中性粒细胞（neutrophils）和巨噬细胞（macrophages）等固有免疫细胞是机体抗病原微生物感染的重要免疫防线，在抗细菌、病毒等病原微生物感染中发挥了重要作用。病原微生物入侵机体，中性粒细胞首先募集到感染部位，通过吞噬、脱颗粒和形成中性粒细胞胞外诱捕网（neutrophil extracellular trap，NET）等，对病原微生物进行杀伤和清除，并及时将病原微生物入侵信息向其他免疫细胞传递，召集更多的免疫细胞发挥抗感染作用。募集而来的免疫细胞中，巨噬细胞是抗病原微生物感染的“主力军”，通过吞噬和细胞因子分泌等功能发挥病原微生物杀伤和清除作用。尽管短时间细胞因子大量分泌常可引起细胞因子风暴（cytokine storm），导致病情加重。但巨噬细胞适度的细胞因子分泌，有助于病情缓解。因此，在某种程度上，巨噬细胞功能的强弱明显相关于病情轻重。先抵达炎症部位的中性粒细胞和随后募集的巨噬细胞都在抗病原微生物感染中发挥重要作用，但是中性粒细胞对巨噬细胞功能的调控机理尚不清楚。前期研究成果显示，离子通道Piezo1在固有免疫细胞树突状细胞（dendritic cells，DC）和巨噬细胞中发挥关键调控作用。但其是否在中性粒细胞中具有调控作用，目前尚未见报道。本论文围绕中性粒细胞Piezo1对巨噬细胞功能调控作用和机制展开研究。

本研究应用急性呼吸道流感病毒感染小鼠模型观察了病毒感染局部免疫细胞浸润变化情况。随着病毒感染时间延长，小鼠病情逐渐加重，病变感染局部支气管肺泡灌洗液中性粒细胞和巨噬细胞局部浸润显著增多。具体表现为疾病临床分数增高，局部浸润中性粒细胞NET形成增多，炎性巨噬细胞分化和促炎细胞因子分泌等功能增强。而且，中性粒细胞NET形成百分率明显与局部集聚的巨噬细胞数目、功能及疾病临床分数呈现正相关。这提示，中性粒细胞NET形成可能与巨噬细胞浸润、功能及病毒感染结局相关联。采用DNase Ⅰ处理阻断中性粒细胞NET形成，观察对急性病毒感染的影响。结果显示，虽然病毒感染病情加重，中性粒细胞NET明显增多，炎性巨噬细胞浸润和功能增强。但是DNase I处理阻断中性粒细胞NET后，疾病临床分数明显降低，巨噬细胞局部浸润减少和炎性巨噬细胞功能受到抑制。这说明，中性粒细胞NET在调控巨噬细胞功能和感染进程中具有重要作用。为明确发挥调节作用的NET的来源，采用体外实验观察了中性粒细胞NET对巨噬细胞功能分化的影响。细菌脂多糖（lipopolysaccharide，LPS）诱导中性粒细胞体外产生NET，分别分离培养上清、细胞沉淀或者两者混合，观察了NET对骨髓来源巨噬细胞功能分化的调控作用。结果显示，培养上清中的NET与中性粒细胞沉淀中NET都可以有效诱导炎性巨噬细胞功能分化，抑制抗炎巨噬细胞功能分化。进一步，分离纯化LPS和病毒感染诱导中性粒细胞NET的DNA成分，直接观察其对巨噬细胞功能分化的调控作用。结果显示，细菌LPS与病毒感染NET的DNA可以直接诱导炎性巨噬细胞功能分化，进而抑制抗炎巨噬细胞功能分化。综合以上结果，中性粒细胞可以通过NET的DNA有效介导炎性巨噬细胞功能分化并在抗急性病毒感染中发挥调控作用。

巨噬细胞可以通过toll样受体9（toll-like receptors 9，TLR9）或者细胞质中的环鸟苷酸-腺苷酸合酶（cyclic GMP-AMP synthase，cGAS），感知胞质中DNA信号，并触发免疫应答。有趣的是，急性病毒感染小鼠肺泡灌洗液中巨噬细胞TLR9和cGAS表达明显升高，而DNase Ⅰ处理阻断NET产生，却可以使巨噬细胞TLR9的cGAS表达明显下调。采用特异性信号阻断剂处理，体外观察了TLR9抑制剂（TLR9i）和cGAS抑制剂（cGASi）在中性粒细胞NET对巨噬细胞功能分化的调控作用。结果显示，虽然NET-DNA可以明显上调TLR9和cGAS表达和促进炎性巨噬细胞功能，但是TLR9i和cGASi处理后，明显下调了TLR9表达信号和cGAS、干扰素基因刺激因子（stimulator of interferon genes，STING）及其下游干扰素调节因子3（IFN regulatory factor 3，IRF3）等信号表达，并明显抑制了炎性巨噬细胞功能。这说明，中性粒细胞NET-DNA就是通过触发巨噬细胞TLR9和cGAS-STING信号途径介导了炎性巨噬细胞功能分化并在抗急性病毒感染中发挥调控作用。

急性感染中，中性粒细胞可以通过产生活性氧（reactive oxygen species，ROS）和表达CXC趋化因子受体2（chemokine C-X-C motif receptor 2，CXCR2）等通过蛋白质精氨酸脱亚胺酶4（protein-arginine deaminase type 4，PAD4）介导NET形成等发挥调控作用。在急性流感病毒感染疾病进程中，PAD4，ROS和CXCR2的表达明显上调。这说明病毒感染后PAD4依赖性ROS/CXCR2信号通路在调控中性粒细胞NET形成中发挥调控作用。生物信息学转录组分析感染小鼠肺泡灌洗液中性粒细胞变化。深入筛选发现离子通道Piezo1基因表达明显升高。采用Piezo1髓系条件敲除小鼠（Piezo1^-/-）开展研究。体外实验发现，给与Piezo1激动剂（Yoda1）处理， Yoda1可以明显上调中性粒细胞Piezo1表达，促进中性粒细胞ROS产生、表达CXCR2细胞增多和NET形成，而Piezo1缺失明显下调了中性粒细胞Piezo1表达和抑制了中性粒细胞ROS产生、表达CXCR2细胞增多和NET形成。这表明，Piezo1在调控中性粒细胞功能和NET形成中发挥重要作用。

流感病毒感染后，中性粒细胞TLR7表达明显升高，而且Piezo1与TLR7表达存在共定位。进一步分析病毒感染后肺泡灌洗液中性粒细胞转录组学调控这些重要功能的关键基因，筛选到18个关键分子。发现去乙酰化酶2（sirtuin2，SIRT2）和缺氧诱导因子1α（hypoxia induced factor 1α，HIF1α）可能在急性病毒感染Piezo1调控中性粒细胞功能和NET形成中发挥调控作用。采用SIRT2和HIF1α髓系条件敲除小鼠（Sirt2^-/-和Hif1α^-/-）开展深入机制研究。结果显示，急性病毒感染，中性粒细胞产生ROS和表达CXCR2及PAD4明显上调，同时NET形成增多。虽然Yoda1上调Piezo1明显促进了中性粒细胞ROS、CXCR2及PAD4上调及NET形成，但是SIRT2或者HIF1α缺失明显逆转了中性粒细胞功能变化和NET形成。这说明，急性病毒感染中性粒细胞Piezo1是通过SIRT2或者HIF1α发挥功能调控作用。

总之，本研究采用急性流感病毒感染小鼠模型，发现急性流感病毒感染触发中性粒细胞Piezo1通过NET介导炎症巨噬细胞功能分化并在抗感染中发挥重要调控作用。中性粒细胞NET-DNA成分是通过巨噬细胞TLR9和cGAS-STING信号指导炎性巨噬细胞功能分化。病毒感染触发中性粒细胞TLR7和Piezo1，通过SIRT2或HIF1α信号途径，调控中性粒细胞PAD4、ROS和CXCR2表达及NET形成等并指导巨噬细胞功能分化在抗急性病毒感染中发挥关键调控作用。该研究为深入理解急性病毒感染中性粒细胞对巨噬细胞功能分化的调控作用提供了实验基础，也为靶向天然免疫细胞干预急性感染治疗策略研究提供了实验依据。

Innate immune cells such as neutrophils and macrophages form an important immune defense line against pathogenic microbial infections, which play an important role in anti-bacterial, viral and other pathogenic microbial infections. When pathogenic microorganisms invade the host, neutrophils are first arriving at the infection site, they can kill and eliminate pathogenic microorganisms via phagocytosis, degranulation, and the formation of neutrophil extracellular trap (NET). Meanwhile, they can also transmit the invasion signals of pathogenic microorganisms to other immune cells in time. So as to recruited to more immune cells to exert anti-infection effects. Among the subsequent immune cells, macrophages are the main force of resistance to pathogenic microbial infection. Generally, macrophages have effects on killing and eliminating pathogenic microorganisms through phagocytosis and cytokine secretion. A short period of high cytokine secretion often causes cytokine storm, leading to an exacerbation of the disease. However, proper macrophage function is helpful to the recovery of disease. Thus, to some extent, the strength of macrophage function is clearly related to the severity of the disease. Therefore, both neutrophils that arrive at the site of inflammation firstly and macrophages that recruited subsequently participate anti-microbial infection. But the mutual regulation mechanism between them is still unclear. Previous studies have shown that the ion channel, like Piezo1, plays an important regulatory role in dendritic cells (DCs) and macrophages. But whether it has a regulatory role in neutrophils is still unclear. This study focused on the regulatory effect and mechanism of neutrophil Piezo1 on the function of macrophages.

We adopted a mouse model of acute respiratory influenza virus infection to study the changes of local immune cell infiltration after virus infection. It was found that with the extension of virus infection time, the disease of the mice gradually aggravated. Besides, the local infiltration of neutrophils and macrophages in the bronchoalveolar lavage fluid of the infected area was significantly increased. It was manifested as up-regulated the clinical scores, increased locally infiltrating neutrophil, as well as promoted formation of NET. The differentiation and cytokine secretion of inflammatory macrophages enhanced. Moreover, the percentage of NET was significantly positively correlated with the number and function of locally aggregated macrophages and clinical scores of diseases. These suggest that NET formation is associated with infiltration and function of macrophages and outcome of viral infection. The regulatory effect on acute viral infection was detected by blocking NET formation with DNase Ⅰ treatment. The results showed that with the aggravation of viral infection, the number of NET and the infiltration and function of inflammatory macrophages significantly increased. However, after blocking the neutrophil NET with DNase I treatment, the disease clinical score was significantly decreased, thelocal macrophage infiltration was reduced and the inflammatory macrophage function was inhibited. This suggested that NET plays an important role in regulating macrophage function and infection progression. To clarify the origin and role of NET, the effect of NET on the functional differentiation of macrophages was detected in vitro. Lipopolysaccharide (LPS) was used to induce the production of NET from neutrophils in vitro. The effects of NET on the differentiation of bone marrow-derived macrophages were observed during the isolation and co-cultured with the supernatant, cell precipitation or mixture of both. These results showed that NET in either culture supernatant or neutrophils could effectively induce the functional differentiation of inflammatory macrophages and inhibit the functional differentiation of anti-inflammatory macrophages. Furthermore, the DNA components of NET induced by LPS and virus infection were isolated and purified. Their regulatory effects on direct functional differentiation of macrophages were observed. The results showed that bacterial LPS or viral infected NET-DNA could directly induce the functional differentiation of inflammatory macrophages but inhibit the functional differentiation of anti-inflammatory macrophages. To sum up, neutrophils can effectively regulate the differentiation of inflammatory macrophages through the DNA of NET and play a regulatory role in anti-acute viral infection.

It has been reported that macrophages can sense DNA signals in the cytoplasm by toll-like receptors 9 (TLR9) or guanosine monophosphate-adenosine monophosphate synthase (cGAS) in the cytoplasm and trigger an immune response. Interestingly, the expression of TLR9 and cGAS in macrophages was significantly increased in bronchoalveolar lavage fluid of mice with acute viral infection. However, blocking NET production by DNase Ⅰ treatment significantly down-regulated TLR9 and cGAS in macrophages. We used specific signal blockers to observe the regulatory effect of TLR9 inhibitor (TLR9i) and cGAS inhibitor (cGASi) on the functional differentiation of macrophages induced by NET in vitro. The results showed that although NET-DNA could significantly up-regulate TLR9 and cGAS expression and promote the function of inflammatory macrophages, after TLR9i and cGASi treatment, the expression of TLR9 and cGAS-stimulator of interferon genes (STING) and its downstream IFN regulatory factor 3 (IRF3) were significantly down-regulated. It also significantly inhibited the function of inflammatory macrophages. These suggest that NET-DNA regulates the differentiation of inflammatory macrophages by triggering TLR9 and cGAS-STING signals and plays a regulatory role in anti-acute viral infection.

In acute infection, neutrophils can produce reactive oxygen species (ROS) and express chemokine C-X-C motif receptor (CXCR2).  Neutrophils can regulate NET formation through protein-arginine deaminase type 4 (PAD4). Therefore, the expression levels of PAD4, ROS and CXCR2 were significantly up-regulated during acute influenza virus infection. This suggests that PAD4-dependent ROS/CXCR2 signaling pathway plays a regulatory role in regulating the formation or NET after virus infection.

Bioinformatics transcriptome analysis was used to analyze the changes of neutrophils in bronchoalveolar lavage fluid of infected mice. It was found that the expression of Piezo1 was significantly increased. We generated Piezo1 myeloid conditional knockout mice (Piezo1^-/-) for study. In vitro experiments showed that Piezo1 agonist (Yoda1) treatment could significantly up-regulate the expression of Piezo1 in neutrophils and promote ROS production and CXCR2 expression, as well as NET formation. Depletion of Piezo1 significantly down-regulated Piezo1 expression in neutrophils and inhibited ROS production and CXCR2 expression, as well as NET formation in neutrophils. These results suggest that Piezo1 plays an important role in regulating neutrophil function and NET formation.

The expression of TLR7 on neutrophils increased significantly after influenza virus infection and the expression of Piezo1 and TLR7 was co-localized. Furthermore, we analyzed the key genes regulating these important functions by the transcriptomics of bronchoalveolar lavage fluid neutrophils after viral infection and screened 18 key molecules. The screening found that sirtuin2 (SIRT2) and hypoxia induced factor 1α (HIF1α) may play a regulatory role in the regulation of neutrophil function and NET formation by Piezo1 in acute virus infection. We generated SIRT2 and HIF1α myeloid conditional knockout mice (Sirt2^-/- and Hif1α^-/-) to carry out in-depth mechanism research. The results showed that ROS production, CXCR2 and PAD4 expression, as well as NET formation were significantly increased in neutrophils after acute viral infection. Although the upregulation of Piezo1 by Yoda1 significantly promoted the up-regulation of neutrophil ROS, CXCR2 and PAD4 and NET formation, SIRT2 or HIF1α deficiency significantly reversed the changes in neutrophil function and NET formation. This suggests that SIRT2 or HIF1α can regulate Piezo1 in neutrophils during acute viral infection.

In conclusion, in this study, with the mouse model of acute influenza virus infection, it was found that acute influenza virus infection triggered neutrophil Piezo1 to regulate the functional differentiation of inflammatory macrophages. NET plays an important regulatory role in anti-infection. NET-DNA components direct the functional differentiation of inflammatory macrophages through macrophage TLR9 and cGAS-STING signals. Viral infection triggers TLR7 and Piezo1 on neutrophils and regulates the expression of PAD4, ROS and CXCR2, as well as the formation of NET through the SIRT2/HIF1α signaling pathway. This study provides an experimental basis for further understanding the regulatory effect of neutrophils on the functional differentiation of macrophages during acute viral infection.