Human samples
Synovial tissues and peripheral blood from RA patients and acute trauma patients were obtained with fully informed consent. Inclusion and exclusion criteria for rheumatoid arthritis were performed according to the American Rheumatism Association rheumatoid arthritis classification criteria [10]. Acute trauma patients should have no history of arthritis. The rheumatoid factor (RF), antistreptolysin (ASO) and DAS28 scores of RA patients were derived from clinical sources. Participants' age, body mass index (BMI), alcohol consumption, smoking status, presence of common chronic conditions (e.g., hypertension, diabetes, and osteoarthritis), and medication use (e.g., angiotensin receptor blockers, metformin, and NSAIDs), did not differ significantly between the two groups (Supplementary Table 1).
Mice
Five- to 8-week female DBA/1JGpt mice were purchased from GemPharmatech Co., Ltd. (Nanjing, China). Esr2fl/fl mice (control) and Esr2fl/fl/Adgre-Cre mice (F4/80+ cell-specific knockout of Esr2 gene) were purchased from GemPharmatech Co., Ltd. (Nanjing, China). Pstpip2CreR26-ZsGreen reporter mice were purchased from Shanghai Model Organisms Center, Inc. (Shanghai, China).
Induction of collagen-induced arthritis
Collagen-induced arthritis (CIA) was induced and assessed as an experimental procedure [11]. Briefly, the time of the primary immunization was defined as day0, and the secondary immunization was performed on day21. Each mouse was injected with 0.1 ml Freund’s complete adjuvant (containing 4mg/ml BCG and 2mg/ml chicken type II collagen) at multiple points on the root of the tail and back. Arthritis score and weight of mice were measured and recorded from day28 onwards. Arthritis scoring criteria: 0: normal, no joint redness or swelling; 1: mild, slight redness and swelling of the ankle or wrist, or obvious redness and swelling of the fingers; 2: moderate, redness and swelling of the ankle or wrist; 3: severe redness and swelling of the entire paw; and 4: severe redness and swelling including multiple joints. The cumulative values for the three paws were calculated as the total clinical arthritis score for each mouse, with a maximum score of 16 for each mouse. Day 35 is the peak of joint inflammation and day56 is the start of remission.
In vivo treatments and cell culture
Animal experiments were performed in three batches. The first grouping was: Normal control, Pstpip2CreR26-ZsGreen, and Wild. These mice were sacrificed on day 35 or day 70. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital and then dislocated to death. Normal control mice did not receive any treatment and did not induce CIA. Pstpip2CreR26-ZsGreen reporter mice and Wild mice were induced to CIA. The second grouping was: CIA, AAV-empty, and AAV-PSTPIP2. These mice were sacrificed on day 42. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital and then dislocated to death. The knee cavities of mice in the AAV-PSTPIP2 group were injected with adeno-associated virus loaded with a plasmid overexpressing the PSTPIP2 gene on day22. Mice in the AAV-empty group were injected with empty adeno-associated virus and the CIA group were injected with saline. The third grouping was: Wild, Esr2fl/f, and Esr2fl/fl/Adgre-Cre. These mice were sacrificed on day 42. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital and then dislocated to death. Wild mice were induced with CIA but without any treatment. Esr2fl/f mice and Esr2fl/fl/Adgre-Cre mice were induced with CIA and treated with PSTPIP2 by injection of adeno-associated virus-loaded overexpression Pstpip2 gene plasmid into the knee joint cavity on day 22.
Synovial tissues from the second and third batches of mice were isolated and then subjected to cell culture. Briefly, the synovial tissues were cut into tiny tissue pieces and then inoculated into culture flasks. After the cells were plastered, it was continued for 24 h, and then F4/80+ cells were further cultured using flow sorting for staining and cell dynamics analysis.
Culture of bone marrow-derived mononuclear cells (BMDMs): Bone marrow cells were obtained by aseptically isolating the femur and tibia of DBA/1 mice. After overnight culture (> 16 h), the non-adherent cells were discarded, and the obtained adherent monocytes were continued to be cultured in a medium containing macrophage-colony stimulating factor (M-CSF), to differentiate into macrophages (M0). M0 polarized into M1-type macrophages (F4/80+CD86+) upon stimulation by IFN-γ and LPS. Under the stimulation of IL-4, M0 polarized into M2-type macrophages (F4/80+CD206+). AZD9496 (MedChemExpress, Cat. HY-12870) was added to the medium to antagonize estrogen receptor alpha (ERα), Prinaberel (MedChemExpress, Cat. HY-14933) was added to antagonize estrogen receptor beta (ERβ), and Estriol (MedChemExpress, Cat. HY-B0412) was added to antagonize the G protein-coupled estrogen receptor (GPER).
Flow cytometry and flow sorting
Human peripheral blood and synovial tissue or mouse synovial tissues were used to make single cell suspensions. According to the operation manual, antibodies were added to the treated single cell suspension (1×106/100 μL) and incubated for 30 min at 4°C protected from light. Centrifuged at 300g for 5 min, resuspended by adding 0.5ml of PBS and finally detected on the machine. Directly labeled antibodies, Hu CD11b APC (Cat. # 553312), Hu CD86 FITC (Cat. # 560958), and Hu CD206 PE (Cat. # 555954), were purchased from BD Pharmingen (America). Ms F4/80 PE (Cat. # 565410), Ms CD86 FITC (Cat. # 561962), and Ms CD206 Alexa (Cat. # 565250) were purchased from BD Pharmingen (America). Indirectly labeled antibodies, Anti-PSTPIP2 antibody (Cat. # ab155543), Anti-Claudin5 antibody (Cat. # ab131259), and Anti-ZO-1 antibody (Cat. # ab190085), were purchased from abcam (UK). Fluorescent secondary antibody: Goat Anti-Mouse IgG H&L Alexa (Cat. # ab150113) or Goat Anti-Mouse IgG H&L FITC (Cat. # ab6758) were purchased from abcam (UK).
Single-cell suspensions of human peripheral blood and synovial tissues or mouse synovial tissues were used in part for flow sorting. Manual sorting reagents: Anti-F4/80 MicroBeads UltraPure, mouse (Miltenyi, Cat. # 130-110-443); MiniMACS Starting Kit (Miltenyi, Cat. # 130-090-312); MS Columns (Miltenyi, Cat. # 130-042-201).
Histology
Hematoxylin-eosin (H&E) staining. The isolated synovial tissues were immediately placed in liquid nitrogen for storage. Frozen tissues were then used for H&E staining, SafraninO-fast green staining, TRAP staining, immunohistochemical staining, and immunofluorescence staining. The antibody used for immunohistochemistry was anti-PSTPIP2 antibody (Cat. # ab155543). The antibodies used for immunofluorescence were anti-CD11b antibody (Cat. # ab52478), anti-PSTPIP2 antibody (Cat. # ab155543), anti-F4/80 antibody (Cat. # ab300421) purchased from abcam (United Kingdom). Isotype control antibodies (Rabbit IgG, monoclonal isotype control, abcam, Cat. # ab172730) were used to eliminate non-specific binding of antibodies in immunohistochemistry and immunofluorescence.
RT-qPCR
F4/80+ synovial macrophages or CD11b+ peripheral blood mononuclear cells (isolated using flow sorting) were used to extract total RNA. Total RNA was used for reverse transcription, and finally, fluorescence quantification was performed. CT values were obtained for each sample after the onboard operation and repeated three times. GAPDH was used as an internal standard. GAPDH primer information: Forward 5′-AATGGATTTGGACGCATTGGT-3′, Reverse 5′-TTTGCACTGGTACGTGTTGAT-3′. PSTPIP2 primer information: Forward 5′-ACAACGTGGCTCAATGTCACA-3′, Reverse 5′-CCGGCACTTTTGCTCATAATTCT-3′. The CT values obtained were transformed using the method ΔΔCT. Shortly, A = CT(target gene, sample to be tested) – CT(internal standard gene, sample to be tested), B = CT(target gene, control sample) – CT(internal standard gene, control sample), K = A – B, relative expression of target gene = 2-K.
Micro-CT analysis
The knee joints were separated from the hip joint and removed as much muscle tissue as possible [9]. The isolated ankle and knee were fixed with 4% paraformaldehyde for 24 h. Machine parameters (model: Bruker SkyScan 1276, Belgium): voltage 50 kV, current 800 μA, scan resolution 12 μm, and a field of view size 1304×1024. Several consecutive slices with a large cross-sectional area were used for 3D image reconstruction (N-Recon software) and 3D analysis was performed with CT-AN software. The bone volume fraction (BV/TV, %) was used to indicate the degree of bone damage.
Construction of adeno-associated virus-loaded overexpression PSTPIP2 gene plasmid
We commissioned Hanheng Biotechnology Ltd. (Shanghai, China) to construct AAV-PSTPIP2 for in vivo overexpression of the Pstpip2 gene [12, 13]. We finally obtained overexpressed adeno-associated virus (AAV) plasmid (AAV-PSTPIP2) and control (AAV-empty). This project used AAV5, which has a high synovial affinity with infection efficiency of >80% and an RT-qPCR detection level of >3× for PSTPIP2.
Time-lapse video microscopy
Isolated synovial macrophages were plated on MatTek cell culture dishes at low density and cultured for 2 days before the experiment. Cells were then stained using Actin (Mouse Anti-Actin antibody, abcam, Cat. # ab11003) and PSTPIP2 (Rabbit Anti-PSTPIP2 antibody, abcam, Cat. # ab155543), and secondary antibodies were used as Goat Anti-Mouse IgG(H+L) (FITC conjugated) (Elabscience, Cat. # E-AB-1015) and Goat Anti-Rabbit IgG (H+L) (AF647 conjugated) (Elabscience, Cat. # E-AB-1075). And then the plates were placed on the stage of an Olympus inverted microscope equipped with a humidified, 5% CO2-containing, 37°C temperature-controlled chamber. Phase-contrast images of five different fields were acquired at 60-s intervals using a Cooke Sensicam QE cooled CCD camera (Auburn Hills, MI) with Scanalytics IPLab software (Fairfax, VA) running on a PC. Sequences were assembled and quantified using ImageJ 1.8.0 software. Filopodia formation was assessed by visual inspection of the time-lapse movies [14].
Scanning electron microscopy
Isolated synovial macrophages were grown on 22-mm circular glass coverslips in six-well cell culture plates to 60–75% confluence. And then the cells were fixed quickly with osmium tetroxide followed by staining with gold-palladium as described [15]. The presence of filopodia and membrane ruffling was examined using a JEOL JSM6400 scanning electron microscope using an accelerating voltage of 5kV. Cell spreading was estimated by manually tracing the cell perimeter and measuring the cell footprint area. Ruffling was estimated by manually tracing the perimeter of the ruffles and measuring the area. The percent ruffling for each cell was obtained by dividing the ruffling area by the sum of the ruffling area and cell area. The number and length of filamentous protrusions were determined by counting and manual tracing, respectively [14].
Laser confocal microscopy
Laser confocal microscopy is used to photograph cells or tissues with fluorescence. Anti-fluorescent bursting agents are added dropwise to the prepared cell crawl or tissue section and photographed on the machine [16]. Olympus laser confocal microscope (FV3000) was used in this study.
Statistical analyses
All data are expressed as mean ± SD. Unpaired t-test was used for two groups, and one-way ANOVA test was used for three groups or more. All data met the assumption of statistical tests. The number of mice in each group is illustrated in the figure. The grouping of mice was randomized. Histological scoring was performed in a blinded fashion. Results were considered significant at P < 0.05. Statistical tests were carried out using GraphPad Prism (version 8.01) Software for Windows.