Inflamed Lung Research Articles

Expression of receptor activator of nuclear factor‐κB (RANK), RANK ligand, and osteoprotegerin in the normal and E. coli lipopolysaccharide‐treated horse lungs

Receptor activator of nuclear factor‐κB ligand (RANKL), its receptor RANK and antagonist osteoprotegerin (OPG) are members of the TNF receptor superfamily. RANKL/RANK signaling mediates TNFα‐induced inflammation and RANKL acts as a chemoattractant for immune cells. OPG interacts with and blocks RANKL signal transduction. There are no or little data on the expression of RANKL, RANK, and OPG in normal or inflamed lungs. Therefore, we studied their expression in control and lipopolysaccharide (LPS) treated horse lungs. Immunohistochemistry showed RANKL expression in alveolar/septal macrophages, vascular endothelium, and alveolar septum but not in airway epithelium in control horses. The LPS treatment increased RANKL expression in airway epithelium and alveolar septum. While a weak staining for RANK was detected in airway epithelium and vascular endothelium of control horse lungs, the expression was increased in airway epithelium of LPS‐treated horses. OPG was expressed in airway epithelium and alveolar/septal macrophages and was increased in LPS‐treated lungs. Immunoelectron microscopy detected all three molecules in pulmonary intravascular and alveolar macrophages. Western blot confirmed expression of all three molecules in the lungs. These data show LPS‐induced changes in the expression of RANK, RANKL, and OPG to suggest their roles in endotoxin‐induced lung inflammation.Grant Funding Source: NSERC

In vivo hydroquinone exposure impairs MCP-1 secretion and monocyte recruitment into the inflamed lung

Alveolar macrophages (AMs) are important cells in the resolution of the inflammatory process and they come into direct contact with inhaled pollutants. Hydroquinone (HQ) is an environmental pollutant and a component of cigarette smoke that causes immunosuppressive effects. In the present work, we showed that mice exposed to low levels of aerosolized HQ (25ppm; 1h/day/5 days) presented impaired mononuclear cell migration to the lipopolysaccharide (LPS)-inflamed lung. This may have been due to reduced monocyte chemoattractant protein-1 (MCP-1) secretion into bronchoalveolar lavage fluid (BALF), and it was not related to alterations to mononuclear cell mobilization into the blood or adhesion molecules expression on mononuclear cell membranes. Corroborating the actions of HQ on MCP-1 secretion, reduced MCP-1 concentrations were also found in the supernatant of ex vivo AM and tracheal tissue collected from HQ-exposed mice. A direct action of HQ on MCP-1 secretion, resulting from impaired gene synthesis, was verified by in vitro incubation of naive AMs or tracheal tissue with HQ. The role of reduced levels of MCP-1 in the BALF on monocyte migration was analysed in the human monocytic lineage THP-1 in in vitro chemotaxis assays, which showed that the reduced concentrations of MCP-1 found in the BALF or cell supernatants from HQ-exposed mice impaired cell migration. Considering the fact that MCP-1 presents a broad spectrum of actions on pathophysiological conditions and that resident mononuclear cells are involved in lung tissue homeostasis and in immune host defence, the mechanism of HQ toxicity presented herein might be relevant to the genesis of infectious lung diseases in smokers and in inhabitants of polluted areas.

Differential Expression of Cytokines in Response to Respiratory Syncytial Virus Infection of Calves with High or Low Circulating 25-Hydroxyvitamin D3

Deficiency of serum levels of 25-hydroxyvitamin D3 has been related to increased risk of lower respiratory tract infections in children. Respiratory syncytial virus (RSV) is a leading cause of low respiratory tract infections in infants and young children. The neonatal calf model of RSV infection shares many features in common with RSV infection in infants and children. In the present study, we hypothesized that calves with low circulating levels of 25-hydroxyvitamin D3 (25(OH)D3) would be more susceptible to RSV infection than calves with high circulating levels of 25(OH)D3. Calves were fed milk replacer diets with different levels of vitamin D for a 10 wk period to establish two treatment groups, one with high (177 ng/ml) and one with low (32.5 ng/ml) circulating 25(OH)D3. Animals were experimentally infected via aerosol challenge with RSV. Data on circulating 25(OH)D3 levels showed that high and low concentrations of 25(OH)D3 were maintained during infection. At necropsy, lung lesions due to RSV were similar in the two vitamin D treatment groups. We show for the first time that RSV infection activates the vitamin D intracrine pathway in the inflamed lung. Importantly, however, we observed that cytokines frequently inhibited by this pathway in vitro are, in fact, either significantly upregulated (IL-12p40) or unaffected (IFN-γ) in the lungs of RSV-infected calves with high circulating levels of 25(OH)D3. Our data indicate that while vitamin D does have an immunomodulatory role during RSV infection, there was no significant impact on pathogenesis during the early phases of RSV infection. Further examination of the potential effects of vitamin D status on RSV disease resolution will require longer-term studies with immunologically sufficient and deficient vitamin D levels.

Effect of betulinic acid on neutrophil recruitment and inflammatory mediator expression in lipopolysaccharide-induced lung inflammation in rats

This study aimed to evaluate the effect of betulinic acid (BA) on acute lung damage induced by bacterial endotoxin (lipopolysaccharide, LPS) in male Sprague–Dawley rats and explore its possible mechanisms. Oral administration of 25 (mg/kg) BA started 7days before LPS or saline nasal installation. Twenty-four hours after LPS or saline installation, samples of lung tissues were collected for determination of level of lipid peroxidation (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), and expression of tumor necrosis-α (TNF-α), transforming growth factor-β1 (TGF-β1) and inducible nitric oxide synthase (iNOS). Histopathology was done to examine pathological changes in lungs. Wet/dry (W/D) ratio and capillary protein leakage were also determined. Bronchoalveolar lavage (BAL) fluid was carried out for quantification of airway cellular inflammation and nitrate/nitrite (NOx) level. In comparison to BAL fluid samples from control animals, LPS-stimulated animals exhibited a higher count of the inflammatory cells and increased NOx levels. Lungs from LPS-treated animals showed increased lipid peroxidation, altered activities of antioxidant enzymes (GSH and SOD) and increased expression of TNF-α, TGF-β1 and iNOS in comparison to lungs from control animals. LPS installation-induced pulmonary edema, manifested by significant increase in lung W/D ratio and Evans blue extravasation in lung tissue. This was supported by the histopathological examination which revealed markedly inflamed lung in LPS-treated animals. Treatment with BA was found to significantly attenuate all these alterations. The present results suggest that BA is endowed with antiinflammatory and antioxidant properties that protect the lung against the deleterious actions of LPS.

دراسة التأثيرات السمية الخلوية للمستخلص المائي لبذور الحلبة ومعقد البلاديوم (II) الجديد على الخط الخلوي لسرطان امعاء الفئران في العراق

treat arthritis inflamed lungs, diabetes, etc. Numerous palladium complexes with promising activity against tumor cell lines have been synthesized. This study involved the evaluation of Aqueous Fenugreek seeds Extract and new palladium (II) Complex with general formula [Pdl2].2ETOH, where L=2-hydroxy phenyl piperonalidine have a promising anitcancer effect on growth cancer cells and were compared to the anti cancer drug cis-ptatinum by utilizing an in vitro system in femal Balb/c mice by using the Inhibitor Rate as a parameter for the cytotoxic effects on Intestine cancer cell line L20B. Cancer cells were treated with four concentrations of cis-platin 31.25,62.5,125, and 250 μg/ml for 48 and 72 hours .The same concentrations were used for the other extract and complex. This study showed that extract and [PdL2]. 2EtOHcomplex have a promising anti cancer cells as could be seen from these effect on Increased of Inhibition cancer cells at different concentrations and these effect were similar to the effect of cis- platin, then the Inhibitory Rate was increased to 44.60,52.32% and 37.93, 42.97% in cancer cells treated with 250 μg/ml of palladium (II) complex and extract respectively on hours exposure with not singnificant differences with compared Cis- platin.

Hypoxia Response in Asthma

Oxygen-sensing prolyl-hydroxylase (PHD)-2 negatively regulates hypoxia-inducible factor (HIF)1-α and suppresses the hypoxic response. Hypoxia signaling is thought to be proinflammatory but also attenuates cellular injury and apoptosis. Although increased hypoxic response has been noted in asthma, its functional relevance is unknown. The objectives of this study were to dissect the mechanisms and role of the hypoxic response in asthma pathophysiology. Experimental studies were conducted in mice using acute and chronic allergic models of asthma. The hypoxic response in allergically inflamed lungs was modulated by using pharmacologic PHD inhibitors (ethyl-3-4-dihydroxybenzoic acid [DHB], 1-10 mg/kg) or siRNA-mediated genetic knockdowns. Increased hypoxia response led to exacerbation of the asthma phenotype, with HIF-1α knockdown being beneficial. Chronically inflamed lungs from mice treated with 10 mg/kg DHB showed diffuse up-regulation of the hypoxia response, severe airway remodeling, and inflammation. Fatal asphyxiation during methacholine challenge was noted. However, bronchial epithelium restricted up-regulation of the hypoxia response seen with low-dose DHB (1 mg/kg) reduced epithelial injury and attenuated the asthmatic phenotype. Up-regulation of the hypoxia response was associated with increased expression of CX3CR1, a lymphocyte survival factor, and increased inflammatory cell infiltrate. This study shows that an exaggerated hypoxia response may contribute to airway inflammation, remodeling, and the development of asthma. However, the hypoxia response may also be protective of epithelial apoptosis at lower levels, and the net effects of modulating the hypoxia response may vary based on the context.

Dangkwisoo-san, an herbal medicinal formula, ameliorates acute lung inflammation via activation of Nrf2 and suppression of NF-κB

Ethnopharmacological relevanceDangkwisoo-san (DS), an herbal medicinal formula, has long been used in Korea for the treatment of inflammatory complications caused by physical trauma. Although the therapeutic effect of DS is likely associated with anti-inflammatory activity, the precise underlying mechanisms are largely unknown. Here we sought to elucidate the possible mechanisms of anti-inflammatory activity of DS. Materials and methodsThe water extract of DS was orally fed to C57BL/6 mice for 14 days prior to LPS intranasal instillation for lung inflammation. The effects of DS on lung inflammation were determined by differential cell counting, lung histology, and semi-quantitative RT-PCR of lung sections. The effects of DS on the activities of Nrf2 and NF-κB were assessed by western blotting, semi-quantitative RT-PCR, and luciferase reporter assays in RAW 264.7, an NF-κB reporter cell line, and HEK 293 transfected with an NF-κB reporter construct. ResultsMice that were treated with a water extract of DS showed significant attenuation of lung inflammation induced by intranasal lipopolysaccharide (LPS) compared to control mice treated with vehicle. In vitro experiments show that DS activated Nrf2, an anti-oxidant transcription factor that protects from various inflammatory diseases, and induced Nrf2-regulated genes including GCLC, NQO-1 and HO-1. In addition, DS suppressed NF-κB activity and reduced the production of pro-inflammatory cytokines. Transfection experiment indicates that inhibition of NF-κB likely occurred upstream of IKK complex. Furthermore, DS enhanced the expression of HO-1 and suppressed that of IL-1β and TNF-α in inflamed mouse lungs. ConclusionsThese results suggest that the therapeutic effects of DS are related with suppression of inflammation, which is, at least in part, mediated by activation of anti-inflammatory factor Nrf2 and inhibition of pro-inflammatory factor NF-κB.

Protective Effect of the Fruit Hull ofGleditsia sinensison LPS-Induced Acute Lung Injury Is Associated with Nrf2 Activation

The fruit hull of Gleditsia sinensis (FGS) has been prescribed as a traditional eastern Asian medicinal remedy for the treatment of various respiratory diseases, but the efficacy and underlying mechanisms remain poorly characterized. Here, we explored a potential usage of FGS for the treatment of acute lung injury (ALI), a highly fatal inflammatory lung disease that urgently needs effective therapeutics, and investigated a mechanism for the anti-inflammatory activity of FGS. Pretreatment of C57BL/6 mice with FGS significantly attenuated LPS-induced neutrophilic lung inflammation compared to sham-treated, inflamed mice. Reporter assays, semiquantitative RT-PCR, and Western blot analyses show that while not affecting NF-κB, FGS activated Nrf2 and expressed Nrf2-regulated genes including GCLC, NQO-1, and HO-1 in RAW 264.7 cells. Furthermore, pretreatment of mice with FGS enhanced the expression of GCLC and HO-1 but suppressed that of proinflammatory cytokines in including TNF-α and IL-1β in the inflamed lungs. These results suggest that FGS effectively suppresses neutrophilic lung inflammation, which can be associated with, at least in part, FGS-activating anti-inflammatory factor Nrf2. Our results suggest that FGS can be developed as a therapeutic option for the treatment of ALI.

Profibrotic potential of Prominin-1+epithelial progenitor cells in pulmonary fibrosis

BackgroundIn idiopathic pulmonary fibrosis loss of alveolar epithelium induces inflammation of the pulmonary tissue followed by accumulation of pathogenic myofibroblasts leading eventually to respiratory failures. In animal models inflammatory and resident cells have been demonstrated to contribute to pulmonary fibrosis. Regenerative potential of pulmonary and extra-pulmonary stem and progenitor cells raised the hope for successful treatment option against pulmonary fibrosis. Herein, we addressed the contribution of lung microenvironment and prominin-1+ bone marrow-derived epithelial progenitor cells in the mouse model of bleomycin-induced experimental pulmonary fibrosis.MethodsProminin-1+ bone marrow-derived epithelial progenitors were expanded from adult mouse lungs and differentiated in vitro by cytokines and growth factors. Pulmonary fibrosis was induced in C57Bl/6 mice by intratracheal instillation of bleomycin. Prominin-1+ progenitors were administered intratracheally at different time points after bleomycin challenge. Green fluorescence protein-expressing cells were used for cell tracking. Cell phenotypes were characterized by immunohistochemistry, flow cytometry and quantitative reverse transcription-polymerase chain reaction.ResultsProminin-1+ cells expanded from healthy lung represent common progenitors of alveolar type II epithelial cells, myofibroblasts, and macrophages. Administration of prominin-1+ cells 2 hours after bleomycin instillation protects from pulmonary fibrosis, and some of progenitors differentiate into alveolar type II epithelial cells. In contrast, prominin-1+ cells administered at day 7 or 14 lose their protective effects and differentiate into myofibroblasts and macrophages. Bleomycin challenge enhances accumulation of bone marrow-derived prominin-1+ cells within inflamed lung. In contrast to prominin-1+ cells from healthy lung, prominin-1+ precursors isolated from inflamed organ lack regenerative properties but acquire myofibroblast and macrophage phenotypes.ConclusionThe microenvironment of inflamed lung impairs the regenerative capacity of bone marrow-derived prominin-1+ progenitors and promotes their differentiation into pathogenic phenotypes.

CX3CR1 is required for airway inflammation by promoting T helper cell survival and maintenance in inflamed lung

CX3CR1 is required for airway inflammation by promoting T helper cell survival and maintenance in inflamed lung

Extracellular DNA Traps Are Associated with Pathogenesis of TRALI in Humans and Mice

Abstract 37Transfusion-related acute lung injury (TRALI) is a serious complication of blood transfusion occurring within 6 hours of transfusion. It is characterized by hypoxemia, respiratory distress and pulmonary infiltrates (Kleinman et al. Transfusion 2004) and is the leading cause of transfusion-induced death. The biological processes contributing to TRALI are still poorly understood. All blood products can cause TRALI, and to date no specific treatment is available. A “two-event model” has been proposed as the trigger (Silliman et al Blood 2007). The first event may include surgery, trauma or infection; the second involves the transfusion of anti-neutrophil antibodies or bioactive lipids within the blood product. Together these events induce neutrophil activation and sequestration in the pulmonary capillaries (Looney et al J Clin Invest 2006) resulting in endothelial damage and capillary leakage (Shaz et al Blood 2011). In response to pathogens or under stress, activated neutrophils have the ability to release neutrophil extracellular traps (NETs) (Brinkmann Science 2004; Fuchs et al J Cell Biol 2007) that are composed of DNA fibers decorated with histones and antimicrobial proteins (Urban PLoS Pathog 2009) originally contained in the neutrophil granules. Although protective against infection, these NETs are injurious to tissue (Xu et al Nat Med 2009, Xu et al J Immunol 2011), activate platelets (Fuchs et al PNAS 2010) and can contribute to the pathology of inflammatory diseases (Kessenbrock et al Nat Med 2009, Garcia-Romo Sci Transl Med 2011). The purpose of this study was to determine whether NETs are formed in patients during TRALI and whether they contribute to TRALI in a mouse model.Here we show that NETs biomarkers are present in TRALI patients' blood and that the antibody responsible for the most severe TRALI cases and that is directed against human neutrophil antigen (HNA)-3a (Davoren et al Transfusion 2003; Greinacher et al Nat Med 2010) stimulates NETs generation from primed human neutrophils when compared to a control antibody. This effect was inhibited when FcgRIIa-binding sites were blocked prior to antibody incubation. Moreover, anti-HNA-3a F(ab')2 fragments had no effect on NETs generation. These results allowed us to conclude that FcgRIIa activation was required in anti-HNA-3a antibody-mediated NETosis.We next used an in vivo model of antibody-induced TRALI in mice (Looney et al J Clin Invest 2006; Looney et al. J Clin Invest 2009; Hidalgo et al Nat Med 2009). In this two-event model, BALB/c mice are injected i.p. with a low-dose of LPS and 24 hours later are infused with an anti-MHC class I monoclonal antibody (anti-H-2Kd). Two hours later, we measured arterial blood oxygen saturation to document lung function and the lung injury markers were quantified. As observed in blood from TRALI patients, we detected a significant increase in the concentration of circulating NETs biomarkers in mice with TRALI compared to mice that were challenged only with LPS. Multiphoton analysis of fixed lung tissue showed strong staining for citrullinated histone H3 (a phenomenon already described to correlate with NETs formation (Wang et al J Cell Biol 2009; Li et al J Exp Med 2010)) collocalizing with DNA streaks in the alveoli of mice with TRALI. Moreover, we found NETs in abundance in the TRALI-affected alveoli of mice by transmission electron microscopy. We demonstrate that intranasal DNaseI pretreatment of the mice prior to antibody infusion prevented NETs accumulation in the alveoli and also improved the lung function of the mice experiencing TRALI by increasing their arterial blood oxygenation. These results support the hypothesis that NETs are indeed formed in the inflamed lungs during TRALI and contribute to the disease process and thus, could be targeted to prevent or treat TRALI. Disclosures:No relevant conflicts of interest to declare.

Interleukin-6 mediates pulmonary vascular permeability in a two-hit model of ventilator-associated lung injury

ABSTRACTTo test the hypothesis that interleukin-6 (IL-6) contributes to the development of ventilator-associated lung injury (VALI), IL-6–deficient (IL6−/–) and wild-type control (WT) mice received intratracheal hydrochloric acid followed by randomization to mechanical ventilation (MV + IT HCl) or spontaneous ventilation (IT HCl). After 4 hours, injury was assessed by estimation of lung lavage protein concentration and total and differential cell counts, wet/dry lung weight ratio, pulmonary cell death, histologic inflammation score (LIS), and parenchymal myeloperoxidase (MPO) concentration. Vascular endothelial growth factor (VEGF) concentration was measured in lung lavage and homogenate, as IL-6 and stretch both regulate expression of this potent mediator of permeability. MV-induced increases in alveolar barrier dysfunction and lavage VEGF were attenuated in IL6−/− mice as compared with WT controls, whereas tissue VEGF concentration increased. The effects of IL-6 deletion on alveolar permeability and VEGF concentration were inflammation independent, as parenchymal MPO concentration, LIS, and lavage total and differential cell counts did not differ between WT and IL6−/− mice following MV + IT HCl. These data support a role for IL-6 in promoting VALI in this two-hit model. Strategies to interfere with IL-6 expression or signaling may represent important therapeutic targets to limit the injurious effects of MV in inflamed lungs.

Allergic inflammation induces a persistent mechanistic switch in thromboxane-mediated airway constriction in the mouse

Actions of thromboxane (TXA(2)) to alter airway resistance were first identified over 25 years ago. However, the mechanism underlying this physiological response has remained largely undefined. Here we address this question using a novel panel of mice in which expression of the thromboxane receptor (TP) has been genetically manipulated. We show that the response of the airways to TXA(2) is complex: it depends on expression of other G protein-coupled receptors but also on the physiological context of the signal. In the healthy airway, TXA(2)-mediated airway constriction depends on expression of TP receptors by smooth muscle cells. In contrast, in the inflamed lung, the direct actions of TXA(2) on smooth muscle cell TP receptors no longer contribute to bronchoconstriction. Instead, in allergic lung disease, TXA(2)-mediated airway constriction depends on neuronal TP receptors. Furthermore, this mechanistic switch persists long after resolution of pulmonary inflammation. Our findings demonstrate the powerful ability of lung inflammation to modify pathways leading to airway constriction, resulting in persistent changes in mechanisms of airway reactivity to key bronchoconstrictors. Such alterations are likely to shape the pathogenesis of asthmatic lung disease.

CCR8 Signaling Influences Toll-Like Receptor 4 Responses in Human Macrophages in Inflammatory Diseases

CCR8 immunity is generally associated with Th2 responses in allergic diseases. In this study, we demonstrate for the first time a pronounced attenuated influx of macrophages in ovalbumin (OVA)-challenged CCR8 knockout mice. To explore whether macrophages in human inflamed lung tissue also were CCR8 positive, human lung tissue from patients with chronic obstructive pulmonary disease (COPD) was evaluated. Indeed, CCR8 expression was pronounced in invading monocytes/macrophages from lungs of patients with Global Initiative for Obstructive Lung Disease (GOLD) stage IV COPD. Given this expression pattern, the functional role of CCR8 on human macrophages was evaluated in vitro. Human peripheral blood monocytes expressed low levels of CCR8, while macrophage colony-stimulating factor (M-CSF)-derived human macrophages expressed significantly elevated surface levels of CCR8. Importantly, CCL1 directly regulated the expression of CD18 and CD49b and hence influenced the adhesion capacity of human macrophages. CCL1 drives chemotaxis in M-CSF-derived macrophages, and this could be completely inhibited by lipopolysaccharide (LPS). Whereas both CCL1 and LPS monotreatment inhibited spontaneous superoxide release in macrophages, CCL1 significantly induced superoxide release in the presence of LPS in a dose-dependent manner. Finally, CCL1 induced production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) and could inhibit LPS-induced cytokine production in a dose-dependent manner. Our data demonstrate, for the first time, the presence of CCR8 on inflammatory macrophages in human COPD lung tissue. Importantly, the functional data from human macrophages suggest a potential cross talk between the CCR8 and the Toll-like receptor 4 (TLR4) pathways, both of which are present in COPD patients.

Asymmetric Dimethylarginine Is Increased in Asthma

Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase (NOS) inhibitor that competes with L-arginine for binding to NOS. It has been suggested that ADMA contributes to inflammation, collagen deposition, nitrosative stress, and lung function in murine models. To test the hypothesis that ADMA is increased in asthma and that NOS inhibition by ADMA contributes to airways obstruction. We assessed alterations of L-arginine, ADMA, and symmetric dimethylarginine (SDMA) levels in a murine model of allergic airways inflammation using LC-tandem mass spectrometry. Based on the levels of ADMA observed in the murine model, we further tested the direct effects of nebulized inhaled ADMA on airways responsiveness in naive control mice. We also assessed alterations of L-arginine, ADMA, and SDMA in humans in adult lung specimens and sputum samples from pediatric patients with asthma. ADMA was increased in lungs from the murine model of allergic airways inflammation. Exogenous administration of ADMA to naive mice, at doses consistent with the levels observed in the allergically inflamed lungs, resulted in augmentation of the airways responsiveness to methacholine. ADMA levels were also increased in human asthma lungs and sputum samples. ADMA levels are increased in asthma and contribute to NOS-related pathophysiology.

IL-17, a new kid on the block of tertiary lymphoid organs

Tertiary lymphoid organs (TLOs) are structures that resemble lymph nodes that can be found in chronically inflamed tissues in a number of inflammatory conditions. TLOs can host ongoing immune responses that can potentially contribute to disease pathogenesis. The development of TLOs has been thought in large part to recapitulate the development of secondary lymphoid organs, but the regulation of TLO development has not been well understood. In a recent article in Nature Immunology, Troy Randall and colleagues1 identify IL-17, a cytokine that contributes to the pathogenesis of a number of inflammatory diseases, as a novel critical player in TLO formation in inflamed lungs.

Stabilizing the VE-cadherin-catenin complex blocks leukocyte extravasation and vascular permeability

To determine whether leukocytes need to open endothelial cell contacts during extravasation, we decided to generate mice with strongly stabilized endothelial junctions. To this end, we replaced VE-cadherin genetically by a VE-cadherin-α-catenin fusion construct. Such mice were completely resistant to the induction of vascular leaks by VEGF or histamine. Neutrophil or lymphocyte recruitment into inflamed cremaster, lung and skin were strongly inhibited in these mice, documenting the importance of the junctional route in vivo. Surprisingly, lymphocyte homing into lymph nodes was not inhibited. VE-cadherin-α-catenin associated more intensely with the actin cytoskeleton as demonstrated by its membrane mobility and detergent extractability. Our results establish the junctional route as the main pathway for extravasating leukocytes in several, although not in all tissues. Furthermore, in these tissues, plasticity of the VE-cadherin-catenin complex is central for the leukocyte diapedesis mechanism.

Antibacterial activity of the essential oil of Mountain Savory (Satureja montana) against Arcanobacterium pyogenes

The pharmaceutical properties of aromatic plants are partially attributed to essential oils (EOs) which are widely used to prevent and treat human diseases [1]. However, little is known about the control of infective animal diseases with EOs. Arcanobacterium pyogenes (AP) is one of the important opportunistic pathogens of the upper respiratory and genital tracts of cattle, sheep, swine, and many other species [2]. Most frequently, this bacteria is isolated from inflamed lung lesions of pigs and cattle, in the samples of uterine mucos of sows and cows with endometritis and the milk from cows with clinical mastitis. Antimicrobial activity of Mountain Savory (Satureja montana) L. (SM) was detected in vitro conditions against Clostridium perfringens type A [3] and Staphylococcus aureus [4]. Therefore, it is assumed that SM can react against AP and therapeutic potential against bacterial infection in animals. The EO of the cultivated SM (Serbia), was extracted by hydrodistillation and analyzed by gas chromatography. According to compositional analysis of the SM EO, 27 chemical compounds were identified, and carvacrol (42.12%), linalool (24.57%) and p-cymene (19.85%) were found as predominant compounds in oil. Antibacterial sensitivity of AP (ATCC 19411) and other 18 isolates (field strain originating from swine-uterus and cattle-uterus and milk) were tested in vitro using an Agar Dilution Test to determine the minimal inhibitory concentration (MIC). The results obtained have shown that EO applied at a concentration of 0.78µl ml-1, which was defined as the MIC, exhibited antimicrobial activity against all AP in the in vitro assays. Keywords: essential oil, Satureja montana, Arcanobacterium pyogenes, antibacterial MIC Acknowledgement: This work was supported by a grant from scientific project TR 031071 of Ministry of Science and Technological Development of Republic of Serbia

IL-22 Is Produced by Innate Lymphoid Cells and Limits Inflammation in Allergic Airway Disease

Interleukin (IL)-22 is an effector cytokine, which acts primarily on epithelial cells in the skin, gut, liver and lung. Both pro- and anti-inflammatory properties have been reported for IL-22 depending on the tissue and disease model. In a murine model of allergic airway inflammation, we found that IL-22 is predominantly produced by innate lymphoid cells in the inflamed lungs, rather than TH cells. To determine the impact of IL-22 on airway inflammation, we used allergen-sensitized IL-22-deficient mice and found that they suffer from significantly higher airway hyperreactivity upon airway challenge. IL-22-deficiency led to increased eosinophil infiltration lymphocyte invasion and production of CCL17 (TARC), IL-5 and IL-13 in the lung. Mice treated with IL-22 before antigen challenge displayed reduced expression of CCL17 and IL-13 and significant amelioration of airway constriction and inflammation. We conclude that innate IL-22 limits airway inflammation, tissue damage and clinical decline in allergic lung disease.

Pretreatment with phosphatase and tensin homolog deleted on chromosome 10 (PTEN) inhibitor SF1670 augments the efficacy of granulocyte transfusion in a clinically relevant mouse model

AbstractThe clinical outcome of granulocyte transfusion therapy is often hampered by short ex vivo shelf life, inefficiency of recruitment to sites of inflammation, and poor pathogen-killing capability of transplanted neutrophils. Here, using a recently developed mouse granulocyte transfusion model, we revealed that the efficacy of granulocyte transfusion can be significantly increased by elevating intracellular phosphatidylinositol (3,4,5)-trisphosphate signaling with a specific phosphatase and tensin homolog deleted on chromosome 10 (PTEN) inhibitor SF1670. Neutrophils treated with SF1670 were much sensitive to chemoattractant stimulation. Neutrophil functions, such as phagocytosis, oxidative burst, polarization, and chemotaxis, were augmented after SF1670 treatment. The recruitment of SF1670-pretreated transfused neutrophils to the inflamed peritoneal cavity and lungs was significantly elevated. In addition, transfusion with SF1670-treated neutrophils led to augmented bacteria-killing capability (decreased bacterial burden) in neutropenic recipient mice in both peritonitis and bacterial pneumonia. Consequently, this alleviated the severity of and decreased the mortality of neutropenia-related pneumonia. Together, these observations demonstrate that the innate immune responses can be enhanced and the severity of neutropenia-related infection can be alleviated by augmenting phosphatidylinositol (3,4,5)-trisphosphate in transfused neutrophils with PTEN inhibitor SF1670, providing a therapeutic strategy for improving the efficacy of granulocyte transfusion.