Endotoxin-induced Acute Lung Injury In Mice Research Articles

Cannabidiol Reduces Systemic Immune Activation in Experimental Acute Lung Injury.

Background: The underlying pathomechanism of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is the immune response to inflammation or infection within the pulmonary microcirculation. Systemic spread of pathogens, activated immune cells, and inflammatory mediators contributes significantly to mortality in patients with ARDS. Objective: The endogenous cannabinoid system is a major modulator of the immune response during inflammation and infection. Phytocannabinoids, such as cannabidiol (CBD), have shown promising anti-inflammatory effects in several pathologies. The overall objective of this study was to evaluate the effects of CBD on local and systemic inflammation in endotoxin-induced ALI in mice. Materials and Methods: ALI was induced by pulmonary endotoxin challenge. Four groups of male C57BL/6 mice were randomized in this study: control, ALI, ALI with CBD treatment, and control with CBD treatment. Analyses of local and systemic cytokine levels, lung histology, and leukocyte activation as visualized by intravital microscopy of the intestinal and pulmonary microcirculation were performed 6 h following intranasal endotoxin administration. Results: Pulmonary endotoxin challenge induced significant inflammation evidenced by local and systemic cytokine and chemokine release, lung histopathology, and leukocyte adhesion. Intraperitoneal CBD treatment resulted in a significant decrease in systemic inflammation as shown by reduced leukocyte adhesion in the intestinal microcirculation and reduced plasma cytokine and chemokine levels. Pulmonary chemokine levels were decreased, while pulmonary cytokine levels were unchanged. Surprisingly, the ALI score was slightly increased by CBD treatment in a manner driven by enhanced neutrophil infiltration of the alveoli. Conclusion: In this model of experimental ALI, CBD administration was associated with reduced systemic inflammation and heterogeneous effects on pulmonary inflammation. Future studies should explore the mechanisms involved as they relate to neutrophil infiltration and proinflammatory mediator production within the lungs.

6-Hydroxy-5,7-dimethoxy-flavone suppresses the neutrophil respiratory burst via selective PDE4 inhibition to ameliorate acute lung injury

Over-activated neutrophils produce enormous oxidative stress and play a key role in the development of acute and chronic inflammatory diseases. 6-Hydroxy-5,7-dimethoxy-flavone (UFM24), a flavone isolated from the Annonaceae Uvaria flexuosa, showed inhibitory effects on human neutrophil activation and salutary effects on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. UFM24 potently inhibited superoxide anion (O2•−) generation, reactive oxidants, and CD11b expression, but not elastase release, in N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLF)-activated human neutrophils. However, UFM24 failed to scavenge O2•− and inhibit the activity of subcellular NADPH oxidase. fMLF-induced phosphorylation of protein kinase B (Akt) was inhibited by UFM24. Noticeably, UFM24 increased cyclic adenosine monophosphate (cAMP) concentration and protein kinase (PK) A activity in activated human neutrophils. PKA inhibitors significantly reversed the inhibitory effects of UFM24, suggesting that the effects of UFM24 were through cAMP/PKA-dependent inhibition of Akt activation. Additionally, activity of cAMP-related phosphodiesterase (PDE)4, but not PDE3 or PDE7, was significantly reduced by UFM24. Furthermore, UFM24 attenuated neutrophil infiltration, myeloperoxidase activity, and pulmonary edema in LPS-induced ALI in mice. In conclusion, our data demonstrated that UFM24 inhibits oxidative burst in human neutrophils through inhibition of PDE4 activity. UFM24 also exhibited significant protection against endotoxin-induced ALI in mice. UFM24 has potential as an anti-inflammatory agent for treating neutrophilic lung damage.

Study of Bone Marrow and Embryonic Stem Cell-Derived Human Mesenchymal Stem Cells for Treatment of Escherichia coli Endotoxin-Induced Acute Lung Injury in Mice.

: Mesenchymal stem cells (MSCs) can be derived from multiple tissue sources. However, the optimal source of MSCs for cell-based therapy for acute lung injury (ALI) is unclear. In the present experiments, we studied bone marrow (BM)-derived and embryonic stem cell-derived human MSC (ES-MSCs) as a therapeutic agent in Escherichia coli endotoxin-induced ALI in mice. We hypothesized that ES-MSCs would be more potent than BM-MSCs owing to its more primitive source of origin. ALI was induced by the intratracheal instillation of endotoxin at 4 mg/kg into 10-12-week-old C57BL/6 mice with or without BM-MSCs, ES-MSCs, or normal human lung fibroblasts as a cellular control. Compared with the endotoxin-injured mice at 48 hours, the administration of ES-MSCs provided results similar to those of BM-MSCs, significantly reducing the influx of white blood cells and neutrophils and decreasing the secretion of the inflammatory cytokines, macrophage inflammatory protein-2 and tumor necrosis factor-α, in the injured alveolus. BM-MSCs also reduced extravascular lung water, a measure of pulmonary edema, by 60% and the total protein levels, a measure of lung permeability, by 66%. However, surprisingly, ES-MSCs did not have these protective effects, which was partially explained by the increased secretion of matrix metallopeptidase 9 by ES-MSCs, an enzyme known to increase lung protein permeability. In conclusion, both BM-MSCs and ES-MSCs markedly decreased endotoxin-induced inflammation. However, ES-MSCs did not show any beneficial effect on reducing pulmonary edema and lung protein permeability compared with BM-MSCs, suggesting that not all MSCs behave in a similar fashion. Our results highlight the need perhaps for a disease-specific potency assay for MSCs. To determine the optimal source of mesenchymal stem cells (MSCs) for cell-based therapy for acute lung injury, bone marrow (BM)- and embryonic stem cell-derived human MSC (ES-MSCs) were compared as therapeutic agents for Escherichia coli endotoxin-induced lung injury in mice. ES-MSCs behaved similarly to BM-MSCs by markedly decreasing the inflammatory response induced by endotoxin. However, unlike BM-MSCs, ES-MSCs provided no protective effects against increasing lung water and protein permeability, in part because of an increase in expression of matrix metallopeptidase 9 by ES-MSCs. In patients with acute respiratory distress syndrome, impaired alveolar fluid clearance (i.e., no resolution of pulmonary edema fluid) has been associated with higher mortality rates. Although ES-MSCs might ultimately be found to have properties superior to those of BM-MSCs, such as for immunomodulation, these results highlight the need for a disease-specific potency assay for stem cell-based therapy.

NOS-2 Inhibition in Phosgene-Induced Acute Lung Injury.

Phosgene exposure via an industrial or warfare release produces severe acute lung injury (ALI) with high mortality, characterized by massive pulmonary edema, disruption of epithelial tight junctions, surfactant dysfunction, and oxidative stress. There are no targeted treatments for phosgene-induced ALI. Previous studies demonstrated that nitric oxide synthase 2 (NOS-2) is upregulated in the lungs after phosgene exposure; however, the role of NOS-2 in the pathogenesis of phosgene-induced ALI remains unknown. We previously demonstrated that NOS-2 expression in lung epithelium exacerbates inhaled endotoxin-induced ALI in mice, mediated partially through downregulation of surfactant protein B (SP-B) expression. Therefore, we hypothesized that a selective NOS-2 inhibitor delivered to the lung epithelium by inhalation would mitigate phosgene-induced ALI. Inhaled phosgene produced increases in bronchoalveolar lavage fluid protein, histologic lung injury, and lung NOS-2 expression at 24 h. Administration of the selective NOS-2 inhibitor 1400 W via inhalation, but not via systemic delivery, significantly attenuated phosgene-induced ALI and preserved epithelial barrier integrity. Furthermore, aerosolized 1400 W augmented expression of SP-B and prevented downregulation of tight junction protein zonula occludens 1 (ZO-1), both critical for maintenance of normal lung physiology and barrier integrity. We also demonstrate for the first time that NOS-2-derived nitric oxide downregulates the ZO-1 expression at the transcriptional level in human lung epithelial cells, providing a novel target for ameliorating vascular leak in ALI. Our data demonstrate that lung NOS-2 plays a critical role in the development of phosgene-induced ALI and suggest that aerosolized NOS-2 inhibitors offer a novel therapeutic strategy for its treatment.

Impact of TREM-2 gene silencing on inflammatory response of endotoxin-induced acute lung injury in mice.

Acute lung injury (ALI) is one of the critical clinical respiratory diseases, of which infection is the main cause and the first risk factor. This study investigated the impact of triggering receptor of myeloid cells expression (TREM)-2 gene silencing on inflammatory response of endotoxin-induced ALI in mice. Lentivirus-mediated TREM-2-shRNA was transfected into healthy male C57BL/6 mice, and the lipopolysaccharide-induced ALI model was established. The immunohistochemistry, immunofluorescence, fluorescence quantitative PCR, western blot, and ELISA were applied to detect the pathological changes of lung tissue and expressions of TREM-2, tumor necrosis factor-α (TNF-α), and interleukin 10 (IL-10) in bronchoalveolar lavage fluid. The lentivirus group, saline control group, ALI model group, blank control group, and negative control group were set up at the same time. Results found that, in lentivirus group, the pathological change of lung tissue was significantly lighter than ALI model group (P < 0.05), and the expression of TREM-2 was significantly reduced compared with all control groups (P < 0.05). The levels of TNF-α and IL-10 were significantly increased than all control groups (P < 0.05), while above indexes in negative control group and blank control group showed no significant difference with ALI group (P > 0.05). This study indicates that TREM-2 has a protective effect on inflammatory response of endotoxin-induced ALI in mice, which has provided new potential targets for prevention and treatment of ALI.

Human Mesenchymal Stem Cell Microvesicles for Treatment of Escherichia coli Endotoxin-Induced Acute Lung Injury in Mice

We previously found that human mesenchymal stem cells (MSC) or its conditioned medium restored lung protein permeability and reduced alveolar inflammation following Escherichia coli endotoxin-induced acute lung injury (ALI) in an ex vivo perfused human lung in part through the secretion of soluble factors such as keratinocyte growth factor (KGF). Recently, MSC were found to release microvesicles (MVs) that were biologically active because of the presence of mRNA or miRNA with reparative properties. MVs are circular fragments of membrane released from the endosomal compartment as exosomes or shed from the surface membranes. These studies were designed to determine if MVs released by human bone marrow derived MSCs would be effective in restoring lung protein permeability and reducing inflammation in E. coli endotoxin-induced ALI in C57BL/6 mice. The intratracheal instillation of MVs improved several indices of ALI at 48 hours. Compared to endotoxin-injured mice, MVs reduced extravascular lung water by 43% and reduced total protein levels in the bronchoalveolar lavage (BAL) fluid by 35%, demonstrating a reduction in pulmonary edema and lung protein permeability. MVs also reduced the influx of neutrophils and macrophage inflammatory protein-2 levels in the BAL fluid by 73% and 49%, respectively, demonstrating a reduction in inflammation. KGF siRNA-pretreatment of MSC partially eliminated the therapeutic effects of MVs released by MSCs, suggesting that KGF protein expression was important for the underlying mechanism. In summary, human MSC-derived MVs were therapeutically effective following E. coli endotoxin-induced ALI in mice in part through the expression of KGF mRNA in the injured alveolus.

The Nitrated Fatty Acid 10-Nitro-oleate Diminishes Severity of LPS-Induced Acute Lung Injury in Mice

Acute lung injury (ALI) is an inflammatory condition culminating in respiratory failure. There is currently no effective pharmacological treatment. Nitrated fatty acids (NFAs) have been shown to exert anti-inflammatory effects. We therefore hypothesized that delivery of NFAs directly to the site of inflammation would reduce the severity of ALI. Pulmonary delivery of 10-nitro-oleate following endotoxin-induced ALI in mice reduced markers of lung inflammation and injury, including capillary leakage, lung edema, infiltration of neutrophils into the lung, and oxidant stress, as well as plasma levels of proinflammatory cytokines. Nitro-oleate delivery likewise downregulated expression of proinflammatory genes by alveolar macrophages, key cells in regulation of lung inflammation. These effects may be accounted for by the observed increases in the activity of PPAR-γ and the PPAR-γ-induced antioxidant transcription factor Nrf2, together with the decreased activity of NF-κB. Our results demonstrate that pulmonary delivery of NFAs reduces severity of acute lung injury and suggest potential utility of these molecules in other inflammatory lung diseases.

Priming With Endotoxin Increases Acute Lung Injury in Mice by Enhancing the Severity of Lung Endothelial Injury

Endotoxin-induced acute lung injury (ALI) is a commonly used model. However, the effect of a priming dose of endotoxin on lung fluid balance has not been well studied. We hypothesized that endotoxin-induced ALI in mice would be enhanced under a priming condition. Mice were intratracheally (IT) instilled with either a priming dose of endotoxin from E. coli (0.5 mg/kg) or equal volume of PBS. Eighteen hours later, a larger challenge dose of endotoxin (5 mg/kg) was given IT. Control mice received PBS only. After 24 hr, the mice were sacrificed and the degree of lung injury and inflammation were measured. Endotoxin priming increased body weight loss and worsened hypothermia. Extravascular lung water and lung endothelial permeability were higher in the primed group. Priming with endotoxin reduced alveolar fluid clearance; however, there was no effect on bronchoalveolar lavage (BAL) levels of receptor for advanced glycation end products (RAGE). The primed group had increased alveolar inflammation as demonstrated by increased numbers of neutrophils in the BAL. There was no significant difference in NF-κB p65 in the lung nuclear extract among the experimental groups. Taken together, priming with a small dose of endotoxin followed by a larger challenge dose of endotoxin induces more systemic illness and increased pulmonary edema in mice, largely due to increased lung endothelial permeability and lung inflammation. This model should be useful to investigators studying ALI who want to simulate the clinical setting in which more than one insult often leads to greater clinical lung injury.

Allogeneic human mesenchymal stem cells for treatment of E. Coli endotoxin‐induced acute lung injury in an ex vivo perfused human lung

Recent studies have indicated that bone marrow derived mesenchymal stem cells (MSC) may have therapeutic applications in multiple clinical disorders. Currently, little is known regarding the effect of MSC in experimental models of acute lung injury (ALI) and pulmonary edema. We recently reported that intrapulmonary (via the trachea) treatment with MSC 4 h after endotoxin delivery to the lung improved survival and reduced the extent of pulmonary edema formation in endotoxin‐induced ALI in mice (J Immunol 2007). Here, we tested the therapeutic potential of human MSC to resolve pulmonary edema from ALI in an ex vivo perfused human lung preparation injured by E. Coli endotoxin. Intra‐bronchial instillation of endotoxin resulted in the loss of alveolar epithelial fluid transport measured as alveolar fluid clearance (AFC). Instillation of allogeneic human MSC or its conditioned medium given 1 h following endotoxin‐induced lung injury completely restored AFC. Using siRNA knockdown of potential paracrine soluble factors, we found that the secretion of keratinocyte growth factor (KGF) was essential for the restorative effect of MSC. In summary, the therapeutic effect of allogeneic human MSC on the resolution of endotoxin‐induced pulmonary edema in an ex vivo perfused human lung was mediated by the secretion of KGF. FAER &amp; NHLBI HL093026, HL51854 and HL51856.

Treatment of Acute Lung Injury: Clinical and Experimental Studies

This presentation at the 2007 Aspen Lung Injury and Repair Conference provided a brief historical perspective from the 1998 Aspen Conference on Acute Lung Injury, highlighting the discussion of clinical definitions. There was also a review of the National Heart, Lung, and Blood Institute ARDS Network clinical trials, with an emphasis on the success of the lung-protective ventilation strategy in reducing mortality. In addition, there was a discussion of the recently completed fluid and catheter treatment trial, which demonstrated no benefit for pulmonary arterial catheterization over central venous catheterization for monitoring patients with acute lung injury (ALI). The trial demonstrated an increase in ventilator-free days with a fluid-conservative protocol. Finally, there was a discussion of recent experimental studies that show promise for cell-based therapy with mesenchymal stem cells for the treatment of endotoxin-induced ALI in mice. There were three objectives for this presentation at the 2007 Lung Injury, Repair, and Remodeling Conference: (1) to provide a brief historical perspective from the 1998 Aspen Conference on Acute Lung Injury; (2) to review major National Heart, Lung, and Blood Institute clinical trials conducted in the past 10 years by the ARDS Network; and (3) to review some of our promising work with cell-based therapy for experimental ALI.