Macrophage-derived exosomal miR-2137 regulates pyroptosis in LPS-induced acute lung injury.

Alveolar macrophages (AMs) play a predominant role in acute lung injury (ALI). However, the role of macrophage-derived exosomal miRNAs in lipopolysaccharide (LPS)-induced ALI has not been determined. We previously reported that exosomes in the bronchoalveolar lavage fluid (BALF) of mice with ALI were derived predominantly from macrophages. Exosomal small RNA sequencing was conducted to identify the miRNA profiles. Exosomes derived from LPS-induced macrophages (LPS-exos) were intravenously administered to C57BL/6J mice, after which lung injury and pyroptosis were assessed. LPS-exos were cultured with alveolar epithelial cells (AECs) to further validate the results of the animal studies. LPS-exos promoted lung inflammation and pyroptosis in vivo and in vitro. MiR-2137 was significantly upregulated in both LPS-exos and in MLE-12 cells. LPS-exos reduced cell viability, promoted the expression of LDH and inflammatory cytokines, and exacerbated vacuolization in MLE-12 cells. The administration of miR-2137 mimics and LPS-treated exosomes further strengthened these effects and enhanced pyroptosis mediated by NLRP3, Caspase1, ASC, and GSDMD. MiR-2137 mediated the effects of LPS-exos by targeting Wnt9a in AECs. In addition, the miR-2137 inhibitor markedly decreased the severity of LPS-exo-induced histological lesions, inflammation and pyroptosis in the lung. Exosomal miR-2137 derived from AMs contributes to LPS-induced ALI by inducing AEC pyroptosis through the targeting of Wnt9a to activate the Wnt signaling pathway. This study revealed that AMs and AECs interact in ALI, providing novel strategies for ALI treatment.

Ye C ，Yang X ，Zhu L ，Chang G ，Hu Y ，Wang W ... -  《-》

Mitoxantrone attenuates lipopolysaccharide-induced acute lung injury via inhibition of NEDD8 activating enzyme.

Lipopolysaccharide (LPS) triggers the activation of nuclear factor kappa B (NF-κB) by interacting with Toll-like receptor 4 (TLR4), leading to the production of various proinflammatory enzymes and cytokines that are crucial in the development of acute lung injury (ALI). Mitoxantrone (MTX) has been demonstrated to mitigate the inflammatory response caused by LPS; however, its precise function in the context of ALI is not fully comprehended. This study aimed to investigate the inhibitory effects and underlying mechanisms of MTX against LPS-induced ALI. ALI was induced in C57BL/6 mice via a single intratracheal administration of LPS (5 mg/kg), followed by an intraperitoneal injection of MTX to evaluate its therapeutic potential. The effects of MTX on lung injury and the progression of inflammation in ALI mice were assessed using a comprehensive range of techniques, including hematoxylin-eosin (H&E) staining, immunohistochemistry (IHC), myeloperoxidase activity measurement, cell enumeration in bronchoalveolar lavage fluid (BALF), Western blotting, and enzyme-linked immunosorbent assay (ELISA). Additionally, IHC, Western blotting, and co-immunoprecipitation were used to elucidate the specific signaling pathways and molecular mechanisms by which MTX exerted its anti-inflammatory effects in ALI mice. Surface plasmon resonance (SPR) and molecular docking were used to examine the target to which MTX binds directly to reduce inflammation. We also established a lung epithelial cell injury model using LPS-treated A549 cells. The polyubiquitination of IκBα and TRAF6 in LPS-induced A549 cells was detected through Western blotting following immunoprecipitation. In mice with LPS-induced ALI, MTX exhibits anti-inflammatory effects by ameliorating histopathological abnormalities caused by LPS, reducing inflammatory cell infiltration, and decreasing the production of proinflammatory enzymes and cytokines. It has been observed that MTX directly binds to the NEDD8 activating enzyme (NAE), thereby inhibiting the transfer of NEDD8 to the substrates UBC12, Cul1, and Cul5. Consequently, the polyubiquitination of IκBα and TRAF6 is disrupted, leading to the suppression of TAK1 activation by TRAF6. This suppression of TAK1 activity hindered the phosphorylation of IKK and MAPK. By stabilizing IκBα through dephosphorylation via IKK inhibition and preventing polyubiquitination, NF-κB activation is reduced. This cascade of events ultimately leads to a reduction in the production of proinflammatory enzymes and cytokines, effectively mitigating the inflammatory response in ALI. In A549 cells, MTX reduces the LPS-induced K48-linked polyubiquitination of IκBα and K63-linked polyubiquitination of TRAF6. This process can be reversed by the overexpression of NEDD8. Additionally, treatment with MG-132, a proteasome inhibitor, can restore the polyubiquitination of IκBα that was inhibited by MTX. These findings confirm the essential role of Cul1/5 neddylation in ALI and suggest that MTX could be a promising therapeutic agent for ALI.

Liu H ，Liu Y ，Lin X ，Fan J ，Huang Z ，Li A ... -  《-》

EGCG targeting STAT3 transcriptionally represses PLXNC1 to inhibit M2 polarization mediated by gastric cancer cell-derived exosomal miR-92b-5p.

M2-polarized tumor-associated macrophages (TAMs) predominate in tumor microenvironment (TME) and serve primary functions in tumor progression, including growth, angiogenesis, metastasis, immunosuppression, chemoresistance, and poor prognosis. The reversal of M2 polarization provides a new treatment strategy for cancer. Presently, the molecular mechanisms of M2 polarization have yet to be fully characterized, and there is a lack of effective therapeutic targets and drugs. Cancer cells initiate an immunosuppressive TME by recruiting macrophages and promoting M2 polarization through the secretion of inflammatory factors. Accordingly, blocking cancer cell-induced TAM M2 polarization may present a more effective strategy from the perspective of cancer cells. Hedyotis diffusa Willd (HDW) possesses immunomodulatory and antitumor properties, and is a precious and direct source of small molecule natural products with a dual function of inhibition of tumor growth and tumor cell-mediated M2 polarization. To identify a new target promoting gastric cancer (GC) cell growth and GC cell-mediated M2 polarization from mRNA profiles of GC cells treated with HDW injection (HDI) and to excavate a natural product from HDI that can regulate related mRNA and inhibit the aforementioned effects. RNA sequencing (RNA-seq) was used to analyze HDI-regulated differentially expressed mRNAs (HRmRNAs) in MKN45 cells. Weighted gene co-expression network analysis (WGCNA), univariate and multivariate Cox regression analysis, KM survival curves, and association analysis between HRmRNA and clinical characteristics/tumor infiltrating immune cells (TIICs) individually were utilized to screen out the target HRmRNA associated with prognosis and M2 macrophage infiltration in GC. shRNA lentiviral vectors were used for stably silencing, and transient overexpressing plasmids were constructed for overexpression. CCK8, EdU, colony formation, migration and invasion assays were used to validate the function of drugs and molecules in GC. HDI constituent analysis was performed using UHPLC-QE-MS. A network of HDI constituent-hub transcription factor (TF)-HRmRNA was constructed based on RNA-Seq, network pharmacology and TFs prediction. Exosome isolation and identification were performed using ultracentrifugation, NTA, TEM and western blot. Apoptosis and macrophage phenotypes were determined by flow cytometric analysis. Small RNA-Seq made exosomal miRNA identification. Small molecule interaction with targets were analyzed using molecular docking, SPR and CETSA. The direct relationship between transcription factors and promoters was verified using ChIP-QPCR and dual-luciferase reporter gene assay. A nude mice xenograft tumor model was established for vivo validation. HDI inhibited MKN45 cell proliferation, migration, invasion and promoted apoptosis. RNA-Seq identified 2583 HRmRNAs. PLXNC1 was screened out as the target HRmRNA associated with prognosis and M2 macrophage infiltration in GC. PLXNC1 promoted GC cell proliferation and facilitated TAMs M2 polarization by transferring GC cell-derived exosomal miR-92b-5p, inhibiting SOCS7-STAT3 interactions and subsequently activating STAT3 in macrophages. M2 TAMs induced by PLXNC1-mediated GC cell-derived exosomes promoted GC cell migration and invasion. PLXNC1 regulated exosomal miR-92b-5p through the MEK1/MSK1/CREB1 pathway. STAT3 could transcriptionally regulate PLXNC1 expression in GC cells. The network of HDI constituent-hub TF-HRmRNA showed epigallocatechin gallate (EGCG) from HDI targeted STAT3 to transcriptionally regulate PLXNC1 expression. EGCG as a natural product directly bound to STAT3 to diminish its nuclear localization, resulting in the transcriptional repression of PLXNC1 and the reversal of M2 polarization induced by PLXNC1-mediated GC cell-derived exosomes. PLXNC1 is a novel target exerting dual effects on GC cell proliferation and GC cell-mediated M2 polarization. EGCG derived from HDI inhibits GC cell proliferation and targets STAT3 to inhibit M2 polarization induced by PLXNC1-mediated exosomes derived from GC cells, which may be a multi-target therapeutic agent for GC cell proliferation and immune microenvironment.

Yi J ，Ye Z ，Xu H ，Zhang H ，Cao H ，Li X ，Wang T ，Dong C ，Du Y ，Dong S ，Zhou W ... -  《-》

Placental mesenchymal stem cells suppress inflammation and promote M2-like macrophage polarization through the IL-10/STAT3/NLRP3 axis in acute lung injury.

Acute lung injury (ALI) is a clinically severe respiratory disorder that currently lacks specific and effective pharmacotherapy. The imbalance of M1/M2 macrophage polarization is pivotal in the initiation and progression of ALI. Shifting macrophage polarization from the proinflammatory M1 phenotype to the anti-inflammatory M2 phenotype could be a potential therapeutic strategy. The intratracheal administration of placental mesenchymal stem cells (pMSCs) has emerged as a novel and effective treatment for ALI. This study aimed to investigate the role and downstream mechanisms of pMSCs in reprogramming macrophage polarization to exert anti-inflammatory effects in ALI. The study used lipopolysaccharide (LPS) to induce inflammation in both cell and rat models of ALI. Intratracheal administration of pMSCs was tested as a therapeutic intervention. An expression dataset for MSCs cultured with LPS-treated macrophages was collected from the Gene Expression Omnibus database to predict downstream regulatory mechanisms. Experimental validation was conducted through in vitro and in vivo assays to assess pMSCs effects on macrophage polarization and inflammation. Both in vitro and in vivo experiments validated that pMSCs promoted M2 macrophage polarization and reduced the release of inflammatory factors. Further analyses revealed that pMSCs activated the signal transducer and activator of transcription (STAT)3 signaling pathway by secreting interleukin (IL)-10, leading to increased STAT3 phosphorylation and nuclear translocation. This activation inhibited NLRP3 inflammasome activation, promoting M2 macrophage polarization and suppressing the inflammatory response. The study concluded that pMSCs alleviated lung injury in an LPS-induced ALI model by inhibiting M1 macrophage polarization and proinflammatory factor secretion, while promoting M2 macrophage polarization. This effect was mediated via the IL-10/STAT3/NLRP3 axis, presenting a novel therapeutic pathway for ALI treatment.

Nie Z ，Fan Q ，Jiang W ，Wei S ，Luo R ，Hu H ，Liu G ，Lei Y ，Xie S ... -  《Frontiers in Immunology》

Cadmium exposure triggers alveolar epithelial cell pyroptosis by inducing mitochondrial oxidative stress and activating the cGAS-STING pathway.

Cadmium is a ubiquitous toxic metal and environmental pollutant. More and more studies have shown that cadmium exposure can damage lung function. Alveolar epithelial cells (AECs) are structural cells that maintain the stability of lung function. The injury of AECs is an essential determinant of many lung diseases. In the lung, cadmium accumulation can cause damage to AECs. However, the specific mechanism is still unclear. This study aimed to explore the key mechanism underlying the injury of AECs caused by cadmium exposure. The main modes of death of AECs induced by cadmium exposure were evaluated in vivo and in vitro. Transcriptomic changes of AECs induced by cadmium exposure were analyzed using RNA-sequence. Mitochondrial ROS scavengers (mitoQ), voltage-dependent anion channel 1 (VDAC1) oligomer inhibitor (VBIT4), and cyclic GMP-AMP synthase (cGAS) inhibitor (RU.521) were used to assess whether cadmium exposure triggered pyroptosis of AECs by inducing mitochondrial stress to activate the cGAS-STING-NLRP3 axis. In this study, the expression of pyroptosis-related proteins was significantly up-regulated in the cadmium-exposed AECs, while the expression of apoptosis, necroptosis, and ferroptosis-related proteins had no significant up-regulated. The pan-caspase inhibitor ZVAD-FMK significantly reduced cell death. Thus, our research indicates that pyroptosis is the primary type of AEC death exported to cadmium. Mechanistically, RNA-seq and Western Blot results showed that cadmium exposure activated the cGAS-STING pathway in AECs and promoted pyroptosis by activating the NLRP3 inflammasome. Further investigation of the mechanism found that cadmium exposure caused mitochondrial oxidative stress, which led to mtDNA leakage into the cytoplasm and activated the cGAS-STING pathway. In addition, inhibition of the cGAS-STING pathway significantly alleviated lung injury induced by cadmium exposure in mice. Our study confirmed that pyroptosis of AECs was a vital mechanism of lung injury after cadmium exposure in a cGAS-STING-dependent manner, which may provide a new target for the treatment of lung diseases induced by cadmium exposure.

Zhang CY ，Ou AJ ，Jin L ，Yang NS ，Deng P ，Guan CX ，Huang XT ，Duan JX ，Zhou Y ... -  《Cell Communication and Signaling》