Mouse Model Of Lung Injury Research Articles

Elevation of IL-6 and IL-33 Levels in Serum Associated with Lung Fibrosis and Skeletal Muscle Wasting in a Bleomycin-Induced Lung Injury Mouse Model.

Weight loss due to skeletal muscle atrophy in patients with chronic pulmonary disease is negatively correlated with clinical outcome. Pulmonary fibrosis is a chronic and progressive interstitial lung disease characterized by the dysregulated deposition of the extracellular matrix (ECM) with the destruction of normal tissue, resulting in end-stage organ failure. BLM-induced fibrosis is one of several different experimental models of pulmonary fibrosis, characterized by inflammation and excessive ECM deposition. We directly induced mouse lung injury by the intratracheal administration of bleomycin and monitored the physiological and biochemical changes in lung and skeletal muscle tissues by using lung function testing, ELISA, Western blotting, and immunohistochemistry. Here, we found that BLM-induced lung fibrosis with thickened interstitial lung tissue, including fibronectin and collagen, was correlated with the increased serum concentrations of IL-6 and IL-33 and accompanied by reduced lung function, including FRC (functional residual capacity), C chord (lung compliance), IC (inspiratory capacity), VC (vital capacity), TLC (total lung capacity), and FVC (forced vital capacity) (p < 0.05). The activity of AKT in lung tissue was suppressed, but conversely, the activity of STAT3 was enhanced during lung fibrosis in mice. In addition, we found that the amount of sST2, the soluble form of the IL-33 receptor, was dramatically decreased in lung fibrosis tissues. The skeletal muscle tissue isolated from lung injury mice increased the activation of STAT3 and AMPK, accompanied by an increased amount of Atrogin-1 protein in BLM-induced lung fibrosis mice. The mouse myoblast cell-based model showed that IL-6 and IL-33 specifically activated STAT3 and AMPK signaling, respectively, to induce the expression of the muscle-specific proteolysis markers MuRF1 and Atrogin-1. These data suggested that increased levels of IL-6 and IL-33 in the serum of mice with BLM-induced lung injury may cause lung fibrosis with thickened interstitial lung tissue accompanied by reduced lung function and muscle mass through the activation of STAT3 and AMPK signals.

The α/β-hydrolase domain 6 inhibitor WWL70 decreases endotoxin-induced lung inflammation in mice, potential contribution of 2-arachidonoylglycerol, and lysoglycerophospholipids.

Lung inflammation plays a crucial role in the pathogenesis of many respiratory diseases that are in need of new therapeutic strategies. Previously, we showed that inhibition of α/β-hydrolase domain 6 (ABHD6) decreased macrophage activation and exerted anti-inflammatory effects. Therefore, we thought to assess the effects of ABHD6 inhibition in a mouse model of acute lung injury (ALI) induced by intratracheal administration of lipopolysaccharides. ABHD6 inhibition with N-methyl-N-{[3-(4-pyridinyl)phenyl]methyl}-carbamic acid 4'-(aminocarbonyl)(1,1'-biphenyl)-4-yl ester (WWL70) decreases most of the hallmarks of ALI, including neutrophil infiltration, cytokine secretion, and protein extravasation. mRNA expression of proinflammatory markers in the cells recovered in the bronchoalveolar lavage was also decreased. Interestingly, ABHD6 inhibition was more efficient than monoacylglycerol lipase inhibition by 4-nitrophenyl-4-[dibenzo(d)(14)dioxol-5-yl(hydroxy)methyl]piperidine-1-carboxylate. We also studied ABHD6 inhibition on primary alveolar macrophages and neutrophils to explore their potential implication in the effects of ABHD6 inhibition in vivo. Moreover, we quantified by high-performance liquid chromatography-mass spectrometry the levels of reported substrates of ABHD6 [i.e., 2-arachidonoylglycerol (2-AG) and lysophospholipids]. The potential implication of these lipid mediators in the effects of WWL70 was further investigated on primary alveolar macrophages. Taken together, these data support ABHD6 inhibition as an interesting anti-inflammatory strategy in acute lung inflammation and assess the possible contribution of 2-AG and lysophospholipids in the observed effects.-Bottemanne, P., Paquot, A., Ameraoui, H., Alhouayek, M., Muccioli, G. G. The α/β-hydrolase domain 6 inhibitor WWL70 decreases endotoxin-induced lung inflammation in mice, potential contribution of 2-arachidonoylglycerol, and lysoglycerophospholipids.

Acute exposure of mice to hydrochloric acid leads to the development of chronic lung injury and pulmonary fibrosis

Objective: Accidental exposure to hydrochloric acid (HCl) is associated with acute lung injury in humans, development of long-term chronic airway obstruction, and fibrosis. However, the mechanisms responsible for the progression to pulmonary fibrosis remain unclear. We utilized a mouse model of progressive lung injury from a single exposure to HCl to investigate the effects of HCl on the lower respiratory tract. Materials and methods: HCl (0.05–0.3 N) or saline was injected intratracheally into male C57Bl/6J mice. At 1, 4, 10 and 30 days post instillation, bronchoalveolar lavage fluid (BALF) and lung tissues were collected and examined for multiple outcomes.Results and discussion: We observed an early inflammatory response and a late mild inflammation present even at 30 d post HCl exposure. Mice treated with HCl exhibited higher total leukocyte and protein levels in the BALF compared to the vehicle group. This was characterized by increased number of neutrophils, monocytes, and lymphocytes as well as pro-inflammatory cytokines during the first 4 d of injury. The late inflammatory response exhibited a predominant presence of mononuclear cells, increased permeability to protein, and higher levels of the pro-fibrotic mediator TGFβ. Pro-fibrotic protein biomarkers, phosphorylated ERK, and HSP90, were also overexpressed at 10 and 30 d following HCl exposure. In vivo lung function measurements demonstrated lung dysfunction and chronic lung injury associated with increased lung hydroxyproline content and increased expression of extracellular matrix (ECM) proteins. The acute inflammation and severity of fibrosis increased in HCl-concentration dependent manner. Conclusions: Our findings suggest that the initial inflammatory response and pro-fibrotic biomarker upregulation may be linked to the progression of pulmonary fibrosis and airway dysfunction and may represent valuable therapeutic targets.

LncRNA analysis of lung tissues after hUC-MSCs and FTY720 treatment of lipopolysaccharide-induced acute lung injury in mouse models

Acute lung injury (ALI), a persistent lung inflammatory response syndrome, may evolve into acute respiratory distress syndrome (ARDS). Characterized by rapid onset, critical features, and a complex etiology, ALI remains a challenging critical respiratory disease. Recently, mesenchymal stem cells (MSCs) have provided a new solution for the treatment of ALI. We built a lipopolysaccharide (LPS)-induced ALI model in mice. After treatment with human umbilical cord mesenchymal stem cells (hUC-MSCs), FTY720, or a combination of hUC-MSCs and FTY207, the lung inflammatory response was apparently attenuated. To understand the mechanism underlying MSCs treatment of ALI at the genetic level, significant differentially expressed long non-coding RNAs (lncRNAs) between the treatment and model groups were analyzed using microarray technology. Moreover, genetic gene prediction, gene ontology (GO) analysis, pathway analysis, and transcription factor (TF) prediction were carried out. The results showed that a total of 66 lncRNAs were differentially expressed in all three treatment groups, including 8 up-regulated and 58 down-regulated lncRNAs. LncRNA A_30_P01029806 and A_30_P01029194, which were down-regulated, were involved in the signaling pathways closely related to ALI. Through further TF analysis, we identified several significant TFs which lay a foundation for revealing the mechanism underlying lncRNAs treatment of ALI. LncRNA A_30_P01029806 and A_30_P01029194 may serve as candidate biomarkers in the diagnosis and treatment of ALI.

Monitoring the Health Status of Mice with Bleomycin-induced Lung Injury by Using Body Condition Scoring.

Well-defined, humane endpoints aid in monitoring animal health status during disease development. Body condition scoring (BCS) is a method for assessing health status in mouse studies where wasting and death are potential endpoints. Whether BCS is useful in monitoring animals with bleomycin-induced lung injury has not been reported. Body weight (BW) is a common humane endpoint for this model, but because the lungs increase in weight as BW decreases, the animal's true physical condition could be masked when using BW as the sole endpoint criterion. Therefore, our goal here was to assess the usefulness of BCS in monitoring health status in a mouse model of lung injury. Lung injury was caused by acute instil- lation of the fibrogenic antibiotic bleomycin into the lungs through the trachea. Male C57BL/6 mice received bleomycin (0.075 U) dissolved in saline or saline alone. Bleomycin instillation led to a doubling of lung weight and decreases in both BW and BCS, compared with saline instillation. The changes in BW and BCS were significantly correlated with lung weight. When the adjusted BW was used (corrected for the increase in lung weight), the correlation was unchanged, suggesting that the increase in lung weight did not significantly mask the decrease in BW. Bleomycin instillation caused decreases in both soleus and visceral epididymal fat masses. The change in BCS was significantly correlated with both soleus and VEF mass, suggesting that BCS is reflective of the systemic loss of muscle and fat mass. Our findings suggest that BW and BCS are significantly correlated to lung injury in the bleomycin model of lung fibrosis and that BCS is an appropriate alternative humane endpoint in this mouse model.

Hydrogel-based delivery of Il-10 improves treatment of bleomycin-induced lung fibrosis in mice

Idiopathic pulmonary fibrosis (IPF) is a life-threatening progressive lung disorder with limited therapeutic options. While interleukin-10 (IL-10) is a potent anti-inflammatory and anti-fibrotic cytokine, its utility in treating lung fibrosis has been limited by its short half-life. We describe an innovative hydrogel-based approach to deliver recombinant IL-10 to the lung for the prevention and reversal of pulmonary fibrosis in a mouse model of bleomycin-induced lung injury. Our studies show that a hyaluronan and heparin-based hydrogel system locally delivers IL-10 by capitalizing on the ability of heparin to reversibly bind IL-10 without bleeding or other complications. This formulation is significantly more effective than soluble IL-10 for both preventing and reducing collagen deposition in the lung parenchyma after 7 days of intratracheal administration. The anti-fibrotic effect of IL-10 in this system is dependent on suppression of TGF-β driven collagen production by lung fibroblasts and myofibroblasts. We conclude that hydrogel-based delivery of IL-10 to the lung is a promising therapy for fibrotic lung disorders.

Selective Inactivation of Intracellular BiP/GRP78 Attenuates Endothelial Inflammation and Permeability in Acute Lung Injury

The role of Endoplasmic Reticulum Chaperone and Signaling Regulator BiP/GRP78 in acute inflammatory injury, particularly in the context of lung endothelium, is poorly defined. In his study, we monitored the effect of SubAB, a holoenzyme that cleaves and specifically inactivates BiP/GRP78 and its inactive mutant SubAA272B on lung inflammatory injury in an aerosolized LPS inhalation mouse model of acute lung injury (ALI). Analysis of lung homogenates and bronchoalveolar lavage (BAL) fluid showed that LPS-induced lung inflammation and injury were significantly inhibited in SubAB- but not in SubAA272B-treated mice. SubAB-treated mice were also protected from LPS-induced decrease in lung compliance. Gene transfer of dominant negative mutant of BiP in the lung endothelium protected against LPS-induced lung inflammatory responses. Consistent with this, stimulation of endothelial cells (EC) with thrombin caused an increase in BiP/GRP78 levels and inhibition of ER stress with 4-phenylbutyric acid (4-PBA) prevented this response as well as increase in VCAM-1, ICAM-1, IL-6, and IL-8 levels. Importantly, thrombin-induced Ca2+ signaling and EC permeability were also prevented upon BiP/GRP78 inactivation. The above EC responses are mediated by intracellular BiP/GRP78 and not by cell surface BiP/GRP78. Together, these data identify intracellular BiP/GRP78 as a novel regulator of endothelial dysfunction associated with ALI.

Cationic Amphipathic Triazines with Potent Anti-bacterial, Anti-inflammatory and Anti-atopic Dermatitis Properties

The emergence of multi-drug resistant bacteria forces the therapeutic world into a position, where the development of new and alternative kind of antibiotics is highly important. Herein, we report the development of triazine-based amphiphilic small molecular antibacterial agents as mimics of lysine- and arginine-based cationic peptide antibiotics (CPAs). These compounds were screened against a panel of both Gram-positive and Gram-negative bacterial strains. Further, anti-inflammatory evaluation of these compounds led to the identification of four efficient compounds, DG-5, DG-6, DL-5, and DL-6. These compounds displayed significant potency against drug-resistant bacteria, including methicillin-resistant S. aureus (MRSA), multidrug-resistant P. aeruginosa (MDRPA), and vancomycin-resistant E. faecium (VREF). Mechanistic studies, including cytoplasmic membrane depolarization, confocal imaging and flow cytometry suggest that DG-5, DG-6, and DL-5 kill bacteria by targeting bacterial membrane, while DL-6 follows intracellular targeting mechanism. We also demonstrate that these molecules have therapeutic potential by showing the efficiency of DG-5 in preventing the lung inflammation of lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model. More interestingly, DL-6 exhibited impressive potency on atopic dermatitis (AD)-like skin lesions in BALB/c mice model by suppressing pro-inflammatory cytokines. Collectively, these results suggest that they can serve a new class of antimicrobial, anti-inflammatory and anti-atopic agents with promising therapeutic potential.

Fine particulate matter alters the microecology of the murine respiratory tract.

Fine particulate matter is a global challenge to human health. We investigated the effects and potential mechanisms of fine particulate matter on respiratory tract microecology in a lung injury mouse model. BALB/c mice were randomized into exposed and control groups. We found that the levels of soluble tumor necrosis factor receptor I was increased following the PM2.5 exposure. 16S rRNA sequencing of respiratory tract lavage fluid confirmed that the composition of the respiratory tract microecology was altered by the exposure. Lactobacillus was the most abundant of bacterial species present. Collectively, these results establish a link between exposure to fine particulate matter and alterations to the respiratory tract microecology. Elucidation of the underlying mechanisms may lead to treatment strategies in lung injury.

Influence of lncRNA MALAT1 on septic lung injury in mice through p38 MAPK/p65 NF-κB pathway.

To investigate the influence of long non-coding ribonucleic acid (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) on septic lung injury in mice and its mechanism, so as to provide references for the clinical prevention and treatment of septic lung injury in the future. A total of 60 male C57 mice were randomly divided into Control group (n=20), lipopolysaccharide (LPS) group (n=20), and LPS+MALAT1 siRNA group (n=20) using a random number table. The mouse model of septic lung injury was established via intraperitoneal injection of LPS (10 mg/kg), and the MALAT1 knockdown model was established via tailintravenousinjection of MALAT1 siRNA. After 12 h, the lung was taken to measure the wet weight/dry weight ratio. Also, the activity of myeloperoxidase (MPO) in lung tissues was detected. The number of neutrophils and macrophages in bronchoalveolar lavage fluid (BALF) was detected via bronchoalveolar lavage. Moreover, the messenger RNA (mRNA) expression levels of inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and IL-6, in lung tissues were detected via Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Finally, the expression level of p38 in lung tissues was detected via immunohistochemical staining, and the expressions of p38 mitogen-activated protein kinase (MAPK)/p65 nuclear factor-κB (NF-κB) signaling pathway-related proteins in lung tissues of mice were detected via Western blotting. The expression of lncRNA MALAT1 in lung tissues of mice with septic lung injury was significantly increased (p<0.05). After knockdown of lncRNA MALAT1, the LPS-induced pathological injury of lungs could be improved, and the wet weight/dry weight ratio of lungs could be reduced (p<0.05). Compared with those in LPS group, the total number of inflammatory cells and the number of neutrophils and macrophages in BALF were significantly decreased in LPS+MALAT1 siRNA group (p<0.05), and the levels of inflammatory cytokines were also significantly inhibited (p<0.05). The immunohistochemical results manifested that the knockdown of lncRNA MALAT1 could inhibit the LPS-induced up-regulation of p38 in lung tissues in mice. According to the results of Western blotting, the p38 MAPK/p65 NF-κB signaling pathway was significantly activated in lung tissues in LPS group (p<0.05), while it was significantly suppressed after inhibition on lncRNA MALAT1 (p<0.05). The knockdown of lncRNA MALAT1 can significantly improve the septic lung injury in mice, whose mechanism may be related to its inhibition on the p38 MAPK/p65 NF-κB signaling pathway.

The Functional Mechanisms of miR-30b-5p in Acute Lung Injury in Children.

BackgroundAcute lung injury in children is a complicated disease linked to the inflammation response. MicroRNA (miRNA) plays a vital role in acute lung injury. However, the role of miR-30b-5p in the pathogenesis of acute lung injury is not clear. The purpose of our study was to investigate the alteration of miR-30b-5p, suppressor of cytokine signaling 3 (SOCS3), in children with acute lung injury, and also in a mouse model of acute lung injury induced by the endotoxin lipopolysaccharide (LPS).Material/MethodsThe levels of miR-30b-5p, SOCS3, FKN (fractalkine), tumor necrosis factor (TNF)-α, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B), interleukin-6 (IL-6), and IL-8 were detected by ELISA (enzyme-linked immunosorbent assay), western blot, and qRT-PCR (quantitative reverse transcription polymerase chain reaction) assay. The alveolar permeability index and the ratio of wet weight/dry weight (W/D) were measured. Then, we examined the inflammation and apoptosis using hematoxylin and eosin (H&E) staining and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay. Additionally, SOCS3 was investigated as a direct target of miR-30a-5p in RAW264.7 cells by dual-luciferase reporter assays.ResultsOur study indicated that the level of miR-30b-5p was decreased and the levels of SOCS3, FKN, TNF-α, NF-κB, IL-6, and IL-8 were increased in lung tissue, serum, and bronchoalveolar lavage fluid of mice with acute lung injury induced by LPS. In addition, LPS increased alveolar permeability index and the ratio of W/D and induced inflammatory responses, including the activation of the NF-κB pathway in a mouse model. Furthermore, SOCS3 was confirmed to be a target of miR-30a-5p in RAW264.7 cells.ConclusionsOur data demonstrated an important role for miR-30b-5p in acute lung injury inflammation and suggested that miR-30b-5p might be an important therapy target in children with acute lung injury.

In Vivo Calpain Knockdown Using Delivery of siRNA.

Calpain is an intracellular Ca2+-dependent non-lysosomal cysteine protease expressed ubiquitously in mammals. In endothelial cells, dysregulation of calpain has been shown to be involved in a wide variety of pathological conditions such as angiogenesis, vascular inflammation, and diabetes. Cell- or tissue-targeted in vivo delivery of small interfering RNA (siRNA) is a powerful research tool in the analysis of protein function and has been proposed as an attractive therapeutic modality that is applicable against a large number of human diseases including cancer. In this chapter we describe a method to knockdown calpain 1 in mouse pulmonary vascular endothelium using delivery of siRNA/cationic liposome complex. This technique results in a greater than 80% reduction in calpain 1 protein levels 48h after a single i.v. injection of calpain 1 siRNA (0.5mg siRNA/kg)/cationic liposome complex. We also describe confocal imaging to verify the loss of calpain 1 expression in pulmonary microvessel endothelial cells and application of this technique in the mouse model of ventilator-induced lung injury.

Blockade of programmed death-ligand 1 attenuates indirect acute lung injury in mice through targeting endothelial cells but not epithelial cells

To examine the expression profile of programmed death-ligand 1 (PD-L1) on lung endothelial or epithelial cells, and to determine the specific role of PD-L1 in mouse model of indirect acute lung injury (i-ALI). Eighty male C57BL/6 mice were randomly divided into two parts (both n = 40). The effects of different administration routes on the expression of PD-L1 were observed. The mice in each part were randomly divided into sham, i-ALI, i-ALI+small interfering RNA (siRNA) random sequence control, and i-ALI+PD-L1 siRNA which could specifically inhibit PD-L1 expression groups, with 10 mice in each group. i-ALI was reproduced in a mouse model of hemorrhagic shock in combination with a subsequent cecal ligation and puncture (CLP). In sham group, only bilateral femoral arteries were ligated without catheterization or bleeding, and only cecum was separated but perforation was not ligated. Intravenous or intratracheal delivery of PD-L1 siRNA was performed 2 hours following the resuscitation to suppress the expression of PD-L1 on lung endothelial or epithelial cells. The mice in i-ALI+siRNA random sequence control group were given siRNA random sequence without inhibition effect on PD-L1 expression, and those in sham group and i-ALI group were given 100 μL phosphate buffered saline (PBS). The mice were sacrificed at 24 hours after CLP, and samples of blood, lung tissue and bronchoalveolar lavage fluid (BALF) were harvested. Expressions of PD-L1 were determined with flow cytometry. Cytokines and chemokines in plasma, lung tissue and BALF were determined by enzyme linked immunosorbent assay (ELISA). The protein concentration in plasma and BALF and the activity of myeloperoxidase (MPO) in lung tissue were quantitatively measured. The pathological changes in lung tissue were observed under light microscope. (1) Compared with sham group, PD-L1 expression on lung endothelial or epithelial cells were significantly elevated in i-ALI group [endothelial cells: (27.88±1.53)% vs. (19.64±1.03)%, epithelial cells: (58.70±8.21)% vs. (29.23±3.94)%, both P < 0.05]. (2) Mice received intravenous delivery of liposomal-encapsulated siRNA had significantly lower expression of PD-L1 on lung endothelial cells as compared with that of i-ALI group [(21.37±0.76)% vs. (27.88±1.53)%, P < 0.05]. Intratracheal delivery of naked PD-L1 siRNA mainly inhibited the PD-L1 expression on epithelial cell as compared with that of i-ALI group [(31.23±4.71) % vs. (58.70±8.21) %, P < 0.05]. The expression of PD-L1 in pulmonary microvascular endothelial cells or pulmonary epithelial cells of i-ALI mice was not affected by siRNA random sequence. (3) PD-L1 silencing on pulmonary endothelial cells induced by intravenous delivery of PD-L1 siRNA led to a lower protein ratio of BALF/plasma [(4.48±0.35)×10-3 vs. (6.11±0.56)×10-3, P < 0.05] and a decreased MPO activity in lung tissue (U×μg-1×min-1: 2.48±0.47 vs. 4.56±0.52, P < 0.05) as compared with that of i-ALI group. Moreover, inflammatory mediator levels such as interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor-α (TNF-α) in lung tissue or plasma were significantly reduced following PD-L1 suppression on endothelial cells as compared with those of i-ALI group [IL-6 (ng/g): 177.4±23.2 vs. 287.9±57.3, MCP-1 (ng/g): 839.6±91.7 vs. 1 395.7±211.9, MIP-2 (ng/g): 923.7±107.3 vs. 1 700.9±240.2 in lung tissue; IL-6 (ng/L): 950.2±192.7 vs. 1 828.2±243.6, TNF-α (ng/L): 258.7±29.1 vs. 443.0±58.1, MCP-1 (ng/L): 2 583.8±302.3 vs. 4 328.1±416.4, MIP-2 (ng/L): 1 512.9±165.6 vs. 2 005.9±85.7 in plasma, all P < 0.05], however, there was no significant change in the levels of inflammatory factors in BALF. It was shown in lung tissue histology that PD-L1 silencing on pulmonary endothelial cells induced by intravenous delivery of PD-L1 siRNA led to lessened pulmonary edema and reduced immune cells emigration. Intratracheal delivery of PD-L1 siRNA for PD-L1 suppression on epithelial cells had minimal effects on protein ratio of BALF/plasma, MPO activity, inflammatory mediator expressions in lung tissue, plasma, and BALF as well as lung tissue histology. PD-L1 silencing on endothelial cells but not epithelial cells protected mice against hemorrhagic shock-sepsis induced i-ALI.

Size-dependent anti-inflammatory activity of a peptide-gold nanoparticle hybrid in vitro and in a mouse model of acute lung injury

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a life-threatening condition of critically-ill patients, characterized by overwhelming inflammatory responses in the lung. Multiple lines of evidence suggest that the excessive activation of Toll-like receptor 4 (TLR4) plays an important role in this detrimental lung inflammation. Recently, we developed a unique class of peptide-gold nanoparticle (GNP) hybrids that act as potent nano-inhibitors of TLR4 signaling by modulating the process of endosomal acidification. In this study, we aimed to identify the key physiochemical factors that could further strengthen the anti-inflammatory activity of these nano-inhibitors, including the nanoparticle size, the density of peptides coating the nanoparticle surface, as well as the number of the effective amino acid phenylalanine (F) residues in the peptide sequence. Among these factors, we found that the nanoparticle size could significantly affect the TLR4 inhibition. Specifically, the peptide-GNP hybrids with a GNP core of 20 nm (P12(G20)) exhibited the most potent inhibitory activity on TLR4 activation and its downstream cytokine production among those with a GNP core of 13 nm (P12(G13)) and 5 nm (P12(G5)) in THP-1 cell-derived macrophages. This size-dependent anti-inflammatory effect of the hybrid P12 was also observed in a lipopolysaccharide (LPS)-induced mouse model of ALI. It was found that P12(G20) was superior to P12(G13) in prolonging the survival of mice experiencing lethal LPS challenge, decreasing the acute lung inflammation, and alleviating diffuse alveolar damage in the lungs. Interestingly, P12(G20) could also promote long-term tolerance to endotoxin. Detailed mechanistic studies demonstrated that when compared to the smaller P12(G13), the larger P12(G20) had higher cellular uptake and a stronger endosomal pH buffering capacity, contributing to its enhanced therapeutic effects on reducing TLR4 activation in vitro and in vivo. Overall, this study suggests that nanoparticle size is one key factor determining the anti-inflammatory potency of the peptide-GNP hybrids, and the hybrid P12 may serve as a promising, novel class of nanotherapeutics for modulating TLR signaling to treat ALI/ARDS. Statement of SignificanceWe have developed a new class of nanoparticle-based inhibitors (i.e., peptide-GNP hybrids) targeting TLR4 signaling in macrophages. Through evidence-based engineering of the nanoparticle size, surface peptide ligand density and effective amino acid (phenylalanine, F) chain length, we identified a peptide-GNP hybrid, P12(G20), with enhanced anti-inflammatory activity. Specifically, P12(G20) was more potent in reducing inflammation in THP-1 cell-derived macrophages and in a LPS-induced ALI mouse model. More interestingly, P12(G20) facilitated long-term protection against lethal LPS challenge in vivo and induced endotoxin tolerance in vitro. We anticipate that these new hybrids would serve as the next generation anti-inflammatory nano-therapeutics for the treatment of ALI/ARDS or other acute and chronic inflammatory diseases.

Comprehensive lipid profiling of bleomycin-induced lung injury.

Drug-induced lung injury is an adverse effect of drug treatment that can result in respiratory failure. Because lipid profiling could provide cutting-edge understanding of the pathophysiology of toxicological responses, we performed lipidomic analyses of drug-induced lung injury. We used a mouse model of bleomycin-induced lung injury and followed the physiological responses at the acute inflammatory (day 2), inflammatory-to-fibrosis (day 7) and fibrosis (day 21) phases. The overall lipid profiles of plasma, lung and bronchoalveolar lavage fluid (BALF) revealed that drastic changes in lipids occurred in the lung and BALF, but not in the plasma, after 7 and 21days of bleomycin treatment. In the lung, the levels of ether-type phosphatidylethanolamines decreased, while those of phosphatidylcholines, bismonophosphatidic acids and cholesterol esters increased on days 7 and 21. In BALF, the global lipid levels increased on days 7 and 21, but only those of some lipids, such as phosphatidylglycerols/bismonophosphatidic acids and phosphatidylinositols, increased from day 2. The lung levels of prostaglandins, such as prostaglandin D2 , were elevated on day 2, and those of 5- and 15-lipoxygenase metabolites of docosahexaenoic acid were elevated on day 7. In BALF, the levels of 12-lipoxygenase metabolites of polyunsaturated fatty acids were elevated on day 7. Our comprehensive lipidomics approach suggested anti-inflammatory responses in the inflammatory phase, phospholipidosis and anti-inflammatory responses in the inflammatory-to-fibrosis phase, and increased oxidative stress and/or cell phenotypic transitions in the fibrosis phase. Understanding these molecular changes and potential mechanisms will help develop novel drugs to prevent or treat drug-induced lung injury.

Endothelial cell-specific anticoagulation reduces inflammation in a mouse model of acute lung injury.

Tissue factor (TF)-dependent coagulation contributes to lung inflammation and the pathogenesis of acute lung injury (ALI). In this study, we explored the roles of targeted endothelial anticoagulation in ALI using two strains of transgenic mice expressing either a membrane-tethered human tissue factor pathway inhibitor (hTFPI) or hirudin fusion protein on CD31+ cells, including vascular endothelial cells (ECs). ALI was induced by intratracheal injection of LPS, and after 24 h the expression of TF and protease-activated receptors (PARs) on EC in lungs were assessed, alongside the extent of inflammation and injury. The expression of TF and PARs on the EC in lungs was upregulated after ALI. In the two strains of transgenic mice, expression of either of hTFPI or hirudin by EC was associated with significant reduction of inflammation, as assessed by the extent of leukocyte infiltration or the levels of proinflammatory cytokines, and promoted survival after LPS-induced ALI. The beneficial outcomes were associated with inhibition of the expression of chemokine CCL2 in lung tissues. The protection observed in the CD31-TFPI-transgenic strain was abolished by injection of an anti-hTFPI antibody, but not by prior engraftment of the transgenic strains with WT bone marrow, confirming that the changes observed were a specific transgenic expression of anticoagulants by EC. These results demonstrate that the inflammation in ALI is TF and thrombin dependent, and that expression of anticoagulants by EC significantly inhibits the development of ALI via repression of leukocyte infiltration, most likely via inhibition of chemokine gradients. These data enhance our understanding of the pathology of ALI and suggest a novel therapeutic strategy for treatment.

Targeted delivery of YSA-functionalized and non-functionalized polymeric nanoparticles to injured pulmonary vasculature

Ephrin type-A receptor 2 (EphA2) is a transmembrane receptor which is upregulated in injured lungs, including those treated with bleomycin. YSA peptide (YSAYPDSVPMMS), a mimic of ephrin ligands, binds to EphA2 receptors on cell surface with high affinity. In this study, we assessed the ability of YSA-functionalized and non-functionalized poly (dl-lactide-co-glycolide) (PLGA) nanoparticles to enhance delivery to bleomycin treated cultured vascular endothelial cells and, in a bleomycin induced lung injury mouse model. Nanoparticles were loaded with a lipophilic fluorescent dye. Human umbilical vein endothelial cells (HUVEC) with or without 2-day bleomycin pretreatment (25 µg/ml) and adult mice with or without intratracheal instillation of bleomycin (0.1 U) were dosed with nanoparticles. Mice received nanoparticles via tail vein injection 4 days after bleomycin treatment. Three days after nanoparticle injection, tissues (lung, heart, kidney, spleen, liver, brain, eyes and whole blood) were harvested and quantified for fluorescence using IVIS imaging. Mean particle uptake increased with time and concentration for both types of particles in HUVEC, with the uptake being higher for YSA-functionalized nanoparticles. Bleomycin treatment increased the 3-h uptake of both types of nanoparticles in HUVEC by about two-fold, with the YSA-functionalized nanoparticle uptake being 1.66-fold compared to non-functionalized nanoparticles (p < .05). In mice, bleomycin injury resulted in 2.3- and 4.7-fold increase in the lung levels of non-functionalized and YSA-functionalized nanoparticles (p < .05), respectively, although the differences between the two particle types were not significant. In conclusion, PLGA nanoparticle delivery to cultured vascular endothelial cells and mouse lungs in vivo is higher following bleomycin treatment, with the delivery tending to be higher for YSA functionalized nanoparticles.

Inhaled TRIM72 Protein Protects Ventilation Injury to the Lung through Injury-guided Cell Repair.

Studies showed that TRIM72 is essential for repair of alveolar cell membrane disruptions, and exogenous recombinant human TRIM72 protein (rhT72) demonstrated tissue-mending properties in animal models of tissue injury. Here we examine the mechanisms of rhT72-mediated lung cell protection in vitro and test the efficacy of inhaled rhT72 in reducing tissue pathology in a mouse model of ventilator-induced lung injury. In vitro lung cell injury was induced by glass beads and stretching. Ventilator-induced lung injury was modeled by injurious ventilation at 30 ml/kg tidal volume. Affinity-purified rhT72 or control proteins were added into culture medium or applied through nebulization. Cellular uptake and in vivo distribution of rhT72 were detected by imaging and immunostaining. Exogenous rhT72 maintains membrane integrity of alveolar epithelial cells subjected to glass bead injury in a dose-dependent manner. Inhaled rhT72 decreases the number of fatally injured alveolar cells, and ameliorates tissue-damaging indicators and cell injury markers after injurious ventilation. Using in vitro stretching assays, we reveal that rhT72 improves both cellular resilience to membrane wounding and membrane repair after injury. Image analysis detected rhT72 uptake by rat alveolar epithelial cells, which can be inhibited by a cholesterol-disrupting agent. In addition, inhaled rhT72 distributes to the distal lungs, where it colocalizes with phosphatidylserine detection on nonpermeabilized lung slices to label wounded cells. In conclusion, our study showed that inhaled rhT72 accumulates in injured lungs and protects lung tissue from ventilator injury, the mechanisms of which include improving cell resilience to membrane wounding, localizing to injured membrane, and augmenting membrane repair.

Laurus nobilis leaf extract controls inflammation by suppressing NLRP3 inflammasome activation.

Laurus nobilis Linn. (Lauraceae), commonly known as Bay, has been used as a traditional medicine in the Mediterranean and Europe to treat diverse immunological disorders. Although the effects of L. nobilis on immunosuppression have been reported, the detailed underlying mechanism remains unclear. In this study, to elucidate the anti-inflammatory mechanism of L. nobilis, we examined the effect of L. nobilis leaf extract on inflammasome activation in mouse bone marrow-derived macrophages. L. nobilis leaf extract inhibited NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome activation, which was associated with caspase-1 activation, interleukin-1β secretion, and apoptosis-associated speck-like protein containing a CARD(ASC) pyroptosome complex formation. We also observed that 1,8-cineole, the major component of L. nobilis extract, consistently suppressed NLRP3 inflammasome activation. Furthermore, L. nobilis leaf extract attenuated the in vivo expression of proinflammatory cytokines in an acute lung injury mouse model. Our results provide the first evidence that L. nobilis leaf extract modulates inflammatory signaling by suppressing inflammasome activation.

Gene silencing of receptor-interacting protein 2 protects against cigarette smoke-induced acute lung injury

Background and purpose: Chronic obstructive pulmonary disease (COPD) is characterized by progressive alveolar damage and generally irreversible airflow limitation. Nuclear factor-κB (NF-κB) plays a critical role in COPD pathogenesis. Receptor-interacting protein 2 (Rip2), a 60 kDa adaptor protein, is a positive regulator of NF-κB pathway and also an inducible transcriptional product of NF-κB activation. We sought to investigate if Rip2 gene silencing could protect against cigarette smoke (CS)-induced acute lung injury.Experimental approach: Gene silencing efficacy of Rip2 siRNA was characterized in mouse macrophage and mouse lung epithelial cell lines, and in a CS-induced acute lung injury mouse model. Bronchoalveolar lavage (BAL) fluid cell counts, levels of pro-inflammatory and oxidative damage markers, lung section inflammatory and epithelium thickness scorings, and nuclear NF-κB translocation were measured.Key results: CS was found to upregulate Rip2 level in mouse lungs. Rip2 siRNA was able to suppress Rip2 levels in both macrophage and lung epithelial cell lines and in mouse lungs, block CS extract (CSE)-induced mediator release by the cultured cells, and abate neutrophil counts in BAL fluid from CS-challenged mice. Rip2 siRNA suppressed CS-induced inflammatory and oxidative damage markers, and nuclear p65 accumulation and transcriptional activation in lung tissues. Besides, Rip2 siRNA was able to disrupt CSE-induced NF-κB activation in a NF-κB reporter gene assay.Conclusions and implications: Taken together, we report for the first time that Rip2 gene silencing ameliorated CS-induced acute lung injury probably via disruption of the NF-κB activity, postulating that Rip2 may be a novel therapeutic target for COPD.