GDF15 ameliorates sepsis-induced lung injury via AMPK-mediated inhibition of glycolysis in alveolar macrophage.

Growth differentiation factor 15 (GDF15) as a stress response cytokine is involved in the development and progression of several diseases associated with metabolic disorders. However, the regulatory role and the underlying mechanisms of GDF15 in sepsis remain poorly defined. Our study analyzed the levels of GDF15 and its correlations with the clinical prognosis of patients with sepsis. In vivo and in vitro models of sepsis were applied to elucidate the role and mechanisms of GDF15 in sepsis-associated lung injury. We observed strong correlations of plasma GDF15 levels with the levels of C-reactive protein (CRP), procalcitonin (PCT), lactate dehydrogenase (LDH), and lactate as well as Sequential Organ Failure Assessment (SOFA) scores in patients with sepsis. In the mouse model of lipopolysaccharide-induced sepsis, recombinant GDF15 inhibited the proinflammatory responses and alleviated lung tissue injury. In addition, GDF15 decreased the levels of cytokines produced by alveolar macrophages (AMs). The anti-inflammatory effect of glycolysis inhibitor 2-DG on AMs during sepsis was mediated by GDF15 via inducing the phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) and the expression of activating transcription factor 4 (ATF4). Furthermore, we explored the mechanism underlying the beneficial effects of GDF15 and found that GDF15 inhibited glycolysis and mitogen-activated protein kinases (MAPK)/nuclear factor-κB (NF-κB) signaling via promoting AMPK phosphorylation. This study demonstrated that GDF15 inhibited glycolysis and NF-κB/MAPKs signaling via activating AMP-activated protein kinase (AMPK), thereby alleviating the inflammatory responses of AMs and sepsis-associated lung injury. Our findings provided new insights into novel therapeutic strategies for treating sepsis.

Impact of Nebulization on the Physicochemical Properties of Polymer-Lipid Hybrid Nanoparticles for Pulmonary Drug Delivery.

Nanoparticles (NPs) have shown significant potential for pulmonary administration of therapeutics for the treatment of chronic lung diseases in a localized and sustained manner. Nebulization is a suitable method of NP delivery, particularly in patients whose ability to breathe is impaired due to lung diseases. However, there are limited studies evaluating the physicochemical properties of NPs after they are passed through a nebulizer. High shear stress generated during nebulization could potentially affect the surface properties of NPs, resulting in the loss of encapsulated drugs and alteration in the release kinetics. Herein, we thoroughly examined the physicochemical properties as well as the therapeutic effectiveness of Infasurf lung surfactant (IFS)-coated PLGA NPs previously developed by us after passing through a commercial Aeroneb® vibrating-mesh nebulizer. Nebulization did not alter the size, surface charge, IFS coating and bi-phasic release pattern exhibited by the NPs. However, there was a temporary reduction in the initial release of encapsulated therapeutics in the nebulized compared to non-nebulized NPs. This underscores the importance of evaluating the drug release kinetics of NPs using the inhalation method of choice to ensure suitability for the intended medical application. The cellular uptake studies demonstrated that both nebulized and non-nebulized NPs were less readily taken up by alveolar macrophages compared to lung cancer cells, confirming the IFS coating retention. Overall, nebulization did not significantly compromise the physicochemical properties as well as therapeutic efficacy of the prepared nanotherapeutics.

Nebulized Inhalation of Peptide-Modified DNA Origami To Alleviate Acute Lung Injury.

Acute lung injury (ALI) is a severe inflammatory lung disease, with high mortality rates. Early intervention by reactive oxygen species (ROS) scavengers could reduce ROS accumulation, break the inflammation expansion chain in alveolar macrophages (AMs), and avoid irreversible damage to alveolar epithelial and endothelial cells. Here, we reported cell-penetrating R9 peptide-modified triangular DNA origami nanostructures (tDONs-R9) as a novel nebulizable drug that could reach the deep alveolar regions and exhibit an enhanced uptake preference of macrophages. tDONs-R9 suppressed the expression of pro-inflammatory cytokines and drove polarization toward the anti-inflammatory M2 phenotype in macrophages. In the LPS-induced ALI mouse model, treatment with nebulized tDONs-R9 alleviated the overwhelming ROS, pro-inflammatory cytokines, and neutrophil infiltration in the lungs. Our study demonstrates that tDONs-R9 has the potential for ALI treatment, and the programmable DNA origami nanostructures provide a new drug delivery platform for pulmonary disease treatment with high delivery efficiency and biosecurity.

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.

Melatonin improves influenza virus infection-induced acute exacerbation of COPD by suppressing macrophage M1 polarization and apoptosis.

BACKGROUND: Influenza A viruses (IAV) are extremely common respiratory viruses for the acute exacerbation of chronic obstructive pulmonary disease (AECOPD), in which IAV infection may further evoke abnormal macrophage polarization, amplify cytokine storms. Melatonin exerts potential effects of anti-inflammation and anti-IAV infection, while its effects on IAV infection-induced AECOPD are poorly understood. METHODS: COPD mice models were established through cigarette smoke exposure for consecutive 24 weeks, evaluated by the detection of lung function. AECOPD mice models were established through the intratracheal atomization of influenza A/H3N2 stocks in COPD mice, and were injected intraperitoneally with melatonin (Mel). Then, The polarization of alveolar macrophages (AMs) was assayed by flow cytometry of bronchoalveolar lavage (BAL) cells. In vitro, the effects of melatonin on macrophage polarization were analyzed in IAV-infected Cigarette smoking extract (CSE)-stimulated Raw264.7 macrophages. Moreover, the roles of the melatonin receptors (MTs) in regulating macrophage polarization and apoptosis were determined using MTs antagonist luzindole. RESULTS: The present results demonstrated that IAV/H3N2 infection deteriorated lung function (reduced FEV20,50/FVC), exacerbated lung damages in COPD mice with higher dual polarization of AMs. Melatonin therapy improved airflow limitation and lung damages of AECOPD mice by decreasing IAV nucleoprotein (IAV-NP) protein levels and the M1 polarization of pulmonary macrophages. Furthermore, in CSE-stimulated Raw264.7 cells, IAV infection further promoted the dual polarization of macrophages accompanied with decreased MT1 expression. Melatonin decreased STAT1 phosphorylation, the levels of M1 markers and IAV-NP via MTs reflected by the addition of luzindole. Recombinant IL-1ß attenuated the inhibitory effects of melatonin on IAV infection and STAT1-driven M1 polarization, while its converting enzyme inhibitor VX765 potentiated the inhibitory effects of melatonin on them. Moreover, melatonin inhibited IAV infection-induced apoptosis by suppressing IL-1ß/STAT1 signaling via MTs. CONCLUSIONS: These findings suggested that melatonin inhibited IAV infection, improved lung function and lung damages of AECOPD via suppressing IL-1ß/STAT1-driven macrophage M1 polarization and apoptosis in a MTs-dependent manner. Melatonin may be considered as a potential therapeutic agent for influenza virus infection-induced AECOPD.

Mechanism of circRNA_SMG6 mediating lung macrophage ECM degradation via miR-570-3p in microplastics-induced emphysema.

Microplastics (MPs) are plastic particles < 5 mm in diameter, of which polystyrene microplastics (PS-MPs) are representative type. The extracellular matrix (ECM) degradation of macrophages is associated with the development of emphysema. Additionally, circular RNAs (circRNAs) have a regulatory role in epigenetic mechanisms related to lung disease. However, the mechanisms of the ECM degradation and circRNAs in MPs-induced emphysema are still unclear. In our study, Sprague-Dawley (SD) rats were treated with 0, 0.5, 1.0 and 2.0 mg/m3 100 nm PS-MPs for 90 days in an inhalation experiment. PS-MPs-exposed rats showed elevated airway resistance and pulmonary dysfunction. Lung histopathology exhibited inflammatory cell infiltration, septal thickening and alveolar dilatation. Exposure to PS-MPs was able to induce elevated levels of ECM degradation-related markers MMP9 and MMP12, as well as reduced levels of elastin in rat lung tissues. CircRNA_SMG6 is a non-coding RNA (ncRNA) with a homologous circular structure in human, rat and mouse. The expression level of circRNA_SMG6 was decreased in both rat lung tissues exposed to PS-MPs and PS-MPs-treated THP-1 cells. The luciferase reporter gene demonstrated that circRNA_SMG6 combined with miR-570-3p and co-regulated PTEN, the target gene of miR-570-3p. Moreover, overexpression of circRNA_SMG6 or inhibition of miR-570-3p attenuated PS-MPs-induced ECM degradation in THP-1 cells. Taken together, circRNA_SMG6 may have a significant function in the deterioration of emphysema caused by PS-MPs-induced macrophage ECM degradation by regulating miR-570-3p. Our findings reveal a novel mechanism of emphysema caused by PS-MPs and provide valuable information for assessing the health risks of MPs.

Caspase-3/Gasdermin E-mediated pyroptosis contributes to Ricin toxin-induced inflammation.

Ricin toxin (RT) is highly cytotoxic and can release a considerable amount of pro-inflammatory factors due to depurination, causing excessive inflammation that may aggravate the harm to the body. Pyroptosis, a type of gasdermin-mediated cell death, is a contributor to the exacerbation of inflammation. Accumulating evidence indicate that pyroptosis plays a significant role in the pathogen infection and tissue injury, suggesting a potential correlation between pyroptosis and RT-induced inflammation. Here, we aim to demonstrate this correlation and explore its molecular mechanisms. Results showed that RT triggers mouse alveolar macrophage MH-S cells pyroptosis by activating caspase-3 and cleaving Gasgermin E (GSDME). In contrast, inhibition of caspase-3 with Z-DEVD-FMK (inhibitor of caspase-3) or knockdown of GSDME attenuates this process, suggesting the essential role of caspase-3/GSDME-mediated pyroptosis in contributing to RT-induced inflammation. Collectively, our study enhances our understanding of a novel mechanism of ricin cytotoxicity, which may emerge as a potential target in immunotherapy to control the RT-induced inflammation.

Targeting BRD4 with PROTAC degrader ameliorates LPS-induced acute lung injury by inhibiting M1 alveolar macrophage polarization.

OBJECTIVES: Acute lung injury (ALI) is a highly inflammatory condition with the involvement of M1 alveolar macrophages (AMs) polarization, eventually leading to the development of non-cardiogenic edema in alveolar and interstitial regions, accompanied by persistent hypoxemia. Given the significant mortality rate associated with ALI, it is imperative to investigate the underlying mechanisms of this condition so as to identify potential therapeutic targets. The therapeutic effects of the inhibition of bromodomain containing protein 4 (BRD4), an epigenetic reader, has been proven with high efficacy in ameliorating various inflammatory diseases through mediating immune cell activation. However, little is known about the therapeutic potential of BRD4 degradation in acute lung injury. METHODS: This study aimed to assess the protective efficacy of ARV-825, a novel BRD4-targeted proteolysis targeting chimera (PROTAC), against ALI through histopathological examination in lung tissues and biochemical analysis in bronchoalveolar lavage fluid (BALF). Additionally, the underlying mechanism by which BRD4 regulated M1 AMs was elucidated by using CUT & Tag assay. RESULTS: In this study, we found the upregulation of BRD4 in a lipopolysaccharide (LPS)-induced ALI model. Furthermore, we observed that intraperitoneal administration of ARV-825, significantly alleviated LPS-induced pulmonary pathological changes and inflammatory responses. These effects were accompanied by the suppression of M1 AMs. In addition, our findings revealed that the administration of ARV-825 effectively suppressed M1 AMs by inhibiting the expression of IRF7, a crucial transcriptional factor involved in M1 macrophages. CONCLUSION: Our study suggested that targeting BRD4 using ARV-825 is a potential therapeutic approach for ALI.

Galectin-3 inhibition ameliorates alveolar epithelial cell pyroptosis in phosgene-induced acute lung injury.

Phosgene is a type of poisonous gas that can cause acute lung injury (ALI) upon accidental exposure. Casualties still occur due to phosgene-induced acute lung injury (P-ALI) from accidents resulting from improper operations. The pathological mechanisms of P-ALI are still understudied. Thus, we performed scRNA-seq on cells isolated from all subpopulations of the BALF in P-ALI and found that Gal3 expression was significantly higher in the gas group than in the control group. Further analysis revealed a ligand-receptor correspondence between alveolar macrophages (AMs) and alveolar epithelial cells (AEC), with Gal3 playing a key role in this interaction. To confirm and elaborate on this discovery, we selected four time points during the previous week: sham (day 0), day 1, day 3, and day 7 in the P-ALI mouse model and found that Gal3 expression was significantly elevated in P-ALI, most abundantly expressed in AM cells. This was further confirmed with the use of a Gal3 inhibitor. The inhibition of Gal3 and elimination of AMs in mice both attenuated epithelial cell pyroptosis, as confirmed in in vitro experiments, and revealed the Gal3/caspase-8/GSDMD signaling pathway. These findings suggest that Galectin-3 inhibition can ameliorate AEC pyroptosis by inhibiting the Gal3/caspase-8/GSDMD signaling pathway, thus reducing alveolar damage in mice with P-ALI. This finding provides novel insights for improving treatment efficacy for P-ALI.

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-1ß), 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. In vivo 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.

Celastrol alleviates airway hyperresponsiveness and inflammation in obese asthma through mediation of alveolar macrophage polarization.

Obese asthma is a unique asthma phenotype that decreases sensitivity to inhaled corticosteroids, and currently lacks efficient therapeutic medication. Celastrol, a powerful bioactive substance obtained naturally from the roots of Tripterygium wilfordii, has been reported to possess the potential effect of weight loss in obese individuals. However, its role in the treatment of obese asthma is not fully elucidated. In the present study, diet-induced obesity (DIO) mice were used with or without ovalbumin (OVA) sensitization, the therapeutic effects of celastrol on airway hyperresponsiveness (AHR) and airway inflammation were examined. We found celastrol significantly decreased methacholine-induced AHR in obese asthma, as well as reducing the infiltration of inflammatory cells and goblet cell hyperplasia in the airways. This effect was likely due to the inhibition of M1-type alveolar macrophages (AMs) polarization and the promotion of M2-type macrophage polarization. In vitro, celastrol yielded equivalent outcomes in Lipopolysaccharide (LPS)-treated RAW264.7 macrophage cells, featuring a reduction in the expression of M1 macrophage makers (iNOS, IL-1ß, TNF-α) and heightened M2 macrophage makers (Arg-1, IL-10). Mechanistically, the PI3K/AKT signaling pathway has been implicated in these processes. In conclusion, we demonstrated that celastrol assisted in mitigating various parameters of obese asthma by regulating the balance of M1/M2 AMs polarization.

Role of macrophage bioenergetics in N-acetylcysteine-mediated mitigation of lung injury and oxidative stress induced by nitrogen mustard.

Nitrogen mustard (NM) is a toxic vesicant that causes acute injury to the respiratory tract. This is accompanied by an accumulation of activated macrophages in the lung and oxidative stress which have been implicated in tissue injury. In these studies, we analyzed the effects of N-acetylcysteine (NAC), an inhibitor of oxidative stress and inflammation on NM-induced lung injury, macrophage activation and bioenergetics. Treatment of rats with NAC (150 mg/kg, i.p., daily) beginning 30 min after administration of NM (0.125 mg/kg, i.t.) reduced histopathologic alterations in the lung including alveolar interstitial thickening, blood vessel hemorrhage, fibrin deposition, alveolar inflammation, and bronchiolization of alveolar walls within 3 d of exposure; damage to the alveolar-epithelial barrier, measured by bronchoalveolar lavage fluid protein and cells, was also reduced by NAC, along with oxidative stress as measured by heme oxygenase (HO)-1 and Ym-1 expression in the lung. Treatment of rats with NAC attenuated the accumulation of macrophages in the lung expressing proinflammatory genes including Ptgs2, Nos2, Il-6 and Il-12; macrophages expressing inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and tumor necrosis factor (TNF)α protein were also reduced in histologic sections. Conversely, NAC had no effect on macrophages expressing the anti-inflammatory proteins arginase-1 or mannose receptor, or on NM-induced increases in matrix metalloproteinase (MMP)-9 or proliferating cell nuclear antigen (PCNA), markers of tissue repair. Following NM exposure, lung macrophage basal and maximal glycolytic activity increased, while basal respiration decreased indicating greater reliance on glycolysis to generate ATP. NAC increased both glycolysis and oxidative phosphorylation. Additionally, in macrophages from both control and NM treated animals, NAC treatment resulted in increased S-nitrosylation of ATP synthase, protecting the enzyme from oxidative damage. Taken together, these data suggest that alterations in NM-induced macrophage activation and bioenergetics contribute to the efficacy of NAC in mitigating lung injury.

Tissue-Resident Alveolar Macrophages Reduce Ozone-induced Inflammation via MerTK-mediated Efferocytosis.

Lung inflammation, caused by acute exposure to ozone (O3), one of the six criteria air pollutants, is a significant source of morbidity in susceptible individuals. Alveolar macrophages (AMØs) are the most abundant immune cells in the normal lung, and their number increases after O3 exposure. However, the role of AMØs in promoting or limiting O3-induced lung inflammation has not been clearly defined. In this study, we used a mouse model of acute O3 exposure, lineage tracing, genetic knockouts, and data from O3-exposed human volunteers to define the role and ontogeny of AMØs during acute O3 exposure. Lineage-tracing experiments showed that 12, 24, and 72 hours after exposure to O3 (2 ppm) for 3 hours, all AMØs were of tissue-resident origin. Similarly, in humans exposed to filtered air and O3 (200 ppb) for 135 minutes, we did not observe at ∼21 hours postexposure an increase in monocyte-derived AMØs by flow cytometry. Highlighting a role for tissue-resident AMØs, we demonstrate that depletion of tissue-resident AMØs with clodronate-loaded liposomes led to persistence of neutrophils in the alveolar space after O3 exposure, suggesting that impaired neutrophil clearance (i.e., efferocytosis) leads to prolonged lung inflammation. Moreover, depletion of tissue-resident AMØs demonstrated reduced clearance of intratracheally instilled apoptotic Jurkat cells, consistent with reduced efferocytosis. Genetic ablation of MerTK (MER proto-oncogene, tyrosine kinase), a key receptor involved in efferocytosis, also resulted in impaired clearance of apoptotic neutrophils after O3 exposure. Overall, these findings underscore the pivotal role of tissue-resident AMØs in resolving O3-induced inflammation via MerTK-mediated efferocytosis.

Rapid conversion of porcine pluripotent stem cells into macrophages with chemically defined conditions.

A renewable source of porcine macrophages derived from pluripotent stem cells (PSCs) would be a valuable alternative to primary porcine alveolar macrophages (PAMs) in the research of host-pathogen interaction mechanisms. We developed an efficient and rapid protocol, within 11 days, to derive macrophages from porcine PSCs (pPSCs). The pPSC-derived macrophages (pPSCdMs) exhibited molecular and functional characteristics of primary macrophages. The pPSCdMs showed macrophage-specific surface protein expression and macrophage-specific transcription factors, similar to PAMs. The pPSCdMs also exhibited the functional characteristics of macrophages, such as endocytosis, phagocytosis, porcine respiratory and reproductive syndrome virus infection and the response to lipopolysaccharide stimulation. Furthermore, we performed transcriptome sequencing of the whole differentiation process to track the fate transitions of porcine PSCs involved in the signaling pathway. The activation of transforming growth factor beta signaling was required for the formation of mesoderm and the inhibition of the transforming growth factor beta signaling pathway at the hematopoietic endothelium stage could enhance the fate transformation of hematopoiesis. In summary, we developed an efficient and rapid protocol to generate pPSCdMs that showed aspects of functional maturity comparable with PAMs. pPSCdMs could provide a broad prospect for the platforms of host-pathogen interaction mechanisms.

Lung adenocarcinoma promotion by air pollutants.

A complete understanding of how exposure to environmental substances promotes cancer formation is lacking. More than 70 years ago, tumorigenesis was proposed to occur in a two-step process: an initiating step that induces mutations in healthy cells, followed by a promoter step that triggers cancer development1. Here we propose that environmental particulate matter measuring ≤2.5 µm (PM2.5), known to be associated with lung cancer risk, promotes lung cancer by acting on cells that harbour pre-existing oncogenic mutations in healthy lung tissue. Focusing on EGFR-driven lung cancer, which is more common in never-smokers or light smokers, we found a significant association between PM2.5 levels and the incidence of lung cancer for 32,957 EGFR-driven lung cancer cases in four within-country cohorts. Functional mouse models revealed that air pollutants cause an influx of macrophages into the lung and release of interleukin-1ß. This process results in a progenitor-like cell state within EGFR mutant lung alveolar type II epithelial cells that fuels tumorigenesis. Ultradeep mutational profiling of histologically normal lung tissue from 295 individuals across 3 clinical cohorts revealed oncogenic EGFR and KRAS driver mutations in 18% and 53% of healthy tissue samples, respectively. These findings collectively support a tumour-promoting role for  PM2.5 air pollutants  and provide impetus for public health policy initiatives to address air pollution to reduce disease burden.

Resolution of post-lung transplant ischemia-reperfusion injury is modulated via Resolvin D1-FPR2 and Maresin 1-LGR6 signaling.

BACKGROUND: Dysregulation of inflammation-resolution pathways leads to postlung transplant (LTx) ischemia-reperfusion (IR) injury and allograft dysfunction. Our hypothesis is that combined treatment with specialized pro-resolving lipid mediators, that is, Resolvin D1 (RvD1) and Maresin-1 (MaR1), enhances inflammation-resolution of lung IR injury. METHODS: Expression of RvD1 and MaR1 was analyzed in bronchoalveolar lavage (BAL) fluid of patients on days 0, 1, and 7 post-LTx. Lung IR injury was evaluated in C57BL/6 (WT), FPR2-/-, and LGR6 siRNA treated mice using a hilar-ligation model with or without administration with RvD1 and/or MaR1. A donation after circulatory death and murine orthotopic lung transplantation model was used to evaluate the protection by RvD1 and MaR1 against lung IR injury. In vitro studies analyzed alveolar macrophages and type II epithelial cell activation after treatment with RvD1 or MaR1. RESULTS: RvD1 and MaR1 expressions in BAL from post-LTx patients was significantly increased on day 7 compared to days 0 and 1. Concomitant RvD1 and MaR1 treatment significantly mitigated early pulmonary inflammation and lung IR injury in WT mice, which was regulated via FPR2 and LGR6 receptors. In the murine orthotopic donation after cardiac death LTx model, RvD1 and MaR1 treatments significantly attenuated lung IR injury and increased PaO2 levels compared to saline-treated controls. Mechanistically, RvD1/FPR2 signaling on alveolar macrophages attenuated HMGB1 and TNF-α secretion and upregulated uptake of macrophage-dependent apoptotic neutrophils (efferocytosis), whereas MaR1/LGR6 signaling mitigated CXCL1 secretion by epithelial cells. CONCLUSIONS: Bioactive proresolving lipid mediator-dependent signaling that is, RvD1/FPR2 and MaR1/LGR6- offers a novel therapeutic strategy in post-LTx injury.

VX-765 attenuates silica-induced lung inflammatory injury and fibrosis by modulating alveolar macrophages pyroptosis in mice.

Silicosis is a diffuse fibrotic lung disease in which excessive inflammatory responses are triggered by silica exposure. Pyroptosis, a pro-inflammatory mode of programmed cell death, is mediated by gasdermin and may play a pivotal role in the development of silicosis. The caspase-1 inhibitor, VX-765, was used in vivo and in vitro to investigate the effects of silica-induced early inflammatory injury and later lung fibrosis. Our findings show that VX-765 reduces inflammatory lung injury by inhibiting silica-induced pyroptosis of alveolar macrophages in a silicosis mouse model. VX-765 limits the infiltration of inflammatory M1 alveolar macrophages, decreasing expression of inflammatory cytokines, including IL-1ß, TNF-α, IL-6, CCL2, and CCL3, and down-regulating endogenous DAMPs and inflammatory immune-related cell pattern recognition receptors TLR4 and NLRP3. Furthermore, VX-765 alleviates fibrosis by down-regulating α-smooth muscle actin (α-SMA), collagen, and fibronectin. In this study, we illustrate that Alveolar macrophages pyroptosis occur in the early stages of silicosis, and VX-765 can alleviate the development of silicosis by inhibiting the pyroptosis signaling pathway. These results may provide new insight into the prevention and treatment of early-stage silicosis.

Pharmacological reversal of post-transcriptional alterations implicated in alcohol-induced alveolar macrophage dysfunction.

Alcohol use disorders (AUD) cause alveolar macrophage (AM) immune dysfunction and increase risk of lung infections. Excessive alcohol use causes AM oxidative stress, which impairs AM phagocytosis and pathogen clearance from the alveolar space. Alcohol induces expression of NADPH oxidases (Noxes), primary sources of oxidative stress in AM. In contrast, alcohol decreases AM peroxisome proliferator-activated receptor gamma (PPARγ), a critical regulator of AM immune function. To explore the underlying molecular mechanisms for these effects of alcohol, we hypothesized that ethanol promotes CCAAT/enhancer-binding protein beta (C/EBPß)-mediated suppression of Nox-related microRNAs (miRs), in turn enhancing AM Nox expression, oxidative stress, and phagocytic dysfunction. We also hypothesized that PPARγ activation with pioglitazone (PIO) would reverse alcohol-induced C/EBPß expression and attenuate AM oxidative stress and phagocytic dysfunction. Cells from the mouse AM cell line (MH-S) were exposed to ethanol in vitro or primary AM were isolated from mice fed ethanol in vivo. Ethanol enhanced C/EBPß expression, decreased Nox 1-related miR-1264 and Nox 2-related miR-107 levels, and increased Nox1, Nox2, and Nox 4 expression in MH-S cells in vitro and mouse AM in vivo. These alcohol-induced AM derangements were abrogated by loss of C/EBPß, overexpression of miRs-1264 or -107, or PIO treatment. These findings identify C/EBPß and Nox-related miRs as novel therapeutic targets for PPARγ ligands, which could provide a translatable strategy to mitigate susceptibility to lung infections in people with a history of AUD. These studies further clarify the molecular underpinnings for a previous clinical trial using short-term PIO treatment to improve AM immunity in AUD individuals.

Alcohol impairs recognition and uptake of Mycobacterium tuberculosis by suppressing toll-like receptor 2 expression.

BACKGROUND: Alcohol impairs pulmonary innate immune function and is associated with an increased risk of tuberculosis (TB). Toll-like receptor 2 (TLR2) is a pattern recognition receptor on alveolar macrophages that recognizes Mycobacterium tuberculosis (Mtb). The expression of TLR2 depends, in part, on granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling. Given our prior work demonstrating the suppression of GM-CSF signaling following chronic alcohol ingestion, we hypothesized that alcohol impairs TLR2 expression via the suppression of GM-CSF and thereby reduces the ability of the macrophage to recognize and phagocytose Mtb. METHODS: Primary alveolar macrophages were isolated from control-fed and alcohol-fed rats. Prior to cell isolation, some alcohol-fed rats were treated with intranasal GM-CSF and then endotracheally inoculated with an attenuated strain of Mtb. Primary macrophages were then isolated and immunofluorescence was used to determine phagocytic efficiency and TLR2 expression in the presence and absence of GM-CSF treatment and phagocytic efficiency in the presence and absence of TLR2 neutralization. RESULTS: TLR2 expression and phagocytosis of Mtb were significantly lower in the alveolar macrophages of alcohol-fed rats than control-fed rats. In parallel, blocking TLR2 signaling recapitulated this decreased phagocytosis of Mtb. In contrast, intranasal GM-CSF treatment restored TLR2 expression and Mtb phagocytosis in the alveolar macrophages of alcohol-fed rats to levels comparable to those of control-fed rats. CONCLUSIONS: Chronic alcohol ingestion reduces TLR2 protein expression and phagocytosis of Mtb, likely due to impaired GM-CSF signaling. GM-CSF restores membrane-bound TLR2 expression and phagocytic function.

Toxicity of curcumin nanoparticles towards alveolar macrophage: Effects of surface charges.

Curcumin has been used for chronic lung diseases management due to its diversified molecular actions. However, the potential cytotoxicity which occurs in cells following the exposure to high concentrations of curcumin has been overlooked. This study evaluated the toxic events of curcumin nanoparticles (Cur-NPs) with alterable surface polarity in alveolar macrophages (NR8383). We aimed to establish the correlation between the toxicity of Cur-NPs with different surface charges and the internalization mechanisms of the NPs. Toxicity data showed that positively charged Cur-NPs (IC50: 9.77 ± 0.5 µg/mL) was the most potent against NR8383, followed by negatively charged Cur-NPs (IC50:13.33 ± 0.9 µg/mL) and neutral Cur-NPs (IC50:18.68 ± 1.2 µg/mL). Results from mitochondrial membrane potential, ATP content and intracellular ROS in NR8383 showed similar ranking to the toxicity assay. The predominant uptake pathway for positively and negatively charged Cur-NPs was via clathrin-mediated endocytosis, while neutral Cur-NPs was internalized via phagocytosis, micropinocytosis and clathrin-mediated endocytosis. Positively charged Cur-NPs mediates the cytotoxicity of NR8383 via lysosomal and mitochondrial-associated destabilization upon entry. In conclusion, the cytotoxicity of Cur-NPs on NR8383 is surface-charge dependent, which in turn is associated to the uptake pathway and localization of Cur-NPs in cells.