Murine Model Of Acute Lung Injury Research Articles

Anti-inflammatory effects and mechanism of the total flavonoids from Artemisia scoparia Waldst. et kit. in vitro and in vivo

Artemisia scoparia Waldst. et Kit. is traditionally used for the treatment of jaundice urinary retention, itching wet sores, infectious icteric hepatitis and influenza in Uighur medicine. This study aimed to further illuminate the anti-inflammatory effects and mechanism of the total flavonoids (ASTF) from Artemisia scoparia Waldst. et Kit. In vitro, RAW 264.7 cells were pretreated with ASTF 1 h before stimulation with LPS (1 μg/mL) for 24 h. Then, the concentrations of NO, PGE2, TNF-α, IL-6 and MCP-1 in the medium were determined. Intracellular oxidative stress was detected using DCFH-DA. Immunofluorescent analysis, western blot and qRT-PCR were carried out to illuminate the mechanism of anti-inflammatory effects of ASTF. In vivo, mice were given an intragastric administration of ASTF 1 h before an intranasal administration of LPS. After 24 h, bronchoalveolar lavage fluid (BALF) was collected to measure the number of total cells, macrophage and neutrophils. The levels of TNF-α and IL-6 in BALF were quantified by ELISA kits. Lung specimens were isolated for histopathological examinations and lung wet-to-dry weight (W/D) ratio. We found that ASTF significantly inhibited the production of NO, PGE2, TNF-α, IL-6, MCP-1 and reactive oxygen species (ROS) in LPS-stimulated RAW 264.7 cells. ASTF can obviously inhibit the degredation of IκBa and inhibit the nucleus translocations of p-NF-κB p65, p-ERK1/2 and p-p38 in RAW 264.7 cells stimulated by LPS. ASTF also markedly decreased the protein and mRNA expression of TNF-α and IL-6 in a dose-dependent manner. When pretreated with ASTF, alveolar hemorrhage and neutrophil infiltration, as well as pulmonary histopathologic changes, were substantially suppressed in lung tissues in the murine acute lung injury model. The lung wet-to-dry weight (W/D) ratio was strongly decreased. These results suggested that ASTF showed important anti-inflammatory activity and might provide protective effects against LPS-induced ALI. The anti-inflammatory effect of ASTF might attribute to its suppression of NF-κB and MAPK signaling pathway.

Tabersonine attenuates lipopolysaccharide-induced acute lung injury via suppressing TRAF6 ubiquitination

Sepsis caused by Gram-negative bacteria is one of major causes for the progression of acute lung injury (ALI) with limited treatment and effective medicines. Tabersonine is an indole alkaloid mainly isolated from Catharanthus roseus, and a potential drug candidate for treatment of cancer and Alzheimer’s disease (AD), however, its anti-inflammatory effect has not been revealed. In this study, we reported that tabersonine ameliorated lipopolysaccharides (LPS)-induced ALI in vivo and inhibited LPS-mediated macrophage activation in vitro. By using murine ALI model, we found that tabersonine significantly attenuated LPS-induced pathological injury in the lung. Tabersonine also inhibited LPS-mediated neutrophil infiltration, elevation of MPO activity and the production of TNF-α, IL-6 and IL-1β. Furthermore, tabersonine inhibited LPS-induced the production of pro-inflammatory mediators such as iNOS, NO and cytokines by suppressing NF-κB and p38 MAPK/MK2 signaling cascades. Tabersonine reduced the K63-linked polyubiquitination of TRAF6. Taken together, these results suggested that tabersonine has anti-inflammatory activities in vitro and in vivo, and is a potential therapeutic candidate for the treatment of ALI/ARDS.

HDAC7 in MLCP‐mediated endothelial barrier preservation

Acute lung injury (ALI) is an acute systemic inflammatory process characterized by pulmonary infiltrates hypoxemia and edema. ALI arises from a wide range of lung injuries such as toxins or inflammatory mediators. Despite the high level of mortality, to date no pharmacological therapies have been proven to improve ALI except from ventilation strategies. A deeper understanding of the pathogenic mechanisms involved remains a barrier to new and effective therapies that will reduce the vascular leak associated with ALI.A major cause of ALI is dysfunction of the pulmonary vascular endothelial barrier. Endothelial cells (EC) are a specialized cell type that line the lumen of the blood vessels serving every major organ system and provide a semi‐selective barrier between the blood and the interstitial space. Epigenetic mechanisms have emerged as a major regulator of enduring changes in cell behavior and the therapeutic potential of inhibiting histone deacetylases (HDACs) for the treatment of cardiovascular and inflammatory diseases is gaining increasing attention. In addition to histones, it has been shown that HDACs can deacetylate many non‐histone proteins to regulate cellular functions such as cytoskeletal acetylation and polymerization, and signal transduction. Further, it has been shown that HDAC inhibition is directly involved in EC barrier preservation, however, the molecular mechanisms of HDAC‐mediated EC barrier regulation remain largely unexplored.Our data indicate that the class IIa histone deacetylases comprising of HDACs 4, 5, 7, 9 are involved in the regulation of pulmonary vascular barrier in vitro and in lipopolysaccharide (LPS)‐induced murine model of ALI. These HDACs are known to function as transcriptional co‐repressors, however, they also are able to shuttle from nucleus to the cytoplasm thus becoming accessible for extra nuclear targets. We demonstrate that HDAC 7 is directly involved in LPS‐induced human lung microvascular EC (HLMVEC) barrier compromise. Further, barrier‐disruptive effects of LPS are accompanied by HDAC7 phosphorylation and its nuclear export. Conversely, agents that protect the EC barrier such as adenosine, decrease its phosphorylation. These data suggest the involvement of phosphatase activity in HDAC7‐mediated EC barrier regulation. Our results show that HDAC7 is a substrate for myosin light chain (MLC) phosphatase (MLCP) in HLMVEC. Conversely, down‐regulation of HDAC7 may affect MLCP activity. However, the physiological role of HDAC7/MLCP crosstalk in EC barrier regulation is currently unexplored. It is known that activation of MLCP leads to dephosphorylation of MLC thus opposing actomyosin contraction. Our newly published results suggest that MLCP activation may be involved in EC barrier preservation via the dephosphorylation of unconventional MLCP cytoskeletal targets such as moesin. Our results demonstrate a direct association between HDAC7 and moesin, suggesting a novel role of a HDAC7/MLCP/moesin signaling complex in the regulation of EC barrier. Collectively, these data demonstrate the involvement of HDAC7 activity in MLCP‐mediated EC barrier preservation and suggest, for the first time, the importance of HDAC7 nuclear export in pulmonary vascular injury induced by edemagenic agents such as LPS.Support or Funding InformationNIH; Contract grant number: HL101902This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phospholipase D isoforms differentially regulate leukocyte responses to acute lung injury.

Phospholipase D (PLD) plays important roles in cellular responses to tissue injury that are critical to acute inflammatory diseases, such as the acute respiratory distress syndrome (ARDS). We investigated the expression of PLD isoforms and related phospholipid phosphatases in patients with ARDS, and their roles in a murine model of self-limited acute lung injury (ALI). Gene expression microarray analysis on whole blood obtained from patients that met clinical criteria for ARDS and clinically matched controls (non-ARDS) demonstrated that PLD1 gene expression was increased in patients with ARDS relative to non-ARDS and correlated with survival. In contrast, PLD2 expression was associated with mortality. In a murine model of self-resolving ALI, lung Pld1 expression increased and Pld2 expression decreased 24h after intrabronchial acid. Total lung PLD activity was increased 24h after injury. Pld1-/- mice demonstrated impaired alveolar barrier function and increased tissue injury relative to WT and Pld2-/- , whereas Pld2-/- mice demonstrated increased recruitment of neutrophils and macrophages, and decreased tissue injury. Isoform-specific PLD inhibitors mirrored the results with isoform-specific Pld-KO mice. PLD1 gene expression knockdown in human leukocytes was associated with decreased phagocytosis by neutrophils, whereas reactive oxygen species production and phagocytosis decreased in M2-macrophages. PLD2 gene expression knockdown increased neutrophil and M2-macrophage transmigration, and increased M2-macrophage phagocytosis. These results uncovered selective regulation of PLD isoforms after ALI, and opposing effects of selective isoform knockdown on host responses and tissue injury. These findings support therapeutic strategies targeting specific PLD isoforms for the treatment of ARDS.

Akap1 genetic deletion increases the severity of hyperoxia-induced acute lung injury in mice.

Critically ill patients are commonly treated with high levels of oxygen, hyperoxia, for prolonged periods of time. Unfortunately, extended exposure to hyperoxia can exacerbate respiratory failure and lead to a high mortality rate. Mitochondrial A-kinase anchoring protein (Akap) has been shown to regulate mitochondrial function. It has been reported that, under hypoxic conditions, Akap121 undergoes proteolytic degradation and promotes cardiac injury. However, the role of Akap1 in hyperoxia-induced acute lung injury (ALI) is largely unknown. To address this gap in our understanding of Akap1, we exposed wild-type ( wt) and Akap1-/- mice to 100% oxygen for 48 h, a time point associated with lung damage in the murine model of ALI. We found that under hyperoxia, Akap1-/- mice display increased levels of proinflammatory cytokines, immune cell infiltration, and protein leakage in lungs, as well as increased alveolar capillary permeability compared with wt controls. Further analysis revealed that Akap1 deletion enhances lung NF-κB p65 activity as assessed by immunoblotting and DNA-binding assay and mitochondrial autophagy-related markers, PINK1 and Parkin. Ultrastructural analysis using electron microscopy revealed that Akap1 deletion was associated with remarkably aberrant mitochondria and lamellar bodies in type II alveolar epithelial cells. Taken together, these results demonstrate that Akap1 genetic deletion increases the severity of hyperoxia-induced acute lung injury in mice.

Incorporation of iloprost in phospholipase-resistant phospholipid scaffold enhances its barrier protective effects on pulmonary endothelium

Correction of barrier dysfunction and inflammation in acute lung injury (ALI) represents an important problem. Previous studies demonstrate barrier-protective and anti-inflammatory effects of bioactive lipid prostacyclin and its stable analog iloprost (ILO). We generated a phospholipase resistant synthetic phospholipid with iloprost attached at the sn-2 position (ILO-PC) and investigated its biological effects. In comparison to free ILO, ILO-PC caused sustained endothelial cell (EC) barrier enhancement, linked to more prolonged activation of Rap1 and Rac1 GTPases and their cytoskeletal and cell junction effectors: cortactin, PAK1, p120-catenin and VE-cadherin. ILO and ILO-PC equally efficiently suppressed acute, Rho GTPase-dependent EC hyper-permeability caused by thrombin. However, ILO-PC exhibited more sustained barrier-protective and anti-inflammatory effects in the model of chronic EC dysfunction caused by bacterial wall lipopolysacharide (LPS). ILO-PC was also more potent inhibitor of NFκB signaling and lung vascular leak in the murine model of LPS-induced ALI. Treatment with ILO-PC showed more efficient ALI recovery over 3 days after LPS challenge than free ILO. In conclusion, this study describes a novel synthetic phospholipid with barrier-enhancing and anti-inflammatory properties superior to existing prostacyclin analogs, which may be used as a prototype for future development of more efficient treatment for ALI and other vascular leak syndromes.

GLP-1 Analogue Liraglutide Enhances SP-A Expression in LPS-Induced Acute Lung Injury through the TTF-1 Signaling Pathway.

The reduction of pulmonary surfactant (PS) is essential for decreased pulmonary compliance and edema in acute lung injury (ALI). Thyroid transcription factor-1 (TTF-1) plays a major role in the regulation of surfactant protein-A (SP-A), the most abundant protein component of PS. Simultaneously, the glucagon-like peptide-1 (GLP-1) analogue can enhance SP-A expression in the lung. However, the underlying mechanism is still unknown. The purpose of this study was to explore whether liraglutide, a GLP-1 analogue, upregulates SP-A expression through the TTF-1 signaling pathway in ALI. In vivo, a murine model of ALI was induced by lipopolysaccharide (LPS). Pulmonary inflammation, edema, insulin level, ultrastructural changes in type II alveolar epithelial (ATII) cells, and SP-A and TTF-1 expression were analyzed. In vitro, rat ATII cells were obtained. SP-A and TTF-1 expression in cells was measured. ShRNA-TTF-1 transfection was performed to knock down TTF-1 expression. Our data showed that LPS-induced lung injury and increase in insulin level, and LPS-induced reduction of SP-A and TTF-1 expression in both the lung and cells, were significantly compromised by liraglutide. Furthermore, we also found that these effects of liraglutide were markedly blunted by shRNA-TTF-1. Taken together, our findings suggest that liraglutide enhances SP-A expression in ATII cells and attenuates pulmonary inflammation in LPS-induced ALI, most likely through the TTF-1 signaling pathway.

Wnt/β-catenin pathway promotes acute lung injury induced by LPS through driving the Th17 response in mice

T helper cell 17 (Th17), one type of CD4+ T cell, plays an important role in regulating the acute lung injury (ALI) inflammatory response. Recent studies showed that Wnt/β-catenin pathway could modulate the differentiation and the function of CD4+ T cell. However, whether Wnt/β-catenin could regulate the differentiation and function of Th17 in the development and progress of ALI induced by lipopolysaccharide (LPS) is still unknown. To test this, we used dickkopf1 (Dkk-1) to block the Wnt/β-catenin pathway and LiCl to activate the Wnt/β-catenin pathway by instillation to the murine model of ALI. Our results revealed that activation of Wnt/β-catenin pathway significantly aggravated the LPS-induced lung inflammation. Meanwhile, we observed that activation of Wnt/β-catenin pathway promoted Th17 response by analyzing CD4+ T cells and the related cytokines secretions. Enhanced Th17 response was responsible for the further neutrophils infiltration and pro-inflammatory cytokines production. In addition, activation of Wnt/β-catenin pathway resulted in induced expression of retinoic acid related orphan receptor-γt (RORγt) via histone acetyltransferase p300. These data suggested that Wnt/β-catenin pathway might be a potential target to treat the LPS-induced inflammation in ALI.

Interleukin-10-Overexpressing Mesenchymal Stromal Cells Induce a Series of Regulatory Effects in the Inflammatory System and Promote the Survival of Endotoxin-Induced Acute Lung Injury in Mice Model.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are life-threatening inflammatory conditions with no effective pharmacological treatment. Previous studies suggested that mesenchymal stromal/stem cell (MSC) infusion resulted in better survival in mouse ALI models and presented low toxicity in human subjects. Therefore, in this study, we investigated the possibility of treating a murine model of ALI using MSCs with constant interleukin-10 overexpression (IL-10-MSC) by retroviral infection. ALI in mice was induced by intratracheal lipopolysaccharides (LPS) instillation. After 96 h, 80% of mice receiving IL-10-MSCs survived, whereas the survival rate of the mice receiving other treatments was only 20-50%. Mice receiving IL-10-MSCs also demonstrated significantly less weight loss (p < 0.01), and lower protein level and TNF concentration in the BAL (p < 0.01). Interestingly, IL-10-MSCs given to mice 3 and 1 day before ALI induction still conferred significant protection against ALI. While direct IL-10 transfusion resulted in an intensive, but transient peak in serum IL-10 level, IL-10-MSCs provided a milder, but more persistent increase in serum IL-10 level, together with significantly higher levels of IL-10-producing T cells and B cells, both in the spleen and in the lung. IL-10-MSCs given 3 days before LPS challenge resulted in higher pulmonary infiltration of IL-10-producing T cells and B cells in mice. On average, mice that survived the LPS challenge for 96 h presented higher pulmonary infiltration of IL-10-producing T cells and B cells than mice that deceased within the experimental period. Together, these results demonstrated that IL-10-MSCs offered superior protection against LPS-induced ALI when given before or at the time of ALI induction, and significantly increased the frequencies of IL-10-expressing T cells and B cells. IL-10-MSCs may thus represent a promising new treatment option in ALI/ARDS.

Rhesus θ-Defensin-1 Attenuates Endotoxin-induced Acute Lung Injury by Inhibiting Proinflammatory Cytokines and Neutrophil Recruitment.

Acute lung injury (ALI) is a clinical syndrome characterized by acute respiratory failure and is associated with substantial morbidity and mortality. Rhesus θ-defensin (RTD)-1 is an antimicrobial peptide with immunomodulatory activity. As airway inflammation and neutrophil recruitment and activation are hallmarks of ALI, we evaluated the therapeutic efficacy of RTD-1 in preclinical models of the disease. We investigated the effect of RTD-1 on neutrophil chemotaxis and macrophage-driven pulmonary inflammation with human peripheral neutrophils and LPS-stimulated murine alveolar macrophage (denoted MH-S) cells. Treatment and prophylactic single escalating doses were administered subcutaneously in a well-established murine model of direct endotoxin-induced ALI. We assessed lung injury by histopathology, pulmonary edema, inflammatory cell recruitment, and inflammatory cytokines/chemokines in the BAL fluid. In vitro studies demonstrated that RTD-1 suppressed CXCL8-induced neutrophil chemotaxis, TNF-mediated neutrophil-endothelial cell adhesion, and proinflammatory cytokine release in activated murine alveolar immortalized macrophages (MH-S) cells. Treatment with RTD-1 significantly inhibited in vivo LPS-induced ALI by reducing pulmonary edema and histopathological changes. Treatment was associated with dose- and time-dependent inhibition of proinflammatory cytokines (TNF, IL-1β, and IL-6), peroxidase activity, and neutrophil recruitment into the airways. Antiinflammatory effects were demonstrated in animals receiving RTD-1 up to 12 hours after LPS challenge. Notably, subcutaneously administered RTD-1 demonstrates good peptide stability as demonstrated by the long in vivo half-life. Taken together, these studies demonstrate that RTD-1 is efficacious in an experimental model of ALI through inhibition of neutrophil chemotaxis and adhesion, and the attenuation of proinflammatory cytokines and gene expression from alveolar macrophages.

Anti-Inflammatory Effect of Anti-TNF-α SiRNA Cationic Phosphorus Dendrimer Nanocomplexes Administered Intranasally in a Murine Acute Lung Injury Model.

Inflammation is an essential component of many lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), or acute lung injury. Our purpose was to design efficient carriers for lung delivery of small interfering RNA (siRNA) targeting tumor necrosis factor (TNF-α) in an acute lung injury model. To achieve this goal, two different types of phosphorus-based dendrimers with either pyrrolidinium or morpholinium as terminal protonated amino groups were selected for their better biocompatibility compared to other dendrimers. Dendriplexes containing pyrrolidinium surface groups demonstrated a stronger siRNA complexation, a higher cellular uptake, and enhanced in vitro silencing efficiency of TNF-α in the lipopolysaccharide (LPS)-activated mouse macrophage cell line RAW264.7, compared to morpholinium-containing dendriplexes. The better performance of the pyrrolidium dendriplexes was attributed to their higher pKa value leading to a stronger siRNA complexation and improved protection against enzymatic degradation resulting in a higher cellular uptake. The superior silencing effect of the pyrrolidinium dendriplexes, compared to noncomplexed siRNA, was confirmed in vivo in an LPS-induced murine model of short-term acute lung injury upon lung delivery via nasal administration. These data suggest that phosphorus dendriplexes have a strong potential in lung delivery of siRNA for treating inflammatory lung diseases.

Ellagic acid protects against LPS-induced acute lung injury through inhibition of nuclear factor kappa B, proinflammatory cytokines and enhancement of interleukin-10

ABSTRACTEllagic acid is a naturally occurring polyphenolic compound which is found in many fruits, nut galls and plant extracts. In the present study, we explored the ability of ellagic acid to modulate lipopolysaccharides (LPS) response using macrophage-mediated inflammatory conditions and acute lung injury (ALI). The data showed that ellagic acid reduced TNF-α, IL-6 and IL-1β secretions, enhance IL-10 production by LPS-stimulated RAW 264.7 macrophages in vitro. In murine ALI model, mice were treated with ellagic acid prior to LPS challenge. The data showed that ellagic acid possess a protective effect on LPS-induced ALI in mice. The underlying mechanism may be through shocking the NF-κB pathway to attenuate the nonspecific pulmonary inflammation induced by LPS administration.

Inhibition of nuclear factor-κB signal by pyrrolidine dithiocarbamate alleviates lipopolysaccharide-induced acute lung injury.

This study mainly studied the effect of inhibition of nuclear factor-κB (NF-κB) signal by pyrrolidine dithiocarbamate (PDTC) on lipopolysaccharide (LPS)-induced inflammatory response, oxidative stress, and mitochondrial dysfunction in a murine acute lung injury model. The results showed that LPS exposure activated NF-κB and its upstream proteins and caused lung inflammation, oxidative stress, and mitochondrial dysfunction in mice. While inhibition of NF-κB by PDTC adminstration alleviated LPS-induced generation of lymphocytes, IL-1β, and TNF-α. Malondialdehyde, a common oxidative product, was markedly reduced after PDTC treatment in LPS-challenged mice. Furthermore, PDTC alleviated LPS-induced mitochondrial dysfunction via improving ATP synthesis and uncoupling protein 2 expression. In conclusion, inhibition of NF-κB by PDTC alleviated LPS-induced acute lung injury via maintaining inflammatory status, oxidative balance, and mitochondrial function in mice.

Hydroxytyrosol Attenuates LPS-Induced Acute Lung Injury in Mice by Regulating Autophagy and Sirtuin Expression.

Recently, the effects of hydroxytyrosol on autophagy during acute lung injury (ALI) have drawn increasing attention. We explored the underlying molecular mechanisms by which hydroxytyrosol exerts its anti-inflammatory effects in a murine model of ALI by up-regulating autophagy. Male BALB/c mice, challenged with intranasal instillations of LPS, were treated with or without hydroxytyrosol (HT, 100 mg/kg, intragastrically) 1 h prior to LPS exposure. Twenty-four hours later, lung and bronchoalveolar lavage (BAL) fluid samples were obtained for the determination of lung wet to dry weight (W/D) ratios, protein leakage levels, and differential counts of inflammatory cells in BAL fluid. LPS-induced cytokine activity, inflammatory factor levels, sirtuin (SIRT1/3/6) expression, mitogenactivated protein kinase (MAPK) activation, and autophagy marker expression in ALImice were examined by western blotting and staining methods. Molecular docking between HT and SIRT and MAPK was studied with a Sybyl/Surflex module. LPS-stimulated SIRT inhibition, MAPK phosphorylation, and autophagy suppression were all notably abolished by HT administration. HT treatment significantly attenuated pulmonary edema and inflammatory cell infiltration into lung tissues, accompanied by decreased lung W/D ratios, protein concentrations, and inflammatory cell levels in BAL fluid. LPS driven release of inflammatory mediators, including TNF-α, IL-1β, IL-6, IL-10, and MCP-1, was strongly regulated by HT. The protective effect of HT on lung inflammation in ALI mice may be attributed to the promotion of autophagy, which is likely associated with the activation of the SIRT/MAPK signaling pathway. Importantly, this study provides new insight into the molecular mechanisms of HT and its therapeutic potential in the treatment of acute respiratory distress syndrome.

Vimentin Directly Interacts with NLRP3 and Regulates Inflammation

Activation of the NLRP3 inflammasome and subsequent maturation of IL‐1β have been implicated in acute lung injury (ALI), which is characterized by inflammation, IL‐1β release, alveolar epithelial cell injury, hyperplasia of alveolar type 2 cells, accumulation of fibroblasts, deposition of collagen, and scar formation. We investigated the role of vimentin, a type III intermediate filament, in this process using three well characterized murine models of ALI which require NLRP3 for inflammasome activation. It was demonstrated that in these ALI models, the pathophysiologic changes are attenuated in Vim−/− mice. We hypothesized that vimentin directly interacts with NLRP3 for normal inflammasome activity. Therefore, we examined, by co‐immunoprecipitation, whether NLRP3 and vimentin interact in a human macrophage cell line, THP‐1, and mouse bone marrow derived macrophages (BMDM). Next, using bio‐layer interferometery we investigated whether NLRP3 and vimentin directly interact. Preliminary results indicated that full length vimentin directly interacts with NLRP3 with a KD around 240 nM. To investigate which region of vimentin interacts with NLRP3 we used peptide arrays. Our results indicate that the vimentin head domain has and a small region of the tail domain interact with NLRP3 while the central rod domain shows very little interaction. Since vimentin has a large number of amino acids which can be phosphorylated, the binding between phosphomimetic vimentin peptides and NLRP3 was also examined. Phosphomimetic peptides of the vimentin head domain inhibit binding to NLRP3 while increased binding was observed in the tail domain when T426D and S430D mutations were examined. To further demonstrate a functional interaction between vimentin and NLRP3, vimentin was deleted from BMDMs using crispr/Cas9 and vimentin domains were expressed in these Vim KO cells using lentiviral infection. These new cell lines were examined for IL‐1β release after activation with LPS and ATP. Additionally, localization of vimentin and vimentin domains near NLRP3 after activation was investigated using proximity ligation assays. Our data strongly indicate that NLRP3 and vimentin directly interact and that this interaction is important for NLRP3 inflammasome activity.Support or Funding InformationThis work was supported by National Heart, Lung, and Blood Institute (HL71643; HL128194)

Protein Kinase Cζ Inhibitor Promotes Resolution of Bleomycin-Induced Acute Lung Injury.

Protein kinase Cζ (PKCζ) is highly expressed in the lung, where it plays several regulating roles in the pathogenesis of acute lung injury (ALI). Proliferation and differentiation of integrin β4+ distal lung epithelial progenitor cells seem to play a key role in proper lung regeneration. We investigated the effects of a myristoylated PKCζ inhibitor (PKCζi) in a murine model of bleomycin-induced ALI. After intratracheal injury, we treated mice three times a week with PKCζi or its vehicle, DMSO. We found that mice injured with bleomycin and then treated with PKCζi for one week showed decreased activation of PKCζ, improved lung compliance, and decreased lung protein permeability compared to injured mice treated with DMSO. Mice treated continuously with PKCζi for 6 weeks showed reduced evidence of lung fibrosis by computed tomographic images, decreased lung collagen deposition, and decreased active transforming growth factor-β in the bronchoalveolar lavage fluid. In addition, we found an increased number of lung β4+ cells compared to DMSO at Week 6. Therefore, we grew isolated integrin β4+ lung progenitor cells in the presence of PKCζi or DMSO and found that β4+ cells treated with PKCζi proliferated more in vitro compared to DMSO. We conclude that the use of a PKCζi promotes resolution of lung fibrosis in a bleomycin ALI model and increases the number of β4+ progenitor cells with regenerative potential in the lung.

The control effects of FLT3 signaling-dependent pulmonary conventional dendritic cells on the initiation of acute lung inflammation response to lipopolysaccharide induced acute lung injury in mice

Objective To clarify the role of FLT3 signaling-dependent pulmonary conventional dendritic cells (cDCs) in the pathogenesis of lipopolysaccharide (LPS)-induced acute lung injury (ALI), and as well as the modulation effects of cDCs in vivo on the inflammatory responses to acute lung injury. Methods Thirty C57BL/6 male mice were divided into normal control group, LPS group, FLT3L pretreatment group, lestaurtinib, (a high efficient and specific blocker in FLT3 signal pathway) pretreatment group and vehicle (DMSD) control group. FLT3L and lestaurtinib were administrated subcutaneously for 5 days. Murine model of ALI was subsequently established by intra-tracheal application of LPS and lung specimens were harvested 6 h or 24 h later. The accumulation and maturation of pulmonary cDCs were assessed by flow cytometry. IL-6 and TNF-α were quantified to evaluate lung inflammation. Lung injury was estimated by lung wet weight/body weight ratio (LWW/BW) and histopathological assessment. Lung myeloperoxidase (MPO) activity was measured to evaluate neutrophil infiltration. Transcription factors T-bet/GATA-3 mRNA ratio was determined to estimate balance of Th1/Th2 response. IFN-γ and IL-4 were quantified to evaluate Th1-specific and Th2-specific cytokine production respectively. Results The accumulation and maturation of pulmonary cDCs peaked at 6h after LPS challenge. FLT3L pretreatment significantly stimulated the accumulation and maturation of pulmonary cDCs (P<0.05), leading to markedly deterioration of LWW/BW and lung histopathological changes. Meanwhile lung MPO activity and T-bet/GATA-3 mRNA ratio were elevated (P<0.05). Furthermore, the production of IL-6, TNF-α and IFN-γ was markedly increased by FLT3L pretreatment (P<0.05). In contrast, lestaurtinib pretreatment markedly inhibited the accumulation and maturation of pulmonary cDCs (P<0.05), leading to significant improvement of LWW/BW and lung histopathological changes. Meanwhile lung MPO activity and T-bet/GATA-3 mRNA ratio were decreased (P<0.05). Furthermore lestaurtinib efficiently suppressed the production of IL-6, TNF-α and IFN-γ(P<0.05). Conclusion This study thus demonstrated that FLT3 signaling-dependent pulmonary cDCs could control the initiation of acute lung inflammation response to LPS-induced ALI through the regulation of neutrophil infiltration and balance of Th1/Th2 response. Key words: Acute lung injury; Conventional dendritic cells; FLT3 signaling; Inflammation; Neutrophils infiltration; T-helper-cell response; Pathogenesis

MicroRNA-7 Deficiency Ameliorates the Pathologies of Acute Lung Injury through Elevating KLF4.

Recent evidence showed that microRNA-7 (miR-7) played an important role in the pathologies of lung-related diseases. However, the potential role of miR-7 in acute lung injury (ALI) still remains poorly understood. Here, we assessed the effect of miR-7 deficiency on the pathology of ALI. We, first, found that the expression of miR-7 was upregulated in lung tissue in murine LPS-induced ALI model. Notably, we generated miR-7 knock down mice by using miRNA-Sponge technique and found that miR-7 deficiency could ameliorate the pathologies of lung as evidenced by accelerated body weight recovery, reduced level of bronchoalveolar lavage (BAL) proinflammatory cytokines and decreased number of BAL cells in ALI mice. Moreover, the proportion and number of various immune cells in BAL, including innate immune cell F4/80+ macrophages, γδT cells, NK1.1+ T cells, and CD11c+DCs, as well as adaptive immune cell CD4+ T cells and CD8+ T cells, also significantly changed, respectively. Mechanistic evidence showed that KLF4, a target molecule of miR-7, was upregulated in lung tissues in ALI model, accompanied by altered transduction of NF-κB, AKT, and ERK pathway. These data provided a previously unknown role of miR-7 in pathology of ALI, which could ultimately aid the understanding of development of ALI and the development of new therapeutic strategies against clinical inflammatory lung diseases.

Repression of a chromatin modifier aggravates lipopolysaccharide-induced acute lung injury in mouse

Local inflammatory responses and alveolar epithelial cells (AECs) apoptosis are both important for the development of the acute lung injury (ALI), a clinically important complication causing high morbidity and mortality, but little is known about the molecular mechanisms underlying the pathogenesis. Herein, we showed for the first time that expression of Metastasis-associated protein 1 (MTA1), a master transcriptional regulator with the ability to regulate divergent cellular pathways by modifying the acetylation status of crucial target genes, was up-regulated in the alveolar cells of the Escherichia coli lipopolysaccharide (LPS)-induced murine ALI model. Inhibition of MTA1 expression by in vivo siRNA treatment exacerbated the pathology of LPS-induced ALI, by selectively promoting the expression of NF-κB-regulated inflammatory cytokines. Moreover, ablation of MTA1 expression promoted the LPS-induced apoptosis in AEC II cells, leaving AEC I cells unaffected. These data collectively underscore an alveolar facet of this important chromatin modifier, which may represent as a novel regulator and a new therapeutic target for the treatment of ALI.

Acute Lung Injury Causes Asynchronous Alveolar Ventilation That Can Be Corrected by Individual Sighs.

Improved ventilation strategies have been the mainstay for reducing mortality in acute respiratory distress syndrome. Their unique clinical effectiveness is, however, unmatched by our understanding of the underlying mechanobiology, and their impact on alveolar dynamics and gas exchange remains largely speculative. To assess changes in alveolar dynamics and associated effects on local gas exchange in experimental models of acute lung injury (ALI) and their responsiveness to sighs. Alveolar dynamics and local gas exchange were studied in vivo by darkfield microscopy and multispectral oximetry in experimental murine models of ALI induced by hydrochloric acid, Tween instillation, or in antibody-mediated transfusion-related ALI. Independent of injury mode, ALI resulted in asynchronous alveolar ventilation characteristic of alveolar pendelluft, which either spontaneously resolved or progressed to a complete cessation or even inversion of alveolar ventilation. The functional relevance of the latter phenomena was evident as impaired blood oxygenation in juxtaposed lung capillaries. Individual sighs (2 × 10 s at inspiratory plateau pressure of 30 cm H2O) largely restored normal alveolar dynamics and gas exchange in acid-induced ALI, yet not in Tween-induced surfactant depletion. We describe for the first time in detail the different forms and temporal sequence of impaired alveolar dynamics in the acutely injured lung and report the first direct visualization of alveolar pendelluft. Moreover, we identify individual sighs as an effective strategy to restore intact alveolar ventilation by a mechanism independent of alveolar collapse and reopening.