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

吸入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是至关重要的。脂多糖（LPS）是革兰氏阴性细菌膜的主要成分，其吸入通过与上皮toll样受体（TLR-4）相互作用，诱导NF-kappaB和促炎基因和细胞因子的转录，模拟细菌吸入。我们发现豚鼠单次吸入LPS可引起炎症细胞流入肺部和立即气道高反应性（AHR）。多次LPS暴露后，出现持续性AHR和杯状细胞增生。单独接种副流感病毒（PIV3）可引起气道炎症和AHR，但没有杯状细胞增生，表明对微生物挑战的不同反应。我们假设LPS和PIV3的结合会引起更高或不同的反应。豚鼠的肺功能、AHR、炎症细胞内流和杯状细胞组织学将确定体内任何改变的微生物反应。流感病毒（H1N1株WSN33和PR8/34）单独和急性或慢性LPS攻击将被检测，以及麻疹（野生型和疫苗病毒）（mWTFB, mEdmonston）和poly[I:C]。这些治疗后的肺组织学将确定大体组织学（血红素和伊红），胶原染色（Gomori染色）和杯状细胞粘蛋白（阿利新蓝/周期性酸性希夫染色）的肺重塑。TLR-4参与LPS后感染和炎症的先天免疫反应，但与流感感染的炎症无关。流感等病毒入侵气道上皮细胞，释放包括IL-8、IL-6和ifn - β在内的促炎和抗病毒产物，我们将测量NF-kappaB水平作为TLR-4受体活性的指标，BAL液IL-8作为病毒感染的标志物。这些测定将取决于豚鼠和人所用试剂之间足够的交叉反应性。研究将通过从肺组织和灌洗液中培养活病毒，并辅以实时PCR检测病毒核酸，确定病情恶化是否与病毒载量变化有关。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加重病毒感染。然而，类固醇有消炎的好处。注射地塞米松和吸入布地奈德对LPS/病毒的功能和组织学反应以及对病毒载量的影响将被检查，以评估任何抗病毒作用。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？非甾体抗炎（NSAI） COX抑制剂吲哚美辛对病毒感染和炎症的作用也将进行研究。粘膜纤毛清除将通过气道功能和伽玛显像检查。吸入粘液促分泌剂（UTP，组胺）引起气道导度的长期降低，其恢复将是粘液纤毛清除的一个指标。UTP是否影响纤毛粘膜清除和粘液分泌？诱导黏液分泌后进行药物干预。伽玛闪烁成像将监测注入麻醉豚鼠气管的放射性标记99mTc-Sn胶体颗粒的清除情况。在单独接受LPS和叠加病毒的豚鼠中吸入UTP后，将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研究体外病毒对细胞因子产生的影响，并将其与体内研究联系起来。先前暴露于IL-13、细菌产物（LPS）或氧化应激源是否会改变病毒反应？重复LPS暴露使toll样受体脱敏。类固醇和非甾体抗炎药将检查病毒进入细胞和随后的复制。

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.