The removal of excessive neutrophil extracellular traps is helpful for delaying the occurrence of chronic obstructive pulmonary disease

doi:10.21203/rs.3.rs-4657470/v1

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The removal of excessive neutrophil extracellular traps is helpful for delaying the occurrence of chronic obstructive pulmonary disease

https://doi.org/10.21203/rs.3.rs-4657470/v1

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Neutrophil extracellular traps (NETs) are elevated in peripheral blood and sputum in patients with COPD, but the effect of NETs on the occurrence of COPD is unknown. There is no effective prevention method for chronic obstructive pulmonary disease (COPD). In order to investigate the role of removing excessive NETs in preventing the occurrence of COPD, we evaluated a mouse model of COPD and the delaying effect of removing excessive NETs by aerosolised inhalation of DNase I. We found that a mouse model of COPD could be successfully established after 12 weeks of LPS + CS intervention; LPS + CS exposure produced excess NETs; DNase I nebulised inhalation was effective in reducing NETs levels; and removal of excessive NETs reduced apoptosis and microthrombus formation in lung epithelial cells, attenuated systemic and pulmonary inflammatory responses, and attenuated lung pathological changes and loss of lung function. These data demonstrate for the first time that removal of excess NETs can effectively protect lung function and delay the onset of COPD.

Health sciences/Diseases/Respiratory tract diseases/Chronic obstructive pulmonary disease

Health sciences/Medical research/Experimental models of disease

Health sciences/Medical research/Pre clinical studies

Neutrophil extracellular traps

delay

Chronic obstructive pulmonary disease

occurrence

nebulizer inhalation

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death and disability worldwide(1). Although COPD is a heterogeneous disease, it is characterized by persistent airflow obstruction and respiratory symptoms (2). Its main causes include tobacco smoke, air pollutants, infection, heredity, development and social environment (3). As the population aging is getting enlarged in many countries and regions, the social burden of COPD is gradually increasing (4). Because most COPD patients have developed irreversible severe lung diseases at diagnosis, palliative approaches are adopted mostly, which can not cure the disease and eliminate social problems of the patients. Therefore, it is critical and practical to adopt effective measures to delay the occurrence of the disease before it becomes serious and irreversible (5).

There are many driving factors involved in the development of COPD, one of which is inflammatory imbalance. It is widely accepted that neutrophils play an important role in inflammatory imbalance as well as in the occurrence of COPD (6, 7). As DNA complexes released by neutrophils into the extracellular space, neutrophil extracellular traps (NETs) have received increasing attention for its role in diseases. NETs are not only associated with infectious diseases, but are also closely related to a variety of non-infectious diseases (8, 9). NETs released by neutrophils in response to external harmful stimuli, have been indicated in killing the pathogenic microorganisms (10). However, prolonged neutrophil activation and excessive NETs production can cause damage to normal tissues (11). Recent clinical studies have found that NETs in the blood and sputum of patients with stable and acute exacerbations of COPD are increased compared with the normal population (12), suggesting that NETs may be associated with the development of COPD, but the relationship between NETs and the occurrence of COPD is still unknown.

Therefore, we speculate that NETs may play an important role in the occurrence of COPD and can cause persistent damage to the lungs. To test this hypothesis, we generated a mouse model by imitating the conditions of COPD and using LPS + CS, and tested the intervention treatment using nebulized inhalation of DNase I to investigate the effect of NETs on the lungs and the benefits of removing excessive NETs.

2.1. Mice and Grouping

We purchased 7-week-old male C57BL/6 mice, weighing 18–22 G, from the Animal House of Army Medical University of the PLA and raised them for 1 week for adaptation to the environment. Mice were housed at a constant temperature of 25 ℃ with a 12-hour dark/light cycle and had free access to standard pelleted rat chow and water. The mice were randomly categorized into 4 groups with 5 mice for each group: (1) control group: the mice of the right age were stored in an independent cage in the SPF animal room without any intervention, and they could move freely and drink water normally; (2) LPS + CS group: the mice were exposed to smoke after intratracheal injection of LPS on time according to the modeling method; (3) LPS + CS + DNase I group: the mice were given aerosol inhalation of DNase I 1 H after daily exposure to smoke; and (4) DNase I group: the mice were treated with aerosolized DNase I without other interventions.

2.2. Molding

LPS plus CS was used for modeling (13, 14). LPS (2 mg/Kg) was given by intratracheal administration on days 1 and 14 with no cigarette smoke exposure on the day of administration. In the rest of time the mice were placed in a 50cm*35cm*30cm Plexiglas box for twice daily cigarette smoke (tar 10 mg, smoke nicotine 1.0 mg, smoke carbon monoxide 9 mg from Chongqing Tobacco Industry Co., Ltd. of China) exposure for 12 weeks(2 cigarettes/12 min, 60 min/time)(15, 16). We anaesthetised each group of mice once every fortnight with 1% sodium pentobarbital and then performed lung function tests on each group of mice.The mice after the lung function tests were euthanised by intraperitoneal injection of a high dose of sodium pentobarbital, whereas the mice were perfused and the lung tissues, bronchoalveolar lavage fluid (BALF) and peripheral blood were collected for further analyses.

2.3. Atomization inhalation

One hour after each smoke inhalation, the mice were fixed with a mouse atomization inhalation device for oronasal inhalation atomization. We performed endpoint analysis in mice after 12 weeks (17) of continuous nebulized DNase I (40 IU/animal, 200 IU/ml, Roche).

2.4. HE Staining

After modeling, the mice were anesthetized and perfused, and then the lung tissue of the mice was fixed in 4% neutral formalin solution. The fixed tissues were dehydrated and paraffin-embedded, sectioned (3 µm), stained with hematoxylin-eosin (HE), and sealed with neutral resin after drying. Olympus microscope was used to observe and record the pathological changes of lung tissue in mice.

2.5. Masson Staining

Paraffin sections of lung tissue were stained with Masson’s trichrome to observe the precipitation of fibrin in lung parenchyma and respiratory tract, which was used to compare the pathological changes of lungs and evaluate the severity of fibrosis (18) .

2.6. Pathological Score

A cross line was drawn in the center of each visual field, and the number of alveolar intervals (Ns) intersecting the cross line and the number of alveoli (Na) in each visual field were calculated. At the same time, the total length of the cross line (L) and the area of each visual field (S) were measured. Lung tissue mean linear intercept (MLI) and mean alveolar number (MAN) were calculated according to the following formula: MLI = L/Ns (its value reflects the mean alveolar diameter), and MAN = Na/S (its value reflects the alveolar density) (19) .

2.7. Pulmonary function

The mice were anesthetized with 1% pentobarbital sodium and tracheostomy was performed. The tracheal cannula of the anesthetized mice was connected to a pulmonary function detector (FlexiVent; SCIREQ, Montreal, Quebec, Canada), and mice were examined for lung function (FEV_0.1 /FVC%) using the FlexiVent FX-Mouse NPFE model.

2.8. Peripheral Blood Plasma Collection

After the mice were anesthetized, the hearts of the mice were exposed to air, and blood was collected from the right ventricle of the mice with a 1ml insulin syringe, centrifuged (4 ℃, 3000 rpm, 10 min) and stored in a refrigerator at -80 ℃ for further analysis (20–23) .

2.9. Collection of BALF

After anesthesia, the trachea of the mouse was separated, the mouse tracheal cannula was fixed in the trachea with a surgical suture, and 1 ml of refrigerated PBS (4 ℃) was drawn with a 1 ml syringe and injected into the lungs for repeated washing for 3 times (24, 25). The collected bronchoalveolar lavage fluid was centrifuged in a centrifuge tube (4 ℃, 3000 rpm, 10 min), and the supernatant was stored in a refrigerator at -80 ℃ for quantitative analysis of NETs and inflammatory factors.

2.10. Immunofluorescence

The free lung tissue was fixed with 4% paraformaldehyde for 24 h and dehydrated with 30% sucrose for 24 h, and then the lung tissue was sectioned (thickness 8 µm). The sections were collected with pathological slides and treated with 0. 5% Triton X-100 (PBS) for 0. 5 h. After blocking with goat serum at room temperature for 2 h, primary antibodies (Anti-CitH3 (1:200; ab5103; Abcam), Anti-Ly6G (1:200; ab25377; Abcam), Anti-Fibrinogen (1:400; Abcam; ab119948)) were used for incubation overnight at 4℃, followed by incubation with secondary antibodies (Alexa Fluor 488 goat anti-rat antibody (1:400; Thermo Fisher Scientific), Alexa Fluor 555 goat anti-rabbit antibody (1:400; Thermo Fisher Scientific), and Alexa Fluor 647 (1:400; Beyotime)) at room temperature for 2 h, and then DNA was stained with 4 ′, 6-diamidino-2-phenylindole (DAPI) (26). Finally, the sections were visualized using a confocal laser scanning microscope (Zeiss 880, Germany).

2.11. Extraction of peripheral blood neutrophils

Extraction of mouse peripheral blood neutrophils We used Solarbio Mouse Peripheral Blood Neutrophil Extract (P9202). Firstly,2ml of fresh anticoagulant mouse blood was mixed with blood diluent and erythrocyte sedimentation at a 1:1:1 ratio and allowed to stand for 30min at room temperature until the supernatant was aspirated and used. Separation reagents A, C and cell suspension were added by gradient density centrifugation at 2:1:1, centrifuged at room temperature (800g, 25min), and the neutrophil layer was removed. The cell wash solution was resuspended and centrifuged (250g, 10min), repeated twice, and then resuspended in DMEM for later use.

2.12. Induction of NETs in vitro

LPS (100ul, 200ug/ml, Sigma-Aldrich) was added to the resuspended neutrophils after resuspension in DMEM medium and co-cultured for 4 h (27) at 37 ℃ in an incubator. The supernatant was taken after centrifugation (3000rpm, 3min).

2.13. TUNEL stain

Pulmonary epithelial cells were categorized into five groups after subculture: PBS group, LPS + N group, LPS + N + DNase I group, LPS group, and DNase I group. One-Step TUNEL kit (C1090, Beyotime) was used for TUNEL detection. Cells were cultured in confocal dishes for 24 h, then fixed with 4% paraformaldehyde for 30 min at room temperature, punched with 0. 5% Triton at room temperature for 10 min, and stained with 50 µl TUNEL staining solution at 37 ℃ for 60 min. A confocal laser scanning microscope (Zeiss 880, Germany) was used for observation and photo taking. Cells were quantified using ImageJ software (28).

2.14. ELISA

NETs, IL-1β, and IL-6 were captured and measured by sandwich assay using the mouse ELISA kit. According to the manufacturer's instructions, the absorbance (OD value) was measured at 450 nm with a microplate reader, and the concentrations of NETs, IL-1β and IL-6 in bronchoalveolar lavage fluid and peripheral blood plasma of mice were calculated by standard curves (29) .

2.15. Statistical analysis

Data of difference analysis were expressed as mean ± SD (standard deviation). Statistical analysis was performed by one-way ANOVA using GraphPad Prism 9. Multiple comparisons were used to compare any two sets of data or one set of data with each other separately. Significant differences between groups were indicated by * (P < 0.05), * * (P < 0.01), * * * (P < 0.001), or * * * * (P < 0.0001). The data of each time point were analyzed and the average value was taken, and the modeling graph and equation were obtained by curve fitting method, and the equation was used to calculate the results.

2.16. Study Approval

All experimental procedures were approved by the Ethics Committee of the Army Medical University (Third Military Medical University) (AMUWEC20207012) and were performed in accordance with the guidelines in the NIH Guide for the Care and Use of Laboratory Animals and complied with the Arrival Guidelines. A total of 225 adult male C57BL/6 mice, weighing 18–22 g, were provided by the Experimental Animal Center of Army Medical University.

2.17. Data availability

All data generated or analysed during this study are included in this published article [and its supplementary.

3.1. The process of COPD occurrence is accompanied by an excessive amount of NETs

During the 12 weeks of modeling, we sampled and detected the NETs in peripheral blood and bronchoalveolar lavage fluid of the mice every 2 weeks, and used ELISA kit to quantitatively analyze the NETs content in peripheral blood and bronchoalveolar lavage fluid of mice. We found that the levels of NETs in plasma and bronchoalveolar lavage fluid increased rapidly after LPS + CS intervention. During the whole process of modeling, the levels of NETs in plasma and BALF in the LPS + CS group were high and maintained within a certain range of fluctuations, without a significant downward or upward trend. The level of NETs in the Control group was maintained at a low level with no significant change. At each time point, NETs concentrations between two groups did not differ from each other significantly (Fig. 2. A, B). At the same time, immunofluorescence staining of NETs in the lung tissue of mice was conducted to find the visual evidence of the presence of NETs in the lungs. We observed the presence of NETs in the lung tissue sections of mice at all time points in the LPS + CS group, and these NETs were scattered or aggregated in the alveoli or pulmonary interstitium. By the end of 2 weeks, the density of neutrophils infiltrating into the lungs was higher in some areas and only a small portion of neutrophils released NETs. The density of infiltrated neutrophils gradually decreased and the proportion of neutrophils releasing NETs gradually increased over time. At 12 weeks, almost all neutrophils infiltrating into the lung tissue released NETs. No evidence for the presence of NETs was observed in the control group (Fig. 1. A-F).

3.2. DNase I nebulized inhalation can effectively remove excessive NETs

We observed the release of abundant NETs in the lungs of LPS + CS mice at different time points through immunofluorescence staining of Ly6G, CitH3, and DNA. At the same time, we also stained the lung tissue sections of LPS + CS + DNase I group mice after DNase I atomization treatment, finding that the neutrophil infiltration density and NETs positive number in the lungs of LPS + CS + DNase I group mice were lower than those of LPS + CS group at each time point. The neutrophils that did not release NETs still accounted for a large proportion even at 12 weeks. NETs were not found in the Control and DNase I groups (Fig. 1.A-F). We used the above method to obtain the supernatant of BALF after centrifugation and peripheral blood plasma of mice, and used ELISA kit to quantitatively analyze soluble NETs. The results showed that NETs concentration in BALF and peripheral blood plasma of mice in LPS + CS group increased rapidly after LPS + CS induction and were significantly higher than that in other groups at all time points, while NETs concentration in LPS + CS + DNase I group decreased significantly at 2 weeks after nebulization of DNase I. After that, although the data of each time node fluctuated in a small range, they were lower than those of LPS + CS group and no obvious change trend was observed, and the NETs concentrations of the two groups were significantly different at each time point. NETs concentrations in Control and DNase I groups were lower than those in LPS + CS + DNase I group and remained at lower levels (Fig. 2.A, B).

3.3. Removal of excessive NETs reduces apoptosis of lung epithelial cells

The experiment was divided into: PBS group, LPS + N group, LPS + N + DNase I group, LPS group and DNase I group. We found a large number of TUNEL-positive signals in the LPS + N group, while the number of TUNEL-positive signals in the LPS + N + DNase I group was significantly reduced, and the number of TUNEL-positive cells in the LPS group was also significantly lower than that in the LPS + N group. There were no obvious TUNEL-positive cells in PBS group and DNase I group (Fig. 2. C). Statistical analysis of the cells in each group also showed that the number of TUNEL positive signals in LPS + N group was the largest, while that in LPS + N + DNase I group and LPS group was lower, and there were significant differences between LPS + N group and other groups (Fig. 2. D).

3.4. Removal of excessive NETs prevents pulmonary microthrombosis

The experimental results showed different sizes of microthrombi in the lung tissue of mice in the LPS + CS group, and a large number of NETs and fibrin clots embedded in them could be clearly seen in the microthrombi. NETs in the microthrombi were at different stages, some of them were in the early stage of formation and were still confined to the membrane of neutrophils. The rest NETs were released to the outside of the cell. At the same time, we compared the immunofluorescence staining of lung sections of mice in LPS + CS + DNase I group, DNase I group and control group. Only a small number of NETs were found in the lung tissue of LPS + CS + DNase I group, and no microthrombus was found. NETs and microthrombi were not found in the control and DNase I groups (Fig. 2. E).

3.5. Removal of excessive NETs reduces pulmonary and systemic inflammatory response

By detecting IL-1β and IL-6 in bronchoalveolar lavage fluid and peripheral blood plasma collected at each time point of four groups of mice, we observed a high level of IL-1β and IL-6 in bronchoalveolar lavage fluid and peripheral blood plasma of mice in LPS + CS group at 2 weeks, and this level was maintained although there were fluctuations at each time point after 2 weeks. The levels of IL-1β and IL-6 in BALF and peripheral blood in LPS + CS + DNase I group were also slightly increased at 2 weeks, but were significantly lower than those in LPS + CS group, and were lower than those in LPS + CS group at other time points. The levels of IL-1β and IL-6 in BALF and peripheral blood plasma in Control group and DNase I group remained at a low level with no significant change. The levels of IL-1β and IL-6 in BALF and peripheral blood of mice in LPS + CS group were significantly different from those in other groups at each time point. (Fig. 3.E-H).

3.6. Removal of excessive NETs can reduce the pathological changes of the lung and delay the occurrence of COPD

HE and Masson staining of the lung tissue of mice in each group revealed that the lung tissue of mice in LPS + CS group gradually showed obvious pathological changes like COPD, with the highest degree of fibrosis. Although the LPS + CS + DNase I group also gradually showed alveolar septal rupture, alveolar fusion, inflammatory cell infiltration and fibrosis, but compared with the LPS + CS group at each time point, the lung texture was cleaner, relatively more complete alveolar structure was preserved, and the fibrosis was also significantly alleviated (Fig. 3. A, B). Pathological analysis gradually showed that the MLI values in LPS + CS group and LPS + CS + DNase I group increased, but the MLI values in LPS + CS group increased more significantly and were greater than those in LPS + CS + DNase I group at each time point. The data of Control group and DNase I group remained stable without significant change, and there were significant differences between LPS + CS group and other groups by the end of 12 weeks (Fig. 3. C). MAN in LPS + CS group and LPS + CS + DNase I group showed a significant decreasing trend, but the value in LPS + CS group decreased more significantly and was lower than that in LPS + CS + DNase I group at each time point. The data of Control group and DNase I group remained stable without significant change。 There were significant differences between LPS + CS group and other groups by the end of 12 weeks (Fig. 3.D). It should be noted that we recorded the lung function test results of mice every two weeks, established the prediction model by processing and analyzing the data, and obtained the trend chart, model chart and equation. The test results showed that the FEV_0.1 /FVC% value of LPS + CS group and LPS + CS + DNase I group showed a significant downward trend. FEV_0.1 /FVC% decreased most rapidly in LPS + CS group, and the decline rate of FEV_0.1 /FVC% was significantly slower in LPS + CS + DNase I group, and the pulmonary function in LPS + CS + DNase I group was better than that in LPS + CS group at all time points. By the end of the 12-week intervention, the mean pulmonary function of mice in the LPS + CS group decreased to below 70%, while the mean pulmonary function of mice in the LPS + CS + DNase I group was more than 70%. The lung function of Control group and DNase I group remained above 90%, and there was no obvious change trend. There were significant differences between LPS + CS group and other groups(Fig. 3.I). By solving the equations corresponding to the LPS + CS group and the LPS + CS + DNase I group, we found that the occurrence of COPD in the LPS + CS + DNase I group was delayed by about 19.2 days compared with the LPS + CS group after 12 weeks of nebulized DNase I treatment (Fig. 4. A,B).

COPD cannot be cured, but can be prevented (3, 13). In addition, as a chronic disease, its prevention is especially important. The previous studies on the pathogenesis of COPD have identified the important role of neutrophils in the pathogenesis of COPD long ago (7); however, neutrophils are a double-edged sword (13). As activated neutrophils can kill pathogenic microorganisms and normal cells indiscriminately, it is necessary to reduce its collateral damage to normal lung tissue.

Firstly, in order to verify whether NETs are closely associated with COPD, we took samples every two weeks during the modeling process. Assessment by ELISA on peripheral blood plasma and BALF supernatant showed that NETs concentration in LPS + CS group was significantly higher than that in control group at all time points, consistent with the results of previous clinical tests (12, 14). The presence of excessive NETs throughout the modeling intervention suggests a close association between NETs and COPD. To have a clear understanding of the generation of NETs in the lungs during the modeling intervention, we performed immunofluorescence staining of the lung tissues in the LPS + CS group at each time point. The staining showed that there were more NETs in the lung tissues of mice in the LPS + CS group at each time point, and the NETs were scattered or aggregated in the pulmonary interstitium and alveolar cavity; however, no NETs were released in the control group. Previous studies have shown that NETs are abundant in the lungs of mice with pulmonary fibrosis (15), acute lung injury (16), and lung transplantation (17), and that these excessive NETs can promote disease progression. Consistent with these studies, we also found that NETs are abundant in the lungs of COPD mice by immunofluorescence of tissue sections. The spatial distribution of NETs was similar to that of mice with pulmonary fibrosis and bacterial pneumonia (18). To sum up, both qualitative and quantitative analyses in our study proved that NETs are closely associated with the occurrence of COPD.

Currently, peptidyl arginine deiminase 4 (PAD4) inhibitors (40) and deoxyribonuclease I (DNase I) are commonly used for removing NETs (19). At present, DNase I aerosol inhalation has been used in the clinical treatment of cystic fibrosis, which is proved to be safe and effective(20–23). Aerosol inhalation is also a common and non-invasive method for clinical treatment of respiratory diseases. Therefore, considering safety and clinical practicability, we chose aerosol inhalation of DNase I to remove excessive NETs in mice. To verify the effectiveness of the method, we conducted sampling and analysis every 2 weeks. In lung tissue sections, we found that more NETs were released in LPS + CS group at each time point, and the positive signals of NETs in LPS + CS + DNase I group were significantly reduced compared with those in LPS + CS group at each time point, and NETs were not found in control group and DNase I group. In addition, we also found that the increased proportion of NETs was accompanied by the aggravation of lung tissue injury and the gradual decline of lung function. The proportion of NETs-positive neutrophils in neutrophils seemed to reflect the severity of the disease. The lower proportion of NETs-positive neutrophils in LPS + CS + DNase I group also proved that a higher proportion of NETs-positive neutrophils infiltrating into tissues is associated with more severe disease progression. It should be noted that the density of neutrophil aggregates and infiltrates in lung tissue decreases over time, which may be explained by the gradual depletion of neutrophils, involving factors such as long-term inflammation (24) and excessive nutrient consumption (25). At the same time, NETs in the peripheral blood plasma and bronchoalveolar lavage fluid of the mice were quantitatively analyzed. It was found that the NETs concentration in the peripheral blood plasma and bronchoalveolar lavage fluid of the LPS + CS + DNase I group was significantly lower than that of the LPS + CS group at each time point. The concentrations of both Control and DNase I groups were low and similar with each other. To sum up, both the visual display by immunofluorescence and the quantitative analysis of ELISA confirmed that nebulized DNase I could effectively remove excessive NETs.

To verify whether removal of excessive NETs can delay the occurrence of COPD, we also used a variety of methods (immunofluorescence, HE, Masson, pulmonary function testing) to explore the effect of removing excessive NETs on the delay of COPD progression structurally and functionally. The results showed that the removal of excessive NETs could effectively reduce the apoptosis of pulmonary epithelial cells, eliminate the formation of microthrombosis, reduce the pulmonary and systemic inflammatory response, delay the pathological changes of lung tissue and the decline of lung function, and effectively delay the occurrence of COPD. Previous studies have shown that the complex components of NETs are cytotoxic and can promote apoptosis (26, 27). Whether excessive NETs produced by lung tissue can aggravate apoptosis in lung tissue is unknown. Therefore, we extracted peripheral blood neutrophils from normal mice. LPS was used to induce neutrophils to produce NETs, and the prepared NETs were added to lung epithelial cells for co-culture. The results showed that LPS + N group had stronger cytotoxicity. Quantitative analysis showed that the number of TUNEL positive signals in LPS + N group was the largest, and that of TUNEL positive signals in LPS + N + DNase I group was significantly reduced, and that of TUNEL positive signals in LPS group was far less than that in LPS + N group. No TUNEL positive signal was found in the other two groups. This result effectively proves that NETs have strong cytotoxicity and can promote apoptosis of lung epithelial cells, and DNase I can effectively reduce the apoptosis of lung epithelial cells by removing excessive NETs. This is extremely important in the self-repair of lung tissue. Apoptosis of a large number of cells in a short time will make the new epithelial cells unable to make up for the loss of existing epithelial cells in time, thus aggravating the intensity of inflammatory response and forming a vicious circle (13), and resulting in structural and functional damage. However, the removal of excessive NETs effectively reduces the apoptosis of cells, gains more time for tissue repair, facilitates the normal tissue repair, and undoubtedly delays the occurrence of organic lesions in lung tissues. In addition, excessive NETs can induce a hypercoagulable state of the blood(28, 29). Microthrombi are formed by activating coagulation factors (30)and platelets(31–34) and providing scaffolds for platelet recruitment (31), and activated platelets can promote the formation of NETs (35). NETs have been found to be associated with microthrombi in brain injury (36), autoimmune diseases (8), and COVID-19 (33), and our experiments also support the idea that excessive NETs are often associated with microthrombi. In the lungs of the LPS + CS group, we observed some microthrombi of different sizes. Although there were a small amount of NETs in the LPS + CS + DNase I group, no microthrombi were observed. Similar results were not observed in the other two groups. These multiple and persistent microthrombi can aggravate the hypoxia and inflammatory reaction of local tissues, and affect the ability of local tissue repair. Although this damage may be local and minor, long-term persistent damage and abnormal repair will inevitably bring about qualitative change, aggravating the pathological changes of lung tissue structure and accelerating the occurrence of COPD. The removal of excessive NETs by DNase I reduced and even eliminated these adverse effects, leading to the delay of COPD occurrence. Tissue destruction caused by excessive NETs will inevitably aggravate the inflammatory response, and whether removal of excessive NETs can alleviate the inflammatory response is unknown. Our study effectively demonstrated that removal of excessive NETs reduced pulmonary and systemic inflammation. ELISA analysis of peripheral blood and bronchoalveolar lavage fluid supernatant at different time points showed that IL-1β and IL-6 in LPS + CS group were at a high level, and the levels of IL-1β and IL-6 in LPS + CS + DNase I group decreased rapidly and significantly after aerosol inhalation of DNase I, lower than that of LPS + CS group at all time points. Previous studies have shown that activated macrophages and neutrophils secrete a large amount of IL-1β, which activates intracellular signaling pathways involving IRAK4, MK2 and NF-κB by binding to IL-R1 in the same or adjacent cells, eventually leading to further expression of inflammatory factors including IL-6 and playing a pro-inflammatory role (37). Removal of excessive NETs obviously inhibited the inflammatory response and weakened the recruitment and activation of neutrophils (31), which could well explain why the density of neutrophil infiltration and the number of NETs in lung tissues of LPS + CS + DNase I group was lower than that of LPS + CS group at different time points. The reduced inflammatory response must play a positive role in delaying the occurrence of COPD.

To display the beneficial effect of removing excessive NETs on lung tissue visually, we stained the lung tissue of four groups of mice with both HE and Masson. HE staining showed that the lung structure of mice in LPS + CS group was the most severely damaged at each time point, while the damage in LPS + CS + DNase I group was relatively alleviated, and the lung structure of the other two groups was normal without obvious pathological changes. The statistical analysis showed that the MLI of mice in LPS + CS group was the largest at each time point, and the upward trend was most obvious in the whole modeling process, while the MLI of mice in LPS + CS + DNase I group was reduced at each time point, and the upward trend was also significantly slowed down in the whole intervention process. The smallest MLI value was observed in the control group and DNase I group with no significant change. The values of MAN in LPS + CS group were the smallest at each time point, and the decreasing trend was the most obvious during the whole modeling period. The values of MAN in LPS + CS + DNase I group increased at each time point, and the decreasing trend was significantly slowed down. The values of MAN in Control group and DNase I group were the largest with no significant change in the whole process. Masson staining showed the most severe fibrosis around the small trachea and pulmonary interstitial at each time point in the LPS + CS group, while the fibrosis of LPS + CS + DNase I group was significantly reduced at each time point, with the difference between the two groups becoming more obvious over time. Abnormally increased fibrosis was not observed in either Control or DNase I group. These results are consistent with the patterns of pathological changes in the occurrence of COPD (38), indicating that DNase I nebulization can effectively delay the pathological changes of normal lung tissue and nebulization of DNase I alone in normal mice will not change the normal structure of the lung. The cytotoxicity of excessive NETs can induce apoptosis of a large number of epithelial cells (26, 27). A lack of timely recruitment of a sufficient number of new epithelial cells will inevitably lead to the destruction of alveolar structure. The removal of excessive NETs maintains alveolar structure when alleviating the loss of epithelial cells, which also explains the presence of more intact alveoli in the LPS + CS + DNase I group at all time points. Previous studies have shown that the presence of excessive NETs can also promote the transformation of smooth muscle cells and macrophages into fibroblasts (30, 39), which is supported by the findings of our study. The differentiation of fibroblasts leads to the production of more fibrin around the small trachea and in the pulmonary interstitium, which exceeds the phagocytic capacity of phagocytes (40, 41) and eventually deposits in the small trachea and the pulmonary interstitium, aggravating the fibrosis of the lung, affecting the ventilation effect of the lung, aggravating tissue hypoxia and affecting tissue repair; thus, contributing to the occurrence of COPD. After excessive NETs were removed, the progress of fibrosis was inhibited to a certain extent, and many negative effects were eliminated, which was of significance for the repair of lung tissue. The alleviation of fibrosis around the small trachea also preserved the diastolic function of the small trachea and alleviated the ventilation disorder.

Most importantly, we established the prediction model of mouse lung function FEV_0.1/FVC% change by analyzing the lung function data at each time node, and obtained the model diagram and equation. Through modeling, we found that the pulmonary function of mice in the LPS + CS + DNase I group was significantly better than that in the LPS + CS group at the same time point, and the pulmonary function of mice in the Control group and DNase I group remained above 90% with no significant change. Based on the equation, we used FEV_0.1 /FVC% ≤ 70% as the cutoff value to calculate the time required for FEV_0.1 /FVC% to decrease to 70% in each group of mice. The Results showed that the time for FEV_0.1 /FVC% to decrease to 70% in LPS + CS + DNase I group was delayed by 19.2 days compared with LPS + CS group. According to the conversion between mouse age and human age and mice, puberty of mice lasts for four weeks (6–10 weeks) and adulthood from 10 to 20 weeks. During our experiment, mice went through 2 weeks of puberty and 10 weeks of adulthood. One day in puberty for mice was equivalent to 99.95 days for human, and one day in adulthood in mice was equivalent to 104.3 days for human; therefore, when the delayed period was converted to human age, it was about 5.44 years (42), which confirms that the removal of excessive NETs by DNase I atomization inhalation has a significant effect on delaying the occurrence of COPD, shedding light on preventing the occurrence of COPD.

Our study has several limitations. First, the relationship between the proportion of NETs-releasing neutrophils among all the neutrophils and disease severity was not confirmed. Second, although we have confirmed that NETs are closely associated the occurrence of COPD, and observed that excessive NETs can affect the occurrence of COPD by inducing apoptosis and microthrombosis, the mechanism is still unclear, which still requires future research. Finally, whether the same effect can be achieved in humans still needs to be verified by further clinical trials.

In conclusion, we have demonstrated for the first time to our knowledge that removal of excessive NETs can delay the occurrence of COPD by effectively inhibiting lung cell apoptosis and microthrombosis, and removal of excessive NETs has a good effect on delaying the occurrence of COPD. Although COPD cannot be cured, delaying the its occurrence by removing excessive NETs is helpful for COPD prevention.

BALF

Bronchoalveolar lavage fluid

Cigarette smoke

COPD

Chronic obstructive pulmonary disease

LPS

Lipopolysaccharide

NETs

Neutrophil extracellular traps

DECLARATION OF INTERESTS

The authors declare no competing interests.

Author Contribution

Wu Li carried out the research design, literature search, data organisation, data collection, data analysis, data interpretation, writing-original drafts, reviewing, and editing; XiaoKe Hao carried out the methodology, survey, literature search; Lei Xue participated in data collation, data collection, and validation; Qiang Zeng participated in data collation, data collection, and validation; Linglin Liu participated in experimental supervision; Cheng Liang participated in data collection and some experimental operations; Weijia Zhou,Yunhua Liu participated in data collection; Xiaotian Dai was responsible for research design, writing-review and editing, data interpretation, and project management; Wei Xiong was responsible for research design, writing-review and editing, and project management; Guohong Deng was responsible for obtaining funds, supervision, and project management.

ACKNOWLEDGMENTS

This study was supported by the 2018 National Key Research and Development Programme (2018YFC2000301) and Chongqing Talent Programme (CQYC20200303153).

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No competing interests reported.

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The removal of excessive neutrophil extracellular traps is helpful for delaying the occurrence of chronic obstructive pulmonary disease

Status:

Version 1

Abstract

Figures

1. Introduction

2. Methods

2.1. Mice and Grouping

2.2. Molding

2.3. Atomization inhalation

2.4. HE Staining

2.5. Masson Staining

2.6. Pathological Score

2.7. Pulmonary function

2.8. Peripheral Blood Plasma Collection

2.9. Collection of BALF

2.10. Immunofluorescence

2.11. Extraction of peripheral blood neutrophils

2.12. Induction of NETs in vitro

2.13. TUNEL stain

2.14. ELISA

2.15. Statistical analysis

2.16. Study Approval

2.17. Data availability

3. Results

3.1. The process of COPD occurrence is accompanied by an excessive amount of NETs

3.2. DNase I nebulized inhalation can effectively remove excessive NETs

3.3. Removal of excessive NETs reduces apoptosis of lung epithelial cells

3.4. Removal of excessive NETs prevents pulmonary microthrombosis

3.5. Removal of excessive NETs reduces pulmonary and systemic inflammatory response

4. Discussion

Abbreviations

Declarations

DECLARATION OF INTERESTS

Author Contribution

ACKNOWLEDGMENTS

References

Additional Declarations

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Version 1