肺脏巨噬细胞在流感病毒感染后对继发革兰氏阴性细菌感染的作用与机制研究

Lung macrophages, mostly alveolar macrophages (AM) but also include interstitial macrophages (IM), and a few recruited macrophage from blood circulation, are the major immune cell types in maintaining lung immune homeostasis and serves as the first line of defense against bacterial infection. Influenza virus infected patients are susceptible to secondary bacterial infections which increase mobility and mortality, suggesting defects in some or all of the resistance and clearance mechanisms to bacteria. Earlier studies were largely concentrated on the role of lung macrophages in modulating disease severity of secondary infection by Gram-positive bacteria such as Streptococcus pneumoniae, but with little knowledge of the role and pathogenic mechanism in secondary infection by Gram-negative bacteria such as Pseudomonas aeruginosa. In this study, we will employ a novel replication competent influenza A virus carrying either Luciferase or GFP, in combination with a new method of using BrdU incorporation to label newly replicated macrophages and using phagocytic labeling dyes to label pulmonary resident alveolar macrophages, as well as confocal microscopy and multi-color flow cytometry, to analyze macrophage in influenza virus infected versus uninfected tissue and to obtain the dynamic changes and roles of macrophages in relation to time and proximity of infection region. Using real time monitor of bioluminescent image in living mice infected with luciferase influenza virus, secondary infection of Gram-negative bacteria Pseudomonas aeruginosa will be analyzed and compared with the secondary infection by Gram-positive bacteria Streptococcus pneumoniae. The role of macrophages on lung immune homeostasis upon influenza virus infection and secondary bacterial infection will further be studied at the sub-organ tissue level. Macrophage subtypes, including alveolar macrophages (AM), interstitial macrophages (IM), and recruited macrophages, as well as their possible functional subtypes such as M1 and M2, will be assessed. We will also evaluate the local administration (e.g. nasal instillation) of cytokines such as GM-CSF, IFN or neutralizing antibodies such as anti-IL10, anti-IFN on modulating lung macrophages. This study will provide the first view on the role of lung macrophages in influenza associated secondary infection of Gram-negative bacteria and also provide more sophisticated information at the sub-organ level on the role of lung macrophages in influenza associated secondary infection of Gram-positive bacteria. The ultimate goal of this study is to provide new insights for strategies in prevention and treatment of influenza associated secondary bacterial infection.

肺泡巨噬细胞对维持肺部免疫稳态、抵御细菌入侵至关重要，其数量及功能失调是流感感染后肺部易继发细菌感染的主要原因。但是，先前研究多集中于巨噬细胞与继发革兰氏阳性菌感染的关系，对其在继发革兰氏阴性菌感染中的作用了解甚少。我们拟用Luciferase及GFP流感病毒，结合使用BrdU标记新生巨噬细胞及应用胞内吞噬染料标记肺泡巨噬细胞，通过活体荧光成像，实时激光共聚焦，多色流式细胞分析等分析流感继发革兰氏阴性菌感染并对比革兰氏阳性菌感染，通过研究肺巨噬细胞在流感继发革兰氏阴性菌感染的亚群数量及功能变化，进而以GM-CSF、抗细胞因子抗体等调控巨噬细胞，验证假说：肺巨噬细胞的亚群数量及功能的失衡与流感病毒感染存在时间及空间的量效关系，调控巨噬细胞的亚群及功能可维持肺脏免疫稳态并抑制继发性革兰氏阴性菌感染。通过阐释巨噬细胞在流感继发革兰氏阴性细菌感染中的作用及病理机制，为流感继发细菌感染提供干预策略。

中性粒细胞是小鼠抵抗铜绿假单胞菌的重要免疫细胞。虽然流感病毒感染诱导中性粒细胞在肺内聚集增多，但流感病毒感染增加小鼠继发铜绿假单胞菌感染的易感性。删除中性粒细胞显著增强小鼠感染铜绿假单胞菌的易感性，进一步研究表明，虽然流感病毒感染后肺内中性粒细胞增多，中性粒细胞的吞噬功能和髓过氧化物酶的活性降低，使中性粒细胞不能有效地吞噬和消化铜绿假单胞菌，从而导致继发铜绿假单胞菌易感。同时，中性粒细胞产生的过量活性氧也可能是流感感染引起肺损伤的重要机制。然而，细胞因子IL-17增加中性粒细胞募集和活化，或盐酸左氧氟沙星与活性氧抑制剂联用治疗并不能明显改善流感继发铜绿假单胞菌感染小鼠的生存率，表明流感继发感染铜绿假单胞菌易感机制的复杂性；用流感病毒中和抗体治疗或免疫铜绿假单胞菌灭活疫苗可有效降低流感感染小鼠对铜绿假单胞菌的敏感性，为流感继发铜绿假单胞菌感染的防治提供重要线索。