Platelet-derived exosomes in situ reprogramming macrophages for rheumatoid arthritis treatment

Platelet-derived exosomes in situ reprogramming macrophages for rheumatoid arthritis treatment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Platelet-derived exosomes in situ reprogramming macrophages for rheumatoid arthritis treatment Dong Yu, Dan Wang, Yuqiu Yu, Yinjin Xu, Wenting Tang, Yuehua Guo, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7169171/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Oct, 2025 Read the published version in Cell Communication and Signaling → Version 1 posted 9 You are reading this latest preprint version Abstract M1 macrophages secrete various pro-inflammatory cytokines and play a pivotal role in the pathogenesis of rheumatoid arthritis (RA). Therefore, strategies aimed at eliminating synovial M1 macrophages or reprogramming them toward an anti-inflammatory M2 phenotype represent critical approaches for RA treatment. In this study, we propose a novel therapeutic strategy using platelet-derived exosomes (PLT-Exos) to induce the polarization of M1 macrophages into the anti-inflammatory M2 phenotype. Our results demonstrate that PLT-Exos are enriched with immunoregulatory proteins associated with M2 macrophage polarization and can effectively stimulate the conversion of M1 to M2 macrophages. Through phagocytosis assays and in vivo imaging, we confirmed that PLT-Exos are efficiently taken up and specifically accumulate in the joints of collagen-induced arthritis (CIA) mice. Treatment with PLT-Exos significantly reduced joint swelling, arthritis scores and synovial inflammation, while alleviating bone erosion and cartilage damage, leading to marked improvement in motor function in CIA mice. Notably, the therapeutic efficacy of PLT-Exos in RA was comparable to that of the clinical drug methotrexate (MTX), with excellent biocompatibility and no observed cytotoxicity. Overall, the use of PLT-Exos to induce M1-to-M2 macrophage polarization represents a promising therapeutic approach for RA and offers substantial potential for the development of anti-inflammatory treatments for various inflammatory diseases. Platelet-derived exosome Macrophage polarization Rheumatoid arthritis Inflammatory cytokines Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Rheumatoid arthritis (RA) is a chronic, inflammatory systemic autoimmune disease that often causes systemic multi-system damage [ 1 ]. It can affect multiple joints of the entire body, leading to the synovial hyperplasia of the joints, gradually destroyed the cartilage and bones, and joint deformity, eventually leading to disability [ 2 ]. This will seriously affect people’s quality of life and increase people’s economic burden. Current treatments for RA typically fall into three major categories: non-steroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), and glucocorticoids (GCs) [ 3 – 5 ]. These medications are usually administered via oral, subcutaneous, or intramuscular routes. However, systemic delivery through the bloodstream often leads to inadequate drug accumulation at the targeted pathological sites [ 6 ]. Frequent administration of large doses or even combined administration are often required to achieve the desired therapeutic effect. Such high-dose regimens can contribute to serious systemic side effects, including gastrointestinal disturbances, liver and kidney impairment, and increased susceptibility to infections [ 7 – 9 ]. Therefore, there is an urgent need for therapies that can effectively target lesions. The primary pathological change in RA involves immune activation that increases infiltration of various inflammatory cells into synovial tissue, resulting in synovitis and exacerbating joint damage [ 10 , 11 ]. Key components of the inflammatory environment include macrophages [ 12 ]. Macrophages are divided into M1 and M2 macrophages according to their phenotypes and functions [ 13 , 14 ]. M1 macrophages promote inflammation through the release of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-1 (IL-1), thereby advancing RA progression [ 15 ]. In contrast, M2 macrophages are anti-inflammatory and secrete cytokines such as transforming growth factor-beta (TGF-β), interleukin-10 (IL-10) and interleukin-13 (IL-13), which help suppress the production of pro-inflammatory factors and alleviate inflammation [ 16 ]. Therefore, promoting the transition of M1 macrophages to M2 macrophages and maintaining a dynamic balance between these two populations has a positive effect on the treatment of RA. Recent studies have explored methods to achieve this balance by facilitating macrophage phenotype switching to treat RA [ 17 – 20 ]. This approach shows promise as a potentially effective alternative that minimizes harm to the immune system. However, many strategies involving extracellular vesicle (EV) and nanoparticles face challenges such as complex engineering, low yield, and poor biocompatibility, limiting their clinical applicability [ 21 , 22 ]. Therefore, there is a significant need to identify drug formulations that are simpler to produce while maintaining adequate yield and effectiveness. Platelet-rich plasma (PRP) is a high-concentration platelet preparation obtained from blood through centrifugation. Known for its tissue repair potential, PRP has been used in both animal and clinical studies for the treatment of arthritis [ 23 – 25 ]. Although PRP has a wide range of potential therapeutic applications, it is susceptible to various environmental factors such as temperature, vibration, and contamination, which can lead to platelet activation [ 26 ]. This activation may result in excessive coagulation [ 27 ], exacerbating inflammatory responses [ 28 – 30 ] and ultimately impacting the therapeutic efficacy of platelets [ 31 – 33 ]. In addition to growth factors and cytokines, platelets also secrete distinct nano-sized cell-derived membrane vesicles known as extracellular exosomes (Exos), which may be crucial contributors to PRP's efficacy[ 34 , 35 ]. Platelet-derived exosomes (PLT-Exos) are membrane-bound vesicles secreted by platelets and range from 40 to 150 nm in size. Compared to PRP, PLT-Exos are noted for their stability, easy transmission in vivo, minimal immunogenicity, and lower oncogenicity [ 36 , 37 ]. Furthermore, PLT-Exos can easily penetrate blood vessels and accumulate at disease sites [ 38 ], with specific targeting ability to inflammatory sites [ 39 , 40 ]. Unlike other exosomes, PLT-Exos can be produced on a large scale, with CaCl 2 -activated platelets yielding more purified exosomes [ 41 ]. Recent years have seen extensive research into PLT-Exos as a drug delivery platform [ 42 – 44 ], with numerous studies indicating their positive effects on inflammation [ 39 , 45 ]. However, whether PLT-Exos have a good therapeutic effect on RA is currently unknown. In this study, we successfully isolated exosomes from platelets and investigated their anti-inflammatory effects and therapeutic potential in RA through both in vitro and in vivo experiments. Our findings revealed that PLT-Exos facilitate the polarization of macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. Proteomic analysis revealed a high expression of CD163 (M130) in PLT-Exos which may relate to their role in macrophage polarization regulation [ 46 , 47 ]. Additionally, we observed that PLT-Exos accumulated effectively at sites of inflammation within the joints, enhancing the secretion of anti-inflammatory factors while inhibiting the production of pro-inflammatory cytokines. Notably, the therapeutic effects of PLT-Exos were comparable to those of methotrexate (MTX), a conventional treatment for RA, but without the adverse side effects associated with MTX, indicating a favorable biosafety profile. Our research presents a promising strategy for the treatment of inflammatory diseases such as RA. 2. Materials and methods 2.1. PLT preparation and activation Whole blood was collected through the orbit of the mouse and placed in an EDTA centrifuge tube, then gently shake it evenly so that it is fully mixed with anticoagulants to prevent platelet activation. The blood was centrifuged three times respectively. The first time was centrifuged at 200 g for 20 min, so that the blood was divided into four layers, including plasma, platelets, leucocytes and red blood cells (RBCs). The top two layers (plasma and platelets) were carefully extracted and transferred to a new EDTA centrifuge tube. After centrifugation at 200 g for 10 min, the second centrifugation was performed to remove the residual RBCs and purify the platelets. Finally, the platelet solution was obtained by centrifugation at 800 g for 20 min. The platelet concentrates were activated with thrombin (2 U/ml, Solarbio, Beijing, China) and CaCl 2 (10%, Beyotime, Shanghai, China) for 30 min at room temperature to promote platelet activation and aggregation. 2.2. Extraction of PLT-Exos PLT-Exos were extracted and purified from platelet concentrates by gradient centrifugation and ultrafiltration [ 48 ]. The activated platelets were subjected to a series of gradient centrifugation at 4 ℃ (500 g for 5 min, 1000 g for 15 min, 16500 g for 30 min), and cell debris was discarded. The supernatant from the last centrifugation was filtrated by a 0.22 µm filter, and the filtrate was ultracentrifuged at 100,000 g for 70 min at 4°C to obtain PLT-Exos, which was washed with sterile PBS. Then the PLT-Exos was centrifuged at the same high speed for 70 min again. Finally, the extracted PLT-Exos were carefully resuspended in sterile PBS and stored at -80 ℃ for subsequent experiments. 2.3. Characterization of PLT-Exos First, the size and concentration distribution data of PLT-Exos were measured by Nanoparticle tracking analysis (NTA, Particle Metrix, GmbH, Ammersee, Germany), and then the morphology of PLT-Exos was observed and photographed using the transmission electron microscopy (TEM, FEI Co, Hillsboro, OR, USA). Finally, western blotting (WB) was carried out as follows: First, proteins were extracted from the platelet or PLT-Exos with RIPA lysis buffer containing protease inhibitor cocktail. Then BCA assay was used to determine the total protein content, and the protein was denatured by heating at 100 ℃ for 5 min. The protein extract was separated on 10% sodium dodecyl sulphate polyacrylamide electrophoresis gel (SDS-PAGE), transferred to polyvinylidene fluoride membranes, and blocked with milk at room temperature (RT) for 2 h. Afterwards, the membranes were incubated with primary antibodies such as anti-CD 9 (Abcam), anti-CD 63 (Abcam), and anti-CD 81 (Abcam) at 4 ℃ overnight, followed by washing in TBST and incubation with secondary antibodies at RT for 2 h. Finally, the labeled proteins were visualized with the Gel Doc XR system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). 2.4. Proteomic analysis Platelets and PLT-Exos were extracted, followed by the addition of an inhibitor (1% protease inhibitor), sonicated. The samples were then centrifuged at 12,000 g for 10 min at 4°C. The supernatant was transferred to new centrifuge tubes, and the protein concentration was measured using a BCA assay kit. Subsequently, the extracted proteins underwent enzymatic digestion. LC-MS/MS analysis was performed on an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) equipped with a nanoelectrospray ion source (Thermo Fisher Scientific). The DIA data were processed using the DIA-NN search engine (v.1.8). Trypsin/P was designated as the cleavage enzyme, allowing for up to one missed cleavage. N-terminal methionine excision and carbamidomethylation of cysteine were specified as fixed modifications, with a false discovery rate (FDR) set to 1.5 and P < 0.05. Further functional analysis of the differentially expressed proteins was conducted using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. 2.5. Preparation and culture of bone marrow-derived macrophages Bone marrow-derived macrophages (BMDM) were isolated as previously reported [ 49 ]. First, the femur and tibia were isolated from healthy C57BL/6 mice, and the bone marrow were washed with 1 mL syringe with cold Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, NY, USA) into a 100 mm culture dish. The collected solution was passed through a 70-µm cell filter to remove other tissues. After, red blood cell lysis solution (Solarbio, Beijing, China) was added to dissolve red blood cells for 5 min. The cells were cultured in DMEM medium with 10% FBS (Gibco, Beijing, China) containing 10 ng/mL macrophage colony-stimulating factor (Peprotech, NJ, USA) for seven days to obtain M0 macrophages. On the 3th day of culture, half of the medium was replaced, and on the 5th day of culture, the entire medium was changed. On the 7th day of culture, 500 ng/mL of LPS (Peprotech, NJ, USA) for polarization of M1 macrophages were added and differentiated for 2 days. 2.6. Cellular uptake of PLT-Exos BMDM were seeded in a confocal culture dish at a density of 1 × 10 5 cells/well and cultured for 48 h in the presence of LPS (500 ng/mL) and IFN-γ (20 ng/mL). PLT-Exos labeled with DiD (10 µM) (Biolegend, CA, USA) were added at 1 × 10 7 per well and incubated with M1 macrophages for 1, 3, 6, 12 and 24 h, respectively. Cell samples were washed three times with PBS for 5 min and fixed with 4% paraformaldehyde (Beyotime, Shanghai, China) for 20 min, and stained with DAPI for 10 min. Finally, cellular uptake of PLT-Exos was observed by confocal laser scanning microscopy (LSM900, ZEISS, USA) and flow cytometry (LSRFortessa, BD, USA). 2.7. Macrophage phenotype transition experiment in vitro BMDMs were seeded in confocal culture plates at a density of 1 × 10 5 cells per well and incubated in DMEM supplemented with LPS at 500 ng/mL and IFN-γ at 20 ng/mL for 48 h. After incubation, PLT-Exos were added to each well at concentrations of 1 × 10 6 , 5 × 10 6 and 1 × 10 7 exosomes per well, followed by an additional incubation period of 48 h. Subsequently, immunofluorescence experiments were conducted. The cells were fixed with 4% paraformaldehyde, permeabilized for 20 min with a cell permeabilization buffer and blocked for 30 min with a blocking buffer. The cells were then treated overnight at 4°C with anti-CD206 antibody Alexa Fluor 647 (Biolegend) and anti-iNOS antibody Alexa Fluor 488 (Invitrogen, Oregon, USA). Afterward, the cells were stained with DAPI and observed using a confocal laser microscope (LSM900, ZEISS, USA). For flow cytometry analysis, the cells were fixed, permeabilized, and blocked using the same procedures as previously described. The acquired cells were then treated with anti-F4/80 antibodies-PE (Biolegend), anti-CD86 antibodies APC/Cyanine7 (Biolegend), anti-CD206 antibodies Alexa Fluor 647 (Biolegend) at 4°C for 30 min. Following this, the cells were washed with DPBS and analyzed using a flow cytometer. Finally, the results were analyzed using FlowJo software (FlowJo LLC, Ashland, OR, USA). 2.8. Quantitative real-time polymerase chain reaction (qRT-PCR) PLT-Exos were added to cultured M1 macrophages at concentrations of 1 × 10 6 , 5 × 10 6 , and 1 × 10 7 vesicles per well. The cells were co-cultured with the PLT-Exos for 24 h. Following incubation, the cells were collected, and total RNA was extracted using TRIzol Reagent (Vazyme). The quantity and purity of the extracted RNA were assessed using a Nanodrop spectrophotometer (Thermo Scientific, USA). Subsequently, complementary DNA (cDNA) templates were synthesized using the HiScript III RT SuperMix for qPCR kit (Vazyme). qRT-PCR was performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme) on a real-time PCR system (QuantStudio 5, Thermo Fisher, USA). The primers used for qRT-PCR are listed in Table S1 . The expression data were normalized to β-actin levels and assessed using the 2 –ΔΔCT method. 2.9. Enzyme-linked immunosorbent assay (ELISA) After incubating M1 macrophages with different concentrations of PLT-Exos for 24 h, the cell supernatants were collected. The samples were then centrifuged at 3000 rpm for 30 min to remove particles and aggregates. The levels of pro-inflammatory cytokines IL-1β and TNF-α, as well as the anti-inflammatory cytokine IL-10, were measured using ELISA kits (Jiangsu Jingmei Biotechnology Co., China) according to the manufacturer’s protocol. 2.10. Animal model of collagen-induced arthritis Healthy male DBA/1 mice were purchased from the Experimental Animal Center of Nantong University. The mice were housed under standard laboratory conditions with appropriate temperature and humidity, allowing for free access to food and water. All animal experiments were conducted in accordance with the experimental protocol approved by the Animal Ethics Committee of Nantong University (S20241129-001). To establish the collagen-induced arthritis (CIA) model, the method previously reported in the literature was utilized [ 50 ]. Firstly, 2 mg/mL of bovine type II collagen (Chondrex, Redmond, WA, USA) was mixed with an equal volume of complete Freund’s adjuvant (Chondrex, USA) and stirred on ice to dissolve and emulsify the mixture. Then 100 µL of the emulsion was administered via subcutaneous injection at the base of the tail. On day 21 post-initial immunization, a booster immunization was performed by preparing an incomplete Freund’s adjuvant (Chondrex, Redmond, WA) mixture with the type II collagen emulsion (Chondrex, Redmond, WA) and injecting it subcutaneously at the base of the tail of the mice. Starting from day 32 after the establishment of the CIA model, when the arthritis score exceeds 4, indicating well-established RA, CIA mice will be randomly assigned to three groups (n = 5): intravenous injection of physiological saline, PLT-Exos, or MTX. The treatment with PLT-Exos, MTX, or saline will begin at this point, and disease progression parameters will be evaluated. The inflammation score for the hind limbs of the CIA mice will be assessed using the following scale: 0 = No signs of erythema or swelling; 1 = Erythema and mild swelling limited to the tarsal or ankle joint; 2 = Erythema and mild swelling extending from the ankle joint to the tarsus; 3 = Erythema and moderate swelling extending from the ankle joint to the metatarsal; 4 = Erythema and severe swelling involving the ankle, foot, and digits or limb rigidity [ 50 ]. The cumulative scores from both hind paws will yield a maximum possible score of 16 for each mouse. During the treatment period, disease progression in RA will be monitored daily, the arthritis score and the thickness of the hind paw were recorded every 3 days, and the weight was measured once a week. 2.11. Motor function analysis On day 32 after the induction of CIA, CIA mice were randomly divided into three groups (n = 5) to evaluate the grip strength of their forepaws and hind paws during treatment with PLT-Exos, MTX or saline. The grip strength test was conducted using a grip strength meter (Bioseb), which was horizontally positioned. The tail of each mouse was gently lifted and placed on a testing mesh, allowing the mouse to grasp the mesh while keeping its body aligned horizontally. The tail was pulled steadily to record grip strength, with an interval of 3 seconds between each test. Each mouse was tested three times. Three weeks post-treatment, a plastic board was set up as a runway for the mice. Ink was applied to the soles of the mice's feet using a paintbrush, and each mouse was placed at the entrance of the runway, where it left paw prints. For each mouse, three distinguishable pairs of paw prints were randomly selected, and three parameters were measured: print length (PL), defined as the distance from the heel to the tip of the third toe; toe spread (TS), measured as the distance from the first to the fifth toe; and intermediate toe spread (IT), the distance from the second to the fourth toe. The Achilles Functional Index (AFI) value was calculated using the formula [ 51 ]: AFI = 74[(NPL-EPL)/EPL] + 161[(ETS-NTS)/NTS] + 48[EIT-NIT)/ NIT]-5 where N and E represent normal and post-treatment paw measurements, respectively. An AFI value of 0 indicates normal function, while negative values indicate impaired function. 2.12. In vivo biodistribution of the PLT-Exos In this study, CIA mice were subjected to intravenous injections of either free DiR or DiR-loaded PLT-Exos. Following administration, the biodistribution of DiR in the knee and ankle joints of the mice was evaluated at several time points: 1, 3, 6, 12 and 24 h post-injection, using an IVIS Spectrum system (Perkin Elmer, Santa Clara, CA, USA) to quantify the fluorescence intensity. After 24 h of in vivo fluorescence imaging, the mice were euthanized using a suitable humane method. Subsequently, the following organs were harvested: heart, liver, spleen, lungs, kidneys and paws. These tissues were analyzed for fluorescence intensity using an IVIS Spectrum system (Perkin Elmer, Santa Clara, CA, USA) to compare the organ-specific distribution of DiR across the treatment groups. 2.13. Micro‑computed tomography (Micro‑CT) analyses of articular bone On day 32 post-induction of CIA, the CIA mice were randomly divided into three groups (n = 3 each): intravenous injection of saline, PLT-Exos and MTX. Treatments with PLT-Exos, MTX or saline were administered every three days, culminating in a total of eight treatments. Following the final treatment, the mice were humanely euthanized. The hind paws were subsequently harvested and fixed in 4% paraformaldehyde for 24 h. After fixation, the samples were scanned using a Bruker SkyScan 1276 micro-computed tomography (micro-CT) system (Bruker, Belgium). A quantitative analysis of various morphological parameters was performed, including bone mineral density (BMD), bone surface to bone volume ratio (BS/BV), trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th). 2.14. Immunohistochemical and histology analysis After the completion of the treatment regimen, the mice from each group were humanely euthanized, and their knee joint tissues were carefully dissected and fixed in 4% paraformaldehyde for 48 h. Subsequently, the fixed tissues were decalcified by immersion in neutral ethylenediaminetetraacetic acid solution (Biosharp, China) for 20 days. The decalcified tissues were then embedded in paraffin and sectioned into 4 µm thick slices for further experimentation. The joint sections were stained using hematoxylin and eosin (H&E) (Servicebio, China) and safranin-O/fast green staining (Servicebio, China), and the morphological features were observed under an optical microscope. Immunohistochemical analysis was conducted on the sections using primary antibodies anti-TNF-α (Abcam), anti-IL-1β (Abcam) and anti-IL-6 (Abcam). The sections were incubated overnight at 4°C with primary antibodies anti-iNOS, anti-CD206 and anti-F4/80, followed by incubation with fluorescently labeled secondary antibodies for 1 h. DAPI staining was performed to visualize cell nuclei, and images were captured using a fluorescence microscope. Lastly, synovial tissues from the knee joints of the mice in each group were collected, The level of pro-inflammatory cytokines IL-1β and TNF-α, as well as the anti-inflammatory cytokine IL-10 and IL-4, were measured using ELISA kits (Jiangsu Jingmei Biotechnology Co., China) according to the manufacturer’s protocol. 2.15. The regulation of immune cells by PLT-Exos Cells were extracted from the blood and spleens of CIA mice that underwent different drug treatments. Cell suspensions were prepared, and red blood cells were lysed using a lysis buffer, followed by washing with PBS. After fixation and permeabilization, flow cytometry analysis was performed using the following fluorescently labeled mouse antibodies: FITC-CD4 (Biolegend), APC-CD25 (Biolegend), APC-CD3 (Biolegend), BV421-Foxp3 (Biolegend) and PE-IFN-γ (Biolegend) for immunostaining the cells. The stained cells were then analyzed using a flow cytometer. Finally, the results were analyzed using FlowJo software (FlowJo LLC, Ashland, OR, USA). 2.16. Safety evaluation Upon completion of the treatment, the mice were humanely euthanized to collect blood samples from each group for analysis. The proportions of lymphocytes (Lymph), monocytes (Mon), granulocytes (Gran), red blood cells (RBC), platelets (PLT), hemoglobin (HGB), and mean corpuscular hemoglobin concentration (MCHC) and content (MCH) were determined to evaluate the safety of PLT-Exos, MTX, and saline. Additionally, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using a biochemical assay kit (Rayto) to assess the hepatic toxicity of the treatments. Renal toxicity was evaluated by measuring creatinine (CREA) and blood urea nitrogen (BUN) levels. Finally, heart, liver, spleen, lung and kidney tissues were harvested, sectioned, and subjected to hematoxylin and eosin (H&E) staining to observe any morphological alterations in response to the treatments. 2.17. Statistical analysis Experimental data were analyzed using GraphPad Prism 9.0 (GraphPad Software, La Jolla, CA, USA). All data are presented as mean ± standard deviation (SD). Differences between two groups were assessed using Student's t-test, while differences among three or more groups were evaluated using one-way analysis of variance (ANOVA) followed by Tukey's post hoc tests. A p-value of < 0.05 was considered statistically significant. Statistical significance is indicated as * P < 0.05, ** P < 0.01, *** P < 0.005, and **** P < 0.001. 3. Results and discussion 3.1. Characterization of PLT-Exos The process for PLT-Exos extraction was shown in Fig. 2 a. To characterize the extracted PLT-Exos, we utilized TEM, NTA and WB. TEM images revealed spherical particles of varying sizes, approximately 100 nm in diameter (Fig. 2 b), which corresponded with the size distribution observed through NTA (Fig. 2 c). WB results showed positive expression of the platelet marker protein CD41 in both the extracted platelets and PLT-Exos. Additionally, positive signals for surface markers CD9, CD63 and CD81 in the extracted PLT-Exos (Fig. 3 d). This further corroborates the identity of the isolated PLT-Exos. 3.2. Proteomic analyses of PLT-Exos To investigate the unique characteristics of PLT-Exos, we performed a proteomic analysis of extracted platelets and PLT-Exos. LC-MS/MS analysis identified a total of 132 differentially-expressed proteins (DEPs) in PLT-Exos compared to platelets ( Fig. S1 a ), with 25 proteins significantly upregulated in the PLT-Exos ( Fig. S1 b ). We performed biological analysis on these DEPs. Enrichment analysis demonstrated that PLT-Exos were associated with the expression of regulatory proteins involved in biosynthetic processes, metabolic processes and immune regulation ( Fig. S1 c-e ). According to the functional annotation based on the Gene Ontology (GO) database, these regulated proteins were arranged into three domains, including biological process, cellular component, and molecular function and biological process, indicating that PLT-Exos are enriched wiqth various functional proteins ( Fig. S1 f ). Furthermore, KEGG pathway analysis indicated that the DEPs in PLT-Exos are linked to “cell movement” and “cell growth and death” within the “Cellular Processes” domain, “signal transduction” within the “Environmental Information Processing” domain, and are associated with “endocrine and metabolic diseases” and “cardiovascular diseases” in the “Human Diseases” domain. Additionally, proteins related to “Global and Overview Maps” in the “Metabolism” domain, as well as those in the “Immune System,” “Endocrine System,” and “Digestive System” categories within the “Organ Systems” domain were also identified ( Fig. S1 g ). These findings suggest that PLT-Exos are rich in a set of functional proteins that are highly relevant to cell behavior, signal transduction, and immune and metabolic regulation. Notably, PLT-Exos are enriched with the protein CD163 (M130), which can induce macrophage polarization toward the anti-inflammatory (M2) phenotype. This enrichment may be related to the immunoregulatory effects mediated by PLT-Exos [ 46 , 47 ]. 3.3. In vitro cellular uptake of PLT-Exos To investigate the uptake of PLT-Exos by M1 macrophages, we activated M0 with lipopolysaccharide (LPS) to induce M1 polarization (Fig. 3 a). Flow cytometry (Fig. 3 b) was employed to validate the activation of M1 macrophages, and 99.1% of the cells confirmed as M1 macrophages compared to those of M0. To track the uptake of PLT-Exos by M1 macrophages, we labeled the exosomes with the fluorescent dye DiD and incubated them with M1 macrophages for 1, 3, 6, 12 and 24 h. Flow cytometry analysis (Fig. 3 c) demonstrated a gradual increase in fluorescence signal within the macrophages starting at 3 h, reaching saturation at 12 h. Furthermore, we confirmed the efficient uptake of PLT-Exos by M1 macrophages using immunofluorescence. Confocal microscopy images (Fig. 3 d) showed strong red fluorescence in several M1 macrophages after 3 h of incubation, indicating substantial uptake. By 12 h, the uptake of PLT-Exos reached saturation. Overall, the results from fluorescence microscopy were highly consistent with those obtained from flow cytometry, both confirming the efficient uptake of PLT-Exos by M1 macrophages. 3.4. PLT-Exos induce the reprogramming of M1 into M2 macrophages in vitro To determine whether treatment with PLT-Exos could facilitate the polarization of macrophages from the M1 to the M2 state, we examined the effects of three different concentrations of PLT-Exos (1 x 10 6 , 5 x 10 6 , and 1 x 10 7 particles) on M1 macrophages polarization by immunofluorescence staining (Fig. 4 b). Compared to untreated M1 macrophages, we observed a gradual increase in CD206 fluorescence signal (red) with increasing concentrations of PLT-Exos, while the fluorescence signal of inducible nitric oxide synthase (iNOS) (green) progressively decreased. Additionally, flow cytometry analysis (Fig. 4 c) confirmed the expression of M2 macrophages markers, showing that the expression of the M2-specific marker CD206 increased with the concentrations of PLT-Exos. Flow cytometry also revealed a gradual enhancement in the expression of CD206 in M0 treated with PLT-Exos (Fig. 4 d), indicating that PLT-Exos promote the polarization of macrophages from M1 to M2. To further validate these findings, we conducted ELISA and RT-qPCR to assess the expression levels of pro-inflammatory and anti-inflammatory cytokines (Fig. 4 e-f and Fig. S2a) . As expected, M1 macrophages treated with PLT-Exos exhibited elevated levels of anti-inflammatory cytokines such as TGF-β, Arginase-1 and IL-10, while the levels of pro-inflammatory cytokines, including TNF-α, IL-1β and IL-6, were significantly reduced compared to untreated M1 macrophages. Collectively, our results demonstrate that PLT-Exos significantly enhance the M1 to M2 polarization of macrophages in vitro. 3.5. The therapeutic efficacy of PLT-Exos in CIA mice Based on the treatment protocol illustrated in Fig. 5 a, we evaluated the efficacy of PLT-Exos therapy in CIA mice. MTX is a potent anti-inflammatory and analgesic agent commonly used to alleviate symptoms in rheumatoid arthritis patients [ 52 ]. We used MTX as a positive control, representing standard clinical treatment and administered PLT-Exos to CIA mice every three days for three weeks. As the disease progressed, CIA mice exhibited reduced food intake, lethargy, and weight loss [ 53 ]. Therefore, body weight was utilized as an indirect indicator of treatment efficacy in RA. Among the various treatment groups, the saline group showed the lowest body weight, while mice treated with PLT-Exos and MTX demonstrated a continuous increase in body weight, nearly reaching the levels of the normal group (Fig. 5 b). To further assess the therapeutic effects, we measured the clinical scores of inflammatory joints and paw thickness in CIA mice (Fig. 5 c and d ). All treatment groups initially exhibited elevated inflammation scores and paw thickness, reflecting disease progression. Subsequently, the scores of the MTX and PLT-Exos treatment groups decreased, indicating therapeutic efficacy. Notably, the average paw thickness and arthritis scores in the saline group progressively increased with disease advancement. In contrast, the MTX and PLT-Exos treatment groups had lower scores, suggesting that both MTX and PLT-Exos effectively alleviated inflammation in the hind limbs of RA. These findings were consistent with our observations from the hind paw photographs. The saline group exhibited pronounced erythema and severe swelling, while the mice treated with PLT-Exos and MTX showed significant reductions in both erythema and swelling ( Fig. 5 e). Additionally, we utilized an infrared thermal imaging camera to measure the temperature of the hind paws in CIA mice to evaluate inflammatory changes (Fig. 5 f). The hind paw temperature of CIA mice in the saline treatment group (31.7°C) was significantly higher than that of the normal group (26.2°C). In comparison, the hind paw temperatures of CIA mice treated with MTX (28.3°C) and PLT-Exos (28.6°C) were significantly lower than saline treatment group. To further evaluate the therapeutic effects of PLT-Exos on inflammatory joints in CIA mice, we employed high-resolution ex vivo micro-CT imaging to monitor the bone tissue in the hind limbs (Fig. 5 g). CIA mice in the saline treatment group exhibited rough bone surfaces and severe bone erosion, whereas the joint structures of CIA mice treated with PLT-Exos and MTX remained intact, with relatively smooth bone surfaces. Quantitative analysis of the hind limb bone tissue revealed that the saline treatment group had significantly reduced bone mineral density (BMD) and trabecular thickness (Tb.Th) (Fig. 5 h-m). In contrast, the PLT-Exos and MTX treatment groups maintain bone quality across various morphological parameters, including BMD, bone volume fraction (BV/TV), bone surface density (BS/BV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp). These data collectively indicate that PLT-Exos can effectively alleviate joint inflammation, inhibit the progression of cartilage damage in CIA mice and promote the repair of bone erosion. In addition, the therapeutic effects of PLT-Exos are comparable to MTX, but without the adverse side effects associated with MTX.[ 54 ] 3.6. Impact of PLT-Exos on Recovery of Motor Function in CIA mice We assessed the extent of recovery in motor function of CIA mice through kinematic and biomechanical evaluations. Grip strength tests of the forepaws and hind paws were conducted ( Figures S3a and S3b ). Healthy mice consistently maintained their grip strength within a stable range, while the grip strength of the forepaws and hind paws in the saline treatment group steadily declined. In contrast, CIA mice treated with PLT-Exos and MTX demonstrated a progressive recovery in grip strength, with values approaching normal levels for three weeks treatment. This indicated that PLT-Exos treatment significantly accelerated the recovery of motor function in injured limbs. Furthermore, we evaluated the recovery of Achilles tendon functionality by analyzing the paw prints of the mice ( Figure S3c ). After three weeks treatment, the saline treatment group exhibited elongated paw prints, but the mice in normal group whose heels did not touch the ground while walking. This observation suggested a decrease in tendon strength and impaired motor function in the injured limbs. In comparison, both MTX and PLT-Exos treatment groups were more likely to walk with elevated heels. Using the footprint data, we calculated the Achilles Functional Index (AFI), where more negative values indicate greater impairment ( Figure S3d ). Our results showed that the AFI scores of CIA mice treated with PLT-Exos were significantly higher than those of the saline group and were closer to the AFI scores of normal mice. This finding reinforces the conclusion that PLT-Exos treatment not only effectively alleviates joint inflammation but also accelerates the recovery of motor function in injured limbs. 3.6. Biodistribution of PLT-Exos in CIA mice To evaluate the accumulation capability of PLT-Exos at the inflammatory sites, we intravenously injected DiR-labeled PLT-Exos or free DiR into CIA mice for in vivo imaging. We measured the fluorescence intensity of DiR in the joints at 1, 3, 6, 12 and 24 h (Fig. 6 a). The fluorescence signals in the inflamed joints of CIA mice treated with free DiR were consistently weaker than those in the DiR-labeled PLT-Exos group at each time point, and the decay rate of the fluorescence signal was faster in the free DiR group. By 24 h, the fluorescence signal in the inflamed joints of the free DiR-treated CIA mice had nearly disappeared. In contrast, the DiR-labeled PLT-Exos group exhibited higher fluorescence in the inflamed joints, particularly in the severely affected paws, indicating significant accumulation. This suggests that PLT-Exos can prolong circulation time, which may be crucial for targeting and accumulating in inflamed joints. Subsequently, we assessed the distribution of fluorescence signals in the heart, liver, spleen, lungs, kidneys, and joints of CIA mice 24 h post-administration (Fig. 6 b and c ). The fluorescence in the inflamed joints of the free DiR group was negligible, while the fluorescence intensity in the inflamed joints of the DiR-labeled PLT-Exos group was significantly higher than that in the heart, lungs, and kidneys. The elevated fluorescence levels in the spleen and liver may be attributed to the spleen being an important immune organ and the liver serving as a key metabolic organ. Collectively, these results indicate that PLT-Exos can effectively target pro-inflammatory macrophages and significantly enhance accumulation in the inflamed joints of CIA mice. 3.7. Histological Study To further confirm the therapeutic effects of PLT-Exos on inflamed joints, we conducted histopathological analysis on knee joint sections. Compared to the healthy group, histological sections from the saline group exhibited severe inflammatory cell infiltration, synovial hyperplasia, and bone destruction (Fig. 7 a). In contrast, the PLT-Exos and MTX groups showed significantly reduced inflammatory cell infiltration and bone erosion, further demonstrating the therapeutic effects of PLT-Exos on inflamed joints. Similar results were observed with Safranin-O Fast Green staining of the joints (Fig. 7 b). In the saline group, most of the cartilage and bone tissue in the joints was disappeared, whereas the joints of mice treated with PLT-Exos and MTX showed stronger red staining on the joint surface, indicating effective protection against cartilage destruction by PLT-Exos. As previously reported, the expression of pro-inflammatory factors TNF-α and IL-1β, along with the anti-inflammatory factor IL-10, can reflect the therapeutic efficacy in RA [ 55 ]. Therefore, we performed immunohistochemical analysis on the joints. Compared to the healthy group, the saline group showed significantly elevated expression of TNF-α and IL-1β, indicating RA disease progression (Fig. 7 c-e). Notably, the PLT-Exos group exhibited decreased levels of the inflammatory factors IL-1β and TNF-α, while the expression of the anti-inflammatory factor IL-10 was significantly increased. These results suggested that PLT-Exos can significantly reduce the production of pro-inflammatory cytokines, increase the secretion of anti-inflammatory cytokines, protect joint cartilage, and alleviate joint inflammation, thereby contributing to the treatment of RA. 3.8. PLT-Exos induce effective in vivo reprogramming of M1 into M2 macrophages To investigate whether PLT-Exos can also induce the phenotypic shift of macrophage in vivo, we detect the macrophage-specific markers such as M1 (iNOS) and M2 (CD206) in arthritic joint sections of CIA mice (Fig. 8 a). Compared to the normal group, the joint tissues of saline-treated CIA mice showed a significant increase in iNOS expression, and the expression of CD206 was significantly decreased. While CIA mice treated with PLT-Exos exhibited a marked decrease in iNOS expression and a significant increase in CD206 expression. These results indicate that PLT-Exos can promote the polarization of M1 macrophages to the M2 phenotype in vivo. To further confirm the effect of PLT-Exos on macrophage phenotypic changes in vivo, we measured the expression levels of pro-inflammatory cytokines TNF-α and IL-1β, as well as anti-inflammatory cytokines IL-10 and IL-4 in the joints of CIA mice (Fig. 8 b). Compared with the normal group, the saline-treated CIA mice exhibited significantly elevated levels of TNF-α and IL-1β, while the expression of IL-10 and IL-4 was not obvious. Notably, in the joints of CIA mice treated with PLT-Exos, the levels of TNF-α and IL-1β were significantly reduced, whereas the secretion of IL-10 and IL-4 was markedly increased. These findings further confirm that PLT-Exos can promote the polarization of M1 macrophages to the M2 phenotype in vivo. 3.9. The regulation of immune cells by PLT-Exos Flow cytometry analysis was employed to evaluate the changes of IFN-γ + CD4 + T and Foxp3 + CD25 + Treg in the blood and spleens of CIA mice. Compared to healthy mice, there was a significant upregulation of IFN-γ + CD4 + T cells and a pronounced downregulation of Foxp3 + CD25 + Treg cells in the spleens of saline-treated CIA mice, indicating an increase in the Th cell population and a decrease in the Treg cell population ( Fig. S4a-d ). Furthermore, we observed that CIA mice treated with PLT-Exos exhibited a reduction in IFN-γ + CD4 + T cells and an increase in Foxp3 + CD25 + Treg cells in their spleens. This finding indicates that PLT-Exos also exert a regulatory effect on immune cells. Additionally, similar results were obtained through analysis of the blood of CIA mice ( Fig. S4e-h ). Overall, these findings indicate that PLT-Exos can modulate the ratio of T cells to Tregs, thereby participating in the immunoregulatory response. 3.10. Safety of PLT-Exos To evaluate the safety of PLT-Exos, we assessed hepatic toxicity by measuring serum levels of liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (Fig. 9 a and b ). Renal toxicity was evaluated by measuring serum levels of blood urea nitrogen (BUN) and creatinine (Cre) (Fig. 9 c and d ). The results indicated that in the PLT-Exos treatment group, serum levels of AST, ALT, BUN, and Cre showed no significant differences compared to the untreated healthy control group and the saline treatment group. This suggests that PLT-Exos exhibit minimal toxicity to the liver and kidneys. Furthermore, we conducted a complete blood count analysis, revealing no significant differences in the proportions of blood cells (RBC), white blood cells (WBC), platelets (PLT), monocytes (Mon), hemoglobin (HGB), mean corpuscular volume (MCV), mean Corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) between the PLT-Exos group and the saline treatment group (Fig. 9 e-l). Histological examination of major organs, including the heart, liver, spleen, lungs, and kidneys, using HE staining showed no significant pathological changes in the PLT-Exos group ( Fig. S5 ). In summary, all these results indicate that PLT-Exos possess good biocompatibility and exhibit minimal toxicity to vital organs. 4. Conclusion In conclusion, our research findings clearly indicate that PLT-Exos can effectively accumulate within inflamed joints and promote the polarization of macrophages from a pro-inflammatory (M1) state to an anti-inflammatory (M2) state, successfully restoring the M1-M2 macrophage balance in RA synovial tissue. The exosome-mediated in situ reprogramming of macrophages toward anti-inflammatory M2 phenotypes significantly alleviated synovial inflammation in CIA mouse models and provided enhanced protection for bone and cartilage. This process involved the downregulation of pro-inflammatory cytokine levels and the upregulation of anti-inflammatory cytokine levels, thereby reducing inflammation associated with joint pathology. Furthermore, the therapeutic efficacy of PLT-Exos for RA was comparable to that of the conventional drug MTX, yet exhibited superior biocompatibility with negligible toxicity in CIA mouse models, unlike the adverse effects often associated with MTX. Thus, PLT-Exos guided in situ macrophage reprogramming presents a highly effective and safe anti-inflammatory treatment strategy, holding significant potential for the management of various inflammation-related diseases, including RA. Declarations Data availability The raw data required to reproduce these findings are available from the authors upon request. CRediT authorship contribution statement Dong Yu : Conceptualization, Methodology, performed the experiments, Data curation, Writing – original draft. Dan Wang : Methodology, Validation, Investigation. Yuqiu Yu: Investigation, Formal analysis, Conceptualization. Yinjin Xu : Investigation, Formal analysis. Wenting Tang : Investigation, Validation. Yuehua Guo : Writing – review & editing, Resources, Formal analysis, Conceptualization. Aidong Deng : Project administration, Conceptualization, Funding acquisition, Formal analysis, Supervision. Declaration of competing interest The authors declare no conflict of interest. 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Supplementary Files SupplementaryFiles.docx Cite Share Download PDF Status: Published Journal Publication published 31 Oct, 2025 Read the published version in Cell Communication and Signaling → Version 1 posted Editorial decision: Revision requested 24 Aug, 2025 Reviews received at journal 18 Aug, 2025 Reviewers agreed at journal 18 Aug, 2025 Reviews received at journal 06 Aug, 2025 Reviewers agreed at journal 03 Aug, 2025 Reviewers invited by journal 02 Aug, 2025 Editor assigned by journal 29 Jul, 2025 Submission checks completed at journal 29 Jul, 2025 First submitted to journal 20 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7169171","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":496767809,"identity":"7d8f3c49-a399-42f2-a358-be3175c53791","order_by":0,"name":"Dong Yu","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Dong","middleName":"","lastName":"Yu","suffix":""},{"id":496767810,"identity":"3048d50e-0158-4c02-bfd2-1aacbd23c3f8","order_by":1,"name":"Dan Wang","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Wang","suffix":""},{"id":496767811,"identity":"f8f49481-5caa-4a3d-957d-7ea656671ca7","order_by":2,"name":"Yuqiu Yu","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Yuqiu","middleName":"","lastName":"Yu","suffix":""},{"id":496767812,"identity":"4f8a2ead-c78c-4af7-9b7b-01ce9bc4f28b","order_by":3,"name":"Yinjin Xu","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Yinjin","middleName":"","lastName":"Xu","suffix":""},{"id":496767813,"identity":"439b1f02-ba7a-4980-85ba-058ea4951e97","order_by":4,"name":"Wenting Tang","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Wenting","middleName":"","lastName":"Tang","suffix":""},{"id":496767814,"identity":"2be81c39-1c5e-4105-9e33-612a51bd355d","order_by":5,"name":"Yuehua Guo","email":"","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Yuehua","middleName":"","lastName":"Guo","suffix":""},{"id":496767815,"identity":"4e00de30-00d6-4885-ba26-c2c78b04f36a","order_by":6,"name":"Aidong Deng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYDACZgYDEMXYAMQPEipqSNPCbPDgzDGi7IFrYZN82MJMWD1/O/PGxwW/GGT7pduvVSQ2sAFFuhPwapE4zFZsPLOPwXjmnDNlNxJ3yDBInDm7Ab81h3nMpHl7GBI33MhJu5F4ho3BQCIXvxZ5mJb9QC0FiW3MhLUYgLTw/ADaIpF+jIEoLYYgv/A2MBjPuJHDLJFw5hgPQb/InT+88THPH2CIzUh/+PFHRY0cf3svAe+DAGPbfyDJA44gHsLKweAPiGB/QKTqUTAKRsEoGGkAADoNSSfhhGG4AAAAAElFTkSuQmCC","orcid":"","institution":"Affiliated Hospital of Nantong University","correspondingAuthor":true,"prefix":"","firstName":"Aidong","middleName":"","lastName":"Deng","suffix":""}],"badges":[],"createdAt":"2025-07-20 10:53:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7169171/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7169171/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12964-025-02473-9","type":"published","date":"2025-10-31T15:57:29+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88649262,"identity":"90db72ab-adb9-4739-bd9f-38bb26fb013a","added_by":"auto","created_at":"2025-08-08 17:01:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":296308,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the strategy for treating RA using PLT-Exos. PLT-Exos improve RA by inducing the conversion of M1 macrophages to M2 macrophages.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7169171/v1/4fa543e041e3ddc2080736a5.png"},{"id":88648645,"identity":"0de1aa29-fc44-4ee2-90f8-156e2244b2b4","added_by":"auto","created_at":"2025-08-08 16:53:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":298766,"visible":true,"origin":"","legend":"\u003cp\u003eExtraction and Characterization of PLT-Exos. a) Extraction of PLT-Exos. b) TEM image of PLT-Exos. c) Size distribution of PLT-Exos. d) WB analysis of surface biomarkers CD9, CD63, and CD81 in PLT-Exos.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7169171/v1/327de15e4569c22b16e6793b.png"},{"id":88648662,"identity":"5a192fa7-b3b0-4f42-8d6a-85d0ff0045b1","added_by":"auto","created_at":"2025-08-08 16:53:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":431298,"visible":true,"origin":"","legend":"\u003cp\u003eEstablishment of M1 Macrophage polarization and In vitro uptake of PLT-Exos by M1 Macrophages. a) Schematic representation of the polarization of M0 to M1 macrophages. b) Flow cytometric analysis of the M1 macrophage marker CD86 in M1 macrophages. c) Flow cytometric analysis of the uptake of DiD-labeled PLT-Exos by macrophages. The negative control (NC) represents M1 macrophages without PLT-Exos. d) Confocal imaging of M1 macrophages incubated with DiD-labeled PLT-Exos at various time points (1, 3, 6, 12 and 24 h).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7169171/v1/219cc64c441b894118366c85.png"},{"id":88648674,"identity":"96fe4ef2-e64d-4950-a5d6-f2fb02fcb686","added_by":"auto","created_at":"2025-08-08 16:54:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":828523,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of PLT-Exos on macrophages polarization in vitro. a) Schematic illustration of the polarization of M1 to M2 macrophages induced by PLT-Exos. b) The images of iNOS (green) and CD206 (red) expression in M1 macrophages incubated with varying concentrations of PLT-Exos. c) Representative flow cytometric analysis of CD206 expression in M1 macrophages treated with different concentrations of PLT-Exos. d) Representative flow cytometric analysis of CD206 expression in M0 macrophages after treatment with varying concentrations of PLT-Exos. e-g) ELISA analysis of inflammatory cytokine levels in the cell-free supernatant of M1 macrophages treated with different concentrations of PLT-Exos, measuring, e) TNF-α, f) IL-1β and g) IL-10 levels (n=3, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7169171/v1/a8379fe2d282a8116bfb8d1d.png"},{"id":88648694,"identity":"c547ec46-34e8-4b05-a537-a57a1974043a","added_by":"auto","created_at":"2025-08-08 16:54:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1293708,"visible":true,"origin":"","legend":"\u003cp\u003eTherapeutic Effect of PLT-Exos on CIA Mice. a) Schematic representation of the RA establishment and treatment protocol. b) Changes in body weight over time for each treatment group (n = 5). c) Changes in clinical scores over time for each treatment group (n = 5). d) Inflammatory joint paw thickness across different treatment methods at various time points. e) Representative photographs of forepaws and hind paws from different treatment groups. f) Representative thermal imaging of the right hind paw. g) Representative micro-CT images of the hind paws following different treatments.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7169171/v1/c4f18edbc8fe3edf537d0961.png"},{"id":88648684,"identity":"04982952-fe86-42c5-a7bd-64e676094520","added_by":"auto","created_at":"2025-08-08 16:54:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":325495,"visible":true,"origin":"","legend":"\u003cp\u003eIn Vivo Biodistribution of PLT-Exos. a) In vivo fluorescence imaging of CIA mice at different time points. b) Biodistribution of free DiR and DiR-labeled PLT-Exos in the paws and major organs. c) Quantitative analysis of the distribution of free DiR and DiR-labeled PLT-Exos.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7169171/v1/ea4e19651db46d062de80f9c.png"},{"id":88648683,"identity":"56592dd5-2214-45bc-ba38-ad60fff8b135","added_by":"auto","created_at":"2025-08-08 16:54:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":549904,"visible":true,"origin":"","legend":"\u003cp\u003eHistological Analysis of Arthritis Inflammation in Different Treatment Groups. a) Hematoxylin and eosin (H\u0026amp;E) staining of knee joints from different treatment groups. b) Safranin O (Saf-O) staining of knee joints under different treatment conditions. c-e) Immunohistochemical analysis of inflammatory cytokines in the joints: c) IL-1β, d) TNF-α, and e) IL-4 and IL-10.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7169171/v1/e6c2e27be613dbe1243df887.png"},{"id":88649271,"identity":"e93264bf-4487-4581-87c2-b08ff7a14cbe","added_by":"auto","created_at":"2025-08-08 17:01:59","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":412937,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of PLT-Exos on Inflammatory Joints in CIA Mice. a) Histological sections of inflamed joints stained for the macrophage marker F4/80 and M1 (iNOS) and M2 (CD206) macrophage markers. b-e) Levels of cytokines measured by ELISA in inflamed joints, b) TNF-α, c) IL-1β, d) IL-4, and e) IL-10.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7169171/v1/b59c853a68eb07868370a8a4.png"},{"id":88648666,"identity":"e4d0c686-61d6-46ed-858b-a0bc88343939","added_by":"auto","created_at":"2025-08-08 16:53:59","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":934937,"visible":true,"origin":"","legend":"\u003cp\u003eThe safety of the PLT-Exos. a-d) Hepatotoxicity and renal toxicity of PLT-Exos assessed by measuring the serum levels, a) ALT, b) AST, c) BUN and d) Cre in CIA mice. Data are presented as the mean ± SD (n = 3). e-l) Blood routine examination parameters after PLT-Exos treatment, e) RBC, f) WBC, g) PLT, h) Mon, i) HGB, j) MCV, k) MCH and i) MCHC in CIA mice. Data are presented as the mean ± SD (n = 4).\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7169171/v1/a3cd9af148ad28ee40ee543e.png"},{"id":95039965,"identity":"12d43e83-06af-49cd-93fe-7ed656063e61","added_by":"auto","created_at":"2025-11-03 16:06:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6533608,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7169171/v1/88910abd-ac2b-4f7b-890d-20cf57f9ff2d.pdf"},{"id":88648638,"identity":"6dc2a667-6a78-4eca-8472-d0388fb7f78e","added_by":"auto","created_at":"2025-08-08 16:53:58","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10860257,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiles.docx","url":"https://assets-eu.researchsquare.com/files/rs-7169171/v1/79e6a97266cdea4dc2ea59df.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Platelet-derived exosomes in situ reprogramming macrophages for rheumatoid arthritis treatment","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eRheumatoid arthritis (RA) is a chronic, inflammatory systemic autoimmune disease that often causes systemic multi-system damage [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It can affect multiple joints of the entire body, leading to the synovial hyperplasia of the joints, gradually destroyed the cartilage and bones, and joint deformity, eventually leading to disability [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This will seriously affect people\u0026rsquo;s quality of life and increase people\u0026rsquo;s economic burden. Current treatments for RA typically fall into three major categories: non-steroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), and glucocorticoids (GCs) [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These medications are usually administered via oral, subcutaneous, or intramuscular routes. However, systemic delivery through the bloodstream often leads to inadequate drug accumulation at the targeted pathological sites [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Frequent administration of large doses or even combined administration are often required to achieve the desired therapeutic effect. Such high-dose regimens can contribute to serious systemic side effects, including gastrointestinal disturbances, liver and kidney impairment, and increased susceptibility to infections [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, there is an urgent need for therapies that can effectively target lesions.\u003c/p\u003e\u003cp\u003eThe primary pathological change in RA involves immune activation that increases infiltration of various inflammatory cells into synovial tissue, resulting in synovitis and exacerbating joint damage [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Key components of the inflammatory environment include macrophages [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Macrophages are divided into M1 and M2 macrophages according to their phenotypes and functions [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. M1 macrophages promote inflammation through the release of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-1 (IL-1), thereby advancing RA progression [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In contrast, M2 macrophages are anti-inflammatory and secrete cytokines such as transforming growth factor-beta (TGF-β), interleukin-10 (IL-10) and interleukin-13 (IL-13), which help suppress the production of pro-inflammatory factors and alleviate inflammation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Therefore, promoting the transition of M1 macrophages to M2 macrophages and maintaining a dynamic balance between these two populations has a positive effect on the treatment of RA.\u003c/p\u003e\u003cp\u003eRecent studies have explored methods to achieve this balance by facilitating macrophage phenotype switching to treat RA [\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This approach shows promise as a potentially effective alternative that minimizes harm to the immune system. However, many strategies involving extracellular vesicle (EV) and nanoparticles face challenges such as complex engineering, low yield, and poor biocompatibility, limiting their clinical applicability [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, there is a significant need to identify drug formulations that are simpler to produce while maintaining adequate yield and effectiveness.\u003c/p\u003e\u003cp\u003ePlatelet-rich plasma (PRP) is a high-concentration platelet preparation obtained from blood through centrifugation. Known for its tissue repair potential, PRP has been used in both animal and clinical studies for the treatment of arthritis [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Although PRP has a wide range of potential therapeutic applications, it is susceptible to various environmental factors such as temperature, vibration, and contamination, which can lead to platelet activation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This activation may result in excessive coagulation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], exacerbating inflammatory responses [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and ultimately impacting the therapeutic efficacy of platelets [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In addition to growth factors and cytokines, platelets also secrete distinct nano-sized cell-derived membrane vesicles known as extracellular exosomes (Exos), which may be crucial contributors to PRP's efficacy[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Platelet-derived exosomes (PLT-Exos) are membrane-bound vesicles secreted by platelets and range from 40 to 150 nm in size. Compared to PRP, PLT-Exos are noted for their stability, easy transmission in vivo, minimal immunogenicity, and lower oncogenicity [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Furthermore, PLT-Exos can easily penetrate blood vessels and accumulate at disease sites [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], with specific targeting ability to inflammatory sites [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Unlike other exosomes, PLT-Exos can be produced on a large scale, with CaCl\u003csub\u003e2\u003c/sub\u003e-activated platelets yielding more purified exosomes [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Recent years have seen extensive research into PLT-Exos as a drug delivery platform [\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], with numerous studies indicating their positive effects on inflammation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. However, whether PLT-Exos have a good therapeutic effect on RA is currently unknown.\u003c/p\u003e\u003cp\u003eIn this study, we successfully isolated exosomes from platelets and investigated their anti-inflammatory effects and therapeutic potential in RA through both in vitro and in vivo experiments. Our findings revealed that PLT-Exos facilitate the polarization of macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. Proteomic analysis revealed a high expression of CD163 (M130) in PLT-Exos which may relate to their role in macrophage polarization regulation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Additionally, we observed that PLT-Exos accumulated effectively at sites of inflammation within the joints, enhancing the secretion of anti-inflammatory factors while inhibiting the production of pro-inflammatory cytokines. Notably, the therapeutic effects of PLT-Exos were comparable to those of methotrexate (MTX), a conventional treatment for RA, but without the adverse side effects associated with MTX, indicating a favorable biosafety profile. Our research presents a promising strategy for the treatment of inflammatory diseases such as RA.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. PLT preparation and activation\u003c/h2\u003e\u003cp\u003eWhole blood was collected through the orbit of the mouse and placed in an EDTA centrifuge tube, then gently shake it evenly so that it is fully mixed with anticoagulants to prevent platelet activation. The blood was centrifuged three times respectively. The first time was centrifuged at 200 g for 20 min, so that the blood was divided into four layers, including plasma, platelets, leucocytes and red blood cells (RBCs). The top two layers (plasma and platelets) were carefully extracted and transferred to a new EDTA centrifuge tube. After centrifugation at 200 g for 10 min, the second centrifugation was performed to remove the residual RBCs and purify the platelets. Finally, the platelet solution was obtained by centrifugation at 800 g for 20 min. The platelet concentrates were activated with thrombin (2 U/ml, Solarbio, Beijing, China) and CaCl\u003csub\u003e2\u003c/sub\u003e (10%, Beyotime, Shanghai, China) for 30 min at room temperature to promote platelet activation and aggregation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Extraction of PLT-Exos\u003c/h2\u003e\u003cp\u003ePLT-Exos were extracted and purified from platelet concentrates by gradient centrifugation and ultrafiltration [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The activated platelets were subjected to a series of gradient centrifugation at 4 ℃ (500 g for 5 min, 1000 g for 15 min, 16500 g for 30 min), and cell debris was discarded. The supernatant from the last centrifugation was filtrated by a 0.22 \u0026micro;m filter, and the filtrate was ultracentrifuged at 100,000 g for 70 min at 4\u0026deg;C to obtain PLT-Exos, which was washed with sterile PBS. Then the PLT-Exos was centrifuged at the same high speed for 70 min again. Finally, the extracted PLT-Exos were carefully resuspended in sterile PBS and stored at -80 ℃ for subsequent experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Characterization of PLT-Exos\u003c/h2\u003e\u003cp\u003eFirst, the size and concentration distribution data of PLT-Exos were measured by Nanoparticle tracking analysis (NTA, Particle Metrix, GmbH, Ammersee, Germany), and then the morphology of PLT-Exos was observed and photographed using the transmission electron microscopy (TEM, FEI Co, Hillsboro, OR, USA). Finally, western blotting (WB) was carried out as follows: First, proteins were extracted from the platelet or PLT-Exos with RIPA lysis buffer containing protease inhibitor cocktail. Then BCA assay was used to determine the total protein content, and the protein was denatured by heating at 100 ℃ for 5 min. The protein extract was separated on 10% sodium dodecyl sulphate polyacrylamide electrophoresis gel (SDS-PAGE), transferred to polyvinylidene fluoride membranes, and blocked with milk at room temperature (RT) for 2 h. Afterwards, the membranes were incubated with primary antibodies such as anti-CD 9 (Abcam), anti-CD 63 (Abcam), and anti-CD 81 (Abcam) at 4 ℃ overnight, followed by washing in TBST and incubation with secondary antibodies at RT for 2 h. Finally, the labeled proteins were visualized with the Gel Doc XR system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Proteomic analysis\u003c/h2\u003e\u003cp\u003ePlatelets and PLT-Exos were extracted, followed by the addition of an inhibitor (1% protease inhibitor), sonicated. The samples were then centrifuged at 12,000 g for 10 min at 4\u0026deg;C. The supernatant was transferred to new centrifuge tubes, and the protein concentration was measured using a BCA assay kit. Subsequently, the extracted proteins underwent enzymatic digestion. LC-MS/MS analysis was performed on an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) equipped with a nanoelectrospray ion source (Thermo Fisher Scientific). The DIA data were processed using the DIA-NN search engine (v.1.8). Trypsin/P was designated as the cleavage enzyme, allowing for up to one missed cleavage. N-terminal methionine excision and carbamidomethylation of cysteine were specified as fixed modifications, with a false discovery rate (FDR) set to \u0026lt;\u0026thinsp;1%. Finally, the identified proteins were quantified, and differentially expressed proteins (DEPs) were filtered based on a fold-change threshold of \u0026gt;\u0026thinsp;1.5 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Further functional analysis of the differentially expressed proteins was conducted using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Preparation and culture of bone marrow-derived macrophages\u003c/h2\u003e\u003cp\u003eBone marrow-derived macrophages (BMDM) were isolated as previously reported [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. First, the femur and tibia were isolated from healthy C57BL/6 mice, and the bone marrow were washed with 1 mL syringe with cold Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) (Gibco, NY, USA) into a 100 mm culture dish. The collected solution was passed through a 70-\u0026micro;m cell filter to remove other tissues. After, red blood cell lysis solution (Solarbio, Beijing, China) was added to dissolve red blood cells for 5 min. The cells were cultured in DMEM medium with 10% FBS (Gibco, Beijing, China) containing 10 ng/mL macrophage colony-stimulating factor (Peprotech, NJ, USA) for seven days to obtain M0 macrophages. On the 3th day of culture, half of the medium was replaced, and on the 5th day of culture, the entire medium was changed. On the 7th day of culture, 500 ng/mL of LPS (Peprotech, NJ, USA) for polarization of M1 macrophages were added and differentiated for 2 days.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Cellular uptake of PLT-Exos\u003c/h2\u003e\u003cp\u003eBMDM were seeded in a confocal culture dish at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well and cultured for 48 h in the presence of LPS (500 ng/mL) and IFN-γ (20 ng/mL). PLT-Exos labeled with DiD (10 \u0026micro;M) (Biolegend, CA, USA) were added at 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e per well and incubated with M1 macrophages for 1, 3, 6, 12 and 24 h, respectively. Cell samples were washed three times with PBS for 5 min and fixed with 4% paraformaldehyde (Beyotime, Shanghai, China) for 20 min, and stained with DAPI for 10 min. Finally, cellular uptake of PLT-Exos was observed by confocal laser scanning microscopy (LSM900, ZEISS, USA) and flow cytometry (LSRFortessa, BD, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Macrophage phenotype transition experiment in vitro\u003c/h2\u003e\u003cp\u003eBMDMs were seeded in confocal culture plates at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well and incubated in DMEM supplemented with LPS at 500 ng/mL and IFN-γ at 20 ng/mL for 48 h. After incubation, PLT-Exos were added to each well at concentrations of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e, 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e and 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e exosomes per well, followed by an additional incubation period of 48 h. Subsequently, immunofluorescence experiments were conducted. The cells were fixed with 4% paraformaldehyde, permeabilized for 20 min with a cell permeabilization buffer and blocked for 30 min with a blocking buffer. The cells were then treated overnight at 4\u0026deg;C with anti-CD206 antibody Alexa Fluor 647 (Biolegend) and anti-iNOS antibody Alexa Fluor 488 (Invitrogen, Oregon, USA). Afterward, the cells were stained with DAPI and observed using a confocal laser microscope (LSM900, ZEISS, USA). For flow cytometry analysis, the cells were fixed, permeabilized, and blocked using the same procedures as previously described. The acquired cells were then treated with anti-F4/80 antibodies-PE (Biolegend), anti-CD86 antibodies APC/Cyanine7 (Biolegend), anti-CD206 antibodies Alexa Fluor 647 (Biolegend) at 4\u0026deg;C for 30 min. Following this, the cells were washed with DPBS and analyzed using a flow cytometer. Finally, the results were analyzed using FlowJo software (FlowJo LLC, Ashland, OR, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Quantitative real-time polymerase chain reaction (qRT-PCR)\u003c/h2\u003e\u003cp\u003ePLT-Exos were added to cultured M1 macrophages at concentrations of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e, 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e, and 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e vesicles per well. The cells were co-cultured with the PLT-Exos for 24 h. Following incubation, the cells were collected, and total RNA was extracted using TRIzol Reagent (Vazyme). The quantity and purity of the extracted RNA were assessed using a Nanodrop spectrophotometer (Thermo Scientific, USA). Subsequently, complementary DNA (cDNA) templates were synthesized using the HiScript III RT SuperMix for qPCR kit (Vazyme). qRT-PCR was performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme) on a real-time PCR system (QuantStudio 5, Thermo Fisher, USA). The primers used for qRT-PCR are listed in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. The expression data were normalized to β-actin levels and assessed using the 2\u003csup\u003e\u0026ndash;ΔΔCT\u003c/sup\u003e method.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Enzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e\u003cp\u003eAfter incubating M1 macrophages with different concentrations of PLT-Exos for 24 h, the cell supernatants were collected. The samples were then centrifuged at 3000 rpm for 30 min to remove particles and aggregates. The levels of pro-inflammatory cytokines IL-1β and TNF-α, as well as the anti-inflammatory cytokine IL-10, were measured using ELISA kits (Jiangsu Jingmei Biotechnology Co., China) according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Animal model of collagen-induced arthritis\u003c/h2\u003e\u003cp\u003eHealthy male DBA/1 mice were purchased from the Experimental Animal Center of Nantong University. The mice were housed under standard laboratory conditions with appropriate temperature and humidity, allowing for free access to food and water. All animal experiments were conducted in accordance with the experimental protocol approved by the Animal Ethics Committee of Nantong University (S20241129-001). To establish the collagen-induced arthritis (CIA) model, the method previously reported in the literature was utilized [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Firstly, 2 mg/mL of bovine type II collagen (Chondrex, Redmond, WA, USA) was mixed with an equal volume of complete Freund\u0026rsquo;s adjuvant (Chondrex, USA) and stirred on ice to dissolve and emulsify the mixture. Then 100 \u0026micro;L of the emulsion was administered via subcutaneous injection at the base of the tail. On day 21 post-initial immunization, a booster immunization was performed by preparing an incomplete Freund\u0026rsquo;s adjuvant (Chondrex, Redmond, WA) mixture with the type II collagen emulsion (Chondrex, Redmond, WA) and injecting it subcutaneously at the base of the tail of the mice. Starting from day 32 after the establishment of the CIA model, when the arthritis score exceeds 4, indicating well-established RA, CIA mice will be randomly assigned to three groups (n\u0026thinsp;=\u0026thinsp;5): intravenous injection of physiological saline, PLT-Exos, or MTX. The treatment with PLT-Exos, MTX, or saline will begin at this point, and disease progression parameters will be evaluated. The inflammation score for the hind limbs of the CIA mice will be assessed using the following scale: 0\u0026thinsp;=\u0026thinsp;No signs of erythema or swelling; 1\u0026thinsp;=\u0026thinsp;Erythema and mild swelling limited to the tarsal or ankle joint; 2\u0026thinsp;=\u0026thinsp;Erythema and mild swelling extending from the ankle joint to the tarsus; 3\u0026thinsp;=\u0026thinsp;Erythema and moderate swelling extending from the ankle joint to the metatarsal; 4\u0026thinsp;=\u0026thinsp;Erythema and severe swelling involving the ankle, foot, and digits or limb rigidity [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The cumulative scores from both hind paws will yield a maximum possible score of 16 for each mouse. During the treatment period, disease progression in RA will be monitored daily, the arthritis score and the thickness of the hind paw were recorded every 3 days, and the weight was measured once a week.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11. Motor function analysis\u003c/h2\u003e\u003cp\u003eOn day 32 after the induction of CIA, CIA mice were randomly divided into three groups (n\u0026thinsp;=\u0026thinsp;5) to evaluate the grip strength of their forepaws and hind paws during treatment with PLT-Exos, MTX or saline. The grip strength test was conducted using a grip strength meter (Bioseb), which was horizontally positioned. The tail of each mouse was gently lifted and placed on a testing mesh, allowing the mouse to grasp the mesh while keeping its body aligned horizontally. The tail was pulled steadily to record grip strength, with an interval of 3 seconds between each test. Each mouse was tested three times. Three weeks post-treatment, a plastic board was set up as a runway for the mice. Ink was applied to the soles of the mice's feet using a paintbrush, and each mouse was placed at the entrance of the runway, where it left paw prints. For each mouse, three distinguishable pairs of paw prints were randomly selected, and three parameters were measured: print length (PL), defined as the distance from the heel to the tip of the third toe; toe spread (TS), measured as the distance from the first to the fifth toe; and intermediate toe spread (IT), the distance from the second to the fourth toe. The Achilles Functional Index (AFI) value was calculated using the formula [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]:\u003c/p\u003e\u003cp\u003eAFI\u0026thinsp;=\u0026thinsp;74[(NPL-EPL)/EPL]\u0026thinsp;+\u0026thinsp;161[(ETS-NTS)/NTS]\u0026thinsp;+\u0026thinsp;48[EIT-NIT)/ NIT]-5\u003c/p\u003e\u003cp\u003ewhere N and E represent normal and post-treatment paw measurements, respectively. An AFI value of 0 indicates normal function, while negative values indicate impaired function.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12. In vivo biodistribution of the PLT-Exos\u003c/h2\u003e\u003cp\u003eIn this study, CIA mice were subjected to intravenous injections of either free DiR or DiR-loaded PLT-Exos. Following administration, the biodistribution of DiR in the knee and ankle joints of the mice was evaluated at several time points: 1, 3, 6, 12 and 24 h post-injection, using an IVIS Spectrum system (Perkin Elmer, Santa Clara, CA, USA) to quantify the fluorescence intensity. After 24 h of in vivo fluorescence imaging, the mice were euthanized using a suitable humane method. Subsequently, the following organs were harvested: heart, liver, spleen, lungs, kidneys and paws. These tissues were analyzed for fluorescence intensity using an IVIS Spectrum system (Perkin Elmer, Santa Clara, CA, USA) to compare the organ-specific distribution of DiR across the treatment groups.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13. Micro‑computed tomography (Micro‑CT) analyses of articular bone\u003c/h2\u003e\u003cp\u003eOn day 32 post-induction of CIA, the CIA mice were randomly divided into three groups (n\u0026thinsp;=\u0026thinsp;3 each): intravenous injection of saline, PLT-Exos and MTX. Treatments with PLT-Exos, MTX or saline were administered every three days, culminating in a total of eight treatments. Following the final treatment, the mice were humanely euthanized. The hind paws were subsequently harvested and fixed in 4% paraformaldehyde for 24 h. After fixation, the samples were scanned using a Bruker SkyScan 1276 micro-computed tomography (micro-CT) system (Bruker, Belgium). A quantitative analysis of various morphological parameters was performed, including bone mineral density (BMD), bone surface to bone volume ratio (BS/BV), trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.14. Immunohistochemical and histology analysis\u003c/h2\u003e\u003cp\u003e After the completion of the treatment regimen, the mice from each group were humanely euthanized, and their knee joint tissues were carefully dissected and fixed in 4% paraformaldehyde for 48 h. Subsequently, the fixed tissues were decalcified by immersion in neutral ethylenediaminetetraacetic acid solution (Biosharp, China) for 20 days. The decalcified tissues were then embedded in paraffin and sectioned into 4 \u0026micro;m thick slices for further experimentation. The joint sections were stained using hematoxylin and eosin (H\u0026amp;E) (Servicebio, China) and safranin-O/fast green staining (Servicebio, China), and the morphological features were observed under an optical microscope. Immunohistochemical analysis was conducted on the sections using primary antibodies anti-TNF-α (Abcam), anti-IL-1β (Abcam) and anti-IL-6 (Abcam). The sections were incubated overnight at 4\u0026deg;C with primary antibodies anti-iNOS, anti-CD206 and anti-F4/80, followed by incubation with fluorescently labeled secondary antibodies for 1 h. DAPI staining was performed to visualize cell nuclei, and images were captured using a fluorescence microscope. Lastly, synovial tissues from the knee joints of the mice in each group were collected, The level of pro-inflammatory cytokines IL-1β and TNF-α, as well as the anti-inflammatory cytokine IL-10 and IL-4, were measured using ELISA kits (Jiangsu Jingmei Biotechnology Co., China) according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.15. The regulation of immune cells by PLT-Exos\u003c/h2\u003e\u003cp\u003eCells were extracted from the blood and spleens of CIA mice that underwent different drug treatments. Cell suspensions were prepared, and red blood cells were lysed using a lysis buffer, followed by washing with PBS. After fixation and permeabilization, flow cytometry analysis was performed using the following fluorescently labeled mouse antibodies: FITC-CD4 (Biolegend), APC-CD25 (Biolegend), APC-CD3 (Biolegend), BV421-Foxp3 (Biolegend) and PE-IFN-γ (Biolegend) for immunostaining the cells. The stained cells were then analyzed using a flow cytometer. Finally, the results were analyzed using FlowJo software (FlowJo LLC, Ashland, OR, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e2.16. Safety evaluation\u003c/h2\u003e\u003cp\u003eUpon completion of the treatment, the mice were humanely euthanized to collect blood samples from each group for analysis. The proportions of lymphocytes (Lymph), monocytes (Mon), granulocytes (Gran), red blood cells (RBC), platelets (PLT), hemoglobin (HGB), and mean corpuscular hemoglobin concentration (MCHC) and content (MCH) were determined to evaluate the safety of PLT-Exos, MTX, and saline. Additionally, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using a biochemical assay kit (Rayto) to assess the hepatic toxicity of the treatments. Renal toxicity was evaluated by measuring creatinine (CREA) and blood urea nitrogen (BUN) levels. Finally, heart, liver, spleen, lung and kidney tissues were harvested, sectioned, and subjected to hematoxylin and eosin (H\u0026amp;E) staining to observe any morphological alterations in response to the treatments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e2.17. Statistical analysis\u003c/h2\u003e\u003cp\u003eExperimental data were analyzed using GraphPad Prism 9.0 (GraphPad Software, La Jolla, CA, USA). All data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Differences between two groups were assessed using Student's t-test, while differences among three or more groups were evaluated using one-way analysis of variance (ANOVA) followed by Tukey's post hoc tests. A p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant. Statistical significance is indicated as \u003csup\u003e*\u003c/sup\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003csup\u003e***\u003c/sup\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.005, and \u003csup\u003e****\u003c/sup\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Characterization of PLT-Exos\u003c/h2\u003e\u003cp\u003eThe process for PLT-Exos extraction was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea. To characterize the extracted PLT-Exos, we utilized TEM, NTA and WB. TEM images revealed spherical particles of varying sizes, approximately 100 nm in diameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), which corresponded with the size distribution observed through NTA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). WB results showed positive expression of the platelet marker protein CD41 in both the extracted platelets and PLT-Exos. Additionally, positive signals for surface markers CD9, CD63 and CD81 in the extracted PLT-Exos (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). This further corroborates the identity of the isolated PLT-Exos.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Proteomic analyses of PLT-Exos\u003c/h2\u003e\u003cp\u003eTo investigate the unique characteristics of PLT-Exos, we performed a proteomic analysis of extracted platelets and PLT-Exos. LC-MS/MS analysis identified a total of 132 differentially-expressed proteins (DEPs) in PLT-Exos compared to platelets (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea\u003c/b\u003e), with 25 proteins significantly upregulated in the PLT-Exos (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb\u003c/b\u003e). We performed biological analysis on these DEPs. Enrichment analysis demonstrated that PLT-Exos were associated with the expression of regulatory proteins involved in biosynthetic processes, metabolic processes and immune regulation (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec-e\u003c/b\u003e). According to the functional annotation based on the Gene Ontology (GO) database, these regulated proteins were arranged into three domains, including biological process, cellular component, and molecular function and biological process, indicating that PLT-Exos are enriched wiqth various functional proteins (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ef\u003c/b\u003e). Furthermore, KEGG pathway analysis indicated that the DEPs in PLT-Exos are linked to \u0026ldquo;cell movement\u0026rdquo; and \u0026ldquo;cell growth and death\u0026rdquo; within the \u0026ldquo;Cellular Processes\u0026rdquo; domain, \u0026ldquo;signal transduction\u0026rdquo; within the \u0026ldquo;Environmental Information Processing\u0026rdquo; domain, and are associated with \u0026ldquo;endocrine and metabolic diseases\u0026rdquo; and \u0026ldquo;cardiovascular diseases\u0026rdquo; in the \u0026ldquo;Human Diseases\u0026rdquo; domain. Additionally, proteins related to \u0026ldquo;Global and Overview Maps\u0026rdquo; in the \u0026ldquo;Metabolism\u0026rdquo; domain, as well as those in the \u0026ldquo;Immune System,\u0026rdquo; \u0026ldquo;Endocrine System,\u0026rdquo; and \u0026ldquo;Digestive System\u0026rdquo; categories within the \u0026ldquo;Organ Systems\u0026rdquo; domain were also identified (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eg\u003c/b\u003e). These findings suggest that PLT-Exos are rich in a set of functional proteins that are highly relevant to cell behavior, signal transduction, and immune and metabolic regulation. Notably, PLT-Exos are enriched with the protein CD163 (M130), which can induce macrophage polarization toward the anti-inflammatory (M2) phenotype. This enrichment may be related to the immunoregulatory effects mediated by PLT-Exos [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.3. In vitro cellular uptake of PLT-Exos\u003c/h2\u003e\u003cp\u003eTo investigate the uptake of PLT-Exos by M1 macrophages, we activated M0 with lipopolysaccharide (LPS) to induce M1 polarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) was employed to validate the activation of M1 macrophages, and 99.1% of the cells confirmed as M1 macrophages compared to those of M0. To track the uptake of PLT-Exos by M1 macrophages, we labeled the exosomes with the fluorescent dye DiD and incubated them with M1 macrophages for 1, 3, 6, 12 and 24 h. Flow cytometry analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec) demonstrated a gradual increase in fluorescence signal within the macrophages starting at 3 h, reaching saturation at 12 h. Furthermore, we confirmed the efficient uptake of PLT-Exos by M1 macrophages using immunofluorescence. Confocal microscopy images (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed) showed strong red fluorescence in several M1 macrophages after 3 h of incubation, indicating substantial uptake. By 12 h, the uptake of PLT-Exos reached saturation. Overall, the results from fluorescence microscopy were highly consistent with those obtained from flow cytometry, both confirming the efficient uptake of PLT-Exos by M1 macrophages.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.4. PLT-Exos induce the reprogramming of M1 into M2 macrophages in vitro\u003c/h2\u003e\u003cp\u003eTo determine whether treatment with PLT-Exos could facilitate the polarization of macrophages from the M1 to the M2 state, we examined the effects of three different concentrations of PLT-Exos (1 x 10\u003csup\u003e6\u003c/sup\u003e, 5 x 10\u003csup\u003e6\u003c/sup\u003e, and 1 x 10\u003csup\u003e7\u003c/sup\u003e particles) on M1 macrophages polarization by immunofluorescence staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Compared to untreated M1 macrophages, we observed a gradual increase in CD206 fluorescence signal (red) with increasing concentrations of PLT-Exos, while the fluorescence signal of inducible nitric oxide synthase (iNOS) (green) progressively decreased. Additionally, flow cytometry analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) confirmed the expression of M2 macrophages markers, showing that the expression of the M2-specific marker CD206 increased with the concentrations of PLT-Exos. Flow cytometry also revealed a gradual enhancement in the expression of CD206 in M0 treated with PLT-Exos (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), indicating that PLT-Exos promote the polarization of macrophages from M1 to M2. To further validate these findings, we conducted ELISA and RT-qPCR to assess the expression levels of pro-inflammatory and anti-inflammatory cytokines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee-f and \u003cb\u003eFig. S2a)\u003c/b\u003e. As expected, M1 macrophages treated with PLT-Exos exhibited elevated levels of anti-inflammatory cytokines such as TGF-β, Arginase-1 and IL-10, while the levels of pro-inflammatory cytokines, including TNF-α, IL-1β and IL-6, were significantly reduced compared to untreated M1 macrophages. Collectively, our results demonstrate that PLT-Exos significantly enhance the M1 to M2 polarization of macrophages in vitro.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.5. The therapeutic efficacy of PLT-Exos in CIA mice\u003c/h2\u003e\u003cp\u003eBased on the treatment protocol illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, we evaluated the efficacy of PLT-Exos therapy in CIA mice. MTX is a potent anti-inflammatory and analgesic agent commonly used to alleviate symptoms in rheumatoid arthritis patients [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. We used MTX as a positive control, representing standard clinical treatment and administered PLT-Exos to CIA mice every three days for three weeks. As the disease progressed, CIA mice exhibited reduced food intake, lethargy, and weight loss [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Therefore, body weight was utilized as an indirect indicator of treatment efficacy in RA. Among the various treatment groups, the saline group showed the lowest body weight, while mice treated with PLT-Exos and MTX demonstrated a continuous increase in body weight, nearly reaching the levels of the normal group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). To further assess the therapeutic effects, we measured the clinical scores of inflammatory joints and paw thickness in CIA mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec and \u003cb\u003ed\u003c/b\u003e). All treatment groups initially exhibited elevated inflammation scores and paw thickness, reflecting disease progression. Subsequently, the scores of the MTX and PLT-Exos treatment groups decreased, indicating therapeutic efficacy. Notably, the average paw thickness and arthritis scores in the saline group progressively increased with disease advancement. In contrast, the MTX and PLT-Exos treatment groups had lower scores, suggesting that both MTX and PLT-Exos effectively alleviated inflammation in the hind limbs of RA. These findings were consistent with our observations from the hind paw photographs. The saline group exhibited pronounced erythema and severe swelling, while the mice treated with PLT-Exos and MTX showed significant reductions in both erythema and swelling \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Additionally, we utilized an infrared thermal imaging camera to measure the temperature of the hind paws in CIA mice to evaluate inflammatory changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). The hind paw temperature of CIA mice in the saline treatment group (31.7\u0026deg;C) was significantly higher than that of the normal group (26.2\u0026deg;C). In comparison, the hind paw temperatures of CIA mice treated with MTX (28.3\u0026deg;C) and PLT-Exos (28.6\u0026deg;C) were significantly lower than saline treatment group. To further evaluate the therapeutic effects of PLT-Exos on inflammatory joints in CIA mice, we employed high-resolution ex vivo micro-CT imaging to monitor the bone tissue in the hind limbs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). CIA mice in the saline treatment group exhibited rough bone surfaces and severe bone erosion, whereas the joint structures of CIA mice treated with PLT-Exos and MTX remained intact, with relatively smooth bone surfaces. Quantitative analysis of the hind limb bone tissue revealed that the saline treatment group had significantly reduced bone mineral density (BMD) and trabecular thickness (Tb.Th) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh-m). In contrast, the PLT-Exos and MTX treatment groups maintain bone quality across various morphological parameters, including BMD, bone volume fraction (BV/TV), bone surface density (BS/BV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp). These data collectively indicate that PLT-Exos can effectively alleviate joint inflammation, inhibit the progression of cartilage damage in CIA mice and promote the repair of bone erosion. In addition, the therapeutic effects of PLT-Exos are comparable to MTX, but without the adverse side effects associated with MTX.[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e3.6. Impact of PLT-Exos on Recovery of Motor Function in CIA mice\u003c/h2\u003e\u003cp\u003eWe assessed the extent of recovery in motor function of CIA mice through kinematic and biomechanical evaluations. Grip strength tests of the forepaws and hind paws were conducted (\u003cb\u003eFigures S3a and S3b\u003c/b\u003e). Healthy mice consistently maintained their grip strength within a stable range, while the grip strength of the forepaws and hind paws in the saline treatment group steadily declined. In contrast, CIA mice treated with PLT-Exos and MTX demonstrated a progressive recovery in grip strength, with values approaching normal levels for three weeks treatment. This indicated that PLT-Exos treatment significantly accelerated the recovery of motor function in injured limbs. Furthermore, we evaluated the recovery of Achilles tendon functionality by analyzing the paw prints of the mice (\u003cb\u003eFigure S3c\u003c/b\u003e). After three weeks treatment, the saline treatment group exhibited elongated paw prints, but the mice in normal group whose heels did not touch the ground while walking. This observation suggested a decrease in tendon strength and impaired motor function in the injured limbs. In comparison, both MTX and PLT-Exos treatment groups were more likely to walk with elevated heels. Using the footprint data, we calculated the Achilles Functional Index (AFI), where more negative values indicate greater impairment (\u003cb\u003eFigure S3d\u003c/b\u003e). Our results showed that the AFI scores of CIA mice treated with PLT-Exos were significantly higher than those of the saline group and were closer to the AFI scores of normal mice. This finding reinforces the conclusion that PLT-Exos treatment not only effectively alleviates joint inflammation but also accelerates the recovery of motor function in injured limbs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e3.6. Biodistribution of PLT-Exos in CIA mice\u003c/h2\u003e\u003cp\u003eTo evaluate the accumulation capability of PLT-Exos at the inflammatory sites, we intravenously injected DiR-labeled PLT-Exos or free DiR into CIA mice for in vivo imaging. We measured the fluorescence intensity of DiR in the joints at 1, 3, 6, 12 and 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The fluorescence signals in the inflamed joints of CIA mice treated with free DiR were consistently weaker than those in the DiR-labeled PLT-Exos group at each time point, and the decay rate of the fluorescence signal was faster in the free DiR group. By 24 h, the fluorescence signal in the inflamed joints of the free DiR-treated CIA mice had nearly disappeared. In contrast, the DiR-labeled PLT-Exos group exhibited higher fluorescence in the inflamed joints, particularly in the severely affected paws, indicating significant accumulation. This suggests that PLT-Exos can prolong circulation time, which may be crucial for targeting and accumulating in inflamed joints. Subsequently, we assessed the distribution of fluorescence signals in the heart, liver, spleen, lungs, kidneys, and joints of CIA mice 24 h post-administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb \u003cb\u003eand c\u003c/b\u003e). The fluorescence in the inflamed joints of the free DiR group was negligible, while the fluorescence intensity in the inflamed joints of the DiR-labeled PLT-Exos group was significantly higher than that in the heart, lungs, and kidneys. The elevated fluorescence levels in the spleen and liver may be attributed to the spleen being an important immune organ and the liver serving as a key metabolic organ. Collectively, these results indicate that PLT-Exos can effectively target pro-inflammatory macrophages and significantly enhance accumulation in the inflamed joints of CIA mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e3.7. Histological Study\u003c/h2\u003e\u003cp\u003eTo further confirm the therapeutic effects of PLT-Exos on inflamed joints, we conducted histopathological analysis on knee joint sections. Compared to the healthy group, histological sections from the saline group exhibited severe inflammatory cell infiltration, synovial hyperplasia, and bone destruction (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). In contrast, the PLT-Exos and MTX groups showed significantly reduced inflammatory cell infiltration and bone erosion, further demonstrating the therapeutic effects of PLT-Exos on inflamed joints. Similar results were observed with Safranin-O Fast Green staining of the joints (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). In the saline group, most of the cartilage and bone tissue in the joints was disappeared, whereas the joints of mice treated with PLT-Exos and MTX showed stronger red staining on the joint surface, indicating effective protection against cartilage destruction by PLT-Exos. As previously reported, the expression of pro-inflammatory factors TNF-α and IL-1β, along with the anti-inflammatory factor IL-10, can reflect the therapeutic efficacy in RA [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Therefore, we performed immunohistochemical analysis on the joints. Compared to the healthy group, the saline group showed significantly elevated expression of TNF-α and IL-1β, indicating RA disease progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec-e). Notably, the PLT-Exos group exhibited decreased levels of the inflammatory factors IL-1β and TNF-α, while the expression of the anti-inflammatory factor IL-10 was significantly increased. These results suggested that PLT-Exos can significantly reduce the production of pro-inflammatory cytokines, increase the secretion of anti-inflammatory cytokines, protect joint cartilage, and alleviate joint inflammation, thereby contributing to the treatment of RA.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e3.8. PLT-Exos induce effective in vivo reprogramming of M1 into M2 macrophages\u003c/h2\u003e\u003cp\u003eTo investigate whether PLT-Exos can also induce the phenotypic shift of macrophage in vivo, we detect the macrophage-specific markers such as M1 (iNOS) and M2 (CD206) in arthritic joint sections of CIA mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). Compared to the normal group, the joint tissues of saline-treated CIA mice showed a significant increase in iNOS expression, and the expression of CD206 was significantly decreased. While CIA mice treated with PLT-Exos exhibited a marked decrease in iNOS expression and a significant increase in CD206 expression. These results indicate that PLT-Exos can promote the polarization of M1 macrophages to the M2 phenotype in vivo. To further confirm the effect of PLT-Exos on macrophage phenotypic changes in vivo, we measured the expression levels of pro-inflammatory cytokines TNF-α and IL-1β, as well as anti-inflammatory cytokines IL-10 and IL-4 in the joints of CIA mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). Compared with the normal group, the saline-treated CIA mice exhibited significantly elevated levels of TNF-α and IL-1β, while the expression of IL-10 and IL-4 was not obvious. Notably, in the joints of CIA mice treated with PLT-Exos, the levels of TNF-α and IL-1β were significantly reduced, whereas the secretion of IL-10 and IL-4 was markedly increased. These findings further confirm that PLT-Exos can promote the polarization of M1 macrophages to the M2 phenotype in vivo.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\u003ch2\u003e3.9. The regulation of immune cells by PLT-Exos\u003c/h2\u003e\u003cp\u003eFlow cytometry analysis was employed to evaluate the changes of IFN-γ\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T and Foxp3\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003e Treg in the blood and spleens of CIA mice. Compared to healthy mice, there was a significant upregulation of IFN-γ\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells and a pronounced downregulation of Foxp3\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003e Treg cells in the spleens of saline-treated CIA mice, indicating an increase in the Th cell population and a decrease in the Treg cell population (\u003cb\u003eFig. S4a-d\u003c/b\u003e). Furthermore, we observed that CIA mice treated with PLT-Exos exhibited a reduction in IFN-γ\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells and an increase in Foxp3\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003e Treg cells in their spleens. This finding indicates that PLT-Exos also exert a regulatory effect on immune cells. Additionally, similar results were obtained through analysis of the blood of CIA mice (\u003cb\u003eFig. S4e-h\u003c/b\u003e). Overall, these findings indicate that PLT-Exos can modulate the ratio of T cells to Tregs, thereby participating in the immunoregulatory response.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003e3.10. Safety of PLT-Exos\u003c/h2\u003e\u003cp\u003eTo evaluate the safety of PLT-Exos, we assessed hepatic toxicity by measuring serum levels of liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea \u003cb\u003eand b\u003c/b\u003e). Renal toxicity was evaluated by measuring serum levels of blood urea nitrogen (BUN) and creatinine (Cre) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec \u003cb\u003eand d\u003c/b\u003e). The results indicated that in the PLT-Exos treatment group, serum levels of AST, ALT, BUN, and Cre showed no significant differences compared to the untreated healthy control group and the saline treatment group. This suggests that PLT-Exos exhibit minimal toxicity to the liver and kidneys. Furthermore, we conducted a complete blood count analysis, revealing no significant differences in the proportions of blood cells (RBC), white blood cells (WBC), platelets (PLT), monocytes (Mon), hemoglobin (HGB), mean corpuscular volume (MCV), mean Corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) between the PLT-Exos group and the saline treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ee-l). Histological examination of major organs, including the heart, liver, spleen, lungs, and kidneys, using HE staining showed no significant pathological changes in the PLT-Exos group (\u003cb\u003eFig. S5\u003c/b\u003e). In summary, all these results indicate that PLT-Exos possess good biocompatibility and exhibit minimal toxicity to vital organs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn conclusion, our research findings clearly indicate that PLT-Exos can effectively accumulate within inflamed joints and promote the polarization of macrophages from a pro-inflammatory (M1) state to an anti-inflammatory (M2) state, successfully restoring the M1-M2 macrophage balance in RA synovial tissue. The exosome-mediated in situ reprogramming of macrophages toward anti-inflammatory M2 phenotypes significantly alleviated synovial inflammation in CIA mouse models and provided enhanced protection for bone and cartilage. This process involved the downregulation of pro-inflammatory cytokine levels and the upregulation of anti-inflammatory cytokine levels, thereby reducing inflammation associated with joint pathology. Furthermore, the therapeutic efficacy of PLT-Exos for RA was comparable to that of the conventional drug MTX, yet exhibited superior biocompatibility with negligible toxicity in CIA mouse models, unlike the adverse effects often associated with MTX. Thus, PLT-Exos guided in situ macrophage reprogramming presents a highly effective and safe anti-inflammatory treatment strategy, holding significant potential for the management of various inflammation-related diseases, including RA.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data required to reproduce these findings are available from the authors upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDong Yu\u003c/strong\u003e: Conceptualization, Methodology, performed the experiments, Data curation, Writing \u0026ndash; original draft. \u003cstrong\u003eDan Wang\u003c/strong\u003e: Methodology, Validation, Investigation. \u003cstrong\u003eYuqiu Yu:\u003c/strong\u003e Investigation, Formal analysis, Conceptualization. \u003cstrong\u003eYinjin Xu\u003c/strong\u003e: Investigation, Formal analysis. \u003cstrong\u003eWenting Tang\u003c/strong\u003e: Investigation, Validation. \u003cstrong\u003eYuehua Guo\u003c/strong\u003e: Writing \u0026ndash; review \u0026amp; editing, Resources, Formal analysis, Conceptualization. \u003cstrong\u003eAidong Deng\u003c/strong\u003e: Project administration, Conceptualization, Funding acquisition, Formal analysis, Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Nantong University (YJXYY202204-YSB45).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAppendix A. Supplementary data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary data to this article can be found online at xxxxxxxx.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eE.M. Gravallese, G.S. Firestein, N. Koscal, E. Ling, D.L. Longo, L.A. Messenger, A. Schubach, What Is Rheumatoid Arthritis?, N Engl J Med 390(13) (2024) e32.\u003c/li\u003e\n\u003cli\u003eA. Di Matteo, J.M. Bathon, P. Emery, Rheumatoid arthritis, The Lancet 402(10416) (2023) 2019-2033.\u003c/li\u003e\n\u003cli\u003eM.N. Faison, A.M. Davis, K.C. Trotter, Disease-Modifying Drugs for Adult-Onset Rheumatoid Arthritis, Jama 331(12) (2024) 1055-1056.\u003c/li\u003e\n\u003cli\u003eE. Harris, Abatacept Could Prevent Progression to Rheumatoid Arthritis, Jama 331(11) (2024) 908.\u003c/li\u003e\n\u003cli\u003eS. Takanashi, T. Takeuchi, Y. 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Mao, Highly effective rheumatoid arthritis therapy by peptide-promoted nanomodification of mesenchymal stem cells, Biomaterials 283 (2022) 121474.\u003c/li\u003e\n\u003cli\u003eB.N. Weber, J.T. Giles, K.P. Liao, Shared inflammatory pathways of rheumatoid arthritis and atherosclerotic cardiovascular disease, Nat Rev Rheumatol 19(7) (2023) 417-428.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Platelet-derived exosome, Macrophage polarization, Rheumatoid arthritis, Inflammatory cytokines","lastPublishedDoi":"10.21203/rs.3.rs-7169171/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7169171/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eM1 macrophages secrete various pro-inflammatory cytokines and play a pivotal role in the pathogenesis of rheumatoid arthritis (RA). Therefore, strategies aimed at eliminating synovial M1 macrophages or reprogramming them toward an anti-inflammatory M2 phenotype represent critical approaches for RA treatment. In this study, we propose a novel therapeutic strategy using platelet-derived exosomes (PLT-Exos) to induce the polarization of M1 macrophages into the anti-inflammatory M2 phenotype. Our results demonstrate that PLT-Exos are enriched with immunoregulatory proteins associated with M2 macrophage polarization and can effectively stimulate the conversion of M1 to M2 macrophages. Through phagocytosis assays and in vivo imaging, we confirmed that PLT-Exos are efficiently taken up and specifically accumulate in the joints of collagen-induced arthritis (CIA) mice. Treatment with PLT-Exos significantly reduced joint swelling, arthritis scores and synovial inflammation, while alleviating bone erosion and cartilage damage, leading to marked improvement in motor function in CIA mice. Notably, the therapeutic efficacy of PLT-Exos in RA was comparable to that of the clinical drug methotrexate (MTX), with excellent biocompatibility and no observed cytotoxicity. Overall, the use of PLT-Exos to induce M1-to-M2 macrophage polarization represents a promising therapeutic approach for RA and offers substantial potential for the development of anti-inflammatory treatments for various inflammatory diseases.\u003c/p\u003e","manuscriptTitle":"Platelet-derived exosomes in situ reprogramming macrophages for rheumatoid arthritis treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-08 16:53:50","doi":"10.21203/rs.3.rs-7169171/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-24T17:52:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-18T08:23:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"311494984535375304271843455482520663027","date":"2025-08-18T08:21:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-06T18:50:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32787111434312003817298916963763451683","date":"2025-08-03T06:12:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-02T21:06:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-29T11:32:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-29T11:29:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Communication and Signaling","date":"2025-07-20T10:37:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"224ac14f-e216-49d6-9476-4b2a57c2e47f","owner":[],"postedDate":"August 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-03T16:00:43+00:00","versionOfRecord":{"articleIdentity":"rs-7169171","link":"https://doi.org/10.1186/s12964-025-02473-9","journal":{"identity":"cell-communication-and-signaling","isVorOnly":false,"title":"Cell Communication and Signaling"},"publishedOn":"2025-10-31 15:57:29","publishedOnDateReadable":"October 31st, 2025"},"versionCreatedAt":"2025-08-08 16:53:50","video":"","vorDoi":"10.1186/s12964-025-02473-9","vorDoiUrl":"https://doi.org/10.1186/s12964-025-02473-9","workflowStages":[]},"version":"v1","identity":"rs-7169171","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7169171","identity":"rs-7169171","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}