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》

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 ... -  《-》

Inhibition of IRE1α/XBP1 axis alleviates LPS-induced acute lung injury by suppressing TXNIP/NLRP3 inflammasome activation and ERK/p65 signaling pathway.

Acute lung injury or acute respiratory distress syndrome (ALI/ARDS) is a devastating clinical syndrome with high incidence and mortality rates. IRE1α-XBP1 pathway is one of the three major signaling axes of endoplasmic reticulum stress that is involved in inflammation, metabolism, and immunity. The role and potential mechanisms of IRE1α-XBP1 axis in ALI/ARDS has not well understood. The ALI murine model was established by intratracheal administration of lipopolysaccharide (LPS). Hematoxylin and eosin (H&E) staining and analysis of bronchoalveolar lavage fluid (BALF) were used to evaluate degree of lung injury. Inflammatory responses were assessed by ELISA and RT-PCR. Apoptosis was evaluated using TUNEL staining and western blot. Moreover, western blot, immunohistochemistry, and immunofluorescence were applied to test expression of IRE1α, XBP1, NLRP3, TXNIP, IL-1β, ERK1/2 and NF-κB p65. The expression of IRE1α significantly increased after 24 h of LPS treatment. Inhibition of the IRE1α-XBP1 axis with 4µ8C notably improved LPS-induced lung injury and inflammatory infiltration, reduced the levels of IL-6, IL-1β, and TNF-α, and decreased cell apoptosis as well as the activation of the NLRP3 inflammasome. Besides, in LPS-stimulated Beas-2B cells, both 4µ8C and knockdown of XBP1 diminished the mRNA levels of IL-6 and IL-1B, inhibited cell apoptosis and reduced the protein levels of TXNIP, NLRP3 and secreted IL-1β. Mechanically, the phosphorylation and nuclear translocation of ERK1/2 and p65 were significantly suppressed by 4µ8C and XBP1 knockdown. In summary, our findings suggest that IRE1α-XBP1 axis is crucial in the pathogenesis of ALI/ARDS, whose suppression could mitigate the pulmonary inflammatory response and cell apoptosis in ALI through the TXNIP/NLRP3 inflammasome and ERK/p65 signaling pathway. Our study may provide new evidence that IRE1α-XBP1 may be a promising therapeutic target for ALI/ARDS.

Wang S ，Hu L ，Fu Y ，Xu F ，Shen Y ，Liu H ，Zhu L ... -  《RESPIRATORY RESEARCH》

SS31 alleviates LPS-induced acute lung injury by inhibiting inflammatory responses through the S100A8/NLRP3/GSDMD signaling pathway.

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is an acute, diffuse, inflammatory lung injury caused by various endogenous or exogenous factors. It is currently widely recognized that an excessive inflammatory response resulting from immune imbalance constitutes a crucial pathogenic mechanism in ALI/ARDS. SS31 is a novel mitochondria-targeted antioxidant peptide. This article validates the role of SS31 in lipopolysaccharide (LPS)-induced ALI. The study applied transcriptome sequencing, immunofluorescence, PCR, immunofluorescence and other methods to explore the mechanism of SS31 in LPS induced ALI. Transcriptome sequencing results indicate that LPS-induced ALI is closely associated with immune regulatory processes, the Toll-like receptor pathway, and the NF-κB signaling pathway. The role of SS31 in acute lung injury is closely related to biological processes, such as immune regulation and cell death. This study demonstrated that SS31 can inhibit the expression of inflammatory factors IL-6, IL-1β, IL-18, and TNF-α, and reduce the expression of pyroptosis-related proteins NLRP3, and GSDMD-N. Further analysis revealed that S100A8 may be a key gene in the effect of SS31. LPS stimulation leads to increased expression of S100A8, while SS31 decreases its expression. Recombinant protein S100A8 can attenuate the inhibitory effect of SS31 on IL-1β, IL-18, NLRP3, and GSDMD-N. The research results indicate that SS31 may inhibit the activation of the NLRP3 inflammasome and suppress inflammatory responses by regulating S100A8, thereby alleviating LPS-induced ALI in mice; this process may be related to pyroptosis.

Luo P ，Gu Q ，Wang J ，Li X ，Li N ，Yang W ，Meng X ，Zhao M ... -  《-》

Shikonin ameliorated LPS-induced acute lung injury in mice via modulating MCU-mediated mitochondrial Ca(2+) and macrophage polarization.

Macrophages play a pivotal role in the development and recovery of acute lung injury (ALI), wherein their phenotypic differentiation and metabolic programming are orchestrated by mitochondria. Specifically, the mitochondrial calcium uniporter (MCU) regulates mitochondrial Ca2+ (mCa2+) uptake and may bridge the metabolic reprogramming and functional regulation of immune cells. However, the precise mechanism on macrophages remains elusive. Shikonin, a natural naphthoquinone, has demonstrated efficacy in mitigating ALI and suppressing glycolysis in macrophages, yet which mechanism remains to be fully elucidated. This study explored whether Shikonin ameliorated ALI via modulating MCU-mediated mCa2+ and macrophage polarization. This study firstly examined the protective effects of Shikonin on LPS-induced ALI mice, and investigated whether it is depends on macrophage by depleting macrophage using clodronate liposomes. The regulatory effect of Shikonin on macrophage polarization and mitochondrial MCU/Ca2+ signal was testified on RAW264.7 cells, and further validated by knocking-down MCU expression or by using RU360, an MCU inhibitor. Additionally, the crucial role of MCU in the therapeutic effect of Shikonin, along with its regulation on macrophage polarization was validated in mice with LPS-induced ALI under the intervention of RU360. Shikonin alleviated LPS-induced mice ALI, down-regulated inflammatory cytokines and inhibited the pro-inflammatory polarization of macrophages. Intravenous injection of clodronate liposomes on mice abolished the protective effects of Shikonin on ALI. On RAW264.7 cells, LPS&IFN decreased the protein expression of MCU, while induced pro-inflammatory polarization and glycolytic metabolism. In contrast, Shikonin increased MCU expression, activated MCU-mediated mCa2+ signal, promoted the polarization of macrophages to anti-inflammatory M2 phenotype, and driven a metabolic shift from glycolysis to oxidative phosphorylation. Either knocking-down MCU expression or pharmacological inhibiting MCU by using RU360 mitigated the effects of Shikonin on Raw 264.7 cells. Furthermore, RU360 counteracted the ameliorative effect of Shikonin on ALI mice. The current data showed that Shikonin alleviated LPS-induced mice ALI by activating mitochondrial MCU/mCa2+ signal and regulating macrophage metabolism.

Bao-Yuan H ，Shu-Ru L ，Le-Xin C ，Liang-Liang B ，Cheng-Cheng L ，Chun-Qi X ，Ming-Jun L ，Jia-Xin Z ，En-Xin Z ，Xiao-Jun Z ... -  《-》