Jing Bi

Deletion of peroxiredoxin 6 potentiates lipopolysaccharide-induced acute lung injury in mice*

Objective:To investigate the role and signaling pathway of peroxiredoxin 6, a newly identified peroxidase, in lipopolysaccharide-induced acute lung injury. Design:Prospective, randomized, controlled study. Setting:Research laboratory. Subjects:Peroxiredoxin 6 (−/−) and wild-type C57BL/6 mice. Interventions:Wild-type or peroxiredoxin 6 (−/−) mice were challenged by intratracheal instillation of lipopolysaccharide (5 mg/kg) for 4 hrs or 24 hrs for lung injury measurement. In other studies, peritoneal macrophages, isolated from wild-type and peroxiredoxin 6 (−/−) mice, were preincubated in presence or absence of mitogen-activated protein kinases inhibitors for 30 mins before being stimulated with lipopolysaccharide (1 &mgr;g/mL) for 4 hrs. Measurements and Main Results:Bronchoalveolar lavage myeloperoxidase activity and the lung injury score were significantly increased in peroxiredoxin 6 (−/−) mice compared with wild-type mice after lipopolysaccharide instillation at both 4 hrs and 24 hrs. Hydrogen peroxide and malondialdehyde levels, as well as nuclear factor-&kgr;B activities, tumor necrosis factor-&agr;, interleukin-1&bgr;, and matrix metalloproteinase-9 messenger RNA, protein concentration, and activities were significantly increased whereas total antioxidative capability was markedly decreased in lungs of peroxiredoxin 6 (−/−) mice compared with wild-type mice. In vitro studies showed intracellular reactive oxygen species levels and release of tumor necrosis factor-&agr;, interleukin-1, and matrix metalloproteinase-9 were significantly increased in macrophages from peroxiredoxin 6 (−/−) mice compared with that from wild-type mice after lipopolysaccharide stimulation. Cytokines release was partially suppressed by extracellular signal-regulated kinase and c-Jun N-terminal kinase inhibitors, but not by the p38 mitogen-activated protein kinase inhibitor. Conclusions:Deletion of peroxiredoxin 6 exaggerates lipopolysaccharide-induced acute lung injury and inflammation with increased oxidative stress, inflammatory responses, and matrix degradation, all of which were partially dependent on nuclear factor-&kgr;B, extracellular signal-regulated kinase, and c-Jun N-terminal kinase pathways.

KGF‐2 targets alveolar epithelia and capillary endothelia to reduce high altitude pulmonary oedema in rats

High altitude pulmonary oedema (HAPE) severely affects non‐acclimatized individuals and is characterized by alveolar flooding with protein‐ rich oedema as a consequence of blood‐gas barrier disruption. Limited choice for prophylactic treatment warrants effective therapy against HAPE. Keratinocyte growth factor‐2 (KGF‐2) has shown efficiency in preventing alveolar epithelial cell DNA damages in vitro. In the current study, the effects of KGF‐2 intratracheal instillation on mortality, lung liquid balance and lung histology were evaluated in our previously developed rat model of HAPE. We found that pre‐treatment with KGF‐2 (5 mg/kg) significantly decreased mortality, improved oxygenation and reduced lung wet‐to‐dry weight ratio by preventing alveolar‐capillary barrier disruption demonstrated by histological examination and increasing alveolar fluid clearance up to 150%. In addition, KGF‐2 significantly inhibited decrease of transendothelial permeability after exposure to hypoxia, accompanied by a 10‐fold increase of Akt activity and inhibited apoptosis in human pulmonary microvascular endothelial cells, demonstrating attenuated endothelial apoptosis might contribute to reduction of endothelial permeability. These results showed the efficacy of KGF‐2 on inhibition of endothelial cell apoptosis, preservation of alveolar‐capillary barrier integrity and promotion of pulmonary oedema absorption in HAPE. Thus, KGF‐2 may represent a potential drug candidate for the prevention of HAPE.

Keratinocyte growth factor-2 intratracheal instillation significantly attenuates ventilator-induced lung injury in rats.

Preservation or restoration of normal alveolar epithelial barrier function is crucial for pulmonary oedema resolution. Keratinocyte growth factor‐2 (KGF‐2), a potent epithelial cell mitogen, may have a role in preventing ventilator‐induced lung injury (VILI), which occurs frequently in mechanically ventilated patients. The aim of the study was to test the role of KGF‐2 in VILI in rats. Forty healthy adult male Sprague‐Dawley rats were randomly allocated into four groups, where rats in Groups HVZP (high‐volume zero positive end‐expiratory pressure) and HVZP+KGF‐2 were given intratracheally equal PBS and 5 mg/kg KGF‐2 72 hrs before 4 hrs HVZP ventilation (20 ml/kg), respectively, while PBS and KGF‐2 were administered in the same manner in Groups Control and KGF‐2, which underwent tracheotomy only with spontaneous breathing. Inflammatory cytokines (tumour necrosis factor‐α, macrophage inflammatory protein 2), neutrophil and total protein levels in bronchoalveolar lavage fluid and surfactant protein mRNA expression in lung tissue were detected; the number of alveolar type II cells, lung water content and lung morphology were also evaluated. The results indicate that pre‐treatment with KGF‐2 showed dramatic improvement in lung oedema and inflammation compared with HVZP alone, together with increased surfactant protein mRNA and alveolar type II cells. Our results suggest that KGF‐2 might be considered a promising prevention for human VILI or other acute lung injury diseases.

Keratinocyte growth factor-2 is protective in lipopolysaccharide-induced acute lung injury in rats.

Keratinocyte growth factor-2 (KGF-2) plays a key role in lung development, but its role in acute lung injury has not been well characterized. Lipopolysaccharide instillation caused acute lung injury, which significantly elevated lung wet-to-dry weight ratio, protein and neutrophils in bronchoalveolar lavage fluid (BALF), inhibited surfactant protein A and C expression in lung tissue, and increased pathological injury. Pretreatment with KGF-2 improved the above lung injury parameters, partially restored surfactant protein A and C expression, and KGF-2 given 2-3 days before LPS challenge showed maximum lung injury improvement. Pretreatment with KGF-2 also markedly reduced the levels of TNF-α, MIP-2, IL-1β and IL-6 in BALF and the levels of IL-1β and IL-6 in lung tissue. Histological analysis showed there was increased proliferation of alveolar type II epithelial cells in lung parenchyma, which reached maximal 2 days after KGF-2 instillation. Intratracheal administration of KGF-2 attenuates lung injury induced by LPS, suggesting KGF-2 may be potent in the intervention of acute lung injury.

Emphysema is an independent risk factor for 5‐year mortality in patients with bronchiectasis

Bronchiectasis is a common disabling respiratory disease in China. However, the literatures that focused on the long‐term prognosis and the risk factors for mortality are limited.

A20 protein regulates lipopolysaccharide-induced acute lung injury by downregulation of NF-κB and macrophage polarization in rats

Modulation of inflammation is a crucial component of the development of acute lung injury. A20, a ubiquitin editing enzyme, may regulate cellular inflammatory reactions, particularly those involving the signaling pathway of nuclear factor NF-κB (NF‑κB). The present study investigated the mechanism by which A20 downregulated NF‑κB and further contributed to macrophage polarization from the M1 to M2 phenotypes in lipopolysaccharide (LPS)‑induced lung injury. Sprague‑Dawley rats injected with LPS were used in the present study. Bronchoalveolar lavage fluid and lung tissue were collected from each experimental rat. A macrophage cell line was used to test the expression levels of A20. Tumor necrosis factor‑α (TNF‑α), interleukin‑1 beta (IL‑1β) and NF‑κB activities were assessed by ELISA and polymerase chain reaction. Macrophage phenotypes were assayed using fluorescence‑activated cell sorting. Elevated levels of TNF‑α, IL‑1β, NF‑κB and A20 were observed in the macrophages of rats treated with LPS. Furthermore, A20 overexpression inhibited NF‑κB DNA binding activity and increased macrophage polarization from the M1 to M2 phenotype in lung macrophages of the NR8383 cell line. It was concluded that the A20 protein in macrophages modulates lung injury induced by LPS. The overexpression of A20 in macrophages may be involved in modulating macrophage polarization. The mechanisms and molecular identification of macrophage polarization activation may provide a basis for the treatment of inflammation in lung injury.

Potential Role of CC Chemokines and their Receptors in the Development of Respiratory Diseases

Increasing evidence suggests that the chemokine system coordinates leukocyte migration in immunity and in- flammation, involving in the pathogenesis of many pulmonary diseases. Chemokines are small proteins which interact with specific receptors to exert their chemotactic functions for inflammatory cells and constitutive cells. Among the com- plex system, CC chemokine receptors (CCRs) represent a subfamily of chemokine receptor, which is considered to play an important role in the pathogenesis of diseases characterized by disordered inflammation and immunity, among which some respiratory diseases, such as asthma, chronic obstructive pulmonary disease, acute lung injury, comprise one impor- tant part. The present paper reviewed the research of possible relationship and progress between this system and human lung diseases. Chemokines and their receptors are essential components of Th1- and Th2-mediated responses via the re- cruitment of lymphocytes and macrophages. The local expression of homeostatic chemokines in pulmonary also plays an important role in protective immune responses. Thus, it is important to understand the potential role of CCRs in respira- tory diseases and it may provide exciting new targets for therapeutic intervention.

Recovery from acute lung injury can be regulated via modulation of regulatory T cells and Th17 cells

Acute lung injury (ALI) is a severe inflammatory disease, for which no specific treatment exists. The decreased ratio of regulatory T cells (CD4+CD25+FoxP3 Tregs) and Th17 cells is implicated in ALI and inflammation. We here investigated whether maintaining the balance of CD4+CD25+Foxp3+Tregs and Th17 cells can alleviate lung injury. For CD4+CD25+FoxP3 Treg depletion, 200 μg of an anti‐CD25 antibody was administered intraperitoneally per mouse on days −3 and −1 before lipopolysaccharide (LPS) instillation. And 150 μg of TGF‐β was administered intraperitoneally per mouse on day 0 after LPS instillation. To down‐regulate of Th17 cells, 200 μg per mouse of isotype, IL‐17 or IL‐22 antibodies were injected intraperitoneally into mice at days 0 after LPS instillation. We detected lung morphology; lung wet‐to‐dry weight ratio; protein concentration, the count of total cells, neutrophils and macrophages, and cytokines in bronchoalveolar lavage fluid (BALF). And we also evaluated the percentage of CD4+CD25+Foxp3+Tregs in lung, and Th17 cells in lung. CD4+CD25+Foxp3+Tregs depletion via anti‐CD25 treatment or TGF‐β neutralization delayed recovery of ALI. The prolonged inflammation was mainly dominated by neutrophils, macrophages and Th17 cells. Furthermore, inhibition of Th17 cells via monoclonal antibodies against IL‐17 and IL‐22 alleviated ALI inflammation by inhibiting the recruitment of neutrophils and macrophages, increasing the number of CD4+CD25+Foxp3+Tregs. Our findings support a critical role for CD4+CD25+Foxp3+Tregs in regulating from ALI pathophysiology, and a potential therapeutic role for the inhibition of Th17 cells in ALI treatment. These findings provide a rationale for treating patients with ALI by modulating CD4+CD25+Foxp3+Tregs and Th17 cells.