNA`:**\ Relative
 expression of **iNOS** and **Arg1** after **LPS/rmGAS6/IL-4** stimulation.
 * **`Figure 4J effero index`:**\ Phagocytosis index in RAW264.7 cells
 quantified by microscopy (n = 6). * **`Figure 4K effero index`:**\
 Phagocytosis index quantified by Flow Cytometry (n = 5). * **`Figure4
 FigureS2B effero index`:**\ Rescue effect of **rmGAS6** on BMDM
 efferocytosis (n = 5). * **Receptor Expression (Supplement 1):** *
 **`Figure4 FigureS1B homo f480 mer`:**\ Quantification of **F4/80-MER+**
 cells in human synovium (n = 6). * **`Figure4 FigureS1D mice f480 mer`:**\
 Quantification of **F4/80-MER+** cells in mouse synovium (n = 6).
 **Figure_5** ``` Figure_5/ ├── A ├── C\ ├── Figure 5-Figure supplement 1
 primary blots ├── Figure 5-Figure supplement 2.png ├── Figure 5-Figure
 supplement 3 primary blots └── Figure 5-Statistical data.xlsx ``` Contains
 underlying data for **Figure 5: GAS6 restored osteoarthritis (OA)
 cartilage loss and decreased apoptotic cell accumulation**, focusing on
 the therapeutic effects of **rmGAS6** and the underlying molecular
 mechanisms in chondrocytes. * **Sub-directories:** A, C * Contains
 original microscopy images evaluating the therapeutic outcome of
 intra-articular treatments at 8 weeks post-surgery. #### Histology (Panel
 A) * **Knee Cartilage:**\ Safranin O and Fast Green staining (Scale bar:
 200 µm). * **Synovial Tissue:**\ H&E staining (Scale bar: 50 µm). *
 **Treatment Groups:**\ DMM mice, DMM + R428 (inhibitor), and Apoe−/− +
 rmGAS6 (recombinant protein). * **Naming Examples:**\ `SFO_C57_OA_R428`,
 `HE_Apoe_OA_rmGAS6`. #### Apoptosis (Panel C) * **Content:**\
 Immunofluorescence staining for **Caspase-3** and **TUNEL** in synovial
 tissues. * **Scale bar:**\ 50 µm. * **Naming Examples:**\
 `Caspase3_Apoe_OA_rmGAS6`, `TUNEL_OA_R428`. #### Figure 5-Figure
 supplement 2 * **Content:**\ Images of human tibial plateau cartilage
 explants stained with Safranin O. * **Groups:**\ Control (Con) vs.
 Recombinant human GAS6 (rhGAS6). #### Sub-directories: Figure 5-Figure
 supplement 1 & 3 primary blots Contains raw, uncropped Western Blot
 images reflecting cartilage health and cellular senescence. * **Biological
 Context:**\ Primary chondrocytes treated with BMDM-derived supernatant
 (co-cultured with apoptotic cells) or direct rhGAS6 treatment. * **Target
 Proteins:** * **Matrix Markers:** COL2, MMP13 * **Senescence Markers:**
 p16, p21 * **Loading Control:** GAPDH #### Figure 5 Statistical Data.xlsx
 Contains the raw quantitative data for therapeutic evaluation and
 molecular assays. * **In Vivo Evaluation (Panels B & D):** * **`Figure
 5B-OARSI score`:**\ Quantitative analysis of cartilage protection. *
 **`Figure 5B-Krenns score`:**\ Quantitative analysis of synovitis
 reduction. * **`Figure 5D-Caspase3 positive`:**\ Quantification of
 apoptosis in the synovial lining. * **`Figure 5D-TUNEL positive`:**\
 Quantification of DNA fragmentation in the synovial lining. ## Human
 subjects data We confirm that written informed consent was obtained from
 all human participants involved in this study. The consent process
 explicitly included permission for the collection of biological samples
 and the publication of resulting de-identified data in the public domain.
 De-identification Method:To protect participant privacy, all direct
 personal identifiers (including names, medical record numbers, dates of
 birth, and contact information) have been removed from the dataset.
 1）Coding: Samples were assigned unique, non-identifiable alphanumeric
 codes (e.g., “HE_Normal_OA_patients”, "SFO_Obese individuals")
 or grouped by clinical phenotype (e.g., "Obese OA"). The key
 linking these codes to original patient identities is stored separately
 and securely by the investigators and is not included in this submission.
 2) Imaging Data: The images provided are high-magnification microscopic
 histology and immunofluorescence fields. They do not contain any facial
 features or other biometric markers that could be used to re-identify
 individuals. 3）Quantitative Data: Clinical data (e.g., age, BMI) presented
 in the dataset has been aggregated or de-identified to prevent deduction
 of participant identities.