Apoptosis Of Alveolar Macrophages Research Articles

Phosphoproteomic analysis reveals changes in A-Raf-related protein phosphorylation in response to Toxoplasma gondii infection in porcine macrophages

BackgroundToxoplasma gondii is an obligate intracellular protozoan parasite that causes severe threats to humans and livestock. Macrophages are the cell type preferentially infected by T. gondii in vivo. Protein phosphorylation is an important posttranslational modification involved in diverse cellular functions. A rapidly accelerated fibrosarcoma kinase (A-Raf) is a member of the Raf family of serine/threonine protein kinases that is necessary for MAPK activation. Our previous research found that knockout of A-Raf could reduce T. gondii-induced apoptosis in porcine alveolar macrophages (3D4/21 cells). However, limited information is available on protein phosphorylation variations and the role of A-Raf in macrophages infected with T. gondii.MethodsWe used immobilized metal affinity chromatography (IMAC) in combination with liquid chromatography tandem mass spectrometry (LC–MS/MS) to profile changes in phosphorylation in T. gondii-infected 3D4/21 and 3D4/21-ΔAraf cells.ResultsA total of 1647 differentially expressed phosphorylated proteins (DEPPs) with 3876 differentially phosphorylated sites (DPSs) were identified in T. gondii-infected 3D4/21 cells (p3T group) when compared with uninfected 3D4/21 cells (pho3 group), and 959 DEPPs with 1540 DPSs were identified in the p3T group compared with infected 3D4/21-ΔAraf cells (p3KT group). Venn analysis revealed 552 DPSs corresponding to 406 DEPPs with the same phosphorylated sites when comparing p3T/pho3 versus p3T/p3KT, which were identified as DPSs and DEPPs that were directly or indirectly related to A-Raf.ConclusionsOur results revealed distinct responses of macrophages to T. gondii infection and the potential roles of A-Raf in fighting infection via phosphorylation of crucial proteins.Graphical

Lnc-Clic5 as a sponge for miR-212–5p to inhibit cow barn PM2.5-induced apoptosis in rat alveolar macrophages

Particulate matter 2.5 (PM2.5) is a highly hazardous airborne particulate matter that poses a significant risk to humans and animals. Urban airborne particulate matter contributes to the increased incidence and mortality of respiratory diseases, such as asthma and chronic obstructive pulmonary disease (COPD), in humans. However, the specific mechanism by which PM2.5 affects animals in barn environments is yet to be elucidated. In this study, we investigated the effect of exposure to cow barn PM2.5 on rat alveolar macrophages (NR8383) and found that it induced apoptosis via the miR-212–5p/RASSF1 pathway. We found that lnc-Clic5 expression was downregulated in NR8383 cells exposed to cow barn PM2.5. Lnc-Clic5 plays a competitive endogenous RNA (ceRNA) regulatory role by sponging miR-212–5p to attenuate the regulation of RASSF1. Moreover, lnc-Clic5 overexpression inhibited NR8383 apoptosis by targeting the miR-212–5p/RASSF1 pathway. Co-treatment with miR-212–5p and lnc-Clic5 in the presence of cow barn PM2.5 revealed that lnc-Clic5 reversed NR8383 cell apoptosis induced by PM2.5 when miR-212–5p was overexpressed. These findings contribute to the study of ncRNAs and ceRNAs regulating PM2.5-induced apoptosis in animal farms, provide therapeutic targets for lung macrophage apoptosis, and may be useful for further evaluating the toxicological effects of PM2.5 in farmhouses on the respiratory systems of humans and animals.

Mechanistic insights into targeting caspase-3 activation and alveolar macrophage pyroptosis by Ephedra and bitter almond compounds for treating pediatric pneumonia via network pharmacology and bioinformatics.

This study investigates the molecular mechanism of Ma Huang-Ku Xing Ren, a traditional Chinese medicine formula, in treating pediatric pneumonia. The focus is on the regulation of caspase-3 activation and reduction of alveolar macrophage necrosis through network pharmacology and bioinformatics analyses of Ephedra and bitter almond components. Active compounds and targets from ephedrine and bitter almond were obtained using TCMSP, TCMID, and GeneCards databases, identifying pediatric pneumonia-related genes. A protein-protein interaction (PPI) network was constructed, and core targets were screened. GO and KEGG pathway enrichment analyses identified relevant genes and pathways. An acute pneumonia mouse model was created using the lipopolysaccharide (LPS) inhalation method, with caspase-3 overexpression induced by a lentivirus. The mice were treated with Ephedra and bitter almond through gastric lavage. Lung tissue damage, inflammatory markers (IL-18 and IL-1β), and cell death-related gene activation were assessed through H&E staining, ELISA, western blot, flow cytometry, and immunofluorescence. The study identified 128 active compounds and 121 gene targets from Ephedra and bitter almond. The PPI network revealed 13 core proteins, and pathway analysis indicated involvement in inflammation, apoptosis, and cell necrosis, particularly the caspase-3 pathway. Invivo results showed that Ephedra and bitter almond treatment significantly mitigated LPS-induced lung injury in mice, reducing lung injury scores and inflammatory marker levels. It also decreased caspase-3 activity and cell death in alveolar macrophages. In conclusion, the active ingredients of Ma Huang-Ku Xing Ren, particularly targeting caspase-3, may effectively treat pediatric pneumonia by reducing apoptosis in alveolar macrophages, as demonstrated by both network pharmacology, bioinformatics analyses, and experimental data.

Circ-phkb promotes cell apoptosis and inflammation in LPS-induced alveolar macrophages via the TLR4/MyD88/NF-kB/CCL2 axis

BackgroundCircular RNAs (CircRNAs) have been associated with acute lung injury (ALI), but their molecular mechanisms remain unclear.MethodsThis study developed a rat model of lipopolysaccharide (LPS)-induced ALI and evaluated the modeling effect by hematoxylin and eosin staining, Masson’s trichrome staining, lung wet-to-dry weight ratio, terminal deoxynucleotidyl transferase UTP nick end labeling (TUNEL), and enzyme-linked immunosorbent assay (ELISA) detection of inflammatory factors (interleukin-1β, tumor necrosis factor alpha, and interleukin-6). Using lung tissues from a rat model of LPS-induced ALI, we then conducted circRNA sequencing, mRNA sequencing, and bioinformatics analysis to obtain differential circRNA and mRNA expression profiles as well as potential ceRNA networks. Furthermore, we performed quantitative real-time polymerase chain reaction (qRT-PCR) assays to screen for circ-Phkb in ALI rat lung tissues, alveolar macrophages, and LPS-induced NR8383 cells. We conducted induction with or without LPS with circ-Phkb siRNA and overexpression lentivirus in NR8383. Cell Counting Kit-8, C5-Ethynyl-2′-deoxyuridine (Edu), TUNEL, and cytometry were used to identify proliferation and apoptosis, respectively. We detected inflammatory factors using ELISA. Finally, we used Western blot to detect the apoptosis-related proteins and TLR4/MyD88/NF-kB/CCL2 pathway activation.ResultsOur results revealed that both circRNA and mRNA profiles are different from those of the Sham group. We observed a significant circ-Phkb upregulation in NR8383 cells and LPS-exposed rats. Apoptosis and inflammation were greatly reduced when circ-Phkb expression was reduced in NR8383 cells, cell proliferation was increased, and circ-Phkb overexpression was decreased.ConclusionsIn terms of mechanism, circ-Phkb suppression inhibits CCL2 expression via the TLR4/MyD88/NF-kB pathway in LPS-induced alveolar macrophages.

MicroRNA miR-212-5p Regulates the MEK/ERK Signaling Pathway by Targeting A-Raf proto-oncogene serine/threonine-protein kinase (ARAF) to Regulate Cowshed PM2.5-Induced NR8383 Apoptosis.

Objective: To investigate the role of miR-212-5p-targeted ARAF during the apoptosis of rat alveolar macrophages induced by cowshed PM2.5. Methods: miRNA and related target genes and pathways were predicted using the KEGG, TargetScan, and other prediction websites. NR8383 macrophages were treated with cowshed PM2.5 to establish an in vitro lung injury model in rats; meanwhile, for the assessment of cell viability, apoptosis, intracellular calcium ions, and mitochondrial membrane potential in NR8383 cells, RT-qPCR was used to detect the expression of miR-212-5p and the target gene ARAF. Results: The bioinformatic analyses showed that miR-212-5p and ARAF were involved in PM2.5-associated cellular damage. Exposure to different concentrations (0 μg/mL, 60 μg/mL, 180 μg/mL, 300 μg/mL) with different durations (0 h, 12 h, 24 h, 48 h) of cowshed PM2.5 resulted in apoptosis, increased intracellular calcium ions, and decreased mitochondrial membrane potential. The miR-212-5p mimic group showed an up-regulation of Bax and cleaved Caspase 3 expression but decreased Bcl2 expression compared to the NC group, and overexpression of ARAF up-regulated the expression of p-MEK1/2 and p-ERK1/2 and simultaneously reversed the above phenomena. Conclusions: miR-212-5p targets ARAF to affect the cowshed PM2.5-induced apoptosis through the MEK/ERK signaling pathway, providing a potential target for relevant farming industry and pathology studies.

Riding apoptotic bodies for cell-cell transmission by African swine fever virus.

African swine fever virus (ASFV), a devastating pathogen to the worldwide swine industry, mainly targets macrophage/monocyte lineage, but how the virus enters host cells has remained unclear. Here, we report that ASFV utilizes apoptotic bodies (ApoBDs) for infection and cell-cell transmission. We show that ASFV induces cell apoptosis of primary porcine alveolar macrophages (PAMs) at the late stage of infection to productively shed ApoBDs that are subsequently swallowed by neighboring PAMs to initiate a secondary infection as evidenced by electron microscopy and live-cell imaging. Interestingly, the virions loaded within ApoBDs are exclusively single-enveloped particles that are devoid of the outer layer of membrane and represent a predominant form produced during late infection. The in vitro purified ApoBD vesicles are capable of mediating virus infection of naive PAMs, but the transmission can be significantly inhibited by blocking the "eat-me" signal phosphatidyserine on the surface of ApoBDs via Annexin V or the efferocytosis receptor TIM4 on the recipient PAMs via anti-TIM4 antibody, whereas overexpression of TIM4 enhances virus infection. The same treatment however did not affect the infection by intracellular viruses. Importantly, the swine sera to ASFV exert no effect on the ApoBD-mediated transmission but can partially act on the virions lacking the outer layer of membrane. Thus, ASFV has evolved to hijack a normal cellular pathway for cell-cell spread to evade host responses.

Cyanidin‐3‐galactoside from Aronia melanocarpa ameliorates PM10‐induced pulmonary inflammation by promoting PINK1/Parkin signaling pathway‐mediated alveolar macrophage mitophagy

AbstractAronia melanocarpa is rich in anthocyanin, among which cyanidin‐3‐galactoside (C3G) is the most abundant. Here, we established an experimental model of mice and alveolar macrophage cell line MH‐S exposed to PM10, and C3G was administered to explore the underlying mechanism of C3G exerting protective effects on PM10‐induced lung inflammation. The results showed C3G alleviated PM10‐induced lung inflammation by reducing matrix metalloproteinases production at both gene and protein levels as well as pro‐inflammatory cytokine levels. Autophagy was activated in PM10‐injured alveolar macrophages (AMs), and C3G promoted autophagy indicated by LC3, BECN1, Atg5, Bcl‐2, and P62 expression, and C3G further resisted PM10‐induced AMs apoptosis. Besides, C3G could promote Pink1/Parkin‐mediated mitochondrial autophagy of AMs to alleviate the overexpression of mitochondrial reactive oxygen species (ROS) so that maintain mitochondrial membrane potential and ATP productions to restrict the distribution of Cyt‐c and further reduce the increasing of caspase 3/7 activity. Therefore, C3G enhanced the clearance of PM10‐damaged mitochondria by promoting AMs mitophagy, thereby reducing the activation of mitochondria‐dependent apoptosis pathway and the production of excess ROS, which reduced AMs apoptosis and secretion of pro‐inflammatory factors to ameliorate PM10‐induced pulmonary inflammation, suggesting that C3G may be as a functional food ingredient with the potential to improve lung health.

TurboID screening of ApxI toxin interactants identifies host proteins involved in Actinobacillus pleuropneumoniae-induced apoptosis of immortalized porcine alveolar macrophages

Actinobacillus pleuropneumoniae (APP) is a gram-negative pathogenic bacterium responsible for porcine contagious pleuropneumonia (PCP), which can cause porcine necrotizing and hemorrhagic pleuropneumonia. Actinobacillus pleuropneumoniae-RTX-toxin (Apx) is an APP virulence factor. APP secretes a total of four Apx toxins, among which, ApxI demonstrates strong hemolytic activity and cytotoxicity, causing lysis of porcine erythrocytes and apoptosis of porcine alveolar macrophages. However, the protein interaction network between this toxin and host cells is still poorly understood. TurboID mediates the biotinylation of endogenous proteins, thereby targeting specific proteins and local proteomes through gene fusion. We applied the TurboID enzyme-catalyzed proximity tagging method to identify and study host proteins in immortalized porcine alveolar macrophage (iPAM) cells that interact with the exotoxin ApxI of APP. His-tagged TurboID-ApxIA and TurboID recombinant proteins were expressed and purified. By mass spectrometry, 318 unique interacting proteins were identified in the TurboID ApxIA-treated group. Among them, only one membrane protein, caveolin-1 (CAV1), was identified. A co-immunoprecipitation assay confirmed that CAV1 can interact with ApxIA. In addition, overexpression and RNA interference experiments revealed that CAV1 was involved in ApxI toxin-induced apoptosis of iPAM cells. This study provided first-hand information about the proteome of iPAM cells interacting with the ApxI toxin of APP through the TurboID proximity labeling system, and identified a new host membrane protein involved in this interaction. These results lay a theoretical foundation for the clinical treatment of PCP.

Repetitive Low-Level Blast Exposure via Akt/NF-κB Signaling Pathway Mediates the M1 Polarization of Mouse Alveolar Macrophage MH-S Cells

Repetitive low-level blast (rLLB) exposure is a potential risk factor for the health of soldiers or workers who are exposed to it as an occupational characteristic. Alveolar macrophages (AMs) are susceptible to external blast waves and produce pro-inflammatory or anti-inflammatory effects. However, the effect of rLLB exposure on AMs is still unclear. Here, we generated rLLB waves through a miniature manual Reddy-tube and explored their effects on MH-S cell morphology, phenotype transformation, oxidative stress status, and apoptosis by immunofluorescence, real-time quantitative PCR (qPCR), western blotting (WB) and flow cytometry. Ipatasertib (GDC-0068) or PDTC was used to verify the role of the Akt/NF-κB signaling pathway in these processes. Results showed that rLLB treatment could cause morphological irregularities and cytoskeletal disorders in MH-S cells and promote their polarization to the M1 phenotype by increasing iNOS, CD86 and IL-6 expression. The molecular mechanism is through the Akt/NF-κB signaling pathway. Moreover, we found reactive oxygen species (ROS) burst, Ca2+ accumulation, mitochondrial membrane potential reduction, and early apoptosis of MH-S cells. Taken together, our findings suggest rLLB exposure may cause M1 polarization and early apoptosis of AMs. Fortunately, it is blocked by specific inhibitors GDC-0068 or PDTC. This study provides a new treatment strategy for preventing and alleviating health damage in the occupational population caused by rLLB exposure.

Inhibition of STAT3 phosphorylation by colchicine regulates NLRP3 activation to alleviate sepsis-induced acute lung injury.

The pharmacotherapeutic mechanism of colchicine, a tricyclic, lipid-soluble alkaloid extracted from the plant of the Lily family Colchicum autumnale, has not been fully understood in diverse disorders, including sepsis-induced acute lung injury (ALI). The study aimed at exploring the impact of colchicine on sepsis-induced ALI and the relevant mechanisms. Colchicine significantly attenuated ALI in mice caused by sepsis by alleviating respiratory dysfunction and pulmonary edema in mice, inhibiting NLRP3 inflammasome formation, and reducing oxidative stress, pyroptosis, and apoptosis of murine alveolar macrophage (J774A.1) cells. The targets of colchicine were predicted in the superPRED database and intersected with the differentially expressed genes in the GSE5883 and GSE129775 datasets. The major targets were subjected to protein-protein interaction network generation and Kyoto Encyclopedia of Genes and Genomes enrichment analysis. It was thus found that colchicine inhibited STAT3 phosphorylation but did not alter STAT3 total protein expression. Phosphorylated STAT3 recruited EP300 to form a complex to promote histone H3 acetylation and histone H4 acetylation of NLRP3 promoter, leading to pyroptosis of J774A.1 cells. In conclusion, inhibition of STAT3 phosphorylation by colchicine represses NLRP3 promoter acetylation via the STAT3/EP300 complex, thereby alleviating ALI caused by sepsis.

Atractylenolide III Ameliorated Autophagy Dysfunction via Epidermal Growth Factor Receptor-Mammalian Target of Rapamycin Signals and Alleviated Silicosis Fibrosis in Mice

Atractylenolide III (ATL-III) is a major active constituent of the natural plant Atractylodes rhizome. Our previous study has shown that ATL-III may alleviate alveolar macrophage apoptosis via the inhibition of the mammalian target of rapamycin (mTOR)-mediated autophagy of human silicosis. Therefore, we aimed to further explore the function of ATL-III in autophagy, apoptosis, and pulmonary fibrosis by establishing the ATL-III–intervened silicosis mouse model in this study. Meanwhile, we sought and then verified potential autophagy-related signaling pathways by matching differentially expressed genes (attained by RNA sequencing) and the autophagy database. In this study, RNA-sequencing results implied that the epidermal growth factor receptor, the crucial upstream activator of mTOR, was seen as a potential autophagy-regulatory molecule in the ATL-III–intervened silicosis mouse model. The finding of this study was that ATL-III might improve the disorder of autophagic degradation via the activation of epidermal growth factor receptor-mTOR signals in the pulmonary tissue of the silicosis mouse model. ATL-III also alleviated cell apoptosis and silicotic fibrosis. Overall, we supposed that ATL-III might be a potential protective medicine, which had a regulatory effect on autophagy, for the intervention of silicotic fibrosis. In the future, the therapeutic drugs for silicosis should be further focused on the development and application of such natural autophagy agents.

FBXO6 regulates the antiviral immune responses via mediating alveolar macrophages survival.

Inducing early apoptosis in alveolar macrophages is one of the strategies influenza A virus (IAV) evolved to subvert host immunity. Correspondingly, the host mitochondrial protein nucleotide-binding oligomerization domain-like receptor (NLR)X1 is reported to interact with virus polymerase basic protein 1-frame 2 (PB1-F2) accessory protein to counteract virus-induced apoptosis. Herein, we report that one of the F-box proteins, FBXO6, promotes proteasomal degradation of NLRX1, and thus facilitates IAV-induced alveolar macrophages apoptosis and modulates both macrophage survival and type I interferon (IFN) signaling. We observed that FBXO6-deficient mice infected with IAV exhibited decreased pulmonary viral replication, alleviated inflammatory-associated pulmonary dysfunction, and less mortality. Analysis of the lungs of IAV-infected mice revealed markedly reduced leukocyte recruitment but enhanced production of type I IFN in Fbxo6-/- mice. Furthermore, increased type I IFN production and decreased viral replication were recapitulated in FBXO6 knockdown macrophages and associated with reduced apoptosis. Through gain- and loss-of-function studies, we found lung resident macrophages but not bone marrow-derived macrophages play a key role in the differences FBXO6 signaling pathway brings in the antiviral immune response. In further investigation, we identified that FBXO6 interacted with and promoted the proteasomal degradation of NLRX1. Together, our results demonstrate that FBXO6 negatively regulates immunity against IAV infection by enhancing the degradation of NLRX1 and thus impairs the survival of alveolar macrophages and antiviral immunity of the host.

MG132 protects against lung injury following brain death in rats.

Brain death (BD) results in injury to organs and induces lung donor dysfunction. Since the 20S proteasome abnormality is associated with a variety of diseases, the present study investigated whether it was involved in lung injury following BD in rats, and the effects of the proteasome inhibitor MG132 on lung injury was also assessed. Rats were assigned to a BD group or a control sham group. The BD group of rats were sacrificed at different time points after BD. Administration of MG132 was performed intraperitoneally 30 min before BD. Arterial blood was drawn to measure the oxygenation index [partial artery pressure of oxygen (PaO2)/fractional concentration of inspired oxygen (FiO2)]. The right lung was used for staining with hematoxylin and eosin, immunohistochemistry, immunofluorescence, western blotting and RT-qPCR analysis. The left lung was used to measure the wet and dry weights. Rat alveolar macrophages (NR8383) were treated with MG132 and hypoxia/reoxygenation (H/R) and used for western blotting and flow cytometry. The PaO2/FiO2 ratio decreased after BD; the wet/dry weight ratio, histological lung injury score and protein expression of 20S proteasome β1 and inducible nitric oxide synthase (iNOS) gradually increased in rats after BD. Colocalization in the immunofluorescence between 20S proteasome β1 and iNOS was observed. MG132 treatment increased the PaO2/FiO2 ratio and decreased the wet/dry weight ratio, histological lung injury score and protein expression of 20S proteasome β1 and iNOS in rats after BD. MG132 was revealed to increase NR8383 apoptosis after H/R and to upregulate the protein expression levels of p-JNK and cleaved-caspase 3. Overall, the proteasome inhibitor MG132 could effectively reduce lung injury, which may be associated with its ability to inhibit the expression of the proteasome and promote the apoptosis of alveolar macrophages.

A novel therapeutic approach for IPF: Based on the “Autophagy - Apoptosis” balance regulation of Zukamu Granules in alveolar macrophages

Ethnopharmacological relevanceZukamu Granules (ZKMG) is one of the representative Uygur patent drugs widely used in China, which is included in the National Essential Drugs List (2018 edition). As the first choice for common cold treatment in Uygur medicine theory, it has unique anti-inflammatory and antitussive efficacy. Aim of the studyAccording to the recent inflammatory hypothesis, the abnormal proliferation, autophagy and apoptosis process of lung cells especially alveolar macrophages (AMs) may play an important role in the progress of idiopathic pulmonary fibrosis (IPF). Therefore, we came up with a novel treatment approach for IPF by regulating the balance of AMs “autophagy - apoptosis”, and took ZKMG as the sample drug for our research. Materials and methodsNetwork pharmacology approach was conducted to predict the active components and intersected targets between ZKMG and inflammation. PPI network, GO and KEGG enrichment analysis were screened and analyzed to predict the anti-inflammatory mechanism of ZKMG. Biological experiment adopted from 128 rats, and hematoxylin-eosin staining, flow cytometry and RT-PCR were performed to examine the pathological morphology, HYP contents in lung tissue, AMs counting, AMs apoptosis, AMs phagocytosis rate, mRNA relative quantity determination of 3 key factors associated with AMs “autophagy - apoptosis” and mRNA relative quantity determination of AMs surface receptor signaling pathway. ResultsThe predicted results showed that the mechanism of ZKMG in anti-inflammatory was related to the response and elimination of inflammatory stimuli, the intervention of apoptosis and surface receptor signaling pathways of cells. The verification experiments showed that excessive apoptosis and insufficient autophagy of AMs always existed in the progression of IPF. ZKMG could inhibit AMs proliferation, significantly reduce AMs apoptosis rate, intervene the binding of the Bcl-2 to Beclin 1, inhibit the Caspase 3 activation, stimulate the enhancement of AMs phagocytosis, and inhibit the high expression of TLR4/MyD88/NF-κB surface receptor signaling pathway, which may partly retard the fibrosis process. ConclusionBy inhibiting proliferation, enhancing phagocytosis, inhibiting the formation of Bcl-2 complex, and inhibiting the high expression of MYD88-dependent TLR4 signaling pathway, ZKMG can regulate the balance of AMs “autophagy - apoptosis” in the alveolitis stage to retard the fibrosis process partly. With a comprehensive strategy of “target prediction - experimental verification”, we have demonstrated that inhibiting the apoptosis and promoting autophagy activity of AMs may suggest a new perspective for IPF treatment, which would provide reference for the subsequent development.

Retraction.

Retraction: "TNF-α-TNFR signal pathway inhibits autophagy and promotes apoptosis of alveolar macrophages in coal worker's pneumoconiosis," by Qingzeng Qian, Xiangke Cao, Bin Wang, Yi Qu, Qingqiang Qian, Zhiqian Sun, Fumin Feng, J Cell Physiol. 2019; 5953-5963: The above article, published online on 23 November 2018 in Wiley Online Library (https://doi.org/10.1002/jcp.27061), has been retracted by agreement between the journal's Editor in Chief, Prof. Dr. Gregg Fields, and Wiley Periodicals LLC. The retraction has been agreed after the authors asked to correct their article, and following an investigation based on allegations raised by a third party. Several flaws and duplications were found in figures 3d, 5a and 6. The authors admit substantial mistakes during figure compilation for this manuscript. After a detailed investigation and analysis of the submitted correction data, the experiments in this article are considered unreliable and to not sufficiently support the conclusions. The authors were not available for a final confirmation of the retraction.

EGCG Restricts PRRSV Proliferation by Disturbing Lipid Metabolism.

ABSTRACTPorcine reproductive and respiratory syndrome virus (PRRSV) infection leads to late-term reproductive failure and respiratory illness that affect the global swine industry. Epigallocatechin gallate (EGCG) is a polyphenolic compound from green tea that exerts antiviral activity against diverse viruses. This study aimed to report an uncharacterized mechanism of how EGCG restricted PRRSV proliferation. EGCG showed no significant effects on cell viability, cell cycle progression, and apoptosis in porcine alveolar macrophages and MARC-145 cells. The treatment of cells with EGCG attenuated the replication of both highly pathogenic and less pathogenic PRRSV in vitro. The viral life cycle analysis demonstrated that EGCG affected PRRSV replication and assembly, but not viral attachment, entry, or release. Interestingly, EGCG treatment abrogated the increased lipid droplets formation and lipid content induced by PRRSV infection. We further demonstrated that EGCG blocked PRRSV-stimulated expression of the key enzymes in lipid synthesis. In addition, EGCG attenuated PRRSV-induced autophagy that is critical for PRRSV proliferation. The supplementation of oleic acid restored PRRSV replication and assembly under EGCG treatment. Together, our results support that EGCG inhibits PRRSV proliferation through disturbing lipid metabolism.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped single-positive-stranded RNA virus that causes acute respiratory distress in piglets and reproductive failure in sows, resulting in huge economic losses to the global swine industry. Several lines of evidence have suggested the crucial roles of lipids in PRRSV proliferation. Our previous report demonstrated that PRRSV activated lipophagy to facilitate viral replication through downregulating the expression of N-Myc downstream-regulated gene 1. The manipulation of lipid metabolism may be a new perspective to prevent PRRSV spread. In the present study, we reported that epigallocatechin-3-gallate (EGCG), the major component of green tea catechins, significantly attenuated PRRSV infection through inhibiting lipid synthesis and autophagy. Given that natural products derived from plants have helped in the prevention and treatment of various infectious diseases, EGCG has a great potential to serve as a safe and environmentally friendly natural compound to treat PRRSV infection.

Bone mesenchymal stem cell-derived extracellular vesicles inhibit DAPK1-mediated inflammation by delivering miR-191 to macrophages

Alveolar macrophage activation and apoptosis are vital contributors to sepsis-associated acute lung injury (ALI). However, the mechanisms of alveolar macrophage activation are yet to be clarified. Death-associated protein kinase 1 (DAPK1) is one of the potential candidates that play crucial roles in regulating alveolar macrophage inflammation. Herein, we found that primary human bone mesenchymal stem cell (BMSC)-derived extracellular vesicles (EVs) antagonize LPS-induced inflammation in the THP-1 human macrophage-like cell line. Mechanistically, LPS stimulation elevates the expression of DAPK1 and the inflammation markers in THP-1 cells, while BMSC-derived EVs inhibit the expression of DAPK1 and inflammation through delivering miR-191, which can target the 3′-UTR of the DAPK1 mRNA and therefore suppress its translation. The importance of DAPK1 in the activation of THP-1 is also stressed in this study. Our findings provide evidence that BMSC-derived EVs regulate the alveolar macrophage inflammation and highlight BMSC-derived EVs as a potential vehicle to deliver biomacromolecules to macrophages.

ISM1 protects lung homeostasis via cell-surface GRP78-mediated alveolar macrophage apoptosis

Alveolar macrophages (AMs) are critical for lung immune defense and homeostasis. They are orchestrators of chronic obstructive pulmonary disease (COPD), with their number significantly increased and functions altered in COPD. However, it is unclear how AM number and function are controlled in a healthy lung and if changes in AMs without environmental assault are sufficient to trigger lung inflammation and COPD. We report here that absence of isthmin 1 (ISM1) in mice (Ism1-/- ) leads to increase in both AM number and functional heterogeneity, with enduring lung inflammation, progressive emphysema, and significant lung function decline, phenotypes similar to human COPD. We reveal that ISM1 is a lung resident anti-inflammatory protein that selectively triggers the apoptosis of AMs that harbor high levels of its receptor cell-surface GRP78 (csGRP78). csGRP78 is present at a heterogeneous level in the AMs of a healthy lung, but csGRP78high AMs are expanded in Ism1-/- mice, cigarette smoke (CS)-induced COPD mice, and human COPD lung, making these cells the prime targets of ISM1-mediated apoptosis. We show that csGRP78high AMs mostly express MMP-12, hence proinflammatory. Intratracheal delivery of recombinant ISM1 (rISM1) depleted csGRP78high AMs in both Ism1-/- and CS-induced COPD mice, blocked emphysema development, and preserved lung function. Consistently, ISM1 expression in human lungs positively correlates with AM apoptosis, suggesting similar function of ISM1-csGRP78 in human lungs. Our findings reveal that AM apoptosis regulation is an important physiological mechanism for maintaining lung homeostasis and demonstrate the potential of pulmonary-delivered rISM1 to target csGRP78 as a therapeutic strategy for COPD.

Long non-coding RNA NEAT1 participates in ventilator-induced lung injury by regulating miR-20b expression.

Long non-coding (lnc)RNA nuclear enriched abundant transcript 1 (NEAT1) has been reported to serve an important role in cancer, but its effects on ventilator-induced lung injury (VILI) remain unclear. The present study aimed to investigate the role of lncRNA NEAT1 in alveolar macrophages (AMs) on ventilator-induced lung injury (VILI). Mouse and cell models were established to detect NEAT1 expression, pathological changes in lung tissues, apoptosis of AMs, expression of the M1 phenotype marker, CD86 and M2 phenotype marker, CD206, and the expression levels of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS). The associations between NEAT1, microRNA (miRNA/miR)-20b and STAT3 were predicted using StarBase and TargetScan, and verified via the dual-luciferase reporter and RIP assays. NEAT1 short hairpin RNA and miR-20b inhibitor were co-transfected into AMs to assess the effect of NEAT1 and miR-20b in VILI. The results demonstrated that NEAT1 was highly expressed in lung tissues of VILI mice and cell stretch (CS) treated AMs. Furthermore, NEAT1 knockdown inhibited lung injury and cell apoptosis induced by VILI. Compared with VILI mice or CS-treated AMs, NEAT1 knockdown accelerated the phenotypic transformation from M1 to M2, and decreased the expression levels of IL-1β, IL-6, TNF-α and iNOS. Notably, miR-20b was identified as the target of NEAT1, and STAT3 was the target of miR-20b. NEAT1 knockdown decreased STAT3 protein expression, the effects of which were reversed following transfection with miR-20b inhibitor. Furthermore, the protective effect of NEAT1 knockdown on VILI was reversed following transfection with miR-20b inhibitor. Taken together, the results of the present study suggest that NEAT1 knockdown promotes phenotypic transformation of AMs from M1 to M2 and alleviates lung injury and apoptosis of VILI by regulating miR-20b expression.

Knockdown of GBP1 inhibits BCG-induced apoptosis in macrophage RAW 264.7 cells via p38/JNK pathway.

Alveolar macrophage apoptosis induced by Mycobacterium tuberculosis (Mtb) plays a significant role in mediating the pathogenesis of tuberculosis. There is growing evidence that guanylate-binding proteins (GBPs) are associated with different pathological processes such as microbial infection. However, it remains unclear whether GBPs can regulate the apoptosis of macrophages induced by Mtb. In this study, we investigated the potential effect of GBP1 on RAW 264.7 cell apoptosis during Bacillus Calmette-Guerin (BCG) infection. The results demonstrated that BCG could induce macrophage apoptosis and GBP1 upregulation. In addition, we explored the role of GBP1 in regulating BCG-induced RAW 264.7 cell apoptosis using small interfering RNAs targeting GBP1. The results showed that knockdown of GBP1 could attenuate BCG-induced apoptosis in RAW 264.7 cells. Moreover, we found that GBP1 knockdown decreased the levels of cleaved-Caspase 3 and cleaved-PARP-1, while decreased those of cleaved-Caspase 9, BAX, Cytochrome C and APAF1. These findings imply that GBP1 knockdown can prevent BCG-induced apoptosis through an endogenous apoptosis pathway. In addition, the mitochondrial membrane potential of macrophages was significantly increased after BCG infection, and GBP1 knockdown could alleviate this phenomenon. Furthermore, downregulation of GBP1 also attenuated BCG-induced accumulation of reactive oxygen species in macrophages. Mechanistically, GBP1 suppressed the phosphorylation of the target molecules in p38/JNK pathway, thus regulating the apoptosis of BGC-infected macrophages. Collectively, these findings reveal a significant role of GBP1 in mediating cell apoptosis in macrophages infected with BCG, and the molecular mechanism underlying its suppressive effect on BCG-induced apoptosis.