Macrophage-derived Microvesicles Coated Nanoparticles Loaded with TGF-β1 Resolve Acute Lung Injury via Regulatory B Cell Activation

Macrophage-derived Microvesicles Coated Nanoparticles Loaded with TGF-β1 Resolve Acute Lung Injury via Regulatory B Cell Activation | 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 Macrophage-derived Microvesicles Coated Nanoparticles Loaded with TGF-β1 Resolve Acute Lung Injury via Regulatory B Cell Activation Ren Jing, Xiaoting Liao, Jianlan Mo, Sheng He, Xianlong Xie, Huijun Dai, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4421925/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 Regulatory B cells (Breg) are vital for inflammation and tissue injury resolution. Here, we investigated the role of transforming growth factor-β1 (TGF-β1)-producing Breg in the murine model of ventilation-induced lung injury (VILI). The percentages of pulmonary CD19 high CD44(+) TGF-β1(+) Breg were increased at PV1d and PV10d in VILI mice. Lung injury and inflammation were attenuated by up-regulating TGF-β1 levels with regulation of T-cell immunity. To prolong and stabilize the effect of exogenous TGF-β1, macrophage-derived microvesicles-coated nanoparticles (MNP) loaded TGF-β1(TMNP) were synthesized, and VILI mice were divided into sham, recombinant TGF-β1 (rTGF-β), MNP, and TMNP groups. TMNP increased the TGF-β1 levels in serum and lung tissues at PV10d. Compared with rTGF-β group, lung injury and inflammation in TMNP group at PV1d were attenuated with Breg proliferation; TMNP induced the reduction of pulmonary CD4(+) T cell proportions and CD4(+)/CD8a(+) T cell ratios, but promoted the proliferation of pulmonary CD8a(+) T cells at PV1d and PV10d. Together, TMNP promote the resolution of inflammatory lung injury, which may be associated with the proliferation of Breg to maintain immunological homeostasis. Ventilation-induced lung injury Transforming growth factor-β1 regulatory B cells nanoparticles Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Summary statement A substantial proportion of patients on mechanical ventilation suffer acute lung injury. Here, macrophage-derived microvesicle-coated nanoparticles packaging TGF-β1 were synthesized to prolong exogenous TGF-β1, which can stable-release TGF-β1 to withhold the cascade acute lung injury via regulatory B cell activation. 1. Introduction Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are caused by severe shock, trauma, and infection, notably during the COVID-19 pandemic, and have a high morbidity and mortality rate[ 1 , 2 ]. Mechanical ventilation is the most often used ARDS respiratory support therapy, although it also causes ALI or worsens the original injury (ventilator-induced lung injury, VILI)[ 3 , 4 ]. The mechanisms of VILI involve three biophysical factors: volutrauma, barotrauma, and atelectrauma[ 5 ]. Cytokines, surfactants, and immune cells are all biochemical components that contribute to the pathogenesis of VILI[ 5 , 6 ]. However, the underlying mechanisms through which immune response contributes to the recovery of lung injury and inflammation are not fully known. Regulatory B cells (Breg) undergo immunosuppression during inflammation and immunological response[ 7 ]. This process is traditionally defined by a release of interleukin (IL)-10, IL-35, and transforming growth factor-β1 (TGF-β1) to induce death ligand-mediated apoptosis and regulate such immune cells as an effector or regulatory T cells (Treg)[ 8 , 9 ]. Despite their inability to entirely eliminate the inflammation, an increase in Breg is beneficial in preventing excessive inflammation and tissue damage, showing that abnormalities in Breg numbers and function are associated with immune homeostasis[ 10 ]. Breg help has long been known to be essential for sustaining IL-10 function during the inhibition of inflammation and immune response[ 11 , 12 ]. Besides, Breg-produced TGF-β1 also has an important role in regulating T-cell immunity in inflammatory diseases[ 13 ]. However, the underlying mechanisms by which Breg-derived TGF-β1 coordinates immune response during VILI remain incompletely understood. TGF-β1 is required for immune cell development and maturation in order to maintain immune homeostasis and regulate tissue repair[ 14 ]. Several studies have reported a critical role of TGF-β1 in the attenuation of hyperoxia-induced ALI[ 15 – 17 ]. In high tidal volume (HTV)-ventilated rats, alveolar TGF-β1 concentrations similarly revealed a significant trend over 24 hours[ 18 ]. To mimic the recovery process of surgical patients undergoing mechanical ventilation, we defined the acute phase as the period between immediate post-ventilation and post-ventilation day 3, and the rehabilitation phase as the period from post-ventilation day 3 to post-ventilation day 10. In this study, we showed that specific Breg phenotype secretes TGF-β1, which is also essential for T-cell immunity; further, it promotes the resolution of VILI. In addition, we synthesized the macrophage-derived microvesicles (MMVs) membrane-coated nanoparticles (MNP) to package TGF-β1 (TMNP) to prolong and stabilize the effect of exogenous TGF-β1. 2. Materials and Methods 2.1 Mice Male or female C57BL/6 mice, aged 4–6 weeks and weighted 25 ± 5 g, were obtained through the Animal Center of Guangxi Medical University (Nanning, China). The study protocol was approved by the Animal Care and Use Committee of Guangxi Medical University (KY-2022-288) and conducted under the guidelines for the care and use of animals set by the Chinese government. 2.2 Preparation of MMVs RAW 264.7 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in dulbecco's modified eagle medium (DMEM, 12491015; Gibco, San Jose, CA, USA) supplemented with 10% fetal bovine serum (FBS,10099; Gibco, San Jose, CA, USA), 1% penicillin (100 IU/mL; Corning, NY, USA) and streptomycin (100 µg/mL; Corning, NY, USA). MMVs were prepared using a previously reported method[ 19 ]. Briefly, the cells were washed twice with phosphate buffer saline (PBS; P3813, Sigma-Aldrich, St. Louis, MO, USA) and incubated in 5 mL of serum-free DMEM with 10 µg/ml cytochalasin B (ab143482; Abcam, Cambridge, UK) for 1 hour at 37°C. The cells were dyed with DiO' (Invitrogen, San Jose, CA, USA) and subjected to confocal laser scanning microscopy to examine the formation of MMVs. The cells without cytochalasin B treatment were applied as a control. After detaching the cells and MMVs from the culture dish, they were rinsed with 5 ml of DMEM and vortexed to isolate the MMVs. Afterward, five milliliters of FBS were added to achieve a final FBS concentration of 50%. The suspension was centrifuged at 5, 000 g for 10 min to remove the cells and large MMVs aggregates. Next, the supernatant was centrifuged at 17, 000 g for 15 min to collect the MMVs. To prepare the purified MMVs, the collected MMVs were washed twice with MilliQ water (IPAKKITA1; Millipore, Burlington, MA, USA) containing 0.25% ethylenedinitrilotetraacetic acid (ED, Sigma-Aldrich, St. Louis, MO, USA). The protein quantity of the final MMVs was assessed by Pierce™ BCA protein assay kit (23225; Thermofisher Scientific, Waltham, MA, USA) and immunoblotting. 2.3 Preparation of nanoparticles (NPs), MNP and TMNP MNP were synthesized using a previously described method[ 20 , 21 ]. First, 0.67 dL/g carboxy-terminated 50:50 poly (lactic-co-glycolic acid) (PLGA; B6001-1, LACTEL Absorbable Polymers, Birmingham, AL, USA) was used to form PLGA NPs cores. PLGA was dissolved in chloroform at a concentration of 20 mg/mL. 2 mL of an aqueous solution containing 2.5 g carrier-free recombinant mouse TGF-β1 (rTGF-β1; 763104, Biolegend, San Diego, CA, USA) were used to create a primary emulsion. Following that, a dropwise addition was undertaken while constantly vortexing the chloroform polymer solution. The formed primary emulsion was sonicated in an ice bath using an Ultrasonic Processor BILON-1000Y (BILON, Shanghai, China) at 60% amplitude for a 10" pulse and three 10" pulses with 20" breaks. Particles were finally hardened for 3 hours at room temperature before being cleaned three times by pelleting at 18, 000 g and resuspension in MilliQ water. Washed NPs were flash-frozen in liquid nitrogen and lyophilized for multiple days to enable long-term storage. The NPs solution was mixed with the MMVs at a protein ratio of 1:10 (w/w, protein to PLGA) and sonicated for 3 minutes in a water bath sonicator (GuTel, Shanghai, China). 2.4 Characterization of NPs, MNP and TMNP The morphology of the MNP and TMNP was observed using a transmission electron microscope (TEM; Hitachi, Japan) after staining with 1% uranyl acetate. The size distribution of the MMVs, NPs, MNP, and TMNP were measured using a ZetaView® Particle Metrix (NTA, Germany), and their zeta potentials were determined with a Malvern Zetasizer (Nano ZS, Malvern). To determine the encapsulation efficiency (EE) and drug loading capacity (DLC) of TGF-β1, TMNP were centrifuged at 15, 000 g for 30 min and dissolved in dimethyl sulfoxide. The amount of TGF-β1 in the TMNP was measured by enzyme linked immunosorbent assay (ELISA) kit (CSB-E04726m; CUSABIO, Wuhan, China) following the manufacturer’s protocol. The EE and DLC were calculated using the following formulas: EE (%) = TGF-β1 encapsulated in TMNP/Total TGF-β1× 100% ; and DLC (%) = TGF-β1 encapsulated in TMNP/Weight of TMNP × 100% . To evaluate the stability of MNP and TMNP in vehicle and blood, the changes in the size of these microspheres were monitored for a week at 4 ℃ and incubated with 50% FBS to observe the changes in the turbidity by comparing the absorbance at 560 nm using a microplate reader, with bare NPs used as a control. Furthermore, to determine the rate of TGF-β1 release from the TMNP, 2 mg of TMNP were suspended in 1 mL of 0.5% (v/v) Tween 80 (in 1×PBS) solution and centrifuged after 1, 2, 6, 12, 24, 48, 72, 96, 144, and 192 hours (3 tubes for each time point) before being tested, with bare MNP used as a control. Finally, the concentration of TGF-β1 was measured using ELISA kits to plot the TGF-β1 release profile. 2.5 Mouse experiments Mice were anesthetized with an intraperitoneal injection of 60 mg/kg sodium pentobarbital and given a third of the initial dosage every 45 minutes to maintain anesthesia. These mice were received oral endotracheal intubation and then allowed to breathe spontaneously (CON) or supported by ventilation using a ventilator (SAR-100, CWE Incorporated, USA). The ventilation rate was set at 80 bpm, and the inspiration to expiration ratio was kept constant at 1:1 in the absence of positive end-expiratory pressure. The HTV and normal tidal volume (NTV) were set to be 20 and 7 mL/kg, respectively. Ventilated mice were randomly assigned to one of five recovery subgroups: 0 hours (end of ventilation, EOV), one day (post-ventilation one day, PV1d), three days (post-ventilation three days, PV3d), seven days (post-ventilation seven days, PV7d), and ten days (post-ventilation ten days, PV10d). There were even numbers of male and female animals in each group ( n = 4). After 4-hours of ventilation, mice in the EOV subgroup were sacrificed. Mice in other subgroups were extubated after spontaneously regaining consciousness and breathing and then sacrificed at 1, 3, 7, and 10 days after extubation, whereas mice in the CON group were euthanized at 10 days after extubation. Bronchoalveolar lavage fluid (BALF) from left lungs, blood serum, and lung tissue was collected and stored at -80 ˚C except for the right-upper and middle lobes, which were obtained for frozen sectioning, TEM examination, and wet/dry (W/D) ratio calculation. 2.6 Administration of Biologics Mice were intravenously administered 40 µg/kg rTGF-β before mechanical ventilation to up-regulate TGF-β1 levels[ 22 , 23 ]. After successfully preparing TMNP or MNP, mice were intravenously injected with 0.5 mg/kg TMNP or MNP. Since the rTGF-β, TMNP, and MNP were all diluted to 50 µL normal saline for injection, the vehicle group of mice was intravenously injected with 50 µL normal saline. According to the result of Breg proportion, we will choose the time points that HTV and NTV-treated mice showed statistical difference on the proportion of Breg in lung for these experiments. 2.7 Cytokine measurements and histological study The W/D weight ratio was calculated to assess the degree of pulmonary edema. The right-upper lung lobe was wiped with filter paper to remove external water before being measured. The lung tissues were dried in an oven at 60˚C for 48 hours before being measured. To investigate changes in pulmonary permeability, the total protein in BALF was quantified with a Pierce™ BCA Protein Assay Kit, and total inflammatory cell infiltration was measured using a cell counter (JIMBIO FILPLUS, Jiangsu, China). IL-1β, IL-6, tumor necrosis factor (TNF)-α, and TGF-β1 levels in serum, BALF, or total lung homogenates were assessed by ELISA kits (CUSABIO, Wuhan, China). The right-lower lung lobe was fixed with 4% paraformaldehyde (30525-89-4; Sigma-Aldrich, St. Louis, MO, USA) and embedded in the Tissue-Tek opti-mum cutting temperature compound (4583, Sakura, Tokyo, Japan). Tissue sections were stained with hematoxylin and eosin. The pathological score of lung injury was determined using previously reported criteria[ 24 ]. To observe the ultrastructural cell changes, appropriate 1 mm 3 lung samples in each group were cut for TEM analysis. 2.8 Single-cell dissociation for flow cytometry (FCM) To prepare a single cell suspension from the lung, the left-upper lung lobe was digested with 0.1 mg/mL Dispase II (4942078001-1g; Roche, Basel, Switzerland), 2000U/mL DNase I (10104159001; Roche, Basel, Switzerland), and 0.2% collagenase (038-22361; Wako, Osaka, Japan) in 10 mM 4-(2-hydroxyerhyl) piperazine-1-erhanesulfonic acid (HEPES) Roswell Park Memorial Institute (RPMI) (22400105; ThermoFisher Scientific, San Jose, CA, USA). The spleen was put in a cell staining buffer (420201; BioLegend, San Diego, CA, USA) and ground by the rough surface of the glass slide to prepare a single cell suspension. After red cell lysis (11814389001; Roche, Basel, Switzerland) and washing twice, cells were passed through a 70-µm strainer and stained with TruStain FcX™ PLUS antibody (S17011E; BioLegend, San Diego, CA, USA). Cells were next incubated with P-phycoerythrin (PE)-conjugated cluster of differentiation (CD) 5 (100608; BioLegend, San Diego, CA, USA), allophycocyanin (APC)-conjugated CD19(11512; BioLegend, San Diego, CA, USA), fluorescein isothiocyanate (FITC)-conjugated CD4 (100510; BioLegend, CA, USA) or APC-conjugated CD8a (100712; BioLegend, CA, USA), CD44- PE-Cyanine7 (PE/Cy7) (560569; BD Biosciences, Franklin Lake, NJ, USA), CD1d- Brilliant™ Blue 515 (BB515) (565956; BD Biosciences, Franklin Lake, NJ, USA), CD5-BB515 (565504; BD Biosciences, Franklin Lake, NJ, USA), C-X3-C Motif Chemokine Receptor 1 (CX3CR1)-PE/Cy7 (149015; BioLegend, San Diego, CA, USA), CD45R/B220-APC/Cy7 (561102; BD Biosciences, Franklin Lake, NJ, USA), CD3e-APC (553066; BD Biosciences, Franklin Lake, NJ, USA), and IgM-PerCP/Cy5.5 (562034; BD Biosciences, Franklin Lake, NJ, USA) on ice for 20 min in the dark. Following fixation and permeabilization, cells were incubated with TGF-β (latency-associated peptide, LAP)-PE (563143; BD Biosciences, Franklin Lake, NJ, USA). Cells were washed twice before being sorted and analyzed using a Beckman Coulter CytoFLEX flow cytometer (Beckman Coulter Commercial Enterprise, Shanghai, China). 2.9 Immunostaining Frozen tissue sections were air dried for 30 min before being fixed with 3.7% paraformaldehyde for 15 min and ice-cold 100% methanol for 15 min. The slides were then blocked with PBS containing 3% bovine serum albumin (SRE0096; Sigma-Aldrich, St. Louis, MO, USA), 3% Goat Serum (16210064; Gibco, San Jose, CA, USA), 0.2% Triton X-100 (X100; Sigma-Aldrich, St. Louis, MO, USA), and 0.02% NaN3 (S2002; Sigma-Aldrich, St. Louis, MO, USA) for 60 min at 37°C, and incubated with anti-CD19 antibody (DF7030; Affinity, Nottingham, UK), Purified anti-mouse LAP (TGF-β1) (141402; BioLegend, San Diego, CA, USA), and CD44 (IM7) Rat mAb (39037, Cell Signaling Technology, MA, USA). Related secondary antibodies included Alexa Fluor 549 Anti-Rabbit IgG H&L antibody (A11037; Invitrogen, Carlsbad, CA, USA), Dylight® 550 Anti-mouse IgG H&L antibody (Ab96880; PythonBio, Guangzhou, China), and Alexa Fluor 488 Anti-Rat IgG H&L antibody (4416, Cell Signaling Technology, MA, USA). Finally, the sections were treated with ProLong®Gold Antifade Reagent with 4', 6-diamidino-2-phenylindole (DAPI) (8961S; Cell Signaling Technology, MA, USA). All sections were analyzed using multiplex confocal microscopy (LSM980, Zeiss, German) 2.10 Bioinformatics The expression matrix of all target genes was uploaded to the Database for Annotation, Visualization, and the Integrated Discovery (DAVID) database for biological processes enrichment and KEGG pathway analysis with count > 5 and P < 0.01. The STRING database was used to assess the interactive relationships of all target genes, and the experimentally validated interactions were defined as statistically different when the combined score ≥ 0.4. 2.11 Statistical analysis. Data were analyzed using StataCorp LP software (STATA Institute, Inc., Collgege Station, TX, USA) and performed using Graphpad Prism 9 (San Diego, CA, USA). One-way ANOVA analyses were used to examine the significance of intergroup differences, and Bonferroni tests were used to analyze pair-wise comparisons. Pearson Correlation analyses between every two factors were performed. A two-tailed P < 0.05 was defined as statistically significant. 3. Results 3.1 CD19 high CD44 high TGF-β1(+) Breg are associated with VILI resolution. To determine the particular Breg phenotype shift that occurred during VILI resolution, wild-type mice were ventilated for 4 hours with NTV or HTV. After 4 hours of post-ventilation, we analyzed the biomarkers of CD19 high TGF-β1 high Breg subsets in the spleen. We found significantly increased expression of CD44 and CX3CR1 but without no significance of CD5, CD45R/B220, and IgM ( Figure S1A ). The function of CD44, a cell-surface receptor involved in T-lymphocyte activation, recirculation, homing, hematopoiesis, inflammation, and response to bacterial infection, was investigated in this study[ 25 ]. CX3CR1 is a receptor for the C-X3-C chemokine fractalkine on many early leukocyte cells, and infiltrating CX3CR1(+) immune cells release mediators to induce injury, stimulate proliferation, and chemoattract inflammatory cell[ 26 ]. We mapped the interaction between CD19, CD44, and TGF-β1 using a protein-protein interaction network, suggesting that these proteins had direct interactions ( Figure S1B ). KEGG pathway analysis showed that these proteins were enriched in the B cell receptor signaling pathway, TGF-β signaling pathway, chronic myeloid leukemia, and pancreatic cancer ( Figure S1C ). These proteins also were enriched in the regulation of epithelial to mesenchymal transition and the TGF-β receptor signaling pathway ( Figure S1D ). Overall, these data suggest that CD19 high CD44 high TGF-β1(+) Breg (abbreviated to TGF-β1(+) Breg) mediates a potent effect in VILI mice. Then, we looked at how TGF-β1(+) Breg altered over time during VILI resolution. Therefore, we assessed the percentage of TGF-β1(+) Bregs in the lung (pBreg) and spleen (sBreg) of EOV, PV1d, PV3d, PV7d, and PV10d subgroups (Fig. 1 A). There was one mouse died in the PV1d subgroup of HTV group, and the probability of survival between the NTV and HTV group was lack of statistical difference ( P = 0.3173). Compared with the mice treated with NTV, the percentages of pBreg in the PV1d and PV10d subgroups of HTV-treated mice were increased (Fig. 1 B and D ). Hence, we choose the PV1d and PV10d as the observation time for the further intervention experiments. Compared with the CON group, the percentages of pBreg at PV3d and PV7d in NTV-treated mice were lower. The percentage of pBreg at PV1d, PV3d, and PV7d in NTV-treated mice was lower than at EOV, but the percentage of pBreg at PV7d was higher than that at PV1d. In the HTV-treated mice, the percentages of pBreg were gradually decreased from EOV to PV7d and minimized at PV7d; and the percentage of pBreg at PV10d was increased to the similar level of EOV. However, sBreg showed higher percentages at the EOV, PV1d, PV3d, and PV10d in HTV-treated mice (Fig. 1 C and D ). The percentage of sBreg at the EOV in NTV-treated mice was higher than that at PV1d, PV3d, and PV10d, as well as the mice of CON group. In the HTV-treated mice, sBreg peaked in the EOV subgroup and then decreased to baseline from PV1d to PV7d. Figure 1 E showed the representative infiltration of pBreg in the PV1d subgroup between NTV or HTV-treated mice. Additionally, we also showed the gating strategy of pBreg and sBreg, as well as the representative FCM plots for the sBreg with statistical differences between NTV or HTV-treated mice in the Figure S2 . 3.2 Resolution of lung injury and inflammation in VILI mice These mice were randomly divided into five groups: CON, NTV1d, HTV1d, NTV10d, and HTV10d. In the mice of HTV1d group, obvious lung tissue injury occurred compared to the other groups. Injured lung tissues showed different degrees of intra-alveolar exudation, extravasated blood, alveolar wall thickening, inflammatory cell accumulation in the lung interstitium, and hyaline membrane formation (Fig. 2 A). Type II alveolar epithelial cells (AEC-II) in the HTV1d group showed different degrees of microvillus disappear, disrupted cytoplasmic and nuclear structure, chromatin margination, increased mitochondrial swelling, and cavitating changes in the lamellar body, as well as cell membrane discontinuities (Fig. 2 B). Compared with CON, NTV1d, NTV10d, and HTV10d groups, mice of HTV1d showed the significant higher pathological scores, W/D ratio, infiltrated cell counts, and total protein levels in BALF. These variables, except W/D ration between the CON and NTV1d group, were both lack of statistical differences across the CON, NTV1d, NTV10d, and HTV10d groups (Fig. 2 C-F). Serum TGF-β1 levels between these five groups were similar, but serum IL-1β level in HTV1d group was higher in comparison with the mice of the CON, NTV10d, and HTV10d groups. The mice of HTV1d showed the significant higher serum IL-6 and TNF-α levels compared with the other groups (Fig. 2 G). TGF-β1 expression in BALF of HTV-treated mice remained higher than in the CON, NTV1d and NTV10d groups. The pulmonary IL-1β and TNF-α levels in the HTV1d group were both higher in comparison with the mice of the other groups; but the pulmonary IL-6 level in the HTV1d group was only higher in comparison with the mice of the CON group (Fig. 2 H). 3.3 HTV induced the proliferation of T cells during VILI resolution. To know the changes in T-cell immunity during VILI resolution, we analyzed the percentage of CD4(+), CD8a(+), CD4(+) CD8a(+) T cells in the lungs and spleen by FCM (Fig. 3 A and B ). When compared to the mice of CON and NTV1d groups, the percentages of pulmonary CD4(+) T cells (pCD4 + TC), pulmonary CD8a (+) T cells (pCD8a + TC), and pulmonary CD4(+) CD8a(+) double positive T cells (pDPTC) in the HTV1d, NTV10d, and HTV10d groups were both increased, and the proportion of these T cells were both peaked in the HTV10d group (Fig. 3 C). Compared with CON group, the percentages of splenic CD4(+) T cells (sCD4 + TC) and splenic CD8a(+) T cells (sCD8a + TC) in NTV10d were lower, yet the percentage of sCD4 + TC in NTV1d was higher than that in the NTV10d and HTV10d groups. Splenic CD4(+) CD8a(+) T cells (sDPTC) proportion in CON group was significantly higher than that in the NTV1d, HTV1d, and NTV10d groups (Fig. 3 D). However, pulmonary and splenic CD4(+)/CD8a(+) T cells ratios in the NTV1d group were both significantly higher than that in the other groups. 3.4 TMNP promotes the resolution of lung injury and inflammation. Exogenous TGF-β1 active ingredient is easily diluted or inactivated, lacks ideal transporter, and has a short biological half-life[ 27 ]. To prolong and stabilize the effect of exogenous TGF-β1, we synthesized the TMNP to package TGF-β1. After cytochalasin B treatment, macrophage actin seemed unstable, and various microvesicles were secreted ( Figure S3A, B) . MMVs maintained the presence of surface proteins such as Flotillin-2; MMVs and MNP both had similar protein banding patterns, but NPs did not contain these proteins ( Figure S3C, D) . NPs, MMVs, and MNP all have a distinct core and shell structure ( Figure S3E) , corresponding with previous research findings[ 28 ]. The size of NPs, MMVs, and MNP was approximately 114.6 ± 36.5 nm, 175.5 ± 96.1 nm, and 190.7 ± 69.6 nm, which both remained constant ( Figure S3F, G) . The zeta potential of MNP was − 26.7 ± 5.4 mV, similar to the natural cell membrane. The EE and DLC of TGF-β1 in TMNP were 35.8% and 8.7%, respectively. TGF-β1 release from TMNP was determined over 24–30 hours ( Figure S3H ), and TMNP were shown to be more stable than NPs ( Figure S3I ). To compare the effect of TMNP and a single dose of rTGF-β1 on regulating lung injury and inflammation in vivo, TMNP and rTGF-β1 were injected intravenously before HTV-ventilation and MNP as control (Fig. 4 A). We found that mice treated with vehicle and MNP had more severe pathological scores, higher W/D ratio, and increased cell counts in BALF at PV1d, while mice treated with TMNP at PV1d had lower pathological scores than mice treated with rTGF-β1. The mice treated with TMNP and rTGF-β1 showed the similar W/D ratio and cell counts in BALF at PV1d. However, the total protein levels across these groups at PV1d were lack of statistical differences. At PV10d, the pathological score, W/D ratio, total protein levels and cell counts in BALF between these groups were both similar (Fig. 4 B-F). As expected, a single administration of rTGF-β1 only induced the elevation of total TGF-β1 levels in serum and lungs at PV1d compared with the mice treated with vehicle ro MNP, but the mice treated with TMNP both showed an increase of total TGF-β1 levels in serum and lungs at PV1d and PV10d (Fig. 4 H). In the TMNP group, serum IL-1β level at PV1d was lower than the vehicle and MNP groups. The mice treated with rTGF-β1 showed the lower serum IL-1β level at PV1d in comparison with the MNP-treated mice. There were no statistical differences in the serum IL-1β level at PV10d across these groups. Mice treated with TMNP showed the lower pulmonary IL-1β level at PV1d and PV10d than the mice of vehicle and MNP groups, and the pulmonary IL-1β level at PV10d in the TMNP group was also lower than rTGF-β group (Fig. 4 I). Mice treated with TMNP showed the lower serum IL-6 level at PV1d and PV10d than the mice of vehicle and MNP groups, but the serum IL-6 level at PV10d was significantly higher than other groups. Pulmonary IL-6 level at PV1d in the TMNP group was lower than vehicle, rTGF-β, and MNP groups, which at PV10d between these groups was lack of statistical differences (Fig. 4 J. Serum TNF-α level in the MNP group was higher than rTGF-β and TMNP groups at PV1d, but pulmonary TNF-α levels at PV1d in the rTGF-β and TMNP groups were both lower than vehicle and MNP groups. There were no statistical differences on serum and pulmonary TNF-α levels at PV1d and PV10d across these groups (Fig. 4 K). 3.5 TMNP play a definite role of Breg proliferation. Since TGF-β1 is the prevalent cytokines that regulates immune cell differentiation, we sought to determine whether TGF-β1 has a role in activating TGF-β1(+) Breg and regulating in T-cell immunity during VILI resolution. We only observed a significant increase of pBreg proportion at PV1d in TMNP group compared with MNP group, and there were no statistical differences noted on the proportions of pBreg at PV10d across these groups (Fig. 5 A). Here we did not find any statistical differences across these groups on the proportions of sBreg at PV1d, but the proportion of sBreg at PV10d in TMNP group was higher than vehicle, rTGF-β, and MNP groups (Fig. 5 B). We also showed the representative FCM plots on the percentages of pBreg and sBreg in the Fig. 5 C, and these results suggested that TMNP play a definite role of Breg proliferation, especially for the multiplication of sBreg at the rehabilitation phase. 3.6 TMNP affects T-cell differentiation. Here we also determined the effect of TMNP on the changes of T-cell immunity. Mice treated with MNP and TMNP showed the reduction of pCD4 + TC proportion and CD4(+)/CD8a(+) T cells ratio, but promoted the proliferation of pCD8a + TC and pDPTC at PV1d and PV10d. Compared with the MNP group, the percentage of pDPTC in the TMNP at PV1d was significantly lower. Compared with the MNP group, TMNP-treated mice showed higher proportion of pCD4 + TC and pDPTC, but lower percentage of pCD8a + TC at PV10d. The pulmonary CD4(+)/CD8a(+) T cells ratio in the rTGF-β group was higher than vehicle group (Fig. 6 A-B). Mice treated with rTGF-β1 showed the higher percentage of sCD4 + and sCD8a + TC at PV1d and PV10d than other three groups. The percentages of sDPTC at PV1d across these groups were similar, but which in the rTGF-β, MNP, and TMNP groups at PV10d were both higher than vehicle group. The splenic CD4(+)/CD8a(+) T cells ratios at PV1d in the MNP and TMNP groups were higher than vehicle group, but the splenic CD4(+)/CD8a(+) T cells ratios at PV10d in the rTGF-β, MNP, and TMNP groups at PV10d were both higher than vehicle group. The percentage of sDPTC and splenic CD4(+)/CD8a(+) T cell ratios at PV10d across the rTGF-β, MNP, and TMNP groups were lack of statistical differences (Fig. 6 C-D). Serum TGF-β level is found to be negatively correlated with IL-6 levels in lung tissue ( Figure S4A) , and sBreg proportion is negatively associated with the pDPTC proportion ( Figure S4B) . pCD8a + TC proportion is negatively correlated with pulmonary CD4(+)/CD8a(+) T cell ratio, but positively associated with the pDPTC proportion ( Figure S4C) , which is negatively associated with the pulmonary CD4(+)/CD8a(+) T cell ratio ( Figure S4D) . sCD4 + TC proportion is positively correlated with sCD8a + TC proportion ( Figure S4E) , but which is negatively associated with splenic CD4(+)/CD8a(+) T cell ratio ( Figure S4F) . sDPTC proportion is both negatively associated with splenic CD4(+)/CD8a(+) T cell ratio ( Figure S4G) . As expected, the W/D ratio is positively associated with the pathological scores ( Figure S4H) , total protein level and cell counts in BALF, as well as serum and pulmonary IL-1β level and serum IL-6 level ( Figure S4I ). Pathological score is positively associated with the total protein level and cell counts in BALF, as well as serum IL-1β level, IL-6 and TNF-α levels ( Figure S4J ). Taken together, sBreg promotes the differentiation of pDPTC that is also produced by the stimulation of pCD8a + TC. sDPTC would restrict the activation of splenic CD4(+) T cells but promote the proliferation of splenic CD8a(+) T cells. Activation of TGF-β1 in lung tissues may be a negative factor of pulmonary IL-6 level. 4. Discussion Breg regulate inflammation and promote tissue repair by releasing chemokines, chemoattractants, and growth factors[ 12 ]. Therefore, effective regulation of Breg via the production of both regulatory cytokines, such as IL-10, IL-35, and TGF-β1, is a prerequisite for the resolution of inflammation and tissue injury[ 29 , 30 ]. Studies have indicated that IL-10-producing Breg promotes the anti-inflammatory process and improve the outcome of inflammation, such as acute myocardial infarction or intestinal inflammation[ 11 , 12 , 31 ]. IL-10-producing Breg are derived from several competent progenitors at unrestricted stages of development to modulate the immune response[ 32 ]. In addition, TGF-β1(+) Breg also produce TGF-β1 to regulate the immune response of Treg or Th1 cells during the development and recovery of inflammatory diseases[ 33 , 34 ]. Although regulating immune cells, cytokines, and other affected materials through Breg are essential for the resolution of inflammation[ 12 , 34 , 35 ], the mechanistic links between Breg phenotype transition and ALI resolution remains unclear. Here, utilizing the murine model of microbiota-independent ALI induced by ventilation, our FCM results and functional characterization of TGF-β1(+) Breg identified a major population of CD19 high CD44(+) TGF-β1(+) Breg that appear highly adjustability and exhibit a definite phenotype during VILI resolution, which were gradually decreased from EOV to PV7d and minimized at PV7d. Due to their ability to regulate T-cell immunity, Breg play an important role in inflammation and management[ 32 – 34 ]. Breg are also essential in that they support Foxp3 + Treg response and population, which depends on the microenvironment and release of specific cytokines such as IL-10 and TGF-β1[ 36 ]. An increased numbers of Breg would convert resting CD4(+) T cells to Treg, preventing host defense against infection while promoting tumor growth and metastasis[ 37 ]. According to the research, IL-10-producing Breg negatively regulate anti-melanoma immunity by reducing Th1 cytokines in tumor-infiltrating CD8(+) T cells[ 38 ]. In this context, we discovered that splenic TGF-β1(+) Breg promote the differentiation of pulmonary CD4(+) CD8a(+) T cells, which are a rarely studied T cell population in immunity to infection or inflammatory diseases[ 39 ]. TGF-β1 is required for the expansion, activation, and effector function of the mature CD4(+) and CD8(+) T cells in the peripheral lymphoid organs and target tissues[ 13 , 14 ]. TGF-β1 has pleiotropic effects on the regulation of regulatory and effector CD4(+) T cell responses[ 40 ]. TGF-β1, on the other hand, would inhibit CD8(+) T cell activation, proliferation, and transcriptional profile[ 41 , 42 ]. TGF-β1 neutralization would also enhance CD8 + T-cell and NK-cell-mediated anti-tumor immune responses[ 43 ]. CD4(+) CD8(+) T cells have the suppressor activity based on the high expression of T cell activity suppressor genes that involved in the TGF-β1 signaling pathway[ 44 ]. Here we did not find any correlation between serum or pulmonary TGF-β1 level and each T cell populations, but pulmonary CD4(+), CD8a (+), and CD4(+) CD8a(+) T cells were increased in the HTV-treated mic at PV1d and PV10d, and these T cells were both peaked at PV10d. NPs can be used in immune therapy to encapsulate active pharmaceutical ingredients and deliver them more efficiently to inflammatory tissues[ 45 ]. MMVs, which are derived from macrophage membranes with rich carbohydrates and transmembrane proteins, exhibit excellent biocompatibility and stability in a physiological environment[ 46 ]. Li R and his colleagues[ 19 ] used MMVs to coat with PLGA NPs encapsulated with tacrolimus, which significantly suppressed the progression of rheumatoid arthritis in mice. Hence, we created MNP to prolong and stabilize the effect of exogenous TGF-β1, and we discovered that TMNP alleviate acute lung injury and inflammation by producing the upregulation of TGF-β1 expression. TMNP play a definite role of Breg proliferation at PV10d, especially for the activation of sBreg, and induced the reduction of pulmonary CD4(+) T cells and CD4(+)/CD8a(+) T cell ratios, but proliferation of pulmonary CD8a + T cells and CD4(+)CD8a(+) T cells. Compared with the MNP group, TMNP-treated mice showed higher pulmonary CD4 + and CD4(+) CD8a(+) T cells, but lower CD8a + T cells in the advanced phase. Notably, our data identify that the non-ignorable effect of TGF-β1(+) Breg was associated with resolution of lung injury and inflammation. Appropriate secretion of TGF-β1 is required to proliferate TGF-β1(+) Breg, reduce inflammation, and resolve lung injury. Furthermore, TMNP can stable-release TGF-β1 to reduce VILI, which may help to prevent cascade inflammation and lung injury in the acute phase. Compared to rTGF-β1 monotherapy, TMNP improved TGF-β1 upregulation and immune homeostasis, which contribute to the attenuation of lung injury and inflammation. However, there were some limitations, such as the absence of adoptive transfer experiments to further explore the role of TGF-β1(+) Breg and the use of Tgfb1 knockout mice to assess the role of endogenous TGF-β1 due to delayed breeding. Unfortunately, we did not evaluate the effect of TNMP or rTGF-β1 on promoting pulmonary fibrosis. The molecular mechanisms between TGF-β1(+) Breg and CD4(+) or CD8(+) T cells require further study. Taken together, TMNP can stable-release TGF-β1 to promote the resolution of inflammatory lung injury, which may be associated with the proliferation of TGF-β1(+) Bregs and regulation of T-cells immunity. These results indicate a potential strategy for preventing the cascade inflammation and lung injury during the acute phase. Abbreviations AEC-II, type II alveolar epithelial cells; ALI, acute lung injury; APC, allophycocyanin; ARDS, acute respiratory distress syndrome; BALF, bronchoalveolar lavage fluid; BB515, Brilliant™ Blue 515; Breg, regulatory B cells; CD, cluster of differentiation; CX3CR1, C-X3-C Motif Chemokine Receptor 1; DLC, drug loading capacity; DAPI, 4', 6-diamidino-2-phenylindole; DMEM, dulbecco's modified eagle medium; EE, encapsulation efficiency; ELISA, enzyme linked immunosorbent assay; EOV, end of ventilation; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HTV, high tidal volume; HEPES, 4-(2-hydroxyerhyl) piperazine-1-erhanesulfonic acid; IL, interleukin; MMVs, macrophage-derived microvesicles; MNP, MMVs membrane-coated nanoparticles; NTV, normal tidal volume; pBreg, TGF-β1(+) Bregs in the lung; pCD4+ TC, pulmonary CD4(+) T cells; pCD8a+ TC, pulmonary CD8a (+) T cells; pDPTC, pulmonary CD4(+) CD8a(+) double positive T cells; PBS, phosphate buffer saline; PE, P-phycoerythrin; PE/Cy7, PE-Cyanine7; PV1d, post-ventilation one day; PV3d, post-ventilation three days; PV7d, post-ventilation seven days; PV10d, post-ventilation ten days; rTGF-β1, recombinant mouse TGF-β1; RPMI, Roswell Park Memorial Institute; sBreg, TGF-β1(+) Bregs in the spleen; sCD4+ TC, splenic CD4(+) T cells; sCD8a+ TC, splenic CD8a (+) T cells; sDPTC, splenic CD4(+) CD8a(+) double positive T cells; TEM, transmission electron microscope; TGF-β1, transforming growth factor-β1; TMNP, MNP to package TGF-β1; TNF, tumor necrosis factor; Treg, regulatory T cells; VILI, ventilator-induced lung injury; W/D, wet/dry ratio. Declarations Disclosure The authors report no conflicts of interest in this work. Consent for Publication All authors have reviewed and agreed to publish this paper. Author Contributions Linghui Pan: Conceptualization, Methodology, Supervision, Funding acquisition, and Writing-review and editing; Ren Jing: Experiments, Data curation, Funding acquisition, and Writing- Original draft, review, and editing; Xiaoting Liao: Experiments, Data curation, Software; Jianlan Mo: Experiments, Data curation, Software; Sheng He: Supervision, Software; Xianlong Xie: Visualization, Investigation; Huijun Dai: Writing- Reviewing and Editing; Zhaokun Hu: Validation, Software. Funding Sources This work was supported by grants from the National Natural Science Foundation of China (81970078), Guangxi Anesthesiology Clinical Medicine Research Center Construction Project (scientific foundation of Guangxi No: 2022AC04002), and Guangdong Province Basic and Applied Basic Research Fund Joint Fund-Youth Project (2023A1515110149). Data Availability Not applicable. References Meyer NJ, Gattinoni L, Calfee CS. Acute respiratory distress syndrome[J]. Lancet (London England). 2021;398(10300):622–37. 10.1016/S0140-6736(21)00439-6 . Perez-Fernandez XL, Sabater-Riera J, Fuset-Cabanes M. COVID-19 ARDS: getting ventilation right[J]. Lancet (London England). 2022;399(10319):22. 10.1016/S0140-6736(21)02439-9 . Nieman GF, Gatto LA, Andrews P, et al. Prevention and treatment of acute lung injury with time-controlled adaptive ventilation: physiologically informed modification of airway pressure release ventilation[J]. Ann Intensive Care. 2020;10(1):3. 10.1186/s13613-019-0619-3 . Villar J, Zhang H, Slutsky AS. Lung Repair and Regeneration in ARDS: Role of PECAM1 and Wnt Signaling[J]. Chest. 2019;155(3):587–94. 10.1016/j.chest.2018.10.022 . Slutsky AS, Ranieri VM. Ventilator-induced lung injury[J]. N Engl J Med. 2013;369(22):2126–36. 10.1056/NEJMra1208707 . Albert RK. Constant Vt Ventilation and Surfactant Dysfunction: An Overlooked Cause of Ventilator-induced Lung Injury[J]. Am J Respir Crit Care Med. 2022;205(2):152–60. 10.1164/rccm.202107-1690CP . Menon M, Hussell T, Ali Shuwa H. Regulatory B cells in respiratory health and diseases[J]. Immunol Rev. 2021;299(1):61–73. 10.1111/imr.12941 . Alhabbab RY, Nova-Lamperti E, Aravena O, et al. Regulatory B cells: Development, phenotypes, functions, and role in transplantation[J]. Immunol Rev. 2019;292(1):164–79. 10.1111/imr.12800 . Cherukuri A, Mohib K, Rothstein DM, Regulatory B. cells: TIM-1, transplant tolerance, and rejection[J]. Immunol Rev. 2021;299(1):31–44. 10.1111/imr.12933 . Habener A, Happle C, Grychtol R et al. Regulatory B cells control airway hyperreactivity and lung remodeling in a murine asthma model[J]. J Allergy Clin Immunol. 2021;147(6):2281-94 e7. 10.1016/j.jaci.2020.09.041 . Radomir L, Kramer MP, Perpinial M, et al. The survival and function of IL-10-producing regulatory B cells are negatively controlled by SLAMF5[J]. Nat Commun. 2021;12(1):1893. 10.1038/s41467-021-22230-z . Wu L, Dalal R, Cao CD, et al. IL-10-producing B cells are enriched in murine pericardial adipose tissues and ameliorate the outcome of acute myocardial infarction[J]. Proc Natl Acad Sci U S A. 2019;116(43):21673–84. 10.1073/pnas.1911464116 . Huai G, Markmann JF, Deng S, et al. TGF-beta-secreting regulatory B cells: unsung players in immune regulation[J]. Clin Transl Immunol. 2021;10(4):e1270. 10.1002/cti2.1270 . Batlle E, Massague J. Transforming Growth Factor-beta Signaling in Immunity and Cancer[J]. Immunity. 2019;50(4):924 – 40. Immunity. 2019;50(4):924 – 40. Xie Y, Qian Y, Wang Y, et al. Mechanical stretch and LPS affect the proliferation, extracellular matrix remodeling and viscoelasticity of lung fibroblasts[J]. Exp Ther Med. 2020;20(5):5. 10.3892/etm.2020.9133 . Huang LT, Chou HC, Chen CM. Inhibition of FABP4 attenuates hyperoxia-induced lung injury and fibrosis via inhibiting TGF-beta signaling in neonatal rats[J]. J Cell Physiol. 2022;237(2):1509–20. 10.1002/jcp.30622 . Chen X, Peng W, Zhou R, et al. Montelukast improves bronchopulmonary dysplasia by inhibiting epithelialmesenchymal transition via inactivating the TGFbeta1/Smads signaling pathway[J]. Mol Med Rep. 2020;22(3):2564–72. 10.3892/mmr.2020 . Curley GF, Contreras M, Higgins B, et al. Evolution of the inflammatory and fibroproliferative responses during resolution and repair after ventilator-induced lung injury in the rat[J]. Anesthesiology. 2011;115(5):1022–32. 10.1097/ALN.0b013e31823422c9 . Li R, He Y, Zhu Y, et al. Route to Rheumatoid Arthritis by Macrophage-Derived Microvesicle-Coated Nanoparticles[J]. Nano Lett. 2019;19(1):124–34. 10.1021/acs.nanolett.8b03439 . Park JS, Park K, Woo DG, et al. PLGA microsphere construct coated with TGF-beta 3 loaded nanoparticles for neocartilage formation[J]. Biomacromolecules. 2008;9(8):2162–9. 10.1021/bm800251x . Hu CM, Zhang L, Aryal S, et al. Erythrocyte membrane-camouflaged polymeric nanoparticles as a biomimetic delivery platform[J]. Proc Natl Acad Sci U S A. 2011;108(27):10980–5. 10.1073/pnas.1106634108 . Zhang XY, Liu ZM, Zhang HF, et al. TGF-beta1 improves mucosal IgA dysfunction and dysbiosis following intestinal ischaemia-reperfusion in mice[J]. J Cell Mol Med. 2016;20(6):1014–23. 10.1111/jcmm.12789 . Taylor RA, Chang CF, Goods BA, et al. TGF-beta1 modulates microglial phenotype and promotes recovery after intracerebral hemorrhage[J]. J Clin Invest. 2017;127(1):280–92. 10.1172/JCI88647 . Lin JY, Jing R, Lin F, et al. High Tidal Volume Induces Mitochondria Damage and Releases Mitochondrial DNA to Aggravate the Ventilator-Induced Lung Injury[J]. Front Immunol. 2018;9:1477. 10.3389/fimmu.2018.01477 . Klement JD, Paschall AV, Redd PS, et al. An osteopontin/CD44 immune checkpoint controls CD8 + T cell activation and tumor immune evasion[J]. J Clin Invest. 2018;128(12):5549–60. 10.1172/JCI123360 . Greiffo FR, Viteri-Alvarez V, Frankenberger M, et al. CX3CR1-fractalkine axis drives kinetic changes of monocytes in fibrotic interstitial lung diseases[J]. Eur Respir J. 2020;55(2):1900460. 10.1183/13993003.00460-2019 . Li M, Zeng L, Liu S, et al. Transforming Growth Factor-beta Promotes Homing and Therapeutic Efficacy of Human Mesenchymal Stem Cells to Glioblastoma[J]. J Neuropathol Exp Neurol. 2019;78(4):315–25. 10.1093/jnen/nlz016 . Sirbulescu RF, Mamidi A, Chan SC, et al. B cells support the repair of injured tissues by adopting MyD88-dependent regulatory functions and phenotype[J]. FASEB J. 2021;35(12):e22019. 10.1096/fj.202101095RR . Wang Z, Zhou Y, Yu Y, et al. Lipopolysaccharide preconditioning increased the level of regulatory B cells in the spleen after acute ischaemia/reperfusion in mice[J]. Brain Res. 2018;1701:46–57. 10.1016/j.brainres.2018.05.036 . Mishima Y, Oka A, Liu B, et al. Microbiota maintain colonic homeostasis by activating TLR2/MyD88/PI3K signaling in IL-10-producing regulatory B cells[J]. J Clin Invest. 2019;129(9):3702–16. 10.1172/JCI93820 . Sun G, Wang Y, Ti Y, et al. Regulatory B cell is critical in bone union process through suppressing proinflammatory cytokines and stimulating Foxp3 in Treg cells[J]. Clin Exp Pharmacol Physiol. 2017;44(4):455–62. 10.1111/1440-1681.12719 . Michee-Cospolite M, Boudigou M, Grasseau A, et al. Molecular Mechanisms Driving IL-10- Producing B Cells Functions: STAT3 and c-MAF as Underestimated Central Key Regulators?[J]. Front Immunol. 2022;13:818814. 10.3389/fimmu.2022.818814 . Gao X, Ren X, Wang Q, et al. Critical roles of regulatory B and T cells in helminth parasite-induced protection against allergic airway inflammation[J]. Clin Exp Immunol. 2019;198(3):390–402. 10.1111/cei.13362 . Horii M, Matsushita T. Regulatory B cells and T cell Regulation in Cancer[J]. J Mol Biol. 2021;433(1):166685. 10.1016/j.jmb.2020.10.019 . Tian J, Zekzer D, Hanssen L, et al. Lipopolysaccharide-activated B cells down-regulate Th1 immunity and prevent autoimmune diabetes in nonobese diabetic mice[J]. J Immunol. 2001;167(2):1081–9. 10.4049/jimmunol.167.2.1081 . Matsushita T, Le Huu D, Kobayashi T et al. A novel splenic B1 regulatory cell subset suppresses allergic disease through phosphatidylinositol 3-kinase-Akt pathway activation[J]. J Allergy Clin Immunol. 2016;138(4):1170-82 e9. 10.1016/j.jaci.2015.12.1319 . Kinker GS, Vitiello GAF, Ferreira WAS, et al. B Cell Orchestration of Anti-tumor Immune Responses: A Matter of Cell Localization and Communication[J]. Front Cell Dev Biol. 2021;9:678127. 10.3389/fcell.2021.678127 . Kobayashi T, Oishi K, Okamura A, et al. Regulatory B1a Cells Suppress Melanoma Tumor Immunity via IL-10 Production and Inhibiting T Helper Type 1 Cytokine Production in Tumor-Infiltrating CD8(+) T Cells[J]. J Invest Dermatol. 2019;139(7):1535–e15441. 10.1016/j.jid.2019.02.016 . Overgaard NH, Jung JW, Steptoe RJ, et al. CD4+/CD8 + double-positive T cells: more than just a developmental stage?[J]. J Leukoc Biol. 2015;97(1):31–8. 10.1189/jlb.1RU0814-382 . Travis MA, Sheppard D. TGF-beta activation and function in immunity[J]. Annu Rev Immunol. 2014;32:51–82. 10.1146/annurev-immunol-032713-120257 . Schwartzkopff S, Woyciechowski S, Aichele U, et al. TGF-beta downregulates KLRG1 expression in mouse and human CD8(+) T cells[J]. Eur J Immunol. 2015;45(8):2212–7. 10.1002/eji.201545634 . Mishra S, Liao W, Liu Y, et al. TGF-beta and Eomes control the homeostasis of CD8 + regulatory T cells[J]. J Exp Med. 2021;218(1):e20200030. 10.1084/jem.20200030 . Yang L, Pang Y, Moses HL. TGF-beta and immune cells: an important regulatory axis in the tumor microenvironment and progression[J]. Trends Immunol. 2010;31(6):220–7. 10.1016/j.it.2010.04.002 . Dai M, Zhao L, Li Z, et al. The Transcriptional Differences of Avian CD4(+)CD8(+) Double-Positive T Cells and CD8(+) T Cells From Peripheral Blood of ALV-J Infected Chickens Revealed by Smart-Seq2[J]. Front Cell Infect Microbiol. 2021;11:747094. 10.3389/fcimb.2021.747094 . Loo YS, Bose RJ, McCarthy JR, et al. Biomimetic bacterial and viral-based nanovesicles for drug delivery, theranostics, and vaccine applications[J]. Drug Discov Today. 2021;26(4):902–15. 10.1016/j.drudis.2020.12.017 . Wang H, Liu Y, He R, et al. Cell membrane biomimetic nanoparticles for inflammation and cancer targeting in drug delivery[J]. Biomater Sci. 2020;8(2):552–68. 10.1039/c9bm01392j . Additional Declarations No competing interests reported. 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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-4421925","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":308075706,"identity":"40c1f87e-811d-420d-82a3-c216a5956936","order_by":0,"name":"Ren Jing","email":"","orcid":"","institution":"Guangxi Medical University Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ren","middleName":"","lastName":"Jing","suffix":""},{"id":308075707,"identity":"1dfeba68-be28-42f3-b139-50b808d72ca7","order_by":1,"name":"Xiaoting Liao","email":"","orcid":"","institution":"Guangxi Maternal and Child Health Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaoting","middleName":"","lastName":"Liao","suffix":""},{"id":308075708,"identity":"aa8d33fc-237d-49f9-8373-2cd5e6620c4c","order_by":2,"name":"Jianlan Mo","email":"","orcid":"","institution":"Guangxi Maternal and Child Health Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jianlan","middleName":"","lastName":"Mo","suffix":""},{"id":308075709,"identity":"4e21fcc2-6e08-4c77-9783-baaedbd9da8e","order_by":3,"name":"Sheng He","email":"","orcid":"","institution":"Guangxi Medical University Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Sheng","middleName":"","lastName":"He","suffix":""},{"id":308075710,"identity":"968b12e0-8906-40e2-84e1-ec9518f5ea6d","order_by":4,"name":"Xianlong Xie","email":"","orcid":"","institution":"Guangxi Medical University Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xianlong","middleName":"","lastName":"Xie","suffix":""},{"id":308075711,"identity":"13243b48-9754-4846-8933-5f053c89495d","order_by":5,"name":"Huijun Dai","email":"","orcid":"","institution":"Guangxi Medical University Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Huijun","middleName":"","lastName":"Dai","suffix":""},{"id":308075712,"identity":"88e1311a-6a55-4c8a-a8bd-0c20476a392a","order_by":6,"name":"Zhaokun Hu","email":"","orcid":"","institution":"Guangxi Medical University Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhaokun","middleName":"","lastName":"Hu","suffix":""},{"id":308075714,"identity":"d0a25ed8-dbad-43b4-8ba5-1e7a040f1fd8","order_by":7,"name":"Linghui Pan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIie3OrwvCQBTA8RsHs9xYncW/4VmUYfBf2WGwHCJY1TjLNLv/QsswPjnQMrEaFcGiQTGtuc0fbZtR8L7hbYz34Y0Qleo309AhxCGU4uuDU2xeRH+vfkOeawy+I/XSBvGwGHRgze5nQ0hilgSQaJFNbK/jIA/XPZBGYPuBJGXvAto4zCaAApC7Kz6LCdxiAjsBVHNzyPbyJuwEPCbNQrJLr/QTQvfpFauA2NP0CnJf6jXND9rMCk/d5TiH1E1RPUTukE+28ng3gkbFHLXm+yjvx54PmQzdigdL3jAbfMgwGfSat6lSqVT/2wO6eFw6qGo6QAAAAABJRU5ErkJggg==","orcid":"","institution":"Guangxi Medical University Cancer Hospital","correspondingAuthor":true,"prefix":"","firstName":"Linghui","middleName":"","lastName":"Pan","suffix":""}],"badges":[],"createdAt":"2024-05-15 01:54:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4421925/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4421925/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57521156,"identity":"9f9103a1-3bb8-498d-a734-3673e2bcdcbe","added_by":"auto","created_at":"2024-05-31 21:54:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3833446,"visible":true,"origin":"","legend":"\u003cp\u003eDefinite Breg phenotype is involved in VILI. (A) Experimental design. (B) Pulmonary proportion of TGF-β1(+) Breg examined by FCM. (C) Splenic proportion of TGF-β1(+) Breg examined by FCM. (D) Representative flow plots showing proportion of pulmonary TGF-β1(+) Breg from mice challenged with NTV or HTV in the PV1d and PV10d subgroups. (E) In vivo imaging of TGF-β1(+) Breg using high-resolution confocal microscopy from mice challenged with NTV or HTV in the PV1d subgroup. (Scale bar, 10 μm). Data shown as mean±SD. \u003cem\u003en\u003c/em\u003e = 4 mice per time point. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 vs. NTV, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 vs. NTV, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001 vs. NTV; *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001. NV or NTV, normal tidal volume; HTV, high tidal volume; PV, post-ventilation with HTV.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4421925/v1/bec0dbd4ceffaeaf7dc34098.png"},{"id":57521155,"identity":"2f9da4e6-8efc-41f9-84f0-cb9914ca3c62","added_by":"auto","created_at":"2024-05-31 21:54:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4490749,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges of lung injury and inflammation after 4-hours ventilation. \u003c/strong\u003e(A) Representative photomicrographs of lung tissues with H\u0026amp;E staining (original magnification×100) in each subgroup. (Scale bar, 100 μm.) (B) Ultrastructural changes of AEC-II evaluated by transmission electron microscope in mice in each group. (Scale bar, 5 μm.) (C) Pathological scores. (D) Wet/dry weight ratios. (E) Total protein levels in BALF. (F) Cell counts in BALF. (G) Total TGF-β1, IL-1β, IL-6, and TNF-α levels in serum. (H) Total TGF-β1, IL-1β, IL-6, and TNF-α levels in BALF. Data shown as mean±SD. \u003cem\u003en\u003c/em\u003e = 4 mice per time point. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001. NTV, normal tidal volume; HTV, high tidal volume; CON, control group; NTV1d, mice treated with 4-hour ventilation with NTV follow by the recovery for 1 day; NTV10d, mice treated with 4-hour ventilation with NTV follow by the recovery for 10 days; HTV1d, mice treated with 4-hour ventilation with HTV follow by the recovery for 1 day; HTV10d, mice treated with 4-hour ventilation with HTV follow by the recovery for 10 days.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4421925/v1/820eb5742708cab65dfdfeb2.png"},{"id":57521278,"identity":"5f8326b9-45ea-41b1-ac58-020ff6af1bc2","added_by":"auto","created_at":"2024-05-31 22:02:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2034676,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChangesof T-cell immunity after 4-hours ventilation. \u003c/strong\u003e(A) Representative flow plots showing proportion of pulmonary T-cells immunity from mice challenged with NTV or HTV at PV1d and PV10d. (B) Representative flow plots showing proportion of splenic T-cells immunity from mice challenged with NTV or HTV at PV1d and PV10d. (C) Pulmonary proportion of CD4(+), CD8a(+), CD4(+)CD8a(+) T cells, and CD4(+)/CD8a(+) T cell ratios. (D) Splenic proportion of CD4(+), CD8a(+), and CD4(+)CD8a(+) T cells. Data shown as mean±SD. \u003cem\u003en\u003c/em\u003e = 4 mice per time point. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001. CON, control group; WT, wild-type; NV or NTV, normal tidal volume; HTV, high tidal volume; NTV1d, mice treated with 4-hour ventilation with NTV follow by the recovery for 1 day; NTV10d, mice treated with 4-hour ventilation with NTV follow by the recovery for 10 days; HTV1d, mice treated with 4-hour ventilation with HTV follow by the recovery for 1 day; HTV10d, mice treated with 4-hour ventilation with HTV follow by the recovery for 10 days.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4421925/v1/8907df8007b381adbf2d8495.png"},{"id":57521158,"identity":"d638a713-eca4-47e9-a058-08d9b34f3932","added_by":"auto","created_at":"2024-05-31 21:54:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3759789,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges of lung injury and inflammation after 4-hours ventilation in rTGF-β1 and TMNP treated mice. \u003c/strong\u003e(A) Experimental design. (B) Representative photomicrographs of lung tissues with H\u0026amp;E staining (original magnification×100) in each subgroup. (Scale bar, 100 μm.) (C) Ultrastructural changes of AEC-II evaluated by transmission electron microscope in mice in each group. (Scale bar, 1 μm.) (D) Pathological scores at PV1d and PV10d. (E) Wet/dry weight ratios at PV1d and PV10d. (F) Total protein levels in BALF at PV1d and PV10d. (G) Cell counts in BALFat PV1d and PV10d. (H) Serum and pulmonary total TGF-β1 levels at PV1d and PV10d. (I) Serum and pulmonary IL-1β levels at PV1d and PV10d. (J) Serum and pulmonary IL-6 levels at PV1d and PV10d. (K) Serum and pulmonary TNF-α levels at PV1d and PV10d. Data shown as mean±SD. \u003cem\u003en\u003c/em\u003e = 4 mice per time point. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001. rTGF-β, micetreated with recombinanttransforming growth factor-β1 before 4-hour ventilation with HTV follow by the recovery for 1 and 10 days; MNP, mice treated with macrophage-derived microvesicles membrane-coated nanoparticles before 4-hour ventilation with HTV follow by the recovery for 1 and 10 days; TMNP, mice treated with macrophage-derived microvesicles membrane-coated nanoparticles loaded TGF-β1 before 4-hour ventilation with HTV follow by the recovery for 1 and 10 days.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4421925/v1/f372e5e92ce1ff5443c676b0.png"},{"id":57521164,"identity":"a26a18f3-7126-44c6-9c31-e53d56d5d446","added_by":"auto","created_at":"2024-05-31 21:54:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2040015,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges of pulmonary and splenic TGF-β1(+) Breg after 4-hours ventilation in rTGF-β1 and TMNP treated mice. \u003c/strong\u003e(A) Pulmonary proportion of TGF-β1(+) Breg examined by FCM at PV1d and PV10d. (B) Splenic proportion of TGF-β1(+) Breg examined by FCM at PV1d and PV10d. (C) Representative flow plots showing proportion of pulmonary and splenic TGF-β1(+) Breg from mice challenged with NTV or HTV in the PV1d and PV10d. Data shown as mean±SD. \u003cem\u003en\u003c/em\u003e = 4 mice per time point. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001. rTGF-β, mice treated with recombinant transforming growth factor-β1 before 4-hour ventilation with HTV follow by the recovery for 1 and 10 days; MNP, mice treated with macrophage-derived microvesicles membrane-coated nanoparticles before 4-hour ventilation with HTV follow by the recovery for 1 and 10 days; TMNP, mice treated with macrophage-derived microvesicles membrane-coated nanoparticles loaded TGF-β1 before 4-hour ventilation with HTV follow by the recovery for 1 and 10 days.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4421925/v1/25a8d7afe8a3da26fdede3a0.png"},{"id":57521522,"identity":"6bf7f029-4bd6-4ca9-87e4-558f45ad926c","added_by":"auto","created_at":"2024-05-31 22:10:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2233130,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eT cells immunity is observed after 4-hours ventilation in rTGF-β1 and TMNP treated mice. \u003c/strong\u003e(A) Representative flow plots and proportion of pulmonary T cells at PV1d. (B) Representative flow plots and proportion of pulmonary T cells at PV10d. (C) Representative flow plots and proportion of splenic T cells at PV1d. (D) Representative flow plots and proportion of splenic T cells at PV10d. Data shown as mean±SD. \u003cem\u003en\u003c/em\u003e = 4 mice per time point. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001. rTGF-β, mice treated with recombinanttransforming growth factor-β1 before 4-hour ventilation with HTV follow by the recovery for 1 and 10 days; MNP, mice treated with macrophage-derived microvesicles membrane-coated nanoparticles before 4-hour ventilation with HTV follow by the recovery for 1 and 10 days; TMNP, mice treated with macrophage-derived microvesicles membrane-coated nanoparticles loaded TGF-β1 before 4-hour ventilation with HTV follow by the recovery for 1 and 10 days.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4421925/v1/3ca494d399a8930f8e3139fe.png"},{"id":86844532,"identity":"80e6f741-51f6-4dd6-b321-8389fd9d7177","added_by":"auto","created_at":"2025-07-16 08:32:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20912227,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4421925/v1/77eae1d5-9b3d-483e-a96c-21b47de4c607.pdf"},{"id":57521160,"identity":"12a9a69e-82f4-4774-9160-7776b925c2aa","added_by":"auto","created_at":"2024-05-31 21:54:29","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":820900,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4421925/v1/f475ccc456574f4156f61fb8.tif"},{"id":57521277,"identity":"e7d97dc6-7af3-4096-894e-d7eb6a4f3fc1","added_by":"auto","created_at":"2024-05-31 22:02:29","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2346748,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-4421925/v1/ea969f030fb82697685fdf51.tif"},{"id":57521163,"identity":"159d3067-e6be-4db6-8580-a8e1a7573367","added_by":"auto","created_at":"2024-05-31 21:54:29","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":6631240,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-4421925/v1/e0bf26de3f6a8869709a228a.tif"},{"id":57521165,"identity":"e57aafe1-894c-46ae-ba69-1daeb5107e5b","added_by":"auto","created_at":"2024-05-31 21:54:29","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":497320,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-4421925/v1/1c5ebb86d5c5e5a2ba63be0e.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Macrophage-derived Microvesicles Coated Nanoparticles Loaded with TGF-β1 Resolve Acute Lung Injury via Regulatory B Cell Activation","fulltext":[{"header":"Summary statement","content":"\u003cp\u003eA substantial proportion of patients on mechanical ventilation suffer acute lung injury. Here, macrophage-derived microvesicle-coated nanoparticles packaging TGF-\u0026beta;1 were synthesized to prolong exogenous TGF-\u0026beta;1, which can stable-release TGF-\u0026beta;1 to withhold the cascade acute lung injury via regulatory B cell activation.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eAcute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are caused by severe shock, trauma, and infection, notably during the COVID-19 pandemic, and have a high morbidity and mortality rate[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Mechanical ventilation is the most often used ARDS respiratory support therapy, although it also causes ALI or worsens the original injury (ventilator-induced lung injury, VILI)[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The mechanisms of VILI involve three biophysical factors: volutrauma, barotrauma, and atelectrauma[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Cytokines, surfactants, and immune cells are all biochemical components that contribute to the pathogenesis of VILI[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, the underlying mechanisms through which immune response contributes to the recovery of lung injury and inflammation are not fully known.\u003c/p\u003e \u003cp\u003eRegulatory B cells (Breg) undergo immunosuppression during inflammation and immunological response[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This process is traditionally defined by a release of interleukin (IL)-10, IL-35, and transforming growth factor-β1 (TGF-β1) to induce death ligand-mediated apoptosis and regulate such immune cells as an effector or regulatory T cells (Treg)[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Despite their inability to entirely eliminate the inflammation, an increase in Breg is beneficial in preventing excessive inflammation and tissue damage, showing that abnormalities in Breg numbers and function are associated with immune homeostasis[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Breg help has long been known to be essential for sustaining IL-10 function during the inhibition of inflammation and immune response[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Besides, Breg-produced TGF-β1 also has an important role in regulating T-cell immunity in inflammatory diseases[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, the underlying mechanisms by which Breg-derived TGF-β1 coordinates immune response during VILI remain incompletely understood.\u003c/p\u003e \u003cp\u003eTGF-β1 is required for immune cell development and maturation in order to maintain immune homeostasis and regulate tissue repair[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Several studies have reported a critical role of TGF-β1 in the attenuation of hyperoxia-induced ALI[\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In high tidal volume (HTV)-ventilated rats, alveolar TGF-β1 concentrations similarly revealed a significant trend over 24 hours[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. To mimic the recovery process of surgical patients undergoing mechanical ventilation, we defined the acute phase as the period between immediate post-ventilation and post-ventilation day 3, and the rehabilitation phase as the period from post-ventilation day 3 to post-ventilation day 10. In this study, we showed that specific Breg phenotype secretes TGF-β1, which is also essential for T-cell immunity; further, it promotes the resolution of VILI. In addition, we synthesized the macrophage-derived microvesicles (MMVs) membrane-coated nanoparticles (MNP) to package TGF-β1 (TMNP) to prolong and stabilize the effect of exogenous TGF-β1.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Mice\u003c/h2\u003e \u003cp\u003eMale or female C57BL/6 mice, aged 4\u0026ndash;6 weeks and weighted 25\u0026thinsp;\u0026plusmn;\u0026thinsp;5 g, were obtained through the Animal Center of Guangxi Medical University (Nanning, China). The study protocol was approved by the Animal Care and Use Committee of Guangxi Medical University (KY-2022-288) and conducted under the guidelines for the care and use of animals set by the Chinese government.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation of MMVs\u003c/h2\u003e \u003cp\u003eRAW 264.7 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in dulbecco's modified eagle medium (DMEM, 12491015; Gibco, San Jose, CA, USA) supplemented with 10% fetal bovine serum (FBS,10099; Gibco, San Jose, CA, USA), 1% penicillin (100 IU/mL; Corning, NY, USA) and streptomycin (100 \u0026micro;g/mL; Corning, NY, USA). MMVs were prepared using a previously reported method[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Briefly, the cells were washed twice with phosphate buffer saline (PBS; P3813, Sigma-Aldrich, St. Louis, MO, USA) and incubated in 5 mL of serum-free DMEM with 10 \u0026micro;g/ml cytochalasin B (ab143482; Abcam, Cambridge, UK) for 1 hour at 37\u0026deg;C. The cells were dyed with DiO' (Invitrogen, San Jose, CA, USA) and subjected to confocal laser scanning microscopy to examine the formation of MMVs. The cells without cytochalasin B treatment were applied as a control. After detaching the cells and MMVs from the culture dish, they were rinsed with 5 ml of DMEM and vortexed to isolate the MMVs. Afterward, five milliliters of FBS were added to achieve a final FBS concentration of 50%. The suspension was centrifuged at 5, 000 g for 10 min to remove the cells and large MMVs aggregates. Next, the supernatant was centrifuged at 17, 000 g for 15 min to collect the MMVs. To prepare the purified MMVs, the collected MMVs were washed twice with MilliQ water (IPAKKITA1; Millipore, Burlington, MA, USA) containing 0.25% ethylenedinitrilotetraacetic acid (ED, Sigma-Aldrich, St. Louis, MO, USA). The protein quantity of the final MMVs was assessed by Pierce\u0026trade; BCA protein assay kit (23225; Thermofisher Scientific, Waltham, MA, USA) and immunoblotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Preparation of nanoparticles (NPs), MNP and TMNP\u003c/h2\u003e \u003cp\u003eMNP were synthesized using a previously described method[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. First, 0.67 dL/g carboxy-terminated 50:50 poly (lactic-co-glycolic acid) (PLGA; B6001-1, LACTEL Absorbable Polymers, Birmingham, AL, USA) was used to form PLGA NPs cores. PLGA was dissolved in chloroform at a concentration of 20 mg/mL. 2 mL of an aqueous solution containing 2.5 g carrier-free recombinant mouse TGF-β1 (rTGF-β1; 763104, Biolegend, San Diego, CA, USA) were used to create a primary emulsion. Following that, a dropwise addition was undertaken while constantly vortexing the chloroform polymer solution. The formed primary emulsion was sonicated in an ice bath using an Ultrasonic Processor BILON-1000Y (BILON, Shanghai, China) at 60% amplitude for a 10\" pulse and three 10\" pulses with 20\" breaks. Particles were finally hardened for 3 hours at room temperature before being cleaned three times by pelleting at 18, 000 g and resuspension in MilliQ water. Washed NPs were flash-frozen in liquid nitrogen and lyophilized for multiple days to enable long-term storage. The NPs solution was mixed with the MMVs at a protein ratio of 1:10 (w/w, protein to PLGA) and sonicated for 3 minutes in a water bath sonicator (GuTel, Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Characterization of NPs, MNP and TMNP\u003c/h2\u003e \u003cp\u003eThe morphology of the MNP and TMNP was observed using a transmission electron microscope (TEM; Hitachi, Japan) after staining with 1% uranyl acetate. The size distribution of the MMVs, NPs, MNP, and TMNP were measured using a ZetaView\u0026reg; Particle Metrix (NTA, Germany), and their zeta potentials were determined with a Malvern Zetasizer (Nano ZS, Malvern). To determine the encapsulation efficiency (EE) and drug loading capacity (DLC) of TGF-β1, TMNP were centrifuged at 15, 000 g for 30 min and dissolved in dimethyl sulfoxide. The amount of TGF-β1 in the TMNP was measured by enzyme linked immunosorbent assay (ELISA) kit (CSB-E04726m; CUSABIO, Wuhan, China) following the manufacturer\u0026rsquo;s protocol. The EE and DLC were calculated using the following formulas: \u003cb\u003eEE (%)\u0026thinsp;=\u0026thinsp;TGF-β1 encapsulated in TMNP/Total TGF-β1\u0026times; 100%\u003c/b\u003e; and \u003cb\u003eDLC (%)\u0026thinsp;=\u0026thinsp;TGF-β1 encapsulated in TMNP/Weight of TMNP \u0026times; 100%\u003c/b\u003e. To evaluate the stability of MNP and TMNP in vehicle and blood, the changes in the size of these microspheres were monitored for a week at 4 ℃ and incubated with 50% FBS to observe the changes in the turbidity by comparing the absorbance at 560 nm using a microplate reader, with bare NPs used as a control. Furthermore, to determine the rate of TGF-β1 release from the TMNP, 2 mg of TMNP were suspended in 1 mL of 0.5% (v/v) Tween 80 (in 1\u0026times;PBS) solution and centrifuged after 1, 2, 6, 12, 24, 48, 72, 96, 144, and 192 hours (3 tubes for each time point) before being tested, with bare MNP used as a control. Finally, the concentration of TGF-β1 was measured using ELISA kits to plot the TGF-β1 release profile.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Mouse experiments\u003c/h2\u003e \u003cp\u003eMice were anesthetized with an intraperitoneal injection of 60 mg/kg sodium pentobarbital and given a third of the initial dosage every 45 minutes to maintain anesthesia. These mice were received oral endotracheal intubation and then allowed to breathe spontaneously (CON) or supported by ventilation using a ventilator (SAR-100, CWE Incorporated, USA). The ventilation rate was set at 80 bpm, and the inspiration to expiration ratio was kept constant at 1:1 in the absence of positive end-expiratory pressure. The HTV and normal tidal volume (NTV) were set to be 20 and 7 mL/kg, respectively. Ventilated mice were randomly assigned to one of five recovery subgroups: 0 hours (end of ventilation, EOV), one day (post-ventilation one day, PV1d), three days (post-ventilation three days, PV3d), seven days (post-ventilation seven days, PV7d), and ten days (post-ventilation ten days, PV10d). There were even numbers of male and female animals in each group (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4). After 4-hours of ventilation, mice in the EOV subgroup were sacrificed. Mice in other subgroups were extubated after spontaneously regaining consciousness and breathing and then sacrificed at 1, 3, 7, and 10 days after extubation, whereas mice in the CON group were euthanized at 10 days after extubation. Bronchoalveolar lavage fluid (BALF) from left lungs, blood serum, and lung tissue was collected and stored at -80 ˚C except for the right-upper and middle lobes, which were obtained for frozen sectioning, TEM examination, and wet/dry (W/D) ratio calculation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Administration of Biologics\u003c/h2\u003e \u003cp\u003eMice were intravenously administered 40 \u0026micro;g/kg rTGF-β before mechanical ventilation to up-regulate TGF-β1 levels[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. After successfully preparing TMNP or MNP, mice were intravenously injected with 0.5 mg/kg TMNP or MNP. Since the rTGF-β, TMNP, and MNP were all diluted to 50 \u0026micro;L normal saline for injection, the vehicle group of mice was intravenously injected with 50 \u0026micro;L normal saline. According to the result of Breg proportion, we will choose the time points that HTV and NTV-treated mice showed statistical difference on the proportion of Breg in lung for these experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Cytokine measurements and histological study\u003c/h2\u003e \u003cp\u003eThe W/D weight ratio was calculated to assess the degree of pulmonary edema. The right-upper lung lobe was wiped with filter paper to remove external water before being measured. The lung tissues were dried in an oven at 60˚C for 48 hours before being measured. To investigate changes in pulmonary permeability, the total protein in BALF was quantified with a Pierce\u0026trade; BCA Protein Assay Kit, and total inflammatory cell infiltration was measured using a cell counter (JIMBIO FILPLUS, Jiangsu, China). IL-1β, IL-6, tumor necrosis factor (TNF)-α, and TGF-β1 levels in serum, BALF, or total lung homogenates were assessed by ELISA kits (CUSABIO, Wuhan, China). The right-lower lung lobe was fixed with 4% paraformaldehyde (30525-89-4; Sigma-Aldrich, St. Louis, MO, USA) and embedded in the Tissue-Tek opti-mum cutting temperature compound (4583, Sakura, Tokyo, Japan). Tissue sections were stained with hematoxylin and eosin. The pathological score of lung injury was determined using previously reported criteria[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. To observe the ultrastructural cell changes, appropriate 1 mm\u003csup\u003e3\u003c/sup\u003e lung samples in each group were cut for TEM analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Single-cell dissociation for flow cytometry (FCM)\u003c/h2\u003e \u003cp\u003eTo prepare a single cell suspension from the lung, the left-upper lung lobe was digested with 0.1 mg/mL Dispase II (4942078001-1g; Roche, Basel, Switzerland), 2000U/mL DNase I (10104159001; Roche, Basel, Switzerland), and 0.2% collagenase (038-22361; Wako, Osaka, Japan) in 10 mM 4-(2-hydroxyerhyl) piperazine-1-erhanesulfonic acid (HEPES) Roswell Park Memorial Institute (RPMI) (22400105; ThermoFisher Scientific, San Jose, CA, USA). The spleen was put in a cell staining buffer (420201; BioLegend, San Diego, CA, USA) and ground by the rough surface of the glass slide to prepare a single cell suspension. After red cell lysis (11814389001; Roche, Basel, Switzerland) and washing twice, cells were passed through a 70-\u0026micro;m strainer and stained with TruStain FcX\u0026trade; PLUS antibody (S17011E; BioLegend, San Diego, CA, USA). Cells were next incubated with P-phycoerythrin (PE)-conjugated cluster of differentiation (CD) 5 (100608; BioLegend, San Diego, CA, USA), allophycocyanin (APC)-conjugated CD19(11512; BioLegend, San Diego, CA, USA), fluorescein isothiocyanate (FITC)-conjugated CD4 (100510; BioLegend, CA, USA) or APC-conjugated CD8a (100712; BioLegend, CA, USA), CD44- PE-Cyanine7 (PE/Cy7) (560569; BD Biosciences, Franklin Lake, NJ, USA), CD1d- Brilliant\u0026trade; Blue 515 (BB515) (565956; BD Biosciences, Franklin Lake, NJ, USA), CD5-BB515 (565504; BD Biosciences, Franklin Lake, NJ, USA), C-X3-C Motif Chemokine Receptor 1 (CX3CR1)-PE/Cy7 (149015; BioLegend, San Diego, CA, USA), CD45R/B220-APC/Cy7 (561102; BD Biosciences, Franklin Lake, NJ, USA), CD3e-APC (553066; BD Biosciences, Franklin Lake, NJ, USA), and IgM-PerCP/Cy5.5 (562034; BD Biosciences, Franklin Lake, NJ, USA) on ice for 20 min in the dark. Following fixation and permeabilization, cells were incubated with TGF-β (latency-associated peptide, LAP)-PE (563143; BD Biosciences, Franklin Lake, NJ, USA). Cells were washed twice before being sorted and analyzed using a Beckman Coulter CytoFLEX flow cytometer (Beckman Coulter Commercial Enterprise, Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Immunostaining\u003c/h2\u003e \u003cp\u003eFrozen tissue sections were air dried for 30 min before being fixed with 3.7% paraformaldehyde for 15 min and ice-cold 100% methanol for 15 min. The slides were then blocked with PBS containing 3% bovine serum albumin (SRE0096; Sigma-Aldrich, St. Louis, MO, USA), 3% Goat Serum (16210064; Gibco, San Jose, CA, USA), 0.2% Triton X-100 (X100; Sigma-Aldrich, St. Louis, MO, USA), and 0.02% NaN3 (S2002; Sigma-Aldrich, St. Louis, MO, USA) for 60 min at 37\u0026deg;C, and incubated with anti-CD19 antibody (DF7030; Affinity, Nottingham, UK), Purified anti-mouse LAP (TGF-β1) (141402; BioLegend, San Diego, CA, USA), and CD44 (IM7) Rat mAb (39037, Cell Signaling Technology, MA, USA). Related secondary antibodies included Alexa Fluor 549 Anti-Rabbit IgG H\u0026amp;L antibody (A11037; Invitrogen, Carlsbad, CA, USA), Dylight\u0026reg; 550 Anti-mouse IgG H\u0026amp;L antibody (Ab96880; PythonBio, Guangzhou, China), and Alexa Fluor 488 Anti-Rat IgG H\u0026amp;L antibody (4416, Cell Signaling Technology, MA, USA). Finally, the sections were treated with ProLong\u0026reg;Gold Antifade Reagent with 4', 6-diamidino-2-phenylindole (DAPI) (8961S; Cell Signaling Technology, MA, USA). All sections were analyzed using multiplex confocal microscopy (LSM980, Zeiss, German)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Bioinformatics\u003c/h2\u003e \u003cp\u003eThe expression matrix of all target genes was uploaded to the Database for Annotation, Visualization, and the Integrated Discovery (DAVID) database for biological processes enrichment and KEGG pathway analysis with count\u0026thinsp;\u0026gt;\u0026thinsp;5 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01. The STRING database was used to assess the interactive relationships of all target genes, and the experimentally validated interactions were defined as statistically different when the combined score\u0026thinsp;\u0026ge;\u0026thinsp;0.4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Statistical analysis.\u003c/h2\u003e \u003cp\u003eData were analyzed using StataCorp LP software (STATA Institute, Inc., Collgege Station, TX, USA) and performed using Graphpad Prism 9 (San Diego, CA, USA). One-way ANOVA analyses were used to examine the significance of intergroup differences, and Bonferroni tests were used to analyze pair-wise comparisons. Pearson Correlation analyses between every two factors were performed. A two-tailed \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was defined as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1 CD19\u003csup\u003ehigh\u003c/sup\u003eCD44\u003csup\u003ehigh\u003c/sup\u003eTGF-β1(+) Breg are associated with VILI resolution.\u003c/h2\u003e \u003cp\u003eTo determine the particular Breg phenotype shift that occurred during VILI resolution, wild-type mice were ventilated for 4 hours with NTV or HTV. After 4 hours of post-ventilation, we analyzed the biomarkers of CD19\u003csup\u003ehigh\u003c/sup\u003eTGF-β1\u003csup\u003ehigh\u003c/sup\u003e Breg subsets in the spleen. We found significantly increased expression of CD44 and CX3CR1 but without no significance of CD5, CD45R/B220, and IgM (\u003cb\u003eFigure S1A\u003c/b\u003e). The function of CD44, a cell-surface receptor involved in T-lymphocyte activation, recirculation, homing, hematopoiesis, inflammation, and response to bacterial infection, was investigated in this study[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. CX3CR1 is a receptor for the C-X3-C chemokine fractalkine on many early leukocyte cells, and infiltrating CX3CR1(+) immune cells release mediators to induce injury, stimulate proliferation, and chemoattract inflammatory cell[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. We mapped the interaction between CD19, CD44, and TGF-β1 using a protein-protein interaction network, suggesting that these proteins had direct interactions (\u003cb\u003eFigure S1B\u003c/b\u003e). KEGG pathway analysis showed that these proteins were enriched in the B cell receptor signaling pathway, TGF-β signaling pathway, chronic myeloid leukemia, and pancreatic cancer (\u003cb\u003eFigure S1C\u003c/b\u003e). These proteins also were enriched in the regulation of epithelial to mesenchymal transition and the TGF-β receptor signaling pathway (\u003cb\u003eFigure S1D\u003c/b\u003e). Overall, these data suggest that CD19\u003csup\u003ehigh\u003c/sup\u003eCD44\u003csup\u003ehigh\u003c/sup\u003eTGF-β1(+) Breg (abbreviated to TGF-β1(+) Breg) mediates a potent effect in VILI mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThen, we looked at how TGF-β1(+) Breg altered over time during VILI resolution. Therefore, we assessed the percentage of TGF-β1(+) Bregs in the lung (pBreg) and spleen (sBreg) of EOV, PV1d, PV3d, PV7d, and PV10d subgroups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). There was one mouse died in the PV1d subgroup of HTV group, and the probability of survival between the NTV and HTV group was lack of statistical difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.3173). Compared with the mice treated with NTV, the percentages of pBreg in the PV1d and PV10d subgroups of HTV-treated mice were increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB \u003cb\u003eand D\u003c/b\u003e). Hence, we choose the PV1d and PV10d as the observation time for the further intervention experiments. Compared with the CON group, the percentages of pBreg at PV3d and PV7d in NTV-treated mice were lower. The percentage of pBreg at PV1d, PV3d, and PV7d in NTV-treated mice was lower than at EOV, but the percentage of pBreg at PV7d was higher than that at PV1d. In the HTV-treated mice, the percentages of pBreg were gradually decreased from EOV to PV7d and minimized at PV7d; and the percentage of pBreg at PV10d was increased to the similar level of EOV. However, sBreg showed higher percentages at the EOV, PV1d, PV3d, and PV10d in HTV-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC \u003cb\u003eand D\u003c/b\u003e). The percentage of sBreg at the EOV in NTV-treated mice was higher than that at PV1d, PV3d, and PV10d, as well as the mice of CON group. In the HTV-treated mice, sBreg peaked in the EOV subgroup and then decreased to baseline from PV1d to PV7d. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eE showed the representative infiltration of pBreg in the PV1d subgroup between NTV or HTV-treated mice. Additionally, we also showed the gating strategy of pBreg and sBreg, as well as the representative FCM plots for the sBreg with statistical differences between NTV or HTV-treated mice in the \u003cb\u003eFigure S2\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Resolution of lung injury and inflammation in VILI mice\u003c/h2\u003e \u003cp\u003eThese mice were randomly divided into five groups: CON, NTV1d, HTV1d, NTV10d, and HTV10d. In the mice of HTV1d group, obvious lung tissue injury occurred compared to the other groups. Injured lung tissues showed different degrees of intra-alveolar exudation, extravasated blood, alveolar wall thickening, inflammatory cell accumulation in the lung interstitium, and hyaline membrane formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Type II alveolar epithelial cells (AEC-II) in the HTV1d group showed different degrees of microvillus disappear, disrupted cytoplasmic and nuclear structure, chromatin margination, increased mitochondrial swelling, and cavitating changes in the lamellar body, as well as cell membrane discontinuities (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCompared with CON, NTV1d, NTV10d, and HTV10d groups, mice of HTV1d showed the significant higher pathological scores, W/D ratio, infiltrated cell counts, and total protein levels in BALF. These variables, except W/D ration between the CON and NTV1d group, were both lack of statistical differences across the CON, NTV1d, NTV10d, and HTV10d groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-F). Serum TGF-β1 levels between these five groups were similar, but serum IL-1β level in HTV1d group was higher in comparison with the mice of the CON, NTV10d, and HTV10d groups. The mice of HTV1d showed the significant higher serum IL-6 and TNF-α levels compared with the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). TGF-β1 expression in BALF of HTV-treated mice remained higher than in the CON, NTV1d and NTV10d groups. The pulmonary IL-1β and TNF-α levels in the HTV1d group were both higher in comparison with the mice of the other groups; but the pulmonary IL-6 level in the HTV1d group was only higher in comparison with the mice of the CON group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3 HTV induced the proliferation of T cells during VILI resolution.\u003c/h2\u003e \u003cp\u003eTo know the changes in T-cell immunity during VILI resolution, we analyzed the percentage of CD4(+), CD8a(+), CD4(+) CD8a(+) T cells in the lungs and spleen by FCM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA \u003cb\u003eand B\u003c/b\u003e). When compared to the mice of CON and NTV1d groups, the percentages of pulmonary CD4(+) T cells (pCD4\u0026thinsp;+\u0026thinsp;TC), pulmonary CD8a (+) T cells (pCD8a\u0026thinsp;+\u0026thinsp;TC), and pulmonary CD4(+) CD8a(+) double positive T cells (pDPTC) in the HTV1d, NTV10d, and HTV10d groups were both increased, and the proportion of these T cells were both peaked in the HTV10d group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Compared with CON group, the percentages of splenic CD4(+) T cells (sCD4\u0026thinsp;+\u0026thinsp;TC) and splenic CD8a(+) T cells (sCD8a\u0026thinsp;+\u0026thinsp;TC) in NTV10d were lower, yet the percentage of sCD4\u0026thinsp;+\u0026thinsp;TC in NTV1d was higher than that in the NTV10d and HTV10d groups. Splenic CD4(+) CD8a(+) T cells (sDPTC) proportion in CON group was significantly higher than that in the NTV1d, HTV1d, and NTV10d groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). However, pulmonary and splenic CD4(+)/CD8a(+) T cells ratios in the NTV1d group were both significantly higher than that in the other groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4 TMNP promotes the resolution of lung injury and inflammation.\u003c/h2\u003e \u003cp\u003eExogenous TGF-β1 active ingredient is easily diluted or inactivated, lacks ideal transporter, and has a short biological half-life[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. To prolong and stabilize the effect of exogenous TGF-β1, we synthesized the TMNP to package TGF-β1. After cytochalasin B treatment, macrophage actin seemed unstable, and various microvesicles were secreted (\u003cb\u003eFigure S3A, B)\u003c/b\u003e. MMVs maintained the presence of surface proteins such as Flotillin-2; MMVs and MNP both had similar protein banding patterns, but NPs did not contain these proteins (\u003cb\u003eFigure S3C, D)\u003c/b\u003e. NPs, MMVs, and MNP all have a distinct core and shell structure (\u003cb\u003eFigure S3E)\u003c/b\u003e, corresponding with previous research findings[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The size of NPs, MMVs, and MNP was approximately 114.6\u0026thinsp;\u0026plusmn;\u0026thinsp;36.5 nm, 175.5\u0026thinsp;\u0026plusmn;\u0026thinsp;96.1 nm, and 190.7\u0026thinsp;\u0026plusmn;\u0026thinsp;69.6 nm, which both remained constant (\u003cb\u003eFigure S3F, G)\u003c/b\u003e. The zeta potential of MNP was \u0026minus;\u0026thinsp;26.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4 mV, similar to the natural cell membrane. The EE and DLC of TGF-β1 in TMNP were 35.8% and 8.7%, respectively. TGF-β1 release from TMNP was determined over 24\u0026ndash;30 hours (\u003cb\u003eFigure S3H\u003c/b\u003e), and TMNP were shown to be more stable than NPs (\u003cb\u003eFigure S3I\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo compare the effect of TMNP and a single dose of rTGF-β1 on regulating lung injury and inflammation in vivo, TMNP and rTGF-β1 were injected intravenously before HTV-ventilation and MNP as control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We found that mice treated with vehicle and MNP had more severe pathological scores, higher W/D ratio, and increased cell counts in BALF at PV1d, while mice treated with TMNP at PV1d had lower pathological scores than mice treated with rTGF-β1. The mice treated with TMNP and rTGF-β1 showed the similar W/D ratio and cell counts in BALF at PV1d. However, the total protein levels across these groups at PV1d were lack of statistical differences. At PV10d, the pathological score, W/D ratio, total protein levels and cell counts in BALF between these groups were both similar (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs expected, a single administration of rTGF-β1 only induced the elevation of total TGF-β1 levels in serum and lungs at PV1d compared with the mice treated with vehicle ro MNP, but the mice treated with TMNP both showed an increase of total TGF-β1 levels in serum and lungs at PV1d and PV10d (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). In the TMNP group, serum IL-1β level at PV1d was lower than the vehicle and MNP groups. The mice treated with rTGF-β1 showed the lower serum IL-1β level at PV1d in comparison with the MNP-treated mice. There were no statistical differences in the serum IL-1β level at PV10d across these groups. Mice treated with TMNP showed the lower pulmonary IL-1β level at PV1d and PV10d than the mice of vehicle and MNP groups, and the pulmonary IL-1β level at PV10d in the TMNP group was also lower than rTGF-β group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Mice treated with TMNP showed the lower serum IL-6 level at PV1d and PV10d than the mice of vehicle and MNP groups, but the serum IL-6 level at PV10d was significantly higher than other groups. Pulmonary IL-6 level at PV1d in the TMNP group was lower than vehicle, rTGF-β, and MNP groups, which at PV10d between these groups was lack of statistical differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ. Serum TNF-α level in the MNP group was higher than rTGF-β and TMNP groups at PV1d, but pulmonary TNF-α levels at PV1d in the rTGF-β and TMNP groups were both lower than vehicle and MNP groups. There were no statistical differences on serum and pulmonary TNF-α levels at PV1d and PV10d across these groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5 TMNP play a definite role of Breg proliferation.\u003c/h2\u003e \u003cp\u003eSince TGF-β1 is the prevalent cytokines that regulates immune cell differentiation, we sought to determine whether TGF-β1 has a role in activating TGF-β1(+) Breg and regulating in T-cell immunity during VILI resolution. We only observed a significant increase of pBreg proportion at PV1d in TMNP group compared with MNP group, and there were no statistical differences noted on the proportions of pBreg at PV10d across these groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Here we did not find any statistical differences across these groups on the proportions of sBreg at PV1d, but the proportion of sBreg at PV10d in TMNP group was higher than vehicle, rTGF-β, and MNP groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). We also showed the representative FCM plots on the percentages of pBreg and sBreg in the Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, and these results suggested that TMNP play a definite role of Breg proliferation, especially for the multiplication of sBreg at the rehabilitation phase.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.6 TMNP affects T-cell differentiation.\u003c/h2\u003e \u003cp\u003eHere we also determined the effect of TMNP on the changes of T-cell immunity. Mice treated with MNP and TMNP showed the reduction of pCD4\u0026thinsp;+\u0026thinsp;TC proportion and CD4(+)/CD8a(+) T cells ratio, but promoted the proliferation of pCD8a\u0026thinsp;+\u0026thinsp;TC and pDPTC at PV1d and PV10d. Compared with the MNP group, the percentage of pDPTC in the TMNP at PV1d was significantly lower. Compared with the MNP group, TMNP-treated mice showed higher proportion of pCD4\u0026thinsp;+\u0026thinsp;TC and pDPTC, but lower percentage of pCD8a\u0026thinsp;+\u0026thinsp;TC at PV10d. The pulmonary CD4(+)/CD8a(+) T cells ratio in the rTGF-β group was higher than vehicle group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). Mice treated with rTGF-β1 showed the higher percentage of sCD4\u0026thinsp;+\u0026thinsp;and sCD8a\u0026thinsp;+\u0026thinsp;TC at PV1d and PV10d than other three groups. The percentages of sDPTC at PV1d across these groups were similar, but which in the rTGF-β, MNP, and TMNP groups at PV10d were both higher than vehicle group. The splenic CD4(+)/CD8a(+) T cells ratios at PV1d in the MNP and TMNP groups were higher than vehicle group, but the splenic CD4(+)/CD8a(+) T cells ratios at PV10d in the rTGF-β, MNP, and TMNP groups at PV10d were both higher than vehicle group. The percentage of sDPTC and splenic CD4(+)/CD8a(+) T cell ratios at PV10d across the rTGF-β, MNP, and TMNP groups were lack of statistical differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSerum TGF-β level is found to be negatively correlated with IL-6 levels in lung tissue (\u003cb\u003eFigure S4A)\u003c/b\u003e, and sBreg proportion is negatively associated with the pDPTC proportion (\u003cb\u003eFigure S4B)\u003c/b\u003e. pCD8a\u0026thinsp;+\u0026thinsp;TC proportion is negatively correlated with pulmonary CD4(+)/CD8a(+) T cell ratio, but positively associated with the pDPTC proportion (\u003cb\u003eFigure S4C)\u003c/b\u003e, which is negatively associated with the pulmonary CD4(+)/CD8a(+) T cell ratio (\u003cb\u003eFigure S4D)\u003c/b\u003e. sCD4\u0026thinsp;+\u0026thinsp;TC proportion is positively correlated with sCD8a\u0026thinsp;+\u0026thinsp;TC proportion (\u003cb\u003eFigure S4E)\u003c/b\u003e, but which is negatively associated with splenic CD4(+)/CD8a(+) T cell ratio (\u003cb\u003eFigure S4F)\u003c/b\u003e. sDPTC proportion is both negatively associated with splenic CD4(+)/CD8a(+) T cell ratio (\u003cb\u003eFigure S4G)\u003c/b\u003e. As expected, the W/D ratio is positively associated with the pathological scores (\u003cb\u003eFigure S4H)\u003c/b\u003e, total protein level and cell counts in BALF, as well as serum and pulmonary IL-1β level and serum IL-6 level (\u003cb\u003eFigure S4I\u003c/b\u003e). Pathological score is positively associated with the total protein level and cell counts in BALF, as well as serum IL-1β level, IL-6 and TNF-α levels (\u003cb\u003eFigure S4J\u003c/b\u003e). Taken together, sBreg promotes the differentiation of pDPTC that is also produced by the stimulation of pCD8a\u0026thinsp;+\u0026thinsp;TC. sDPTC would restrict the activation of splenic CD4(+) T cells but promote the proliferation of splenic CD8a(+) T cells. Activation of TGF-β1 in lung tissues may be a negative factor of pulmonary IL-6 level.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eBreg regulate inflammation and promote tissue repair by releasing chemokines, chemoattractants, and growth factors[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, effective regulation of Breg via the production of both regulatory cytokines, such as IL-10, IL-35, and TGF-β1, is a prerequisite for the resolution of inflammation and tissue injury[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Studies have indicated that IL-10-producing Breg promotes the anti-inflammatory process and improve the outcome of inflammation, such as acute myocardial infarction or intestinal inflammation[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. IL-10-producing Breg are derived from several competent progenitors at unrestricted stages of development to modulate the immune response[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition, TGF-β1(+) Breg also produce TGF-β1 to regulate the immune response of Treg or Th1 cells during the development and recovery of inflammatory diseases[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Although regulating immune cells, cytokines, and other affected materials through Breg are essential for the resolution of inflammation[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], the mechanistic links between Breg phenotype transition and ALI resolution remains unclear. Here, utilizing the murine model of microbiota-independent ALI induced by ventilation, our FCM results and functional characterization of TGF-β1(+) Breg identified a major population of CD19\u003csup\u003ehigh\u003c/sup\u003eCD44(+) TGF-β1(+) Breg that appear highly adjustability and exhibit a definite phenotype during VILI resolution, which were gradually decreased from EOV to PV7d and minimized at PV7d.\u003c/p\u003e \u003cp\u003eDue to their ability to regulate T-cell immunity, Breg play an important role in inflammation and management[\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Breg are also essential in that they support Foxp3\u003csup\u003e+\u003c/sup\u003e Treg response and population, which depends on the microenvironment and release of specific cytokines such as IL-10 and TGF-β1[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. An increased numbers of Breg would convert resting CD4(+) T cells to Treg, preventing host defense against infection while promoting tumor growth and metastasis[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. According to the research, IL-10-producing Breg negatively regulate anti-melanoma immunity by reducing Th1 cytokines in tumor-infiltrating CD8(+) T cells[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In this context, we discovered that splenic TGF-β1(+) Breg promote the differentiation of pulmonary CD4(+) CD8a(+) T cells, which are a rarely studied T cell population in immunity to infection or inflammatory diseases[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTGF-β1 is required for the expansion, activation, and effector function of the mature CD4(+) and CD8(+) T cells in the peripheral lymphoid organs and target tissues[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. TGF-β1 has pleiotropic effects on the regulation of regulatory and effector CD4(+) T cell responses[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. TGF-β1, on the other hand, would inhibit CD8(+) T cell activation, proliferation, and transcriptional profile[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. TGF-β1 neutralization would also enhance CD8\u0026thinsp;+\u0026thinsp;T-cell and NK-cell-mediated anti-tumor immune responses[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. CD4(+) CD8(+) T cells have the suppressor activity based on the high expression of T cell activity suppressor genes that involved in the TGF-β1 signaling pathway[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Here we did not find any correlation between serum or pulmonary TGF-β1 level and each T cell populations, but pulmonary CD4(+), CD8a (+), and CD4(+) CD8a(+) T cells were increased in the HTV-treated mic at PV1d and PV10d, and these T cells were both peaked at PV10d.\u003c/p\u003e \u003cp\u003eNPs can be used in immune therapy to encapsulate active pharmaceutical ingredients and deliver them more efficiently to inflammatory tissues[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. MMVs, which are derived from macrophage membranes with rich carbohydrates and transmembrane proteins, exhibit excellent biocompatibility and stability in a physiological environment[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Li R and his colleagues[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] used MMVs to coat with PLGA NPs encapsulated with tacrolimus, which significantly suppressed the progression of rheumatoid arthritis in mice. Hence, we created MNP to prolong and stabilize the effect of exogenous TGF-β1, and we discovered that TMNP alleviate acute lung injury and inflammation by producing the upregulation of TGF-β1 expression. TMNP play a definite role of Breg proliferation at PV10d, especially for the activation of sBreg, and induced the reduction of pulmonary CD4(+) T cells and CD4(+)/CD8a(+) T cell ratios, but proliferation of pulmonary CD8a\u0026thinsp;+\u0026thinsp;T cells and CD4(+)CD8a(+) T cells. Compared with the MNP group, TMNP-treated mice showed higher pulmonary CD4\u0026thinsp;+\u0026thinsp;and CD4(+) CD8a(+) T cells, but lower CD8a\u0026thinsp;+\u0026thinsp;T cells in the advanced phase.\u003c/p\u003e \u003cp\u003eNotably, our data identify that the non-ignorable effect of TGF-β1(+) Breg was associated with resolution of lung injury and inflammation. Appropriate secretion of TGF-β1 is required to proliferate TGF-β1(+) Breg, reduce inflammation, and resolve lung injury. Furthermore, TMNP can stable-release TGF-β1 to reduce VILI, which may help to prevent cascade inflammation and lung injury in the acute phase. Compared to rTGF-β1 monotherapy, TMNP improved TGF-β1 upregulation and immune homeostasis, which contribute to the attenuation of lung injury and inflammation. However, there were some limitations, such as the absence of adoptive transfer experiments to further explore the role of TGF-β1(+) Breg and the use of \u003cem\u003eTgfb1\u003c/em\u003e knockout mice to assess the role of endogenous TGF-β1 due to delayed breeding. Unfortunately, we did not evaluate the effect of TNMP or rTGF-β1 on promoting pulmonary fibrosis. The molecular mechanisms between TGF-β1(+) Breg and CD4(+) or CD8(+) T cells require further study.\u003c/p\u003e \u003cp\u003eTaken together, TMNP can stable-release TGF-β1 to promote the resolution of inflammatory lung injury, which may be associated with the proliferation of TGF-β1(+) Bregs and regulation of T-cells immunity. These results indicate a potential strategy for preventing the cascade inflammation and lung injury during the acute phase.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAEC-II, type II alveolar epithelial cells; ALI, acute lung injury; APC, allophycocyanin; ARDS, acute respiratory distress syndrome; BALF, bronchoalveolar lavage fluid; BB515, Brilliant\u0026trade; Blue 515; Breg, regulatory B cells; CD, cluster of differentiation; CX3CR1, C-X3-C Motif Chemokine Receptor 1; DLC, drug loading capacity; DAPI, 4\u0026apos;, 6-diamidino-2-phenylindole; DMEM, dulbecco\u0026apos;s modified eagle medium; EE, encapsulation efficiency; ELISA, enzyme linked immunosorbent assay; EOV, end of ventilation; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HTV, high tidal volume; HEPES, 4-(2-hydroxyerhyl) piperazine-1-erhanesulfonic acid; IL, interleukin; MMVs, macrophage-derived microvesicles; MNP, MMVs membrane-coated nanoparticles; NTV, normal tidal volume; pBreg, TGF-\u0026beta;1(+) Bregs in the lung; pCD4+ TC, pulmonary CD4(+) T cells; pCD8a+ TC, pulmonary CD8a (+) T cells; pDPTC, pulmonary CD4(+) CD8a(+) double positive T cells; PBS, phosphate buffer saline; PE, P-phycoerythrin; PE/Cy7, PE-Cyanine7; PV1d, post-ventilation one day; PV3d, post-ventilation three days; PV7d, post-ventilation seven days; PV10d, post-ventilation ten days; rTGF-\u0026beta;1, recombinant mouse TGF-\u0026beta;1; RPMI, Roswell Park Memorial Institute; sBreg, TGF-\u0026beta;1(+) Bregs in the spleen; sCD4+ TC, splenic CD4(+) T cells; sCD8a+ TC, splenic CD8a (+) T cells; sDPTC, splenic CD4(+) CD8a(+) double positive T cells; TEM, transmission electron microscope; TGF-\u0026beta;1, transforming growth factor-\u0026beta;1; TMNP, MNP to package TGF-\u0026beta;1; TNF, tumor necrosis factor; Treg, regulatory T cells; VILI, ventilator-induced lung injury; W/D, wet/dry ratio.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDisclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no conflicts of interest in this work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have reviewed and agreed to publish this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLinghui Pan:\u0026nbsp;Conceptualization, Methodology, Supervision, Funding acquisition, and Writing-review and editing; Ren Jing: Experiments, Data curation, Funding acquisition, and Writing- Original draft, review, and editing; Xiaoting Liao: Experiments, Data curation, Software; Jianlan Mo: Experiments, Data curation, Software; Sheng He:\u0026nbsp;Supervision, Software; Xianlong Xie: Visualization, Investigation; Huijun Dai:\u0026nbsp;Writing- Reviewing and Editing; Zhaokun Hu: Validation, Software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (81970078), Guangxi Anesthesiology Clinical Medicine Research Center Construction Project (scientific foundation of Guangxi No: 2022AC04002), and Guangdong Province Basic and Applied Basic Research Fund Joint Fund-Youth Project (2023A1515110149).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMeyer NJ, Gattinoni L, Calfee CS. Acute respiratory distress syndrome[J]. Lancet (London England). 2021;398(10300):622\u0026ndash;37. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0140-6736(21)00439-6\u003c/span\u003e\u003cspan address=\"10.1016/S0140-6736(21)00439-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerez-Fernandez XL, Sabater-Riera J, Fuset-Cabanes M. COVID-19 ARDS: getting ventilation right[J]. Lancet (London England). 2022;399(10319):22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0140-6736(21)02439-9\u003c/span\u003e\u003cspan address=\"10.1016/S0140-6736(21)02439-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNieman GF, Gatto LA, Andrews P, et al. Prevention and treatment of acute lung injury with time-controlled adaptive ventilation: physiologically informed modification of airway pressure release ventilation[J]. Ann Intensive Care. 2020;10(1):3. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13613-019-0619-3\u003c/span\u003e\u003cspan address=\"10.1186/s13613-019-0619-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVillar J, Zhang H, Slutsky AS. Lung Repair and Regeneration in ARDS: Role of PECAM1 and Wnt Signaling[J]. Chest. 2019;155(3):587\u0026ndash;94. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.chest.2018.10.022\u003c/span\u003e\u003cspan address=\"10.1016/j.chest.2018.10.022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSlutsky AS, Ranieri VM. Ventilator-induced lung injury[J]. N Engl J Med. 2013;369(22):2126\u0026ndash;36. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1056/NEJMra1208707\u003c/span\u003e\u003cspan address=\"10.1056/NEJMra1208707\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlbert RK. Constant Vt Ventilation and Surfactant Dysfunction: An Overlooked Cause of Ventilator-induced Lung Injury[J]. Am J Respir Crit Care Med. 2022;205(2):152\u0026ndash;60. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1164/rccm.202107-1690CP\u003c/span\u003e\u003cspan address=\"10.1164/rccm.202107-1690CP\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMenon M, Hussell T, Ali Shuwa H. Regulatory B cells in respiratory health and diseases[J]. Immunol Rev. 2021;299(1):61\u0026ndash;73. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/imr.12941\u003c/span\u003e\u003cspan address=\"10.1111/imr.12941\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlhabbab RY, Nova-Lamperti E, Aravena O, et al. Regulatory B cells: Development, phenotypes, functions, and role in transplantation[J]. Immunol Rev. 2019;292(1):164\u0026ndash;79. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/imr.12800\u003c/span\u003e\u003cspan address=\"10.1111/imr.12800\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCherukuri A, Mohib K, Rothstein DM, Regulatory B. cells: TIM-1, transplant tolerance, and rejection[J]. Immunol Rev. 2021;299(1):31\u0026ndash;44. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/imr.12933\u003c/span\u003e\u003cspan address=\"10.1111/imr.12933\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHabener A, Happle C, Grychtol R et al. Regulatory B cells control airway hyperreactivity and lung remodeling in a murine asthma model[J]. J Allergy Clin Immunol. 2021;147(6):2281-94 e7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jaci.2020.09.041\u003c/span\u003e\u003cspan address=\"10.1016/j.jaci.2020.09.041\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRadomir L, Kramer MP, Perpinial M, et al. The survival and function of IL-10-producing regulatory B cells are negatively controlled by SLAMF5[J]. Nat Commun. 2021;12(1):1893. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41467-021-22230-z\u003c/span\u003e\u003cspan address=\"10.1038/s41467-021-22230-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu L, Dalal R, Cao CD, et al. IL-10-producing B cells are enriched in murine pericardial adipose tissues and ameliorate the outcome of acute myocardial infarction[J]. Proc Natl Acad Sci U S A. 2019;116(43):21673\u0026ndash;84. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.1911464116\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1911464116\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuai G, Markmann JF, Deng S, et al. TGF-beta-secreting regulatory B cells: unsung players in immune regulation[J]. Clin Transl Immunol. 2021;10(4):e1270. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/cti2.1270\u003c/span\u003e\u003cspan address=\"10.1002/cti2.1270\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBatlle E, Massague J. Transforming Growth Factor-beta Signaling in Immunity and Cancer[J]. Immunity. 2019;50(4):924\u0026thinsp;\u0026ndash;\u0026thinsp;40. Immunity. 2019;50(4):924\u0026thinsp;\u0026ndash;\u0026thinsp;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie Y, Qian Y, Wang Y, et al. Mechanical stretch and LPS affect the proliferation, extracellular matrix remodeling and viscoelasticity of lung fibroblasts[J]. Exp Ther Med. 2020;20(5):5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3892/etm.2020.9133\u003c/span\u003e\u003cspan address=\"10.3892/etm.2020.9133\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang LT, Chou HC, Chen CM. Inhibition of FABP4 attenuates hyperoxia-induced lung injury and fibrosis via inhibiting TGF-beta signaling in neonatal rats[J]. J Cell Physiol. 2022;237(2):1509\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/jcp.30622\u003c/span\u003e\u003cspan address=\"10.1002/jcp.30622\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen X, Peng W, Zhou R, et al. Montelukast improves bronchopulmonary dysplasia by inhibiting epithelialmesenchymal transition via inactivating the TGFbeta1/Smads signaling pathway[J]. Mol Med Rep. 2020;22(3):2564\u0026ndash;72. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3892/mmr.2020\u003c/span\u003e\u003cspan address=\"10.3892/mmr.2020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCurley GF, Contreras M, Higgins B, et al. Evolution of the inflammatory and fibroproliferative responses during resolution and repair after ventilator-induced lung injury in the rat[J]. Anesthesiology. 2011;115(5):1022\u0026ndash;32. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1097/ALN.0b013e31823422c9\u003c/span\u003e\u003cspan address=\"10.1097/ALN.0b013e31823422c9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi R, He Y, Zhu Y, et al. Route to Rheumatoid Arthritis by Macrophage-Derived Microvesicle-Coated Nanoparticles[J]. Nano Lett. 2019;19(1):124\u0026ndash;34. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/acs.nanolett.8b03439\u003c/span\u003e\u003cspan address=\"10.1021/acs.nanolett.8b03439\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark JS, Park K, Woo DG, et al. PLGA microsphere construct coated with TGF-beta 3 loaded nanoparticles for neocartilage formation[J]. Biomacromolecules. 2008;9(8):2162\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/bm800251x\u003c/span\u003e\u003cspan address=\"10.1021/bm800251x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu CM, Zhang L, Aryal S, et al. Erythrocyte membrane-camouflaged polymeric nanoparticles as a biomimetic delivery platform[J]. Proc Natl Acad Sci U S A. 2011;108(27):10980\u0026ndash;5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.1106634108\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1106634108\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang XY, Liu ZM, Zhang HF, et al. TGF-beta1 improves mucosal IgA dysfunction and dysbiosis following intestinal ischaemia-reperfusion in mice[J]. J Cell Mol Med. 2016;20(6):1014\u0026ndash;23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/jcmm.12789\u003c/span\u003e\u003cspan address=\"10.1111/jcmm.12789\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaylor RA, Chang CF, Goods BA, et al. TGF-beta1 modulates microglial phenotype and promotes recovery after intracerebral hemorrhage[J]. J Clin Invest. 2017;127(1):280\u0026ndash;92. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1172/JCI88647\u003c/span\u003e\u003cspan address=\"10.1172/JCI88647\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin JY, Jing R, Lin F, et al. High Tidal Volume Induces Mitochondria Damage and Releases Mitochondrial DNA to Aggravate the Ventilator-Induced Lung Injury[J]. Front Immunol. 2018;9:1477. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2018.01477\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2018.01477\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKlement JD, Paschall AV, Redd PS, et al. An osteopontin/CD44 immune checkpoint controls CD8\u0026thinsp;+\u0026thinsp;T cell activation and tumor immune evasion[J]. J Clin Invest. 2018;128(12):5549\u0026ndash;60. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1172/JCI123360\u003c/span\u003e\u003cspan address=\"10.1172/JCI123360\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreiffo FR, Viteri-Alvarez V, Frankenberger M, et al. CX3CR1-fractalkine axis drives kinetic changes of monocytes in fibrotic interstitial lung diseases[J]. Eur Respir J. 2020;55(2):1900460. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1183/13993003.00460-2019\u003c/span\u003e\u003cspan address=\"10.1183/13993003.00460-2019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi M, Zeng L, Liu S, et al. Transforming Growth Factor-beta Promotes Homing and Therapeutic Efficacy of Human Mesenchymal Stem Cells to Glioblastoma[J]. J Neuropathol Exp Neurol. 2019;78(4):315\u0026ndash;25. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/jnen/nlz016\u003c/span\u003e\u003cspan address=\"10.1093/jnen/nlz016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSirbulescu RF, Mamidi A, Chan SC, et al. B cells support the repair of injured tissues by adopting MyD88-dependent regulatory functions and phenotype[J]. FASEB J. 2021;35(12):e22019. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1096/fj.202101095RR\u003c/span\u003e\u003cspan address=\"10.1096/fj.202101095RR\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Z, Zhou Y, Yu Y, et al. Lipopolysaccharide preconditioning increased the level of regulatory B cells in the spleen after acute ischaemia/reperfusion in mice[J]. Brain Res. 2018;1701:46\u0026ndash;57. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.brainres.2018.05.036\u003c/span\u003e\u003cspan address=\"10.1016/j.brainres.2018.05.036\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMishima Y, Oka A, Liu B, et al. Microbiota maintain colonic homeostasis by activating TLR2/MyD88/PI3K signaling in IL-10-producing regulatory B cells[J]. J Clin Invest. 2019;129(9):3702\u0026ndash;16. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1172/JCI93820\u003c/span\u003e\u003cspan address=\"10.1172/JCI93820\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun G, Wang Y, Ti Y, et al. Regulatory B cell is critical in bone union process through suppressing proinflammatory cytokines and stimulating Foxp3 in Treg cells[J]. Clin Exp Pharmacol Physiol. 2017;44(4):455\u0026ndash;62. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/1440-1681.12719\u003c/span\u003e\u003cspan address=\"10.1111/1440-1681.12719\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMichee-Cospolite M, Boudigou M, Grasseau A, et al. Molecular Mechanisms Driving IL-10- Producing B Cells Functions: STAT3 and c-MAF as Underestimated Central Key Regulators?[J]. Front Immunol. 2022;13:818814. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2022.818814\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2022.818814\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao X, Ren X, Wang Q, et al. Critical roles of regulatory B and T cells in helminth parasite-induced protection against allergic airway inflammation[J]. Clin Exp Immunol. 2019;198(3):390\u0026ndash;402. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/cei.13362\u003c/span\u003e\u003cspan address=\"10.1111/cei.13362\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHorii M, Matsushita T. Regulatory B cells and T cell Regulation in Cancer[J]. J Mol Biol. 2021;433(1):166685. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jmb.2020.10.019\u003c/span\u003e\u003cspan address=\"10.1016/j.jmb.2020.10.019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian J, Zekzer D, Hanssen L, et al. Lipopolysaccharide-activated B cells down-regulate Th1 immunity and prevent autoimmune diabetes in nonobese diabetic mice[J]. J Immunol. 2001;167(2):1081\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4049/jimmunol.167.2.1081\u003c/span\u003e\u003cspan address=\"10.4049/jimmunol.167.2.1081\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatsushita T, Le Huu D, Kobayashi T et al. A novel splenic B1 regulatory cell subset suppresses allergic disease through phosphatidylinositol 3-kinase-Akt pathway activation[J]. J Allergy Clin Immunol. 2016;138(4):1170-82 e9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jaci.2015.12.1319\u003c/span\u003e\u003cspan address=\"10.1016/j.jaci.2015.12.1319\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKinker GS, Vitiello GAF, Ferreira WAS, et al. B Cell Orchestration of Anti-tumor Immune Responses: A Matter of Cell Localization and Communication[J]. Front Cell Dev Biol. 2021;9:678127. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fcell.2021.678127\u003c/span\u003e\u003cspan address=\"10.3389/fcell.2021.678127\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKobayashi T, Oishi K, Okamura A, et al. Regulatory B1a Cells Suppress Melanoma Tumor Immunity via IL-10 Production and Inhibiting T Helper Type 1 Cytokine Production in Tumor-Infiltrating CD8(+) T Cells[J]. J Invest Dermatol. 2019;139(7):1535\u0026ndash;e15441. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jid.2019.02.016\u003c/span\u003e\u003cspan address=\"10.1016/j.jid.2019.02.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOvergaard NH, Jung JW, Steptoe RJ, et al. CD4+/CD8\u0026thinsp;+\u0026thinsp;double-positive T cells: more than just a developmental stage?[J]. J Leukoc Biol. 2015;97(1):31\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1189/jlb.1RU0814-382\u003c/span\u003e\u003cspan address=\"10.1189/jlb.1RU0814-382\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTravis MA, Sheppard D. TGF-beta activation and function in immunity[J]. Annu Rev Immunol. 2014;32:51\u0026ndash;82. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1146/annurev-immunol-032713-120257\u003c/span\u003e\u003cspan address=\"10.1146/annurev-immunol-032713-120257\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchwartzkopff S, Woyciechowski S, Aichele U, et al. TGF-beta downregulates KLRG1 expression in mouse and human CD8(+) T cells[J]. Eur J Immunol. 2015;45(8):2212\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/eji.201545634\u003c/span\u003e\u003cspan address=\"10.1002/eji.201545634\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMishra S, Liao W, Liu Y, et al. TGF-beta and Eomes control the homeostasis of CD8\u0026thinsp;+\u0026thinsp;regulatory T cells[J]. J Exp Med. 2021;218(1):e20200030. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1084/jem.20200030\u003c/span\u003e\u003cspan address=\"10.1084/jem.20200030\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang L, Pang Y, Moses HL. TGF-beta and immune cells: an important regulatory axis in the tumor microenvironment and progression[J]. Trends Immunol. 2010;31(6):220\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.it.2010.04.002\u003c/span\u003e\u003cspan address=\"10.1016/j.it.2010.04.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDai M, Zhao L, Li Z, et al. The Transcriptional Differences of Avian CD4(+)CD8(+) Double-Positive T Cells and CD8(+) T Cells From Peripheral Blood of ALV-J Infected Chickens Revealed by Smart-Seq2[J]. Front Cell Infect Microbiol. 2021;11:747094. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fcimb.2021.747094\u003c/span\u003e\u003cspan address=\"10.3389/fcimb.2021.747094\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLoo YS, Bose RJ, McCarthy JR, et al. Biomimetic bacterial and viral-based nanovesicles for drug delivery, theranostics, and vaccine applications[J]. Drug Discov Today. 2021;26(4):902\u0026ndash;15. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.drudis.2020.12.017\u003c/span\u003e\u003cspan address=\"10.1016/j.drudis.2020.12.017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang H, Liu Y, He R, et al. Cell membrane biomimetic nanoparticles for inflammation and cancer targeting in drug delivery[J]. Biomater Sci. 2020;8(2):552\u0026ndash;68. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/c9bm01392j\u003c/span\u003e\u003cspan address=\"10.1039/c9bm01392j\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\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":"[email protected]","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":"Ventilation-induced lung injury, Transforming growth factor-β1, regulatory B cells, nanoparticles","lastPublishedDoi":"10.21203/rs.3.rs-4421925/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4421925/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRegulatory B cells (Breg) are vital for inflammation and tissue injury resolution. Here, we investigated the role of transforming growth factor-β1 (TGF-β1)-producing Breg in the murine model of ventilation-induced lung injury (VILI). The percentages of pulmonary CD19\u003csup\u003ehigh\u003c/sup\u003eCD44(+) TGF-β1(+) Breg were increased at PV1d and PV10d in VILI mice. Lung injury and inflammation were attenuated by up-regulating TGF-β1 levels with regulation of T-cell immunity. To prolong and stabilize the effect of exogenous TGF-β1, macrophage-derived microvesicles-coated nanoparticles (MNP) loaded TGF-β1(TMNP) were synthesized, and VILI mice were divided into sham, recombinant TGF-β1 (rTGF-β), MNP, and TMNP groups. TMNP increased the TGF-β1 levels in serum and lung tissues at PV10d. Compared with rTGF-β group, lung injury and inflammation in TMNP group at PV1d were attenuated with Breg proliferation; TMNP induced the reduction of pulmonary CD4(+) T cell proportions and CD4(+)/CD8a(+) T cell ratios, but promoted the proliferation of pulmonary CD8a(+) T cells at PV1d and PV10d. Together, TMNP promote the resolution of inflammatory lung injury, which may be associated with the proliferation of Breg to maintain immunological homeostasis.\u003c/p\u003e","manuscriptTitle":"Macrophage-derived Microvesicles Coated Nanoparticles Loaded with TGF-β1 Resolve Acute Lung Injury via Regulatory B Cell Activation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-31 21:54:24","doi":"10.21203/rs.3.rs-4421925/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","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":"ed017b0b-0cd2-4c10-95e3-9b83b7b33cfd","owner":[],"postedDate":"May 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-16T08:23:47+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-31 21:54:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4421925","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4421925","identity":"rs-4421925","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}