目的：

铜绿假单胞菌是临床中常见的机会致病菌，是囊性纤维化患者气道内常见的致病菌，感染后严重者会出现肺功能下降甚至呼吸衰竭。也是细菌性角膜炎中常见的致病菌，感染后严重者会出现角膜溃疡穿孔甚至失明。已有研究表明这两种疾病中出现的严重后果主要是由于形成了难以清除的生物膜。中性粒细胞作为抵御病原体入侵的第一道防线，在肺部和角膜发生感染时会大量募集到感染部位发挥作用。目前的研究主要集中在对生物膜的表征和对铜绿假单胞菌免疫逃逸机制的探究，但是中性粒细胞与铜绿假单胞菌形成的生物膜之间的相互作用机制仍然不清楚。本研究旨在建立相应的模拟囊性纤维化和角膜炎的小鼠模型，并在这两种动物模型中探究中性粒细胞与铜绿假单胞菌生物膜的相互作用机制。探究敲除中性粒细胞的相关基因后对于中性粒细胞抵抗生物膜作用（促进作用、抑制作用或是没有影响）。探究生物膜抵御中性粒细胞杀伤的机制。探究清除铜绿假单胞菌生物膜的潜在治疗方式。

方法：

建立用琼脂糖包埋菌体外形成生物膜的方法；将体外形成的生物膜与分离的中性粒细胞进行体外共孵育观察二者的相互作用；体内则利用肺炎模型滴注包埋MPAO1菌株的琼脂糖珠，冲洗肺泡灌洗液、取肺制作冰冻切片和病理切片观察生物膜与中性粒细胞的相互作用；采用不能形成完整生物膜的敲除菌株MP-ΔPf4Pf6core做对照，肺炎模型中观察两种菌的琼脂糖包埋珠形成的生物膜与中性粒细胞的相互作用情况，以及对小鼠的影响；采用敲除中性粒细胞三种丝氨酸蛋白酶（中性粒细胞弹性蛋白酶（NE）、组织蛋白酶G（CG）和蛋白酶3（PR3））的敲除鼠，在肺炎模型中观察三种丝氨酸蛋白酶对中性粒细胞与生物膜相互作用的影响；采用同时敲除Caspase1和Caspase11、同时敲除GSDMD和GSDME以及只敲除GSDMD的全敲鼠，在肺炎模型中观察焦亡相关基因对中性粒细胞与生物膜相互作用的影响；建立角膜炎模型，观察该模型中生物膜的形成情况及生物膜与中性粒细胞的相互作用情况；采用不能形成完整生物膜的敲除菌株MP-ΔPf4Pf6core做对照，在角膜炎模型中观察两种菌形成的生物膜与中性粒细胞的相互作用情况以及对小鼠角膜炎严重程度的影响；采用敲除中性粒细胞GSDMD的条件敲除鼠，角膜炎模型中感染后的1d和7d分别观察中性粒细胞与生物膜的相互作用情况以及对小鼠角膜炎严重程度的影响；骨髓分离纯化的中性粒细胞或巯基乙醇酸（TG）诱导的腹腔中性粒细胞与体外琼脂糖包埋菌形成的生物膜共孵育后，Western Blot检测GSDMD的剪切情况，PI染色观察统计中性粒细胞死亡的情况；采用敲除GSDME的全敲鼠和敲除中性粒细胞GSDME的条件敲除鼠，角膜炎模型中感染后观察中性粒细胞与生物膜的相互作用情况以及对小鼠角膜炎严重程度的影响。

结果：

1）制作的包埋MPAO1菌株的琼脂糖珠在体外培养12 h可以形成生物膜，培养36 h后可以形成大量生物膜；

2）体外共孵育中性粒细胞聚集在包埋MPAO1菌株的琼脂糖珠形成的生物膜周围，体内肺炎模型中性粒细胞聚集在包埋MPAO1菌株的琼脂糖珠形成的生物膜周围，位置在气管；

3）肺炎模型中相比感染敲除菌株MP-ΔPf4Pf6core的包埋珠，感染形成完整生物膜的MPAO1菌株包埋珠小鼠的生存率明显更低。肺炎模型中相比感染敲除菌株MP-ΔPf4Pf6core的包埋珠，感染形成完整生物膜的MPAO1菌株包埋珠明显增加肺部菌载量、炎症因子水平、中性粒细胞募集和肺损伤；

4）肺炎模型感染游离MPAO1后，敲除NE、CG和PR3的小鼠肺部菌载量、肺泡灌洗液炎症因子水平、肺泡灌洗液总蛋白水平、肺部募集中性粒细胞数量都明显比野生型小鼠低。体内肺炎模型滴注包埋MPAO1菌株的琼脂糖珠后，敲除NE、CG和PR3的小鼠肺部菌载量、肺泡灌洗液炎症因子水平、肺泡灌洗液总蛋白水平、肺部白细胞数量和肺部中性粒细胞数量都明显比野生型鼠高；

5）肺炎模型感染游离MPAO1或包埋MPAO1琼脂糖珠，同时敲除Caspase1和Caspase11的小鼠肺部菌载量、肺泡灌洗液总蛋白水平、肺部募集中性粒细胞数量都明显比野生型小鼠高。肺炎模型感染游离MPAO1或包埋MPAO1琼脂糖珠，敲除GSDMD的小鼠生存率明显比野生型小鼠低。肺炎模型感染游离MPAO1，同时敲除GSDMD和GSDME的小鼠肺部菌载量明显低于野生型小鼠，生存率也明显比野生型小鼠高；肺炎模型感染包埋MPAO1琼脂糖珠，同时敲除GSDMD和GSDME的小鼠肺部菌载量明显高于野生型小鼠，生存率也明显比野生型小鼠低；

6）角膜炎模型角膜募集大量中性粒细胞且中性粒细胞聚集在铜绿假单胞菌生物膜周围；

7）角膜炎模型感染形成完整生物膜的MPAO1菌株后明显比感染敲除菌株MP-ΔPf4Pf6core后眼睛菌载量、炎症因子IL-1β水平、中性粒细胞数量和角膜炎严重程度高；8）角膜炎模型感染MPAO1一天时，敲除中性粒细胞GSDMD的小鼠眼睛菌载量、炎症因子水平、白细胞数量、中性粒细胞数量和角膜炎严重程度明显比同窝未敲除小鼠低。角膜炎模型感染七天时敲除中性粒细胞GSDMD的小鼠角膜炎严重程度、眼睛菌载量和外周血菌载量都明显低于同窝未敲除的小鼠；

9）体外包埋MPAO1菌株的琼脂糖珠形成的生物膜与骨髓分离纯化中性粒细胞共孵育后用Western Blot检测，不仅有全长GSDMD条带，也有剪切后的GSDMD-NT条带。共孵育后用PI阳性中性粒细胞比例远大于只有中性粒细胞的对照组。体外包埋MPAO1菌株的琼脂糖珠形成的生物膜与TG诱导的腹腔中性粒细胞共孵育后用Western Blot检测，不仅有全长GSDMD条带，也有剪切后的GSDMD-NT条带；

10）角膜模型感染MPAO1一天后，敲除GSDME的全敲鼠与野生型小鼠角膜炎严重程度、眼睛菌载量、炎症因子水平、总蛋白水平、白细胞数量、中性粒细胞数量和中性粒细胞死亡情况均未见明显差异。角膜炎模型感染MPAO1一天后，敲除中性粒细胞GSDME的小鼠角膜炎严重程度、眼睛菌载量、白细胞数量、中性粒细胞数量和中性粒细胞死亡情况与同窝未敲除小鼠相比均未见明显差异。

结论：

本研究首先发现中性粒细胞聚集在琼脂糖包埋菌形成的生物膜周围，中性粒细胞不能有效清除铜绿假单胞菌生物膜。体内肺炎模型滴注琼脂糖包埋菌不仅可以作为体内形成生物膜的模型，也可以模拟囊性纤维化患者体内病理，作为模拟囊性纤维化的小鼠模型。模拟囊性纤维化的肺炎模型中揭示了针对铜绿假单胞菌生物膜的宿主防御机制。肺炎模型中NE、CG和PR3三种丝氨酸蛋白酶可能通过水解生物膜或形成NETs在中性粒细胞对抗铜绿假单胞菌生物膜中发挥积极作用。肺炎模型中焦亡相关基因可能通过让细胞焦亡促进中性粒细胞对抗铜绿假单胞菌生物膜。建立的小鼠角膜炎模型可以研究生物膜与中性粒细胞的相互作用。角膜炎模型中揭示了铜绿假单胞菌针对中性粒细胞的免疫逃逸机制。角膜炎模型中铜绿假单胞菌生物膜通过刺激GSDMD剪切进而导致中性粒细胞焦亡逃避中性粒细胞的杀伤。角膜炎模型中GSDME不影响中性粒细胞与铜绿假单胞菌生物膜的相互作用。本研究为临床治疗囊性纤维化和角膜炎提供新的治疗方向。

Objective: 

Pseudomonas aeruginosa is a common opportunistic pathogen in clinical settings and a prevalent pathogen in the airways of cystic fibrosis patients, where severe infections can lead to decreased lung function and even respiratory failure. It is also a frequent causative agent of bacterial keratitis, with severe cases resulting in corneal ulceration, perforation, and even blindness. Existing research has shown that the severe consequences observed in these two diseases are primarily due to the formation of difficult-to-eradicate biofilm. As the first line of defense against pathogen invasion, neutrophils are massively recruited to the site of infection in the lungs and cornea to exert their effects. Current research primarily focuses on the characterization of biofilm and the exploration of P. aeruginosa's immune evasion mechanisms; however, the interaction mechanisms between neutrophils and P. aeruginosa biofilm remain unclear. We aim to establish corresponding mouse models that mimic cystic fibrosis and keratitis, and within these animal models, investigate the interaction mechanisms between neutrophils and P. aeruginosa biofilm. We will investigate the impact of knocking out specific neutrophil-related genes on their ability to combat biofilm (promotion, inhibition, or no effect). We will investigate the mechanisms by which biofilm resist neutrophil-mediated killing. Additionally, we will investigate potential therapeutic approaches for eradicating P. aeruginosa biofilm.

Methods:

We established a method to embed bacteria in agarose to form biofilm in vitro; the biofilm were then co-incubated with isolated neutrophils to observe their interactions. In pneumonia model, we intratracheally instilled MPAO1-laden agarose beads, and subsequently washed out the bronchoalveolar lavage fluid, harvested lungs for frozen sections and histopathological sections to observe the interactions between biofilm and neutrophils. As a control, we used a knockout strain, MP-ΔPf4Pf6core, which is unable to form complete biofilm, to observe the interactions between the biofilm formed by the MPAO1/MP-ΔPf4Pf6core-laden agarose beads and neutrophils, as well as their effects on mice, in the pneumonia model. Additionally, we employed knockout mice lacking three serine proteases (NE, CG, and PR3) in neutrophils to investigate the impact of these proteases on the interactions between neutrophils and biofilm in the pneumonia model. Using knockout mice with simultaneous deletion of Caspase1 and Caspase11, simultaneous deletion of GSDMD and GSDME, and mice with only GSDMD knocked out, the effects of pyroptosis-related genes on the interaction between neutrophils and biofilms were observed in a pneumonia model. Furthermore, we established a keratitis model to observe biofilm formation and the interactions between biofilm and neutrophils in this context. Using the MP-ΔPf4Pf6core strain as a control, we examined the interactions between the biofilm formed by the two strains and neutrophils, as well as their effects on the severity of keratitis in mice, in the keratitis model. We also utilized conditional knockout mice lacking GSDMD in neutrophils to observe the interactions between neutrophils and biofilm, as well as their impact on the severity of keratitis, at one day and seven days post-infection in the keratitis model. After co-incubation of bone marrow-derived or TG-induced peritoneal neutrophils with biofilm formed by MPAO1-laden agarose beads, Western Blot was used to detect the cleavage of GSDMD, and PI staining was employed to observe and quantify neutrophil death. Finally, we used full knockout mice lacking GSDME and conditional knockout mice lacking GSDME in neutrophils to investigate the interactions between neutrophils and biofilm, as well as their effects on the severity of keratitis, in the keratitis model.

Results: 

1) The agarose beads embedded with the MPAO1 strain can form a biofilm after 12 hours of cultivation and a significant amount of biofilm after 36 hours of cultivation.

2) In vitro, neutrophils aggregate around the biofilm formed by MPAO1-laden agarose beads. In the pneumonia model, neutrophils also aggregate around the biofilm formed by MPAO1-laden agarose beads, located in the trachea.

3) In the pneumonia model, mice infected with agarose beads embedded with the MPAO1 strain that forms a complete biofilm had significantly lower survival rates compared to those infected with agarose beads embedded with the knockout strain MP-ΔPf4Pf6core. Furthermore, in the pneumonia model, infection with agarose beads embedded with the MPAO1 strain that forms a complete biofilm significantly increased lung bacterial load, inflammation factor levels, neutrophil recruitment, and lung damage compared to infection with agarose beads embedded with the knockout strain MP-ΔPf4Pf6core.

4) In the pneumonia model, after infection with free MPAO1, the lung bacterial load, levels of inflammatory cytokines in bronchoalveolar lavage fluid (BALF), total protein levels in BALF, and the number of recruited neutrophils in the lungs were significantly lower in mice knockout for NE, CG and PR3 compared to wild-type mice. After intratracheal instillation of agarose beads embedded with the MPAO1 strain in the pneumonia model, the lung bacterial load, levels of inflammatory cytokines in BALF, total protein levels in BALF, the number of leukocytes in the lungs and the number of neutrophils in the lungs were significantly higher in mice knockout for NE, CG and PR3 compared to wild-type mice.

5) In the pneumonia model infected with either free MPAO1 or MPAO1-laden agarose beads, mice with simultaneous knockout of Caspase1 and Caspase11 exhibited significantly higher lung bacterial loads, total protein levels in bronchoalveolar lavage fluid, and numbers of recruited neutrophils in the lungs compared to wild-type mice. In the pneumonia model infected with either free MPAO1 or MPAO1-laden agarose beads, mice with GSDMD knocked out had significantly lower survival rates than wild-type mice. In the pneumonia model infected with free MPAO1, mice with simultaneous knockout of GSDMD and GSDME had significantly lower lung bacterial loads and significantly higher survival rates compared to wild-type mice. However, in the pneumonia model infected with MPAO1-embedded agarose beads, mice with simultaneous knockout of GSDMD and GSDME had significantly higher lung bacterial loads and significantly lower survival rates compared to wild-type mice.

6) In the keratitis model, a large number of neutrophils are recruited to the cornea and aggregate around P. aeruginosa biofilm.

7) In the keratitis model, infection with the MPAO1 strain that forms a complete biofilm results in significantly higher eye bacterial load, IL-1β inflammatory cytokine levels, neutrophil counts and severity of keratitis compared to infection with the knockout strain MP-ΔPf4Pf6core.

8) One day after infection with MPAO1 in the keratitis model, mice with knockout of neutrophil GSDMD have significantly lower eye bacterial load, inflammatory cytokine levels, leukocyte counts, neutrophil counts and severity of keratitis compared to their littermate controls without the knockout. Seven days after infection in the keratitis model, mice with knockout of neutrophil GSDMD have significantly lower severity of keratitis, eye bacterial load and peripheral blood bacterial load compared to their littermate controls without the knockout.

9) In vitro, after co-incubation of the biofilm formed by agarose beads embedded with the MPAO1 strain with purified neutrophils isolated from bone marrow, Western Blot analysis revealed not only the full-length GSDMD band but also the cleaved GSDMD-NT band. The proportion of PI-positive neutrophils after co-incubation was significantly higher than that of the control group containing only neutrophils. Similarly, after co-incubation of the biofilm formed by agarose beads embedded with the MPAO1 strain with TG-induced peritoneal neutrophils, Western Blot analysis also showed both the full-length GSDMD band and the cleaved GSDMD-NT band.

10) One day after corneal infection with MPAO1, there were no significant differences in the severity of keratitis, eye bacterial load, inflammatory cytokine levels, total protein levels, leukocyte count, neutrophil count and neutrophil death between the full knockout mice with GSDME knocked out and the wild-type mice. Similarly, one day after corneal infection with MPAO1, there were no significant differences in the severity of keratitis, eye bacterial load, leukocyte count, neutrophil count and neutrophil death between the mice with neutrophil-specific GSDME knockout and their littermate controls without the knockout.

Conclusion: 

We firstly found that neutrophils aggregate around the biofilm formed by agarose-embedded bacteria, but neutrophils are unable to effectively clear the P. aeruginosa biofilm. The instillation of agarose-embedded bacteria in the pneumonia model not only serves as a model for biofilm formation in vivo but also mimics the pathology observed in cystic fibrosis patients, thus acting as a mouse model for cystic fibrosis. The host defense mechanism against P. aeruginosa biofilm has been revealed in the pneumonia model mimicking cystic fibrosis. In the pneumonia model, the three serine proteases NE, CG, and PR3 may play a positive role in neutrophils' combat against P. aeruginosa biofilm by hydrolyzing the biofilm or forming NETs. Pyroptosis-related genes in the pneumonia model may promote neutrophils' resistance against P. aeruginosa biofilm by inducing cell pyroptosis. The established mouse keratitis model allows for the investigation of the interaction between biofilm and neutrophils. In the keratitis model, the bacterial immune evasion mechanism targeting neutrophils has been revealed. The P. aeruginosa biofilm in the keratitis model stimulates the cleavage of GSDMD, subsequently leading to neutrophil pyroptosis and evading neutrophil-mediated killing. GSDME does not affect the interaction between neutrophils and P. aeruginosa biofilm in the keratitis model. This research provides new therapeutic directions for the clinical treatment of cystic fibrosis and keratitis.