{"paper_id":"3092fba1-b99b-4644-8417-e5144e3c1dd9","body_text":"Inhalation of mesenchymal stromal cell‐derived extracellular vesicles activates macrophage polarization through the miR-22-3p/NLRP3/IL-1β pathway, ameliorating lung ischemia‒reperfusion injury | 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 Inhalation of mesenchymal stromal cell‐derived extracellular vesicles activates macrophage polarization through the miR-22-3p/NLRP3/IL-1β pathway, ameliorating lung ischemia‒reperfusion injury Tao Wang, Guodong Wu, Peigen Gao, Fenghui Zhuang, Zeyu Wang, Ziheng Zhou, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6121171/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background： Lung ischemia/reperfusion injury (LIRI) is a primary contributing factor to the occurrence of primary graft dysfunction. Extracellular vesicles derived from mesenchymal stem cells (MSC-EVs) can ameliorate tissue damage and promote recovery in animal models of inflammatory diseases. However, the capacity of MSC-EVs to induce an anti-inflammatory effect in LIRI remains unclear. Methods: In this study, we used two administration methods, inhalation and intravenous injection, to investigate the role and activity of MSC-EVs in pulmonary ischemia‒reperfusion injury. Furthermore, through in vivo and in vitro experiments to explored the role and mechanism of MSC-EVs in LIRI. Results: we elucidated that MSC-EVs alleviate LIRI by promoting the polarization of macrophages from the M1 to M2 phenotype. Mechanistically, we revealed that miR-22-3pwithin MSC-EVs directly targets and inhibits the expression of NLRP3, consequently suppressing the NLRP3/caspase-1/IL-1β pathway and facilitating the transition of macrophages toward the M2 phenotype. Conclusions: Collectively, our data show that Inhalation of MSC-EVs activates macrophage polarization through the miR-22-3p/NLRP3/IL-1β pathway, ameliorating pulmonary IRI. Lung ischemia/reperfusion injury MSC-EVs primary graft dysfunction Macrophage NLRP3 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Lung transplantation represents a singular efficacious intervention for the management of end-stage respiratory system diseases, post-transplantation primary graft dysfunction (PGD) is an acute lung injury that manifests during the lung transplantation process and is attributable to ischemia‒reperfusion injury, with an incidence rate ranging between 25% and 30%[ 1 , 2 ]. Lung IRI is a pathological manifestation within pulmonary tissue involving the accelerated progression of damage after a defined period of ischemia, followed by the reinstatement of blood flow (reperfusion) and is histologically characterized by the increased permeability of blood vessels and the death of pulmonary epithelial and endothelial cells[ 1 , 3 ]. During the ischemia‒reperfusion injury process, macrophages tend to polarize toward the M1 phenotype, secreting a substantial amount of pro-inflammatory cytokines that exacerbate vascular permeability and damage endothelial and epithelial cells[ 4 , 5 ]. Therefore, the shift in the balance between M1 and M2 macrophages, leading to earlier and increased infiltration of M2 macrophages, could serve as a therapeutic target for the treatment of lung ischemia‒reperfusion injury. Mesenchymal stem cells possess pluripotent differentiation capabilities and exhibit regulatory effects on immune function, inflammatory response, and tissue repair within the body[ 6 ]. Extracellular vesicles, particularly exosomes, are secretory components of mesenchymal stem cells and contain crucial biological effectors such as microRNAs (miRNAs)[ 7 , 8 ]. Recent studies have indicated that exosomes derived from MSCs (MSC-EVs) can ameliorate tissue damage and promote recovery in animal models of inflammatory diseases[ 9 , 10 ]. However, the capacity of MSC-EVs to induce macrophages to establish an anti-inflammatory milieu in the context of lung ischemia‒reperfusion injury remains unclear. In this study, we used two administration methods, inhalation and intravenous injection, to investigate the role and activity of MSC-EVs in pulmonary ischemia‒reperfusion injury. Through in vivo and in vitro experiments, we elucidated that MSC-EVs alleviate LIRI by promoting the polarization of macrophages from the M1 to M2 phenotype. Mechanistically, we revealed that miR-22-3p within MSC-EVs directly targets and inhibits the expression of NLR family pyrin domain containing 3 (NLRP3), consequently suppressing the NLRP3/caspase-1/IL-1β pathway and facilitating the transition of macrophages toward the M2 phenotype. The aim of this study was to provide insights into the therapeutic mechanisms of MSC-EVs in the context and a reference for future studies on IRI treatment mechanisms. 2. Methods 2.1. Animals Approval for all animal experiments was granted by the Animal Care and Use Committee of Shanghai Pulmonary Hospital (FKDS-22-1-067), adhering to the Guide for the Institutional Animal Care and Use Committee (IACUC). 8-week-old male C57BL/6 wild type (WT) mice were purchased from GemPharmatech company in Jiangsu, China. All animals were kept in a pathogen-free environment at the Shanghai Pulmonary Hospital Animal Facility (Shanghai, China), with unrestricted access to water and standard laboratory food. 2.2. Mouse left hilar-clamp model for lung IRI A mouse model of lung IRI was established by clamping the left hilar for one hour and releasing for three hours. Briefly, mice were subjected to anesthesia by intraperitoneal injection of sodium pentobarbital. Subsequently, orotracheal intubation was performed using a 20-G catheter, and a ventilator was connected with ambient air at a tidal volume of 0.8 ml and a respiratory rate of 100 breaths per minute. After left thoracotomy in the fourth intercostal space, the left pulmonary artery, vein, and bronchus of mice in the IRI group was clamped by a microvascular clamp. After one-hour ischemia, the microvascular was removed, the chest was closed, and mice were re-moved from the ventilator, and allowed for the 3-hour reperfusion period, five mice in each group. The mice were euthanized by cervical dislocation. 2.3. Exosome isolation Exosomes were provided by Cellular Biomedicine Group. (CBMG, Shanghai, China), and the detailed production method was as reported in previous literature[ 11 ]. In brief, upon achieving 70–80% confluency, MSCs underwent medium replacement with 5% exosome-depleted fetal bovine serum and were cultured for 48 hours. Exosomes were isolated through differential centrifugation, employing previously established protocols with minor adjustments. Veh represents exosomes extracted from fibroblasts as control. 2.4. Western blotting Primary antibodies against CD9 (1:2000; ab92726, Abcam, USA), TSG101 (1:2000; ab125011, Abcam, USA), calnexin(1:2000; ab92573, Abcam, USA), NLRP3 (1:1000; PA5–27882, Invitrogen, IL, USA), ASC (1:1000; 10500-1-AP, Proteintech, China ), caspase-1 (1:1000; 81482-1-RR, Proteintech, China ), iNOS (1:1000; 80517-1-RR, Proteintech, China), Arg1 (1:10,000; 16,001–1-AP, Proteintech, China ), IL-1β (1:1000; 31202S, CST, MA, ) and β-actin (1:100,000; AC026, ABclonal, China) overnight at 4°C. The membranes were incubated with secondary antibodies at room temperature for 1 hour. 2.5. Dual luciferase reporter assay Bioinformatics analysis of the Target-Scan dataset revealed that NLRP3 is a potential miRNA target. RAW.2.4 cells were transfected with 100 ng of either a wild-type (WT) or mutant (MUT) 3'-UTR NLRP3 vector using Lipofectamine 2000 reagent (Invitrogen, USA) following the manufacturer's protocol. After 48 h, Renilla luciferase activity was assessed using the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized relative to the Renilla signal. 2.6. Statistical analysis Data from a minimum of five independent experiments are presented as the mean ± SD, unless otherwise specified. Statistical analyses for group differences at a specific time point involved either a two-tailed, unpaired Student’s t test or one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. For group differences across multiple time points, two-way ANOVA followed by Bonferroni’s multiple comparisons test was employed. Prism 8 software (GraphPad) was used for all analyses, with statistical significance established at a P value less than 0.05. The work has been reported in line with the ARRIVE guidelines 2.0. 3. Results 3.1. Characterization of MSC-EVs To verify the characterization of the MSC-EVs, we used nanoparticle tracking analysis (NTA) to determine that the concentration of the particles and the diameters of the particles were within the range of 50–150 nm (Figure 1A). Transmission electron microscopy (TEM) revealed that the particles had a double-layer membrane structure and a cup-shaped canonical EV morphology (Figure 1B). Furthermore, western blot analysis confirmed that the particles expressed TSG101, CD9 and calnexin. which are widely recognized molecular markers for exosomes (Figure 1C). 3.2. MSC-EVs administered via inhalation performed better than those administered via tail vein injection in terms of anti-inflammatory effects and tissue repair in a lung IRI mouse model We used immunofluorescence technology to track PKH67-labeled exosomes that entered the lung tissue. Two hours after the inhalation or tail vein injection of MSC-EVs, more MSC-EVs were located in the lung tissue of mice that inhaled MSC-EVs than in the lung tissue of mice that received MSC-EVs via tail vein injection (Figure 1D). To investigate the role of MSC-EVs in lung IRI injury, we subjected mice to 1 h of ischemia followed by reperfusion, MSC-EVs were delivered to lung tissue 2 h before ischemia (Figure 2A). First, pathologic examination by HE staining displayed less severe edema and reduced infiltration of inflammatory cells across the interalveolar septum in the lungs of mice in MSC-EVs treatment group compared with mice in the IRI+PBS group. Furthermore, compared with the LIRI-iv + MSC-EVs group, the LIRI-inh + MSC-EVs group displayed less severe edema and reduced infiltration of inflammatory cells, these changes were verified by the increased pathological scores (Figure 1B, C). Lung function was measured as airway resistance and lung compliance. Compared to that in the LIRI + Veh group, impairment of lung function in the MSC-EVs treatment groups, as evidenced by increased elasticity and resistance, was significantly alleviated, and lower airway resistance and lung compliance were observed in the lung IRI-inh + MSC-EVs group compared with those in the lung IRI-iv + MSC-EVs group (Figure 1D,E). In addition, we assessed the lung wet/dry weight ratio as an indicator of lung permeability damage. As shown in Figure 1F, compared with that in the lung IRI-iv group, a lower wet/dry weight ratio was observed in the lung IRI-inh + EVs group. Additionally, the expression of cytokines in lung tissue was assessed using Elisa. Our results showed that both the inhalation (IRI-inh + MSC-EVs) and tail vein injection (IRI-iv + MSC-EVs) of MSC-EVs reduced the levels of the pro-inflammatory cytokines TNFα and IL-6 and increased the level of the anti-inflammatory cytokine IL-10 compared with those in the LIRI +Veh group. Notably, compared with those in the LIRI-iv + MSC-EVs group, lower levels of TNFα and IL-6 and higher level of IL-10 were observed in the LIRI-inh + MSC-EVs group (Figure G-I). these data indicated that MSC-EVs ameliorated pathological alterations, lung dysfunction and inflammatory responses and that the effects of MSC-EVs against LIRI were greater for MSC-EVs administered via inhalation than for MSC-EVs administered via tail vein injection. 3.3. MSC-EVs modulate macrophage polarization and pyroptosis following LIRI Since previous studies demonstrated that macrophages play a critical role in mediating tissue injury after IRI[12, 13]. We investigated the role of macrophages in MSC-EVs therapy. First, we examined the quantity of macrophages within lung tissue after MSC-EVs treatment by flow cytometry. The results showed that the proportion of CD11b + F4/80 + macrophages was significantly greater in the lung tissue of the Veh-treated group than in that of the sham-operated group, whereas MSC-EVs treatment did not alter the total macrophage population compared with that in the Veh-treated group (Figure 3A, B). Macrophages could convert between the M1 and M2 phenotypes in response to microenvironmental changes, therefore, we investigated whether MSC-EVs could regulate macrophage polarization. The populations of M1 and M2 macrophages were identified using flow cytometry analysis. Interestingly, the number of M1 macrophages (CD11c + CD206 - ) was markedly lower in the MSC-EVs-treated mice than in the Veh-treated mice. Moreover, the number of M2 macrophages (CD11c - CD206 + ) increased after MSC-EVs treatment (Figure 3C-E). TUNEL staining demonstrated that MSC-EV significantly alleviated cell apoptosis induced by ischemia-reperfusion injury in the LIRI model. Moreover, immunofluorescence analysis revealed that MSC-EVs notably reduced the elevation of M1 macrophages resulting from LIRI (Figure 3F). Furthermore, we sorted lung tissue macrophages by flow cytometry, and western blot analysis also revealed that the M1 marker iNOS was markedly lower in lung macrophages from MSC-EVs treated mice than in lung macrophages from Veh-treated mice. Additionally, the level of the M2 marker Arg1 increased after MSC-EVs treatment (Figure 3G, H), a finding that was consistent with the quantitative real–time PCR results (Figure 3I). In addition, the rate of lung tissue macrophage pyroptosis was greater in the sham group than in the sham group, and it was lower in the IRI + MSC-EVs group than in the IRI + Veh group (Figure 3J, K). These results indicated that MSC-EVs promote the transition of macrophages from the M1 phenotype toward an M2-like state and reduce pyroptosis during lung IRI injury. 3.4. MSC-EVs regulate macrophage polarization and pyroptosis in vitro Further substantiating the direct impacts of MSC-EVs on macrophages in vitro, PKH67‐labeled MSC-EVs were internalized by macrophages and localized to the cytoplasm within 6 h (Figure 4A). Hypoxia-reoxygenation (H/R) was induced in RAW 264.7 cells to simulate ischemia‒reperfusion injury. MSC-EVs or Veh was administered to the stimulated macrophages. After approximately 6 h of hypoxia and 12 h of reoxygenation, the concentrations of TNF-α, IL-6 and IL-10 in the culture supernatants were assessed. Compared with those in the control group, the levels of both TNF-α, IL-6 and IL-10 were significantly greater in the H/R treatment group, and the level of IL-10 was greater and the level of TNF-α, IL-6 was lower in the H/R+MSC-EVs group than in the H/R + Veh group (Figure 4B-D). Furthermore, flow cytometry analysis revealed that the ratio of M1 macrophages was significantly lower in the H/R+MSC-EVs group than in the H/R+Veh group (Figure 4E, F). Then, we assessed the expression of iNOS and Arg 1 by western blotting and qPCR, and the results showed that MSC-EVs inhibited H/R-induced iNOS expression and promoted the upregulation of Arg1 expression (Figure 4G-I). 3.5. MSC-EVs-mediated macrophage polarization was mediated by miR-22-3p Exosomes play an important role in cellular communication by exchanging miRNAs or proteins between cells. To understand how MSC-EVs regulate macrophage polarization. We analyzed the miRNA composition of MSC-EVs from miRNA chip. Based on the miRNA profiles of the MSC-EVs, we searched for miRNAs reported to be involved in ischemia‒reperfusion injury (miR-21, miR-125-5p, miR-221-3p, miR-22-3p, miR-20a-5p, miR-144-3p, miR-760-3p, and miR-1)[14-20] and macrophage polarization (miR-21, miR-125-5p, miR-221-3p, miR-22-3p, miR-30d-5p, miR-155, miR-210, and miR-223)[21-28]and enriched in MSC-EVs; miR-221-3p and miR-22-3p were candidates enriched in MSC-EVs that are likely responsible for macrophage polarization (Figure 5A). Subsequently, we assessed miR-221-3p and miR-22-3p expression levels in MSC-EVs and Veh. The results showed that miR-22-3p was significantly enriched in MSC-EVs, with no difference in miR-221-3p (Figure 5B). Next, we examined the effects of miR-22-3p on macrophages. Following transfection with miRNA mimics, we investigated the expression level of miR-22-3p in RAW264.7 cells. To investigate the effects of miR-22-3p on macrophage polarization, the levels of M1 and M2 markers were detected by flow cytometry analysis, and the results showed that miR-22-3p significantly reduced the proportion of the M1 phenotype under H/R conditions (Figure 5D, E). To confirm the role of miR-22-3p in MSC-EVs, we inhibited the expression of miR-22-3p in MSC-EVs by transfecting MSCs with a miR-22-3p inhibitor and subsequently isolated the exosomes from the culture supernatants, qRT‒PCR analysis revealed that miR-22-3p levels were significantly lower in the miR-22-3p inhibitor MSC-EVs than in the negative control inhibitor MSC-EVs (Figure 5C). Then, we stimulated RAW264.7 cells under H/R conditions, treated them with NC inhibitor MSC-EVs or miR-22-3p inhibitor MSC-EVs for 48 h, and subsequently collected the cells for flow cytometry analysis. The results showed that the polarization of macrophages from the M1 to M2 phenotype simulated by H/R conditions was reversed by NC inhibitor MSC-EVs but was not significantly affected by miR-22-3p inhibitor MSC-EVs (Figure 5F, G). Overall, we confirmed that MSC-EVs-mediated macrophage polarization was mediated by miR-22-3p. 3.6. miR-22-3p directly targets NLRP3. We performed bioinformatic analysis using the TargetScan dataset to explore the molecular mechanism by which miR-22-3p facilitates macrophage polarization and found that NLRP3 was a potential target (Figure 6A). The results of the luciferase assay, which was used to study the binding of miR-22-3p to the 3’-UTR of NLRP3, revealed significantly decreased luciferase activity compared to that of the negative controls (Figure 6B). Then, we overexpressed miR-22-3p in RAW264.7 cells using a miR-22-3p mimic (Figure 6C). The corresponding NLRP3 mRNA expression significantly decreased (Figure 6D). In addition, attenuating miR-22-3p expression rescued the suppression of NLRP3 by miR-22-3p (Figure 6E, F), a finding that was confirmed by the immunofluorescence (Figure 6G) and western blot results (Figure 6H, I). These results indicated that NLRP3 was a direct downstream target of miR-22-3p in macrophages. The NLRP3 molecule serves as a critical regulatory component in cellular pyroptosis. we hypothesized that exosomes regulate the expression of NLRP3 and inhibit macrophage pyroptosis, thereby promoting macrophage polarization toward the M2 phenotype. We initially assessed the impact of exosome treatment on the expression of NLRP3 in macrophages in a lung IRI model. The findings revealed a significant reduction in the expression of NLRP3 in macrophages in the MSC-EVs treated group compared to that in the Veh group (Figure 6J, K). 3.7. miR-22-3p shuttling by MSC-EVs modulated macrophage phenotype through mediating pyroptosis via the NLRP3/Caspase-1/IL-1β pathway. The inhibition of NLRP3-mediated signaling might increase NLRP3/Caspase-1/IL-1β signaling pathway activation, which is important for the conversion of M1 macrophages to anti-inflammatory M2 macrophages. Thus, we investigated the NLRP3/Caspase-1/IL-1β signaling pathway after the transfection of macrophages with miR-22-3p. Western blot analysis revealed that the levels of NLRP3 and IL-1β were significantly decreased. Moreover, the expression of Arg-1 was markedly upregulated after transfection with the miR-22-3p mimic (Figure 7A, B). Immunofluorescence analysis demonstrated that MSC-EVs alleviated the elevation of pyroptosis-related molecules NLRP3 and IL-1β induced by H/R (Figure 7C). Changes in mitochondrial membrane potential and the production of ROS are key processes in pyroptosis. MitoTracker staining revealed that MSC-EVs can improve mitochondrial function to alleviate cellular damage (Figure 7D). Additionally, MSC-EVs were found to reduce the production of ROS during hypoxia-reoxygenation (Figure 7E). The rate of macrophage pyroptosis was lower in the H/R +MSC-EVs group than in the H/R + Veh group (Figure 7F, G). These results suggest that MSC-EVs, through the shuttling of miR-22-3p, modulate macrophage phenotype, specifically by mediating pyroptosis via the NLRP3/Caspase-1/IL-1β pathway. 4. Discussion In contrast to MSCs, extracellular vesicles originating from stem cells do not exhibit immediate responsiveness to the compromised microenvironment. Because of their distinctive characteristics, such as reduced size and low immunogenicity, extracellular vesicles from stem cells exert pivotal biological functions. These include the mitigation of cellular apoptosis, attenuation of inflammatory responses, facilitation of angiogenesis, inhibition of fibrosis, and enhancement of tissue repair potential[ 29 ]. Consequently, stem cell-derived extracellular vesicles have emerged as promising entities in the medical landscape because of their potential for precise and effective regulation of tissue regeneration[ 30 ]. Macrophages serve as crucial mediators of the inflammatory response, and their polarization plays a fundamental regulatory role in both the initiation and resolution of inflammation[ 31 ]. NLRP3, a key component of the inflammasome complex, plays a pivotal role in pyroptosis, an inflammatory form of programmed cell death, and in the subsequent polarization of macrophages[ 32 ]. The inflammatory milieu generated by NLRP3-mediated pyroptosis significantly impacts the polarization of macrophages, which are key players in the innate immune response. The release of IL-1β and IL-18, along with other inflammatory mediators, promotes the differentiation of macrophages toward the proinflammatory M1 phenotype[ 33 , 34 ]. Conversely, the absence or inhibition of NLRP3 can skew macrophage polarization toward an anti-inflammatory M2 phenotype, characterized by the production of anti-inflammatory cytokines and the promotion of tissue repair and regeneration[ 35 ]. While compelling evidence suggests the potential involvement of NLRP3 as a downstream effector, it is apparent that extracellular vesicles, along with their miRNA cargo, exhibit protein effector functions, suggesting potential synergistic mechanisms. The selection of a single candidate may oversimplify the intricate biological processes at play. While our data underscore the pivotal role of extracellular vesicle miR-22-3p in the immunomodulatory and cardioprotective effects of MSC-EVs, we acknowledge the plausible contributions of other extracellular vesicle cargoes. Numerous other components within the vesicles are undeniably biologically active, collectively contributing to the overall functional benefits. 5. Conclusion In this study, we observed that treatment with MSC-EVs effectively ameliorated pulmonary ischemia‒reperfusion injury. Notably, compared to intravenous injection, inhalation administration resulted in superior lung tissue permeability and enhanced injury mitigation rates in the context of pulmonary ischemia‒reperfusion injury. Mechanistically, MSC-EVs exerted their effects on macrophages within injured tissue. Specifically, MSC-EVs promoted the transition of macrophages within injured tissue toward the M2 phenotype, resulting in the reduced local secretion of proinflammatory cytokines and the increased secretion of the anti-inflammatory cytokine. This modulation mitigates local pathological damage, thereby improving pulmonary function. Consistent findings were verified through in vitro cell experiments. Furthermore, we discovered that MSC-EVs can enter macrophages and that miR-22-3p can regulate the expression of the NLRP3 molecule, thereby influencing the ASC/caspase-1/IL-1β signaling axis and promoting the macrophage polarization of macrophages. 6. Declarations 6.1 Ethics approval and consent to participate Approval for all animal experiments was granted by the Animal Care and Use Committee of Shanghai Pulmonary Hospital (FKDS-22-1-067, 2022/01/16) named Study of MSC-EVs alleviating pulmonary ischemia and reperfusion, adhering to the Guide for the Institutional Animal Care and Use Committee (IACUC). 6.2 Consent for publication The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 6.3 Availability of data and materials Not applicable. 6.4 Acknowledgements The authors declare that they have not use AI-generated work in this manuscript. 6.5 Fund This work was supported by the Science and Technology Commission of Shanghai Municipality (22Y21900500). 6.6 Author contributions Tao Wang conducted the experiments, analyzed the data and wrote the paper. Guodong Wu and Peigen Gao conducted the experiments and supervised the research. Fenghui Zhuang, Ziheng Zhou and Zeyu Wang conducted the experiments. Chongwu Li supervised the research and wrote the paper. Junqi Wu, Wenxin He and Deping Zhao designed the study, supervised the research and wrote the paper. All authors read and approved the final manuscript. 6.7 Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 6.8 Consent for Publication declaration All authors consent for Publication. References Capuzzimati M, Hough O, Liu M. Cell death and ischemia-reperfusion injury in lung transplantation. J Heart Lung Transpl. 2022;41:1003–13. Squiers JJ, DiMaio JM, Van Zyl J, Lima B, Gonzalez-Stawisnksi G, Rafael AE, Meyer DM, Hall SA. Long-term outcomes of patients with primary graft dysfunction after cardiac transplantation. 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Zimmermann JA, Hettiaratchi MH, McDevitt TC. Enhanced Immunosuppression of T Cells by Sustained Presentation of Bioactive Interferon-γ Within Three-Dimensional Mesenchymal Stem Cell Constructs. Stem Cells Transl Med. 2017;6:223–37. Du L, Lin L, Li Q, Liu K, Huang Y, Wang X, Cao K, Chen X, Cao W, Li F, et al. IGF-2 Preprograms Maturing Macrophages to Acquire Oxidative Phosphorylation-Dependent Anti-inflammatory Properties. Cell Metab. 2019;29:1363–e13751368. He S, Li L, Chen H, Hu X, Wang W, Zhang H, Wei R, Zhang X, Chen Y, Liu X. PRRSV Infection Induces Gasdermin D-Driven Pyroptosis of Porcine Alveolar Macrophages through NLRP3 Inflammasome Activation. J Virol. 2022;96:e0212721. Burdette BE, Esparza AN, Zhu H, Wang S. Gasdermin D in pyroptosis. Acta Pharm Sin B. 2021;11:2768–82. Frank D, Vince JE. Pyroptosis versus necroptosis: similarities, differences, and crosstalk. Cell Death Differ. 2019;26:99–114. Liu X, Zhang M, Liu H, Zhu R, He H, Zhou Y, Zhang Y, Li C, Liang D, Zeng Q, Huang G. Bone marrow mesenchymal stem cell-derived exosomes attenuate cerebral ischemia-reperfusion injury-induced neuroinflammation and pyroptosis by modulating microglia M1/M2 phenotypes. Exp Neurol. 2021;341:113700. Supplementary Files ARRIVEChecklist.pdf originalblots.pdf Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6121171\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":437608487,\"identity\":\"2539693d-b7af-46fe-b600-f0210205b6cb\",\"order_by\":0,\"name\":\"Tao Wang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Tongji University Affiliated Shanghai Pulmonary Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Tao\",\"middleName\":\"\",\"lastName\":\"Wang\",\"suffix\":\"\"},{\"id\":437608488,\"identity\":\"fb4a2476-0d6c-4606-863b-dd0be2d80420\",\"order_by\":1,\"name\":\"Guodong Wu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Tongji University Affiliated Shanghai Pulmonary Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Guodong\",\"middleName\":\"\",\"lastName\":\"Wu\",\"suffix\":\"\"},{\"id\":437608489,\"identity\":\"e908c6f7-89ed-4644-9bf4-c586e6ca768a\",\"order_by\":2,\"name\":\"Peigen Gao\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Tongji University Affiliated Shanghai Pulmonary Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Peigen\",\"middleName\":\"\",\"lastName\":\"Gao\",\"suffix\":\"\"},{\"id\":437608490,\"identity\":\"43badca9-48a9-48cf-b1dd-3b9742c4f2fe\",\"order_by\":3,\"name\":\"Fenghui Zhuang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Tongji University Affiliated Shanghai Pulmonary Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Fenghui\",\"middleName\":\"\",\"lastName\":\"Zhuang\",\"suffix\":\"\"},{\"id\":437608491,\"identity\":\"555eafdc-e200-4f38-ac4c-daf01403fa14\",\"order_by\":4,\"name\":\"Zeyu Wang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Tongji University Affiliated Shanghai Pulmonary Hospital Department of Pulmonary Function Test\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Zeyu\",\"middleName\":\"\",\"lastName\":\"Wang\",\"suffix\":\"\"},{\"id\":437608492,\"identity\":\"5e6e107e-d53e-438b-ae98-ef9a5ec73ff0\",\"order_by\":5,\"name\":\"Ziheng Zhou\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Tongji University Affiliated Shanghai Pulmonary Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Ziheng\",\"middleName\":\"\",\"lastName\":\"Zhou\",\"suffix\":\"\"},{\"id\":437608493,\"identity\":\"cf7aaf29-24fc-4bca-a988-04a4d36acabd\",\"order_by\":6,\"name\":\"chongwu li\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Tongji University Affiliated Shanghai Pulmonary Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"chongwu\",\"middleName\":\"\",\"lastName\":\"li\",\"suffix\":\"\"},{\"id\":437608494,\"identity\":\"59389d53-9584-4add-9bea-e0a2d8de0dab\",\"order_by\":7,\"name\":\"Junqi Wu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Tongji University Affiliated Shanghai Pulmonary Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Junqi\",\"middleName\":\"\",\"lastName\":\"Wu\",\"suffix\":\"\"},{\"id\":437608495,\"identity\":\"d4377286-0d91-43dc-88b7-0ef7bf1c78ba\",\"order_by\":8,\"name\":\"Wenxin He\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Tongji University Affiliated Shanghai Pulmonary Hospital\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Wenxin\",\"middleName\":\"\",\"lastName\":\"He\",\"suffix\":\"\"},{\"id\":437608496,\"identity\":\"9c5838dc-59d0-4e54-acdc-7b0e3c5c25bb\",\"order_by\":9,\"name\":\"Deping Zhao\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYDCCAwxsQNIGzDZIYGBgbCBSS5oEyVoOS8D4hLXw3W5/9uBHxfk6fun2CwUPGGxkNxxgfvYAnxbJOwfSDXvO3JaQnHOmAOiwNOMNB9jMDfBpMbiRcEyCt+22hMGNnASglsOJGw7wsEng15LYJvm37RxMy39itCSzSfO2HQBqST8A1HKAsBbJO8fYpGXOJEvOnJEDDGSDZOOZh9nM8GoBhZjkmwo7fn6J9GeGPyrsZPuONz/Dq4UBIctjZsAACipmvOpRtLA/fkBI8SgYBaNgFIxMAAABtEyGsTUJOgAAAABJRU5ErkJggg==\",\"orcid\":\"\",\"institution\":\"Tongji University Affiliated Shanghai Pulmonary Hospital\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Deping\",\"middleName\":\"\",\"lastName\":\"Zhao\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-02-27 12:59:20\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-6121171/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-6121171/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":81289950,\"identity\":\"107c132c-1c17-4d17-90eb-35c1b4be45ca\",\"added_by\":\"auto\",\"created_at\":\"2025-04-24 11:41:52\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":4731961,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eCharacterization of MSC-EVs.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA. Nanoparticle Tracking Analysis (NTA) shows the mean size distribution of the collected EVs; B. Transmission electron microscope (TEM) of MSC-EVs , left bar = 1μm, right bar = 500 nm; C. Western blotting showed that, compared with MSCs, the nanovesicles strongly express the membrane protein TSG 101 and CD 9, and weakly express calnexin, Full-length blots/gels are presented in Supplementary Figure; D. Representative images show the location of PKH-27 labeled MSC-EVs (red) in the lung tissues of lung IRI model, bar = 15μm, the square area appears with higher magnification, bar = 5μm.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6121171/v1/0be389bdf448d4731ecab272.png\"},{\"id\":81289953,\"identity\":\"6bf42c40-e44c-4da4-ba98-6ac99d490039\",\"added_by\":\"auto\",\"created_at\":\"2025-04-24 11:41:52\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":937874,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eMSC-EVs administered via inhalation performed better than those administered via tail vein injection regarding anti-inflammation and tissue repair in lung IRI mouse model.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA. Flow chart of animal model; B. Representative images showing morphological changes in Lung IRI treated with tail vein injection of MSC-EVs or MSC-EVs inhalation, bar = 200 μm; C. Histogram showing the pathological scores in different groups; D-E. The lung function airway resistance (D) and lung compliance (E) following the treatment of LIRI mice with vehicle or MSC-EVs via tail vein or inhalation; F. Wet/dry ratio of lung following the treatment of LIRI mice with vehicle or MSC-EVs via tail vein or inhalation; G-I. The concentration of pro-inflammatory cytokines TNF-α (G), IL-6 (H) and the anti-inflammatory cytokine IL-10 (I) following the treatment of LIRI mice with vehicle or MSC-EVs via tail vein or inhalation.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6121171/v1/7ba48cc8eccb37977d8cbf8d.png\"},{\"id\":81290191,\"identity\":\"1ff7ac1e-17b4-4e53-8e78-b8345992b283\",\"added_by\":\"auto\",\"created_at\":\"2025-04-24 11:49:52\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":827726,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eMSC-EVs modulate macrophage polarization and pyroptosis following LIRI.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA. Representative flow cytometry plots showing the gating strategy used to determine total macrophage in lung tissue. B. Quantification of total macrophages. C. Representative flow cytometry plots showing the percentages of M1 (CD11c\\u003csup\\u003e+\\u003c/sup\\u003eCD206\\u003csup\\u003e−\\u003c/sup\\u003e) and M2 (CD11c\\u003csup\\u003e−\\u003c/sup\\u003eCD206\\u003csup\\u003e+\\u003c/sup\\u003e) phenotype in Lung IRI model macrophages. D,E. Quantification of flow cytometry data in (C). F. Representative images of HE, TUNEL and Immunofluorescence of lung tissue of mice treated with Veh or MSC-EVs after Lung IRI. G.Representative images of western blot to assess levels of iNOS and Arg1 in the lung tissue of mice treated with Veh or MSC-EVs after Lung IRI, Full-length blots/gels are presented in Supplementary Figure. H. Quantification of flow cytometry data in (G); I. mRNA level of iNOS and Arg1; J. Representative flow cytometry plots showing the percentages of pyroptosis; K. Quantification of flow cytometry data in (J).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6121171/v1/268262c6bcf42f25b483966b.png\"},{\"id\":81289949,\"identity\":\"81d6f67b-ba56-4110-b988-0aeef4cc3de2\",\"added_by\":\"auto\",\"created_at\":\"2025-04-24 11:41:52\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":376199,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eMSC-EVs regulate macrophage polarization and pyroptosis in vitro.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA. Representative images of the uptake of PKH-67-labelled MSC-EVs by RAW264.7 cells (DAPI blue) and fluorescence uptake with Veh; B-D. Concentration of cytokine TNF-α，IL-6 and IL-10 in supernatants of H/R-stimulated RAW 264.7 cells after culturing with MSC-EVs or Veh; E. Representative flow cytometry plots showing the percentages of M1(CD11c+CD206−) phenotype in H/R-stimulated macrophages after treated with MSC-EVs or Veh; F. Quantification of flow cytometry data in (E); G. Western blot for iNOS and Arg1 expression in H/R-stimulated RAW 264.7 cells after treated with MSC-EV, Full-length blots/gels are presented in Supplementary Figure; G. Quantitative analysis of iNOS and Arg1 levels in (G); I. mRNA level of iNOS and Arg1.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6121171/v1/53a19ee3e49550d7fce4249a.png\"},{\"id\":81289947,\"identity\":\"2a33d555-8404-4c3d-9b1c-539f92436996\",\"added_by\":\"auto\",\"created_at\":\"2025-04-24 11:41:52\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":348155,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eMSC-EVs-mediated macrophage polarization was mediated by miR-22-3p.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA. Main reported miRNAs participated in IRI, macrophage polarization, and miRNAs abundant in MSC-EVs; B. Real-time PCR analysis of miR-221-3p and miR-22-3p levels in exosomes derived from MSCs and Veh; C. Real-time PCR analysis of miR-22-3p levels in MSC-EVs and MSCs with miR-22-3p inhibitor-EVs. D. Representative flow cytometry plots showing the percentages of M1 (CD11c\\u003csup\\u003e+\\u003c/sup\\u003eCD206\\u003csup\\u003e−\\u003c/sup\\u003e) phenotype in H/R-stimulated RAW264.7 cells after transfection with NC mimic or miR-22-3p mimic; E. Quantification of flow cytometry data in (D); F. Representative flow cytometry plots showing the percentages of M1(CD11c\\u003csup\\u003e+\\u003c/sup\\u003eCD206\\u003csup\\u003e−\\u003c/sup\\u003e) phenotype in H/R-stimulated RAW264.7 cells treated with miR-22-3p inhibitor MSC-EVs or NC inhibitor MSC-EVs; G. Quantification of flow cytometry data in (F).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6121171/v1/793417488a0c71b226009cc9.png\"},{\"id\":81289951,\"identity\":\"5cbb2beb-bceb-4180-a68c-25c7f703016e\",\"added_by\":\"auto\",\"created_at\":\"2025-04-24 11:41:52\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":433471,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003emiR-22-3p directly targets NLRP3.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA. miR-22-3p and its putative binding sequence in the wild-type and mutant 3′-UTR of NLRP3; B. miR-22-3p significantly decreased the luciferase \\u0026nbsp;activity of constructs with negative control but not mutant (MUT) NLRP3 3-UTR; C-D. RT-qPCR showed that mRNA of NLRP3 was significantly decreased using miR-22-3p mimics; E-F. RT-qPCR showed that attenuating miR-22-3p expression reduced the expression of NLRP3 ; G. Immunofluorescence showed that attenuating miR-22-3p expression rescued the suppression of NLRP3. H. Western blot showed that attenuating miR-22-3p expression rescued the suppression of NLRP3,Full-length blots/gels are presented in Supplementary Figure; I. Quantitative analysis of NLRP3 levels in (H); J. Protein levels of NLRP3 in lung macrophage of mice treated with Veh or MSC-EVs after LIRI, Full-length blots/gels are presented in Supplementary Figure; K. Quantitative analysis of NLRP3 levels in (J).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6121171/v1/d319a1c52a56999898082601.png\"},{\"id\":81290925,\"identity\":\"6a3a0e24-ec54-4f56-92aa-cf3a92882305\",\"added_by\":\"auto\",\"created_at\":\"2025-04-24 11:57:52\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":674342,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003emiR-22-3p shuttling by MSC-EVs modulated macrophage phenotype through mediating pyroptosis via the NLRP3/Caspase-1/IL-1β pathway.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA. Representative images of western blots for NLRP3 and downstream ASC/caspase-1 and IL-1β signaling pathways in H/R-stimulated RAW264.7 cells after transfection with miR-22-3p mimic or NC mimic,Full-length blots/gels are presented in Supplementary Figure; B. Representative images of western blots for NLRP3 and downstream ASC/caspase-1 and IL-1β signaling pathways in H/R-stimulated RAW264.7 cells after transfection with miR-22-3p inhibitor or NC inhibitor,Full-length blots/gels are presented in Supplementary Figure; C. Immunofluorescence showed that MSC-EVs rescued the expression of NLRP3 and IL-1β; D. Mitotrack stain; E. Probe detects reactive oxygen species (ROS); F. Representative flow cytometry plots showing the percentages of pyrotosis; G. Quantification of flow cytometry data in (F).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6121171/v1/2b0d1d8ac6c3b7978f85a8e7.png\"},{\"id\":85523629,\"identity\":\"d2f959a7-d3f0-446f-a806-212b5f3a2de2\",\"added_by\":\"auto\",\"created_at\":\"2025-06-26 22:01:46\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":8881017,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6121171/v1/57d60c89-6777-424a-b22a-5a5370669aa8.pdf\"},{\"id\":81289961,\"identity\":\"6e41a76a-1d4b-47c8-ab61-0d3a83697558\",\"added_by\":\"auto\",\"created_at\":\"2025-04-24 11:41:52\",\"extension\":\"pdf\",\"order_by\":12,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":132572,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"ARRIVEChecklist.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6121171/v1/3746c76858d49518a4f812d8.pdf\"},{\"id\":81290202,\"identity\":\"7039b74e-6168-4a68-b9ae-817cee422d1f\",\"added_by\":\"auto\",\"created_at\":\"2025-04-24 11:49:52\",\"extension\":\"pdf\",\"order_by\":13,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":362516,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"originalblots.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6121171/v1/148df13e513029bb7e635ac1.pdf\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Inhalation of mesenchymal stromal cell‐derived extracellular vesicles activates macrophage polarization through the miR-22-3p/NLRP3/IL-1β pathway, ameliorating lung ischemia‒reperfusion injury\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eLung transplantation represents a singular efficacious intervention for the management of end-stage respiratory system diseases, post-transplantation primary graft dysfunction (PGD) is an acute lung injury that manifests during the lung transplantation process and is attributable to ischemia‒reperfusion injury, with an incidence rate ranging between 25% and 30%[\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eLung IRI is a pathological manifestation within pulmonary tissue involving the accelerated progression of damage after a defined period of ischemia, followed by the reinstatement of blood flow (reperfusion) and is histologically characterized by the increased permeability of blood vessels and the death of pulmonary epithelial and endothelial cells[\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. During the ischemia‒reperfusion injury process, macrophages tend to polarize toward the M1 phenotype, secreting a substantial amount of pro-inflammatory cytokines that exacerbate vascular permeability and damage endothelial and epithelial cells[\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. Therefore, the shift in the balance between M1 and M2 macrophages, leading to earlier and increased infiltration of M2 macrophages, could serve as a therapeutic target for the treatment of lung ischemia‒reperfusion injury.\\u003c/p\\u003e \\u003cp\\u003eMesenchymal stem cells possess pluripotent differentiation capabilities and exhibit regulatory effects on immune function, inflammatory response, and tissue repair within the body[\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e]. Extracellular vesicles, particularly exosomes, are secretory components of mesenchymal stem cells and contain crucial biological effectors such as microRNAs (miRNAs)[\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. Recent studies have indicated that exosomes derived from MSCs (MSC-EVs) can ameliorate tissue damage and promote recovery in animal models of inflammatory diseases[\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. However, the capacity of MSC-EVs to induce macrophages to establish an anti-inflammatory milieu in the context of lung ischemia‒reperfusion injury remains unclear.\\u003c/p\\u003e \\u003cp\\u003eIn this study, we used two administration methods, inhalation and intravenous injection, to investigate the role and activity of MSC-EVs in pulmonary ischemia‒reperfusion injury. Through in vivo and in vitro experiments, we elucidated that MSC-EVs alleviate LIRI by promoting the polarization of macrophages from the M1 to M2 phenotype. Mechanistically, we revealed that miR-22-3p within MSC-EVs directly targets and inhibits the expression of NLR family pyrin domain containing 3 (NLRP3), consequently suppressing the NLRP3/caspase-1/IL-1β pathway and facilitating the transition of macrophages toward the M2 phenotype. The aim of this study was to provide insights into the therapeutic mechanisms of MSC-EVs in the context and a reference for future studies on IRI treatment mechanisms.\\u003c/p\\u003e\"},{\"header\":\"2. Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1. Animals\\u003c/h2\\u003e \\u003cp\\u003e Approval for all animal experiments was granted by the Animal Care and Use Committee of Shanghai Pulmonary Hospital (FKDS-22-1-067), adhering to the Guide for the Institutional Animal Care and Use Committee (IACUC). 8-week-old male C57BL/6 wild type (WT) mice were purchased from GemPharmatech company in Jiangsu, China. All animals were kept in a pathogen-free environment at the Shanghai Pulmonary Hospital Animal Facility (Shanghai, China), with unrestricted access to water and standard laboratory food.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2. Mouse left hilar-clamp model for lung IRI\\u003c/h2\\u003e \\u003cp\\u003eA mouse model of lung IRI was established by clamping the left hilar for one hour and releasing for three hours. Briefly, mice were subjected to anesthesia by intraperitoneal injection of sodium pentobarbital. Subsequently, orotracheal intubation was performed using a 20-G catheter, and a ventilator was connected with ambient air at a tidal volume of 0.8 ml and a respiratory rate of 100 breaths per minute. After left thoracotomy in the fourth intercostal space, the left pulmonary artery, vein, and bronchus of mice in the IRI group was clamped by a microvascular clamp. After one-hour ischemia, the microvascular was removed, the chest was closed, and mice were re-moved from the ventilator, and allowed for the 3-hour reperfusion period, five mice in each group. The mice were euthanized by cervical dislocation.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3. Exosome isolation\\u003c/h2\\u003e \\u003cp\\u003eExosomes were provided by Cellular Biomedicine Group. (CBMG, Shanghai, China), and the detailed production method was as reported in previous literature[\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. In brief, upon achieving 70\\u0026ndash;80% confluency, MSCs underwent medium replacement with 5% exosome-depleted fetal bovine serum and were cultured for 48 hours. Exosomes were isolated through differential centrifugation, employing previously established protocols with minor adjustments. Veh represents exosomes extracted from fibroblasts as control.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.4. Western blotting\\u003c/h2\\u003e \\u003cp\\u003ePrimary antibodies against CD9 (1:2000; ab92726, Abcam, USA), TSG101 (1:2000; ab125011, Abcam, USA), calnexin(1:2000; ab92573, Abcam, USA), NLRP3 (1:1000; PA5\\u0026ndash;27882, Invitrogen, IL, USA), ASC (1:1000; 10500-1-AP, Proteintech, China ), caspase-1 (1:1000; 81482-1-RR, Proteintech, China ), iNOS (1:1000; 80517-1-RR, Proteintech, China), Arg1 (1:10,000; 16,001\\u0026ndash;1-AP, Proteintech, China ), IL-1β (1:1000; 31202S, CST, MA, ) and β-actin (1:100,000; AC026, ABclonal, China) overnight at 4\\u0026deg;C. The membranes were incubated with secondary antibodies at room temperature for 1 hour.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.5. Dual luciferase reporter assay\\u003c/h2\\u003e \\u003cp\\u003eBioinformatics analysis of the Target-Scan dataset revealed that NLRP3 is a potential miRNA target. RAW.2.4 cells were transfected with 100 ng of either a wild-type (WT) or mutant (MUT) 3'-UTR NLRP3 vector using Lipofectamine 2000 reagent (Invitrogen, USA) following the manufacturer's protocol. After 48 h, Renilla luciferase activity was assessed using the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized relative to the Renilla signal.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.6. Statistical analysis\\u003c/h2\\u003e \\u003cp\\u003eData from a minimum of five independent experiments are presented as the mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SD, unless otherwise specified. Statistical analyses for group differences at a specific time point involved either a two-tailed, unpaired Student\\u0026rsquo;s t test or one-way analysis of variance (ANOVA) followed by Tukey\\u0026rsquo;s multiple comparisons test. For group differences across multiple time points, two-way ANOVA followed by Bonferroni\\u0026rsquo;s multiple comparisons test was employed. Prism 8 software (GraphPad) was used for all analyses, with statistical significance established at a P value less than 0.05. The work has been reported in line with the ARRIVE guidelines 2.0.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"3. Results\",\"content\":\"\\u003cp\\u003e\\u003cem\\u003e3.1. Characterization of MSC-EVs\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo verify the characterization of the MSC-EVs, we used nanoparticle tracking analysis (NTA) to determine that the concentration of the particles and the diameters of the particles were within the range of 50–150 nm (Figure 1A). Transmission electron microscopy (TEM) revealed that the particles had a double-layer membrane structure and a cup-shaped canonical EV morphology (Figure 1B). Furthermore, western blot analysis confirmed that the particles expressed TSG101, CD9 and calnexin. which are widely recognized molecular markers for exosomes (Figure 1C).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e3.2. MSC-EVs\\u003c/em\\u003e\\u003cem\\u003e administered via inhalation performed better than those administered via tail vein injection in terms of anti-inflammatory effects and tissue repair in a lung IRI mouse model\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe used immunofluorescence technology to track PKH67-labeled exosomes that entered the lung tissue. Two hours after the inhalation or tail vein injection of MSC-EVs, more MSC-EVs were located in the lung tissue of mice that inhaled MSC-EVs than in the lung tissue of mice that received MSC-EVs via tail vein injection (Figure 1D).\\u003c/p\\u003e\\n\\u003cp\\u003eTo investigate the role of MSC-EVs in lung IRI injury, we subjected mice to 1 h of ischemia followed by reperfusion, MSC-EVs were delivered to lung tissue 2 h before ischemia (Figure 2A). First, pathologic examination by HE staining displayed less severe edema and reduced infiltration of inflammatory cells across the interalveolar septum in the lungs of mice in MSC-EVs treatment group compared with mice in the IRI+PBS group. Furthermore, compared with the LIRI-iv + MSC-EVs group, the LIRI-inh + MSC-EVs group displayed less severe edema and reduced infiltration of inflammatory cells, these changes were verified by the increased pathological scores (Figure 1B, C). Lung function was measured as airway resistance and lung compliance. Compared to that in the LIRI + Veh group, impairment of lung function in the MSC-EVs treatment groups, as evidenced by increased elasticity and resistance, was significantly alleviated, and lower airway resistance and lung compliance were observed in the lung IRI-inh + MSC-EVs group compared with those in the lung IRI-iv + MSC-EVs group (Figure 1D,E). In addition, we assessed the lung wet/dry weight ratio as an indicator of lung permeability damage. As shown in Figure 1F, compared with that in the lung IRI-iv group, a lower wet/dry weight ratio was observed in the lung IRI-inh + EVs group. Additionally, the expression of cytokines in lung tissue was assessed using Elisa. Our results showed that both the inhalation (IRI-inh + MSC-EVs) and tail vein injection (IRI-iv + MSC-EVs) of MSC-EVs reduced the levels of the pro-inflammatory cytokines TNFα and IL-6 and increased the level of the anti-inflammatory cytokine IL-10 compared with those in the LIRI +Veh group. Notably, compared with those in the LIRI-iv + MSC-EVs group, lower levels of TNFα and IL-6 and higher level of IL-10 were observed in the LIRI-inh + MSC-EVs group (Figure G-I). these data indicated that MSC-EVs ameliorated pathological alterations, lung dysfunction and inflammatory responses and that the effects of MSC-EVs against LIRI were greater for MSC-EVs administered via inhalation than for MSC-EVs administered via tail vein injection.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e3.3. MSC-EVs modulate macrophage polarization and pyroptosis following LIRI \\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eSince previous studies demonstrated that macrophages play a critical role in mediating tissue injury after IRI[12, 13]. We investigated the role of macrophages in MSC-EVs therapy. First, we examined the quantity of macrophages within lung tissue after MSC-EVs treatment by flow cytometry. The results showed that the proportion of CD11b\\u003csup\\u003e+\\u003c/sup\\u003eF4/80\\u003csup\\u003e+\\u003c/sup\\u003e macrophages was significantly greater in the lung tissue of the Veh-treated group than in that of the sham-operated group, whereas MSC-EVs treatment did not alter the total macrophage population compared with that in the Veh-treated group (Figure 3A, B). Macrophages could convert between the M1 and M2 phenotypes in response to microenvironmental changes, therefore, we investigated whether MSC-EVs could regulate macrophage polarization. The populations of M1 and M2 macrophages were identified using flow cytometry analysis. Interestingly, the number of M1 macrophages (CD11c\\u003csup\\u003e+\\u003c/sup\\u003eCD206\\u003csup\\u003e-\\u003c/sup\\u003e) was markedly lower in the MSC-EVs-treated mice than in the Veh-treated mice. Moreover, the number of M2 macrophages (CD11c\\u003csup\\u003e-\\u003c/sup\\u003eCD206\\u003csup\\u003e+\\u003c/sup\\u003e) increased after MSC-EVs treatment (Figure 3C-E). TUNEL staining demonstrated that MSC-EV significantly alleviated cell apoptosis induced by ischemia-reperfusion injury in the LIRI model. Moreover, immunofluorescence analysis revealed that MSC-EVs notably reduced the elevation of M1 macrophages resulting from LIRI (Figure 3F). Furthermore, we sorted lung tissue macrophages by flow cytometry, and western blot analysis also revealed that the M1 marker iNOS was markedly lower in lung macrophages from MSC-EVs treated mice than in lung macrophages from Veh-treated mice. Additionally, the level of the M2 marker Arg1 increased after MSC-EVs treatment (Figure 3G, H), a finding that was consistent with the quantitative real–time PCR results (Figure 3I). In addition, the rate of lung tissue macrophage pyroptosis was greater in the sham group than in the sham group, and it was lower in the IRI + MSC-EVs group than in the IRI + Veh group (Figure 3J, K).\\u003c/p\\u003e\\n\\u003cp\\u003eThese results indicated that MSC-EVs promote the transition of macrophages from the M1 phenotype toward an M2-like state and reduce pyroptosis during lung IRI injury.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e3.4. MSC-EVs regulate macrophage polarization and \\u003c/em\\u003epyroptosis\\u003cem\\u003e in vitro\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFurther substantiating the direct impacts of MSC-EVs on macrophages in vitro, PKH67‐labeled MSC-EVs were internalized by macrophages and localized to the cytoplasm within 6 h (Figure 4A). Hypoxia-reoxygenation (H/R) was induced in RAW 264.7 cells to simulate ischemia‒reperfusion injury. MSC-EVs or Veh was administered to the stimulated macrophages. After approximately 6 h of hypoxia and 12 h of reoxygenation, the concentrations of TNF-α, IL-6 and IL-10 in the culture supernatants were assessed. Compared with those in the control group, the levels of both TNF-α, IL-6 and IL-10 were significantly greater in the H/R treatment group, and the level of IL-10 was greater and the level of TNF-α, IL-6 was lower in the H/R+MSC-EVs group than in the H/R + Veh group (Figure 4B-D). Furthermore, flow cytometry analysis revealed that the ratio of M1 macrophages was significantly lower in the H/R+MSC-EVs group than in the H/R+Veh group (Figure 4E, F). Then, we assessed the expression of iNOS and Arg 1 by western blotting and qPCR, and the results showed that MSC-EVs inhibited H/R-induced iNOS expression and promoted the upregulation of Arg1 expression (Figure 4G-I). \\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e3.5. MSC-EVs-mediated macrophage polarization was mediated by miR-22-3p\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eExosomes play an important role in cellular communication by exchanging miRNAs or proteins between cells. To understand how MSC-EVs regulate macrophage polarization. We analyzed the miRNA composition of MSC-EVs from miRNA chip. Based on the miRNA profiles of the MSC-EVs, we searched for miRNAs reported to be involved in ischemia‒reperfusion injury (miR-21, miR-125-5p, miR-221-3p, miR-22-3p, miR-20a-5p, miR-144-3p, miR-760-3p, and miR-1)[14-20] and macrophage polarization (miR-21, miR-125-5p, miR-221-3p, miR-22-3p, miR-30d-5p, miR-155, miR-210, and miR-223)[21-28]and enriched in MSC-EVs; miR-221-3p and miR-22-3p were candidates enriched in MSC-EVs that are likely responsible for macrophage polarization (Figure 5A). Subsequently, we assessed miR-221-3p and miR-22-3p expression levels in MSC-EVs and Veh. The results showed that miR-22-3p was significantly enriched in MSC-EVs, with no difference in miR-221-3p (Figure 5B).\\u003c/p\\u003e\\n\\u003cp\\u003eNext, we examined the effects of miR-22-3p on macrophages. Following transfection with miRNA mimics, we investigated the expression level of miR-22-3p in RAW264.7 cells. To investigate the effects of miR-22-3p on macrophage polarization, the levels of M1 and M2 markers were detected by flow cytometry analysis, and the results showed that miR-22-3p significantly reduced the proportion of the M1 phenotype under H/R conditions (Figure 5D, E). To confirm the role of miR-22-3p in MSC-EVs, we inhibited the expression of miR-22-3p in MSC-EVs by transfecting MSCs with a miR-22-3p inhibitor and subsequently isolated the exosomes from the culture supernatants, qRT‒PCR analysis revealed that miR-22-3p levels were significantly lower in the miR-22-3p inhibitor MSC-EVs than in the negative control inhibitor MSC-EVs (Figure 5C). Then, we stimulated RAW264.7 cells under H/R conditions, treated them with NC inhibitor MSC-EVs or miR-22-3p inhibitor MSC-EVs for 48 h, and subsequently collected the cells for flow cytometry analysis. The results showed that the polarization of macrophages from the M1 to M2 phenotype simulated by H/R conditions was reversed by NC inhibitor MSC-EVs but was not significantly affected by miR-22-3p inhibitor MSC-EVs (Figure 5F, G).\\u003c/p\\u003e\\n\\u003cp\\u003eOverall, we confirmed that MSC-EVs-mediated macrophage polarization was mediated by miR-22-3p.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e3.6. miR-22-3p directly targets NLRP3.\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe performed bioinformatic analysis using the TargetScan dataset to explore the molecular mechanism by which miR-22-3p facilitates macrophage polarization and found that NLRP3 was a potential target (Figure 6A). The results of the luciferase assay, which was used to study the binding of miR-22-3p to the 3’-UTR of NLRP3, revealed significantly decreased luciferase activity compared to that of the negative controls (Figure 6B). Then, we overexpressed miR-22-3p in RAW264.7 cells using a miR-22-3p mimic (Figure 6C). The corresponding NLRP3 mRNA expression significantly decreased (Figure 6D). In addition, attenuating miR-22-3p expression rescued the suppression of NLRP3 by miR-22-3p (Figure 6E, F), a finding that was confirmed by the immunofluorescence (Figure 6G) and western blot results (Figure 6H, I). These results indicated that NLRP3 was a direct downstream target of miR-22-3p in macrophages. The NLRP3 molecule serves as a critical regulatory component in cellular pyroptosis. we hypothesized that exosomes regulate the expression of NLRP3 and inhibit macrophage pyroptosis, thereby promoting macrophage polarization toward the M2 phenotype.\\u003c/p\\u003e\\n\\u003cp\\u003eWe initially assessed the impact of exosome treatment on the expression of NLRP3 in macrophages in a lung IRI model. The findings revealed a significant reduction in the expression of NLRP3 in macrophages in the MSC-EVs treated group compared to that in the Veh group (Figure 6J, K).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e3.7. miR-22-3p shuttling by MSC-EVs modulated macrophage phenotype through mediating pyroptosis via the NLRP3/Caspase-1/IL-1β pathway.\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe inhibition of NLRP3-mediated signaling might increase NLRP3/Caspase-1/IL-1β signaling pathway activation, which is important for the conversion of M1 macrophages to anti-inflammatory M2 macrophages. Thus, we investigated the NLRP3/Caspase-1/IL-1β signaling pathway after the transfection of macrophages with miR-22-3p. Western blot analysis revealed that the levels of NLRP3 and IL-1β were significantly decreased. Moreover, the expression of Arg-1 was markedly upregulated after transfection with the miR-22-3p mimic (Figure 7A, B). Immunofluorescence analysis demonstrated that MSC-EVs alleviated the elevation of pyroptosis-related molecules NLRP3 and IL-1β induced by H/R (Figure 7C). Changes in mitochondrial membrane potential and the production of ROS are key processes in pyroptosis. MitoTracker staining revealed that MSC-EVs can improve mitochondrial function to alleviate cellular damage (Figure 7D). Additionally, MSC-EVs were found to reduce the production of ROS during hypoxia-reoxygenation (Figure 7E). The rate of macrophage pyroptosis was lower in the H/R +MSC-EVs group than in the H/R + Veh group (Figure 7F, G).\\u003c/p\\u003e\\n\\u003cp\\u003eThese results suggest that MSC-EVs, through the shuttling of miR-22-3p, modulate macrophage phenotype, specifically by mediating pyroptosis via the NLRP3/Caspase-1/IL-1β pathway.\\u003c/p\\u003e\"},{\"header\":\"4. Discussion\",\"content\":\"\\u003cp\\u003eIn contrast to MSCs, extracellular vesicles originating from stem cells do not exhibit immediate responsiveness to the compromised microenvironment. Because of their distinctive characteristics, such as reduced size and low immunogenicity, extracellular vesicles from stem cells exert pivotal biological functions. These include the mitigation of cellular apoptosis, attenuation of inflammatory responses, facilitation of angiogenesis, inhibition of fibrosis, and enhancement of tissue repair potential[\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. Consequently, stem cell-derived extracellular vesicles have emerged as promising entities in the medical landscape because of their potential for precise and effective regulation of tissue regeneration[\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eMacrophages serve as crucial mediators of the inflammatory response, and their polarization plays a fundamental regulatory role in both the initiation and resolution of inflammation[\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. NLRP3, a key component of the inflammasome complex, plays a pivotal role in pyroptosis, an inflammatory form of programmed cell death, and in the subsequent polarization of macrophages[\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. The inflammatory milieu generated by NLRP3-mediated pyroptosis significantly impacts the polarization of macrophages, which are key players in the innate immune response. The release of IL-1β and IL-18, along with other inflammatory mediators, promotes the differentiation of macrophages toward the proinflammatory M1 phenotype[\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e]. Conversely, the absence or inhibition of NLRP3 can skew macrophage polarization toward an anti-inflammatory M2 phenotype, characterized by the production of anti-inflammatory cytokines and the promotion of tissue repair and regeneration[\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e]. While compelling evidence suggests the potential involvement of NLRP3 as a downstream effector, it is apparent that extracellular vesicles, along with their miRNA cargo, exhibit protein effector functions, suggesting potential synergistic mechanisms. The selection of a single candidate may oversimplify the intricate biological processes at play. While our data underscore the pivotal role of extracellular vesicle miR-22-3p in the immunomodulatory and cardioprotective effects of MSC-EVs, we acknowledge the plausible contributions of other extracellular vesicle cargoes. Numerous other components within the vesicles are undeniably biologically active, collectively contributing to the overall functional benefits.\\u003c/p\\u003e\"},{\"header\":\"5. Conclusion\",\"content\":\"\\u003cp\\u003eIn this study, we observed that treatment with MSC-EVs effectively ameliorated pulmonary ischemia‒reperfusion injury. Notably, compared to intravenous injection, inhalation administration resulted in superior lung tissue permeability and enhanced injury mitigation rates in the context of pulmonary ischemia‒reperfusion injury. Mechanistically, MSC-EVs exerted their effects on macrophages within injured tissue. Specifically, MSC-EVs promoted the transition of macrophages within injured tissue toward the M2 phenotype, resulting in the reduced local secretion of proinflammatory cytokines and the increased secretion of the anti-inflammatory cytokine. This modulation mitigates local pathological damage, thereby improving pulmonary function. Consistent findings were verified through in vitro cell experiments. Furthermore, we discovered that MSC-EVs can enter macrophages and that miR-22-3p can regulate the expression of the NLRP3 molecule, thereby influencing the ASC/caspase-1/IL-1β signaling axis and promoting the macrophage polarization of macrophages.\\u003c/p\\u003e\"},{\"header\":\"6. Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003e6.1 Ethics approval and consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eApproval for all animal experiments was granted by the Animal Care and Use Committee of Shanghai Pulmonary Hospital (FKDS-22-1-067, 2022/01/16) named Study of MSC-EVs alleviating pulmonary ischemia and reperfusion, adhering to the Guide for the Institutional Animal Care and Use Committee (IACUC).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e6.2\\u0026nbsp;Consent for publication\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e6.3 Availability of data and materials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e6.4 Acknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they have not use AI-generated work in this manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e6.5 Fund\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported by the Science and Technology Commission of Shanghai Municipality (22Y21900500).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e6.6 Author contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTao Wang conducted the experiments, analyzed the data and wrote the paper. Guodong Wu and Peigen Gao conducted the experiments and supervised the research. Fenghui Zhuang, Ziheng Zhou and Zeyu Wang conducted the experiments. Chongwu Li supervised the research and wrote the paper. Junqi Wu, Wenxin He and Deping Zhao designed the study, supervised the research and wrote the paper. All authors read and approved the final manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e6.7 Competing interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e6.8 Consent for Publication declaration\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll authors consent for Publication.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eCapuzzimati M, Hough O, Liu M. 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Exp Neurol. 2021;341:113700.\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Lung ischemia/reperfusion injury, MSC-EVs, primary graft dysfunction, Macrophage, NLRP3\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-6121171/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-6121171/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e\\u003cstrong\\u003eBackground：\\u003c/strong\\u003eLung ischemia/reperfusion injury (LIRI) is a primary contributing factor to the occurrence of primary graft dysfunction. Extracellular vesicles derived from mesenchymal stem cells (MSC-EVs) can ameliorate tissue damage and promote recovery in animal models of inflammatory diseases. However, the capacity of MSC-EVs to induce an anti-inflammatory effect in LIRI remains unclear.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMethods:\\u003c/strong\\u003e \\u0026nbsp;In this study, we used two administration methods, inhalation and intravenous injection, to investigate the role and activity of MSC-EVs in pulmonary ischemia‒reperfusion injury. Furthermore, through in vivo and in vitro experiments to explored the role and mechanism of MSC-EVs in LIRI.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eResults: \\u003c/strong\\u003ewe elucidated that MSC-EVs alleviate LIRI by promoting the polarization of macrophages from the M1 to M2 phenotype. Mechanistically, we revealed that miR-22-3pwithin MSC-EVs directly targets and inhibits the expression of NLRP3, consequently suppressing the NLRP3/caspase-1/IL-1β pathway and facilitating the transition of macrophages toward the M2 phenotype.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConclusions:\\u003c/strong\\u003e Collectively, our data show that Inhalation of MSC-EVs activates macrophage polarization through the miR-22-3p/NLRP3/IL-1β pathway, ameliorating pulmonary IRI.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Inhalation of mesenchymal stromal cell‐derived extracellular vesicles activates macrophage polarization through the miR-22-3p/NLRP3/IL-1β pathway, ameliorating lung ischemia‒reperfusion injury\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-04-24 11:41:47\",\"doi\":\"10.21203/rs.3.rs-6121171/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"c5faf65e-986f-4e8f-98fa-97bad682b382\",\"owner\":[],\"postedDate\":\"April 24th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-06-26T21:53:35+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-04-24 11:41:47\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-6121171\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-6121171\",\"identity\":\"rs-6121171\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}