Murine Model Of Acute Lung Injury Research Articles

Core genes involved in the regulation of acute lung injury and their association with COVID-19 and tumor progression: A bioinformatics and experimental study.

Acute lung injury (ALI) is a specific form of lung damage caused by different infectious and non-infectious agents, including SARS-CoV-2, leading to severe respiratory and systemic inflammation. To gain deeper insight into the molecular mechanisms behind ALI and to identify core elements of the regulatory network associated with this pathology, key genes involved in the regulation of the acute lung inflammatory response (Il6, Ccl2, Cat, Serpine1, Eln, Timp1, Ptx3, Socs3) were revealed using comprehensive bioinformatics analysis of whole-genome microarray datasets, functional annotation of differentially expressed genes (DEGs), reconstruction of protein-protein interaction networks and text mining. The bioinformatics data were validated using a murine model of LPS-induced ALI; changes in the gene expression patterns were assessed during ALI progression and prevention by anti-inflammatory therapy with dexamethasone and the semisynthetic triterpenoid soloxolone methyl (SM), two agents with different mechanisms of action. Analysis showed that 7 of 8 revealed ALI-related genes were susceptible to LPS challenge (up-regulation: Il6, Ccl2, Cat, Serpine1, Eln, Timp1, Socs3; down-regulation: Cat) and their expression was reversed by the pre-treatment of mice with both anti-inflammatory agents. Furthermore, ALI-associated nodal genes were analysed with respect to SARS-CoV-2 infection and lung cancers. The overlap with DEGs identified in postmortem lung tissues from COVID-19 patients revealed genes (Saa1, Rsad2, Ifi44, Rtp4, Mmp8) that (a) showed a high degree centrality in the COVID-19-related regulatory network, (b) were up-regulated in murine lungs after LPS administration, and (c) were susceptible to anti-inflammatory therapy. Analysis of ALI-associated key genes using The Cancer Genome Atlas showed their correlation with poor survival in patients with lung neoplasias (Ptx3, Timp1, Serpine1, Plaur). Taken together, a number of key genes playing a core function in the regulation of lung inflammation were found, which can serve both as promising therapeutic targets and molecular markers to control lung ailments, including COVID-19-associated ALI.

CXCR7 Antagonism Reduces Acute Lung Injury Pathogenesis.

Loss of control in the trafficking of immune cells to the inflamed lung tissue contributes to the pathogenesis of life-threatening acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS). Targeting CXCR7 has been proposed as a potential therapeutic approach to reduce pulmonary inflammation; however, its role and its crosstalk with the two chemokine receptors CXCR3 and CXCR4 via their shared ligands CXCL11 and CXCL12 is not yet completely understood. The present paper aimed to characterize the pathological role of the CXCR3/CXCR4/CXCR7 axis in a murine model of ALI. Lipopolysaccharide (LPS) inhalation in mice resulted in the development of key pathologic features of ALI/ARDS, including breathing dysfunctions, alteration in the alveolar capillary barrier, and lung inflammation. LPS inhalation induced immune cell infiltration into the bronchoalveolar space, including CXCR3+ and CXCR4+ cells, and enhanced the expression of the ligands of these two chemokine receptors. The first-in-class CXCR7 antagonist, ACT-1004-1239, increased levels of CXCL11 and CXCL12 in the plasma without affecting their levels in inflamed lung tissue, and consequently reduced CXCR3+ and CXCR4+ immune cell infiltrates into the bronchoalveolar space. In the early phase of lung inflammation, characterized by a massive influx of neutrophils, treatment with ACT-1004-1239 significantly reduced the LPS-induced breathing pattern alteration. Both preventive and therapeutic treatment with ACT-1004-1239 reduced lung vascular permeability and decreased inflammatory cell infiltrates. In conclusion, these results demonstrate a key pathological role of CXCR7 in ALI/ARDS and highlight the clinical potential of ACT-1004-1239 in ALI/ARDS pathogenesis.

Dehydrocostus Lactone Attenuates Methicillin-Resistant Staphylococcus aureus-Induced Inflammation and Acute Lung Injury via Modulating Macrophage Polarization.

Dehydrocostus lactone (DHL), a natural sesquiterpene lactone isolated from the traditional Chinese herbs Saussurea lappa and Inula helenium L., has important anti-inflammatory properties used for treating colitis, fibrosis, and Gram-negative bacteria-induced acute lung injury (ALI). However, the effects of DHL on Gram-positive bacteria-induced macrophage activation and ALI remains unclear. In this study, we found that DHL inhibited the phosphorylation of p38 MAPK, the degradation of IκBα, and the activation and nuclear translocation of NF-κB p65, but enhanced the phosphorylation of AMP-activated protein kinase (AMPK) and the expression of Nrf2 and HO-1 in lipoteichoic acid (LTA)-stimulated RAW264.7 cells and primary bone-marrow-derived macrophages (BMDMs). Given the critical role of the p38 MAPK/NF-κB and AMPK/Nrf2 signaling pathways in the balance of M1/M2 macrophage polarization and inflammation, we speculated that DHL would also have an effect on macrophage polarization. Further studies verified that DHL promoted M2 macrophage polarization and reduced M1 polarization, then resulted in a decreased inflammatory response. An in vivo study also revealed that DHL exhibited anti-inflammatory effects and ameliorated methicillin-resistant Staphylococcus aureus (MRSA)-induced ALI. In addition, DHL treatment significantly inhibited the p38 MAPK/NF-κB pathway and activated AMPK/Nrf2 signaling, leading to accelerated switching of macrophages from M1 to M2 in the MRSA-induced murine ALI model. Collectively, these data demonstrated that DHL can promote macrophage polarization to an anti-inflammatory M2 phenotype via interfering in p38 MAPK/NF-κB signaling, as well as activating the AMPK/Nrf2 pathway in vitro and in vivo. Our results suggested that DHL might be a novel candidate for treating inflammatory diseases caused by Gram-positive bacteria.

Haloperidol Attenuates Lung Endothelial Cell Permeability In Vitro and In Vivo.

We previously reported that claudin-5, a tight junctional protein, mediates lung vascular permeability in a murine model of acute lung injury (ALI) induced by lipopolysaccharide (LPS). Recently, it has been reported that haloperidol, an antipsychotic medication, dose-dependently increases expression of claudin-5 in vitro and in vivo, in brain endothelium. Notably, claudin-5 is highly expressed in both brain and lung tissues. However, the effects of haloperidol on EC barrier function are unknown. We hypothesized that haloperidol increases lung EC claudin-5 expression and attenuates agonist-induced lung EC barrier disruption. Human pulmonary artery ECs were pretreated with haloperidol at variable concentrations (0.1–10 μM) for 24 h. Cell lysates were subjected to Western blotting for claudin-5, in addition to occludin and zona occludens-1 (ZO-1), two other tight junctional proteins. To assess effects on barrier function, EC monolayers were pretreated for 24 h with haloperidol (10 µM) or vehicle prior to treatment with thrombin (1 U/mL), with measurements of transendothelial electrical resistance (TER) recorded as a real-time assessment of barrier integrity. In separate experiments, EC monolayers grown in Transwell inserts were pretreated with haloperidol (10 µM) prior to stimulation with thrombin (1 U/mL, 1 h) and measurement of FITC-dextran flux. Haloperidol significantly increased claudin-5, occludin, and ZO-1 expression levels. Measurements of TER and FITC-dextran Transwell flux confirmed a significant attenuation of thrombin-induced barrier disruption associated with haloperidol treatment. Finally, mice pretreated with haloperidol (4 mg/kg, IP) prior to the intratracheal administration of LPS (1.25 mg/kg, 16 h) had increased lung claudin-5 expression with decreased lung injury as assessed by bronchoalveolar lavage (BAL) fluid protein content, total cell counts, and inflammatory cytokines, in addition to lung histology. Our data confirm that haloperidol results in increased claudin-5 expression levels and demonstrates lung vascular-protective effects both in vitro and in vivo in a murine ALI model. These findings suggest that haloperidol may represent a novel therapy for the prevention or treatment of ALI and warrants further investigation in this context.

Oxypaeoniflorin Prevents Acute Lung Injury Induced by Lipopolysaccharide through the PTEN/AKT Pathway in a Sirt1-Dependent Manner.

Acute lung injury (ALI) is featured by pulmonary edema, alveolar barrier injury, inflammatory response, and oxidative stress. The activation of Sirt1 could relieve lipopolysaccharide- (LPS-) induced murine ALI by maintaining pulmonary epithelial barrier function. Oxypaeoniflorin (Oxy) serves as a major component of Paeonia lactiflora Pall., exerting cardioprotection by activating Sirt1. However, the role of Oxy in ALI induced by LPS remains unclear. The aim of the present study is to illustrate the modulatory effects and molecular mechanisms by which Oxy operates in ALI induced by LPS. The intraperitoneal injection of LPS was performed to establish the murine ALI model while LPS-treated alveolar epithelial cells were used to mimic the in vitro ALI model. Levels of lung injury, oxidative stress, and inflammatory response were detected to observe the potential effects of Oxy on ALI. Oxy treatment mitigated lung edema, inflammatory response, and oxidative stress in mouse response to LPS, apart from improving 7-day survival. Meanwhile, Oxy also increased the expression and activity of Sirt1. Intriguingly, Sirt1 deficiency or inhibition counteracted the protective effects of Oxy treatment in LPS-treated mice or LPS-treated alveolar epithelial cells by regulating the PTEN/AKT signaling pathway. These results demonstrated that Oxy could combat ALI in vivo and in vitro through inhibiting inflammatory response and oxidative stress in a Sirt1-dependent manner. Oxy owns the potential to be a promising candidate against ALI.

Airway epithelial integrin β4-deficiency exacerbates lipopolysaccharide-induced acute lung injury.

Airway epithelial cells, the first barrier of the respiratory tract, play an indispensable role in innate immunity. Integrin β4 (ITGB4) is a structural adhesion molecule that is involved in the pathological progression of acute inflammatory diseases and is downregulated in asthmatic patients. Research has shown that endothelial ITGB4 has proinflammatory properties in acute lung injury (ALI). However, the role of epithelial ITGB4 in a murine ALI model is still unknown. This study investigated the role of ITGB4 in lipopolysaccharide (LPS)-induced ALI. We found that ITGB4 in the airway epithelium had remarkably increased after the introduction of LPS in vivo and in vitro. Then, we constructed airway epithelial cell-specific ITGB4 knockout (ITGB4-/- ) mice to study its role in ALI. At a time point of 12 h after the tracheal injection of LPS, ITGB4-/- mice showed increased macrophages (mainly M1-type macrophages) and neutrophil infiltration into the lungs; inflammation-related proteins including interleukin (IL)-6, tumor necrosis factor, and IL-17A were significantly elevated compared to their levels in ITGB4+/+ mice. Furthermore, we investigated the role of ITGB4 in the anti-inflammatory response. Intriguingly, in the ITGB4-/- + LPS group, we found significantly reduced expression of anti-inflammatory factors, including IL-10 messenger RNA (mRNA) and ARG-1 mRNA. We also observed that monocyte chemotactic protein (MCP-1) increased significantly both in vivo and in vitro. Airway epithelium activates macrophages, most likely driven by MCP-1, which we confirmed in the coculture of epithelia and macrophages. These phenomena indicate that ITGB4 in airway epithelial cells plays an important role in the process of inflammation and activation of macrophages in ALI. Overall, these data demonstrated a novel link between airway epithelial ITGB4 and the inflammatory response in LPS-induced ALI.

The receptor for advanced glycation end products mediates dysfunction of airway epithelial barrier in a lipopolysaccharides-induced murine acute lung injury model

BackgroundAirway epithelial cells (AECs) act as the first barrier protecting against invasion of environment agents and maintain integrity of lung structure and function. Dysfunction of airway epithelial barrier has been shown to be involved in ALI/ARDS pathogenesis. Yet, the exact mechanism is still obscure. Our study evaluated whether the receptor for advanced glycation end products (RAGE) mediates impaired airway epithelial barrier in LPS-induced murine ALI model. MethodsMale BALB/c mice were subjected to intratracheal instillation of LPS to generate an ALI model. Inhibitors of RAGE, FPS-ZM1 and Azeliragon were respectively given to the mice through intraperitoneal injection. Bronchoalveolar lavage fluid (BALF) and lung tissues were collected for further analysis. ResultsLPS exposure led to markedly increased expression of RAGE and its ligands HMGB1, HSP70, S100b. Treatment of FPS-ZM1 or Azeliragon not only effectively descended the expression of RAGE and its ligands but also attenuated LPS-induced neutrophil-predominant airway inflammation and injury, decreased levels of IL-6, IL-1β and TNF-α in BALF, alleviated increased alveolar-capillary permeability and pulmonary edema. LPS stimulation significantly impaired the integrity of airway epithelium, paralleled with dislocation of adheren junction (AJ) protein E-cadherin at cell-cell contacts and down-expression of both AJ and tight junction (TJ) proteins Claudin-2 and occludin, all of which were dramatically rescued by RAGE inhibition. ConclusionRAGE signaling mediates airway epithelial barrier dysfunction in a LPS-induced ALI murine model.

Circulating BMP9 Protects the Pulmonary Endothelium during Inflammation-induced Lung Injury in Mice.

Rationale: Pulmonary endothelial permeability contributes to the high-permeability pulmonary edema that characterizes acute respiratory distress syndrome. Circulating BMP9 (bone morphogenetic protein 9) is emerging as an important regulator of pulmonary vascular homeostasis.Objectives:To determine whether endogenous BMP9 plays a role in preserving pulmonary endothelial integrity and whether loss of endogenous BMP9 occurs during LPS challenge.Methods: A BMP9-neutralizing antibody was administrated to healthy adult mice, and lung vasculature was examined. Potential mechanisms were delineated by transcript analysis in human lung endothelial cells. The impact of BMP9 administration was evaluated in a murine acute lung injury model induced by inhaled LPS. Levels of BMP9 were measured in plasma from patients with sepsis and from endotoxemic mice.Measurements and Main Results: Subacute neutralization of endogenous BMP9 in mice (N = 12) resulted in increased lung vascular permeability (P = 0.022), interstitial edema (P = 0.0047), and neutrophil extravasation (P = 0.029) compared with IgG control treatment (N = 6). In pulmonary endothelial cells, BMP9 regulated transcriptome pathways implicated in vascular permeability and cell-membrane integrity. Augmentation of BMP9 signaling in mice (N = 8) prevented inhaled LPS–induced lung injury (P = 0.0027) and edema (P < 0.0001). In endotoxemic mice (N = 12), endogenous circulating BMP9 concentrations were markedly reduced, the causes of which include a transient reduction in hepatic BMP9 mRNA expression and increased elastase activity in plasma. In human patients with sepsis (N = 10), circulating concentratons of BMP9 were also markedly reduced (P < 0.0001).Conclusions: Endogenous circulating BMP9 is a pulmonary endothelial-protective factor, downregulated during inflammation. Exogenous BMP9 offers a potential therapy to prevent increased pulmonary endothelial permeability in lung injury.

Transcription factor NF-κB promotes acute lung injury via microRNA-99b-mediated PRDM1 down-regulation

Acute lung injury (ALI), is a rapidly progressing heterogenous pulmonary disorder that possesses a high risk of mortality. Accumulating evidence has implicated the activation of the p65 subunit of NF-κB [NF-κB(p65)] activation in the pathological process of ALI. microRNAs (miRNAs), a group of small RNA molecules, have emerged as major governors due to their post-transcriptional regulation of gene expression in a wide array of pathological processes, including ALI. The dysregulation of miRNAs and NF-κB activation has been implicated in human diseases. In the current study, we set out to decipher the convergence of miR-99b and p65 NF-κB activation in ALI pathology. We measured the release of pro-inflammatory cytokines (IL-1β, IL-6, and TNFα) in bronchoalveolar lavage fluid using ELISA. MH-S cells were cultured and their viability were detected with cell counting kit 8 (CCK8) assays. The results showed that miR-99b was up-regulated, while PRDM1 was down-regulated in a lipopolysaccharide (LPS)-induced murine model of ALI. Mechanistic investigations showed that NF-κB(p65) was enriched at the miR-99b promoter region, and further promoted its transcriptional activity. Furthermore, miR-99b targeted PRDM1 by binding to its 3'UTR, causing its down-regulation. This in-creased lung injury, as evidenced by increased wet/dry ratio of mouse lung, myeloperoxidase activity and pro-inflammatory cytokine secretion, and enhanced infiltration of inflammatory cells in lung tissues. Together, our findings indicate that NF-κB(p65) promotion of miR-99b can aggravate ALI in mice by down-regulating the expression of PRDM1.

Sophoricoside attenuates lipopolysaccharide-induced acute lung injury by activating the AMPK/Nrf2 signaling axis

Sophoricoside (SOP), an isoflavone glycoside isolated from seed of Sophora japonica L., has been reported to have various pharmacological activities, including anti-cancer, anti-allergy and anti-inflammation. However, the effect of SOP on lipopolysaccharides (LPS)-acute lung injury (ALI) is completely unclear. Here, we found that SOP pretreatment significantly ameliorated LPS-induced pathological damage, tissue permeability, neutrophil infiltration and the production of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) in a murine model of ALI. Besides, SOP reduced the production of pro-inflammatory mediators such as iNOS, NO and inflammatory cytokines including TNF-α, IL-1β and IL-6 in LPS-stimulated RAW264.7 cells and bone marrow derived macrophages. Interestingly, treatment with SOP exhibited no effect on the activation of NF-κB and MAPKs in macrophages but prominently accelerated the expression and nuclear translocation of Nrf2. By using ML385, a specific Nrf2 inhibitor, we found that inhibition of Nrf2 abolished the inhibitory effect of SOP on LPS-induced iNOS expression, NO production as well as pro-inflammatory cytokine generation. SOP also activated AMPK, an upstream protein of Nrf2, under LPS stimuli. Furthermore, we demonstrated that the accelerated expression of Nrf2 induced by SOP was reversed by interference with the AMPK inhibitor Compound C. Taken together, our results suggested that SOP attenuated LPS-induced ALI in AMPK/Nrf2 dependent manner and indicated that SOP might be a potential therapeutic candidate for treating ALI/ARDS.

Abstract 12651: Fine-Tuning of Lung Thrombosis is a Therapeutic Strategy Against Inflammatory Lung Injury

Introduction: Lung thrombosis (LT) and endothelial cell (EC) death are positively associated with mortality in sepsis-induced acute lung injury (ALI) patients, but anti-coagulants have failed in clinical trials of sepsis. Hypothesis: We hypothesized that different levels of LT mediate ALI by regulating lung EC viability. Methods: To assess the impact of different levels of LT on ALI, we used murine models of LT and ALI. To explore how LT level might alter lung EC viability and ALI, we assessed survival gene expression by RNA sequencing. To identify the mechanism(s) responsible for changes in ALI after varying levels of LT, we assessed lung EC viability and ALI in mice with cell-specific knockout of different survival genes. Results: In platelet-depleted mice, LT was reduced while ALI was increased. Intra-venous microbeads were then administered to induce LT in a dose-dependent manner, showing that restoration of LT to the level found in platelet-replete mice protected against thrombocytopenia-induced ALI. We next showed in wild type mice, that while excessive increases in LT worsened ALI, induction of a mild level of LT conversely protected against ALI. The opposing impact of diminished or excessive versus mild LT on ALI was associated with changes in lung EC apoptosis. Subsequent studies showed a panel of candidate survival factors were differentially expressed in the lungs of ALI mice with or without mild or excessive LT. Mechanistically, we found that the protective impact of mild LT on ALI is dependent upon hypoxia-inducible factor (HIF) 1α in Tie2-expressing cells. Remarkably, the same mild level of LT retained its protective impact in platelet-depleted and platelet-replete mice even when induced up to 8 hours after sepsis. Conclusions: ALI is enhanced by diminished or excessive LT but suppressed by mild LT through HIF1α. The control of LT represents a therapeutic strategy for the induction of a pro-survival response that protects against inflammatory lung injury.

RAC1 nitration at Y32 IS involved in the endothelial barrier disruption associated with lipopolysaccharide-mediated acute lung injury

Acute lung injury (ALI), a devastating illness induced by systemic inflammation e.g., sepsis or local lung inflammation e.g., COVID-19 mediated severe pneumonia, has an unacceptably high mortality and has no effective therapy. ALI is associated with increased pulmonary microvascular hyperpermeability and alveolar flooding. The small Rho GTPases, RhoA and Rac1 are central regulators of vascular permeability through cytoskeleton rearrangements. RhoA and Rac1 have opposing functional outcome: RhoA induces an endothelial contractile phenotype and barrier disruption, while Rac1 stabilizes endothelial junctions and increases barrier integrity. In ALI, RhoA activity is increased while Rac1 activity is reduced. We have shown that the activation of RhoA in lipopolysaccharide (LPS)-mediated ALI, is dependent, at least in part, on a single nitration event at tyrosine (Y)34. Thus, the purpose of this study was to determine if the inhibition of Rac1 is also dependent on its nitration. Our data show that Rac1 inhibition by LPS is associated with its nitration that mass spectrometry identified as Y32, within the switch I region adjacent to the nucleotide-binding site. Using a molecular modeling approach, we designed a nitration shielding peptide for Rac1, designated NipR2 (nitration inhibitor peptide for the Rho GTPases 2), which attenuated the LPS-induced nitration of Rac1 at Y32, preserves Rac1 activity and attenuates the LPS-mediated disruption of the endothelial barrier in human lung microvascular endothelial cells (HLMVEC). Using a murine model of ALI induced by intratracheal installation of LPS we found that NipR2 successfully prevented Rac1 nitration and Rac1 inhibition, and more importantly attenuated pulmonary inflammation, reduced lung injury and prevented the loss of lung function. Together, our data identify a new post-translational mechanism of Rac1 inhibition through its nitration at Y32. As NipR2 also reduces sepsis induced ALI in the mouse lung, we conclude that Rac1 nitration is a therapeutic target in ALI.

Adenosine and ATP\u03b3S protect against bacterial pneumonia-induced acute lung injury

Lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, disrupts the alveolar-capillary barrier, triggering pulmonary vascular leak thus inducing acute lung injury (ALI). Extracellular purines, adenosine and ATP, protected against ALI induced by purified LPS. In this study, we investigated whether these purines can impact vascular injury in more clinically-relevant E.coli (non-sterile LPS) murine ALI model. Mice were inoculated with live E. coli intratracheally (i.t.) with or without adenosine or a non-hydrolyzable ATP analog, adenosine 5′-(γ-thio)-triphosphate (ATPγS) added intravenously (i.v.). After 24 h of E. coli treatment, we found that injections of either adenosine or ATPγS 15 min prior or adenosine 3 h after E.coli insult significantly attenuated the E.coli-mediated increase in inflammatory responses. Furthermore, adenosine prevented weight loss, tachycardia, and compromised lung function in E. coli-exposed mice. Accordingly, treatment with adenosine or ATPγS increased oxygen saturation and reduced histopathological signs of lung injury in mice exposed to E. coli. Lastly, lung-targeting gene delivery of adenosine or ATPγS downstream effector, myosin phosphatase, significantly attenuated the E. coli-induced compromise of lung function. Collectively, our study has demonstrated that adenosine or ATPγS mitigates E. coli-induced ALI in mice and may be useful as an adjuvant therapy in future pre-clinical studies.

Corticosteroids alleviate lipopolysaccharide-induced inflammation and lung injury via inhibiting NLRP3-inflammasome activation.

The role of corticosteroids in acute lung injury (ALI) remains uncertain. This study aims to determine the underlying mechanisms of corticosteroid treatment for lipopolysaccharide (LPS)‐induced inflammation and ALI. We used corticosteroid treatment for LPS‐induced murine ALI model to investigate the effect of corticosteroid on ALI in vivo. Moreover, LPS‐stimulated macrophages were used to explore the specific anti‐inflammatory effects of corticosteroids on NLRP3‐inflammasome in vitro. We found corticosteroids attenuated LPS‐induced ALI, which manifested in reduction of the alveolar structure destruction, the infiltration of neutrophils and the inflammatory cytokines release of interleukin‐1β (IL‐1β) and interleukin‐18 (IL‐18) in Lung. In vitro, when NLRP3‐inflammasome was knocked out, inflammatory response of caspase‐1 activation and IL‐1β secretion was obviously declined. Further exploration, our results showed that when corticosteroid preprocessed macrophages before LPS primed, it obviously inhibited the activation of caspase‐1 and the maturation of IL‐1β, which depended on inhibiting the nuclear factor‐κB (NF‐κB) signal pathway activation. However, when corticosteroids intervened the LPS‐primed macrophages, it also negatively regulated NLRP3‐inflammasome activation through suppressing mitochondrial reactive oxygen species (mtROS) production. Our results revealed that corticosteroids played a protection role in LPS‐induced inflammation and ALI by suppressing both NF‐κB signal pathway and mtROS‐dependent NLRP3 inflammasome activation.

Inhibition of Class IIa HDACs improves endothelial barrier function in endotoxin-induced acute lung injury.

Acute lung injury (ALI) is an acute inflammatory process arises from a wide range of lung insults. A major cause of ALI is dysfunction of the pulmonary vascular endothelial barrier but the mechanisms involved are incompletely understood. The therapeutic potential of histone deacetylase (HDAC) inhibitors for the treatment of cardiovascular and inflammatory diseases is increasingly apparent, but the mechanisms by which HDACs regulate pulmonary vascular barrier function remain to be resolved. We found that specific Class IIa HDACs inhibitor, TMP269, significantly attenuated the lipopolysaccharide (LPS)-induced human lung microvascular endothelial cells (HLMVEC) barrier compromise in vitro and improved vascular barrier integrity and lung function in murine model of ALI in vivo. TMP269 decreased LPS-induced myosin light chain phosphorylation suggesting the role for Class IIa HDACs in LPS-induced cytoskeleton reorganization. TMP269 did not affect microtubule structure and tubulin acetylation in contrast to the HDAC6-specific inhibitor, Tubastatin A suggesting that Class IIa HDACs and HDAC6 (Class IIb) regulate endothelial cytoskeleton and permeability via different mechanisms. Furthermore, LPS increased the expression of ArgBP2 which has recently been attributed to HDAC-mediated activation of Rho. Depletion of ArgBP2 abolished the ability of LPS to disrupt barrier function in HLMVEC and both TMP269 and Tubastatin A decreased the level of ArgBP2 expression after LPS stimulation suggesting that both Class IIa and IIb HDACs regulate endothelial permeability via ArgBP2-dependent mechanism. Collectively, our data strongly suggest that Class IIa HDACs are involved in LPS-induced ALI in vitro and in vivo via specific mechanism which involved contractile responses, but not microtubule reorganization.

MicroRNA‑93 contributes to the suppression of lung inflammatory responses in LPS‑induced acute lung injury in mice via the TLR4/MyD88/NF‑κB signaling pathway.

Acute lung injury (ALI) is a severe inflammatory lung disease with a rapid onset. The anti-inflammatory functions of microRNA-93 (miRNA/miR-93) have been described in various types of tissue injury and disease. However, the biological role of miR-93 and its molecular mechanisms underlying the initiation and progression of ALI have not yet been reported, at least to the best of our knowledge. The present study aimed to investigate the regulatory effects exerted by miR-93 in ALI. Using an in vivo murine model of ALI induced by lipopolysaccharide (LPS), miR-93 expression was found to be downregulated in the lung tissues and bronchoalveolar lavage fluid (BALF) compared with the control group. Following agomiR-93 injection, it was observed that agomiR-93 attenuated lung injury, as evidenced by decreased lung permeability, a reduced lung wet/dry weight ratio and an increased survival rate of the mice. Concomitantly, agomiR-93 significantly reduced LPS-induced the interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α levels in BALF. Of note, Toll-like receptor 4 (TLR4), an upstream regulator of the nuclear factor (NF)-κB signaling pathway, was directly suppressed by miR-93 in RAW 264.7 cells. Importantly, agomiR-93 induced a significant suppression of the TLR4/myeloid differentiation primary response 88 (MyD88)/NF-κB signaling pathway, as demonstrated by the downregulation of MyD88, and the phosphorylation of IκB-α and p65 in the lung tissues of mice with ALI. Taken together, the findings of the present study indicate that miR-93 attenutes LPS-induced lung injury by regulating the TLR4/MyD88/NF-κB signaling pathway, suggesting that miR-93 may prove to be a potential therapeutic target for ALI.

Levonadifloxacin, a Novel Benzoquinolizine Fluoroquinolone, Modulates Lipopolysaccharide-Induced Inflammatory Responses in Human Whole-Blood Assay and Murine Acute Lung Injury Model.

Fluoroquinolones are reported to possess immunomodulatory activity; hence, a novel benzoquinolizine fluoroquinolone, levonadifloxacin, was evaluated in lipopolysaccharide-stimulated human whole-blood (HWB) and mouse acute lung injury (ALI) models. Levonadifloxacin significantly mitigated the inflammatory responses in an HWB assay through inhibition of proinflammatory cytokines and in the ALI model by lowering lung total white blood cell count, myeloperoxidase, and cytokine levels. The immunomodulatory effect of levonadifloxacin, along with promising antibacterial activity, is expected to provide clinical benefits in the treatment of infections.

Mesenchymal stromal cells ameliorate acute lung injury induced by LPS mainly through stanniocalcin-2 mediating macrophage polarization.

BackgroundAcute lung injury (ALI) is a devastating syndrome with no effective pharmacological therapies in the clinic. Mesenchymal stromal cells (MSCs) have been demonstrated to promote inflammation resolution and tissue repair in ALI. However, the specific mechanisms of this have not been clearly elucidated. Stanniocalcin-2 (STC2) is a stress-responsive protein that has anti-oxidative properties. Our previous study found that STC2 is a highly expressed stanniocalcin in MSCs, which may be involved in immunomodulatory activities. However, the role of STC2 in MSCs to resolve ALI has never been elucidated.MethodsSpecific shRNA was used to downregulate STC2 in MSCs. We detected ROS, cell apoptosis, and paracrine factors changes in MSCs. STC2-associated antioxidant genes were also investigated by Co-immunoprecipitation (Co-IP) and immunofluorescence. Macrophage (THP1 cells) phenotype transitions were measured by flow cytometry after coculturing with MSCs in vitro. Then, we used MSCs to treat LPS-induced ALI in mice, and assessed injury scores inflammation, and antioxidant activities in the lungs of the mice. Alveolar macrophage (AM) phenotypes and CFSE-labeled MSC apoptosis in collected bronchoalveolar fluids (BALF) were also analyzed by flow cytometry.ResultsAfter the STC2 knockdown, MSCs increased ROS generation and cell apoptosis after PX12 pretreatment. The antioxidant protein Nrf2 was colocalized with STC2 in the nucleus. A lack of STC2 expression in MSCs produced less interleukin 10 (IL10) and blunted macrophage polarization in THP1 cells. Furthermore, in the murine LPS-induced ALI model, the STC2 knockdown counteracted the inflammatory resolution and antioxidative effect of MSCs in the lungs. MSCshSTC2-treated mice had a higher lung injury score than the controls, which may be attributed to diminished AM polarization and increased apoptosis of MSCs in vivo.ConclusionsCollectively, these results suggested that STC2 is essential to the anti-oxidative and anti-inflammation properties of MSCs and could prove to be crucial for stem cell therapies for ALI.

Inhibition of the endoplasmic reticulum (ER) stress-associated IRE-1/XBP-1 pathway alleviates acute lung injury via modulation of macrophage activation.

BackgroundBoth endoplasmic reticulum (ER) stress and macrophage diversity contribute to inflammatory processes in lung injury. However, the interaction between ER stress and macrophage M1/M2 imbalance in lung inflammation remains unclear. The present study, thus, aimed to evaluate the role of ER stress-mediated macrophage phenotype changes in lipopolysaccharide (LPS)-induced acute lung injury (ALI).MethodsLung inflammation and injury were examined in a murine model of LPS-induced ALI with or without ER stress inhibitors. Alveolar macrophage (AM) polarization was determined by flow cytometry. Bone marrow-derived macrophages (BMDMs) were treated with either an ER stress inducer, inhibitor, or an IRE-1 endonuclease inhibitor before being polarized to an M1 and M2 phenotype. The macrophage polarization status was examined via RT-PCR and flow cytometry.ResultsOur results indicated that ER stress and IRE-1/XBP-1 signaling are activated in LPS-induced ALI. Furthermore, we observed that AM polarizes to an inflammatory phenotype upon exposure to LPS in the induction phase and an anti-inflammatory phenotype in the resolution phase of lung inflammation. Inhibition of ER stress attenuated the pathophysiological features of LPS-induced lung inflammation/injury, as evidenced by a decrease in bronchoalveolar lavage (BAL) protein levels, the number of inflammatory cells, and the expression level of inflammatory mediators. In addition, the ER stress inducer promoted M1 polarization and the switch from M2 to M1 in BMDMs, whereas inhibition of ER stress and XBP-1 splicing suppressed M1 but did not promote M2, both in vivo and in vitro.ConclusionsOur results demonstrated that inhibition of the ER stress-associated IRE-1/XBP-1 signaling pathway suppresses M1 polarization and ameliorates LPS-induced lung injury. This indicates that the interaction between ER stress and macrophage polarization might be a novel therapeutic target for endotoxin-induced lung inflammatory disorders.

LncRNA TUG1 alleviates sepsis-induced acute lung injury by targeting miR-34b-5p/GAB1

BackgroundSepsis-induced acute lung injury (ALI) is a clinical syndrome characterized by the injury of alveolar epithelium and pulmonary endothelial cells. This study aimed to investigate the regulation of long noncoding RNA (lncRNA) taurine up-regulated gene 1 (TUG1) in a murine ALI model and in primary murine pulmonary microvascular endothelial cells (PMVECs) stimulated with lipopolysaccharide (LPS).MethodsAdult C57BL/6 mice were intravenously injected with or without TUG1-expressiong adenoviral vector or control vector 1 week before the establishment of ALI model. PMVECs were transfected with TUG1-expressiong or control vectors followed by LPS stimulation. MiR-34b-5p was confirmed as a target of TUG1 using dual-luciferase reporter assay. GRB2 associated binding protein 1 (GAB1) was confirmed as a downstream target of miR-34b-5p using the same method. In the rescue experiment, PMVECs were co-transfected with TUG1-expressing vector and miR-34b-5p mimics (or control mimics) 24 h before LPS treatment.ResultsALI mice showed reduced levels of TUG1, pulmonary injury, and induced apoptosis and inflammation compared to the control group. The overexpression of TUG1 in ALI mice ameliorated sepsis-induced pulmonary injury, apoptosis and inflammation. TUG1 also showed protective effect in LPS-treated PMVECs. The expression of MiR-34b-5p was negatively correlated with the level of TUG1. TUG1-supressed apoptosis and inflammation in LPS-stimulated PMVECs were restored by miR-34b-5p overexpression. GAB1 was inversely regulated by miR-34b-5p but was positively correlated with TUG1 expression.ConclusionTUG1 alleviated sepsis-induced inflammation and apoptosis via targeting miR-34b-5p and GAB1. These findings suggested that TUG1 might be served as a therapeutic potential for the treatment of sepsis-induced ALI.