In vivo examination of viral exacerbations of airways inflammation function mucus hypersecretion and mucociliary clearance

吸入的微生物可能是肺部炎症和功能的驱动因素，了解气道对微生物挑战的反应将为识别新药提供见解。因此，气道反应的体内建模至关重要。脂多糖（LPS）是革兰氏阴性菌膜的主要成分，其吸入通过与上皮toll样受体（TLR-4）相互作用而模拟细菌吸入，导致NF-κ B的诱导以及促炎基因和细胞因子的转录。我们发现，豚鼠单次吸入LPS可导致炎性细胞流入肺部和立即气道高反应性（AHR）。反复LPS暴露后，AHR持续存在，杯状细胞增生。单独接种副流感病毒3型（PIV 3）可引起气道炎症和AHR，但无杯状细胞增生，表明对微生物挑战的不同反应。我们假设LPS和PIV 3的组合将诱导增强的或不同的反应。豚鼠的肺功能、AHR、炎性细胞流入和杯状细胞组织学将鉴定体内任何修饰的微生物应答。将检查流感病毒（H1N1毒株WSN 33和PR 8/34）单独和急性或慢性LPS攻毒以及麻疹（野生型和疫苗病毒）（mWTFB，mEdmonston）和聚[I：C]。这些处理后的肺组织学将确定大体组织学（苏木精和伊红）、来自胶原染色（Gomori染色）和杯状细胞粘蛋白（阿尔新蓝/高碘酸希夫染色）的肺重塑。TLR-4参与LPS后对感染和炎症的先天免疫应答，但不参与流感感染的炎症。流感病毒等病毒侵入气道上皮细胞，释放促炎和抗病毒产物，包括IL-8、IL-6和IFN-β，我们将测量NF-κ B水平作为TLR-4受体活性的指标，并测量BAL液IL-8作为病毒感染的标志物。这些试验将取决于豚鼠和人用试剂之间的充分交叉反应性。研究将使用来自肺组织和灌洗液的活病毒培养物，通过真实的时间PCR检测病毒核酸来确定急性加重是否与病毒载量变化相关。在病毒性炎症加重中使用皮质类固醇是违反直觉的，因为它们会降低免疫反应并使病毒感染恶化。然而，类固醇具有抗炎的优点。将针对对LPS/病毒的功能和组织学应答以及针对病毒载量检查注射地塞米松和吸入布地奈德的作用，以评估任何促病毒或抗病毒作用。类固醇是否影响病毒进入上皮细胞和随后的复制，如果是，其机制是什么？还将检查非甾体抗炎药（NSAI）考克斯抑制剂吲哚美辛对病毒感染和炎症的作用。将通过气道功能和伽马射线照相术检查粘膜纤毛清除率。吸入粘液促分泌素（UTP，组胺）导致气道传导性延长降低，其恢复将是粘液纤毛清除的指标。UTP是否影响粘液纤毛清除以及粘液分泌？诱导粘液分泌后，将检查药物干预。γ射线照相术将监测注入麻醉豚鼠气管的放射性标记99 mTc-Sn胶体颗粒的清除。在接受单独LPS和叠加病毒的豚鼠中，在吸入UTP后测量粘膜纤毛清除率。在诺华，人类和豚鼠上皮细胞的原代培养将使学生能够检查体外病毒对细胞因子产生的影响，以与体内研究相关。预先暴露于IL-13、细菌产物（LPS）或氧化应激物是否会改变病毒反应？重复LPS暴露使Toll样受体脱敏。将在病毒进入细胞和随后复制时检查类固醇和NSAID。

Inhaled microbes are likely drivers of lung inflammation and function and understanding airway responses to microbial challenge will provide insights to identify novel drugs. In vivo modelling of the airway responses is therefore critical. Lipopolysaccharide (LPS) is a major component of gram negative bacterial membranes, whose inhalation mimics bacterial inhalation by interacting with epithelial toll-like receptors (TLR-4) resulting in induction of NF-kappaB and transcription of proinflammatory genes and cytokines. We showed that single LPS inhalations by guinea-pigs cause inflammatory cell influx to the lungs and immediate airways hyperreactivity (AHR). After repeated LPS exposure, there was persistent AHR and goblet cell hyperplasia. Parainfluenza3 virus (PIV3) inoculation alone caused airways inflammation and AHR but no goblet cell hyperplasia indicating differential responses to microbial challenges. We hypothesise that combining LPS and PIV3 will induce heightened or differential responses. Lung function, AHR, inflammatory cell influx and goblet cell histology in guinea-pigs will identify any modified microbial responses in vivo. Influenza virus (H1N1 strains WSN33 and PR8/34) alone and with acute or chronic LPS challenge will be examined as well as measles (wildtype and vaccine virus) (mWTFB, mEdmonston) and poly[I:C]. Lung histology after these treatments will determine gross histology (haematoxylin and eosin), lung remodelling from collagen staining (Gomori's stain) and goblet cell mucin (Alcian blue/periodic acid Schiff stain). TLR-4 are implicated in innate immune responses to infection and inflammation after LPS, but not inflammation from influenza infection. Invasion of airways epithelial cells by viruses like influenza, release proinflammatory and antiviral products including IL-8, IL-6 and IFN-beta and we will measure NF-kappaB levels as an index of TLR-4 receptor activity and BAL fluid IL-8 as a marker of viral infection. These assays will depend upon sufficient cross-reactivity between reagents for guinea-pig and human. Studies will determine whether exacerbations are associated with changes in viral load using culture of live virus from lung tissue and lavage fluid, supported by real time PCR to detect viral nucleic acids. Corticosteroid use in viral inflammatory exacerbations is counter-intuitive since they should reduce immune responses and worsen viral infection. However, steroids have the advantage of being anti-inflammatory. The effects of injected dexamethasone and inhaled budesonide will be examined against functional and histological responses to LPS/virus and against viral load to assess any pro- or antiviral effects. Do steroids affect viral entry to epithelial cells and subsequent replication, and if so, what is the mechanism? A non-steroidal anti-inflammatory (NSAI) COX inhibitor, indomethacin, on viral infection and inflammation will also be examined. Mucociliary clearance will be examined from airway function and gamma scintigraphy. Inhaled mucus secretagogues (UTP, histamine) cause prolonged reduction in airways conductance, recovery of which will be an index of mucociliary clearance. Does UTP affect mucociliary clearance as well as mucus secretion? Drug interventions will be examined after inducing mucus secretion. Gamma scintigraphy will monitor clearance of radiolabelled 99mTc-Sn colloid particles instilled into anaesthetized guinea-pig trachea. Mucociliary clearance will be measured following UTP inhalation in guinea-pigs receiving LPS alone and superimposed virus. At Novartis, primary cultures of human and guinea-pig epithelial cells will enable the student to examine in vitro viral effects on cytokine production to relate to in vivo studies. Do prior exposure to IL-13, bacterial products (LPS) or oxidant stressors alter viral responses? Repeated LPS exposure desensitizes toll-like receptors. Steroids and NSAIs will be examined on viral entry into cells and subsequent replication.