Lung Leak Research Articles

Assessment of Spinster homologue 2 (Spns2)-dependent transport of sphingosine-1-phosphate as a therapeutic target.

Sphingosine-1-phosphate (S1P) receptor modulator (SRM) drugs suppress immune system function by disrupting lymphocyte trafficking, but SRMs are broadly immunosuppressive with on-target liabilities. Another strategy to modulate the immune system is to block S1P transport. This study tests the hypothesis that blockers of S1P transport (STBs) mediated by Spinster homologue 2 (Spns2) approximate the efficacy of SRMs without their adverse events. We have discovered and optimized STBs to enable investigations of S1P biology and to determine whether S1P transport is a valid drug target. The STB SLF80821178 was administered to rodents to assess its efficacy in a multiple sclerosis model and to test for toxicities associated with SRMs or Spns2-deficient mice. Further, potential biomarkers of STBs, absolute lymphocyte counts (ALCs) in blood and S1P concentrations in plasma and lymph, were measured. SLF80821178 resembles SRMs in that it is efficacious in a standard multiple sclerosis model but does not evoke bradycardia or lung leakage, common to the SRM drug class. Also, chronic SLF80821178 administration does not affect auditory responses in adult mice despite the neurosensorial hearing defect observed in Spns2-null mice. While both SRM and STB administration decrease ALCs, the maximal effect is less with an STB (45% vs. 90%). STBs have minimal effects on S1P concentration in plasma or thoracic duct lymph. We found nothing to invalidate Spns2-dependent S1P transport as a drug target. Indeed, STBs could be superior to SRMs as a therapy to modulate immune system function.

Effect of Refined Nursing on Wound Complications After Thoracoscopic Surgery for Lung Cancer.

Lung cancer thoracoscopic postoperative wound complications bring great pain and inconvenience to patients. To provide clinical nurses with a more scientific and effective nursing plan, this study evaluated the effect of refined nursing on wound complications after thoracoscopic surgery for lung cancer. Two-hundred thirty patients undergoing thoracoscopic radical resection for lung cancer were randomly divided into two groups according to the random number table method. The study group received refined nursing intervention (115 cases), and the control group received routine nursing intervention (115 cases). The effects of the two groups' nursing modes on the wound complications (pleural effusion, incision infection, lung leakage, and wound bleeding) after thoracoscopic surgery for lung cancer were compared. According to the study results, the study group experienced a shorter intraoperative blood loss, lower extubation time, shorter hospital stay, and lower wound complication rate than the control group (p < 0.05). Compared to the control group, the study group had significantly lower Hamilton Anxiety Scale (HAMA), Hamilton Depression Scale (HAMD) and visual analog scale (VAS) scores, and higher the short form 36 health survey questionnaire (SF-36) scores (p < 0.05). Compared with the control group, the implementation of refined nursing intervention for patients with thoracoscopic radical resection of lung cancer has fewer postoperative wound complications and can improve patients' nursing satisfaction and quality of life, which is worthy of clinical promotion and application.

Outer membrane vesicles from X-ray-irradiated Pseudomonas aeruginosa alleviate lung injury caused by P. aeruginosa infection-mediated sepsis.

Pseudomonas aeruginosa infection causes pneumonia and sepsis. Previous research found that X-ray radiation can induce P. aeruginosa to release outer membrane vesicles (OMVs) of relatively consistent sizes. This study found that OMVs derived from X-ray-irradiated P. aeruginosa can significantly inhibit lung leakage, inflammatory cell infiltrating into lung, and the production of pro-inflammatory cytokines, IL-1β and TNFα caused by P. aeruginosa infection under preventive and therapeutic administration conditions. Under the same conditions, OMVs also significantly alleviated pathological characteristics of lung injury, including pulmonary edema, pulmonary hemorrhage, and alveolar wall thickening. OMVs also significantly reduced bacterial burdens in peritoneal cavity, accompanied by a reduction in the number of viable bacteria capable of forming bacterial colonies. Pretreating macrophages and neutrophils with OMVs enhances their bactericidal ability. When bacteria were cocultured with treated cells, the number of viable bacteria capable of forming bacterial colonies was significantly reduced. OMVs themselves have not been shown to cause any lung injury or affect bacterial viability. Therefore, OMVs derived from X-ray-irradiated P. aeruginosa may not only be applied in prevention and treatment of diseases associated with P. aeruginosa infection, but also served as an excellent vaccine development platform.

Bradykinin release following trauma and hemorrhagic shock causes pulmonary alveolar leak in a rodent model.

Hemorrhage accounts for 40% of the preventable death following severe injury. Activation of systemic coagulation produces bradykinin (BK), which may cause leak from the plasma to the extravascular space and to the tissues, which is part of the complex pathophysiology of trauma-induced end-organ injury. We hypothesize that BK, released during activation of coagulation in severe injury, induces pulmonary alveolar leak. Isolated neutrophils (PMNs) were pretreated with a specific BK receptor B2 antagonist HOE-140/icatibant and BK priming of the PMN oxidase was completed. Rats underwent tissue injury/hemorrhagic shock (TI/HS), TI/icatibant/HS, and controls (no injury). Evans blue dye was instilled, and the percentage leak from the plasma to the lung was calculated from the bronchoalveolar lavage fluid (BALF). CINC-1 and total protein were measured in the BALF, and myeloperoxidase was quantified in lung tissue. The BK receptor B2 antagonist HOE140/icatibant inhibited (85.0 ± 5.3%) BK priming of the PMN oxidase ( p < 0.05). The TI/HS model caused activation of coagulation by increasing plasma thrombin-antithrombin complexes ( p < 0.05). Versus controls, the TI/HS rats had significant pulmonary alveolar leak: 1.46 ± 0.21% versus 0.36 ± 0.10% ( p = 0.001) and increased total protein and CINC-1 in the BALF ( p < 0.05). Icatibant given after the TI significantly inhibited lung leak and the increase in CINC-1 in the BALF from TI/icatibant/HS rats versus TI/HS ( p < 0.002 and p < 0.05) but not the total protein. There was no PMN sequestration in the lungs. Conclusions: This mixed injury model caused systemic activation of hemostasis and pulmonary alveolar leak likely due to BK release. This mixed injury model caused systemic activation of hemostasis and pulmonary alveolar leak likely due to BK release. Original Article, Basic Science.

Heterotypic Influenza Infections Mitigate Susceptibility to Secondary Bacterial Infection.

Influenza-associated bacterial superinfections have devastating impacts on the lung and can result in increased risk of mortality. New strains of influenza circulate throughout the population yearly, promoting the establishment of immune memory. Nearly all individuals have some degree of influenza memory before adulthood. Due to this, we sought to understand the role of immune memory during bacterial superinfections. An influenza heterotypic immunity model was established using influenza A/Puerto Rico/8/34 and influenza A/X31. We report in this article that influenza-experienced mice are more resistant to secondary bacterial infection with methicillin-resistant Staphylococcus aureus as determined by wasting, bacterial burden, pulmonary inflammation, and lung leak, despite significant ongoing lung remodeling. Multidimensional flow cytometry and lung transcriptomics revealed significant alterations in the lung environment in influenza-experienced mice compared with naive animals. These include changes in the lung monocyte and T cell compartments, characterized by increased expansion of influenza tetramer-specific CD8+ T cells. The protection that was seen in the memory-experienced mouse model is associated with the reduction in inflammatory mechanisms, making the lung less susceptible to damage and subsequent bacterial colonization. These findings provide insight into how influenza heterotypic immunity reshapes the lung environment and the immune response to a rechallenge event, which is highly relevant to the context of human infection.

IL‐6 Signaling is Required for LPS‐induced Barrier Function Loss

Interleukin‐6 (IL‐6) is a major mediator of the cytokine storm in response to severe systemic inflammation and sepsis. This cytokine promotes the activation of the transcription factor STAT3 via Janus kinases (JAK)‐dependent phosphorylation on tyrosine 705. Prior work in our lab demonstrated that overactivation of this pathway within the endothelium via an endothelial‐specific deletion of the negative regulator SOCS3 (SOCS3iEKO) leads to kidney failure and acute mortality after a single lipopolysaccharides (LPS) challenge that was non‐lethal in wild‐type mice. This mortality was associated with increased systemic IL‐6 levels and vascular leak in lungs and brain of SOCS3iEKO mice. Furthermore, translating ribosome affinity purification studies showed that in response to LPS, the kidney and brain endothelia expressed high levels of IL‐6. These findings suggested that IL‐6 signaling within the endothelium might be required to promote vascular leak. To sought to test this hypothesis in vitro. We found that LPS promoted a mild increase in monolayer permeability in HUVEC that was abrogated by pretreatment with the JAK inhibitor ruxolitinib. This loss of barrier function by LPS was associated with an increase in IL‐6 expression and a modest increase in STAT3 phosphorylation. Endothelial cells express very low levels of the gp80 subunit of the IL‐6 receptor. Thus, to test whether an IL‐6 autocrine loop induced barrier function loss, we treated cells with LPS in the presence or absence of a soluble form of gp80 (herein, LPS+R). LPS+R‐treated cells showed increased STAT3 phosphorylation and severe barrier function loss. A similar increase in STAT3 phosphorylation and monolayer permeability was observed when gp80 was added to TNF‐treated cells. In contrast, addition of gp80 to LPS‐ or TNF‐treated cells did not alter the levels of p65RelA or p38 phosphorylation. Pretreatment of LPS+R‐treated cells with the p38 inhibitor SB203580 showed that activation of p38 signaling was not required for STAT3 phosphorylation or barrier function loss. In summary, our data strongly suggest that the endothelium responds to strong inflammatory challenges with an increase in IL‐6 expression that may act in an autocrine fashion to promote strong barrier function loss, potentially explaining the systemic vascular leak observed in critically ill patients with high circulating IL‐6 levels.

Local nebulization of 1\u03b1,25(OH)2D3 attenuates LPS-induced acute lung inflammation

BackgroundEvidence supports a critical role of vitamin D status on exacerbation in chronic obstructive pulmonary disease, indicating the need to avoid vitamin D deficiency in these patients. However, oral vitamin D supplementation is limited by the potential risk for hypercalcemia. In this study, we investigated if local delivery of vitamin D to the lungs improves vitamin D-mediated anti-inflammatory action in response to acute inflammation without inducing hypercalcemia.MethodsWe studied vitamin D sufficient (VDS) or deficient (VDD) mice in whom 1α,25(OH)2D3 (0.2 μg/kg) or a vehicle followed by lipopolysaccharide (LPS 25 µg) were delivered to the lung as a micro-spray.ResultsLocal 1α,25(OH)2D3 reduced LPS-induced inflammatory cells in bronchoalveolar lavage (BAL) in VDS (absolute number of cells: − 57% and neutrophils − 51% p < 0.01) and tended to diminish LPS-increased CXCL5 BAL levels in VDS (− 40%, p = 0.05) while it had no effect on CXCL1 and CXCL2 in BAL and mRNA in lung of VDS and VDD. It also significantly attenuated the increased IL-13 in BAL and lung, especially in VDD mice (− 41 and − 75%, respectively). mRNA expression of Claudin-18 in lung was significantly lower in VDS mice with local 1α,25(OH)2D3 while Claudin-3, -5 and -8 mRNA levels remained unchanged. Finally, in VDD mice only, LPS reduced lung mRNA expression of adhesion junction Zona-occludens-1, in addition to increasing uric acid and total protein in BAL, which both were prevented by local 1α,25(OH)2D3.ConclusionUnder normal levels of vitamin D, local 1α,25(OH)2D3 nebulization into the lung efficiently reduced LPS induction of inflammatory cells in BAL and slightly attenuated LPS-increase in CXCL5. In case of severe vitamin D deficiency, although local 1α,25(OH)2D3 nebulization failed to significantly minimize cellular inflammation in BAL at this dose, it prevented epithelial barrier leakage and damage in lung. Additional research is needed to determine the potential long-term beneficial effects of local 1α,25(OH)2D3 nebulization on lung inflammation.

Gene transfer of MRCK\u03b1 rescues lipopolysaccharide-induced acute lung injury by restoring alveolar capillary barrier function

Acute Lung Injury/Acute Respiratory Distress Syndrome (ALI/ARDS) is characterized by alveolar edema accumulation with reduced alveolar fluid clearance (AFC), alveolar-capillary barrier disruption, and substantial inflammation, all leading to acute respiratory failure. Enhancing AFC has long been considered one of the primary therapeutic goals in gene therapy treatments for ARDS. We previously showed that electroporation-mediated gene delivery of the Na+, K+-ATPase β1 subunit not only increased AFC, but also restored alveolar barrier function through upregulation of tight junction proteins, leading to treatment of LPS-induced ALI in mice. We identified MRCKα as an interaction partner of β1 which mediates this upregulation in cultured alveolar epithelial cells. In this study, we investigate whether electroporation-mediated gene transfer of MRCKα to the lungs can attenuate LPS-induced acute lung injury in vivo. Compared to mice that received a non-expressing plasmid, those receiving the MRCKα plasmid showed attenuated LPS-increased pulmonary edema and lung leakage, restored tight junction protein expression, and improved overall outcomes. Interestingly, gene transfer of MRCKα did not alter AFC rates. Studies using both cultured microvascular endothelial cells and mice suggest that β1 and MRCKα upregulate junctional complexes in both alveolar epithelial and capillary endothelial cells, and that one or both barriers may be positively affected by our approach. Our data support a model of treatment for ALI/ARDS in which improvement of alveolar-capillary barrier function alone may be of more benefit than improvement of alveolar fluid clearance.

Gene Delivery of MRCKα Repairs the Alveolar‐Capillary Barrier and Rescues the Experimental Lipopolysaccharide‐Induced Acute Lung Injury

Rationale Acute Respiratory Distress Syndrome (ARDS) is a devastating condition which is characterized by the pulmonary accumulation of protein-rich edema fluids and insufficient gas exchange, leading to acute hypoxemic respiratory failure. During ARDS, the net fluid clearance is undermined not only due to the impaired alveolar fluid clearance (AFC) which osmotically drives water following the ion gradient out of alveoli, but also the disrupted alveolar-capillary barrier. The previous data from our lab indicated that electroporation-mediated gene delivery of the β1 subunit of the Na+, K+-ATPase into mouse lungs treats LPS induced acute lung injury (ALI), showing significantly decreased lung edema, and improved overall outcome of lung injury. Overexpression of β1 increased AFC in healthy and injured murine lungs, and more importantly, gene delivery of β1 restored pulmonary barrier function, demonstrated by upregulated tight junction (TJ) proteins, and decreased lung permeability, total protein and cellularity in BAL fluid. MRCKα (CDC42 binding protein kinase alpha) was identified by our lab as an interacting partner of the β1 subunit through mass spectrometry. MRCKα is a Rho GTPase effector kinase, regulating diverse cell behaviors, including junction formation. Our previous in vitro data indicate that MRCKα mediated β1’s upregulation of TJ proteins and epithelial barrier integrity. Overexpression of MRCKα alone sufficiently increased the trans-epithelial electrical resistance (TEER) in cell culture. Thus, it is intriguing to investigate whether MRCKα has the therapeutic potential to treat LPS induced ALI by restoring pulmonary barrier function in vivo. This study tested whether electroporation-mediated gene transfer of MRCKα could upregulate TJ protein and barrier function and treat LPS induced ALI in mice. Methods ALI was induced by LPS intratracheal administration and 24 hours later, plasmids encoding MRCKα, β1- Na+, K+-ATPase, or nothing (control) were delivered individually or in combination to mouse lungs by oropharyngeal aspiration and electroporation. Two days after gene delivery, various endpoint assays were performed to evaluate lung edema, permeability, inflammation and histological injury. In addition, AFC was performed on mice with MRCKα overexpression to evaluate the fluid clearance. Results Gene transfer of MRCKα alone did not enhance AFC, but alone or in combination with β1- Na+, K+-ATPase attenuated LPS-increased Wet/Dry ratio, lung leakage, and BAL cellularity and protein concentration, restored TJ protein expression, and improved overall outcome of lung injury. Conclusions Gene delivery ofMRCKα repair alveolar capillary barrier integrity without promoting ion transport, and could benefit the pre-injured lungs by reducing pulmonary edema, restoring lung barrier function and reducing inflammation. Moreover, the data also suggest that improving barrier function alone may be of equal or even more benefit than improving alveolar fluid clearance in order to treat ALI/ARDS.

Pneumothorax

A pneumothorax is defined by the presence of free air between the pleura visceralis and the pleura partietalis. The lung separates from the chest wall, which then, depending on several parameters, leads to a slight or clinically threatening impairment of lung function. Non-specific signs such as thoracic pain or coughing are common and do not correlate with the extent of the pneumothorax. Almost without exception, the cause of this accumulation of air is a leakage in the lung's surface, which then results in air escaping into the pleural space. Depending on the cause of the "lung leakage", a distinction is made between a primary (idiopathic) spontaneous pneumothorax (PSP) that can be triggered without direct cause, and a secondary spontaneous pneumothorax (SSP) in case of an underlying known lung disease. Further between an iatrogenic pneumothorax in connection with a lung injury caused by medical measures, and a traumatic pneumothorax in the case of an accident-related lung tear. The relevant therapeutic goals are the elimination of the acute symptoms, the reliable achievement of re-expansion of the lungs, and, after appropriate information gathering about the probability and clinical significance of a pneumothorax recurrence and depending on the patient's wish, avoiding a recurrence by means of surgical measures. The therapy options range from a "wait-and-see" procedure, that merely monitors the findings, to a primary video-assisted thoracoscopic surgical therapy with detection and resection of the superficial lung lesion, as well as a measurement to obliterate the pleural cavity that prevents relapse. Regarding "follow-up care" or even behavioral recommendations after a pneumothorax, there are no recommendations that reduce the risk of recurrence.

Abstract 17211: Targeting Mitogen-Activated Kinase Alleviates Free Heme-Induced Endothelial Barrier Dysfunction and Vascular Leakage in Lungs

Introduction: Several studies confirm that free heme in circulation due to hemolysis causes endothelial barrier dysfunction. We recently demonstrated that hemolysis-induced vascular leakage with barrier dysfunction was a contributory factor to the development of pulmonary hypertension (PH). However, the precise molecular mechanisms involved in the pathology of heme induced barrier disruption still remains to be elucidated. Hypothesis: Previous studies by us showed that free heme activated the p38/MAPK pathway. Therefore, we hypothesized that targeting mitogen-activated protein kinase kinase 3 (MKK3) a key regulator of this pathway would alleviate heme induced vascular leakage. Methods: Barrier dysfunction in human micro-vascular endothelial cells (HLMVEC) was monitored using noninvasive electrical impedance and immunostaining. We used an MKK3 knockout mouse model to assess the efficacy of targeting the p38/MAPK pathway. Results: We found a rapid drop in the HLMVEC barrier integrity with heme, in a dose dependent manner (p&lt;0.05). Investigating the barrier proteins showed that heme significantly affected the tight junction proteins, zona occludens-1, claudin1, and claudin5 (p&lt;0.05). We also found the p38MAPK/HSP27 pathway, involved in regulating the endothelial cytoskeleton remodeling, to be significantly altered with heme treatment, both in the HLMVEC and mice (p&lt;0.05). However, heme treated mice showed no significant change in E-selectin, ICAM1 and VCAM1, indicating that the primary rapid target of heme was the p38/MAPK pathway and not the inflammatory pathways. Finally, injecting mice with heme-FITC-dextran and then following its release into the lungs demonstrated that the MKK3 KO significantly prevented heme induced vascular leakage (p&lt;0.05). Conclusion: We demonstrate that heme induces a rapid barrier dysfunction by disruption of endothelial barrier proteins via the p38/MAPK pathway. Also, knocking out MKK3, a crucial regulator of the p38/MAPK pathway significantly decreased heme induced vascular leakage, a contributory factor to PH. Taken together, our results show that targeting the MKK3/p38MAPK axis represents a decisive treatment strategy in alleviating heme induced barrier dysfunction in cardiovascular diseases.

Fibrinogen Activates PAK1/Cofilin Signaling Pathway to Protect Endothelial Barrier Integrity.

We recently demonstrated that fibrinogen stabilizes syndecan-1 on the endothelial cell (EC) surface and contributes to EC barrier protection, though the intracellular signaling pathway remains unclear. P21 (Rac1) activated kinase 1 (PAK1) is a protein kinase involved in intracellular signaling leading to actin cytoskeleton rearrangement and plays an important role in maintaining endothelial barrier integrity. We therefore hypothesized that fibrinogen binding to syndecan-1 activated the PAK1 pathway. Primary human lung microvascular endothelial cells were incubated in 10% lactated Ringers (LR) solution or 10% fibrinogen saline solution (5 mg/mL). Protein phosphorylation was determined by Western blot analysis and endothelial permeability measured by fluorescein isothiocyanate (FITC)-dextran. Cells were silenced by siRNA transfection. Protein concentration was measured in the lung lavages of mice. Fibrinogen treatment resulted in increased syndecan-1, PAK1 activation (phosphorylation), cofilin activation (dephosphorylation), as well as decreased stress fibers and permeability when compared with LR treatment. Cofilin is an actin-binding protein that depolymerizes F-actin to decrease stress fiber formation. Notably, fibrinogen did not influence myosin light chain activation (phosphorylation), a mediator of EC tension. Silencing of PAK1 prevented fibrinogen-induced dephosphorylation of cofilin and barrier integrity. Moreover, to confirm the in vitro findings, mice underwent hemorrhagic shock and were resuscitated with either LR or fibrinogen. Hemorrhage shock decreased lung p-PAK1 levels and caused significant lung vascular leakage. However, fibrinogen administration increased p-PAK1 expression to near sham levels and remarkably prevented the lung leakage. We have identified a novel pathway by which fibrinogen activates PAK1 signaling to stimulate/dephosphorylate cofilin, leading to disassembly of stress fibers and reduction of endothelial permeability.

Cytokine pre-activation of cryopreserved xenogeneic-free human mesenchymal stromal cells enhances resolution and repair following ventilator-induced lung injury potentially via a KGF-dependent mechanism

BackgroundHuman mesenchymal stem/stromal cells (hMSCs) represent a promising therapeutic strategy for ventilator-induced lung injury (VILI) and acute respiratory distress syndrome. Translational challenges include restoring hMSC efficacy following cryopreservation, developing effective xenogeneic-free (XF) hMSCs and establishing true therapeutic potential at a clinically relevant time point of administration. We wished to determine whether cytokine pre-activation of cryopreserved, bone marrow-derived XF-hMSCs would enhance their capacity to facilitate injury resolution following VILI and elucidate mechanisms of action.MethodsInitially, in vitro studies examined the potential for the secretome from cytokine pre-activated XF-hMSCs to attenuate pulmonary epithelial injury induced by cyclic mechanical stretch. Later, anaesthetised rats underwent VILI and, 6 h following injury, were randomized to receive 1 × 107 XF-hMSC/kg that were (i) naive fresh, (ii) naive cryopreserved, (iii) cytokine pre-activated fresh or (iv) cytokine pre-activated cryopreserved, while control animals received (v) vehicle. The extent of injury resolution was measured at 24 h after injury. Finally, the role of keratinocyte growth factor (KGF) in mediating the effect of pre-activated XF-hMSCs was determined in a pulmonary epithelial wound repair model.ResultsPre-activation enhanced the capacity of the XF-hMSC secretome to decrease stretch-induced pulmonary epithelial inflammation and injury. Both pre-activated fresh and cryopreserved XF-hMSCs enhanced resolution of injury following VILI, restoring oxygenation, improving lung compliance, reducing lung leak and improving resolution of lung structural injury. Finally, the secretome of pre-activated XF-hMSCs enhanced epithelial wound repair, in part via a KGF-dependent mechanism.ConclusionsCytokine pre-activation enhanced the capacity of cryopreserved, XF-hMSCs to promote injury resolution following VILI, potentially via a KGF-dependent mechanism.

Prophylactic efficacy of Quercetin in ameliorating the hypoxia induced vascular leakage in lungs of rats.

The objective of the study was to find out the prophylactic efficacy of Quercetin in ameliorating the hypoxia induced vascular leakage in lungs of rats. Male SD rats received different doses of quercetin @ 25mg, 50mg, 100mg and 200mg/Kg BW, 1h prior to hypobaric hypoxia exposure (7,620m, for 6h). Quercetin 50 mg/kg BW supplemented orally 1h prior to hypoxia exposure was considered to be the optimum dose, due to significant reduction (p<0.001) in lung water content and lung transvascular leakage compared to control (hypoxia, 6h). Further, biochemical analysis (ROS, MDA, GSH, GPx, LDH, and albumin) and differential expressions of proteins (IKK-α/β, NFĸB, Nrf-2,TNF-α, ICAM-1, VCAM, P-selectin, Hif-1α, VEGF, TNF-α, TGF-β, INF-γ and IL-4) were determined by western blotting and ELISA. Changes in lung parenchyma were assessed by histopathology. Quercetin (50 mg/kg BW) prophylaxis under hypoxia showed significant reduction in oxidative stress (ROS and MDA), concomitant increase in antioxidants (GSH, GPx and SOD) followed by decreased LDH and albumin extravasation in BAL fluid over hypoxia. Quercetin prophylaxis significantly down regulated hypoxia induced increase in IKKα/β and NFĸB expressions leading to reduction in the levels of pro-inflammatory cytokines (TNF-α and INF-γ) followed by up regulation of anti-inflammatory cytokines (IL-4 and INF-γ) in lungs. Further, hypoxia mediated increase in HIF-1α was stabilized and VEGF levels in lungs were significantly down regulated by quercetin supplementation, leading to reduction in vascular leakage in lungs of rats under hypoxia. However, Quercetin has also enacted as Nrf-2 activator which significantly boosted up the synthesis of GSH under hypoxic condition compared to hypoxia. Histopathological observations further confirmed that quercetin preconditioning has an inhibitory effect on progression of oxidative stress and inflammation via attenuation of NFκB and stabilization HIF-1α in lungs of rats under hypoxia.These studies indicated that quercetin prophylaxis abrogates the possibility of hypobaric hypoxia induced pulmonary edema in rats.

Enhancing immunity prevents virus-induced T-cell-mediated immunopathology in B cell-deficient mice.

Hyper‐activated or deviated immune responses can result in immunopathological diseases. Paradoxically, immunodeficiency represents a frequent cause of such immune‐mediated pathologies. Immunopathological manifestations are commonly treated by immunosuppression, but in situations in which immunodeficiency is the basis of disease development, enhancing immunity may represent an alternative treatment option. Here, we tested this counterintuitive concept in a preclinical model using infection of mice with lymphocytic choriomeningitis virus (LCMV). Firstly, we demonstrate that infection of B‐cell‐deficient (B−/−) but not of wild‐type (WT) mice with the LCMV strain Docile induced a rapid and fatal CD8+ T‐cell‐mediated immunopathological disease. Similar to WT mice, LCMV‐infected B−/− mice generated a potent, functional LCMV‐specific CD8+ T‐cell response but exhibited prolonged viral antigen presentation and increased vascular leakage in liver and lungs. Secondly, we were able to prevent this virus‐induced immunopathology in B−/− mice by active or passive T‐cell immunizations or by treatment with LCMV‐specific virus neutralizing or non‐neutralizing monoclonal antibodies (mAb). Thus, boosting antiviral immunity did not aggravate immunopathology in this model, but prevented it by decreasing the formation of target structures for damage‐causing CD8+ T cells.

Novel Role for PD-1:PD-L1 as Mediator of Pulmonary Vascular Endothelial Cell Functions in Pathogenesis of Indirect ARDS in Mice.

Deficiency of the co-inhibitory receptor, Programmed cell death receptor (PD)-1, provides a survival benefit in our murine shock/sepsis model for the development of indirect acute respiratory distress syndrome (iARDS). Further, of clinical significance, patients that develop ARDS express increased PD-1 on their blood leukocytes. While PD-1 expression and its regulatory role have been associated with mainly T-cell responses, the contribution of its primary ligand, PD-L1, broadly expressed on non-immune cells such as lung endothelial cells (ECs) as well as immune cells, is less well-understood. Here we show that a “priming insult” for iARDS, such as non-lethal hemorrhagic shock alone, produced a marked increase in lung EC PD-L1 as well as blood leukocyte PD-1 expression, and when combined with a subsequent “trigger event” (polymicrobial sepsis), not only induced marked iARDS but significant mortality. These sequelae were both attenuated in the absence of PD-L1. Interestingly, we found that gene deficiency of both PD-1 and PD-L1 improved EC barrier function, as measured by decreased bronchoalveolar lavage fluid protein (i.e., lung leak). However, PD-L1 deficiency, unlike PD-1, significantly decreased EC activation through the Angiopoietin/Tie2 pathway in our iARDS mice. Additionally, while PD-1 gene deficiency was associated with decreased neutrophil influx in our iARDS mice, EC monolayers derived from PD-L1 deficient mice showed increased expression of EC junction proteins in response to ex vivo TNF-α stimulation. Together, these data suggest that ligation of PD-1:PD-L1 may play a novel role(s) in the maintenance of pulmonary EC barrier regulation, beyond that of the classic regulation of the leukocyte tolerogenic immune response, which may account for its pathogenic actions in iARDS.

Endogenous cannabinoid, anandamide mitigates acute lung injury by altering the microbiome and metabolome profile in the gut-lung axis with the induction of an anti-inflammatory response

Abstract Staphylococcus Enterotoxin B (SEB) is a superantigen that can activate ~30% of T cells. Inhalation of SEB can lead to toxic shock syndrome and death. We have shown that anandamide (AEA) is effective in ameliorating SEB-induced ALI. Cross-talk between microbiome through the gut-lung axis (GLA) can influence the immune and respiratory functions. In the current study, we found that AEA treatment alleviated SEB-induced ALI in mice by alteration in the microbiome and the metabolome profile especially short-chain fatty acid (SCFA) production in the GLA. Sequencing of the V3–V4 region of 16S rRNA of colon, cecal flushes and lungs was performed and analysis showed that AEA treatment led to a significant increase, starting from the phylum level in Bacteroidetes until genus Aldercreutzia. Mass Spectrometric (MS) analysis revealed that there was a significant increase in the SCFAs in AEA treatment group, especially iso-butyrate, which may be responsible for induction of T regulatory cells (T regs). Flow cytometry data showed that AEA increased CD4+FOXP3+ in the lung compared to vehicle-treated SEB-induced disease group. Our study also showed that AEA treatment significantly decreased the infiltration of inflammatory cells in the lungs. Furthermore, AEA treatment led to a significant decrease in gut and lung leakage, and pro-inflammatory cytokines (IL-2 and TNFα) while increasing anti-inflammatory cytokines (IL-10). Collectively, these data suggest that AEA ameliorates ALI and prevents leakage through modulation of microbiome profile and the metabolome in the GLA while promoting an anti-inflammatory response to include induction of Tregs and decrease in pro-inflammatory cytokines.

Plasma surfactant proteins and lipid mediators in the airway associate with different stages of viral infection and resolution in a sub-lethal mouse Influenza A model

Abstract Viral quantification serves as a valid biomarker for detecting Influenza A infection however viral burden is not closely associated with disease severity or clinical outcomes. Biomarkers reflecting the status of the host immune response are needed to monitor and/or predict disease outcomes in a hospitalized setting. Here, we utilized a sub-lethal mouse model of Influenza A infection to explore plasma and airway cytokines, plasma surfactant proteins (SP-D, SP-A and CC16), and airway bioactive lipid mediators as potential biomarkers of lung leakage and host response, respectively. This study aimed to assess the pharmacodynamic response of these biomarkers after treatment with an efficacious dose of an anti-HA stalk-binding antibody and to assess association of these biomarkers with disease severity metrics. Plasma SP-D and IP-10 correlated with lung viral titers while SP-A and CC16 tracked more closely with changes in bronchial albumin levels and body weight. Bioactive lipid precursors Arachidonic Acid (AA), Docosahexaenoicacid (DHA) and Eicosapentaenoic acid (EPA), reported to have both pro- and anti-inflammatory properties, increase in the lung with infection. 17-HDHA, a DHA metabolite reported to be pro-resolving, was induced later during disease resolution. With anti-HA antibody treatment, all plasma surfactant proteins and precursor lipids in the airway were reduced and 17-HDHA production was accelerated. In summary, plasma SP-D may serve as an early biomarker of host response and/or potentially indicate a decline in lung epithelium integrity. Plasma SP-A, CC16, and pro-inflammatory lipid mediators such as AA may reflect the ongoing host response and may therefore be more closely associated with disease outcomes.

DUB activity of Endothelial A20 maintains and repairs endothelial barrier after inflammatory lung injury

Vascular endothelial cadherin (VE‐cad) expression at endothelial adherens junctions (AJs) stabilizes endothelial barrier. Inflammatory mediators induced VE‐cad phosphorylation and ubiquitination promotes its degradation; which in turn destabilizes endothelial barrier to trigger vascular inflammatory responses. However, the intrinsic molecular mechanisms involved in maintaining the basal endothelial barrier stability and the repair of endothelial barrier after vascular injury are poorly understood. Here, we generated endothelial cell (EC)‐restricted Tnfaip3 (A20) knockout (A20ΔEC) mice and showed the pivotal role of the deubiquitinase (DUB) function of A20 in regulating the expression of VE‐cad at AJs. A20ΔEC mice failed to survive sub‐lethal dose of LPS challenge. Consistent with this observation, A20ΔEC mice displayed uncontrolled lung vascular leak and persistent PMN sequestration in lungs in response to LPS. In support of these findings, we observed markedly reduced VE‐cad expression in lungs of A20ΔEC mice. Liposome mediated in vivo delivery of WT‐A20 but not DUB‐inactive (C103A‐A20) mutant construct restored VE‐cad expression in lungs, suppressed LPS induced lung vascular leak and sequestration of PMNs in lungs of A20ΔEC mice. Further, A20 knockdown in human lung microvascular ECs resulted increased VE‐cad phosphorylation on Y685 and Y731, and loss VE‐cad expression at AJs. Forced expression of WT‐A20 but not DUB‐inactive mutant in ECs prevented VE‐cad ubiquitination and degradation. Since induced A20 expression promotes resolution of inflammation, we studied the mechanism of A20 induction in response to TLR4 activation in ECs. Here we observed that IRAK‐M‐mediated NF‐κB activation independent of TAK1 is essential for A20 expression in ECs. Importantly, silencing of IRAK‐M in mouse lung microvessels suppressed expression of A20 and VE‐cad and augmented LPS induced lung vascular inflammatory responses. These findings support that targeting endothelial‐A20 expression is a potential therapeutic strategy to restore endothelial barrier integrity in acute lung injury.Support or Funding InformationNational Institute of Health Grants R01 HL‐128539 and R01 GM‐117028This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Vasculotide, an Angiopoietin-1 Mimetic, Restores Microcirculatory Perfusion and Microvascular Leakage and Decreases Fluid Resuscitation Requirements in Hemorrhagic Shock

Microcirculatory dysfunction is associated with multiple organ failure and unfavorable patient outcome. We investigated whether therapeutically targeting the endothelial angiopoietin/Tie2 system preserves microvascular integrity during hemorrhagic shock. Rats were treated with the angiopoietin-1 mimetic vasculotide and subjected to hemorrhagic shock and fluid resuscitation. Microcirculatory perfusion and leakage were assessed with intravital microscopy (n = 7 per group) and Evans blue dye extravasation (n = 8 per group), respectively. The angiopoietin/Tie2 system was studied at protein and RNA level in plasma, kidneys, and lungs. Hemorrhagic shock significantly reduced continuously perfused capillaries (7 ± 2 vs. 11 ± 2) and increased nonperfused vessels (9 ± 3 vs. 5 ± 2) during hemorrhagic shock, which could not be restored by fluid resuscitation. Hemorrhagic shock increased circulating angiopoietin-2 and soluble Tie2 significantly, which associated with microcirculatory perfusion disturbances. Hemorrhagic shock significantly decreased Tie2 gene expression in kidneys and lungs and induced microvascular leakage in kidneys (19.7 ± 11.3 vs. 5.2 ± 3.0 µg/g) and lungs (16.1 ± 7.0 vs. 8.6 ± 2.7 µg/g). Vasculotide had no effect on hemodynamics and microcirculatory perfusion during hemorrhagic shock but restored microcirculatory perfusion during fluid resuscitation. Interestingly, vasculotide attenuated microvascular leakage in lungs (10.1 ± 3.3 µg/g) and significantly reduced the required amount of volume supplementation (1.3 ± 1.4 vs. 2.8 ± 1.5 ml). Furthermore, vasculotide posttreatment was also able to restore microcirculatory perfusion during fluid resuscitation. Targeting Tie2 restored microvascular leakage and microcirculatory perfusion and reduced fluid resuscitation requirements in an experimental model of hemorrhagic shock. Therefore, the angiopoietin/Tie2 system seems to be a promising target in restoring microvascular integrity and may reduce organ failure during hemorrhagic shock.