Bronchoalveolar Neutrophils Research Articles

Prevalence and clinical features of progressive pulmonary fibrosis in patients with unclassifiable idiopathic interstitial pneumonia: A post hoc analysis of prospective multicenter registry.

Idiopathic interstitial pneumonias (IIPs) may remain unclassifiable owing to inadequate, nonspecific, or conflicting clinical, radiological, or histopathological findings despite multidisciplinary discussion (MDD). Unclassifiable IIP (UCIIP) is a heterogeneous disease that can present with progressive pulmonary fibrosis (PPF). This study aimed to investigate the prevalence and clinical features of PPF in patients with UCIIP. In this post hoc analysis of a prospective multicenter registry of 222 patients with IIPs, 71 with UCIIP diagnosed using MDD were enrolled. PPF was defined based on worsening symptoms and radiological and physiological progression using the guideline criteria within 12 months or the criteria from the INBUILD trial within 24 months. The median age was 72 years, and surgical lung biopsy was performed in 19.7%. Of the 66 patients with adequate follow-up data, 30 (45.5%) met either criterion and were diagnosed with PPF. UCIIP patients with PPF had significantly higher serum surfactant protein-D level and percentage of bronchoalveolar fluid neutrophils, lower %forced vital capacity and %diffusing capacity for carbon monoxide, and a higher proportion of honeycombing on high-resolution computed tomography and desaturation on exertion than those without PPF. Additionally, they had significantly more anti-fibrotic therapy and long-term oxygen therapy, a higher incidence of acute exacerbation, and a poorer prognosis than those without PPF. Cox proportional hazards analysis revealed that PPF was a significant poor prognostic factor, regardless of the criteria. PPF is common and associated with poor prognosis in patients with UCIIP. Appropriate evaluation and management of PPF are essential for UCIIP.

Deficient neutrophil responses early in influenza infection promote viral replication and pulmonary inflammation.

Neutrophils play key protective roles in influenza infections, yet excessive neutrophilic inflammation is a hallmark of acute lung injury during severe infections. Phenotypic heterogeneity is increasingly recognized in neutrophil populations; however, how functional variation in neutrophils between individuals determine the diverse outcomes of influenza remains unclear. To examine immunologic responses that may drive varying outcomes in influenza, we infected C57BL/6 (B6) and A/J mice with mouse-adapted influenza A virus A/PR/8/34 H1N1. A self-resolving dose in B6 mice was lethal in A/J mice, which had increased viral load throughout infection accompanied by prominent bronchoalveolar neutrophilia and pulmonary vascular leakage preceding mortality. Notably, the B6 mice heavily recruited neutrophils to lungs early in infection while A/J mice failed to do so. Neutrophils from A/J mice additionally displayed reduced neutrophil extracellular trap (NET) release and reactive oxygen species (ROS) generation compared to B6 mice early in infection, suggesting the failure to control virus in A/J mice was a product of deficient neutrophil response. To determine if variation in neutrophils between strains governed viral control and inflammation, we adoptively transferred bone marrow neutrophils from B6 or A/J donors to A/J recipients early in infection and found that the transfer of B6 neutrophils enhanced viral clearance and abrogated the dissemination of CXCL1 and IL-6. The transfer of A/J neutrophils, however, failed to achieve either. Furthermore, B6 neutrophils were capable of greater levels of viral killing in vitro than their A/J counterparts. These results suggest that a key moderator of inflammation in influenza infection is the control of virus by neutrophils early in infection. Thus, host-specific differences in both the recruitment of these cells as well as interindividual variation in neutrophil ability to support viral clearance may in part dictate differing susceptibility to respiratory viral infections.

Neutrophils and secondary infections in COVID-19 induced acute respiratory distress syndrome

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is the cause of the current global pandemic and has affected more than 188 countries worldwide. Infection by the virus can have diverse clinical manifestations, with one of the most severe clinical manifestation being respiratory failure and the development of acute respiratory distress syndrome. Clinical manifestations of acute respiratory distress syndrome secondary to SARS-CoV-2 are also diverse with a lack of diagnostic tools to distinguish between primary viral infection and secondary bacterial infections. This was a single-centre, retrospective case-control study of bronchoalveolar lavage fluid cell counts, flow cytometry and culture results from mechanically ventilated patients with SARS-CoV-2 (COVID-19) pneumonia and acute respiratory distress syndrome. Neutrophils were the predominant cell type in bronchoalveolar fluid samples up to 2 weeks into mechanical ventilation. There also was a strong correlation between positive respiratory cultures and significant elevation in bronchoalveolar fluid neutrophil counts/percentages and serum C-reactive protein levels. Absolute levels of T cell subtypes correlated with reduced lung compliance measurements. Patients with SARS-CoV-2 and severe respiratory disease are at risk for secondary infections. In some COVID-19 patients, serum C-reactive protein and bronchoalveolar fluid neutrophils may be correlated with a secondary infection.

Inhaled nitric oxide protects transgenic SAD mice from sickle cell disease–specific lung injury induced by hypoxia/reoxygenation

AbstractCentral to the pathophysiology of sickle cell disease are the vaso-occlusive events that lead to tissue damages and life-threatening complications. Lungs are particularly vulnerable to vaso-occlusion because of their specific vasculature. We developed a mouse model of hypoxia/reoxygenation lung injury closely mimicking the lung pathology of patients with sickle cell disease. This model involves the exposure of transgenic sickle cell (SAD) mice to hypoxia (8β oxygen) for 4, 10, and 46 hours followed by 2 hours of reoxygenation. Gene expression profiling of SAD lung tissue pointed to the specific induction of genes involved in the response to ischemic stress and microcirculation remodeling: Hspcb, Hsp86-1, Nfe2l2, Ace, and Fgf7. Hypoxia/reoxygenation also induced a marked increase in bronchoalveolar (BAL) total leukocyte and neutrophil counts, BAL total protein content, and BAL tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), IL-1α, and macrophage inflammatory protein 2 (MIP-2) levels, all indicators of enhanced inflammatory response as compared with control mice. Nitric oxide (NO) was administered to SAD mice. NO (40 ppm) inhalation protected SAD mice from the histopathologic lesions of ischemic/reperfusion lung injury with corresponding normalization and/or modulation of tissue gene expression profiles. Inhaled NO (1) significantly reduced the increase in BAL total protein content, BAL total leukocyte, and neutrophil counts; (2) modulated BAL cytokine network; and (3) did not affect hemoglobin and methemoglobin levels. The present study provides evidences for the beneficial effects of inhaled NO in pulmonary injury induced by hypoxia/reoxygenation in a mouse model of sickle cell disease (SCD) and opens new avenues in drug design based on tissue gene expression profiling.

Increasing the Resistance of the Body to Adverse Cytotoxic Effects of Amorphous Silicon Dioxide Nanoparticles

Introduction: Amorphous silicon dioxide nanoparticles of (SiNPs) are among the most common nanomaterials today. Besides, industrial aerosols formed by condensation and containing a significant proportion of SiNPs are spontaneously produced by a number of pyrometallurgical and welding processes. A significant number of individuals are therefore exposed to SiNPs in the occupational setting or polluted ambient air and while using this nanomaterial. The purpose of our experimental study was to develop a preventive method promoting the resistance of the organism to adverse cytotoxic effects of amorphous silicon dioxide nanoparticles. Materials and methods: White laboratory rats received a monthly course of a specially developed bioprophylactic complex (BPC) before a single intratracheal instillation of a SiNPs suspension at a concentration of 0.5 mg/mL. The suspension was obtained by laser ablation of thin sheet targets of the appropriate material of 99.99 % purity in sterile deionized water. The average diameter of SiNPs was 43 ± 11 nm. Cytological (the number of bronchoalveolar macrophages and neutrophils and their ratio) and cytochemical indices of the bronchoalveolar lavage (BAL) fluid (alkaline phosphatase, alanine aminotransferase, gamma-glutamyl transpeptidase, amylase, and lactate dehydrogenase) were evaluated at 24 hours after the injection. The bioprophylactic complex was administered to the animals with feed and drink and included monosodium glutamate, fish oil rich in omega-3 polyunsaturated fatty acids (PUFAs), iodine, and an antioxidant complex of selenium, quercetin (rutoside), and vitamins A, E, and C. Conclusions: Our findings show that changes in both cytological and biochemical BAL parameters proved a positive health effect of premedication that helped reduce cytotoxicity of SiNPs exposure.

Quantification of bronchoalveolar neutrophil extracellular traps and phagocytosis in murine pneumonia.

The past two decades have witnessed a resurgence in neutrophil research, inspired in part by the discovery of neutrophil extracellular traps (NETs) and their myriad roles in health and disease. Within the lung, dysregulation of neutrophils and NETosis have been linked to an array of diseases including pneumonia, cystic fibrosis, acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), and severe asthma. However, our understanding of pathologic neutrophil responses in the lung remains incomplete. Two methodologic issues have contributed to this gap: first, an emphasis on studying neutrophils from blood rather than the lung and second, the technical difficulties of interrogating neutrophil responses in mice, which has largely restricted basic murine research to specialized laboratories. To address these limitations, we have developed a suite of techniques for studying neutrophil effector functions specifically in the mouse lung. These include ex vivo assays for phagocytosis and NETosis using bronchoalveolar neutrophils and in situ evaluation of NETosis in a murine model of pneumonia. Throughout, we have prioritized technical ease and robust, quantitative readouts. We hope these assays will help to standardize research on lung neutrophils and improve accessibility to this burgeoning field.

Neutrophil Extracellular Traps Are Elevated in Patients with Pneumonia-related Acute Respiratory Distress Syndrome.

Neutrophil extracellular traps have been associated with tissue damage. Whether these are involved in the pathogenesis of human acute respiratory distress syndrome (ARDS) and could be a potential therapeutic target is unknown. The authors quantified bronchoalveolar and blood neutrophil extracellular traps in patients with pneumonia-related ARDS and assessed their relationship with ventilator-free days. Immunocompetent patients with pneumonia and moderate or severe ARDS (n = 35) and controls (n = 4) were included in a prospective monocentric study. Neutrophil extracellular trap concentrations were quantified (as DNA-myeloperoxidase complexes) in bronchoalveolar lavage fluid and serum by enzyme-linked immunosorbent assay. The relationship between bronchoalveolar lavage neutrophil extracellular trap concentrations and the primary clinical endpoint (i.e., the number of live ventilator-free days at day 28) was assessed using linear regression analyses. There was no significant relationship between bronchoalveolar lavage neutrophil extracellular trap concentrations and ventilator-free days by multiple regression analysis (β coefficient = 2.40; 95% CI, -2.13 to 6.92; P = 0.288). Neutrophil extracellular trap concentrations were significantly higher in bronchoalveolar lavage than in blood of ARDS patients (median [first to third quartiles]:154 [74 to 1,000] vs. 26 [4 to 68] arbitrary units, difference: -94; 95% CI, -341 to -57; P < 0.0001). Bronchoalveolar concentrations of patients were higher than those of controls (154 [74 to 1,000] vs. 4 [4 to 4] arbitrary units, difference: -150; 95% CI, -996 to -64; P < 0.001) and associated with bronchoalveolar interleukin-8 (Spearman's ρ = 0.42; P = 0.012) and neutrophil concentrations (ρ = 0.57; P < 0.0001). Intensive care unit mortality (12%, n = 2 of 17 vs. 17%, n = 3 of 18; P > 0.99) and the number of ventilator-free days at day 28 (22 [14 to 25] vs. 14 [0 to 21] days; difference: -5; 95% CI, -15 to 0; P = 0.066) did not significantly differ between patients with higher (n = 17) versus lower (n = 18) bronchoalveolar neutrophil extracellular trap concentrations. Bronchoalveolar neutrophil extracellular trap concentration was not significantly associated with mechanical ventilation duration in pneumonia-related ARDS.

In vitro/in vivo investigation on the potential of Pluronic® mixed micelles for pulmonary drug delivery

In this paper, we shed light on the potential of Pluronic® mixed micelles in lung delivery of poorly water-soluble drugs. To this purpose, Pluronic® P123/F127 mixed micelles (PMM), exhibiting superior stability in biological fluids, were loaded with budesonide (BUD), a model hydrophobic corticosteroid, and fully investigated focusing on their stability in pulmonary-relevant media, transport through the mucus barrier and aerodynamic behaviour in vitro. Then, lung bio-distribution and efficacy were evaluated in vivo, after intra-tracheal administration in rats. PMM showed excellent stability in saline, mucin, artificial airway mucus and simulated interstitial lung fluid. Likely due to their small size coupled with the hydrophilic biofouling shell, PMM did not interact with mucin and consequently diffused through artificial mucus. BUD was loaded with high efficiency in PMM and released at sustained rate in artificial mucus. BUD-PMM dispersion in saline was efficiently delivered through a common jet nebulizer without aggregation. After intratracheal administration in rats, PMM labelled with Rhodamine B persisted in the lung up to 24 h, while serum levels rapidly dropped. Finally, the effects of BUD-PMM in a rat model of lung inflammation induced by intra-tracheal aerosolization of lipopolysaccharide (LPS) from E. coli were investigated. Of note, a single intra-tracheal aerosolization of BUD-PMM significantly reduced bronchoalveolar neutrophil infiltration and the expression of protein/enzymes derived from the arachidonic acid cascade induced by LPS, whereas a control BUD aqueous suspension showed a weaker effect. Overall, this study demonstrates that inhalable formulations of PMM can be considered as a platform for local delivery of hydrophobic drugs at lungs worth of further consideration.

Effects of positive end-expiratory pressure and recruitment maneuvers in a ventilator-induced injury mouse model.

BackgroundPositive-pressure mechanical ventilation is an essential therapeutic intervention, yet it causes the clinical syndrome known as ventilator-induced lung injury. Various lung protective mechanical ventilation strategies have attempted to reduce or prevent ventilator-induced lung injury but few modalities have proven effective. A model that isolates the contribution of mechanical ventilation on the development of acute lung injury is needed to better understand biologic mechanisms that lead to ventilator-induced lung injury.ObjectivesTo evaluate the effects of positive end-expiratory pressure and recruitment maneuvers in reducing lung injury in a ventilator-induced lung injury murine model in short- and longer-term ventilation.Methods5–12 week-old female BALB/c mice (n = 85) were anesthetized, placed on mechanical ventilation for either 2 hrs or 4 hrs with either low tidal volume (8 ml/kg) or high tidal volume (15 ml/kg) with or without positive end-expiratory pressure and recruitment maneuvers.ResultsAlteration of the alveolar-capillary barrier was noted at 2 hrs of high tidal volume ventilation. Standardized histology scores, influx of bronchoalveolar lavage albumin, proinflammatory cytokines, and absolute neutrophils were significantly higher in the high-tidal volume ventilation group at 4 hours of ventilation. Application of positive end-expiratory pressure resulted in significantly decreased standardized histology scores and bronchoalveolar absolute neutrophil counts at low- and high-tidal volume ventilation, respectively. Recruitment maneuvers were essential to maintain pulmonary compliance at both 2 and 4 hrs of ventilation.ConclusionsSigns of ventilator-induced lung injury are evident soon after high tidal volume ventilation (as early as 2 hours) and lung injury worsens with longer-term ventilation (4 hrs). Application of positive end-expiratory pressure and recruitment maneuvers are protective against worsening VILI across all time points. Dynamic compliance can be used guide the frequency of recruitment maneuvers to help ameloriate ventilator-induced lung injury.

Toll-like receptor 2 has a prominent but nonessential role in innate immunity to Staphylococcus aureus pneumonia.

Staphylococcus aureus is an important cause of acute bacterial pneumonia. Toll‐like receptor 2 (TLR2) recognizes multiple components of the bacterial cell wall and activates innate immune responses to gram‐positive bacteria. We hypothesized that TLR2 would have an important role in pulmonary host defense against S. aureus. TLR null (TLR2−/−) mice and wild type (WT) C57BL/6 controls were challenged with aerosolized S. aureus at a range of inocula for kinetic studies of cytokine and antimicrobial peptide expression, lung inflammation, bacterial killing by alveolar macrophages, and bacterial clearance. Survival was measured after intranasal infection. Pulmonary induction of most pro‐inflammatory cytokines was significantly blunted in TLR2−/− mice 4 and 24 h after infection in comparison with WT controls. Bronchoalveolar concentrations of cathelicidin‐related antimicrobial peptide also were reduced in TLR2−/− mice. Lung inflammation, measured by enumeration of bronchoalveolar neutrophils and scoring of histological sections, was significantly blunted in TLR2−/− mice. Phagocytosis of S. aureus by alveolar macrophages in vivo after low‐dose infection was unimpaired, but viability of ingested bacteria was significantly greater in TLR2−/− mice. Bacterial clearance from the lungs was slightly impaired in TLR2−/− mice after low‐dose infection only; bacterial elimination from the lungs was slightly accelerated in the TLR2−/− mice after high‐dose infection. Survival after high‐dose intranasal challenge was 50–60% in both groups. TLR2 has a significant role in early innate immune responses to S. aureus in the lungs but is not required for bacterial clearance and survival from S. aureus pneumonia.

Fluticasone/salmeterol reduces remodelling and neutrophilic inflammation in severe equine asthma

Asthmatic airways are inflamed and undergo remodelling. Inhaled corticosteroids and long-acting β2-agonist combinations are more effective than inhaled corticosteroid monotherapy in controlling disease exacerbations, but their effect on airway remodelling and inflammation remains ill-defined. This study evaluates the contribution of inhaled fluticasone and salmeterol, alone or combined, to the reversal of bronchial remodelling and inflammation. Severely asthmatic horses (6 horses/group) were treated with fluticasone, salmeterol, fluticasone/salmeterol, or with antigen avoidance for 12 weeks. Lung function, central and peripheral airway remodelling, and bronchoalveolar inflammation were assessed. Fluticasone/salmeterol and fluticasone monotherapy decreased peripheral airway smooth muscle remodelling after 12 weeks (p = 0.007 and p = 0.02, respectively). On average, a 30% decrease was observed with both treatments. In central airways, fluticasone/salmeterol reversed extracellular matrix remodelling after 12 weeks, both within the lamina propria (decreased thickness, p = 0.005) and within the smooth muscle layer (p = 0.004). Only fluticasone/salmeterol decreased bronchoalveolar neutrophilia (p = 0.03) to the same extent as antigen avoidance already after 8 weeks. In conclusion, this study shows that fluticasone/salmeterol combination decreases extracellular matrix remodelling in central airways and intraluminal neutrophilia. Fluticasone/salmeterol and fluticasone monotherapy equally reverse peripheral airway smooth muscle remodelling.

Myeloid but not epithelial tissue factor exerts protective anti‐inflammatory effects in acid aspiration‐induced acute lung injury

Essentials Tissue factor (TF) represents a central link between hemostasis and inflammation.We studied the roles of myeloid and airway epithelial TF in acid‐caused acute lung injury (ALI).TF on myeloid cells displays a non‐coagulatory role regulating the inflammatory response in ALI.Airway epithelial TF contributes to hemostatic functions, but is dispensable in ALI pathogenesis. SummaryIntroductionAcute lung injury (ALI) is a life‐threatening condition characterized by damaged alveolar–capillary structures and activation of inflammatory and hemostatic processes. Tissue factor (TF) represents a crucial link between inflammation and coagulation, as inflammatory mediators induce myeloid TF expression, and TF initiates extrinsic coagulation.ObjectiveAs pulmonary inflammation stimulates TF expression and TF modulates immune responses, we aimed to elucidate its impact on ALI. In particular, we wanted to distinguish the contributions of TF expressed on airway epithelial cells and TF expressed on myeloid cells.MethodsMice with different cell type‐specific TF deficiency and wild‐type littermates were intratracheally treated with hydrochloric acid, and leukocyte recruitment, cytokine levels, thrombin–antithrombin (TAT) complexes and pulmonary protein‐rich infiltrates were analyzed.ResultsOur data demonstrate that a lack of epithelial TF did not influence acute responses, as bronchoalveolar neutrophil accumulation 8 h after ALI induction was unaltered. However, it led to mild, prolonged inflammation, as pulmonary leukocyte and erythrocyte numbers were still increased after 24 h, whereas those in wild‐type mice had returned to basal levels. In contrast, myeloid TF was primarily involved in regulating the acute phase of ALI without affecting local coagulation, as indicated by increased bronchoalveolar neutrophil infiltration, pulmonary interleukin‐6 levels, and edema formation, but equal TAT complex formation, 8 h after ALI induction. This augmented inflammatory response associated with myeloid TF deficiency was confirmed in vitro, as lipopolysaccharide‐stimulated TF‐deficient alveolar macrophages released increased levels of chemokine (C‐X‐C motif) ligand 1 and tumor necrosis factor‐α as compared with wild‐type macrophages.ConclusionWe conclude that myeloid TF dampens inflammation in acid‐induced ALI.

Impact of Pulmonary Vein Cryoballoon Ablation on Bronchial Injury.

There is a paucity of data on the mechanisms of cough and hemoptysis that sometimes ensue from cryoballoon ablation of pulmonary veins (Cryo-PV). This study specifically examined the impact of ultra-cold (≤-60 °C, 3 minutes), prolonged (>-55 °C, 6 minutes), and conventional (>-55 °C, 3 minutes) Cryo-PV on lung/bronchial injury. Four healthy adult swine underwent Cryo-PV. Each animal received Cryo-PV to the inferior common trunk and the right superior PV. In 2 animals, 1 PV was treated with 2 ultra-cold (Cryo-AUltra-cold ) and the other with 2 conventional (Cryo-AConventional ) cryoapplications. In the other 2 animals, 1 PV was ablated using 2 prolonged (Cryo-BProlonged ) and the other with 2 conventional (Cryo-BConventional ) applications. The nadir cryoballoon temperatures were lower in Cryo-AUltra-cold versus Cryo-AConventional (-66 ± 6 °C vs. -45 ± 5 °C; P = 0.001), but did not differ between Cryo-BProlonged and Cryo-BConventional (-46 ± 3 °C vs. -49 ± 3 °C; P = 0.123). Post-ablation bronchoscopy revealed immediate mucosal edema and erythema with/without bleeding in the adjacent bronchi in 100% of Cryo-AUltra-cold and 50% of Cryo-AConventional /Cryo-BConventional and Cryo-BProlonged . At 4 hours post-ablation, there were marked increases in bronchoalveolar macrophages (P <0.001), lymphocytes (P = 0.035) and neutrophils (P = 0.001). Furthermore, Cryo-AUltra-cold yielded the largest increase in the macrophage (P = 0.005) and neutrophil (P = 0.034) cell counts. While similar trends were seen in Cryo-BProlonged , these did not reach statistical significance. Cryo-PV can elicit acute bronchial inflammation, bleeding, and mucosal injury. While this was further augmented by ultra-cold cryoapplications, it was also evident to a lesser degree with prolonged and even conventional cryoapplications. The mechanism for this appears to be direct collateral injury.

Lung transcriptional profiling: insights into the mechanisms of ozone-induced pulmonary injury in Wistar Kyoto rats

Acute ozone-induced pulmonary injury and inflammation are well characterized in rats; however, mechanistic understanding of the pathways involved is limited. We hypothesized that acute exposure of healthy rats to ozone will cause transcriptional alterations, and comprehensive analysis of these changes will allow us to better understand the mechanism of pulmonary injury and inflammation. Male Wistar Kyoto rats (10–12 week) were exposed to air, or ozone (0.25, 0.5 or 1.0 ppm) for 4 h and pulmonary injury and inflammation were assessed at 0-h or 20-h (n = 8/group). Lung gene expression profiling was assessed at 0-h (air and 1.0 ppm ozone, n = 3–4/group). At 20-h bronchoalveolar lavage, fluid protein and neutrophils increased at 1 ppm ozone. Numerous genes involved in acute inflammatory response were up-regulated along with changes in genes involved in cell adhesion and migration, steroid metabolism, apoptosis, cell cycle control and cell growth. A number of NRF2 target genes were also induced after ozone exposure. Based on expression changes, Rela, SP1 and TP3-mediated signaling were identified to be mediating downstream changes. Remarkable changes in the processes of endocytosis provide the insight that ozone-induced lung injury and inflammation are likely initiated by changes in cell membrane components and receptors likely from oxidatively modified lung lining lipids and proteins. In conclusion, ozone-induced injury and inflammation are preceded by changes in gene targets for cell adhesion/migration, apoptosis, cell cycle control and growth regulated by Rela, SP1 and TP53, likely mediated by the process of endocytosis and altered steroid receptor signaling.

Erythropoietin reduces acute lung injury and multiple organ failure/dysfunction associated to a scald-burn inflammatory injury in the rat.

Erythropoietin (EPO) is an endogenous regulator of erythropoiesis and is given exogenously as a replacement therapy for selected red blood cell disorders. In the past years, EPO has been emerging as a multifunctional, cytoprotective cytokine with anti-apoptotic, anti-inflammatory, and immunomodulatory properties. We aimed to evaluate the cytoprotective effect of rhEPO (recombinant human EPO) treatment on a rat model of multiorgan dysfunction induced by thermal injury. rhEPO was administered at 1000 U/kg (i.v.) 5 min prior to induction of injury and significantly reduced multiorgan dysfunction markers (liver, kidney, lung, serum cytokine levels). In the lung, rhEPO reduced: histological signs of tissue injury, inflammatory/injury markers on the bronchoalveolar fluid, neutrophil chemotaxis/infiltration, GSK-3β activation, and apoptosis. Our study showed that erythropoietin has the potential to exhibit pleiotropic cytoprotective effects and that it might be an interesting pharmacological strategy in the modulation of acute lung injury, such as the one associated to severe burn.

Anti-inflammatory functions of protein C require RAGE and ICAM-1 in a stimulus-dependent manner.

By binding β 2-integrins both ICAM-1 and the receptor for advanced glycation end products (RAGE) mediate leukocyte recruitment in a stimulus-dependent manner. Using different inflammatory mouse models we investigated how RAGE and ICAM-1 are involved in anti-inflammatory functions of protein C (PC; Ceprotin, 100 U/kg). We found that, depending on the stimulus, RAGE and ICAM-1 are cooperatively involved in PC-induced inhibition of leukocyte recruitment in cremaster models of inflammation. During short-term proinflammatory stimulation (trauma, fMLP, and CXCL1), ICAM-1 is more important for mediation of anti-inflammatory effects of PC, whereas RAGE plays a major role after longer proinflammatory stimulation (TNFα). In contrast to WT and Icam-1−/− mice, PC had no effect on bronchoalveolar neutrophil emigration in RAGE−/− mice during LPS-induced acute lung injury, suggesting that RAGE critically mediates PC effects during acute lung inflammation. In parallel, PC treatment effectively blocked leukocyte recruitment and improved survival of WT mice and Icam-1-deficient mice in LPS-induced endotoxemia, but failed to do so in RAGE-deficient mice. Exploring underlying mechanisms, we found that PC is capable of downregulating intracellular RAGE and extracellular ICAM-1 in endothelial cells. Taken together, our data show that RAGE and ICAM-1 are required for the anti-inflammatory functions of PC.

A Decoy Oligonucleotide to NF-κB Delivered through Inhalable Particles Prevents LPS-Induced Rat Airway Inflammation

The inflammatory process plays a crucial role in the onset and progression of several lung pathologies, including cystic fibrosis (CF), and the involvement of NF-κB is widely recognized. The specific inhibition of NF-κB by decoy oligonucleotides delivered within the lung may be beneficial, although rationally designed systems are needed to optimize their pharmacological response. Prompted by this need, we have developed and tested in vivo an inhalable dry powder for the prolonged delivery of a decoy oligodeoxynucleotide to NF-κB (dec-ODN), consisting of large porous particles (LPPs) based on poly(lactic-co-glycolic) acid. First, LPPs containing dec-ODN (dec-ODN LPPs) were engineered to meet the aerodynamic criteria crucial for pulmonary delivery, to gain an effective loading of dec-ODN, to sustain its release, and to preserve its structural integrity in lung lining fluids. We then investigated the effects of dec-ODN LPPs in a rat model of lung inflammation induced by the intratracheal aerosolization of LPS from Pseudomonas aeruginosa. The results show that a single intratracheal insufflation of dec-ODN LPPs reduced the bronchoalveolar neutrophil infiltration induced by LPS for up to 72 hours, whereas naked dec-ODN was able to inhibit it only at 6 hours. The persistent inhibition of neutrophil infiltrate was associated with reduced NF-κB/DNA binding activity, as well as reduced IL-6, IL-8, and mucin-2 mRNA expression in lung homogenates. We consider it noteworthy that the developed LPPs, preventing the accumulation of neutrophils and NF-κB-related gene expression, may provide a new therapeutic option for the local treatment of inflammation associated with lung disease.

Ticagrelor reduces neutrophil recruitment and lung damage in abdominal sepsis

Platelets play an important role in abdominal sepsis and P2Y12 receptor antagonists have been reported to exert anti-inflammatory effects. Herein, we assessed the impact of platelet inhibition with the P2Y12 receptor antagonist ticagrelor on pulmonary neutrophil recruitment and tissue damage in a model of abdominal sepsis. Wild-type C57BL/6 mice were subjected to cecal ligation and puncture (CLP). Animals were treated with ticagrelor (100 mg/kg) or vehicle prior to CLP induction. Edema formation and bronchoalveolar neutrophils as well as lung damage were quantified. Flow cytometry was used to determine expression of platelet–neutrophil aggregates, neutrophil activation and CD40L expression on platelets. CLP-induced pulmonary infiltration of neutrophils at 24 hours was reduced by 50% in ticagrelor-treated animals. Moreover, ticagrelor abolished CLP-provoked lung edema and decreased lung damage score by 41%. Notably, ticagrelor completely inhibited formation of platelet–neutrophil aggregates and markedly reduced thrombocytopenia in CLP animals. In addition, ticagrelor reduced platelet shedding of CD40L in septic mice. Our data indicate that ticagrelor can reduce CLP-induced pulmonary neutrophil recruitment and lung damage suggesting a potential role for platelet antagonists, such as ticagrelor, in the management of patients with abdominal sepsis.

Glutathione (GSH) and the GSH synthesis gene Gclm modulate plasma redox and vascular responses to acute diesel exhaust inhalation in mice

Context: Inhalation of fine particulate matter (PM2.5) is associated with acute pulmonary inflammation and impairments in cardiovascular function. In many regions, PM2.5 is largely derived from diesel exhaust (DE), and these pathophysiological effects may be due in part to oxidative stress resulting from DE inhalation. The antioxidant glutathione (GSH) is important in limiting oxidative stress-induced vascular dysfunction. The rate-limiting enzyme in GSH synthesis is glutamate cysteine ligase and polymorphisms in its catalytic and modifier subunits (GCLC and GCLM) have been shown to influence vascular function and risk of myocardial infarction in humans.Objective: We hypothesized that compromised de novo synthesis of GSH in Gclm−/+ mice would result in increased sensitivity to DE-induced lung inflammation and vascular effects.Materials and methods: WT and Gclm−/+ mice were exposed to DE via inhalation (300 μg/m3) for 6 h. Neutrophil influx into the lungs, plasma GSH redox potential, vascular reactivity of aortic rings and aortic nitric oxide (NO•) were measured.Results: DE inhalation resulted in mild bronchoalveolar neutrophil influx in both genotypes. DE-induced effects on plasma GSH oxidation and acetylcholine (ACh)-relaxation of aortic rings were only observed in Gclm−/+ mice. Contrary to our hypothesis, DE exposure enhanced ACh-induced relaxation of aortic rings in Gclm−/+ mice.Discussion and conclusion: These data support the hypothesis that genetic determinants of antioxidant capacity influence the biological effects of acute inhalation of DE. However, the acute effects of DE on the vasculature may be dependent on the location and types of vessels involved. Polymorphisms in GSH synthesis genes are common in humans and further investigations into these potential gene-environment interactions are warranted.

Bronchoalveolar neutrophilia inversely correlates with DLCO at diagnosis in asbestosis but not lung function decline at 1 year.

The role of bronchoalveolar lavage (BAL) in the assessment of interstitial lung disease (ILD) remains controversial. Previous studies have demonstrated that BAL cell differential is useful in predicting disease progression in many forms of ILD. We wished to investigate whether BAL had a similar use in predicting disease progression in asbestosis. 21 patients who had significant asbestos exposure, findings of UIP radiologically and BAL performed as part of their investigation were reviewed. There was a significant inverse correlation between percentage BAL neutrophils and percentage predicted DLCO at diagnosis (n=21; P=0.02; r(2)=(-)0.25; CI, (-)0.77(-)0.08), but not with DLCO decline over 1 year. Unlike previous reports in IPF, BAL cell differential is not predictive of decline in classic asbestosis with a UIP pattern and its routine use in this cohort of patients provides little if any additional benefit.