~目的：流感病毒是引起人类呼吸道疾病的主要原因，世界卫生组织也将流感病毒感染确定为全球重大公共卫生问题之一。近年来，随着天然免疫细胞“记忆”功能的发现，天然免疫和获得性免疫在病原体感染炎症中的作用机制成为宿主抗感染领域的热点。作为承接天然免疫和获得性免疫的关键衔接点，趋化因子在炎症部位淋巴细胞的转运和招募中有着重要的作用，尤其是CXCL5在中性粒细胞募集到炎症部位的过程中起关键作用，因此筛选流感感染介导的急性肺损伤相关的关键蛋白分子或细胞调控因子，揭示甲型流感病毒感染导致肺损伤的具体分子机制，将为流感治疗提供潜在创新药物靶点，这不仅具有临床治疗意义，也将为今后小蛋白分子在抗感染中的研究提供重要指示。
方法：在本课题中，我们以野生型WT小鼠和趋化因子CXCL5敲除小鼠作为研究对象，麻醉后以滴鼻的方式感染H1N1流感毒株，通过临床症状的观察、免疫组化实验以及ELISA实验来探索趋化因子CXCL5在H1N1感染中的作用;继而使用质谱流式技术筛选到趋化因子CXCL5敲除小鼠肺组织中突出的免疫细胞亚群；通过免疫荧光实验和病理切片分析H1N1感染后的WT和CXCL5敲除小鼠肺组织滞留B细胞的积聚特征；最后我们通过二代测序技术对CXCL5敲除小鼠肺组织中形成iBALT结构的因素进行分析，鉴定CXCL5缺陷小鼠体内对iBALT结构形成和维持有促进作用的差异基因，为进一步了解iBALT结构的形成和维持奠定基础。
结果：流感病毒H1N1感染后，CXCL5敲除小鼠肺组织中性粒细胞募集显著下降，作为免疫细胞的“先头兵”，中性粒细胞是解决原发性甲型流感病毒感染的关键。本课题趋化因子CXCL5的敲除虽然减少了中性粒细胞的募集，但是缺陷小鼠肺组织病毒颗粒的清除能力不降反增，我们猜测它可能是通过调控IL-6的表达来抵抗流感病毒的感染；同时我们发现CXCL5敲除小鼠肺组织中肺滞留B细胞显著增多，且主要聚集在支气管上皮的基部或肺血管周围，形成有明显T细胞和B细胞分区的三级淋巴结构-iBALT。在验证中发现趋化因子CXCL5的缺失，导致肺组织中很多调控因子如B细胞趋化因子CXCL13的表达量大幅升高，促进B细胞在肺组织中的积聚，进而创造了一个更易于形成iBALT结构的良好微环境，并且在数量、面积和完整性上不同于WT小鼠组，而CXCL5敲除小鼠肺组织形成的iBALT结构在流感感染后能够快速启动局部肺免疫应答从而抵抗流感的感染。
结论：趋化因子CXCL5在H1N1感染起着重要的的作用，而H1N1的感染导致CXCL5敲除小鼠的肺组织中易于形成一种能够快速启动的三级淋巴组织iBALT结构，iBALT结构在机体内抗流感感染过程中发挥重要作用。
创新：我们发现趋化因子CXCL5的敲除导致炎性细胞的积聚减少，伴随着趋化因子CXCL13的高表达；CXCL13是B细胞的关键驱动因子，促进B细胞在缺陷小鼠肺组织中大量积聚，形成一种可先于获得性免疫系统抗病毒反应的免疫应答结构-诱导性支气管相关淋巴组织（iBALT），由此设想趋化因子CXCL5的敲除很可能创造了一种易于形成iBALT结构的微环境，这种结构能够在流感感染过程中快速被启动用以保护机体。但是目前对于肺部感染或慢性炎症诱导形成iBALT结构的机制以及维持iBALT存在的关键因素尚不明朗。
 

~Objective Influenza virus is the main cause of human respiratory diseases. The World Health Organization has also identified influenza virus infection as one of the major global public health problems. In recent years, with the discovery of the "memory" function of innate immune cells, the mechanism of action of innate and acquired immunity in pathogen infection inflammation has become a hot spot in the field of host anti-infection. As the key part of innate and acquired immunity in join point, chemokines in arthritic lymphocyte plays an important role in their transfer and recruitment, especially CXCL5 raised parts of inflammation in neutrophils play a key role in the process, so the screening of influenza infection mediated acute lung injury related key protein molecules or cellular control factor, revealing the influenza a virus infection lead to specific molecular mechanism of lung injury, will provide creative potential drug targets for the treatment, the clinical significance, not only will also be small protein molecules for the future research in anti-infection provides important instructions.
Method In this study, wild-type WT mice and chemokine CXCL5 -/- mice were used as research objects. After anesthesia, the H1N1 influenza strain was infected by nasal drops. The role of the chemokine CXCL5 in H1N1 infection was explored through observation of clinical symptoms, immunohistochemical experiment and ELISA experiment. Then, mass spectrometry was used to screen the prominent immune cell subsets in the lung tissues of mice with CXCL5 -/-. Immunofluorescence assay and pathological sections were used to analyze the accumulation characteristics of retained B cells in the lung tissues of WT and CXCL5 -/- mice infected with H1N1. Finally, we analyzed the factors of iBALT structure formation in the lung tissues of CXCL5 -/- mice through second-generation sequencing technology, and identified the differential genes that promote the formation and maintenance of iBALT structure in CXCL5 deficient mice, laying a foundation for further understanding of the formation and maintenance of iBALT structure.
Results: The recruitment of neutrophils in the lung tissues of CXCL5 -/- mice significantly decreased after the infection of influenza A H1N1 virus. Neutrophils were the key to solve the primary influenza A virus infection as the "first frontier" of immune cells. In this study, the knockout of CXCL5, a chemokine, reduced the recruitment of neutrophils, but the clearance ability of virus particles in the lung tissues of defective mice increased instead of decreased. We speculated that it might be to resist the infection of influenza virus by regulating the expression of IL-6. At the same time, we found that pulmonary b-cell retention in the lung tissues of CXCL5 -/- mice significantly increased, and mainly gathered at the base of bronchial epithelium or around pulmonary vessels, forming a tertiary lymphoid structure -iBALT with distinct T cells and B cell divisions. Found in the validation chemokines CXCL5, bring many regulatory factors such as B cells in the lung tissue chemokine expression of CXCL13 quantity, promote accumulation of B cells in the lung tissue, which creates an easier to form iBALT structure of micro environment, and on the number, size and integrity differs from that of WT mice group, while CXCL5 -/-  mice iBALT structure of lung tissue formation after flu infection can kick-start local lung immune response against influenza infection.
Conclusion The chemokine CXCL5 plays an important role in H1N1 infection, which leads to the formation of a fast-starting tertiary lymphoid tissue iBALT structure in the lung tissue of CXCL5 -/- mice. IBALT plays an important role in the body's resistance to influenza infection.
Innovate Induced broncho-associated lymphoid tissue (iBALT) is formed by the body under the stimulation of pathogen infection or inflammation and other factors. It is generally distributed near the base of bronchial epithelium or around pulmonary vessels, and there are obvious T cells and B cells in it, including stromal cells, lymphoid vessels and high endothelial venules. It has been reported that iBALT plays an important role in lung infection, interstitial lung disease, non-small cell lung cancer and other diseases and may be related to its pathogenesis. IBALT is a tertiary lymphoid tissue similar to the secondary lymphoid organ in structure. It has been proven to initiate a similar natural immune response and then regulate the adaptive immune process. However, iBALT has two sides. It can also exacerbate local tissue damage in autoimmune diseases. At present, the mechanism of iBALT formation induced by pulmonary infection or chronic inflammation and the key factors to maintain the presence of iBALT are still unclear.