Association between high-flow nasal cannula use and mortality in patients with sepsis-induced acute lung injury: a retrospective propensity score-matched cohort study.

BACKGROUND: High-flow nasal cannula (HFNC) has emerged as a promising noninvasive method for delivering oxygen to critically ill patients, particularly those with sepsis and acute lung injury. However, uncertainties persist regarding its therapeutic benefits in this specific patient population. METHODS: This retrospective study utilized a propensity score-matched cohort from the Medical Information Mart in Intensive Care-IV (MIMIC-IV) database to explore the correlation between HFNC utilization and mortality in patients with sepsis-induced acute lung injury. The primary outcome was 28-day all-cause mortality. RESULTS: In the propensity score-matched cohort, the 28-day all-cause mortality rate was 18.63% (95 out of 510) in the HFNC use group, compared to 31.18% (159 out of 510) in the non-HFNC group. The use of HFNC was associated with a lower 28-day all-cause mortality rate (hazard ratio [HR] = 0.53; 95% confidence interval [CI] = 0.41-0.69; P < 0.001). HFNC use was also associated with lower ICU mortality (odds ratio [OR] = 0.52; 95% CI = 0.38-0.71; P < 0.001) and lower in-hospital mortality (OR = 0.51; 95% CI = 0.38-0.68; P < 0.001). Additionally, HFNC use was found to be associated with a statistically significant increase in both the ICU and overall hospitalization length. CONCLUSIONS: These findings indicate that HFNC may be beneficial for reducing mortality rates among sepsis-induced acute lung injury patients; however, it is also associated with longer hospital stays.

Manufacturing, quality control, and GLP-grade preclinical study of nebulized allogenic adipose mesenchymal stromal cells-derived extracellular vesicles.

BACKGROUND: Human adipose stromal cells-derived extracellular vesicles (haMSC-EVs) have been shown to alleviate inflammation in acute lung injury (ALI) animal models. However, there are few systemic studies on clinical-grade haMSC-EVs. Our study aimed to investigate the manufacturing, quality control (QC) and preclinical safety of clinical-grade haMSC-EVs. METHODS: haMSC-EVs were isolated from the conditioned medium of human adipose MSCs incubated in 2D containers. Purification was performed by PEG precipitation and differential centrifugation. Characterizations were conducted by nanoparticle tracking analysis, transmission electron microscopy (TEM), Western blotting, nanoflow cytometry analysis, and the TNF-α inhibition ratio of macrophage [after stimulated by lipopolysaccharide (LPS)]. RNA-seq and proteomic analysis with liquid chromatography tandem mass spectrometry (LC-MS/MS) were used to inspect the lot-to-lot consistency of the EV products. Repeated toxicity was evaluated in rats after administration using trace liquid endotracheal nebulizers for 28 days, and respiratory toxicity was evaluated 24 h after the first administration. In vivo therapeutic effects were assessed in an LPS-induced ALI/ acute respiratory distress syndrome (ARDS) rat model. RESULTS: The quality criteria have been standardized. In a stability study, haMSC-EVs were found to remain stable after 6 months of storage at - 80°C, 3 months at - 20 °C, and 6 h at room temperature. The microRNA profile and proteome of haMSC-EVs demonstrated suitable lot-to-lot consistency, further suggesting the stability of the production processes. Intratracheally administered 1.5 × 108 particles/rat/day for four weeks elicited no significant toxicity in rats. In LPS-induced ALI/ARDS model rats, intratracheally administered haMSC-EVs alleviated lung injury, possibly by reducing the serum level of inflammatory factors. CONCLUSION: haMSC-EVs, as an off-shelf drug, have suitable stability and lot-to-lot consistency. Intratracheally administered haMSC-EVs demonstrated excellent safety at the tested dosages in systematic preclinical toxicity studies. Intratracheally administered haMSC-EVs improved the lung function and exerted anti-inflammatory effects on LPS-induced ALI/ARDS model rats.

Bone mesenchymal stem cell-derived exosomal miR-26a-3p promotes autophagy to attenuate LPS-induced apoptosis and inflammation in pulmonary microvascular endothelial cells.

Acute lung injury (ALI) is a serious lung disease. The apoptosis and inflammation of pulmonary microvascular endothelial cells (PMVECs) are the primary reasons for ALI. This study aimed to explore the treatment effect and regulatory mechanism of bone mesenchymal stem cell-derived exosomes (BMSC-expos) on ALI. PMVECs were stimulated by Lipopolysaccharide (LPS) to imitate ALI environment. Cell viability was determined by CCK-8 assay. Cell apoptosis was evaluated by TUNEL and flow cytometry. ELISA was utilized for testing the contents of TNF-α, IL-1ß, IL-6, and IL-17. Western blot was applied for testing the levels of autophagy-related proteins LC3, p62, and Beclin-1. RNA interaction was determined by luciferase reporter assay. The ALI rat model was established by intratracheal injection of LPS. Evans blue staining was utilized for detecting pulmonary vascular permeability. Our results showed that LPS stimulation notably reduced cell viability, increased cell apoptosis rate, and enhanced the contents of inflammatory factors in PMVECs. However, BMSC-exo treatment significantly abolished the promoting effects of LPS on cell injury. In addition, we discovered that BMSC-exo treatment notably activated autophagy in LPS-induced PMVECs. Furthermore, BMSC-expos upregulated miR-26a-3p expression and downregulated PTEN in PMVECs. MiR-26a-3p was directly bound to PTEN. MiR-26a-3p overexpression reduced cell apoptosis, and inflammation and promoted autophagy by silencing PTEN. Animal experiments proved that miR-26a-3p overexpression effectively improved LPS-induced lung injury in rats. The results proved that BMSC-expos promotes autophagy to attenuate LPS-induced apoptosis and inflammation in pulmonary microvascular endothelial cells via miR-26a-3p/PTEN axis.

MSCs alleviate LPS-induced acute lung injury by inhibiting the proinflammatory function of macrophages in mouse lung organoid-macrophage model.

Acute lung injury (ALI) is an inflammatory disease associated with alveolar injury, subsequent macrophage activation, inflammatory cell infiltration, and cytokine production. Mesenchymal stem cells (MSCs) are beneficial for application in the treatment of inflammatory diseases due to their immunomodulatory effects. However, the mechanisms of regulatory effects by MSCs on macrophages in ALI need more in-depth study. Lung tissues were collected from mice for mouse lung organoid construction. Alveolar macrophages (AMs) derived from bronchoalveolar lavage and interstitial macrophages (IMs) derived from lung tissue were co-cultured, with novel matrigel-spreading lung organoids to construct an in vitro model of lung organoids-immune cells. Mouse compact bone-derived MSCs were co-cultured with organoids-macrophages to confirm their therapeutic effect on acute lung injury. Changes in transcriptome expression profile were analyzed by RNA sequencing. Well-established lung organoids expressed various lung cell type-specific markers. Lung organoids grown on spreading matrigel had the property of functional cells growing outside the lumen. Lipopolysaccharide (LPS)-induced injury promoted macrophage chemotaxis toward lung organoids and enhanced the expression of inflammation-associated genes in inflammation-injured lung organoids-macrophages compared with controls. Treatment with MSCs inhibited the injury progress and reduced the levels of inflammatory components. Furthermore, through the nuclear factor-κB pathway, MSC treatment inhibited inflammatory and phenotypic transformation of AMs and modulated the antigen-presenting function of IMs, thereby affecting the inflammatory phenotype of lung organoids. Lung organoids grown by spreading matrigel facilitate the reception of external stimuli and the construction of in vitro models containing immune cells, which is a potential novel model for disease research. MSCs exert protective effects against lung injury by regulating different functions of AMs and IMs in the lung, indicating a potential mechanism for therapeutic intervention.

Alveolar-capillary endocytosis and trafficking in acute lung injury and acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is associated with high morbidity and mortality but lacks specific therapeutic options. Diverse endocytic processes play a key role in all phases of acute lung injury (ALI), including the initial insult, development of respiratory failure due to alveolar flooding, as a consequence of altered alveolar-capillary barrier function, as well as in the resolution or deleterious remodeling after injury. In particular, clathrin-, caveolae-, endophilin- and glycosylphosphatidyl inositol-anchored protein-mediated endocytosis, as well as, macropinocytosis and phagocytosis have been implicated in the setting of acute lung damage. This manuscript reviews our current understanding of these endocytic pathways and subsequent intracellular trafficking in various phases of ALI, and also aims to identify potential therapeutic targets for patients with ARDS.

The efficacy of extracellular vesicles for acute lung injury in preclinical animal models: a meta-analysis.

BACKGROUND: With the increasing research on extracellular vesicles (EVs), EVs have received widespread attention as biodiagnostic markers and therapeutic agents for a variety of diseases. Stem cell-derived EVs have also been recognized as a new viable therapy for acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). To assess their efficacy, we conducted a meta-analysis of existing preclinical experimental animal models of EVs for ALI treatment. METHODS: The database was systematically interrogated for pertinent data encompassing the period from January 2010 to April 2022 concerning interventions involving extracellular vesicles (EVs) in animal models of acute lung injury (ALI). The lung injury score was selected as the primary outcome measure for statistical analysis. Meta-analyses were executed utilizing RevMan 5.3 and State15.1 software tools. RESULTS: The meta-analyses comprised 31 studies, exclusively involving animal models of acute lung injury (ALI), categorized into two cohorts based on the presence or absence of extracellular vesicle (EV) intervention. The statistical outcomes from these two study groups revealed a significant reduction in lung injury scores with the administration of stem and progenitor cell-derived EVs (SMD = -3.63, 95% CI [-4.97, -2.30], P < 0.05). Conversely, non-stem cell-derived EVs were associated with an elevation in lung injury scores (SMD = -4.34, 95% CI [3.04, 5.63], P < 0.05). EVs originating from stem and progenitor cells demonstrated mitigating effects on alveolar neutrophil infiltration, white blood cell counts, total cell counts in bronchoalveolar lavage fluid (BALF), lung wet-to-dry weight ratios (W/D), and total protein in BALF. Furthermore, pro-inflammatory mediators exhibited down-regulation, while anti-inflammatory mediators demonstrated up-regulation. Conversely, non-stem cell-derived EVs exacerbated lung injury. CONCLUSION: In preclinical animal models of acute lung injury (ALI), the administration of extracellular vesicles (EVs) originating from stem and progenitor cells demonstrably enhances pulmonary function. This ameliorative effect is attributed to the mitigation of pulmonary vascular permeability and the modulation of immune homeostasis, collectively impeding the progression of inflammation. In stark contrast, the utilization of EVs derived from non-stem progenitor cells exacerbates the extent of lung injury. These findings substantiate the potential utility of EVs as a novel therapeutic avenue for addressing acute lung injury.

Engineered extracellular vesicles carrying let-7a-5p for alleviating inflammation in acute lung injury.

BACKGROUND: Acute lung injury (ALI) is a life-threatening respiratory condition characterized by severe inflammation and lung tissue damage, frequently causing rapid respiratory failure and long-term complications. The microRNA let-7a-5p is involved in the progression of lung injury, inflammation, and fibrosis by regulating immune cell activation and cytokine production. This study aims to use an innovative cellular electroporation platform to generate extracellular vesicles (EVs) carring let-7a-5p (EV-let-7a-5p) derived from transfected Wharton's jelly-mesenchymal stem cells (WJ-MSCs) as a potential gene therapy for ALI. METHODS: A cellular nanoporation (CNP) method was used to induce the production and release of EV-let-7a-5p from WJ-MSCs transfected with the relevant plasmid DNA. EV-let-7a-5p in the conditioned medium were isolated using a tangential flow filtration (TFF) system. EV characterization followed the minimal consensus guidelines outlined by the International Society for Extracellular Vesicles. We conducted a thorough set of therapeutic assessments, including the antifibrotic effects using a transforming growth factor beta (TGF-ß)-induced cell model, the modulation effects on macrophage polarization, and the influence of EV-let-7a-5p in a rat model of hyperoxia-induced ALI. RESULTS: The CNP platform significantly increased EV secretion from transfected WJ-MSCs, and the encapsulated let-7a-5p in engineered EVs was markedly higher than that in untreated WJ-MSCs. These EV-let-7a-5p did not influence cell proliferation and effectively mitigated the TGF-ß-induced fibrotic phenotype by downregulating SMAD2/3 phosphorylation in LL29 cells. Furthermore, EV-let-7a-5p regulated M2-like macrophage activation in an inflammatory microenvironment and significantly induced interleukin (IL)-10 secretion, demonstrating their modulatory effect on inflammation. Administering EVs from untreated WJ-MSCs slightly improved lung function and increased let-7a-5p expression in plasma in the hyperoxia-induced ALI rat model. In comparison, EV-let-7a-5p significantly reduced macrophage infiltration and collagen deposition while increasing IL-10 expression, causing a substantial improvement in lung function. CONCLUSION: This study reveals that the use of the CNP platform to stimulate and transfect WJ-MSCs could generate an abundance of let-7a-5p-enriched EVs, which underscores the therapeutic potential in countering inflammatory responses, fibrotic activation, and hyperoxia-induced lung injury. These results provide potential avenues for developing innovative therapeutic approaches for more effective interventions in ALI.

Mesenchymal Stem Cells-Derived Exosomes Alleviate Acute Lung Injury by Inhibiting Alveolar Macrophage Pyroptosis.

Acute lung injury (ALI) is an important pathological process of acute respiratory distress syndrome, yet there are limited therapies for its treatment. Mesenchymal stem cells-derived exosomes (MSCs-Exo) have been shown to be effective in suppressing inflammation. However, the effects of MSCs-Exo on ALI and the underlying mechanisms have not been well elucidated. Our data showed that MSCs-Exo, but not exosomes derived from MRC-5 cells (MRC-5-Exo), which are human fetal lung fibroblast cells, significantly improved chest imaging, histological observations, alveolocapillary membrane permeability, and reduced inflammatory response in ALI mice model. According to miRNA sequencing and proteomic analysis of MSCs-Exo and MRC-5-Exo, MSCs-Exo may inhibit pyroptosis by miRNAs targeting caspase-1-mediated pathway, and by proteins with immunoregulation functions. Taken together, our study demonstrated that MSCs-Exo were effective in treating ALI by inhibiting the pyroptosis of alveolar macrophages and reducing inflammation response. Its mechanism may be through pyroptosis-targeting miRNAs and immunoregulating proteins delivered by MSCs-Exo. Therefore, MSCs-Exo may be a new treatment option in the early stage of ALI.

Umbilical cord MSC-derived exosomes improve alveolar macrophage function and reduce LPS-induced acute lung injury.

Acute lung injury (ALI) is a severe condition that can progress to acute respiratory distress syndrome (ARDS), with a high mortality rate. Currently, no specific and compelling drug treatment plan exists. Mesenchymal stem cells (MSCs) have shown promising results in preclinical and clinical studies as a potential treatment for ALI and other lung-related conditions due to their immunomodulatory properties and ability to regenerate various cell types. The present study focuses on analyzing the role of umbilical cord MSC (UC-MSC))-derived exosomes in reducing lipopolysaccharide-induced ALI and investigating the mechanism involved. The study demonstrates that UC-MSC-derived exosomes effectively improved the metabolic function of alveolar macrophages and promoted their shift to an anti-inflammatory phenotype, leading to a reduction in ALI. The findings also suggest that creating three-dimensional microspheres from the MSCs first can enhance the effectiveness of the exosomes. Further research is needed to fully understand the mechanism of action and optimize the therapeutic potential of MSCs and their secretome in ALI and other lung-related conditions.

Pulmonary RNA interference against acute lung injury mediated by mucus- and cell-penetrating nanocomplexes.

Trans-mucosal delivery of anti-inflammatory siRNA into alveolar macrophages represents a promising modality for the treatment of acute lung injury (ALI). However, its therapeutic efficacy is often hurdled by the lack of effective carriers that can simultaneously overcome the mucosal barrier and cell membrane barrier. Herein, we developed mucus/cell membrane dual-penetrating, macrophage-targeting polyplexes which enabled efficient intratracheal delivery of TNF-α siRNA (siTNF-α) to attenuate pulmonary inflammation against lipopolysaccharide (LPS)-induced ALI. P-G@Zn, a cationic helical polypeptide bearing both guanidine and zinc dipicolylamine (Zn-DPA) side charged groups, was designed to condense siTNF-α and promote macrophage internalization due to its helicity-dependent membrane activity. Coating of the polyplexes with charge-neutralizing carboxylated mannan (Man-COOH) greatly enhanced the mucus penetration potency due to shielding of the electrostatic adhesive interactions with the mucus, and it cooperatively enabled active targeting to alveolar macrophages to potentiate the intracellular delivery efficiency of siTNF-α. As such, intratracheally administered Man-COOH/P-G@Zn/siTNF-α polyplexes provoked notable TNF-α silencing by ∼75 % in inflamed lung tissues at 500 µg siRNA/kg, and demonstrated potent anti-inflammatory performance to treat ALI. This study provides an effective tool for the synchronized trans-mucosal delivery of siRNA into macrophages, and the unique properties of the polyplexes render remarkable potentials for anti-inflammatory therapy against ALI. STATEMENT OF SIGNIFICANCE: siRNA-mediated anti-inflammatory management of acute lung injury (ALI) is greatly challenged by the insufficient delivery across the mucus layer and cell membrane. To address such critical issue, mucus/cell membrane dual-penetrating, macrophage-targeting polyplexes are herein developed, which are comprised of an outer shell of carboxylated mannan (Man-COOH) and an inner nanocore formed by TNF-α siRNA (siTNF-α) and a cationic helical polypeptide P-G@Zn. Man-COOH coating endowed the polyplexes with high mucus-penetrating capability and macrophage-targeting ability, while P-G@Zn bearing both guanidine and zinc dipicolylamine afforded potent siTNF-α condensation capacity and high intracellular delivery efficiency with reduced cytotoxicity. Intratracheally administered polyplexes solicit pronounced TNF-α silencing and anti-inflammatory efficiencies in ALI mice. This study renders an effective example for overcoming the multiple barriers against trans-mucosal delivery of siRNA into macrophages, and holds profound potentials for gene therapy against ALI.

High-intensity intermittent training ameliorates methotrexate-induced acute lung injury.

Inflammation and oxidative stress are recognized as two primary causes of lung damage induced by methotrexate, a drug used in the treatment of cancer and immunological diseases. This drug triggers the generation of oxidants, leading to lung injury. Given the antioxidant and anti-inflammatory effects of high-intensity intermittent training (HIIT), our aim was to evaluate the therapeutic potential of HIIT in mitigating methotrexate-induced lung damage in rats. Seventy male Wistar rats were randomly divided into five groups: CTL (Control), HIIT (High-intensity intermittent training), ALI (Acute Lung Injury), HIIT+ALI (pretreated with HIIT), and ALI + HIIT (treated with HIIT).HIIT sessions were conducted for 8 weeks. At the end of the study, assessments were made on malondialdehyde, total antioxidant capacity (TAC), superoxide dismutase (SOD), glutathione peroxidase (Gpx), myeloperoxidase (MPO), interleukin 10 (IL-10), tumor necrosis factor-alpha (TNF-α), gene expression of T-bet, GATA3, FOXP3, lung wet/dry weight ratio, pulmonary capillary permeability, apoptosis (Caspase-3), and histopathological indices.Methotrexate administration resulted in increased levels of TNF-α, MPO, GATA3, caspase-3, and pulmonary edema indices, while reducing the levels of TAC, SOD, Gpx, IL-10, T-bet, and FOXP3. Pretreatment and treatment with HIIT reduced the levels of oxidant and inflammatory factors, pulmonary edema, and other histopathological indicators. Concurrently, HIIT increased the levels of antioxidant and anti-inflammatory factors.

Bone marrow mesenchymal stem cells-derived exosomes ameliorate LPS-induced acute lung injury by miR-223-regulated alveolar macrophage M2 polarization.

Numerous studies have shown that the M2 polarization of alveolar macrophages (AM) plays a protective role in acute lung injury (ALI). Mesenchymal stem cells (MSCs) secreted exosomes have been reported to be involved in inflammatory diseases by the effects of polarized M1/M2 macrophage populations. However, whether bone marrow mesenchymal stem cells (BMMSCs) derived exosomes could protect from ALI and its mechanisms are still unclear. Here, we explored the role of exosomes from BMMSC in rat AM polarization and the lipopolysaccharide- (LPS-) induced ALI rat model. Furthermore, the levels of exosomal miR-223 in BMMSCs were measured by RT-qPCR. Additionally, miR-223 mimics and its inhibitors were used to verify the vital role of miR-223 of BMMSCs-derived exosomes in the polarization of M2 macrophages. The results showed that BMMSCs-derived exosomes were taken up by the AM. Exosomes derived from BMMSCs promoted M2 polarization of AM in vitro. BMMSCs exosomes effectively mitigated pathological injuries, lung edema, and the inflammation of rats from LPS-induced ALI, accompanied by an increase of M2 polarization of AM in lung tissue. Interestingly, we also found that miR-223 was enriched in BMMSCs-derived exosomes, and overexpression of miR-223 in BMMSCs-derived exosomes promoted M2 polarization of AM while depressing miR-223 showed opposite effects in AM. The present study demonstrated that BMMSCs-derived exosomes triggered alveolar M2 polarization to improve inflammation by transferring miR-223, which may provide new therapeutic strategies in ALI.

Endothelium-Derived Engineered Extracellular Vesicles Protect the Pulmonary Endothelial Barrier in Acute Lung Injury.

Acute lung injury (ALI) is a severe respiratory disease with a high mortality rate. The integrity of the pulmonary endothelial barrier influences the development and prognosis of ALI. Therefore, it has become an important target for ALI treatment. Extracellular vesicles (EVs) are promising nanotherapeutic agents against ALI. Herein, endothelium-derived engineered extracellular vesicles (eEVs) that deliver microRNA-125b-5p (miRNA-125b) to lung tissues exerting a protective effect on endothelial barrier integrity are reported. eEVs that are modified with lung microvascular endothelial cell-targeting peptides (LET) exhibit a prolonged retention time in lung tissues and targeted lung microvascular endothelial cells in vivo and in vitro. To improve the efficacy of the EVs, miRNA-125b is loaded into EVs. Finally, LET-EVs-miRNA-125b is constructed. The results show that compared to the EVs, miRNA-125b, and EVs-miRNA-125b, LET-EVs-miRNA-125b exhibit the most significant treatment efficacy in ALI. Moreover, LET-EVs-miRNA-125b is found to have an important protective effect on endothelial barrier integrity by inhibiting cell apoptosis, promoting angiogenesis, and protecting intercellular junctions. Sequencing analysis reveals that LET-EVs-miRNA-125b downregulates early growth response-1 (EGR1) levels, which may be a potential mechanism of action. Taken together, these findings suggest that LET-EVs-miRNA-125b can treat ALI by protecting the endothelial barrier integrity.

A heterogenous population of extracellular vesicles mobilize to the alveoli postinjury.

BACKGROUND: Acute lung injury and subsequent resolution following severe injury are coordinated by a complex lung microenvironment that includes extracellular vesicles (EVs). We hypothesized that there is a heterogenous population of EVs recruited to the alveoli postinjury and that we could identify specific immune-relevant mediators expressed on bronchoalveolar lavage (BAL) EVs as candidate biomarkers of injury and injury resolution. METHODS: Mice underwent 30% TBSA burn injury and BAL fluid was collected 4 hours postinjury and compared with sham. Extracellular vesicles were purified and single vesicle flow cytometry (vFC) was performed using fluorescent antibodies to quantify the expression of specific cell surface markers on individual EVs. Next, we evaluated human BAL specimens from injured patients to establish translational relevance of the mouse vFC analysis. Human BAL was collected from intubated patients following trauma or burn injury, EVs were purified, then subjected to vFC analysis. RESULTS: A diverse population of EVs were mobilized to the alveoli after burn injury in mice. Quantitative BAL vFC identified significant increases in macrophage-derived CD44+ EVs (preinjury, 10.8% vs. postinjury, 13%; p < 0.05) and decreases in IL-6 receptor alpha (CD126) EVs (preinjury, 19.3% vs. postinjury, 9.3%, p < 0.05). Bronchoalveolar lavage from injured patients also contained a heterogeneous population of EVs derived from myeloid cells, endothelium, and epithelium sources, with CD44+ EVs being highly detected. CONCLUSION: Injury causes mobilization of a heterogeneous population of EVs to the alveoli in both animal models and injured patients. Defining EV release after injury will be critical in identifying diagnostic and therapeutic targets to limit postinjury acute lung injury.

Neutrophil membrane-engineered Panax ginseng root-derived exosomes loaded miRNA 182-5p targets NOX4/Drp-1/NLRP3 signal pathway to alleviate acute lung injury in sepsis: experimental studies.

BACKGROUND: The purpose of this study was to prepare neutrophil membrane-engineered Panax ginseng root-derived exosomes (N-exo) and investigate the effects of N-exo microRNA (miRNA) 182-5p (N-exo-miRNA 182-5p) on acute lung injury (ALI) in sepsis. METHODS: Panax ginseng root-derived exosomes were separated by differential centrifugation. Neutrophil membrane engineering was performed on exo to obtain N-exo. miRNA182-5p was transmitted into N-exo by electroporation technology to obtain N-exo-miRNA 182-5p. LPS was used to establish an in-vivo and in-vitro model of ALI of sepsis to evaluate the anti-inflammatory effect of N-exo-miRNA 182-5p. RESULTS: The results of transmission electron microscope showed that exo was a double-layer membrane structure like a saucer. Nanoparticle size analysis showed that the average particle size of exo was 129.7 nm. Further, compared with exo, the level of miRNA182-5p was significantly increased in N-exo. The experimental results showed that N-exo-miRNA 182-5p significantly improved ALI via target regulation of NOX4/Drp-1/NLRP3 signal pathway in vivo and in vitro . CONCLUSION: In conclusion, this study prepared a novel engineered exosome (N-exo and N-exo-miRNA 182-5p significantly improved ALI in sepsis via target regulation of NOX4/Drp-1/NLRP3 signal pathway, providing new ideas and methods for treatment of ALI in sepsis.

Apoptotic bodies inhibit inflammation by PDL1-PD1-mediated macrophage metabolic reprogramming.

Apoptosis triggers immunoregulation to prevent and suppress inflammation and autoimmunity. However, the mechanism by which apoptotic cells modulate immune responses remains largely elusive. In the context of allogeneic mesenchymal stem cells (MSCs) transplantation, long-term immunoregulation is observed in the host despite the short survive of the injected MSCs. In this study, utilizing a mouse model of acute lung injury (ALI), we demonstrate that apoptotic bodies (ABs) released by transplanted human umbilical cord MSCs (UC-MSCs) convert the macrophages from a pro-inflammatory to an anti-inflammatory state, thereby ameliorating the disease. Mechanistically, we identify the expression of programmed cell death 1 ligand 1 (PDL1) on the membrane of UC-MSCs-derived ABs, which interacts with programmed cell death protein 1 (PD1) on host macrophages. This interaction leads to the reprogramming of macrophage metabolism, shifting from glycolysis to mitochondrial oxidative phosphorylation via the Erk-dependent pathway in ALI. Importantly, we have reproduced the PDL1-PD1 effects of ABs on metabolic switch using alveolar macrophages from patients with ALI, suggesting the potential clinical implications of developing therapeutic strategies for the patients.

ISX-9 Promotes KGF Secretion From MSCs to Alleviate ALI Through NGFR-ERK-TAU-ß-Catenin Signaling Axis.

BACKGROUND: Mesenchymal stem cells (MSCs) have been widely studied to alleviate acute lung injury (ALI) due to their paracrine function. However, the microenvironment of inflammatory outbreaks significantly restricted the factors secreted from MSCs like keratinocyte growth factor (KGF). KGF is a growth factor with tissue-repaired ability. Is there a better therapeutic prospect for MSCs in combination with compounds that promote their paracrine function? Through compound screening, we screened out isoxazole-9 (ISX-9) to promote MSCs derived KGF secretion and investigated the underlying mechanisms of action. METHODS: Compounds that promote KGF secretion were screened by a dual-luciferase reporter gene assay. The TMT isotope labeling quantitative technique was used to detect the differential proteins upon ISX-9 administrated to MSCs. The expressions of NGFR, ERK, TAU, and ß-catenin were detected by Western blot. In the ALI model, we measured the inflammatory changes by HE staining, SOD content detection, RT-qPCR, immunofluorescence, etc. The influence of ISX-9 on the residence time of MSCs transplantation was explored by optical in vivo imaging. RESULTS: We found out that ISX-9 can promote the expression of KGF in MSCs. ISX-9 acted on the membrane receptor protein NGFR, upregulated phosphorylation of downstream signaling proteins ERK and TAU, downregulated phosphorylation of ß-catenin, and accelerated ß-catenin into the nucleus to further increase the expression of KGF. In the ALI model, combined ISX-9 with MSCs treatments upgraded the expression of KGF in the lung, and enhanced the effect of MSCs in reducing inflammation and repairing lung damage compared with MSCs alone. CONCLUSIONS: ISX-9 facilitated the secretion of KGF from MSCs both in vivo and in vitro. The combination of ISX-9 with MSCs enhanced the paracrine function and anti-inflammatory effect of MSCs compared with MSCs applied alone in ALI. ISX-9 played a contributive role in the transplantation of MSCs for the treatment of ALI.

Extracellular Vesicles Derived from Human Adipose-Derived Mesenchymal Stem Cells Alleviate Sepsis-Induced Acute Lung Injury through a MicroRNA-150-5p-Dependent Mechanism.

Extracellular vesicles (EVs) derived from human adipose mesenchymal stem cells (hADSCs) may exert a therapeutic benefit in alleviating sepsis-induced organ dysfunction by delivering cargos that include RNAs and proteins to target cells. The current study aims to explore the protective effect of miR-150-5p delivered by hADSC-EVs on sepsis-induced acute lung injury (ALI). We noted low expression of miR-150-5p in plasma and bronchoalveolar lavage fluid samples from patients with sepsis-induced ALI. The hADSC-EVs were isolated and subsequently cocultured with macrophages. It was established that hADSC-EVs transferred miR-150-5p to macrophages, where miR-150-5p targeted HMGA2 to inhibit its expression and, consequently, inactivated the MAPK pathway. This effect contributed to the promotion of M2 polarization of macrophages and the inhibition of proinflammatory cytokines. Further, mice were made septic by cecal ligation and puncture in vivo and treated with hADSC-EVs to elucidate the effect of hADSC-EVs on sepsis-induced ALI. The in vivo experimental results confirmed a suppressive role of hADSC-EVs in sepsis-induced ALI. Our findings suggest that hADSC-EV-mediated transfer of miR-150-5p may be a novel mechanism underlying the paracrine effects of hADSC-EVs on the M2 polarization of macrophages in sepsis-induced ALI.

Bone Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Ameliorated Lipopolysaccharide-Induced Lung Injury Via the miR-21-5p/PCSK6 Pathway.

Acute lung injury (ALI) is a life-threatening disease that currently lacks a cure. Although stem cell-derived small extracellular vesicles (sEVs) have shown promising effects in the treatment of ALI, their underlying mechanisms and responsible components have yet to be identified. Proprotein convertase subtilisin/kexin type 6 (PCSK6) is a gene involved in inflammation and a potential target of miR-21-5p, a microRNA enriched in stem cell-derived sEVs. The current study investigated the role of PCSK6 in lipopolysaccharide (LPS)-induced ALI and its interaction with miR-21-5p. Notably, our results showed that PCSK6 expression was positively correlated with LPS stimulation. Knockdown of PCSK6 ameliorated LPS-induced inhibition of proliferation and upregulation of permeability in human BEAS-2B cells, whereas PCSK6 overexpression displayed the opposite effects. BEAS-2B cells were able to actively internalize the cocultured bone mesenchymal stem cell (MSC)-derived sEVs (BMSC-sEVs), which alleviated the cell damage caused by LPS. Overexpressing PCSK6, however, eliminated the therapeutic effects of BMSC-sEV coculture. Mechanistically, BMSC-sEVs inhibited PCSK6 expression via the delivery of miR-21-5p, which is directly bound to the PCSK6 gene. Our work provides evidence for the role of PCSK6 in LPS-induced ALI and identified miR-21-5p as a component of BMSC-derived sEVs that suppressed PCSK6 expression and ameliorated LPS-induced cell damage. These results reveal a novel molecular mechanism for ALI pathogenesis and highlight the therapeutic potential of using sEVs released by stem cells to deliver miR-21-5p for ALI treatment.